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Energy Metabolism and Autism: The Ameliorative Potential of Carnosine and Agmatine

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DOI: 10.31038/JNNC.2018111

Abstract

Recent studies have revealed that autistic spectrum disorders (ASD) is associated with enhanced glycolysis (i.e. establishment of the Warburg effect) accompanied by increased formation of glycated proteins in sera and urine. Both carnosine and agmatine levels in sera of autistic individuals are reported to be lower than in control subjects. Carnosine and agmatine can influence cellular energy metabolism, in part via effects on mTOR, thereby decreasing glycolysis and enhancing mitochondrial activity and thus countering onset of Warburg-like metabolism: other mechanisms including suppressing methylglyoxal toxicity are also discussed. Dietary supplementation studies with carnosine and arginine (agmatine precursor) indicate ameliorative activity towards behaviour in ASD subjects. It is suggested that co-administration of carnosine and agmatine should be explored as a potential route for ASD amelioration.

Key words

Carnosine, Agmatine, Autism, Glycolysis, Glycation, Mitochondria, Methylglyoxal, Propionic Acid, Rapamycin, Mtor

Introduction

A recent publication has suggested that Autism Spectrum Disorders (ASD) is accompanied, associated and/or related to changes in energy metabolism, more specifically the imposition of enhanced aerobic glycolysis, coupled with a suppression of mitochondrial ATP synthesis, also known as the Warburg effect [1]. Another recent paper has revealed the presence of elevated amounts of oxidized, nitrated and glycated proteins in the plasma of some ASD subjects, as well as a disturbance in arginine metabolism and/or clearance [2]. The objective of the present piece is to attempt to integrate these findings by highlighting the possible ameliorative roles of carnosine and agmatine (decarboxylated arginine), both of which are diminished in sera of some ASD subjects [3–5].

Energy metabolism and ASD

The Vallée and Vallée hypothesis [1] proposes that ASD is strongly associated with “a shift in energy production from mitochondrial oxidative phosphorylation to aerobic glycolysis – despite the availability of oxygen” i.e. the imposition of the Warburg effect. Plausible mechanistic routes proposed include the WNT/beta-catenin pathway, and activation of the regulatory complex PI3Akt/mTOR [1]. It is uncertain whether the induction of the predominantly glycolytic metabolism is caused primarily by dysfunction of the PI3AktmTOR regulatory complex, provoked perhaps by glycated protein (also called advanced glycation end-products i.e. AGEs) [6] or whether the imposition of the Warburg-type metabolism is a response to some other causative event or events, such as mitochondrial dysfunction. Indeed, it has been claimed that ASD is associated with mitochondrial dysfunction [7–10], and a three-fold decline in oxidative phosphorylation has been detected in ASD subjects’ granulocytes [10]. It would obviously be informative to determine if this deficit is systemic and also occurring in the CNS, or exhibited solely in granulocytes.

There is evidence suggesting that formation and/or accumulation of propionic acid is associated with some cases of ASD [11–13], possibly originating in the gut tissue or more likely in the microbiome (mostly Clostridia bacterial species) [14]. It is thought that, in the brain, propionic acid inhibits GABA breakdown causing its accumulation thereby affecting brain function. Interestingly, raised levels of β-alanine have been detected in the urine of some autistic subjects [4], whilst in other autistic individuals a decrease was detected [3] These observations, although seemingly contradictory, may reflect differences in β-alanine generation and utilisation by the micro-organisms in the gut. β-Alanine is a precursor pantothenic acid which, in turn, is a precursor of Co-enzyme-A (CoA) (synthesized by the gut micro-organisms). The microbiome is the predominant source of pantothenic acid in the human body. One speculative suggestion is that propionic acid accumulates as a result of a failure in its carboxylation, which requires functional CoA, hence propionic acid accumulates if synthesis of Co-A is compromised. General deficiency in CoA availability would also decrease fatty acid oxidation, as found in ASD [7,8,10,16,17]. The resultant mitochondrial dysfunction would have two important consequences which directly impact ASD. First, the decreased supply of electrons (i.e. acetyl units attached to CoA) will provoke an increase in the generation of incompletely reduced oxygen molecules, i.e. oxygen free-radicals [18], the presence of which will provoke formation of deleterious Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). Such a mechanism may account for the raised levels of protein oxidation and nitration recently detected in autistic patients’ urine and plasma [2]. A second consequence of insufficient mitochondrial ATP synthesis may be a compensating upregulation of glycolysis in an attempt to maintain ATP levels [1]. Indeed enhanced glycolysis accompanied by mitochondrial abnormalities have been detected in ASD subjects (compared to siblings and controls) [19]. Importantly, upregulated glycolysis would enhance generation of the highly toxic bicarbonyl compound, Methylglyoxal (MG), produced following spontaneous decomposition of the glycolytic intermediates, dihydroxyacetone-phosphate and glyceraldehyde-3-phosphate. MG is a strong glycating agent and well-recognised as a major source of the post-synthetic protein modifications which characterise both type-2 diabetes and ageing [20,21]. The notion that ASD is associated with enhanced MG generation is supported by the detection in some autistic subjects of gene polymorphisms in the MG detoxification enzyme, glyoxalase-1 [22–25], which could result in decreased MG elimination and increased macromolecular glycation. The increased glycolytic activity could therefore account for the raised levels of glycated proteins detected in autistic patients’ plasma and urine [2], especially if glyoxalase-1 activity was insufficient to meet the increased generation of MG. However it must be pointed out that the suggestion that ASD is associated with glyoxalase dysfunction has been disputed [26,27]. Never-the-less, it is interesting that (i) changes in glyoxalase-1 expression in white blood cells seems to influence mood in human subjects [28], and (ii) it has recently been reported that erythrocytes of autistic boys possess lower levels of the detoxification enzyme retinal dehydrogenase (RALDH1), than was present in controls [29], observations which suggest that detoxification deficiency may influence behaviour in ASD individuals. The possibility that autism is associated with aldehyde toxicity generally and acetaldehyde in particular, has been proposed [30]. Furthermore, it has recently been shown that MG readily reacts with β-alanine [31], a reaction which would decrease pantothenate synthesis and further compromise formation of the CoA in the microbiome, as outlined above. Additionally the microbiome can also generate a well-studied neurotoxin, 3-Nitropropionic Acid (3NPA), presumably from propionic acid, which can induce a range of neuropathologies in model animals [32], although no specific claims for ASD have been made. Biochemically, 3NPA inhibits the TCA cycle enzyme succinate dehydrogenase, thereby compromising mitochondrial ATP synthesis and so could induce a Warburg-like metabolic state. It is likely that any gut organism in which propionic acid accumulates (as discussed above) may also increase the potential for 3NPA generation following attack by Reactive Nitrogen Species (RNS); it is noteworthy ASD sera is enriched with nitrated proteins [2]. Consequently, it is possible to integrate a number of observations associated with ASD, including mitochondrial dysfunction, propionic acid accumulation, increased urinary β-alanine levels, decreased pantothenate levels, decreased glyoxalase-1 activity, and raised levels of sera oxidized, nitrated and glycated proteins.

There is additional evidence that ASD may be associated directly with changes in energy metabolism. A number of studies showing that the anti-aging agent rapamycin, which suppresses mTOR signalling activity to decrease glycolysis and upregulate oxidative phosphorylation, also suppresses autism-like behaviour in animal models [33,34]. Glycated proteins (AGEs) have been shown to activate mTOR [6] and elevated mTOR activity was detected in cells obtained from ASD children [35,36], suggesting a possible causative relationship between these phenomena. Animals exposed to valproic acid have been used as an animal model of ASD see Nicolini & Fahnestock, 2018 for recent review [37]: amongst the resultant effects of valproic acid is a dose dependent stimulation of glycolysis [38] and, perhaps even more importantly, it has previously been observed that resveratrol, an anti-diabetic agent which inhibits non-enzymic glycosylation (glycation) of proteins, prevents valproic acid-induced social impairment in these animals [40]. Furthermore, ketogenic diets (presumably provoking very little glycolysis) have been shown to be somewhat effective in controlling ASD behavioural symptoms in human subjects [41]. Although it is uncertain whether these effects are mediated via the microbiome or specifically in the cells of CNS, these findings are nevertheless consistent with the suggestion that ASD is associated with increased protein glycation resulting from enhanced glycolysis and MG generation.

Deficiency in vitamin-D has also been proposed to play a role in ASD [42–44] and it has been claimed that vitamin D supplementation in children may improve symptoms of ASD [45]. It is interesting to note that vitamin D appears to play a role in controlling the reaction between advanced glycation end-products (AGEs) with their cellular receptors (RAGEs) [46–48], again observations consistent with the findings of elevated levels of protein glycation in ASD subjects.

Carnosine and ASD

The dipeptide carnosine (β-alanyl-L-histidine), when given as a dietary supplement to autistic children, has been shown to exert beneficial effects on behaviour [49,50]. Furthermore the levels of carnosine in urine [51] and sera [17,52 ] of autistic subjects are reported to be substantially lower (by up to 75%) than in controls. Although first described more than 100 years ago [53], carnosine was regarded as “enigmatic” [54]; its precise physiological function still remains uncertain. Amongst the variety of suggestions, all supported with evidence using model and/or cell and animal studies, carnosine can behave as a hydrogen ion buffer, anti-oxidant, anti-glycator, wound-healing agent, metal ion chelator, whilst beneficial effects towards diabetes, atherosclerosis, heart failure, tumour cell growth and cellular ageing have also been reported [55–57]. Interestingly, dietary supplementation studies in human subjects have revealed improvement in cognition and/or behaviour in schizophrenics [58], elderly subjects [59], Gulf War veterans [60] and as well as autistic children [3, 52].

There are a number of possible mechanisms by which carnosine might ameliorate aspects of ASD. First, the additional presence of dietary dipeptide carnosine could, following its hydrolysis, provide a supply of β-alanine and thus allow pantothenate and CoA synthesis in the microbiome, and thereby permit effective oxidative phosphorylation and perhaps additionally ensuring removal of the propionic acid via its carboxylation using acetyl-CoA kinase. Secondly, as outlined above, in order to maintain ATP levels, a compensating response to mitochondrial dysfunction would be enhanced glycolysis, despite the presence of oxygen (i.e. Warburg effect). There is evidence that carnosine can partially suppress glycolysis and decrease glycolytic ATP synthesis in yeast [61] and in transformed cells [62–64 ], which may decrease synthesis of triose phosphates and MG formation. Carnosine can also directly react with methylglyoxal [65] and other reactive carbonyl compounds [66], as well as inhibit formation of glycated proteins as shown in whole animal studies [67,68] and in humans [69]. Carnosine has also been shown to exert regulatory effects on mitochondrial function [70,71] as well as activate the Nrf2 transcription factor (regulator of the antioxidant response) and thereby enhance oxidative defence [72,73]. It is relevant to note that autism in young boys is associated with alteration in Nfr2 expression and/or function [10,74]. Furthermore it should be noted that carnosine may mimic rapamycin to some degree in its ability to inhibit mTOR activity [75]; as noted above, rapamycin is a well-recognised mTOR inhibitor that exerts beneficial effects towards ASD subjects and in animal models [33–35].

These properties (inhibitory effects on mTOR and glycolysis, suppression of MG-induced macromolecular modifications and enhancement of anti-oxidant defence) exhibited by carnosine would appear to counter the onset of the Warburg effect and might account for the beneficial effects of carnosine towards at least some aspects of ASD. Furthermore carnosine has been shown to suppress acetaldehyde-mediated toxicity towards cultured cells [76] and DNA-protein cross-linking in a model system [77], observations consistent with the proposal that ASD is somehow associated with acetaldehyde-mediated dysfunction [30]. It is interesting to note that carnosine seems to possess many of the properties which are likely to suppress generation of the changes exhibited by sera and urinary proteome detected in ASD subjects [2].

There is also a study showing that carnosine can ameliorate the deleterious effect of propionic acid in an animal model of ASD, although the mechanisms responsible have not been explored [78]. Recent studies have suggested that the DJ-1 protein complex can facilitate protein deglycation [31], including glycated β-alanine (induced by MG). Many years ago it was suggested that carnosine might participate the repair of glycated proteins (via deglycation and/or transglycation), perhaps acting as a recipient of the detached glycating agent [79]. However, the possibility that carnosine might participate in protein deglycation has not been explored experimentally.

Carnosine can also inhibit protein nitration by forming adducts such as NO-carnosine and carnosine nitrite [80]. Given that raised levels of protein nitration have been detected in ASD plasma and urine [2], as well as in hair and nails [81], this may partly explain carnosine’s ability to moderate aspects ASD behaviour [49]. More recently it has been shown that romidepsin can ameliorate autism-like behavioural symptoms in a mouse model of ASD [82] by binding to zinc ions in the zinc pocket of histone deacetylase and thus altering gene expression. As carnosine is a well-known zinc chelator, one wonders if the dipeptide might also bind the zinc in histone deacetylase in a manner similar to romidepsin.

Agmatine and ASD

There is evidence from an animal study that agmatine (decarboxylated arginine) can be beneficial towards valproic acid-induced autism-like symptoms of ASD in an animal model [83,84] and that some ASD subjects possess decreased levels of agmatine in their sera [5]. While there is evidence that agmatine possesses anti-inflammatory properties [84,85], there is little direct evidence of any anti-glycation activity of agmatine, although the structure of the molecule (an amino group plus the guanidino group) resembles the strong but toxic anti-glycator, aminoguanidine. Consequently, it is suggested that agmatine should be very readily glycated by a variety of reactive aldehydes, including MG and acetaldehyde, although this property does not appear to have been investigated. Never-the-less it is very relevant to note that agmatine can bind ADP-ribose [86] which may indicate agmatine’s possible inhibitory action towards protein modification by ADP-ribose, or its participation in reversible protein modification (e.g. NAD-dependent histone deacetylation or polyADP ribosylation). The fact that agmatine activity has been likened to that of the anti-aging agent rapamycin [87], including mTOR inhibition, suppression of glycolysis and activation of mitochondrial activity [88], supports this idea. The findings that ASD is associated with changes in arginine metabolism [34] and its intracellular distribution [2] reinforces the proposal that arginine’s decarboxylation product, agmatine, might be ameliorative [89].

It is perhaps also interesting to note that agmatine can promote an increase in cyclic-AMP levels in tissues [88], but cyclic-AMP has been reported to suppress carnosine synthesis [90]. Such observations might suggest that while agmatine can suppress carnosine synthesis, but upon its glycation agmatine may not suppress carnosine synthesis, which could indicate a possible regulatory mechanism of carnosine production in response to endogenous and exogenous glycating agents. Agmatine has been shown to inhibit polyamine synthesis, but whether this property is suppressed following agmatine glycation has not been investigated. However it has been proposed that polyamines generally can, by being readily glycated themselves [91], behave protectively and thereby prevent glycation of polypeptides and nucleic acids.

Conclusions

ASD causation is undoubtedly complex [92]; amongst the factors so far recognised are changes in the microbiome, enhanced glycolytic activity, mitochondrial dysfunction and alteration in redox activity, all of which, presumably together with unrecognised metabolic and exogenous agents, contribute to varying degrees to the changes in behaviour and social interaction which characterise autism. Amongst these factors are agents such as AGEs which affect energy metabolism directly or indirectly, especially glycolysis, oxidative phosphorylation and their potentially dysfunctional, glycated, by-products.

The proposal that ASD is associated with mTOR activation leading to enhanced glycolytic activity as exemplified by the establishment of the Warburg effect (as proposed by Vallée & Vallée, [1] is supported by the findings that not only is a ketogenic diet beneficial towards ASD [93], but that glycated proteins (i.e. AGEs) can indeed activate mTOR to provoke onset of the Warburg effect [6]. Thus carnosine and possibly agmatine, both being pluripotent and essentially non-toxic endogenous molecules which can decrease glycolysis, possibly via effects on mTOR [75,87], plus their reactivity towards reactive carbonyls such as MG, may inhibit protein glycation and thereby ameliorate some of the consequences of increased glycolytic activity and exert beneficial effects on aspects of behaviour in ASD children. Although the specific mechanisms by which some of these effects are mediated may differ; for example control of protein nitration may occur via carnosine’s direct reaction with the nitrating agent whereas agmatine may inhibit nitric oxide synthesis, such complementary mechanisms could conceivably be therapeutically efficacious. That changes in both carnosine and agmatine may be connected to ASD is also supported by the findings that their serum levels are substantially lower in ASD subjects and that they both can also ameliorate the effects of propionic acid, which is known to sometimes accumulate in ASD. It is also suggested that the ability of carnosine and agmatine to ameliorate the effects of MG, either directly or following upregulation of antioxidant defences may also contribute to their efficacy towards ASD. It is interesting to note that two of propionic acid’s likely metabolites, 3-nitropropionate [94] and propionaldehyde [95], have also been associated with ASD; both carnosine and agmatine, could theoretically antagonise either their formation and/or toxicity via inhibiting propionate nitration or promoting aldehyde scavenging.

Whether combined treatment with both carnosine and agmatine is therapeutic towards ASD has not been explored. However, it has been noted that co-administration of carnosine and arginine (agmatine precursor) was more effective in combating hypoxic stress in rats than when either agent was supplied singly [96], an observation at least consistent with the above suggestion. More generally, as both carnosine and agmatine [97] when administered separately seem to exert beneficial effects towards aspects of both Parkinson’s disease [70, 98, 99] and Alzheimer’s disease [100–102] in cellular and animal models, then perhaps their co-administration should be also explored towards these age-related neurodegenerative conditions.

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Is Cervical Cancer a Defeated Enemy?

