Neuroprotective effects of feeding Centella asiatica (L) Urb. – Study of hippocampal neurons in pups born to alcohol-fed female Wistar rats

 

Mitha KV1, Saraswati Jaiswal Yadav2, Rashmi K S3, Ganaraja Bolumbu3*

1Assistant Professor of Physiology, Kannur Medical College, Anjarkandi, Kannur, Kerala. India.

2Post-Doctoral Fellow, Centre of Behavioural and Cognitive Sciences,

University of Allahabad, Allahabad - 211008. India.

3Department of Physiology, Kasturba Medical College,

(Constituent of Manipal Academy of Higher Education) Mangalore.

*Corresponding Author E-mail: mithamukundan@gmail.com, saras.jaiswal@gmail.com, rashmi.ks@manipal.edu

 

ABSTRACT:

Objectives: To evaluate the neuroprotective function of C. asiatica on the offsprings in maternal alcohol abuse.  Centella Asiatica (C. asiatica) has been known to Indian traditional medicine Ayurveda as an effective brain tonic. Alcohol is an abused substance and poses a health risk to all in society, including pregnant women. We force-fed alcohol to pregnant rats and studied its effect on rat pups' hippocampus, which were fed with C. asiatica. The results were also correlated to the cognitive performance of the animals. Methods: Adult female rats, confirmed of pregnancy, were fed with 30% (w/v) alcohol at a dosage of 20g/kg body weight, daily oral gavage. The pups were divided into seven groups (n=6 each) as control and experimental/treated. Results: Hippocampus was isolated, and the slices were stained, and the cell count was done by applying appropriate techniques. The pup quality, cognitive parameters showed differences in alcohol-treated groups.  The cell count was performed and compared among the groups. A significant increase in the cell count and the hippocampal neuron population's size was observed in the rats fed with C. asiatica extract.  The pup quality was also better. Their cognitive performance was significantly better. Conclusion: This study revealed the adverse effects of fetal alcohol exposure, which reversed after treatment with C. asiatica. This study confirms the role of C. asiatica as an effective neuroprotective agent, and it could be useful to treat the patients suffering from the effects of exposure to alcohol in fetal life and early childhood.

 

KEYWORDS: Alcohol, Centella asiatica, Hippocampus, Neuronal damage, Neurotropic agent.

 

 


INTRODUCTION:

Alcohol is one of the most abused of all addictive substances due to its wide availability. There is no gender bias in alcohol addiction, and it is seen in both males and females. It was stated that there is a steady increase in alcohol consumption in India.1

 

In the US, sixty percent of women of childbearing age occasionally consumed alcohol.2 But the rate of alcohol consumption among Indian women is relatively low at 5.8% in the population and up to 28 to 45% among tribal communities.3

 

Alcohol is readily transported across the cell membrane; hence all tissues are affected by it. Alcohol also affects cell function by acting on the DNA and cause cancer.4 Alcohol can enter the placental barrier and affect fetal metabolism, and cause fetal alcohol spectrum disorder (FASD) characterized by several congenital abnormalities and decreased mental growth.5,6,7  Several brain regions are affected by exposure to alcohol, namely the frontal lobe, cerebellum, basal ganglia, corpus callosum, hippocampus, hypothalamus, etc.6 We reported that the pup quality was adversely affected by maternal alcohol consumption in Wistar rats.8  We found reduced postnatal weight gain, increased mortality rate by about 10% though there was no statistically significant difference in the birthweight, crown length, and litter size.

Maternal alcohol consumption during pregnancy affects the migration, proliferation, and organization of the fetal brain cells in different specific brain regions, mainly the hippocampus, cerebellum, prefrontal cortex, etc., leading to FSAD.9-12 Studies conducted in humans using Magnetic Resonance Imaging (MRI) also reported the size of different brain regions like basal ganglia, cerebellum, corpus callosum, hippocampus, diencephalon, caudate nucleus was decreased in offspring whose mothers were fed with alcohol during pregnancy.6,13,14 According to researchers, structural damages in parts of the brain caused deficits like poor planning, cognitive inflexibility,  reduced response inhibition with impaired spatial memory observed in FASD patients.15,16,17 Our study also confirmed these observations.18 Centella Asiatica, also commonly known as Mandukaparni in Ayurvedic literature, is a medicinal plant, very widely seen all over Asia and known by different names. https://indiabiodiversity.org/species/show/229119).

