Neurotoxicity in pre-eclampsia involves oxidative injury,exacerbated cholinergic activity and impaired proteolytic and purinergic activities in cortex and cerebellum

Abstract

Women with a history of pre-eclampsia (PE) tend to have a higher risk of developing cardiovascular and neurological diseases later in life. Imbalance in oxidative markers and purinergic enzymes have been implicated in the pathogenesis of neurological disease. This study investigated the effect of PE on oxidative imbalance, purinergic enzyme inhibitory activity, acetylcholinesterase and chymotrypsin activities in the brain of PE rat model at post-partum/post-natal day (PP/PND) 60. Pregnant rats divided into early-onset and late-onset groups were administered with Nω-nitro-l-arginine methyl through drinking water at gestational days 8–17. Rats were allowed free access to water throughout the pregnancy and allowed to deliver on their own. The mother and the pups were euthanized at PP and PND 60, respectively, the cortex and the cerebellum excised, homogenized and stored for analyses of the enzymes. Results showed an increase in nitric oxide and malondialdehyde with a concomitant decrease in reduced glutathione and superoxide dismutase, an indication of oxidative damage. Also, there was an increase in acetylcholinesterase activity with a decrease in chymotrypsin, adenylpyrophosphatase and ecto-nucleoside triphosphate diphosphohydrolase activities in both the cortex and the cerebellum of the mother and the pups at PND 60. These results indicate the involvement of oxidative stress, increased cholinergic activity and depleted proteolytic and purinergic activities in PE-induced neurotoxicity.

Keywords

Neurotoxicity pre-eclampsia oxidative imbalance acetylcholinesterase chymotrypsin purinergic enzymes

Introduction

Pre-eclampsia (PE) is defined as new onset of hypertension (>140/90 mm hg) after 20 weeks of gestation in a previously normotensive women associated with at least 1+ proteinuria on urinary dipstick measurement.^1,2 Notwithstanding the severity and the incidence, the pathophysiology of this disease is not fully understood.³ Also despite knowing that the consequence of PE ends after the delivery of placenta, it is now apparent that women with PE and their offspring have greater susceptibility to develop cardiovascular complications such as heart disease, stroke and venous thromboembolism over a 5–15-year period post-delivery. Moreover, there is greater risk of dying from cerebrovascular diseases such as stroke and vascular dementia after pregnancy with PE than with healthy pregnancy.⁴

The long-term consequence of PE on the maternal brain and the developing brain of the offspring requires investigation. More recently, a change in brain size years after the index pre-eclamptic pregnancy has been reported.^5,6 Nonetheless PE is associated with development of white and grey matter lesions^7
–9 together with cognitive impairment later in life.^5,6,10
–12

Oxidative stress is a disproportion between the production of reactive oxygen species (free radicals) emanating from normal metabolic processes and anti-oxidants in the cell. The brain is especially susceptible to oxidative injury due to its high oxygen metabolism, its rich lipid milieu and its low antioxidant enzyme content. Oxidative stress is involved in the aetiology of early developmental brain injury and where it also triggers cell death.¹³ It is a neurogenic pathway that is involved in almost all the central nervous system pathologies.¹⁴ In aged humans, oxidative damage leads to concomitant suppression of the endogenous anti-oxidants viz., superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase in cognitive sites such as the hippocampus and cerebral cortex thereby contributing to cognitive decline.^15,16

Purinergic enzymes are enzymes anchored to the cell surface and control major physiological processes in the body.¹⁷ Increase in adenylpyrophosphatase (ATPase) has been reported in conditions that involve inflammation, hypoxia or ischemic. Furthermore, PE is an inflammatory immune imbalance condition.^18,19 Acetylcholine, a major parasympathetic neurotransmitter, inhibits the release of pro-inflammatory cytokines from macrophages and microglia and has been linked to pathogenesis and progression of inflammatory neurodegenerative diseases.²⁰

Currently, it is controversial whether a previous history of PE predisposes to an increased risk of Alzheimer’s disease (AD) development. Growing evidence, however, supports the hypothesis that oxidative stress plays a major role in the cognitive impairment,²¹ emanating from the increased lipid peroxidation (LPO), protein and DNA oxidation in neurons.²²