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DOI: 10.31038/IGOJ.2018114

Editorial

Cervical screening by Pap-test has been on the top of public health agenda for several decades. This is not because of the epidemiological weight of the disease but because we have all tools for its prevention in hand. The primary prevention of cervical cancer by vaccination against human papillomvirus (HPV) infection is an increasingly widespread practice; however, we still do not know enough how long the protection will last. The secondary prevention by method of proven effectiveness for early detection of both premalignant lesion of uterine cervix, and cervical cancer itself has long been widely available for women. Screening tests, by definition, sort out apparently well persons who probably have the target disease those who have not. Cervical screening is to substantially reduce the burden of disease in terms of mortality, morbidity, and improve quality of life. Primary and secondary prevention of cervical cancer could be the “success story” of health care system. Unfortunately, they are not so.

In the last 50–60 years, the clinical spread of screening was followed by the need for its public health application. In 1960’s, expert groups established the concept of “organized screening”, as opposed to “opportunistic” one, meaning actions initiated and financed by the provider health care system, and individually inviting of those women to be screened [1]. In the development of such population screening, the Nordic Countries have shown a good example [2]. Population screening is most effective if most invited women in the eligible population choose to participate. The participation rate is the Achiles’heel of population screening.

In fact, not everyone benefits equally from the screening due to inequalities of various kind, such as diversity in health care systems, access to screening services, socioeconomic and demographic status, lack of knowledge and education, and last but not least, due to differences in geopolitical status [3].  Screening programmes are much better developed in Nordic and Western Europe as compared to the Central-Eastern Europe, where the burden of the disease is much higher due to a history of mostly opportunistic cervical screening practices, and due to the strong influence of political and economic changes in post-communist transition; as a result the screening facilities are underdeveloped [4].

As far as Europe is concerned, in 2003, the Council of the European Union recommended to its Member States to implement organized, population-based cervical screening programmes [5]. In 2017, in the second report of the implementation of the Council Recommendation, out of 27 member states not more than nine countries reported “complete rolling out”, the rest of the countries “piloting” or “planning” organised cervical screening programmes [6]. The up-to-date estimates of cancer burden in Europe shows that cervical cancer mortality is inversely proportional to the intensity of the cancer screening activities in the respective countries [7].

The gynaecological community has a lot to contribute with to the impact of cervical screening, as the smear-taking for cytological analysis is their task in all those countries where the task is not delegated to paramedical personnel, as midwifes, praxis nurses, public health nurses. In such a situation, the gynaecologists are the “gate-keepers” of the screening which tends to be opportunistic rather than organized one; opportunistic screening is much less effective than the organized one [8]. The insistence of gynaecological community on their “historical role” seems to be a major obstacle to be overcome.

Cytological screening every three to five years can potentially prevent up to four out five cases of cervical cancer, and can reduce cervical cancer incidence up to 80% at population level [9]. Such benefits can only be achieved if screening is provided in organised population-based programmes with optimal attendance rate, and quality assurance at all levels [10]. Following this protocol, the cervical cancer might become a defeated enemy [11].

References

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The impact of P-glycoprotein and Midkine on Paclitaxel / Cisplatin Chemoresistance in Ovarian Cancer

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DOI: 10.31038/CST.2018333

Abstract

Chemoresistance is one of the most important factors leading to high mortality in ovarian cancer (OC). Overexpression of P-glycoprotein (P-gp) in OC cells may results in resistance to paclitaxel treatment by pumping the drug out of the cells, which in turn decreases the intracellular drug concentration. Additionaly overproduction of midkine (MK) can also affect the development of chemoresistance in OC. Although, the mechanisms of action of P-gp and MK are not the same, overexpression of both proteins in OC may intensify chemoresistance to paclitaxel treatment. Therefore, simultaneously inhibition of P-gp and MK in overcoming chemoresistance to drugs may improve treatment results in OC.

Introduction

Ovarian cancer (OC) is the fourth most common type of gynecological cancers worldwide and has the highest mortality rates among female genital tract malignancies [1–3]. Even patients with same clinical characteristics, such as cancer stage, histological type and grade display different disease progression and treatment results [3–5]. Due to absence of specific symptoms in the early stage, OCs are diagnosed at the advanced stages in two thirds of the patients [6]. The overall 5 year survival rate is still less than 40% despite some advances in the treatment of OC, including the combination of surgery, radiation and chemotherapy. This may be attributed to the late stage diagnosis, poor prognosis and resistance to chemotherapy, which is one of the major problems to controlling malignant tumors [3, 7, 8]. The first-line treatment of OC is cytoreductive surgery followed by adjuvant chemotherapy, including paclitaxel and cisplatin [3, 9–11]. Paclitaxel, administered as monotherapy or in combination with cisplatin, is potentially effective therapeutic regimen in OC. Paclitaxel may be regarded as a mitotic poison and affects the cellular microtubule network. It inhibits chromosome alignment and segregation and then trigger the apoptosis pathway [10, 11].

Initial response rates to chemotherapy vary between 40 and 80% in OCs. However, majority of these patients who respond to chemotherapy at first, eventually have recurrence following the development of chemoresistance. Thus, acquired resistance is the main cause of unsuccessful treatment in OC. The molecular mechanisms behind chemoresistance is multifactorial and involves multiple processes, including drug transport and metabolism, DNA repair and apoptosis. Currently, the factors that affect the development of chemoresistance in OC has not been completely understood [6, 12]. Chemoresistance is usually attributed to the overproduction of P-gp. It has been reported that overexpression of P-gp is the major factor for reduced chemo-sensitivity in a lot of malignancies, including OC [6, 12–14]. It has been demonstrated that the overexpression of P-gp in aggressive OC cells results in the development of resistance to paclitaxel treatment [10, 11, 15]. Although the mechanism of P-gp-induced chemoresistance is not fully known, it is considered to acts essentially as an efflux pump and plays an important role in the exclusion of drugs from tumor cells, resulting in decreased accumulation of chemotherapy drugs within cancer cells [8, 10, 11, 15].

Another important protein, MK, is overexpressed in many cancers, including OC and induces the growth and survival of tumors. On the other hand, overproduction of MK can also affect the development of chemoresistance. The chemoresistance caused by MK is mainly due to its inhibitory action on the apoptosis process.

Our proposal is that both proteins, namely P-gp and MK, may protect tumor cells against chemotherapeutic drugs more effectively by a synergistically way than they do one by one and they could increase chemoresistance [3, 16–19]. Therefore, it can be speculated that inhibition of both proteins may enhance the effectiveness of paclitaxel chemosensitivity in OC.

The role of P-glycoprotein in chemoresistance to paclitaxel /cisplatin in ovarian cancer

ATP-binding cassette transporter B1 (ABCB1), also known as P-gp or multidrug resistance protein 1 (MDR1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types [20]. It is a 170 kD transmembrane glycoprotein and has unusually broad polyspecificity for structurally different substances, including anticancer drugs such as paclitaxel and cisplatin. Most of these substances are hydrophobic, thus, P-gp acts like a ‘’hydrophobic vacuum cleaner’’ [20].

P-gp leads to chemoresistance by pumping drugs out of the cells and decreases the intracellular drug concentration [9]. P-gp is also associated with a more progressed malignant phenotype in carcinogenesis. The function of P-gp in relation to cellular differentiation may be pleiotropic, depending on the origins from which the cancer arises [8]. P-gp is localized in the membrane of epithelial cells in the intestine, liver, proximal tubule of the kidney and in the capillary endothelial cells. It functions as a blood–brain barrier, blood–placenta barrier and blood-testis barrier and protects them from toxic xenobiotics [20]. This transporter may affect the pharmacological treatment of numerous diseases by changing drug pharmacokinetics and inhibiting accumulation of anticancer drugs in cancer cells. Cancer cells of some tissues also produce very large amount of P-gp, which lead to chemoresistance by transfering chemotherapeutic agents out of cancer cells. Additionally, increased intestinal expression of P-gp can inhibit the absorption of orally administered drugs, promotes their biliary and renal elimination and as a result, decreases plasma concentrations of these drugs, which causes unsuccesful treatment [6, 19, 20].

Fojo et al. have reported that the MDR1 gene is overexpressed in many cancers arising from some tissues in which the MDR1 gene is expressed at high levels. Most of these cancers are resistant to chemotherapy, and the MDR1 gene plays an important role in intrinsic and acquired chemoresistance [8, 21]. Approximately 40% of OCs after chemotherapy produce P-gp at high level, suggesting chemoresistance in OCs may be most likely acquired [8, 22]. However, some OC cases before chemotherapy are intrinsically multidrug resistant, which can be determined by MDR1 gene expression, and this phenotype should be taken into account for effective chemotherapy of ovarian epithelial carcinomas [8]. It has been revealed that the overexpression of P-gp in aggressive OC cells is associated with the development of resistance to paclitaxel treatment [10, 11, 15]. In contrast, downregulation of P-gp increases the effectiveness of certain chemotherapeutic agents. For example, myricetin (a dietary-flavonoid) enhances the chemotherapeutic potential of paclitaxel in OC cells by downregulating P-gp and inhibits the migratory properties of OC cells [10]. Alike, microRNAs (miRNA), which are endogenous, noncoding RNAs may regulate the ABCB1 gene. Recently, Sun et al. have demonstrated that miR-186 overexpression may sensitize OC cells to paclitaxel and cisplatin by downregulating P-gp in the OC cell lines [9]. Another study has demonstrated that miR-21 may regulate the production of MDR1/P-gp, by targeting hypoxia-inducible factor-1α (HIF-1α, ) which influences the development of drug resistance in paclitaxel-resistant OC A2780/taxol cell lines. Furthermore, the inhibition of miR-21 may sensitize A2780/taxol cells to paclitaxel [12]. Aditionally, upregulation of miR-27a expression results in inhibition of P-gp expression and decreases paclitaxel-resistance in OC cell line [15].

As the expression of P-gp in cancer cells usually results in multidrug resistance (MDR) to chemotherapeutic drugs, which is the main cause of chemotherapy failure in cancer treatment, it is important to develop new treatment strategies, which target P-gp [11]. Some MDR reversal agents that inhibit the drug efflux activity of P-gp could increase the intracellular drug levels [11]. It has been demonstrated that MDR1 expression levels after promethazine (an antihistaminic agent) administration is significantly reduced and verapamil (a calcium channel antagonist) leads to a significant decrease in MDR1 mRNA levels and downregulates P-gp activity [23].

The role of midkine in chemoresistance to paclitaxel /cisplatin in ovarian cancer

Midkine (MK), a heparin-binding growth factor, was firstly found to be the product of a retinoic acid-responsive gene during embryogenesis [24, 25]. Despite its high expression during embryogenesis, MK is downregulated to neglible levels in healthy adults and only re-expressed in some pathological processes [16, 25, 26]. MK promotes many cellular functions including survival, growth, migration, reproduction and repair, and gene expression while inhibiting apoptosis [27]. Due to its multiple functions, MK has significant impact on the pathogenesis of neurological, cardiovascular and inflammatory diseases and malignancies [19, 25]. It induces several signal transduction pathways including phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK), therefore participates in the regulation of diverse biological processes. Recent studies showed that MK expression is influenced by hypoxia, growth factors, and cytokines through a nuclear factor-κB (NF-κB) dependent pathway. The precise regulatory mechanisms behind MK expression is not fully understood [25, 28, 29]. MK plays significant roles as a growth factor during carcinogenesis, such as transformation, fibrinolysis, cell invasiveness, cell survival, anti-apoptosis, and angiogenesis processes [24, 27, 29–33].

It has been shown that MK is overexpressed in various human malignancies, including oral, lung, thyroid, bladder, prostate, cervical and OCs [18, 25, 35–37]. MK is also a plasma-secreted protein, and its levels in blood may increase in patients with malignant diseases [25]. Nakanish et al. have demonstrated that the expression of MK in germ cell ovarian tumors is significantly lower than in epithelial ovarian tumors, and expression in malignant epithelial tumors is significantly higher than in benign ones [18]. MK not only induces carcinogenesis but also contributes to chemoresistance [34]. It is considered that MK-induced chemoresistance is mainly due to inhibitory impact on apoptosis mediated by the Janus-activated kinases (JAKs) and STAT1 by activating the Akt-mediated survival pathway and senescence of tumor cells [19, 31]. On the other hand, it appears that some of the mechanisms of its chemoresistance actions are partially similar to those of P-gp [19].

MK, has been verified overexpressed in many cancers, including OC. It has been shown that MK is increased in the serum of patients with epithelial OC. MK may also be an indicator of the response to paclitaxel and/or cisplatin in the clinical treatment of OC [3, 16–18]. Zhang et al. have demonstrated that cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) may lead to the high level of MK in tumours and that CAF-derived MK can induce cisplatin resistance via inhibition of the cell apoptosis in the TME by increasing production of lncRNA ANRIL. CAF-derived MK increases lncRNA ANRIL expression in tumour cells and thus promoting the up-regulation of ABC family proteins, multidrug resistance-associated protein 1 (MRP1) and ABCC2, which ultimately cause resistance to cisplatin. These findings related to the source of MK in tumour tissues, may serve as a novel therapeutic approach for cancer [34]. Further evidence is that a novel midkine inhibitor (iMK) has antitumor effect against oral squamous cell carcinoma and it has been demonstrated that iMK inhibits the expression of MK and suggested that iMK can be effectively used for the treatment of oral squamous cell carcinoma [19, 25, 38].

On the contrary, Wu et al. have suggested that the MK expression has a positive correlation with the predicted survival time and chemosensitivity of OC to paclitaxel/cisplatin. This study proposed that MK could down-regulate the expression of multidrug resistance-associated protein 3 (MRP3), and in turn increases the cytotoxicity of paclitaxel and/or cisplatin [3]. Despite this contrary opinion, it is generally considered that MK increases chemoresistance and decreases effective treatment during chemotherapy. On the other hand, due to its biological significance in carcinogenesis, it is suggested that MK can be regarded as a candidate molecular target for therapy against human carcinomas [25].

Conclusion

Chemoresistance is one of the important factors leading to high mortality in OC. At present paclitaxel and cisplatin are the most used drugs to treat OC. However, numerous patients with OC frequently relapse following the development of chemoresistance to chemotherapeutic agents, including paclitaxel and cisplatin. Overexpression of P-gp and MK have important impacts on chemoresistance in many cancer types, including OCs. Therefore, inhibition of both P-gp and MK may overcome chemoresistance in OCs. However, whether they act synergistically or in contrary remains unclear and further investigations are needed to clarify the interplay of these proteins in cancer cells and in the treatment of malignancies.

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  29. You Z, Dong Y, Kong X, Beckett LA, Gandour-Edwards R, et al. (2008) Midkine is a NF-kappaB-inducible gene that supports prostate cancer cell survival. BMC Med Genomics 1: 6. [crossref]
  30. Kojima S, Muramatsu H, Amanuma H, Muramatsu T. Midkine enhances fibrinolytic activity of bovine endothelial cells. J Biol Chem. 1995; 270(16): 9590–6.
  31. Ratovitski EA, Kotzbauer PT, Milbrandt J, Lowenstein CJ, Burrow CR. Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases. J Biol Chem. 1998; 273(6): 3654–60.
  32. Huang Y, Hoque MO, Wu F, Trink B, Sidransky D, Ratovitski EA. Midkine induces epithelial-mesenchymal transition through Notch2/Jak2-Stat3 signaling in human keratinocytes. Cell Cycle. 2008; 7(11): 1613–22.
  33. Huang Y, Sook-Kim M, Ratovitski E. Midkine promotes tetraspanin-integrin interaction and induces FAK-Stat1alpha pathway contributing to migration/invasiveness of human head and neck squamous cell carcinoma cells. Biochem Biophys Res Commun. 2008; 377(2): 474–478.
  34. Zhang D, Ding L, Li Y, Ren J, Shi G, Wang Y, Zhao S, Ni Y, Hou Y. Midkine derived from cancer-associated fibroblasts promotes cisplatin-resistance via up-regulation of the expression of lncRNA ANRIL in tumour cells. Sci Rep. 2017; 7(1): 16231
  35. Ruan M, Ji T, Wu Z, Zhou J, Zhang C (2007) Evaluation of expression of midkine in oral squamous cell carcinoma and its correlation with tumour angiogenesis. Int J Oral Maxillofac Surg 36: 159–164.
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Providing Sand Rats (Psammomys Obesus) Environmental Enrichment is not Inhibiting their Diabetes Development and Use as an Animal Model for Human Diet Induced Type 2 Diabetes

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DOI: 10.31038/IJVB.2018232

Abstract

The gerbil, Psammomys obesus, commonly known as the fat sand rat, is a well-defined animal model for human type 2 diabetes (T2D). Captive housed fat sand rats often develop serious digging- and gnawing stereotypies but historically, little has been done to improve the housing conditions for the animals by providing environmental enrichment and thereby minimizing or eliminating this unwanted behaviour. Although not scientifically proven, it is generally believed that providing environmental enrichment might inhibit the development of diabetes in the fat sand rats, mainly due to raised activity levels.

This study compared the development of T2D in fat sand rats housed in standard housing conditions and sand rats housed in various enriched environments. The study included 51 fat sand rats in five groups, of which one group acted as the control. The remaining four groups were housed in four different enriched environments for 37 days; including various combinations of provided mazes/burrows, nuts, seeds, maize and barley plus access to salt water. No significant differences were found in the development of diabetes in the five groups. It is concluded that provision of the tested environmental enrichment has no effect on the development of T2D in Psammomys obesus, and hence there are no reasons for not providing captive housed fat sand rats with species-specific environmental enrichment like the tested items to fulfil their natural needs and enhance their welfare.

