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ORIGINAL ARTICLE
Year : 2012  |  Volume : 2  |  Issue : 2  |  Page : 111-120

Neuroprotective effect of cow colostrum and tetramethylpyrazine against global cerebral ischemia reperfusion injury


Department of Pharmacology, Pune District Education Association's Seth Govind Raghunath Sable College of Pharmacy, Saswad, SNJB's Shriman Sureshdada Jain College of Pharmacy, Chandwad, Nashik, Maharashtra, India

Date of Submission01-Aug-2011
Date of Acceptance13-Oct-2011
Date of Web Publication9-May-2012

Correspondence Address:
Vaishali R Undale
Department of Pharmacology, PDEA's SGRS College of Pharmacy, Saswad, Maharashtra - 412 301
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.95947

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   Abstract 

Aim: Cerebral ischemia is the third leading cause of death in the western world. According to the WHO 15 million of the world's population suffer cerebral ischemia each year. The aim of the present study was to investigate the effect of tetramethylpyrazine (TMP) and cow colostrum in bilateral common carotid artery occlusion (BCCAO)-induced global ischemia-reperfusion injury. Cow colostrum is claimed to have antioxidant effects and TMP is popularly used in the treatment of cardiovascular diseases. Materials and Methods: Male Sprague-Dawley rats weighing 300350 g were treated with cow colostrum (125, 250, and 500 mg/kg) and TMP (12.5, 25, and 50 mg/kg) for 7 days after cerebral ischemia induced by BCCAO for 30 minute followed by reperfusion. Results: A significant decrease in body weights and marked weakness in the ischemic animals were observed. These effects were attenuated by cow colostrum (500 mg/kg) and TMP (25 and 50 mg/kg). There was significant increase in the level of malondialdehyde (MDA) and attenuation in the levels of reduced glutathione (GSH) and catalase in ischemic animals, indicating oxidative stress. Elevated levels of MDA and attenuated levels of GSH and catalase were compensated by treatment with cow colostrum and TMP dose dependently. Overall, the results of the study indicate that at doses of 25 mg/kg and 50 mg/kg TMP markedly improves cognition. The results also indicate that both TMP and cow colostrum have antioxidant activity. Conclusion: This study demonstrates the neuroprotective potential of cow colostrum and TMP. They appear to be promising drugs for the prophylaxis and treatment of global cerebral ischemia.

Keywords: Bilateral common carotid artery occlusion, cow colostrum, global cerebral ischemia, tetramethylpyrazine


How to cite this article:
Undale VR, Desai SS, Sangamnerkar SK, Upasani CD. Neuroprotective effect of cow colostrum and tetramethylpyrazine against global cerebral ischemia reperfusion injury. Int J Nutr Pharmacol Neurol Dis 2012;2:111-20

How to cite this URL:
Undale VR, Desai SS, Sangamnerkar SK, Upasani CD. Neuroprotective effect of cow colostrum and tetramethylpyrazine against global cerebral ischemia reperfusion injury. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2019 Sep 15];2:111-20. Available from: http://www.ijnpnd.com/text.asp?2012/2/2/111/95947


   Introduction Top


Cerebrovascular diseases include some of the most common and devastating disorders, e.g., ischemic stroke, hemorrhagic stroke, and cerebrovascular anomalies such as intracranial aneurysms, arteriovenous malformations (AVMs) etc. The incidence of cerebrovascular diseases increases with age, and the number of strokes is projected to double as the population of the elderly grows in numbers. [1] Stroke is estimated to be responsible for 9.5% of all deaths. In the United States more than 700000 new cases of strokes occur annually. In India, the occurrence of stroke is estimated to be 203 per 100000 population, which amounts to about 1 million cases annually. [2]

Cerebral ischemia is defined as reduction in blood flow to the brain that lasts longer than several seconds. It is the third leading cause of death in the western world. Clinically, it manifests as rapidly developing clinical signs of focal and at times global loss of cerebral function, with symptoms lasting more than 24h or leading to death and no apparent cause other than a vascular crisis. According to the WHO, 15 million of the world's population suffer from strokes each year. Of these, one-third do not survive and one-third remain disabled; only the remaining one-third recover sufficiently to lead satisfactory lives. [3]

Cerebral ischemia-hypoxia can be divided into three types: Syncope, global ischemia, and focal ischemia. Syncope is defined as transient loss of consciousness due to reduced cerebral blood flow (for example, due to cardiac arrhythmia, myocardial infarction, or hemorrhagic shock). If low cerebral blood flow persists for a longer duration, then infarction in the border zones between the major cerebral artery distributions may develop as global ischemia. Focal ischemia or infarction, on the other hand, is usually caused by thrombosis of the cerebral vessels themselves or by emboli from a proximal arterial source or the heart, leading to damage to the particular area of the brain. More severe instances of global hypoxia-ischemia cause widespread brain injury, and the constellation of cognitive sequelae that ensue is called hypoxic-ischemic encephalopathy. [1] Global ischemia leads to deficits in a number of neurological domains, leading to a broad spectrum of neuropsychiatric complications, including emotional, behavioral, and cognitive disorders. [4],[5]