Notably, hypertension in pregnancy is associated with utero-placenta ischemia leading to fetal hypoxia and intra-uterine growth restriction.²³ Fetal hypoxia is associated with oxidative stress in preterm infants.²⁴ Oxidative stress occurs at post-natal day (PND) 7 in preterm children with or without hypoxia. Shoji et al. reported an increase in 8-hydroxy-2″-deoxyguanosine which is a marker of oxidative DNA damage in the urine of infant with low birth weight and concluded that this marker is correlated to mental development. Therefore, it can be a predictive marker of neurodevelopmental outcome in low birth weight infants.²⁵

Animal models that can mimic hypertension in pregnancy have been developed by researchers using different methods.^3,26

–29 One of these models produces a dose-dependent hypertension like in pregnant rodent which is the use of Nω-nitro-l-arginine methyl (l-NAME) to produce a PE-like syndrome.^30,31 Also, the use of l-NAME had previously been used to establish the induction of PE and its effect on neuroinflammatory markers in our laboratory.³² Therefore, we adopted the model for this study. This study aims at investigating the effect of PE on oxidative imbalance, purinergic enzymes inhibition, acetylcholinesterase and chymotrypsin activities in the brain at PND 60.

Materials and methods

Animals

Fifteen pregnant albino female rats (Sprague-Dawley strain) weighing 180–200 g were obtained from the Biomedical Research Unit (BRU), University of KwaZulu-Natal, Durban, South Africa. The rats were fed on pelletized chows, and water given ad libitum, while acclimatizing for 7 days under natural photoperiods of 12-h light–dark cycle. They were maintained under the guidelines and approval of the Animal Ethics Committee of the University of KwaZulu-Natal, Durban, South Africa (AREC/055/017D).

The rats were divided into three groups of five animals each: control, early-onset (EOPE) and late-onset (LOPE) PE. The EOPE and LOPE groups were given l-NAME at 0.3 g/L in the drinking water ad libitum at gestational days (GD) 8–12 and 12–16, respectively. Although, water consumption level was not measured for but sufficient water was given, which they rarely finished before another was prepared. Blood pressure was measured at GD 12 and 17 using a tail-cuff BP monitor (MRBP IITC Life Sciences Inc., Woodland Hills, CA, USA). The rats were allowed to give birth on their own and the pups were left with the mother till 21 days. After weaning, the pups were separated from the mother.

Euthanization and collection of organs

Forty-two male and females comprising of 14 pups from each group were euthanatized with the mothers at PND 60 and post-partum day 60, respectively, using Isofor. The frontal cortex and cerebellum were carefully excised and rinsed in 0.9% NaCl to remove blood stains. They were homogenized in 50 mM sodium phosphate buffer (with 10% triton X-100, pH 7.5) in a ratio of 1:1 (i.e. 1 g of tissue to 1 mL of buffer) in a test tube using a tissue homogenizer (Omni, Kennesaw, GA, USA). The homogenized samples were then centrifuged at 20,000 rcf for 10 min at 4°C. The supernatant was collected and stored at −20°C for subsequent analysis.

For all assays, the frozen supernatants were allowed to thaw on ice and all experiments were carried out on ice to minimize loss of enzyme activity

Determination of oxidative stress

The supernatants were analysed for oxidative stress biomarkers which covers for GSH³³ and SOD³⁴ activities, and malondialdehyde (MDA) level.³⁵

Reduced GSH level

This was carried out using the Ellman’s method 33. Briefly, after deproteinizing with an equal volume of 10% trichloroacetic acid (TCA), the supernatants were centrifuged for 5 min at 4700 rcf. Two hundred microlitres of the supernatant together with 50 µL of Ellman reagent was thereafter added to a 96-well plate. The reaction mixture was allowed to stand for 5 min, and absorbance was read at 415 nm. The GSH concentration was extrapolated from a standard curve.

SOD activity

The SOD activity was determined using a method based on the principle that 6-hydroxydopamine (6-HD) is oxidized by hydrogen peroxide (H₂O₂) from SOD catalysed dismutation of $O_{2}^{\cdot -}$ , which produces a coloured product.³⁶ Briefly, 15 µL of the supernatants were dissolved in 170 µL of 0.1 mM diethylenetriaminepentaacetic acid in a 96-well plate. Fifteen microlitres of 1.6 mM 6-HD was thereafter added. Absorbance of the reactant mixture was measured at 492 nm for 5 min at 1 min interval.