Introduction

Psammomys obesus, the fat sand rat, of the genus Psammomys, which also often is referred to as the desert sand rat [1], the Israeli sand rat [2] or simply the sand rat [3–5] has since the 1960s been used as an animal model for diet induced T2D [6]. In the sand rat’s natural habitat – the arid regions of North Africa and Eastern Mediterranean – the animals mainly feed on succulent leaves that are relatively low in energy and high in water and electrolytes [7]. Sand rats living in this habitat are lean and normoglycemic and do not naturally develop T2D. However, when fed a regular or high energy laboratory rodent diet in captivity, the animals gain weight and develop insulin resistance, hyperinsulinemia and eventually hyperglycaemia. As the disease development progresses, the sand rats lose their functional pancreatic beta cell mass and become hypoinsulinemic eventually leading to death if insulin therapy is not initiated. This is very similar to the T2D development seen in humans [8–12]. In the early 1970s, sand rats were caught in the wild in the desert areas north of the Dead Sea in Israel. A breeding colony was then successfully established at the Hebrew University Hadassah Medical School, Israel [13]. In captivity, the colony developed four distinct phenotypes; 32% of the animals were normoglycemic and normoinsulinemic, 26% were moderately obese, normoglycemic and hyperinsulinemic, 36% were hyperglycaemic and hyperinsulinemic, and about 6% developed hypoinsulinemia and hyperglycaemia with weight loss and ketosis [14, 15]. These different phenotypes led to the establishment of two distinct breeding lines: A diabetes prone (DP) line, in which more than 70% of the animals develop T2D and a diabetes resistant (DR) line in which 60–70% of the animals remain normoglycemic despite intake of a high calorie diet [16]. Heled et al [17] have documented that exercise training in sand rats could prevent or postpone the progression of T2D.

It is generally believed that environmental enrichment for laboratory animals will increase activity associated with exploring and manipulating inanimate enrichment items [18], and hence it could be a concern, that enriching the environment of the sand rat would inhibit or delay the development of T2D due to raised activity levels. Accordingly, very little has historically been done to improve the housing conditions. Even though sand rats, being gerbils, differ notably from mice and rats in their way of living e.g. in being great diggers and building extensive tunnel systems with several entrances leading to different foraging sites and spending only 2–3 hours above ground every day [25], sand rats are traditionally housed in the same way as laboratory rats.

The present study aimed at investigating if sand rats can be housed under environmentally enriched conditions without there being an effect on the development and onset of T2D.

Material And Methods

Animals

For this study, a total of 98 diabetes prone (DP) sand rats (49 males and 49 females) (Psammomys obesus), aged nine weeks on arrival, were used. All animals originated from the same commercial breeding colony (Harlan, Jerusalem, Israel) which twice a year is health monitored based on the recommendations of the FELASA Working Group on Health Monitoring of Rodent and Rabbit Colonies (FELASA 2002). The breeding colony is historically and by the use of mice and rat sentinels tested positive for Pneumonia Virus of Mice (PVM) and Helicobacter spp. only. On arrival, the animals were micro chipped, randomly assigned to single-sex groups with two or three animals per cage and acclimatized for two weeks on a low-energy diet (LE) (3084 Teklad Low Energy Sand Rat Diet, Harlan, Jerusalem, Israel) with 2.4 cal/g of total digestible energy and consisting of 70% carbohydrate, 3.1% fat, 16.7% protein, and 10.2% ash in the form of hard-pressed pellets. On this diet, the sand rats maintain a non-fasting blood glucose (BG) level of 4–6 mmol/L. The study consisted of two parts; a pre-study and the main study on the effect of enrichment (the enrichment study). After two weeks of acclimatisation, the pre-study, lasting 10 days, was done to identify diabetes prone animals (DP) to be used in the enrichment study. After the selection of DP animals, a two-week period for normalisation of blood glucose was initiated prior to the enrichment study, which lasted 37 days.

Housing

The sand rats were housed in yellow, semi-transparent type IV macrolon cages (Scanbur A/S, Karlslunde, Denmark) throughout the study. For details on environmental enrichment please refer to Table 1. All cages had two water bottles. Citric acid was added to the drinking water in a 0.4% solution (pH 2–3) to prevent bacterial growth. All cages were changed once a week. When the animals became diabetic and developed polyuria, cages were changed more frequently. The animals were housed in an animal room with a 12 hour light-dark cycle providing light from 6: 00 to 18: 00. The temperature was
22–25oC, and the humidity 30–70% (norm. 45–65%). The animals had free access to food and water. All animals were observed and cared for by experienced animal caretakers at least once daily and all animals included in the study were weighed twice a week. At the end of the study, all animals were euthanized using a gradually filled 85% CO2 / 15% O2 chamber. Throughout the study, all animals were housed and cared according to current Danish and European legislation and guidelines, and the actual study was both approved by the Danish Animal Experiments Inspectorate and the Novo Nordisk Ethical Review Committee (ERC).

Table 1. Housing of the sand rats prior to study start (SH = standard housing) and during the study.

Study

SH

G1

G2

G3

G4

G5

N

51

11

10

10

10

10

Yellow semi-transparent type IV macrolon cage (595 x 380 x 200 mm; floor area 1820 cm²) (Scanbur A/S, Karlslunde, Denmark)

X

X

X

X

X

X

Standard lid (total cage height 25 cm)

X

X

X

X

X

X

3 cm layer of aspen bedding (Tapvei, Kortteinen, Finland) or enough to cover maze/burrow

X

X

X

X

X

X

Paper based nesting material “EnviroDri” (Lillico, Surrey, UK) and two aspen biting sticks, size medium (Tapvei, Kortteinen, Finland).

These sticks followed the animals, when the cages were changed

X

X

X

X

X

X

Food enrichment*

X

X

X

X

Saltwater (3% saline/sea salt)

X

X

X

Novo Nordisk shelter

X

X

X

X

Plastic maze/burrow

X

Metal maze/burrow

X

N = number of animals. G1 = Group 1 (control), G2 = enrichment group 2,
G3 = enrichment group 3 etc. *Food enrichment = peanuts and hazelnuts with shells, sunflower seeds, maize and barley.

Blood sampling

Blood samples for determination of whole BG and HbA1C levels in both the pre-study and the enrichment study were drawn from the tip of the tail of non-sedated animals, using an “Assistant” blood lancet (Bie & Berntsen, Rødovre, Denmark). Samples for measurement of BG were collected in 10 μl glass Na-heparinized capillary tubes (Vitrex, Herlev, Denmark), immediately suspended in 500 μl Biosen analysis buffer (EKF Diagnostics, Cardiff, UK) and analysed for BG concentration expressed as mmol/L. Blood samples for HbA1Cwere transferred to a freezer (-20oC) until analysis on a Hitachi 912 (Roche HbA1cII; Tina-quant Hemoglobin A1c II, Roche/Hitachi 912, Mannheim, Germany) and expressed as percentage glycated haemoglobin of the total haemoglobin.

Pre-study

After two weeks of acclimatization, all animals were transferred for 10 days to a high energy diet (HE) (Purina LabDiet 5008, Brogaarden, Gentofte, Denmark) with 3.1 cal/g of total digestible energy and consisting of 66.6% carbohydrate, 2.1% fat, 22.4% protein, and 6.9% ash. BG was measured on days 0, 3, 6, 7, 8, 9 and 10. Animals that developed BG levels above 10 mmol/L for two consecutive days during the 10-day period were classified as diabetes prone (DP) and included in the study (19 DP females and 32 DP males). Animals with BG levels below10 mmol/L were classified as diabetes resistant (DR) and were not included in the study. If a DP animal was pair housed with a DR animal, the DR animal remained in the cage as a companion animal for animal welfare reasons. For this study, 9 females and 8 males were kept as companions for a DP cage mate. If both pair housed animals were DR, they were excluded from the study and used for other purposes. The study animals were after selection transferred back to the LE diet for two weeks to lower their BG to normal levels before start of the actual study.

Enrichment study

The animals were housed in one of five environments (Table 1) for 37 days and fed HE diet. BG was measured on day 0 and twice per week in the morning thereafter (a total of 12 samples). Samples for HbA1C were drawn on day 0 and once a week (a total of 6 samples) thereafter. All the blood samples were taken at the same time point, early in the morning, from overnight fed animals. A variety of enrichment items were used in the four enrichment groups (Table 1). The used shelter was a small hideout used as standard enrichment for rats at Novo Nordisk (Mikkelsen 2010). A multi-entranced labyrinth-like tunnel-house was designed and fabricated (“the maze/burrow”; Novo Nordisk, Maaloev, Denmark). Two different types were used; one produced in stainless steel (Group 4) and one produced in non-transparent plastic (Group 5) (Figure 1). A 3% saltwater (sodium chloride; ATLANTIS sea salt from Portugal) solution was provided as enrichment in groups 3, 4 and 5.

IJVB2018-113-LarsDenmark_F1

Figure 1. The non-transparent plastic maze/borrow.

To satisfy the animal’s need for gnawing and searching for food, several food items commonly used as enrichment for other species of rodents were used. Those selected were peanuts and hazelnuts with shells, sunflower seeds, maize and barley (all from Brogaarden, Gentofte, Denmark). All were given once daily in a very limited amount to all enrichment groups (groups 2, 3, 4 and 5).

Data

Data was analysed with PASW Statistics 18, 2009 (SPSS Inc., Chicago, Illinois, USA) and graphs made with Graph Pad Prism version 5.00 for Windows (Graph Pad Software, San Diego, California, USA).

The data sets were continuous and found to be normally distributed, hence the differences between the groups were analysed using the one-way analysis of variance. Dichotomous data (e.g. the number of animals developing diabetes or not) were analysed using the Chi-square test. A two-sided 5% level of significance was used in all the analysis.

Results

The five groups did not differ with respect to either HbA1C (p=0.28) or BG levels (p=0.87) at the start of the study (Table 2). The changes in measured variables over time did not differ significantly between the groups either with respect to HbA1C (p=0.40) or BG levels (p=0.83) (Table 2). In total, 38 out of 51 (75%) sand rats developed T2D (defined as a BG reading above 10 mmol/L on two consecutive days) which is in accordance with our previous experiences where 60 to 85% of the animals develop diabetes. Furthermore, the percentage of sand rats developing T2D did not differ between groups (p=0.16). However, the fact that not all sand rats developed diabetes resulted in a rather large standard deviation in glycaemic levels.

Table 2. mean (+/- standard deviation) blood glucose (mmol/l) and HbA1C (% of total hemoglobin) values on day 0 and day 37 (start and end of study).

Blood Glucose mmol/L

HbA1C (% )

Day 0

Day 37

Day 0

Day 37

Group 1 (control)

7.4 (+/- 4.5)

18.4 (+/- 10.8)

5.3 (+/- 0.5 )

7.7 (+/- 0.7)

Group 2

7.5 (+/- 4.8)

18.0 (+/- 10.0)

5.2 (+/- 0.9 )

7.7 (+/- 1.9)

Group 3

8.7 (+/- 4.4)

16.6 (+/- 4.5)

5.0 (+/- 0.6 )

8.0 (+/- 0.9)

Group 4

7.3 (+/- 4.0)

15.5 (+/- 9.2)

5.1 (+/- 0.5 )

8.0 (+/- 1.6 )

Group 5

5.0 (+/- 3.2)

14.2 (+/- 8.7)

4.8 (+/- 0.5 )

6.6 (+/- 1.3 )

P = 0.87

P = 0.83

P = 0.28

P = 0.40

Discussion

This study has demonstrated that is possible to provide environmental enrichment to sand rats without having significant effects on the development of T2D. The four test-groups were enriched in four different ways and none of the groups showed any significant difference in either BG levels or HbA1C, as compared to the control group.

Housing systems containing items used by the animals to satisfy species-specific basic needs contribute to improved animal welfare [19]. Even though not quantified, it was observed that the animals used the maze/burrow, when provided; they would dig under it and they would arrange their shelters in it (Figure 2) and they seemed to have a lower level of stereotypies (personal observations). Hence the constructed maze/burrow seemed to be a valuable enrichment item in the cage with a positive effect on the animals’ welfare. The animal caretakers preferred the plastic maze/burrow, as it was easier to handle than the stainless-steel maze/burrow. The 3% saltwater solution as drinking water seemed to be preferred by the sand rats, especially in the beginning of the study. As the animals became diabetic they seemed to increase their intake of normal water (personal observations). Sand rats may show adrenal pathology and increased mortality, when not given extra sodium chloride [20, 21] and hence this supply may be valuable for the health of the animals. Further studies could establish the need as well as the preference for sodium chloride water compared to fresh water in sand rats. The sand rats ate the sunflower seeds, the maize and the barley. However, the hazelnuts with shells did not seem to be highly valued by the sand rats [21–25].

IJVB2018-113-LarsDenmark_F2

Conclusion

As there were no significant differences in the development of diabetes, there are no reasons to withhold species-specific environmental enrichments, like the items tested in this study, for fat sand rats. Based on the results and observations made in this study, a plastic maze/burrow, a 3% salt water solution and sunflower seeds, maize and barley would be excellent choices for adding environmental complexity to the cages of sand rats, allowing the sand rats to express natural behaviours such as exploration and foraging, thereby increasing the welfare of the fat sand rat.

References

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  2. Collier G, Walder K, De Silva A, Tenne-Brown J, Sanigorski A, et al. (2002) New approaches to gene discovery with animal models of obesity and diabetes: Lipids and Insulin Resistance: the Role of Fatty Acid Metabolism and Fuel Partitioning. Annals of the New York Academy of Sciences 967: 403–413.
  3. Borenshtein D, Ofri R, Werman M, Stark A, Tritschler HJ, et al. (2001) Cataract development in diabetic sand rats treated with alpha-lipoic acid and its gamma-linolenic acid conjugate. Diabetes Metab Res Rev 17: 44–50. [crossref]
  4. Gruber HE, Johnson T, Norton HJ, Hanley EN (2002) The sand rat model for disc degeneration: Radiologic characterization of age-related changes – Cross-sectional and prospective analyses. Spine 27: 230–234.
  5. Shafrir E (1996) Development and consequences of insulin resistance: Lessons from animals with hyperinsulinaemia. Diabetes & Metabolism 22: 122–131.
  6. Fine J, Quimby FW, Greenhouse DD (1986) Annotated bibliography on uncommonly used laboratory animals: Mammals. ILAR NEWS 29: 1A-38A.
  7. Daly M, Daly S (1975) Behavior of Psammomys Obesus (Rodentia: Gerbillinae) in the Algerian Sahara. Zietschrift fur Tierpsychologie 37: 298–321.
  8. Kaiser N, Cerasi E, Leibowitz G (2012) Diet-Induced Diabetes in the Sand Rat (Psammomys obesus). Animal Models in Diabetes Research, Methods in Molecular Biology. 933, 89–102.
  9. Donath MY, Gross DJ, Cerasi E, Kaiser N (1999) Hyperglycemia-induced beta-cell apoptosis in pancreatic islets of Psammomys obesus during development of diabetes. Diabetes 48: 738–744.
  10. Barnett M, Collier GR, Collier F, McL, Zimmet P, O´Dea K (1994) A cross-sectional and short-term longitudinal characterisation of NIDDM in Psammomys obesus. Diabetologia 37: 671–676.
  11. Hackel DB, Mikat E, Lebovitz HE, Schmidt-Nielsen K, Horton ES, Kinney TD (1967) The sand rat (Psammomys Obesus) as an experimental animal in studies of diabetes mellitus. Diabetologia 3: 130–134.
  12. Schmidt-Nielsen K, Haines Hb, Hackel Db (1964) Diabetes Mellitus In The Sand Rat Induced By Standard Laboratory Diets. Science 143: 689–690. [crossref]
  13. Shafrir E, Ziv E, Kalman R (2006) Nutritionally induced diabetes in desert rodents as models of type 2 diabetes: Acomys cahirinus (spiny mice) and Psammomys obesus (desert gerbil). Ilar Journal 47: 212–224.
  14. Shafrir E, Gutman A (1993) Psammomys obesus of the Jerusalem colony: A model for nutritionally induced, non-insulin-dependent diabetes. Journal of Basic & Clinical Physiology & Pharmacology 4: 83–99.
  15. Kalderon B, Gutman A, Levy E, Shafrir E, Adler JH (1986) Characterization of stages in development of obesity-diabetes syndrome in sand rat (Psammomys obesus). Diabetes 35: 717–724. [crossref]
  16. Kaiser N, Nesher R, Donath MY, Fraenkel M, Behar V, et al. (2005) Psammomys obesus, a model for environment-gene interactions in type 2 diabetes. Diabetes 54 Suppl 2: S137–144. [crossref]
  17. Heled Y, Shapiro Y, Shani Y, Moran DS, Langzam L, et al. (2002) Physical exercise prevents the development of type 2 diabetes mellitus in Psammomys obesus. Am J Physiol Endocrinol Metab 282: E370-E375.
  18. Bayne K (2005) Potential for Unintended Consequences of Environmental Enrichment for Laboratory Animals and Research Results. ILAR 46: 129–139.
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The Effect of Age on Spinal Range of Motion: A Review

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DOI: 10.31038/ASMHS.2018231

Abstract

Reduced spinal mobility may result in activity limitations and participation restrictions, which could subsequently affect quality of life. This literature review examined the effects of aging on spinal range of motion (ROM). Two databases (PubMed and Google Scholar) were searched using the MeSH terms spine, aging, range of motion, athlete, human and collagen. Two hundred twenty-four articles were identified; 210 of these were rejected as not directly relevant with the current review. The accepted articles (n=14) were categorized into four participant groups (athletes, clinical, elderly, and general).  Each of the studies was analyzed and assigned a quality grade using the GRADE system provided by the American Dietetic Association. The results suggested that aging causes increased risk for spinal fractures and loss of ROM and bone density.  For women, spinal deformity and vertebral compression fractures may lead to impaired mobility and quality of life.  More research is needed on the effects of the aging spine in relation to overall health, quality of life and socio-economic status.

Keywords

aging, spinal, range of motion, human, athlete, collagen

Introduction

Musculoskeletal function is determined by range of motion (ROM), strength, endurance, coordination, and sensation 1]. The majority of these physiological parameters (e.g. aerobic power, strength, endurance, coordination, and sensation) peak in late adolescence and then gradually decline with age 2]. Within the musculoskeletal system, part of the aging effect is the increase in intramuscular connective tissue stiffness which results in decreased ROM and a gradual performance decline in Activities of Daily Living (ADL) 3].