Multiple pathways are involved in cerebral ischemic injury. It includes excitotoxicity; [6] ionic imbalance; mitochondrial dysfunctions; [7],[8] poly (ADP-ribose) polymerase over-activation; [9] inflammatory reaction; [10],[11] activation of mitogen-activated protein kinases; [8],[12] death-associated protein kinases; [13] apoptosis [14] and oxidative stress by generation of reactive oxidative species (ROS); and oxidization of various micro and macro components of the cellular system, such as lipid, proteins, and nucleotides, etc.

The present therapy for ischemic injury involves two distinct approaches, i.e., vascular reperfusion and neuroprotection. A wide variety of neuroprotective agents have been studied but they are found to be effective only in animal models of acute ischemic stroke and fail in clinical trials. Currently, there is no neuroprotective drug available in the market. At present, the only drug available for the treatment of cerebral ischemic injury is rt-PA (reverse transcriptase plasminogen activator), which acts through vascular reperfusion. [15]

The most adequate treatment for acute stroke in humans is immediate canalization of the occluded arteries. However, it has been found that reperfusion of the ischemic region may paradoxically exacerbate brain damage via reperfusion injury. [16],[17] Ischemia/reperfusion (I/R)-induced brain damage results from NMDA (N-methyl D-aspartate)-induced excitotoxicity and altered intracellular Ca 2+ homeostasis. [18] Secondary injury can result from ROS formation, inflammatory cytokine release, and failed energy metabolism. [19],[20] This neuronal cell death cascade continues for several days.

Cow colostrum (the first milk produced by cows after the birth of a calf) contains many biologically active substances with various functions; these include immunoglobulins, anti-bacterial peptides, and various growth substances. [21] Cow colostrum is known to be effective in improving or preventing lapses in diseases such as short bowel syndrome, inflammatory bowel disease, necrotizing enterocolitis, non-steroidal anti-inflammatory drugs (NSAIDs)-induced intestinal injury, liver disease, and infective diarrhea. [22] Tetramethylpyrazine (TMP) is a biologically active alkaloid that was isolated in 1957 from Ligusticum wallichii Franchat, which is used in traditional Chinese herbal medicine. Its pharmacological actions include vasodilatation, [23] inhibition of platelet aggregation, [24] and a strong antioxidant effect. [25] TMP has been verified to be a newtype antagonist of the calcium channel. [26] Synthetic TMP is available in the market and has been traditionally used in China for the treatment of ischemic cerebrovascular diseases [27] and cardiovascular diseases. [28] However, its exact mechanism of action is still unclear.

Cow colostrum and TMP have been screened for neuroprotective activity after focal cerebral ischemia-reperfusion injury. Indications are that the neuroprotective activity of TMP might be due to suppression of inflammatory reaction, reduction of neuronal apoptosis, and prevention of neuronal loss. [29] In rats, the neuroprotective activity of cow colostrum may be due to reduction of serum pro-inflammatory cytokines and suppression of brain infarct volume. [30] However, the possible effect of cow colostrum and TMP on global cerebral ischemia has not been studied either in vivo or in vitro.

Cow colostrum has recently received considerable attention as a dietary supplement (antioxidant). TMP has received marked attention as a potent traditional drug for cardiovascular diseases. Traditional medicines are currently drawing a lot of attention as they are believed to have less toxicity compared to synthetic medicines, and they are being studied to find out the scientific basis of their therapeutic actions. The aim of the present study is to investigate the effect of TMP and cow colostrum in global type of cerebral ischemia-reperfusion injury. The present study was designed to explore the mechanism of the neuroprotective action of cow colostrum and TMP activity after global cerebral ischemia and to examine whether there is a ground for clinical trials of such traditional medicines to assess their value for neuronal protection in human beings.


   Materials and Methods Top


The cow colostrum used in this study was a gift sample from Dhanwantari Distributers Pvt. Ltd. and the TMP was gifted by Mohanish Chemicals Pvt. Ltd.