LPO levels

This was determined by measuring the thiobarbituric acid (TBA) reactive substances, expressed as MDA equivalent in the supernatants.³⁵ Briefly, a reaction mixture consisting of 100 µL of the supernatants, 100 µL of 8.1% SDS solution, 375 µL of 20% acetic acid, 1 mL of 0.25% TBA and 425 µL of distilled water was heated at 95°C for 1 h in a water bath. Two hundred microlitres of the heated mixture was thereafter pipetted into 96-well plate, and absorbance read at 532 nm.

Determination of nitric oxide level

Tissue nitric oxide (NO) levels were determined using the Griess method.³⁷ One hundred microlitres of the samples or distilled water (blank) was incubated with an equal volume of Griess reagent for 30 min at 25°C in the dark. Absorbance was read at 548 nm and the result was calculated using the formula:

Nitric oxide conc.= (Absorbance of sample - Absorbance of blank) ×0.1305

Determination of purinergic enzymes activities

Determination of ATPase activity

The ATPase activity within tissue was determined according to an established protocol.^38,39 Briefly, a reaction mixture consisting of 200 µL of the tissues’ supernatants, 200 µL of 5 mM KCl, 1300 µL of 0.1 M Tris-HCl buffer and 40 µL of 50 mM ATP was incubated at 37°C in a shaker for 30 min. The reaction was stopped by adding 1 mL of distilled water and 1.25% ammonium molybdate. Thereafter, 1 mL of freshly prepared 9% ascorbic acid was added to the reaction mixture and allowed to stand for 30 min. Absorbance was read at 660 nm.

Determination of ecto-nucleoside triphosphate diphosphohydrolase activity

Tissue ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity was determined according to a modified established protocol.^40,41 Briefly, 20 µL of the supernatants were incubated with 200 µL of the reaction buffer (1.5 mM CaCl₂, 5 mM KCl, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM Tris-HCl) at 37°C for 10 min. Twenty microlitres of 50 mM ATP was thereafter added to the reaction mixture and further incubated at 37°C in a shaker for 20 min. Two hundred microlitres of 10% TCA was added to the reaction mixture to stop the reaction. The reaction was incubated on ice for 10 min and absorbance was read 600 nm.

Acetylcholinesterase activity

Tissue acetylcholinesterase activity was determined using the Ellman’s method.⁴² Briefly, 20 µL of the supernatants was mixed with 10 µL of 3.3 mM Ellman’s reagent (pH 7.0) and 50 µL of 0.1 M phosphate buffer (pH 8). The reaction mixture was incubated for 20 min at 25°C. The reaction was stopped by adding 10 µL of 0.05 M acetylcholine iodide to the reaction mixture. Absorbance was read at 412 nm at 3 min intervals.

Determination of proteolytic activity

This was carried out by determining the α-chymotrypsin activity in the tissue according to a previous method,⁴³ with slight modifications. Briefly, a reaction mixture consisting of 15 µL of supernatants and 60 µL Tris-HCl buffer (50 mM pH 7.6) was pre-incubated at 37°C for 20 min. The reaction was initiated by the addition of 15 µL 1.3 mM N-succinyl phenyl-alanine-P-nitroanilide. This reaction mixture was incubated at 37°C for 30 min, and absorbance read at 410 nm.

Statistical analysis

Data were subjected to analysis of variance, followed by Bonferroni multiple comparison and significant difference established at p < 0.05, with results presented as mean ± SEM using Graph Pad Prism, version 5.01.

Results

NO in the cerebral cortex and the cerebellum at PND 60

As shown in Figure 1(a) and (b), there was a significant increase in the level of NO within the cortex of both the EOPE and LOPE groups compared to control (p < 0.05), likewise within the cerebellum of both the EOPE and LOPE groups compared to the control (p < 0.01).

Figure 1.

NO and MDA levels of (a) cortex and (b) cerebellum of maternal rats at PND 60. Values = mean ± SEM; n = 5. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01 compared to control. NO: nitric oxide; MDA: malondialdehyde; PND: post-natal day.