Range of Motion in Different Populations

The changes that occur with aging, such as loss of lumbar flexion, extension and lateral flexion, may be responsible for decreases in spinal ROM 3,4]. The motion profile of physically active or athletic populations is more difficult to evaluate than the profile of less active populations because age-associated differences in the degree of muscle damage after exercise in well-trained humans have yet to be clearly demonstrated in the literature 5].

Yukawa et al reported mean spinal flexion of 53.0° and hyperextension of 23.4° with no difference between the genders 6]. Another study reported females decreasing extension and flexion ROM slightly more than males between the ages of 20–70 years (13.9° and 9.0° vs 16.3° and 8.0°) [4]. Age-related reductions in lumbar flexion, extension and lateral flexion were most evident after approximately 40 years of age.

The role of collagen

It is important to understand the role of collagen and how age-related changes to collagen matrices are linked to the declining mechanical properties of aging bone and joints [4,7]. Physical and biochemical changes occur to collagen with increasing age, resulting in decreased extensibility. These changes include an increased formation of intramolecular and intermolecular cross-links that restricts the ability of the collagen fibres to move past each other as tissue length changes [3]. Cross-linking involves two different mechanisms, one a precise and enzymatically controlled cross-linking during development and maturation and the other an adventitious non-enzymatic mechanism following maturation of the tissue.  This non-enzymatic cross-linking, known as glycation, is the major cause of dysfunction of the collagenous tissues in old age.

The process of cross-linking and the presence of advanced glycation end products (AGEs) seem to be major determinants in the loss of ROM and strength [8]. AGEs naturally form inside the body when proteins or fats combine with sugars (glycation). This non-enzymatic reaction affects the normal function of cells, making them more susceptible to damage and premature aging. The effect of glycation on cell-matrix interactions may be an equally important aspect of aging collagen. It is interesting to note that this process is accelerated in diabetic individuals due to higher blood glucose levels [9]. Although there are 19 genetically distinct human collagens, the functions of the more minor collagens have yet to be clarified. Types I and II collagen are found in intervertebral discs.  Aging causes this type of collagen to transition into a more fibrotic tissue.

This increased fibrosis, which is associated with degeneration, contributes to changes in material properties of the nucleus pulposus from a fluid-like to a solid-like material, thus contributing to a more brittle, fragile disc [10].  Fragility of aging bone may be related to changes in collagen as evidence suggests that altered collagen molecules have a detrimental effect on the mechanical properties of bone. When bone collagen is damaged due to non-enzymatic cross linking, also known as glycation, the bone exhibits increased stiffness, ROM, decreased bone strength and reduced stability [6].

Aging and hyaline cartilage

The aging process similarly affects muscles, hyaline cartilage and joint motion.  Muscle fibers atrophy and cartilage dehydrates, causing a loss in elasticity and joint motion restriction that leads to flexibility decrease and loss of ROM. The articular surfaces of the human spine’s facet joints are covered by hyaline cartilage that serves as an elastic load-bearing material responsible for the frictionless movement of the surfaces of articulating joints. As the structure of hyaline cartilage changes, there is an increased risk of joint inflammation and arthritis [11]. The decrease in tensile strength after the third decade of life, along with inflammation from repeated injury, overuse in sport, and congenital defects may lead to increased risk of osteoporosis [10,12].

Aging and loss of bone mass

The aging spine is characterized by two parallel but independent processes: development of degenerative discogenic changes and bone mass reduction. osteoporosis, or reduced bone mineral density, increases the risk of stress fractures [13]. In focusing on the relationship between these two processes, the American College of Sports Medicine underlines the need for further research on osteoporosis [14]. One study evaluated factors related to spinal mobility in patients with postmenopausal osteoporosis [15]. The researchers found that skeletal fractures are an important clinical manifestation of the disease, with older female patients the most severely affected. Multiple vertebral fractures can result in postural deformities, which could cause significant functional impairments in ADLs [15,16] and have a significant impact on quality of life.

Joint Hypermobility

Another important consideration connected to bone health is joint hypermobility, which is defined as excessive range of motion with a global, whole-body score of 4 or higher on the 9-point Beighton scale [17,18]. When considering spinal ROM and aging, available motion in the lumbar spine drops by approximately 30% between youth and age 70 [2,19]. According to Day et al. [20], available hypermobility data in general populations are conflicting; they state that some findings report reduced bone mineral density in hypermobile participants, while others report increases. A study on hypermobile 34-year old women not only found significantly lower bone mineral density measurements, but some of the participants had already reached osteoporotic levels [20].

Characteristics of the aging spine

Important characteristics of the aging spine include a decrease in collagen and proteoglycan content of the annulus fibrosus and nucleus pulposus [21], damage to collagen from cross-linking [22] and atrophy of type II muscle fibres.  This damage results in a decrease in elasticity and joint motion restriction that leads to a decrease in flexibility and loss of ROM [11]. In addition, increased intramuscular connective tissue stiffness can result in decreased ROM [3]. Long-term complications associated with aging affect spinal health and can cause significant functional impairments in activities of daily living [15,16]. Since spinal health and mobility are key determinants of whole body function, an increase in participation restrictions may result in a perceived quality of life change that is usually detrimental [23].

To our knowledge, no systematic review exists that examines the relationship between aging and spinal ROM. Therefore, the aim of this review was to investigate the role of aging and its effects on spinal range of motion, with a deliberate focus on athletic, elderly, clinical and general populations.

Methods

Two databases were searched (PubMed and Google Scholar) between July and September 2013 and again in September 2017, using the following Medical Subject Heading (MeSH) terms:  spine, aging, athlete, range of motion, human, collagen.  Research studies were rejected if they did not meet the one of following criteria: English language, human participant, observational study, longitudinal study and case study. The initial search produced 224 articles of which 21 were immediately removed for being animal studies.  A further 132 were removed for being unrelated to the MeSH terms and 11 articles were removed because they were not applicable or did not meet the quality requirements of the ADA (American Dietetic Association) Evidence Manual. The remaining 60 studies were rated on a scale of 1 to 3, with 3 being the lowest quality (Limited), 2 being studies with minor methodological concerns (Fair) and 1 being the highest quality with strong design and free from bias (Good). Studies not meeting these criteria were excluded. An additional 46 articles were eliminated as they either did not pertain to the question being addressed, or were inconclusive in their findings (Table 1). Of the 14 retained papers, 6 were review articles and were manually checked to identify any missed studies that may have related to the subject; none were found. The accepted articles were categorized into the following populations: athletes, clinical, general and elderly.  The athlete group consisted of all studies mentioning the word athlete, sports or exercise. The clinical group included clinical trials or research and the elderly group included studies mentioning the term elderly or aging populations.  The general group consisted of all other studies which did not fall into the athlete, clinical, or elderly categories (Figure 1).

Results

Athlete population

Although the athlete category yielded four papers specifically mentioning athletes, exercise, sport, competition and sports injuries, useful information was gleaned from only one paper relating to ROM or exercise in relation t.o biomechanical function and aging. The review by Benjamin et al. [24] discussed the structure-function correlations of entheses on both the hard and soft tissues with attention paid to mechanical factors that influence form and function.   It explored the relationship between entheses and exercise, and emphasized the degenerative, rather than inflammatory nature of most enthesopathies (pathological changes at an enthesis) in sport. This study is relevant because, as stated by the authors, the tendon-ligament complex response to loading allows for multi-axis bending, such as in the lumbar spine.  It applies to diseases associated with the spondyloarthritides (SpA) including ankylotic spondylitis, psoriatic arthritis, reactive arthritis and undifferentiated SpA, all of which may have deleterious effects on ROM [24]. The removed papers addressed bone formation and fracture healing, evaluation of changes in T1rho and T2 relaxation time in the meniscus using 3.0 T MRI in asymptomatic knees of marathon runners, tissue engineered strategies for skeletal muscle injury and post-meniscectomy qualitative risk analysis considering high BMI and pre-existing osteoarthritis.

ASMHS2018-104-Janine Bryant UK_F1

Figure 1. Prisma and exclusion flow chart here.

Table 1. Categories table

Article

Participants

Method

Quality Grade

Significance

Conclusion

8

A

R

II

Focus on degenerative

Role of enthesopathies in ROM changes

27

G

MR

II

Poor cellular nutrition

Tissue degeneration leads to osteoarthritis

40

E

O

II

Focus on aging spinal disorders

Link spinal disorders to ROM loss

16

E

R

II

Focus on structural changes due to age

Further study is crucial for understanding the unique biomechanical function of the aging spine

23

E

R

II

Age-associated conditions

Implications on elderly limited mobility and Quality of Life

43

E

OBS

I

Compares healthy and ageing degenerated discs

Connects endplate damage to Degenerative Disk Disease

13

E

OBS

I

Aging disc and collagen changes

Both collagen and proteoglycans undergo age-related changes

33

E

R

II

Age is a primary risk for dev. Of OA

More data is needed to understand age-related changes that lead to Osteoarthritis

49

C

R

II

Collagen changes and joint function relating to OA

Aging impacts reparative abilities that can lead to Osteoarthritis and loss of ROM

42

C

OBS

I

Links muscle atrophy with low back pain

Pilates improves ROM in trunk and pelvic segments

Key: A: Athletes G: General E: Elderly C: Clinical

Methodology: R: Review MR: Mini-review O: Overview OBS: Observational

Clinical population

In the clinical category, osteoarthritis (OA) was discussed in one of the three retained papers.  In their review, authors Sinkov and Cymet [25] discuss imbalance of joint function as an initiator of the disease process worsened through changes in the collagen in the joint. The authors describe OA as a non-inflammatory disease characterized by progressive loss of joint articular cartilage resulting in pain and deformity and most present in populations over the age of 65, an element that can significantly affect quality of life (QoL). Some risk factors for primary OA include increasing age or history of injury to the joint from trauma, repetitive stress or inflammation.

The prevailing explanation for the onset of OA is a progressive fatigue failure, or prolonged wear and tear [12,15,25,26]. This explains the increase in incidence of OA with age, as well as its prevalence in joints that are overloaded or overused, such as the ankle in ballet dancers [10], or the elbow in baseball pitchers [25].

The second paper in this category discusses lumbopelvic flexibility and stability as affected by Pilates training utilizing forty healthy male and female volunteers with a mean age of 31.65 ± 6.21 yrs [27]. The study was retained not because it utilized Pilates as a therapeutic measure, but because it examined asymptomatic individuals exhibiting an inability to control lumbo-pelvic stability; this may be an early detection sign for spinal problems [27]. This study indicated that Pilates could be used as an adjunctive exercise program to improve flexibility, enhance control-mobility of the trunk and pelvic segments and, more relatedly, may also prevent and attenuate the predisposition to axial musculoskeletal injury.

The third study [8] examines the effect of strenuous exercise on the turnover rate of collagen and included a discussion on the molecular mechanisms involved in the aging of collagen, increase in stiffness and the process of enzymatic and non-enzymatic collagen cross-links.  The authors reported age-related changes in bone, tendon, articular cartilage and the matrix protein glycation leading to formation of intermolecular cross-linking, thereby affecting optimal mechanical functioning of tissue. This process clearly has relevance to aging and exercise because the slow turnover of aging collagen results in an accumulation of advanced glycation end-products. This also can be described as an oxidation rendering the collagen fibres too stiff for optimal functioning. This publication is somewhat limited in that its findings showed that, although strenuous sports training regimes increase tensile strength of bone and tendon, further understanding of the mechanisms of collagen turnover and cross-linking are needed to improve understanding of the problems caused by exercise and injury recovery [8]. The paper was ultimately retained in our study for its focus on glycation and the aging of connective tissue via the process of collagen cross-linking.

General population

The first of two studies retained in this category, identified as mini-review, addresses transport properties of cartilaginous tissues in relation to their cellular nutrition as it applies to articular cartilage. Poor cellular nutrition in cartilaginous tissues is believed to be a primary source of tissue degeneration that results in OA or disc degeneration [4]. Transport properties include:

Solute diffusivities that are significant because, due to the avascular nature of cartilaginous tissue, diffusion of solutes through the tissue extracellular matrix plays an integral role in cellular nutrition;

Hydraulic permeability as an important property of cartilage because water is the major component of the tissue and is an important factor governing the rate of fluid transport; and,

Effect of mechanical loading of tissue that significantly affects the transport of fluids and solutes through the tissue, but is dependent on the type of loading (i.e., dynamic vs. static loading).

Intolo et al. [10] focused in their review on the effect of age on lumbar ROM.  They stated that, although lumbar ROM reduces with advancing age, it is still unclear how this reduction occurs across different age categories. Furthermore, they stressed the importance of determining if movement reduces with age and whether it does so consistently across different age strata.  There were several limitations in this study including that some relevant published studies were not identified due to either alternative keywords or poorly worded abstracts.  However, the paper was retained herein for its potential value to clinicians by providing normative data on the expected loss of lumbar ROM in healthy aging individuals. The article demonstrated age-related reductions in lumbar flexion, extension and lateral flexion, losses in flexibility that are most evident after approximately 40 years of age.

Elderly population

Eight papers were found on the aging process of the spine or musculoskeletal system.  The first was an overview discussing aging on intervertebral discs (IVDs), endplates, facet joints, muscles and ligaments, and the vertebral body. Papadakis et al. [28] linked a number of painful disorders to aging of the spine, including loss of bone mass, disc degeneration, facet degeneration, disc bulging, facet hypertrophy and ligamentum flavum hypertrophy. These may contribute to compromised biomechanics and ROM loss.

The second paper [29], reported an overview of the mechanisms of aging in the spine that cause structural changes and injury risk affecting biomechanics and ROM. However, the review did not address other mechanisms of degeneration beyond advancing age, stating that further study is required to understand the mechanisms of degeneration and the unique biomechanical function of the aging spine.

The third paper retained from the elderly category focused on aging in the musculoskeletal system [30]. The paper focused mainly on age-associated conditions involving the bones, muscles and peripheral joints; the research was broader in that it included musculoskeletal disorders and a range of interactive conditions such as fibromyalgia and tendinopathy that affect soft tissues, tendons and ligaments, bones and osteoporosis, IVD, and muscular conditions like polymyalgia and myopathies. Implications of musculoskeletal disorders on the public health of elderly persons from the perspectives of physical and social impacts caused by pain was presented, including limited mobility and reduced QOL [30].

An observational study was found [22] which aimed to investigate the presence, localization and abundance of cells expressing notochordal cell markers in human lumbar discs during degeneration. Postembryonic vestiges of the notochord were found in the nucleus pulposus of human IVDs. This research suggests a correlation between cells with an immuno-histochemical notochordal phenotype that do not exhibit typical morphology of notochordal cells and early degenerative changes, particularly granular matrix changes. The researchers studied two groups of specimens, the first being lumbar motion segments that were removed from 30 deceased individuals between 26 weeks of fetal gestation and 86 years of age.

None in this group had a known history of back problems or pain. The second group was comprised of 38 disc samples that had been obtained during surgery for painful lumbar disc degeneration and/or disc herniation (protrusion, extrusion, or sequestration). The samples were obtained from individuals (23 males, 15 females; age range 26–69 years) with known clinical symptoms, radiological features, and histological degree of disc degeneration.

The loss of cells with typical notochordal phenotype (physaliferous) and the coincident onset with signs of disc degeneration leads to speculations about their role in the preservation of disc function. Although interspecies comparison—premature loss of notochordal cells from chondrodystrophic breeds with higher incidence of intervertebral disc degeneration—gave some support for this idea. This study, was promising as the first study analyzing the presence of cells with notochordal phenotype and age-related changes of adult human discs. However, the authors state that conclusive evidence for this hypothesis is still missing and, therefore, this paper was ultimately removed.

According to Rajasekaran et al. [31], chronic overuse of the immature spine is related to endplate damage leading to degenerative disk disease (DDD). This study was observational in nature, focused on DDD and discussed decreased nutrition as the final common pathway for DDD and endplate (EP) damage. EP damage affects diffusion and, therefore, disc nutrition. The authors found that damage to the endplate may be the initiating factor for disc degeneration by both altering the mechanical environment and affecting the nutritional pathways. This study is also the first in literature to document the feasibility of pharmacological modulation of endplate vascularity and disc diffusion, but is purely a radiological assessment of degeneration, thus, the clinical symptoms have not been considered. Another limitation of the research is that disc degeneration, being an ongoing phenomenon, requires a serial longitudinal and in-vivo study, which, as stated by the authors, was not performed. It would have been useful to have histologically supportive data to explain the changes in the endplate and nucleus pulposus.

Research by Singh et al. [32] discussed age-related changes in the human intervertebral disc. The aim of this study was to characterize age-related changes in the matrix of human intervertebral discs from the third to eighth decade of life with a focus on collagen and proteoglycan composition.  It utilized background data of disc degeneration as associated with changes in the concentration and fragmentation of matrix molecules. Forty-six discs of human thoracolumbar spines (T11-L5) aged 32 through 80 years were analyzed. However, the authors did not include the youngest age group (31–40-years old) in their analysis because it is difficult to obtain IVD specimens in this age group due to the relatively low rate of mortality. DNA, collagen and proteoglycan contents were measured using chemical assays while small non-aggregating proteoglycan levels were analyzed by comparative Western blotting.  The paper concludes that large proteoglycans play a major role in water retention, while small proteoglycans regulate formation of the extracellular matrix. During aging, proteoglycan and collagen levels decrease, while some small proteoglycans show differing patterns of changes in both the inner and outer nucleus pulposus and annulus fibrosus. The concentration of biglycan increases in all three disc compartments with age, while decorin content declines. The decrease in total collagen and proteoglycan content may increase susceptibility in IVD degeneration. The authors concluded that the functional significance of these changes needs further investigation.