Animals

Male Sprague-Dawley rats breed in animal house under conventional facilities. (certificate number of the animal breeder: 311/CPCSEA, dated 15 th December, 2000) weighing 300-350 g were used in this study. They were procured from the animal house facility of the Department of Pharmacology, PDEA's Seth Govind Raghunath Sable College of Pharmacy, Saswad, Maharashtra. The rats were maintained in a 12-hour dark/light cycle and had free access to standard rat feed and water. They were housed in polypropylene cages at room temperature throughout the study. The experimental protocol was approved by the Animal Ethics Committee of PDEA's Seth Govind Raghunath Sable College of Pharmacy, Saswad, Maharashtra (IAEC No. SGRS/IAEC/04/10-11).

Induction of ischemia

Induction of ischemia [31],[32],[33] was performed by BCCAO for 30 minutes, using bulldog clamps. After the 30-minute period, the clamps were removed and reflow was verified visually. The neck incision area was then sutured and antiseptic cream (Soframycin™ cream) was applied. The body temperature of the animals was maintained at about 37°C until recovery to prevent postischemic hypothermia. Sham-operated animals underwent the same surgical procedure, except that occlusion of the common carotid arteries was not done. Treatment was started from the second day. On the second and sixth days, the animals were assessed for behavioral parameters. On the seventh day, the body weights were recorded and the animals were then sacrificed for biochemical assessment.

Groups and treatment

Forty male Sprague-Dawley rats weighing 300-350 g were divided randomly into eight groups of five rats each. In all the groups except the control group (sham-operated group) ischemic-reperfusion injury was done by 30 minutes of BCCAO followed by reperfusion for 7 days. From the second day onwards the animals were given the test substances: Cow colostrum (125, 250, and 500 mg/kg) or TMP (12.5, 25, and 50 mg/kg) or vehicle. In all these groups the test substances were given per orally early in the morning for 7 days. After administration of the doses the animals were assessed for behavioral parameters. The detailed treatment schedule as as follows:

  • Group I: No ischemia; received normal saline (10 ml/kg) (sham-operated, control).
  • Group II: BCCAO for 30 minutes; received 1% solution of carboxy methyl cellulose (CMC) in normal saline saline (10 ml/kg) (vehicle control).
  • Group III: BCCAO for 30 min, followed by reperfusion for 7 days; received cow colostrum (125 mg/kg).
  • Group IV: BCCAO 30 min, followed by reperfusion for 7 days; received cow colostrum (250 mg/kg).
  • Group V: BCCAO for 30 min, followed by reperfusion for 7 days; received cow colostrum (500 mg/kg).
  • Group VI: BCCAO for 30 min, followed by reperfusion for 7 days; received TMP (12.5 mg/kg).
  • Group VII: BCCAO for 30 min, followed by reperfusion for 7 days; received TMP (25 mg/kg).
  • Group VIII: BCCAO for 30 min, followed by reperfusion for 7 days; received TMP (50 mg/kg).
Behavioral parameters

The animals were housed in a temperature-regulated room with a reversed 12 hour/12 hour light/dark cycle. Rats (n=5) underwent psychometric testing as follows:

Elevated plus-maze test for spatial memory

The Elevated plus-maze (EPM) test is based on the natural aversion of rodents to the open arms and high spaces. The animals prefer the enclosed arm and try to reach it as quickly as possible because they experience fear in open arms of the maze. Itoh et al., in 1990, demonstrated that the time taken by an animal to move from an open arm to an enclosed arm is significantly reduced if the animal had previous experience of entering the enclosed arm.

The EPM, consisting of two open arms (35 × 6 cm) and two enclosed arms (35 × 6 × 15 cm), was elevated to a height of 50 cm. Acquisition of memory was assessed by placing the animals individually at the end of either of the open arms, facing away from the central platform. The time taken by each animal to move from the open arm to either of the closed arms was recorded. This duration of time was called transfer latency (TL). If the animal did not enter into any of the enclosed arms within 600 seconds, it was gently pushed into any of the enclosed arms and the TL was assigned as 600 seconds. Later, the animal was allowed to explore the plus-maze for 5 minutes and then placed back in the home cage. The TL was then recorded on the second and seventh day after surgery. The TL measured on the first day served as a parameter for acquisition (learning) while the TL measured on the second and seventh days indicated retention (memory).

Biochemical tests

After assessment of behavioral parameters on the seventh day, the animal was weighed and then decapitated. Following decapitation, the brain was removed and washed in cooled 0.9% saline, kept on ice, and subsequently blotted on filter paper; it was then weighed and homogenized (10% w/v) in cold phosphate buffer (0.05M; pH 7.4). The homogenate was centrifuged at 1000g for 10 minutes at 4°C. The supernatant was separated and aliquots were used for biochemical estimations of malondialdehyde (MDA), reduced glutathione (GSH), and catalase. [34],[35],[36]