As represented in Figure 2(a) and (b), there was no significant increase (p > 0.05) in the level of NO in the cortex of both male and female pups at PND 60. In contrast, there was significant difference (p < 0.05) of NO between the control and the LOPE groups of the female cerebellum.

Figure 2.

NO and MDA levels of (a) cortex and (b) cerebellum of pups at PND 60. Values = mean ± SEM; n = 7. Comparison of differences across treatment groups indicated as *p < 0.05 compared to control. NO: nitric oxide; MDA: malondialdehyde; PND: post-natal day.

LPO in the cerebral cortex and the cerebellum at PND 60

MDA level of the maternal rat at PND 60 (Figure 1(a) and (b)) was significantly increased in the cortex (p < 0.01) and cerebellum (p < 0.05) of EOPE and LOPE groups versus control groups.

As shown in Figure 2(a) and (b), the MDA level displayed the same trend as that of NO, in that there was no significant difference (p > 0.05) within the cortex. A significant difference of MDA within the cerebellum was noted between the control versus the LOPE in the female (p < 0.05) pups compared to their male counterparts (p > 0.05) at PND 60.

GSH and SOD activity in the cerebral cortex and the cerebellum at PND 60

Both GSH and SOD activity declined within the cerebral cortex and the cerebellum of the EOPE and LOPE groups compared to control as represented in Figure 3(a) and (b). A greater expression occurred within the cerebellum (p < 0.001) compared to the cortex (p < 0.05).

Figure 3.

GSH level and SOD activity of (a) cortex and (b) cerebellum of maternal rats at PND 60. Values = mean ± SEM; n = 5. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control. GSH: glutathione; SOD: superoxide dismutase; PND: post-natal day.

As represented in Figure 4(a) and (b), more specifically, the GSH within the cerebral cortex of the pups at PND 60 was significantly down-regulated between control versus EOPE (p < 0.001) and between control versus LOPE (p < 0.001) irrespective of the sex. In the cerebellum, there was significant decrease (p < 0.001) between control versus EOPE group and between control versus LOPE in the female compared to their male counterpart (p > 0.05).

Figure 4.

GSH level and SOD activity of (a) cortex and (b) cerebellum of pups at PND 60. Values = mean ± SEM; n = 7. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control. GSH: glutathione; SOD: superoxide dismutase; PND: post-natal day.

Acetylcholinesterase activity and α-chymotrypsin activity in the cerebral cortex and the cerebellum at PND 60

There was significant elevation of acetylcholinesterase activity (p < 0.05), with concomitant suppression of α-chymotrypsin activity in both the maternal cortex and cerebellum of the EOPE group compared to the control. Likewise, acetylcholinesterase activity within both cerebral and cerebellum was up-regulated (p < 0.01) in the LOPE group compared to the control as represented by Figure 5(a) and (b).

Figure 5.

Acetylcholinesterase and α-chymotrypsin activities of (a) cortex and (b) cerebellum of maternal rats at PND 60. Values = mean ± SEM; n = 5. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01 as compared to control. PND: post-natal day.

There was significant increase (p < 0.01) in acetylcholinesterase activity with concomitant significant decrease (p < 0.001) in the activity of α-chymotrypsin between LOPE compared to control groups in both the male and female pup cortex at PND 60. In contrast, there was no significant difference between EOPE versus control in the female but significantly increased in the male cortex (p < 0.05). Meanwhile, there was significant increase (p < 0.01) in acetylcholinesterase activity only in the female between EOPE and LOPE compared to control, but no significant in the male cerebellum at PND 60. Likewise, no significant difference in the activity of α-chymotrypsin was noted between in both male and female cerebellum of EOPE, LOPE versus control at PND 60 as shown in Figure 6(a) and (b).

Figure 6.

Acetylcholinesterase and α-chymotrypsin activities of (a) cortex and (b) cerebellum of pups at PND 60. Values = mean ± SEM; n = 7. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control. PND: post-natal day.