The seventh study found in this category was a review that discussed age related changes in the musculoskeletal system and the development of osteoarthritis [12]. Although this paper duplicated much of what was collected from other studies in this category, this is the first paper to mention Vitamin D deficiency as a risk factor for OA (possibly contributing to oxidative stress symptoms).  In addition, this study links formation of advanced glycation end products (AGEs) to the modification of collagen resulting in increased cross-linking of collagen molecules. Formation of excessive collagen cross-links affects the biomechanical properties of cartilage resulting in increased stiffness, more brittle cartilage,and increased susceptibility of the tissue to fatigue failure.

The eighth and final study [33] aimed to establishing radiographic standard values for cervical spine morphometry, alignment, and ROM, and included 1,230 asymptomatic male and female subjects between ages 30 and 80.  Subjects underwent anteroposterior (AP), lateral, flexion and extension radiography of the cervical spine. AP diameters of the spinal canal, vertebral body and disc were measured at each level from the 2nd to the 7th cervical vertebra (C2-C7), with sagittal alignment and ROM during flexion and extension calculated using a computer digitizer. Findings included the AP diameter of the spinal canal and disc height decreased gradually with increasing age as well as extension ROM decreasing more than the flexion ROM, and lordotic alignment progressing with increasing age. In addition, the study found there was a significant difference in C2-C7 alignment and ROM between males and females, with cervical lordosis and thoracic kyphosis increasing more with age in females than in males.  Although the study had several limitations, including possible measurement errors, difficulty in achieving uniform positions of the vertebrae in relation to the X-ray beams in different positions of motion and measurement being performed only once due to the large sample size, it was ultimately retained in our review as it establishes standard values and age-related changes in cervical anatomy, alignment and ROM (Table 1).

Discussion

Reduced spinal mobility may result in activity limitations and participation restrictions, which could subsequently affect quality of life. This literature review examined the effects of aging on spinal range of motion (ROM). This research relates to current available research and offers a deeper inquiry into spinal ageing specifically.  When investigating how the spine ages, there were several significant subcategories found in the literature.  Among those specific to aging, were aging and articular cartilage, aging and flexibility/ROM, aging of specific spinal regions (cervical, thoracic, lumbar), special considerations of the aging athlete, aging and osteoarthritis, aging and muscle strength, bone and aging, and the role of collagen in aging and ROM.  In considering ROM and aging, entheses were discussed as common sites of overuse, exploring the relationship between entheses, enthesopathies and exercise drawing attention to degeneration rather than inflammation as histological evidence of the most common enthesopathies rarely demonstrates evidence of inflammation within the affected entheses.  In this respect, spinal ROM is affected because the tendon-ligament response to loading allows for multi-axis bending, such as in the lumbar spine [26]. With regards to specific pathologies related to ROM and aging, osteoarthritis, being non-inflammatory, causes joint pain and damage which is progressively degenerative, the clinical presentation being deep localized pain with stiffness, especially in OA of the spine, which also can result in pain and weakness.

Findings, especially in the elderly population, focused on degenerative disorders of the aging spine including disc and facet degeneration, facet hypertrophy and loss of bone mass over time as contributors to compromised biomechanics and ROM loss [26]. Finally, in the general category, degeneration was again a theme with focus on poor cellular nutrition in cartilaginous tissues being the primary cause of tissue degeneration resulting in OA (in the case of articular cartilage degeneration) or disc degeneration especially of the intervertebral discs [34], both leading to back pain and loss of ROM.

The process of aging affects all of the body systems including the spine. The literature links loss of bone density and flexibility to increased risk for postural changes and disc fractures that contribute to loss of range of motion and participation in activities of daily living. Quality of life is affected as aging populations experience decreased mobility due to age-related changes in spinal health. Information found, especially relating to collagen, points to physical and biochemical changes to collagenous frameworks with increased age resulting in decreased extensibility especially in aging skeletal muscle. Collagenous structural changes, regardless of type, cause degenerative effects in the mechanical properties of bone, tendons, ligaments and cartilage.

Aging affects intervertebral disks, endplates, facet joints, muscles and ligaments. This can lead to degenerative conditions such as disc degeneration, loss of bone mass, facet degeneration, bulging discs, facet hypertrophy and ligamentum flavum hypertrophy. Athletic, clinical, general and elderly populations experience these changes in various ways depending on age, activity level, and genetic disposition, with the major commonality being compromised biomechanics and loss of range of motion. Aging bone in shows an increased risk for development of osteoporosis thereby increasing the risk for stress fractures, especially in older females.

The limitations to the present study was the lack of available research into the ageing human spine as related to different populations and ROM. Continued research into the process of spinal aging, and how it affects range of motion and quality of life, with particular focus on the spinal segments, surrounding muscles, vertebrae and discs, is warranted based on this review. Further research will enable an increasingly aging population worldwide to benefit from findings. The overall goal is to promote spinal health and identify preventive and therapeutic interventions that will increase or maintain spinal range of motion, thereby allowing individuals to continue participation in activities of daily living and to enjoy an overall increased quality of life.

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Introductory Teaching Tool Utilizing Immunohistochemistry to Explain the Placenta

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DOI: 10.31038/UGFM.2018111

“The womb may be more important than the home”
J.P. Barker

Abstract

Introduction: The placenta occupies an important place in science, medicine, and law; yet, it remains a difficult organ to explain and understand because of its unique characteristics.

Methods and Materials: A descriptive observational study was carried out on placentas. Placental components were illustrated with diagrams and corresponding microphotographs were highlighted by immunohistochemical staining to identify cell-types and structures.

Results: The placenta was diagrammatically demonstrated by dividing it into chorionic plate with umbilical cord, basal plate and placental disc. In highlighting the histological components, approximately 80 immunohistochemical preparations were taken into account

Conclusion: Placental complexity continues to be the critical interface for maternal-fetal dialogue.  Better educational methods are needed to explain the complex pathophysiology of this essential organ. Improving placental education is the key to attracting young researchers dedicated to this field.

Key words

Histology; immunohistochemistry; medical education; placenta; trophoblast.

Introduction

The placenta is a complex and misunderstood organ not only because it is exclusively vascular in nature and possesses allograft-like characteristics, but also because it ceases to function at week 40 of gestation. The placenta is temporary because it is unable to be efficient after reaching 40 weeks of gestation; it is no longer able to sustain the rapidly growing fetus. Its vascular features are unique because the placenta manages two individual circulatory systems belonging to two different people (mother and fetus). It is allograft-like because it comes from two individuals with unique genotypes from the same species. .

The placenta has been described as “the most important organ of the body, but paradoxically the most poorly understood” [1,2]. Examination of the placenta by the pathologist has been defined as a “gestation diary” [3]identifying conditions that are likely to recur in subsequent pregnancies, separating clinical syndromes into distinct pathological phenotypes for further investigation, and uncovering the underlying cause of unexpected adverse outcomes. Classification of placental lesions has evolved from being a purely descriptive exercise through a stage in which the major pathophysiological processes such as disorders of maternal implantation and the amniotic fluid infection syndrome were first described to a recently proposed comprehensive classification system that includes all of the major maternal and fetal vascular and infectious and idiopathic/immune inflammatory processes (Amsterdam Placental Workshop Group; assessment of stillbirth could not be complete without the  study of the placenta and vice versa [4]. Therefore, in medical-legal cases, “the placenta has played a pivotal role, and at times, stood in the forefront, not only as participant but also as witness”[5]. These types of cases have recently increased, in which the placenta is like a “black box” in a trial involving a medical malpractice lawsuit [6].

Odd as it may seem, study of the placenta in pathology departments is not mandatory, on the premise that most babies will be born healthy and therefore the placenta must also be healthy [7]. Most of the organs or pieces of tissue removed from any patient must be sent to the pathology lab for examination; the placenta is the most common exception to this requirement. Incongruous, as well, is the fact that the placenta is currently one of the most frequently examined specimens in pathology departments due to the large number of deliveries. The importance of the placenta is now a recognized factor in newborn prognosis, essential to explaining the causes for stillbirth; and, in the long-term it acquires medico-legal aspects when  later childhood neurodevelopmental delay are diagnosed [8, 9].

Additionally, the importance of placental examination in clarifying failed maternal reproduction has been on the rise in recent decades. Placental examination can also be directly requested by families themselves who want to know how etiology, prognosis, future outcomes, probabilities and any other information could be useful in analyzing future gestations among their own relatives. Therefore, in order to obtain the greatest amount of answers for families, the joint interpretation of the binomial fetus and placenta is mandatory [10, 11]. Furthermore, science appears to be rediscovering the placenta. The trophoblast and the other placental components are now being widely researched [12]. New techniques in molecular biology have brought about significant advances in epigenetics, imprinting, cell culture, genetic diseases, complete genome sequencing, and immunology, all of which have helped to expand our knowledge about placental components and their functions. Diseases like preeclampsia, with its enormous impact on fetal and maternal morbidity and mortality, are now being focused on, but despite the existence of a multitude of theories [13, 14], benefits to clinical application have yet to be seen. We still do not know enough about interactions between trophoblast and maternal immune cells [histiocytes and lymphocytes], the role of trophoblast in decidual and spiral arterial invasion, and other related issues.

  • The concept of two different circulatory systems belonging to two different individuals in the same organ is unique.
  • Understanding how each joined individual pumps its own circulation in not easy, even among students in advanced specialized medical studies.
  • The importance of the umbilical cord [UC] as a structure that carries fetal blood, which is akin to having the aorta and the cava vein outside the body, is not sufficiently recognized [Figure 1 highlights the placental tree as a continuity of fetal circulation].
  • The presence of a variety of special, temporary and unique cells –the trophoblast- with tumor-like behavior and tumor-like appearance  [invasive capacity, anisonucleosis, nucleomegaly, nuclear hyperchromasia and pleomorphic nuclei] [9], whose subdivisions in appearance and shape can be difficult to understand and even to pronounce [cytotrophoblast, syncytiotrophoblast, endovascular extravillous trophoblast, interstitial extravillous trophoblast].
  • The placenta does not respond in a single way to injuries, for example, to hypoxia [10]297 with uterine type of chronic hypoxic placental injury (group 2
  • The variety of normal and abnormal aspects that depend upon the age of gestation, its maturation [19]: nucleated fetal red blood cells [20], fibrin, syncytial knots, multinucleated extravillous trophoblast, amount of spiral artery muscle [21]and for this purpose they are remodelled into highly dilated vessels by the action of invading trophoblast (physiological change.
  • The placenta is an organ that plays many roles; no organ can match the placenta for functional diversity [1].

The principal aim of this article was to propose a clear simple method to describe the placenta to those individuals who have had little or no previous exposure; additionally, some specific aspects have been emphasized so that the significance, continuity and wholeness of the fetal-placental unit may be completely understood.

Methods And Materials

We performed a descriptive observational study on the placenta and used immunohistochemical staining to highlight its components; diagrams and microphotographs were referred to when explaining placental structures and functions.   Placental cases were collected that corresponded to samples studied at the PUJ-HUSI Department of Pathology from 2007 to 2017. A diagram of the placenta which illustrated its components, was created. This was accompanied by corresponding microphotographs, highlighted by immunohistochemistry staining, which differentiated cells and structures. Placental components were divided into fetal and maternal directions and vice versa, following the tree [Figure 1]. Then, slides were chosen from the available cases that had been collected for both diagnosis and research, and that had been tested with conventional immunohistochemistry on paraffin embedded tissue  [BAX, FAS, bcl2, p57, cMyc, IGF2, FGF2, VEGFA, VEGF R1, MMP1, VEGFB, TGFB3, Ki67, PLGF, thrombomoduline]. Antibodies that best highlight specific placental cell types were chosen for this purpose. Microphotographs were taken and included with explanatory diagrams.

UGFM2018-101-MercedesOlaya Colombia_F1

Figure 1. Unit fetus, umbilical cord and placenta

The figure represents the continuity from the fetal heart to the distal villi in the placenta. Blood is pumped from the fetal heart through the entire fetal body which also needs to be impulsed outside the body and  pass through the umbilical cord, as long as it can be. When the blood reaches the chorionic plate in the placenta, it is distributed in many branches, up to the last villi where the capillaries are close to the mother’s blood:  her blood functions like air moving around leaves. Afterwards, oxygenated blood must return to the fetal heart.

In the close up, a villous is shown. Fetal capillaries are carrying fetal circulation (F); around it, maternal blood is present (M).

Results

An overall diagram of the placenta was created [Figure 2] which was subsequently divided into the chorionic plate with umbilical cord, basal plate and placental disc. Ordinary placentas and placentas previously chosen for other studies were collected to illustrate structures, especially those that have received scant recognition. A work checklist was created and filled with data for future use with this collection; microphotographs were taken and selected for the abovementioned purpose . Approximately 80 immunohistochemical preparations were taken into account when spotlighting histological components. Some of said antibodies have not been described in the placenta. Different trophoblast cell types exhibit different expressions; for example, villous syncytiotrophoblast does not have the same expression as subchorionic plate syncytiotrophoblast or basal plate syncytiotrophoblast, for the MMP1 antibody, the explanation of which is beyond this paper’s goal.

UGFM2018-101-MercedesOlaya Colombia_F2

Figure 2. The placenta

The figure represents the placenta. Spiral arteries (A) are shown in the basal plate. Decidual cells (red hexagons) are mixed with endovascular extravillous trophoblasts (light green stars), interstitial extravillous trophoblasts (dark green rectangles); these cells also form columns of trophoblasts (B).

The trophoblast is always green in this figure, regardless of type. Syncytiotrophoblast overcoats the whole placental tree (light green rectangles) (D) and completely separates both circulatory systems (maternal and fetal); syncytiotrophoblast is also seen in maternal and fetal borders. Cytotrophoblast is beneath the villous syncytiotrophoblast. Fibrin is represented by a pink line (C). The amnion is on the fetal side (gray rectangles). In the chorionic plate, arterial (E) and venous vessels (F) can be seen, connected to the UC (G).

However, we suggest that cells of the same origin and denomination could have very different functions, but these have not been studied in-depth nor are they well understood. Simultaneously, the expressions of some antibodies were surprisingly high; as was the case of FAS, with regards to apoptosis; others were surprisingly low, like bcl2, with regards to anti-apoptosis. Protein expression analysis via immnunohistochemistry shows that cells normally seen as equals, are not; furthermore, their activities include unexpected biochemical pathways. The best antibodies chosen to be photographed and described are listed in Table 1.

Table 1. Antibodies in Figures:

[ ]: Concentration of primary antibody; C: clonality, M: monoclonal, P: polyclonal.

FIG

ANTIBODIES

CODE

LAB

[]

C

Control

Fig 3

 cMyc

(9E11): sc-47694

Santa Cruz Biotechnology Inc

1:1,000

M

Colon

Fig 3

 cMyc

(9E11): sc-47694

Santa Cruz Biotechnology Inc

1:1,000

M

Colon

Fig 3

 cMyc

(9E11): sc-47694

Santa Cruz Biotechnology Inc

1:1,000

M

Colon

Fig 3

FAS

(C-20): sc- 715

Santa Cruz Biotechnology Inc

01:25

P

Liver

Fig 4

VEGF-A

GTX102643

GeneTex

P

Riñón

Fig 4

VEGF-R1

GTX15294

GeneTex

P

Placenta

Fig 5

VEGF-R1

GTX15294

GeneTex

P

Placenta

Fig 5

p 57

SPM308:sc-56456

Santa Cruz Biotechnology Inc

01:50

M

Placenta

Fig 5

VEGF-A

GTX102643

GeneTex

P

Riñón

Fig 6

MMP1

GTX100534

GeneTex

P

Endometrial carcinoma

Fig 6

VEGF-R1

GTX15294

GeneTex

P

Placenta

Fig 6

VEGF-B

(J-14I): sc-80442

Santa Cruz Biotechnology Inc

01:50

M

Placenta /Heart

Fig 7

VEGF-R1

GTX15294

GeneTex

P

Placenta

Discussion

The complexity of the placenta can be simplified by following the blood flow through the fetal extracorporeal circulation from the umbilical cord, where the umbilical vein and arteries are crucial for fetal life, and on toward the maternal bed with its remodeled spiral arteries. Maternal arteries and their abnormal remodeling are now recognized as linked to some of the most important adverse outcomes. In order to better understand the placenta, it should be divided into the:

  • Fetal surface-umbilical cord
  • Fetal surface-chorionic plate
  • Placental disc
  • Maternal surface-basal plate

Fetal surface- umbilical cord

The placenta is an extension of the fetal cardiovascular system [Figure 1]. The umbilical cord [UC] is the bridge between the intra and the extracorporeal circulations. The UC protects the whole fetal blood volume by means of coiling and with Wharton’s jelly. Starting from the outside, the first layer of the UC is the amnion, which is firmly attached to the cord that protects Wharton’s jelly [Figure 3D]. Beneath the amniotic epithelial cells, Wharton’s jelly is interspersed with stromal cells [Figures 3C and 3D]; these are few, and they are spread haphazardly between the amnion and the blood vessels. The vessels are surrounded by myofibroblast, which are inconspicuous. The umbilical arteries and the vein lack a real adventitia, which is expected because of their size; therefore, the muscular layer is interlaced with Wharton’s jelly structures [Figure 3B]. This is why the umbilical vessels cannot be dissected. The venous and arterial walls contain smooth muscle [Figure 3B], perivascular and endothelial cells [Figure 3B]. The vein also has an unusual subintimal elastic layer [23]. The umbilical arteries, on the contrary, do not have an inner elastic layer as do the other great arteries within the body [24]porous Wharton’s jelly, two umbilical arteries, and one umbilical vein, are designed to protect blood flow to the fetus during a term pregnancy. The outer amnion layer may regulate fluid pressure within the umbilical cord. The porous, fluid filled Wharton’s jelly likely acts to prevent compression of the vessels. Blood flow is regulated by smooth muscle surrounding the arteries that is intermingled with a collagen based extracellular matrix (ECM; furthermore, in spite of their caliber, UC arteries have a much thinner outer elastic layer than would be expected [23]. The venous diameter is double that of the arterial diameter [Figures 4B] [25].