 

It was being used in traditional medicine for a variety of conditions of lungs, skin, etc. C.asiatica is one of the drugs used in medhyarasayana (which can promote neuronal regeneration and cognitive functions) described by Acharya Charaka in Charaka Samhitha, the ancient Indian pharmacological scripture.19,20 It contains triterpenes and asiatic acid, and madecassic acid apart from other chemical agents. Further, it is non-toxic, and even at a dosage of 1g/kg body weight (IP), did not cause any long-term adverse effects in mice. (European medicines agency, 25 November 2010, EMA/HMPC/291177/2009). It was known as "Brain Food" in India.21,22,23 It contains apart from triterpenes, asiatic acid, madecassic acid, it is also rich in terminolic acid, centic acid, centoic acid, glycosides such as asiaticoside A, B, C, D, E and F, madecassoside and centelloside, alkaloids, flavonoids, other volatile acids such as Palmitic, stearic, lignoceric, oleic, linoleic.24 C. asiatica extract has been shown to have antioxidant property,25 and neuroprotective properties,26,27, and cell proliferation in Sprague –Dawley rats.28

 

In the present study, we evaluated the neuronal damage and neuronal population in prenatally alcohol-exposed rats (Alcohol fed during pregnancy). We compared it in those animals fed with C. asiatica extract during development. The study revealed the depletion of neuronal population in untreated animals and higher neuronal population following the treatment in the alcohol-exposed group. This study provides enough evidence for the neuroprotective action of the extract of C. asiatica.

 

MATERIALS AND METHODS:

Animals: Wistar albino rats, bred in-house, adults of reproductive age group, weighing about 200±20g at the time of procurement were selected for breeding. They were housed in a light and temperature-controlled laboratory, with rat chow (Amruth laboratory, Maharashtra, India), potable water was made available ad.lib. They were maintained standard housing conditions with paddy husk bedding Plexiglas cages following strict ethical guidelines as per CPCSEA, Government of India. Ethical clearance was obtained from the Institutional Ethical Committee (Dated: 01/02/2013) before the project. 

 

The female rats were allowed to mate with the male (One with one for 24 hours), and the vaginal observation for the copulatory plug confirmed the mating. Female rats in the alcohol group were fed using oral gavage daily with 30% w/v with distilled water (Hayman Ltd, England, absolute alcohol made to 30%w/v) alcohol at a dose of 5g/kg/day using oral gavage, for 22 (gestation period) days during pregnancy and 60 days after delivery, apart from free access to rat feed and drinking water.29 Both male and female pups born were used for the experiment.

 

Grouping: rat pups were grouped as follows: (n= 6 each); Group 1: Control group 1a: Offspring from normal pregnant rats fed with water. Control – alcohol: 1b. Offspring from alcohol-fed pregnant rats

 

Group 2: Experimental group 2a: Offspring exposed to alcohol both pre and early postnatal life, i.e., mother rats of these offspring were fed with alcohol during gestation and lactation period. Experimental group 2b: Offspring exposed to alcohol only during prenatal life, i.e., mother rats of these offspring were fed alcohol only during the gestation period. Group 3: Experimental group 3a: Offspring of alcohol-fed female rats were exposed to the treatment only during prenatal life, i.e., mother rats of these offspring were fed C. Asiatica and alcohol during gestation period; the duration of treatment was 22 days. Experimental group 3b: Offspring from alcohol-fed female rats treated with the extract of C.asiatica during postnatal life. Offspring were treated with the water extract immediately after the weaning period and continued till the 90th day of postnatal life; the duration of treatment  60 days. Experimental group 3c: Offspring from alcohol-fed female rats treated with the extract of C.asiatica both pre and postnatal life, i.e., alcohol-fed pregnant rats were treated with C.asiatica during the gestation period, and the treatment was continued in their offspring during postnatal life. Offspring were treated with the extract immediately after the weaning period and continued till the 90th day of postnatal life; the duration of treatment 80 days.

