International Journal of Anatomical Sciences 2012, 3(2): 26-32

Research Paper

Bacopa  monniera  a  Potent  Neuroprotector  Against  Transient  Global

Cerebral Ischemia Induced Hippocampal Damage and Memory Function

Ramesh Kumar R, Kathiravan K, Muthusamy R.

Department of Anatomy, Dr.A.L.M. Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani campus Chennai 600 113, Tamil Nadu, India.

Key words: Global cerebral ischemia, Bacopa monniera, Radial arm maze

Abstract: Cerebral ischemia is the major complication associated with the vascular health, next to cardiovascular disease, resulting in irreversible damage to the brain tissues and permanent loss of specific brain functions. Hippocamous is one of the most vulnerable regions of brain for global cerebral ischemic insult with impaired memory. Bacopa monniera (BM) is a well known traditional herbal medicine extensively used as an adjuvant to enhance cognitive development, as well to treat various neurological disorders. The present study emphasis the effect of the prophylactic administration of BM against hippocampal damage and spatial learning induced by Transient Global Cerebral Ischemia (TGCI) by 4VO method in wistar albino rats. Earlier the rats were trained for spatial navigation task in an eight arm radial maze for 10 days and were simultaneously treated with the ethanolic extract of BM, at a dose of 100mg/kg body weight/ ml /day orally for 30 days and then the animals were subjected to TGCI for 15 minutes. After TGCI the rats were tested for the retention of spatial learning and memory in the radial arm maze and the reference memory error,  working  memory error  and  latency were  determined.  The  normal pyramidal cell density in the CA1 region of dorsal hippocampus was also counted. The outcome of this study suggests that BM could protect pyramidal cell damage of CA1 region of hippocampus and retention of hippocampal associated spatial learning and memory in the rats pretreated with BM extract, this finding confirms the neuroprotective role of BM against cerebral ischemia.

Transient global cerebral ischemia (TGCI) is a major consequence of cardiac arrest resulting in sudden circulatory block to complete brain. TGCI leads to selective damage of neurons in the vulnerable brain regions, especially CA1 region of hippocampus, few cortical neurons located in layer 3, 5 and 6 and few striatal neurons (Hartman et al, 2005; Kubo et al, 2009) and resulting in long term learning and memory deficits(  Farkas  et  al,  2001).  Among  the brain regions hippocampus and its subfields are more vulnerable to TGCI and an extensive neuronal death was found in the CA1 region of rodent hippocampus starting from 2 to 4 days after ischemia and reaching a maximal effect within 1–2 weeks dependending   on   the   duration   of   the ischemic insult (Bendel et al., 2005; Colbourne  et  al.,  1999;  Pulsinelli  et  al.,1982; Smith et al., 1984). The pronounced deletion  of the CA1 pyramidal  neurons  is associated with severe impairments of hippocampal-dependent  brain  functions, such as spatial learning and memory (Bendel et al., 2005; Block, 1999; Hartman et al.,2005).

Several experimental studies have been carried out to combat the deleterious effect of cerebral ischemia and to promote a better therapeutic module which can benefit the brain function by protecting the neurons against the ischemic damage. Natural products, especially medicinal plants, could be an ideal source to develop safe and effective agents for neuroprotection against cerebral ischemia (Kim, 2005). Bacopa monniera (BM) has been a major constituent of   traditional   Indian   medicine   and   its encrypts were seen in Charaka Samitha, a  medicinal book of 5th century A.D. The biological  effects  of  BM  are  also documented in various  other traditional as well as scientific literatures.   It is a rich source of antioxidant and has been reported to  reduce  oxidation  of  fats  in  the  blood stream  (Stough  et  al.,  2001).  It  has  been used for centuries in traditional medicinal practice to help benefit against epilepsy, memory capability, to increase concentration and  to  reduce stress-induced  anxiety,  It  is also listed as a nootropic, a drug that enhances cognitive ability (Singh and Dhawan, 1992; Dhawan and Singh 1996; Russo, et al, 2006).

This present study is aimed to understand the effect of BM on the TGCI induced learning impairment and memory disorder and the neurodegenerative consequences.

 Materials and Methods

Animals

Twelve healthy adult Male Wistar albino rats weighing about 200 – 250 gms were used for this study. They were maintained in an optimum environment of constant  temperature  (21◦C),  humidity and 12 hours of day and night cycle. Animals were  fed  with  standard  food  pellets  and water ad libitum. The experiments were conducted in accordance with the standard guidelines    of    the    Institutional    Animal Ethical Committee (IAEC).