Once the UC contacts the placental disc on the fetal surface, ideally on the center or close to it and at a 90-degree angle, the chorionic plate has been reached [Figure 4A].

Fetal surface- chorionic plate

To reiterate, the closest layer to the fetus is the amnion [Figures 4C, E and F], but this amnion is not firmly attached here, as it is on the cord; conversely, the union is virtual. Grossly the chorionic plate is bluish in color, but up close, transparent,  which allows the vessels branching out on the surface to be seen. It should also be possible to distinguish veins from arteries [Figure 4A]: the arteries are on top of the veins. Histologically, the amnion looks like the UC, with a single organized layer of cuboidal cells.

Located under the amnion is the chorionic tissue [Figures 4D, E and F]; it also looks like the UC: hypocellular and with large fetal vessels. Each fetal vessel should not be more than three times larger than the adjacent vessels [24]such as true knots (TK. Behind the chorionic layer the trophoblastic cell layer is found [Figures 4E and F [green]], the trophoblast cell layer covers the inner part of the placenta including each small terminal villi [25], which has also been described in mice [26]. The human placenta is hemochorial, the maternal blood has contact with the trophoblast, but the trophoblast isolates the branched tree, including the chorionic and basal plates inside the disc [Figures 2, 4, 5 and 6].

UGFM2018-101-MercedesOlaya Colombia_F3

Figure 3. The umbilical cord

A – The UC clearly exhibits the amnion surrounding it; in the center, the umbilical artery (cMyc 4x). B- In the umbilical artery, the smooth muscle with the nondefined border (long white arrow) can be seen; in the center of the vessel, darker cells, the endothelium (short brown arrow) (cMyc 10x). C- Stromal cells in the Wharton´s Jelly (cMyc 40x). D- From left to right, vascular wall, Wharton´s Jelly with stromal cells and in the border, the amnion (brown arrow) (FAS 10X).

UGFM2018-101-MercedesOlaya Colombia_F4

Figure 4. The chorionic plate

A – Gross placental morphology on the fetal side. UC Insertion is close to the center and placed at a 90o angle; the placenta is covered by the amnion; fetal surface is bluish in color. The chorionic vessels can be seen on the surface. The membranes are upside down (white arrow). B- A histological microphotograph of the UC with two arteries and one vein; the vein is twice the size of the arteries. C- The amnion covering the UC surface can be seen at the top and the artery exhibits its thick smooth muscular layer (VEGF-A 2x). D- The chorionic vessel is shown and the villi are immediately below it (H&E 2X). E- The figure represents the chorionic plate and its continuity with the UC and with the villi. F- As in Figure D, the amnion can be recognized at the top, the artery is in the chorionic plate. Here, the fibrin is clear and the syncytiotrophoblast is highlighted (green arrow) (VEGF-R1 10X); this syncytiotrophoblast is covering the chorionic plate, isolating the maternal and fetal circulatory systems.

UGFM2018-101-MercedesOlaya Colombia_F5

Figure 5. The placental disc

A – Gross placental morphology of the villi, which resemble tiny fingers. B- The figure represents the inside of the placenta; each villous carries capillaries and is covered by syncytiotrophoblast. C-Transversal view of the villous placental tree; the syncytiotrophoblast is highlighted; note the syncytial knots (VEGF-R1 20X). D- Transversal view of the villous placental tree; villi are separated; in the space between them, maternal blood circulates (H&E 10X). E- Transversal view of the villous placental tree; the cytotrophoblast is highlighted, behind the syncytiotrophoblast (p57 10X). F- Transversal view of the villous placental tree; fetal capillaries are highlighted insight the villi. The surrounding blue colored nucleuses correspond to stromal cells (VEGF-A 20X).

UGFM2018-101-MercedesOlaya Colombia_F6

Figure 6. The basal plate 1

A – Gross placental morphology of maternal surface; the placental cotyledons are discreetly outlined. Membranes were removed. The UC is seen on top. B- The figure represents some contact between the anchoring villi and the basal plate. The syncytiotrophoblast continues isolating both circulations, now covering behind the decidua (C), like a line. Also, the trophoblast columns can be distinguished (dark green). The interstitial extravillous trophoblast (dark green squares) and intravascular extravillous trophoblast (light green stars) in spiral artery walls are represented. C- The syncytiotrophoblast behind the basal plate is seen (MMP1 20X). D- The basal plate with its mixed cell population is exposed: decidualized (maternal) cells and interstitial extravillous trophoblast (arrows) (H&E 10X). E-Immunohistochemistry microphotograph highlights the interstitial extravillous trophoblast (green arrows) (VEGF-R1 10X). F- Immunohistochemistry staining highlights the endovascular extravillous trophoblast (VEGF-B 10X). G- The endovascular extravillous trophoblast can be easily seen in routine preparations (H&E 40X).

Placental disc

Trophoblast cells that protect the contact area, which extends all around the internal placental disc, between the two circulations, were highlighted in Figure 2 [light green rectangles]. Figures 2 to 6 show this trophoblast cell layer, and demonstrate their different roles in different locations since their respective protein expression is not the same. The placental disc can be represented as a villous tree, whose branches only distribute fetal necessities, moving from the fetal heart to the terminal villi upstream and downstream. The maternal blood supply in the intervillous spaces is distributed like air among branches and leaves [27], coming from remodeled arteries which have lost their muscle and have larger lumens and are less resistant to flow, and, hence, have less pressure [Figures 1, 2, 5 and 7]. The intervillous space should only contain maternal blood, without obstructions [Figures 5]. Syncytiotrophoblast cells are multinucleated fused cells whose surfaces are covered by a dense network of branched microvilli that encompass an area of 12 m2 in the final stage of human gestation [28] [Figure 5].

UGFM2018-101-MercedesOlaya Colombia_F7

Figure 7. The basal plate 2

A – Gross placental morphology of maternal surface, bilobate shape is recognized. B- The figure represents some contact between anchoring villi and basal plate. Maternal spiral arteries can be seen. C- Decidua cell are pale, with bland nucleus (H&E 10X). D-The decidua is delineated by immunohistochemistry staining (VEGF-R1 40X). E- A remodeled artery is shown, the once normal thick wall is now, during normal gestation, vena-like (H&E 10X). F-The anchoring villous is observed in contact with the decidua  (H&E 4X).

Fetal and maternal circulations are separated. The fetal system is located in the villi and is separated from the maternal system by:  the vasculosyncytial membrane;(this membrane decreases in thickness as pregnancy progresses; it is composed of:  the endothelium inside the villi, the basement membrane and some connective tissue), by a layer of discontinuous cytotrophoblast cells that also decreases progressively, and by the syncytiotrophoblast and its basal membrane [29].

There are other special structures, the anchoring villi, which attach the placental tree to the decidualized endometrium, forming columns of trophoblast [Figure 7]. Other specialized septa divide placental regions into cotelydons.

Maternal surface- basal plate

The above mentioned cotyledons are easily discernible, grossly recognizable circular structures [Figures 6A and 7A]. They are inconspicuously covered by decidua, called basal decidua. Microscopically, the basal plate represents the other fetus-maternal interface, where the immunological dialogue occurs. The invasive trophoblast penetrates the decidua at the implantation site and remodels the maternal arterioles, including the endovascular extravillous trophoblast and the interstitial extravillous trophoblast, both kinds of trophoblast work together in vascular remodeling [30] [Figure 6]. Beneath the decidua, the above mentioned insulation trophoblast is also present [Figure 6C]. This part of the placenta is currently the most interesting because of its link to gestational diseases. The interaction between histiocytes and lymphocytes has not been determined; we know only that it is important. Large numbers of histiocytes and lymphocytes in the decidua, especially in the presence of plasma cells, suggest pathology. Endovascular extravillous trophoblast, interstitial extravillous trophoblast, columns of trophoblast and anchoring villi play a major role in fetal-placental interaction and correct gestational course, but their different behaviors deserve further study. The decidual component is also important, including spiral arteries; however, their active function in supplying blood remains poorly understood [Figures 6 and 7].

Limitations of the present study are related to population type; selection bias is due to our hospital´s highly complex population, which means that a larger number of complicated gestations with abnormal placentas exist than in the number shown; many of these gestations also belong to the Hypertensive Disorders of Pregnancy context. We would like to have access to before-and-after studies in order to better compare our students’ comprehension of the information they have been provided.

Conclusion

The placenta is now, more than ever, the focus of multiple investigations related to the evolution, development, health and etiology of disease. New and improved approaches are needed to further our knowledge on the placenta.

Acknowledgements

We wish to thank the Hospital Universitario San Ignacio- Pontificia Universidad Javeriana for its indispensable support and participation in this study. We also extend our thanks to the parents who participated in this study and to Steven W. Bayless for the English text correction.

Financial Disclosure

This study makes up part of the research project entitled, “Factors that determine umbilical cord length”, [ID PPTA 00005140] financed by the Pontificia Universidad Javeriana and its Hospital Universitario San Ignacio. Bogota, Colombia.

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The Need of Sealants in Oral Health

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DOI: 10.31038/JDMR.2018111

Abstract

A good oral health education program starts from early ages, and the most important time to initiate this it is when the first permanent tooth (the first permanent molar) erupts, and it lies on a correct diet and hygiene habits, including a healthy diet, a correct brushing technique, and a preventive therapeutic approach. The beneficial effect of a preventive program is a good oral health and the therapeutic approach is the dental sealing. Of course it is important to motivate small patients to maintain a good oral health, through the correct use of oral hygiene products, individual brushing techniques, and good eating habits. The prevention programs and the children’s receptiveness in maintaining proper oral hygiene can improve the oral health. If there is good communication between the physician and the patient then we can talk about the triad: theoretical presentation, practical exercises and individual biofilm removal techniques, which are indispensable factors in achieving an appropriate oral health status. When dental education programs can’t provide the proper prophylaxis of caries lesion, especially at the occlusal surfaces of molars and premolars with deep and retentive pits and grooves, dental sealing is an ideal option.

Keywords

Dental Plaque, Dental Sealing, Tooth Decay, Plaque Indexes, Biofilm

Introduction

Risk assessment for the disease development is an essential component of any oral disease prevention program. Susceptibility risks can be determined by various methods including the person, the whole community and of course the teeth, and all dental surfaces. Tooth decay is a bacterial-dependent disease so dental sealing is a preventive measure that can be implemented when the patient is at risk. There are many risk factors that can influence the formation of carious lesions which includes the eating habits and dental hygiene habits, regarding brushing techniques in sense of frequency and time of dental brushing. Of course that periodic consultation and early detection of caries are also important factors that hold the key of success in dental health. Oral disorders, starting from the incipient lesions and leading to the most complicated clinical cases, depend on many factors including the dietetic and hygiene habits of the patient. When talking about dietetic habits, the evolution of the carious lesion depends to a great extent, of the presence in the diet of sugars, carbohydrates (juices) etc., but also it depends on regular meals without eating between meals. Also, an important factor in the appearance and evolution of the carious lesion is the patient’s hygiene habits, such as how he performs dental brushing, how many times a day, etc. In other words good hygiene habits must be learned from an early age, and this can be done through dental education programs, which can be implemented in schools. Tooth brushing is an essential part of an effective dental education program, and achieving optimal oral health depends on the efficiency of the method used to remove the biofilm from the surface of the teeth. Tooth brushing aims, to remove, by mechanical means, the food residues and the biofilm from dental surfaces. The efficiency of the biofilm removal depends on several factors: the time of dental brushing, its frequency, the technique used, the tools used, and the quality of the toothpastes. Associating oral hygiene with a healthy diet will help reduce the risk of carious lesions.

Even if all of these rules are respected by patients, this may not be enough, if the teeth have deep and retentive pits and fissures, because the toothbrush bristle can’t get inside those grooves, and clean the biofilm, which can accumulate and determine the lesion on the occlusal surface of the premolars and molars. Dental sealing is considered by the World Health Organization to be a major factor in the prevention of carious lesions, especially in schools with children from low-income families, so through dental sealing programs it can be reduce the incidence of caries, especially when focusing on sealing permanent molars in children. Preventive dental sealing treatment has a beneficial effect on the patient, from all points of view. Maintaining a good oro-dental health is one of the most important therapeutic attitudes in all dentistry branches. The most beautiful feeling as a dentist is when he succeeds in motivating the patient about oral health, because it is easier to prevent than to treat a disease.

Methods & Materials

In our study we conducted a preventive school program, which included theoretical presentation, practical exercises, individual biofilm removal techniques and dental sealing. We randomly selected a group of 107 children who received specialized consultations. So, the study was conducted on a group of 107 patients, with age range between 6–8 years, from an urban school, class 0 and 1 students. We evaluated the ways in which living habits, influence the oral health, so we gathered data about hygiene habits and eating habits. In this part of the study, we evaluated the foods that the patient usually consumes and which may have repercussions on oral health, but also if the patient consumes sugars, sweet snacks, carbohydrate beverages, sweets (chocolate), fruits, fats and vegetables, and the way they do it. (Figure 1)

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Figure 1. Eating habits.

It is noted that from the entire group of patients, the majority of children consume carbonated beverages, which is not suited for a good oral health status. However, a very small percentage of children do not consume carbonated beverages. Drinking this kind of beverage, is not a good thing for oral health, especially if dental brushing is not done right after, so we have gathered data on sweets consumption, and we noticed that all children in the batch eat sweets except for one boy, which is worrying in the absence of dental brushing. It’s a concerning result for children’s oral hygiene, but if teeth brushing is performed after eating sweets, then the oral health of small children will not be affected. We next correlated the data on fruit consumption, which is a part of the category of foods that are beneficial to both the oro-dental health and the health of the whole organism. It is observed that most of the patients included in the lot consume fruits, almost in the same way that they consume carbonated beverage (Figure1). The data collected about fat consumption, which, at some point is beneficial to dental health, but can also have a negative effect to oral health, is as follow: an increased percentage of patients consume non-fat foods, and a small number of patient have fat in the diet. The consumption of vegetables, which are recognized as being good for oral health, is found in more than half of the patients; in fact a third does not consume them against the two-thirds who consume them. This result is a positive one, because there are more children who consume vegetables, but it would have been better if that number would have been even higher.

After correlating all the outcomes on the patients’ diet, we assessed the way in which the small children consume sugars, so we evaluated whether eating sugary foods is done between or during meals (Figure 2).

JDMR-18-101-Onisei Doina Romania_F2

Figure 2. Eating habits-consumed sugars.

Sugar is consumed between meals by most patients in the group, regardless of gender, representing a high percentage, 63%, with a total of 67 patients, compared with 40 patients who consume sugar during meals, representing 37% of the children. This result is not satisfactory because the consumption of sugary foods increases the risk of carious lesions in absence of dental brushing after consumption. We have noticed that patients or their parents give a great importance to oral hygiene products by choosing different products on the market but they do not put as much emphasis on the brush technique and its frequency. It is noted that all patients include in their hygiene habits, the toothbrush and toothpaste to perform everyday dental brushing. Most of the patients tend to complete their daily dental hygiene with the mouth wash, which chemically removes the biofilm from dental surfaces, so there was a 62% of children, male and female that uses mouthwash, this being a relatively satisfactory percentage, but it would have been better if this percentage were higher. Regarding dental floss, things are different, and it appears that 85% of patients do not use it. This result is not satisfactory one because the use of dental floss reduces the risk of carious lesions on the proximal surfaces of the teeth (Figure 3).

JDMR-18-101-Onisei Doina Romania_F3

Figure 3. Products used in dental hygiene habits.

Although all the patients included in the study use the toothpaste and the toothbrush, a special importance should be given to the frequency of dental brushing. It is noted that most patients perform dental brushing twice a day, accounting for 57 patients of both sexes, namely 28% of male patients and 25% of female patients, followed by 29% of patients performing dental care after each meal, 13% patients performing dental brushing once a day, and the smallest percentage is 5% representing patients who do not perform dental brushing every day (Figure 4).

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Figure 4. The frequency of dental brushing.

Following the correlation of all the results obtained so far, the tendency of the entire group of patients relating to oral hygiene habits and diet habits are in ascending parameters, towards good oral health, but some improvements should be made. The gaps that still exist in the habits of the patients had repercussions on oral health, so initial patient evaluation did not necessarily have good results. In the initial examination of the patients, they presented different degrees of oral hygiene, as can be seen in the chart below, 11% boys and 7% girls were with unsatisfactory hygiene, 25% boys and 22% girls, had an average grade of hygiene and good hygiene was achieved by 16% boys and19% of the girls. According to the chart below (Figure 5), the majority of patients, 47%, had average oral hygiene, followed by the 35% with good hygiene, and the lowest percentage was for unsatisfactory hygiene, 18%.

JDMR-18-101-Onisei Doina Romania_F5

Figure 5. The degree of oral hygiene.

The idea deduced from this series of arguments or findings is that the percentage of patients with unsatisfactory grade of hygiene was 18% patients, and 82% of children had average or good hygiene (Figure5). By correlating all data relating to dietary habits, dental brushing with its frequency, the auxiliary means used in removing dental plaque and the evaluation of oral hygiene, we tried to relate them to the incidence of caries in permanent teeth of this children, namely the first molars.

From the data provided in the graph below (Figure 6), it is found that a number of 226 teeth are without lesions or with clinically undetected lesions and it represents 53% of teeth, more than half of the teeth examined. Also we found that 19% of the molars had incipient lesions. Tooth decay was present in an28% of the molars examined, but 9% of molars were treated before our examination, and 19% still presented caries lesions. In other word we can say that tooth decay was present in 15% of males and 13% females.

JDMR-18-101-Onisei Doina Romania_F6

Figure 6. Incidence of caries lesions at first molars.