C. asiatica aqueous extract preparation:

The plant has been identified and certified by Dr. Krishna Kumar G, Professor of Botany, Mangalore University, Mangalore, as Centella asiatica (L) Urb. (Apiaceae). Specimens are available in the Mangalore University Herbarium. We carried out tests for major components of the plant extract; namely, flavonoids were tested using 'ferric chloride test.' Triterpenoids were tested by ‘Salkowski’s test,' and saponins were tested by the 'froth test.' The water extract of C. asiatica was prepared by the standard method using Soxhelt's apparatus as per the procedure.30 The extract was used to feed the rats at 20mg/kg body weight daily for 22 days in the prenatal period and 60 days in the postnatal period orally as per the protocol for each group. Thirty percent bioavailability of major components has been reported on oral feeding of the water extract.  

 

Histology:

At the end of the study, the rats were sacrificed by cardiac puncture under ether anesthesia. Their brain was carefully dissected and preserved in formol saline. Hippocampus was dissected in ice-cold condition using a rat brain slicer (Zovic, Germany) and kept in 10% formalin for 48 hrs. Coronal sections of 5μm thickness were cut in the dorsal hippocampus using a rotary microtome (Jung Biocutt 2035, Leica, Germany). Subsequently, the slices were histologically processed and stained with cresyl violet stain, and the cells were counted.

 

In the hippocampal section, the CA3 region, Cornu ammonis 300μm length, and 200μm2 of the dentate gyrus were selected using imaging software (NIS Elements Br version 4.30), and the viable neurons were counted. Four sections from each rat were considered (darkly stained, shrunken, and fragmented nuclei were excluded from the count). Results were expressed as the number of cells per unit length of the cell field.31 

 

RESULTS:

Histological studies (Fig. 2 photomicrographs): In the hippocampus, the number of neurons in the dentate gyrus (DG) and Hippocampal CA3 areas of 90 days (3 months) old rat was counted (group mean±SD).

 

Statistical analysis:

Statistical analysis for multiple comparisons was performed by one-way analysis of variance (ANOVA) followed by post hoc test or Kruskal -Wallis test followed by post-hoc test and Tukey’s multiple comparison tests as per the requirements.

 

Number of neurons in the Dentate Gyrus (Fig 1): Results of the present study showed offspring exposed to alcohol continuously during pre and early postnatal life (group 2a) and prenatal life only (group 2b) had significantly less number of neurons in the DG area of the hippocampus as compared to the offspring from control (1a) and positive control (1b) groups, (p<0.001). Further comparison between the experimental group 2a and 2b showing that the number of neurons was less in offspring exposed to alcohol both pre and early postnatal life (2a) compared to the offspring exposed to alcohol only during prenatal life (2b), but it was not statistically significant. No significant difference among alcohol-fed rats treated with C.asiatica during prenatal life compared to the offspring from experimental groups 2a and 2b. Offspring from alcohol-fed female rats treated with C.asiatica only during postnatal life (3b) showed significantly more number of neurons in the DG of the hippocampus when compared to the offspring from experimental groups 2a and 2b (p<0.05). Those treated during pre and postnatal life (3c) showed significantly more number of neurons in the DG area of the hippocampus when compared to the offspring from experimental groups 2a and 2b (p<0.05). But the number of neurons was significantly less in offspring from treatment group 3a than the progeny from treatment groups 3b and 3c (p<0.05) and control (p<0.001). No significant difference was observed in neuronal count between the offspring from treatment group 3b and 3c.

 

 

Figure 1: Neuronal assay – the number of neurons in the DG of hippocampus – Male offspring rats.

Statistical analysis was done by the Kruskal-Wallis test (p˂0.001), followed by Tukey’s multiple comparison tests. 1a Vs 2a  and  2b ***p<0.001; 1a Vs 3a ***p<0.001; 1b Vs 2a  and G2b ###p<0.001; 1a Vs 3a ###p<0.001; 2a Vs 3b  and  3c $p<0.05; 2b Vs 3b  and  3c Ɵp<0.05; 3a Vs 3b  and  3c ¥p<0.05.