Transient Global Cerebral Ischemic Model

 Forebrain  ischemia  was  performed  using the four-vessel occlusion method (4VO) i.e. Bilateral vertebral and common carotid artery, as previously described by Pulsinelli and Brierley (1979); Lee et al (2012).  Briefly the  animals  were anesthetized with Ketamine & Xylazine (80 mg and 10 mg/kg body weight, intraperitonealy). The surgical procedure consists of two steps,     Step 1. Includes electro-cauterization   of  the  bilateral vertebral arteries on day one, followed by Step 2. Bilateral common carotid artery occlusion   for   15   minutes   using   micro vascular clips (FST, USA) on next day. For sham group the above mentioned surgical procedures were performed, except bilateral common carotid artery occlusion. This study includes three experimental groups i.e. (i) Sham,  (ii)  TGCI and  (iii)  Pretreatment  of Bacopa monniera (BM) for 30 days followed by  TGCI.  After  surgical  procedures  rats were  maintained  for  a  week  under  proper post operative care.

 Bacopa monniera (BM) extract

Ethanolic extract of BM with 40% Bacoside A (The active compound responsible for its cognitive function) was procured  from  Bayer’s,  Bangalore,  India. BM extract was dissolved in distilled water and made as a suspension. BM suspension was administered to the animals for 30 days orally at a dose of 100 mg/Kg body weight before they were exposed to TGCI.

Radial arm maze Test

To assess the spatial learning and memory in rats the radial arm maze test was performed. The radial arm maze (RAM) consisted of an octagonal platform (26cm) with eight arms (42 x 12 cm) and    placed 60cm above the floor. Each animal was trained individually prior to the surgery, the training  module  started  with  three  baited arms (numbers 3, 5 and 7) and the remaining arms  were  un-baited.  The  trial  continued until all four baits had  been  consumed or until 5 minutes had elapsed. Reference Memory Errors (RME) i.e. Entering an arm that was not baited and Working Memory Errors (WME) i.e. Re-entering an arm containing food and latency were assessed after one week of regular training and post surgical assessment was made on day 1 & 7.

Histology

At the end of post operative experimental trials the rats were euthanized with Thiopentone sodium 80mg/Kg body weight   and   transcardially   perfused   with 4% paraformaldehyde in phosphate buffered saline. The animals were decapitated and brains were removed. The fore brain was processed and embedded in paraffin wax and the tissue blocks were sectioned in to 10 micron thick sections using a rotary microtome (Weswox, India). The sections obtained  were  stained  using  Cresyl  fast violet (CFV).   Density of normal neuronal population in the granular cells of the CA1 region within the 400µm2 of bilateral dorsal hippocampus was counted at defined coronal in blinded fashion with reticule incorporated eyepiece at a magnification of 200X  using  a  light  microscope.  A quantitative estimation of cell damage was made by direct visual counting of apparently normal neurons in the bilateral CA1 area of all   the   three   animal   groups   within   the reticule area. Cells showing dark cytoplasm and shrinkage were not counted.

Statistical analysis

All data were expressed as mean ± standard  error  mean  (SEM)  and  the statistical analysis of the results was performed by one-way analysis of variance (ANOVA) followed by turkey’s test using SPSS. P values ≤ 0.05 was considered significant.

Observations

The effect of pretreatment of BM on the transient Global cerebral ischemic rats was well documented in this study through spatial learning and memory behavior in RAM task and the histological assessments.

Radial arm maze (RAM) Test

In    this    study    the    hippocampal associated spatial learning and  memory of the rats was assessed in the radial arm maze. On day 1 after 4VO in the RAM task the group II rats showed a significant increase in the reference memory error (RME) i.e. 10.5 ± 0.86 (Mean ± SEM) than the group I (1.25  ± 0.25), similarly the group III rats also showed a significant increase (7.75 ± 0.85), when compared with group I. The group III rats  showed  moderate  decrease  in  RME when  compared  with  group  II,  but statistically non significant. On Day 7 after4VO and reperfusion the RME of group II (5.75 ± .025) was significant higher than group I (0.75 ± .25) and RME of group III (4.0± 0.40) was significantly less, when compared with group II (Fig 1a).

 Fig.  1a  Reference Memory Error  Assessment

Using Radial Arm Maze

The working memory error (WME) on day1 after 4VO and reperfusion was significantly higher in group II (2.75±0.25) and group III (1.75 ± 0.25) when compared to the group I rats, which performed without any errors. The group III rats showed a significant  decrease  in  WME  when compared with group II.   On day 7 after lesion the WME of group II (1.75±0.50) and group   III   (1.50±0.59)   was   significantly higher when compared to group I, which performed without any WME. The group III showed a marginal reduction in WME when compared to group II, But statistically non significant (Fig 1b).