From the category of teeth with no clinically detectable lesions, 121 of them had a risk of carious lesions, representing 28% of the whole teeth we examined, and it is shown in the chart below (Figure 7).This percentage does not differ significantly from that of teeth without risk to caries, which is 25% of examined teeth.

JDMR-18-101-Onisei Doina Romania_F7

Figure 7. Teeth at risk of carious lesions.

The fact that there is not a very big difference between these percentages suggests that there is a high risk of tooth decay among children with age range between 6–8 year old. Following the results of the incidence of carious lesions and the risk of their occurrence, all the teeth presenting a risk of carious lesions have been sealed. Those teeth also presented deep retentive pits and fissures on the occlusal surfaces or incipient lesions, undetectable on X-ray, in the form of demineralized enamel, shown as white spots. Teeth sealed who presented deep retentive pits and fissures were 89 and there were 32 teeth sealed with white spots, incipient lesions, undetectable on X-ray, in the form of demineralized enamel. From the whole number of teeth that had dental sealing there were 74 % with deep and retentive pits and fissures, and 26% of teeth sealed were with white spots. (Figure 8)

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Figure 8. Teeth examined and sealed.

From the graph above, it is noted that the total number of 428 teeth examined, only 226 did not show treated or untreated dental lesions, so only this half was evaluated for dental sealing. The other half who experienced lesions with or without treatment was examined and either the incorrect dental restorations were replaced or the present lesions were treated. From the evaluated teeth for dental sealing, which is, half of the teeth evaluated and examined in the entire group of patients, the need for dental sealing was at half of them. This means that of the 226 teeth evaluated in terms of prophylactic and preventive treatment, 121 teeth, permanent first molars, had an indication of sealing.

The teeth which received dental sealing were half of the total teeth not affected by lesions, and this is a very high number, because in fact, these teeth actually have a tooth decay predisposition, and in the absence of dental sealing they lose their morpho structural integrity. Following control, 6 months after application, most of the seals remained unchanged, however 3 dental seals were no longer present on the teeth, and in the control after 1 year, 5 teeth lost their sealing. The loss of dental sealing may vary, depending on whether the patients followed the indication for a good oral health or simply because of local factors. It is noted that the number of un affected dental sealing is high, so the purpose of prevention is met. There was also an improvement in oral health through better diet habits and oral hygiene habits with better brushing techniques and increasing brushing frequency, and also using more auxiliary means for removing the biofilm.

Conclusion

Dental sealing has had a good result because as long as it remained in the pits and fissures, its preventive purpose was fulfilled. The need for dental sealing, as shown in our study is quite high, and depends on many factors, such as better brushing techniques, increasing brushing frequency, using more auxiliary means such as dental floss and mouthwash, in removing the biofilm, through both local as well as general means.

References

  1. American Academy of Pediatric Dentistry (2012) Guideline on caries-risk assessment and management for infants, children, and adolescents. Pediatr Dent 34: 118–125.
  2. Amin HE1 (2008) Clinical and antibacterial effectiveness of three different sealant materials. J Dent Hyg 82: 45. [crossref]
  3. Dye BA, Mitnik GL, Iafolla TJ, Vargas CM (2017) Trends in dental caries in children and adolescents according to poverty status in the United States from 1999 through 2004 and from 2011 through 2014. J Am Dent Assoc 148: 550–565. [crossref]
  4. Dye BA, Thornton-Evans G, Li X, Iafolla TJ (2015) Dental caries and sealant prevalence in children and adolescents in the United States, 2011–2012. NCHS Data Brief 191: 1–8. [crossref]
  5. Griffin SO1, Gray SK, Malvitz DM, Gooch BF (2009) Caries risk in formerly sealed teeth. J Am Dent Assoc 140: 415–423. [crossref]
  6. Lile IE, Freiman PC, Hosszu T, Vasca E, Vasca V, et al. (2015) A Subsidiary Physical Research of Glass Ionomers. Jurnal Medical Aradean 52: 175–179.
  7. Tagliaferro EP, Pardi V, Ambrosano GM, Meneghim Mde C, da Silva SR, et al. (2011) Occlusal caries prevention in high and low risk schoolchildren. A clinical trial. Am J Dent 24: 109–114. [crossref]
  8. Tellez M, Gray SL, Gray S, Lim S, Ismail AI (2011) Sealants and dental caries: dentists’ perspectives on evidence-based recommendations. JADA 142: 1033–1040.
  9. Vaida L, Moldovan L, Lile IE, Todor BI, Porumb A, et al. (2015) A Comparative Study on Mechanical Properties of Some Thermoplastic and Thermo Set Resins Used for Orthodontic Appliances, MaterialePlastice 52: 364–367
  10. Wright JT, Tampi MP, Graham L (2016) Sealants for preventing and arresting pit-and-fissure occlusal caries in primary and permanent molars: a systematic review of randomized controlled trials a report of the American Dental Association and the American Academy of Pediatric Dentistry. JADA 147: 631–645.

Assessment of Quality and Safety of Medical Care in Russia

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DOI: 10.31038/NAMS.2018131

Abstract

Goals: This study is aimed to analyze the results of external audits in medical facilities in Russia.

Design: Analysis of the results of audits in terms of the sections: “Epidemiologic safety. Preventing and Controlling Healthcare Associated Infections”, “Drug safety. Pharmacovigilance”, “Control of quality and safety of medical devices circulation”, and “Surgical safety. Preventions of risks associated with surgical intervention” in medical facilities of Russia.

Setting: 10 medical facilities in which the quality management system had not been implemented before.

Results: Nowadays the absence of unified approaches to the management of quality and safety of medical care is one of the most complicated and debatable issues in the medical society in Russia. Within the framework of this study, we have analyzed the results of external audits of quality and safety of medical care conducted in accordance with several sections of the Guidelines in medical facilities in which the quality management system had not been implemented before. The lowest level of conformity (15,9%) was found for the “Epidemiologic safety. Preventing and Controlling Healthcare Associated Infections”. The organizational problems were found in medical devices circulation, microbiologic monitoring systems, and systems of registration and collection of information concerning severe and unexpected adverse drug reactions. The practice of audits in medical facilities revealed essential structural problems with medical care quality and safety management in Russia.

Key words

medical care, Russia, audit, quality and safety, quality management system

Introduction

Nowadays the absence of unified approaches to the management of quality and safety of medical care is one of the most complicated and debatable issues in the medical society. There is no developed unified regulatory standard for management of medical care in medical facilities in Russia now.

In order to solve this problem, in 2015 Federal State Budgetary Institution “Center for Monitoring and Clinical and Economic Expert Evaluation” of Federal Service for Surveillance in Healthcare developed Roszdravnadzor’s Practical Guidelines (Recommendations) on the internal system of quality and safety control of medical care in medical facilities [1]. These Guidelines became the prototype of the national safety and quality healthcare standard for hospitals in Russia. The Guidelines were developed with due consideration of the requirements of current worlds standards: Joint Commission International Standards for Hospital (USA), National Safety and Quality Health Service Standards (Australia), Canadian Council on Health Services Accreditation (Canada), and others.

The Guidelines provided the basis for the System of the voluntary certification of medical facilities “Quality and Safety of Medical Care”, which was registered in 2016 [3].

Audit is the form of evaluation of the conformity of the medical facility to the requirements of the Guidelines [4]. Audits are to be carried out by specialists from a separate independent organization who are experts in the field.

This system implies external evaluations (audits) of medical facilities regarding the compliance with the requirements of the Guidelines.

The Guidelines include the following main fields of concern:

  1. Human resources management;
  2. Patient Identification;
  3. Epidemiologic safety. Preventing and Controlling Healthcare Associated Infections;
  4. Drug safety. Pharmacovigilance;
  5. Control of quality and safety of medical devices circulation;
  6. Emergency care in inpatient facilities;
  7. Managing clinical responsibility. Patient internal and external transfer;
  8. Surgical safety. Preventions of risks associated with surgical intervention;
  9. Blood management;
  10. Safe environment for the delivery of care. Patient care management. Preventing and managing falls, pressure injuries;

Methods

In this article, we will analyze the results of external audits of quality and safety of medical care conducted in accordance with several sections of the Guidelines in medical facilities in which the quality management system had not been implemented before. The audits were carried out by multidisciplinary work groups of experts under the supervision of experts from the Federal State Budgetary Institution “Center for Monitoring and Clinical and Economic Expert Evaluation” of Federal Service for Surveillance in Healthcare by the unified procedure based on the Guidelines.

Within the framework of this study, we have analyzed the results of audits carried out in terms of the following sections: “Epidemiologic safety. Preventing and Controlling Healthcare Associated Infections”, “Drug safety. Pharmacovigilance”, “Control of quality and safety of medical devices circulation”, and “Surgical safety. Preventions of risks associated with surgical intervention”.

The assessment sheet for these sections includes the list of criteria combined into groups. The assessment system is binary; it determines the conformity or non-conformity to one or another criterion. The non-conformity to any criterion in the group is the reason to consider the whole group of parameters non-conforming. For example, when evaluating the requirement concerning the availability of the microbiology testing system the experts checked the conformity to the following criteria: availability of an own microbiology laboratory or an agreement, necessary conditions for material sampling 24 hours per day, 7 days per week, 365 days per year, including the availability of transport media, thermostats and procedures of material sampling for all possible cases for a certain medical facility, personnel’s knowledge of procedures (interviewing) and practical skills (observation), and following the procedures, which was assessed using the method of studying medical records, and the criteria of timeliness of getting the results of cultures and their proper use for changing the empiric regimen of antimicrobial therapy for another regimen taking into account the sensitivity. Only positive answers to all the questions provided a positive result of evaluation according to one (!) requirement.

The sources of information described on the Figure 1.

NAMS 2018-103-Igor Russia_F1

Figure 1. Sources of information

The article uses the results of external audits of medical facilities, which are super specialty hospitals that deliver both elective and emergency care including high-tech medical care. The average hospital bed capacity was 500 beds (up to 1000); the average number of the personnel (both healthcare professionals and allied health personnel) was about 2000 people in each facility. The main criterion for choosing a medical facility for this study was the fact that there was not the quality management system based on the ISO 9000 standards.

The summarized results of audits are shown in the Table 1.

Table 1. summarized results of audits

NAMS 2018-103-Igor Russia_F2

Initiators of conducting audits were the authorities of medical facilities. All the members of expert teams adhered to the principles of privacy and goodwill. Experts made a point of the fact that the authorities of the medical facilities in question had ensured the personnel that they would not be punished after the audit in any case which made the personnel more open. According to the conditions of the agreement between medical facilities and Federal State Budgetary Institution “Center for Monitoring and Clinical and Economic Expert Evaluation” of Federal Service for Surveillance in Healthcare the experts had access to all the rooms of the facilities and to all medical and organizational records.

Results

As it is shown in Table 1, the lowest level of conformity (15,9%) was found for the “Epidemiologic safety. Preventing and Controlling Healthcare Associated Infections” section. Almost all medical facilities in question had no effective microbiologic monitoring systems, no microbiology studies, and prudent use of antimicrobial drugs was not provided there. It is also necessary to make profound changes in revealing, recording, registration and analysis of infections associated with healthcare delivery.

When evaluating the “Surgical safety” section (the level of conformity – 22,9%), experts found organizational problems. For example, all the medical facilities in question have no functional surgical safety management system: a surgical check-list, procedures of transferring clinical responsibility in a post-surgery period, evaluation of anesthesia and pain management effectiveness in a post-surgery period were not developed and are not used. Many facilities do not use pain assessment scales which help to customize approaches to pain management.

The “Drug safety” section has the level of conformity equal to 28,9%. All the medical facilities in question have no effective systems of registration and collection of information concerning severe and unexpected adverse drug reactions. The labeling of vials with infusion solutions did not conform to the established criteria. There are still unsolved problems with the knowledge of procedures and the quality of verbal drug administration. Moreover, it is almost impossible to assess the conformity of the drug selection and dosage to clinical recommendations (treatment protocols) as there are no such recommendations at most workplace, though they can be found in the federal electronic medical library.

The procedure for medical devices circulation is assessed in the “Circulation of medical devices” section and has the level of conformity equal to 59,6%. Organizational problems with medical devices circulation were revealed during audits. In 9 of the 10 investigational medical facilities the system of medical devices circulation quality and safety monitoring is fragmentary, and a system approach is not used properly. The personnel are not being trained regarding issues of medical devices circulation quality and safety, and no internal audits are conducted. The personnel of medical facilities do not work properly with instruction manuals of medical devices. The requirements for correct use, maintenance, storage and disposal stated by manufacturers are just partially adhered to in all the assessed medical facilities.

Conclusion

The practice of conducting audits in medical facilities, where the quality management system had not been implemented before, revealed essential structural problems with medical care quality and safety management. The approach to the assessment of the medical care quality described in the Guidelines gives the opportunity to assess a medical facility in an integrated manner.

In contrast with the approved Russian practice of evaluating mainly submitted documents, the assessment in accordance with the Guidelines helps to reveal system problems based on several sources of information (profound observation over the processes of medical care, interviewing the personnel and patients). The evaluation process is more successful when the expert is maximally immersed in the clinical environment instead of documents.

The importance of studying the issues of epidemiologic, drug and surgical safety, the issues of medical devices circulation, the importance of management of risks associated with these spheres of medical care have met with support of the medical personnel and are regarded promising in Russia.

The further use of the Guidelines will help to improve approaches to solving these problems in order to change the situation for the better.

References

  1. Proposals for arrangement of inner quality and safety control of medical activity in medical organization (hospital) // Bulletin of Roszdravnadzor. — 2016. — N 2. — P. 35, 36.
  2. Ivanov I.V., Shvabskii O.R., Minulin I.B., Shchesyul A.G. Medical activity: quality and safety // Standards and Quality. – 2017. – N 3. – P.72–74.
  3. Ivanov I.V., Shvabskii O.R., Minulin I.B., Emanuel A.V. Audit as a tool of healthcare quality assessment // Standards and Quality. – 2017. – N 11. – P.27–29.
  4. Ivanov I.V., Shvabskii O.R., Minulin I.B., Shcheblykina A.A. Results of audits of quality and safety of medical activity in hospital. // Quality Management in Healthcare. – 2018. – N 1. – P. 18–22.

Drug discovery: science and/or art?

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DOI: 10.31038/JPPR.2018112

Editorial

Pharma companies have been among the most profitable ones at least until the new era of the Info companies like Google, but are still quite interesting, and in fact not so different in principle: it is always question of information, a drug is useful to restore the lost info in an organism, trying not to override to much of what it is still working!

One of the reason for such healthy and wealthy attitude is that drug discovery is not obvious nor simple nor cheap nor fast, as a rule: a good chemical could imply a fortune even in the few years of validity of the patent. A thorough knowledge of pathophysiology is required in order to design biochemistry able to heal without damaging to much. Intuition and experience are paramount, but nowadays at least a couple of approaches are keen to help.

On one side, machine learning, able to identify the statistical properties of the known, may be of great help in forecasting at least simple but necessary properties of the candidate drug, like hydrophilic or hydrophobic behavior.

On the other side, esascale high performance computing is nowadays able to assist physical chemists in simulate and forecast the properties of a big molecule taking into account the properties of every single atom composing its structure.

Such approaches, nowadays seen as opposite, probably because inherited by different communities not sharing a common background, are instead keen to be complementary, as often in science and even in life, when almost perfect but still not sufficient great approaches are combined.

The best would of course be to have an algorithm already able to combine both, but this is still a kind of Graal in the minds of the many scientist both theoreticians and in other applications, dreaming of, and/or working on, such important direction, that would imply quite a revolution not just in drug discovery

Pharmacokinetics of Acetaminophen in the Hypothalamus of Rats Based on in vivo Microdialysis

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DOI: 10.31038/JPPR.2018111

Abstract

To explore the studying method for pharmacokinetics in the target site of drugs, the pharmacokinetic process of acetaminophen in the hypothalamus of rats was investigated. Male Sprague-Dawleyrats were anaesthetized and secured in a stereotaxic frame. A microdialysis probe was implanted into the hypothalamus and perfused with artificial cerebrospinal fluid at a flow rate of 2 µL/min. Adaptation for 1 h, rats were administrated with acetaminophen (150 mg/kg, i.p.) and microdialysates were collected continuously at 12-min intervals for 6 h. The acetaminophen concentrations in microdialysates were determined by HPLC-Ultraviolet detection (HPLC-UV), and the concentration-time profile and pharmacokinetic parameters of acetaminophen were calculated by DAS software. The results showed that the concentration-time curve of acetaminophen in the hypothalamus of rats was fitted to a one-compartment open model. The main pharmacokinetic parameters t1/2, Tmax, Cmax and AUCinf were (1.95 ± 0.59) h, (1.26 ± 0.22) h, (11.39 ± 2.17) µg/mL and (58.04 ± 18.39) µg·h/mL, respectively. In conclusion, by means of in vivo microdialysis approach, the pharmacokinetic process of acetaminophen in the hypothalamus of rats is investigated and an experimental method for studying pharmacokinetics of drugs in the target site is established, which is simple, feasible and reliable.

Keywords

Acetaminophen, HPLC, in vivo Microdialysis, Pharmacokinetics, Rats

Introduction

In pharmacokinetic studies, the traditional method is to measure drugs or their metabolites concentrations in blood, and calculate pharmacokinetic parameters based on the plasma drug and/or its metabolite concentrations, which are used for guiding drug administration and dosing regimens in clinic. However, the biochemical events and pharmacological effects do not usually take place in the bloodstream, but in target organs and/or tissues [1, 2]. Therefore, it is unreasonable to use the plasma drug or its metabolite concentration to in place of the target organ/tissue drug concentration for calculating pharmacokinetic parameters. For example, a study by Konings et al. [3] found that the 5-Fuorouracil concentrations in the extracellular fluid (ECF) of tumors were lower than the unbound plasma concentrations. Furthermore, especially for central nervous system (CNS) drugs, there exists significant difference between the plasma drug concentration and the CNS drug concentration because of the influence of the blood-brain barrier (BBB). Bostrom et al. [4] showed that the unbound concentrations of oxycodone in brain were higher than those in blood, due to the presence of active influx of oxycodone at the BBB.