 

Fig 2: Photomicrograph of the DG region of the hippocampus under the magnification of 20X (Cresyl violet stain).

 

Neuronal assay in CA3 of the hippocampus (Fig 3):

Alcohol exposure during pre and early postnatal life and only during their prenatal life showed a statistically significant decrease (p<0.001) in the number of neurons in the CA3 of the hippocampus when compared to the offspring in the control and positive control group. No significant difference was observed between the untreated groups. Offspring from alcohol-fed rats were treated with extract of C. asiatica at a dose of 20ml/kg/day during their prenatal life (G3a), postnatal life (G3b), both pre and postnatal life (G3c). Offsprings in and showed a statistically significant increase in the neuronal number when compared to the offspring in untreated groups G2a (p<0.01 and  p<0.001). In comparison, treatment with C.asiatica only during their prenatal life (G3a) showed a slight increase, but it was not significant.

 

Offspring in G3a showed a statistically significant (p<0.05) decrease in the number of neurons in CA3 of the hippocampus when compared to the offspring in G3b and G3c. Offspring in G3a showed a statistically significant (p<0.001) decrease in the number of neurons when compared to the offsprings in control and the positive control. Offspring in G3b and G3c also had a decreased number of neurons than the offspring in the control and positive control group, but it was not significant. (Table 1).

 

 

Figure 3: Neuronal assay in the CA3 of hippocampus – Male offspring rats

G1a- offspring from the control group, G1b- offspring from the positive control group, G2a- offspring from the untreated group (2a), G2b- offspring from the untreated group (2b), G3a- offspring from treatment group (3a), G3a- offspring from treatment group (3b), G3a- offspring from the treatment group. 3c Kruskal-Wallis test (P˂0.001) was done, followed by Tukey's multiple comparison tests.

G1a Vs G2a, G2b and G3a *** p<0.001; G1b Vs G2a, G2b and G3a ### p<0.001; G2a Vs G3b $$ p<0.01 and G2a Vs G3c $$$ p<0.001; G2b Vs G3b ββ p<0.01 G2b Vs G3c βββ p<0.001; G3aVs G3b and G3c ¥ p<0.05.


 

Table 1: Hippocampal neuronal distribution in male and female rats:

Groups

DG (Mean ± SE)

CA3 (Mean ± SE)

G1a

Male

108±4.2

p-0.302

F-1.182

21±0.85

p-0.228

F-1.65

Female

113±7.07

25.5±11.47

G1b

Male

105±2.5

p-0.099

F-3.301

17±0.68

p-0.160

F-2.3

Female

91±3.34

20±1.35

G2a

Male

16.25±2.5

p-0.313

F-1.126

8±0.68

p-0.311

F-.852

Female

14±1.15

8.8±0.82

G2b

Male

15.16±1.27

p-0.319

F-1.1

8.5±0.42

p-0.372

F-.872

Female

19.83±1.81

8.88±0.65

G3a

Male

14.8±1.9

p-0.042

F-5.43

9.6±0.91

p-0.401

F-.769

Female

15.3±0.84

17±1.23

G3b

Male

62.7±2.62

p-0.851

F-0.037

17±1.23

p-1

F-0.12

Female

67.3±2.48

16±1.15

G3c

Male

53.16±5.5

p-0.063

F-4.37

19.5±0.71

p-0.746

F-.111

Female

73±2.01

16.16±0.6

 

 


DISCUSSION:

Dentate gyrus plays a major role in learning and memory. Their synaptic connections are the basis for the establishment of memory. Our previous study on pups born to alcohol-fed mother rats stated that treatment with Centella asiatica for 60 days following weaning, produced a statistically significant increase in the performance in cognitive tests.32 Other reports30 also have stated improved cognitive performance following the administration of C. asiatica extracts, when used at the higher dosage at 200mg/kg bodyweight for 14 days, as compared to our study, which spanned the whole life term of the animals, we used low dosage at 20mg/kg body weight. We found detectable cognitive benefits in the animals.