ATPase and E-NTPDase in the cerebral cortex and the cerebellum at PND 60

There was significant decrease in the activities of ATPase and E-NTPDase within the cortex and the cerebellum of the EOPE and LOPE groups compared to control (Figure 7(a) and (b)). Cerebellar expression of both ATPase and E-NTPDase within the cerebellum (p < 0.01) of the EOPE group than in the LOPE group (p < 0.05).

Figure 7.

ATPase and E-NTPDase activities of (a) cortex and (b) cerebellum of maternal rats at PND 60. Values = mean ± SEM; n = 5. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01 as compared to control. PND: post-natal day.

There was significant decrease (p < 0.001) in E-NTPDase activity across the EOPE, LOPE and control groups in both the male and female pup cortex at PND 60. In contrast, there was no significant difference between EOPE versus control in the male but significantly reduced in the female (p < 0.01) for the cerebellum. Likewise, no significant difference of E-NTPDase activity was noted between LOPE versus control in the male pups. In contrast, a significant difference was noted between LOPE and controls in the female pups (Figure 8(a) and (b)).

Figure 8.

ATPase and E-NTPDase activities of (a) cortex and (b) cerebellum of pups at PND 60. Values = mean ± SEM; n = 7. Comparison of differences across treatment groups indicated as *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control. PND: post-natal day.

Discussion

Women with a pre-eclamptic index pregnancy are reported to be at high risk of developing hypertension, cardiovascular disease, cognitive deficit, stroke and dementia later in life.^5,6,8,9,12 Systemic oxidative stress, a balance shift in favour of reactive oxygen species generation leads to oxidative or cellular damage occurs in mild cognitive impairment and late-onset AD.⁴⁴ Oxidative stress is also one of the mechanisms underlying neuronal damage associated with deep brain microstructural changes such as white matter lesion.⁴⁵ Oxidative stress seems to be the main factor responsible for low cognitive performance⁴⁶ and is associated with most neurodevelopment disorders.⁴⁷

In this present study, we report a significant increase in the oxidative stress marker, MDA with concomitant suppression of GSH level and SOD activity in both the maternal cerebellum and the cortex of the l-NAME-treated rats compared to control. The high MDA level indicates LPO, and these may be attributed to the decreased GSH level and SOD activity (Figures 1 to 4). A decreased GSH and SOD activity has been implicated in increased production of H₂O₂, which if not mopped by CAT contributes to LPO.⁴⁸ The high level of NO in the maternal cortex and the cerebellum tissue of l-NAME groups compared with the control (Figures 1 and 2) also indicates pro-inflammation. NO in the presence of low SOD activity reacts with superoxide anion ( $O_{2}^{\cdot -}$ ) that leads to the production of the potent radical, peroxynitrite (ONOO⁻), which triggers nitrosative stress.⁴⁹ Women with history of PE has been reported to develop white matter lesion years after index pregnancy^9,50 and oxidative stress is one of the underlying mechanism in white matter lesion.⁴⁵ Therefore, alteration in oxidative stress markers present in the cerebellum and the cortex of l-NAME induced PE in the present study may be correlated with white matter lesion reported in women with history of PE. Cognitive impairments such as short term memory loss,⁵¹ attention deficit,¹⁰ slower motor speed with poor score in cognitive questionnaire^12,52 have been reported in women with a history of severe or mild form of PE and the mechanism underlying this is unclear, although peripheral inflammation can affect the function of CNS alongside memory and cognition.⁵³ Previously in our lab, we reported long term sensorimotor and cognitive deficit in PE animal model.⁵⁴ One of the mechanism involve in low cognitive performance has been reported to be oxidative stress.⁴⁶ In the present study, we report that oxidative stress is present in the cerebral cortex and cerebellum of l-NAME-induced PE rats at PND 60. These findings may be one of the mechanisms through which cognitive impairment occurs in women with a history of PE although we did not check for baseline of oxidative stress markers nor any cognitive impairment test in the present study. Nonetheless, Revel and colleagues reported an increase in systemic GSH in relation to cognitive decline in AD patient in a 6-month follow-up which they reported to be paradoxical since high intracellular GSH content is considered protective against cell damage caused by free radical.