Acetaminophen, (N – (4 – Hydroxyphenyl) acetamide), an antipyretic-analgesic drug, is widely used for the treatment of mild pain and fever. The mechanism of acetaminophen hypothermia is not fully understood, but is assumed to be related to inhibit cyclooxygenase in the CNS [5, 6] and the target site locates in the hypothalamus [7]. At recommended therapeutic doses, acetaminophen is safe and effective, however, excessive intake of acetaminophen may cause acute liver failure and even death [8–10]. And acetaminophen overdose is a major cause of liver injury in the USA and Europe [8]. Hence, more accurate and precise pharmacokinetics data of acetaminophen in the CNS (target site) should be obtained. However, so far little research has been done on the pharmacokinetics of acetaminophen at the target site.

Microdialysis, a semi-invasive probe-based sampling technique, is able to measure the unbound drug or endogenous substance concentrations in the ECF of target tissues [11], which is widely used to pharmacokinetics, metabolic as well as tissue distribution studies [1, 12, 13]. Moreover, compared with traditional sampling methods such as tissue biopsy, saliva sampling, skin blister, etc., microdialysis is currently the most appropriate, highly efficient and well-established sampling method [12, 14, 15]. The purpose of this study is to utilize the microdialysis method coupled with high performance liquid chromatography-Ultraviolet detection (HPLC-UV) to analyze the pharmacokinetic process of acetaminophen in the ECF of rats’ hypothalamus. And the results would provide suitable references for the clinical development of dosing schedules of acetaminophen. At the same time, it may also establish a new studying method for pharmacokinetics of drugs in the target tissue.

Materials and Methods

Chemicals

Acetaminophen was purchased from Anhui Yongan Pharmaceutical Co. Ltd. Urethane (ethyl carbamate) and propylene glycol were obtained from Shanghai Chemical Reagent Co. Ltd (Shanghai, China) and Hunan Erkang Pharmaceutical Co. Ltd. (Hunan, China), respectively. HPLC-grade methanol andacetic acid were purchased from Tianjin Fu Chen Chemical Reagent Factory (Tianjin, China) and Nanjing Chemical Reagent Co. Ltd. (Nanjing, China), respectively. Artificial cerebrospinal fluid (aCSF) included KCl 3.0 mM, MgCl2 1.0 mM, CaCl2 1.3 mM, NaCl 140 mM, Na2HPO4 2.0 mM and NaH2PO4 0.2 mM. Purified water from an AHJZ water purification system was used throughout the experiment. All other chemicals and reagents were of analytical grade.

Animals

Healthy male Sprague-Dawley rats, weighing 250–320g, were purchased from Qing Longshan Animal Breeding Laboratory (Nanjing, China). All animals had free access to food and tap water and were housed at a constant temperature (25 ± 2°C) with a relative humidity (60 ± 2%) under a 12 h light/dark cycle. All experimental protocols were performed in accordance with the principles of animal use and care approved by the ethnical committee of Wannan Medical College.

Chromatographic System

The chromatographic analysis was carried out on Agilent 1100 LC system (Agilent, USA), coupled to an ultraviolet detection (G1314A). The chromatographic system was used under the following condition: Ultimate XB-C18 column (4.6 × 150 mm, particle size 5μm, USA); mobile phase consisting of phosphate buffer-methanol-glacial acetic acid (90: 10: 0.25, v/v/v) at a flow rate of 1.0 mL/min; injection volume of 20 µL; Column temperature of 25°C and UV detection at 248 nm.

Microdialysis System

The microdialysis system consisted of a microinjection pump (KD Scientific, USA) connected to a 5.0 mL plastic syringe (Hamilton, USA), a MAB 85 fraction collector (Stockholm, Sweden) and a microdialysis probe (EICOM, Japan) which was inserted into the target site.

Preparation of Standard Solutions and Quality Control Samples

The acetaminophen was dissolved in aCSF for obtaining standard final concentrations of 0.25, 0.5, 2.5, 5.0, 10.0, 25.0 µg/mL. Quality control (QC) samples with low (0.5 µg/mL), medium (5.0 µg/mL) and high (25 µg/mL) were also prepared. And then 20 µL of microdialysate samples were injected into the chromatographic system for analyzing.

Selectivity, Linearity and Sensitivity

The selectivity was determined by comparing the chromatograms of blank aCSF sample, blank aCSF spiked with acetaminophen, and microdialysate sample obtained after administration of acetaminophen. To calculate the linearity, the calibration curve with six points in the range of 0.25–25.0 µg/mL was built using peak area of acetaminophen versus acetaminophen concentration. The limit of detection (LOD) was defined as the lowest concentration of analyte and calculated by signal/noise (S/N) ratio equal to 3.

Precision and Relative Recovery

Inter- and intra-day precision were calculated from replicate analysis (n = 5) of QC samples for microdialysate samples, on five consecutive days. The relative recovery was also determined by analyzing the same QC samples in replicate analysis (n = 5). The relative recovery (mean ± SD) was estimated by comparing the measured concentrations to the known concentrations. The relative standard deviation (R.S.D.%) was used to judge the precision.

In vivo Microdialysis Experiment

After rats (n = 5) were anaesthetized by the 20% urethane solution (1.2 g/kg, i.p.) and placed in a stereotaxic apparatus, a microdialysis probe was implanted stereotaxically into the hypothalamus zone (from lambda 4.0 mm posterior, 1.0 mm lateral, 8.0 mm ventral) according to the atlas of George Paxinos & Charles Wastson[16]. The probe was perfused with aCSF solution at a flow rate of 2 μL/min by a microinjection pump. After the probe was allowed to equilibrate for 1 h, the rat was treated with acetaminophen (150 mg/kg, i.p.). Then, the microdialysate samples were collected at 12-min intervals (24 µL) for 6 h and preserved at – 40°C refrigerator until analysis.

Recovery of Microdialysis Probes

The in vivo microdialysis probe recovery was determined by using a retrodialysis method [17]. For in vivo recovery, the microdialysis probe was implanted into the hypothalamus zone (above mentioned) of urethane anaesthetized rats. The microdialysis probe was perfused with aCSF solution containing acetaminophen (0.5, 5 and 10 µg/mL, respectively) at a constant flow rate of 2 μL/min by a microinjection pump. Following equilibration 1 h after probe implanted, microdialysates were collected at 12 min intervals for 1 h. And the concentrations of acetaminophen in the perfusate (Cin) and dialysate (Cout) were determined by the HPLC-UV system. The in vivo recovery was calculated by following equation: R = 1 – (Cout/Cin) × 100%.

Pharmacokinetics data

The concentrations of acetaminophen in rat microdialysates were determined from the calibration curve. The actual concentration in the ECF of hypothalamus (CHypo) were calculated from the concentrations in microdialysates (CMdia) by following equation: CHypo= CMdia/R. The observed data was used for the calculation of pharmacokinetic parameters by a one-compartment model method using DAS2.0 software. The main pharmacokinetic parameters: elimination of half-life (t1/2), peak time (Tmax), peak concentration (Cmax), and area under the concentration-time curve (AUClast, AUCinf), etc., were determined. The results are presented as mean ± standard deviation (mean ± SD). All data were analyzed by SPSS 13.0 software.

Results

Selectivity, Linearity and Sensitivity

The acetaminophen’s retention time was 7.9 min. The high selectivity was proved by the absence of interfering peak of endogenous compounds around the retention time of acetaminophen (Fig. 1). The calibration curve for acetaminophen in microdialysates (0.25–25.0 µg/mL) was fitted to a linear equation which was A = 57.467 C – 1.9726 (r = 0.9993, n = 5), where A represents the peak area of acetaminophen, and C represents the concentration of acetaminophen. The LOD of acetaminophen for the microdialysate method was 0.25 µg/mL.

JPPR 2018-101-Zong-Yuan Hong China-F1

Figure 1. Chromatograms of (A) blank aCSF sample, (B) blank aCSF spiked with acetaminophen (peak 1), and (C) microdialysate sample obtained after administration of acetaminophen (peak 1). aCSF, artificial cerebrospinal fluid.

Precision and Relative Recovery

The inter- and intra-day precision and the relative recovery of the method were shown in Table 1. The inter- and intra-day precision of QC samples were 2.13%, 3.98% ,4.78%, and 2.56%, 3.44%, 6.37%, respectively. The relative recoveries of QC samples were 99.13 ± 2.17%, 99.53 ± 3.49% and 98.20 ± 5.24%, respectively. These results showed that the method had good precision and accuracy.

Recovery of Microdialysis Probe

The probe recovery determined by retrodialysis was showed in Table 2. At the concentrations of 0.5, 5.0 and 10.0 µg/mL (n = 5), the average recovery rate of microdialysis probe was 18.3%.

Table 1. The recovery rate and precision of acetaminophen in ECF (mean ± SD, n = 5)

Marked Conc. (µg/mL)

Measured Conc. (µg/mL)

Relative recovery rate (%)

Precision (RSD, %)

Inter-day

Intra-day

25.0

24.78 ± 0.54

99.13 ± 2.17

2.13%

2.56%

5.0

4.98 ± 0.17

99.53 ± 3.49

3.98%

3.44%

0.5

0.49 ± 0.03

98.20 ± 5.24

4.78%

6.37%

All means presented are arithmetic.

ECF: Extracellular fluid; SD: Standard deviation; Conc.: Concentration; RSD: Relative standard deviation.

Table 2. The recovery rate of microdialysis probe (mean ± SD, n = 5)

 Cin (μg/mL)

 Cout (μg/mL)

 R (%)

0.50

0.41 ± 0.00

18.27 ± 0.02

5.00

4.08 ± 0.00

18.34 ± 0.02

10.00

8.17 ± 0.00

18.28 ± 0.01

All means presented are arithmetic.

SD, standard deviation; Cin, concentration in perfusate; Cout, concentration in dialysate.

Pharmacokinetic Process of Acetaminophen

The mean concentration vs. time profile of acetaminophen in the ECF of rats’ hypothalamus after administration (150 mg/kg. i.p.) was presented in Figure 2. The concentration-time curve of acetaminophen was fitted to a one-compartment open model, and the main pharmacokinetic parameter estimates were t1/2 = (1.95 ± 0.59) h, Tmax = (1.26 ± 0.22) h, Cmax = (11.39 ± 2.17) μg/mL, AUClast = (42.93 ± 5.39) μg·h/mL and AUCinf = (58.04 ± 18.39) μg·h/mL, as shown in Table 3.

JPPR 2018-101-Zong-Yuan Hong China-F2

Figure 2. Mean C-T curves of acetaminophen in the hypothalamus extracellular fluid of rats after administration of acetaminophen (150 mg/kg, i.p., n = 5). (A) arithmetical C-T curve, (B) logarithmic C-T curve. C-T, concentration-time, error bars indicate standard deviation.

Table 3. Main pharmacokinetic parameters of acetaminophen (150 mg/kg, i.p.) in the ECF of rats (mean ± SD, n = 5)

Parameter

Hypothalamus

t1/2 (h)

1.95 ± 0.59

Cmax (μg/mL)

11.39 ± 2.17

AUClast (μg·h/mL)

42.93 ± 5.39

AUCinf ( μg·h/mL)

58.04 ± 18.39

Tmax (h)

1.26 ± 0.22

All means presented are arithmetic.

ECF, extracellular fluid; SD standard deviation; t1/2, terminal elimination half-life, Cmax, maximum concentration; AUClast, area under the concentration–time profile to the last measurable concentration; AUCinf, area under the concentration–time profile from the time of dosing extrapolated to infinity; tmax, time to reach maximum concentration.

Discussion

In the present study, we used in vivo microdialysis sampling method combined with HPLC-UV to investigate the pharmacokinetics process of acetaminophen in the hypothalamus of rats which is the target site (active-site ) of acetaminophen, and obtained some main pharmacokinetic parameters, such as t1/2, Tmax, Cmax, etc. Compared with data in plasma (t1/2, Tmax and Cmax were 1.20 ± 0.30 h, 0.58 ± 0.13 h and 97.09 ± 11.08 μg/mL, respectively), the t1/2 of acetaminophen in the hypothalamus was prolonged significantly, indicating that acetaminophen eliminated more slowly in the hypothalamus. While the Tmax in the hypothalamus was 2-fold longer than that in plasma, suggesting the delayed distrib ution of acetaminophen into the hypothalamus, which might be attributed to the presence of the BBB. It is apparent that the Cmax in plasma was higher than that in the hypothalamus as the acetaminophen concentration in plasma consisted of the free (unbound) and bund concentrations of acetaminophen. All these results suggested that there were significant differences in pharmacokinetic processes between the plasma and target organs/tissues.

In general, the biochemical events and pharmacological effects do not usually take place in the bloodstream, but in target tissues [1, 2]. And active site concentrations of unbound substances are better predictors of drug effects than total plasma or whole tissue concentrations. This is partly due to the presence of active transporters at tissues, but is also due to differences in plasma protein binding and non-specific tissue binding [11]. The active-site concentrations can be defined as the concentrations of unbound, pharmacologically active substances at the site of action. In contrast, the total concentrations of the drug in plasma ⁄ organ ⁄ tissue also include the protein- or tissue-bound molecules that are pharmacologically inactive [11].

Traditionally, plasma and whole tissue concentrations are used as predictors of effects and side effects, as well as calculating pharmacokinetic parameters because of their ease of sampling, while the concentrations of unbound drug in tissue are more difficult to measure. But just as mentioned above, better predictors of drug effects are the active site concentrations of unbound substances. The results in the present study suggested that there were significant differences between pharmacokinetic parameters based on the plasma drug concentrations and active-site drug concentrations, implicating that the dosing schedule of acetaminophen in clinic should be designed according to the pharmacokinetic parameters based on the active-site drug concentration for decreasing acetaminophen-causing side effects, i.e. hepatotoxicity. Therefore, it is not accurate and precise to calculate pharmacokinetic parameters using the plasma or whole tissue concentrations for dosing schedule of drugs in clinic.

With the introduction of microdialysis, the first technique with which unbound concentrations could be easily measured in vivo. Compared with plasma or tissue sampling, the in vivo microdialysis method possesses many advantages. Firstly, in vivo microdialysis method can obtain the active site concentrations of unbound drugs [1], which can more accurately predict drug effects and calculate pharmacokinetic parameters. Secondly, the microdialysate samples obtained by in vivo microdialysis method can be directly detected by HPLC or HPLC/MS/MS because only low-molecular weight substances can be diffusible through the semi-permeable membrane [2, 18]. Yet, before detecting, the plasma or tissue samples must be dealt with by series of pretreatment, such as protein precipitation, homogenization and centrifugation, etc. These procedures were relatively complex and resulted in unreliable drug concentrations. Finally, in vivo microdialysis method can be continuously sampling without loss of the body fluid during the experiment [19], especially small animals such as mice, rats, etc. In brief, in vivo microdialysis is a simple, feasible and reliable sampling method, which is of unique advantages in pharmacokinetics study.

For in vivo microdialysis method, it is essential to obtain the recovery rate of probe for determining the actual concentration of endogenous drugs or substances, which is determined by several factors such as physicochemical properties of the analyte, semi-permeable membrane materials, diffusion coefficient and temperature [20–23]. The flow rate of microdialysis probes is also a significant factor on the recovery rate of probe increasing with lower flow rate [20]. Generally, the perfusate is usually at a flow rate from 0.1 to 5.0 μL/min. In this study, taking several factors, such as sample volume, sampling time and sensitivity to detect the analyte, into account, we thought that the ideal flow rate was 2 μL/min. Moreover, there are many methods to calibrate the recovery rate of probe, for instance, retrodialysis method [17], external standard method [24], internal standard method [25] and zero-net flux method [26]. In this study, the recovery rate of probe was determined by using the retrodialysis method, the most common calibration method.

This study provided one sample of in vivo microdialysis applications in acquiring drug concentration at the target sites for studying pharmacokinetics in rats. In fact, in vivo microdialysis has been also widely applied in humans for obtain unbound drug or endogenous substance concentrations in the ECF of target tissues/organs. La Favor et al. [27] successfully utilized microdialysis to measure in vivo reactive oxygen species in human skeletal muscle, demonstrating the feasibility of measuring both in vivo H2O2 and superoxide in the extracellular environment of human skeletal muscle. Simmel et al. [28] provided the proof of principle of long-term subcutaneous microdialysis in humans in which they developed a special setting to ensure good clinical practice compliance, tolerability, and convenience for participants and personnel. Moreover, Prolonged microdialysis sampling over several days has been used for endogenous compounds and/or drugs in humans in neonatal [29–31] and adult [32–35] diabetic patients, in patients admitted for breast reconstruction with transverse rectus abdominis muscle flaps after mastectomy [36], in patients with ischemic heart disease [37], and in patients for neurochemical monitoring or on the neurosurgical ICU [38]. Hence, unquestionably, with the development of experimental science and technology, in vivo microdialysis would be more wide and deep application in basic and clinical pharmacokinetics studies.

Conclusion

Our results reveal that there are significant differences between pharmacokinetic parameters based on the plasma drug concentration and the active-site drug concentration, implicating that the dosing schedule of drugs in clinic should be designed according to the pharmacokinetic parameters based on the active-site drug concentration. And this study provides a simple, feasible and reliable experimental method for studying pharmacokinetics of drug in the target.

Acknowledgement: This work was supported in part by grants-in-aid for the National Natural Science Foundation of China (81671318, 81171255) and the Programs for Science and Technology Development of Anhui Province (1501041157).

Conflict of interest: All authors declare no conflict of interest

Ethical approval: All experimental protocols were performed in accordance with the principles of animal use and care approved by the ethnical committee of Wannan Medical College.

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