 

Basic synaptic transmission in the hippocampus is believed to be a trisynaptic circuit, which played an important role in the regulatory functions. Studies showed that interruption within this circuit causes behavioral changes in rodents.17,33 This circuit contains the granule cells in DG, pyramidal cells in CA3 and CA1 areas of the hippocampus. Granule cells in DG and pyramidal cells in CA receive information from Entorhinal Cortex (EC) through the perforant path. The DG neurons then synapse with the pyramidal cells in the CA3 area through the mossy fibers (one of the largest and influential synapses in the brain). Axons from CA3 project to CA1 region through Schaffer collaterals or associational commissural pathway. CA1 receives these axons either from the ipsilateral hippocampus or from the contralateral hippocampus. Axons from CA3 extend to the subiculum and on to the entorhinal cortex. This CA1-subiculum-EC form the principal output from the hippocampus.34 In the present study, we found decreased neuronal density, which suggested lesser synaptic connections in the hippocampus in alcohol-exposed animals. This finding was in line with the articles previously reported. Several studies showed that prenatal exposure to alcohol decreases the volume of DG, alters the dendritic arborization, neuritic outgrowth of granule and pyramidal cells, reduces the concentration of neurotrophic growth factors Brain-Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) as well as the stem cell proliferation of DG.33,34,35 It also decreases the apical and basilar dendritic spine densities in all CA regions of the hippocampus of rats.36 These appear to be reversed in C Asiatica treated groups.

 

Our previous studies have reported low pup quality following alcohol force-feeding of mother rats during pregnancy.7 However, when these animals were treated with C. asiatica water extracts, the cognitive parameters showed measurable improvements, suggesting a strong case for neuroprotective functions of C. asiatica.17 However, we did not find notable differences between gender, as evident from Table 1.

 

Studies done in animals showed that maternal alcohol consumption during gestation decreases the Long Term Potentiation (LTP), persistent increase in synaptic strength following high-frequency stimulation in the chemical synapse, in CA and DG regions of the hippocampus.37,38 Kaneko et al. (1993) studied the auditory event-related potentials (ERPS), the measured brain response to the specific sensory, motor, or cognitive event, in rats exposed to alcohol during their prenatal life. They observed long latencies of P1 and N1 ERP components in the hippocampus of these rats, suggesting the hippocampus's impaired functions due to the toxic actions of alcohol.

 

Researchers have found a relationship between structural damage in the hippocampus and cognitive dysfunctions in those offspring who were exposed to alcohol during their intrauterine life.39 In the present study, feeding the C.asiatica extracts in juvenile rats and maintaining the treatment for up to 60 days (Group 3c; Fig 2), we found a significant increase in the neuronal population untreated animals. This is a clear indication of the beneficial effects of C. asiatica as a natural herbal tonic, which can produce significant improvement in learning and memory. Studies showed that maternal alcohol consumption during pregnancy decreased spatial learning and memory in rats by affecting synaptogenesis; they showed poor performance in the Morris Water Maze test, place learning test, etc.40 We reported a significant improvement in the cognitive parameters in rat models treated with C. asiatica,17 which appears to be accomplished by the increased number of neurons and also increased synaptic connections in the Hippocampal region.

 

CONCLUSION:

Alcohol is teratogenic and produced harmful effects on the body. It affected fetal brain growth, as evidenced by the offspring's cognitive deficits born to the alcohol-fed female rats. In our study, there was a decrease in the number of neurons in the hippocampal region of rats born to such alcohol-treated mother rats. On long term treatment with C. asiatica extract, there was a significant increase in the neuronal population, suggesting the beneficial effect of C. asiatica as an effective neurotropic agent.

 

ACKNOWLEDGMENT:

We acknowledge the support from the Kasturba Medical College, Mangalore, for the Laboratory facility and the support to the research fellow.

 

FUNDING SOURCES:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 18.09.2020            Modified on 24.04.2021

Accepted on 12.08.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(3):1047-1052.

DOI: 10.52711/0974-360X.2022.00175