Also, in this present study, the pups born to pre-eclamptic rat group shows an increase in LPO and NO in both cerebral cortex and the cerebellum in male and female at PND 60 though the increase is more pronounced in the LOPE. Likewise, the present study shows a decrease in SOD and GSH in the cerebral cortex and cerebellum tissue of PND 60 pups in both the EOPE and LOPE male and female. This result shows the same trend as seen in the mother. Veronica et al. hypothesis that change observed in anti-oxidant status of PE mother is similar to that of their new-borns.⁵⁵ Likewise, infants born to PE mother are associated with increased oxidative stress, low activities of anti-oxidant activity and increased LPO and protein oxidation.⁵⁶ The fetal and neonatal brain are vulnerable to the effect of oxygen and nitrogen-based free radicals. Oxidative stress is implicated in the pathogenesis of most neurological disease such as hypoxic-ischemic injury,⁵⁷ epilepsy,⁵⁸ haemorrhagic and cerebral injury,⁵⁹ therefore oxidative stress serves as a component of early ageing process.⁶⁰ The imbalance in oxidative stress in this study might be the reason for developmental and neurological deficit reported in children born to PE mother. This study is the first to demonstrate oxidative stress at PND in pups born to pre-eclamptic mothers.

Acetylcholine, a major parasympathetic neurotransmitter inhibits the release of pro-inflammatory cytokines from macrophages and microglia.²⁰ It is hydrolysed by acetylcholinesterase. Patients with AD display an elevation of plasma and tissue activity of acetylcholinesterase; hence it is linked to the pathogenesis and the progression of neurodegenerative disease.^61,62 In our study, there was significant increase in the activity of acetylcholinesterase in the cerebral cortex and the cerebellar tissue at PND 60 of the l-NAME-induced PE groups compared to control in both the mother and the pups. An elevation of acetylcholinesterase activity is also associated with systemic inflammation and with an impaired cognitive and motor neuron dysfunction.⁶³ Our findings are corroborated by the induction of neuroinflammation concomitant with elevated acetylcholinesterase activity⁶⁴ and also corroborated with the presence of peripheral inflammation that leads to an increased number of neuronal damage with an elevation of acetylcholinesterase activity in the cortex.⁶⁵ Moreover, one should be cognizant of the fact that PE is a hyper exaggerated inflammatory condition. It is therefore plausible that there may be a higher tendency of neurodegenerative disease in relation to PE.

Likewise, our study shows a suppression of α-chymotrypsin activity in both the maternal cortex and cerebellum of the EOPE group compared to the control. Meanwhile, there is only significant difference in the pups cortex but not in the cerebellum in both l-NAME PE-induced groups compared to control. Chymotrypsin is a proteolytic enzyme with anti-inflammatory activity.⁶⁶ In PE, chymotrypsin is known to be implicated in the endothelial expression of P-selectin and E-selectin that are usually expressed on endothelial activation.⁶⁷ It is also involved in the facilitation of tissue repair by providing better resolution of inflammatory symptoms.⁶⁸ The decreased activity in the present study (Figure 5) is suggestive of neurodegeneration.

The neuroprotective functions of purinergic enzymes and signalling have been reported.^69,70 These enzymes catalyse the production of adenosines which facilitates the suppression of inflammation and tissue injury.⁴¹ The decreased ATPase and E-NTPDase activities in cerebellums and cortexes of l-NAME PE-induced treated groups compared to control in both the mother and the pups at PND 60 in the present study (Figures 7 and 8) therefore, insinuates neurotoxicity. These decreased activities also indicate a decreased level of adenosine, which may also portray an induction of proinflammation.⁴¹ Impairments of these enzymes have been implicated in the pathogenesis and progression of neurotoxicity.^41,70

Conclusion

These results indicate the involvement of oxidative stress, increased cholinergic activity and depleted proteolytic and purinergic activities in PE-induced neurotoxicity. Modulation of these activities may be therapeutic in the management and treatment of neurotoxicity associated with PE.

Footnotes

Acknowledgement

OKI acknowledges the support of National Research Funding in collaboration with the World Academy of Science (NRF-TWAS) doctoral fellowship 2016.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Research Funding in collaboration with the World Academy of Science (NRF-TWAS), and College of Heath Sciences (CHS) Bursary, University of KwaZulu-Natal, Durban, South Africa.

ORCID iDs

OK Ijomone

OL Erukainure

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