Fig.  1b  Working  Memory  Error  Assessment

Using Radial Arm Maze

*p<0.05 compared to sham, #p<0.05 compared to lesion group.

The count of normal pyramidal neurons within the 400µm2  of defined CA1 region of bilateral hippocampus in the group I rats was 118.31± 1.46 (Mean ± SE). The cell count in group II (43.43 ± 1.33) and in the group III (72.37± 1.29) was significantly reduced when compared with group I rats. The   group   III   rats   showed   significant increase in the number  of CA1 pyramidal neurons,   when   compared   with   group   II (Fig.3).

Discussion

The  transient  global  cerebral ischemia by 4VO methods in Wistar albino rat have induced a progressive bilateral damage to the CA1 pyramidal neurons of the rat hippocampus and have significantly reduced the percentage of surviving pyramidal neurons (Pulsinelli et al., 1982). The outcome of this present study correlates with the above data resulting in permanent selective damage to the CA1 region of the hippocampus with retarded performance in the radial arm maze task.

Fig. 2 Histological appearance of hippocampus in animals of experimental groups

Cresyl fast violets stained brain sections, Fig 2a, 2b represents Group I (Sham), the arrow indicates the prominent surviving neurons. Fig 2c, d represents Group II (Lesion) arrow indicates the dead shrunken neurons with pyknotic nucleus. Fig 2e, f represents group III (BM treated) arrow represents few dead neurons. Py – pyramidal cell layer, D- Dentate gyrus.

Fig. 3 Number of surviving pyramidal neurons in stress, exitotoxicity, glial cells mediated inflammatory reactions, apoptotic cell death, etc (Mattson et al., 2000; Lo et al., 2003). The ethanolic extract of BM was found to inhibit calcium influx via voltage and receptor operated calcium channels of cell membrane in the intestinal smooth muscles.

The extent of the pyramidal cell damage by counting the surviving neurons in predetermined CA1 region hippocampus.  *p<0.05 compared to sham, #p<0.05 compared to lesion group.

BM a traditional medicinal plant, which was well known as a potent nervine tonic,  enhancing  cognitive  function     and well documented in the studies conducted in normal subjects both experimental and clinical  trials  (Singh  et  al.,  1997).  Apart from cognitive enhancing property BM’s potential on various disease conditions, especially neurological disorders were experimentally proved (Russo et al., 2003; Jyoti et al 2006) considering this versatile role of BM on brain functions especially on cognitive function, an effort was made through this study to understand whether the prophylactic administration of BM for 30 days prior to the induction of TGCI in wistar albino rat. The BM treated rats could retain the spatial memory and  could execute the RAM  task  with  less  error  and  reduced latency than the untreated rats.    The histological  observations  also  showed  that the density of surviving neurons in the CA1 region of BM treated rats was significantly higher than the lesion group. By this study it was ascertained that pretreatment of BM made tremendous contribution to protect the pyramidal cells from the adverse effect of TGCI and supported to retain the spatial memory in the  RAM task.

Guinea pig (Dar et al., 1999).   During ischemic and reperfusion phase, a significantly increased levels of several free- radical species are generated in brain, that degrade cell and capillary membranes have been postulated (Safar, et al, 1986). In the previous study, B. monniera has been documented to provide neuroprotection against cigarette smoke induced apoptosis (Anbarasi   et   al,   2006)   and   aluminium induced oxidative stress (Jyoti et al, 2007). Neuroprotective role of BM was also demonstrated in transient bilateral internal carotid artery occluded rats by administering variable dosages (Saraf, et al, 2010).

This neuroprotective role of BM in TGCI induced 4 vessel model of Wistar albino rat may be due to its rich antioxidant resource it posses (Stough et al., 2001; Bhattacharya, et al., 2000) might have controlled the free radical generated during cerebral ischemia and prevented neuronal death or its active compound might have evoked an effective resistance shield against various other mechanism which operates the ischemic cascade.

Conclusion

In conclusion, this study has given a viable   data   that   BM,   traditionally   well known drug for memory enhancement could also protect one of the most vulnerable population  of  brain  regions,  the hippocampus  against  the deleterious  effect of transient global cerebral ischemia and retained the hippocampus mediated memory functions. The core mechanism of BM which works against the ischemic cascade operated by TGCI was not completely understood through this study. So a future detailed investigation could explore the unknown potentiality of Bacopa monniera.