Determination of lipid peroxidation

The amount of MDA served as a measure of lipid peroxidation. It was determined by reaction with thiobarbituric acid (TBA) at 512 nm. Briefly, 0.5 ml of the above homogenate was incubated with 15% trichloroacetic acid (TCA), 0.375% TBA, and 5N HCl at 95°C for 15 minutes. The mixture was cooled and centrifuged, and the absorbance of the supernatant was measured at 512 nm against an appropriate blank. The amount of lipid peroxidation was determined by using the formula ε = 1.56 × 10 5 M/1 cm/1 and expressed as nanomoles of MDA formed/milligram of protein. [37]

Estimation of catalase

Catalase was estimated using the method developed by Aebi et al. [38] In this test, the breakdown of hydrogen peroxide (H 2 O 2 ) is measured at 240 nm. Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05M; pH 7.0), 1.0 ml hydrogen peroxide (0.019M), and 0.05 ml homogenate (10% w/v) in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of nanoMoles of H 2 O 2 consumed/minute/milligram of protein.

Estimation of GSH

GSH was determined by the method described by Moron et al. [39] Equal volumes of tissue homogenate (supernatant) and 20% TCA were mixed. The precipitated fraction was centrifuged. To 0.25 ml of the supernatant, 2 ml of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reagent was added. The final volume was made up to 3 ml with phosphate buffer. The color developed was read at 412 nm against a reagent blank. Different concentrations (10-50 mg) of standard glutathione was taken and processed as above to get a standard graph. The amount of GSH was expressed as micrograms of GSH per gram of wet tissue.

Statistical analysis

All the values are expressed as mean±SEM. The data of all the experiments was analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. In all the tests, the criterion for statistical significance was P/i>.05.


   Results Top


Effect on body weight

In the vehicle control group, after the 30 minutes of BCCAO followed by reperfusion, the average food intake decreased. Severe reduction in the body weight was observed in this group. On the first day all the groups showed reduction in food intake, but from the second day onwards the food intake was normalized. In group II animals (vehicle control) significant (P/i>.001) reduction in body weights was seen on the seventh day after the induction of ischemia. No significant change in body weights was observed in the animals of group III, IV, V, VI, VII, and VIII after induction of ischemia [Table 1].
Table 1: Effect of treatment on body weight

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Effect on spatial memory

No significant difference in initial transfer latency (ITL) was found in the animals treated with vehicle, cow colostrum, and TMP, compared to the sham-operated control group. After BCCAO followed by reperfusion, in group II (vehicle control) there was significant (P>.001) increase in retention transfer latency (RTL) on the second day, compared to the corresponding RTL of the sham-operated control group. The RTL was further increased on the seventh day. Animals treated with the test drugs in groups III, IV, V, VI, VII, and VIII showed dose-dependent attenuation in RTLs on the second and seventh day, as compared to the vehicle control group. On the seventh day, the RTLs of groups V and VIII decreased drastically and became nearly equal to the RTL of the sham-operated control group [Table 2]. The RTL was increased on the second day and then decreased sharply on the seventh day to become statistically equivalent to the ITL in the sham-operated control group, which indicates retention of memory. In the vehicle control group, the RTL was increased significantly (P>.001) on the second as well as the seventh days after BCCAO and reperfusion.
Table 2: Effect of treatment on spatial memory

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In the animals treated with cow colostrum, doses of 125 mg/kg and 250 mg/kg produced significant increases in RTL (P>.001) on the second as well as the seventh days. In contrast, with the dose of 500 mg/ kg the RTL increased significantly (P>.001) on the second day, but on the seventh day the RTL became equal to the ITL.

In the animals treated with TMP, the dose of 12.5 mg/ kg produced significant increase in RTL (P>.001) on the second day as well as the seventh day. However, with the doses of 25 mg/kg and 50 mg/kg the RTL on the second day increased very slightly and on the seventh day significantly decreased compared to ITL [Figure 1].
Figure 1: Effect of cow colostrum (125, 250, and 500 mg/kg) and tetramethylpyrazine (12.5, 25, and 50 mg/kg) on spatial memory after induction of ischemia. The values are expressed as mean±SEM (n=5). Data was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. *P>.05, **P>.01, ***P>.001, and ns (nonsignificant) as compared to ITL of the respective groups

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Effect on oxidative stress parameters

After BCCAO followed by reperfusion, in group II (vehicle control) there was significant (P>.001) increase in oxidative stress. This proves that 30 minutes of BCCAO followed by reperfusion caused ischemic damage, as indicated by increased MDA and decreased catalase and GSH activity in the brain as compared to group I (sham-operated control) animals.

In the groups treated with cow colostrum and TMP, dose-dependent restoration of GSH and catalase activity and attenuation of MDA was observed as compared to group I (sham-operated control). The changes in oxidative stress parameters were as follows:

Effect on lipid peroxidation (MDA) level

The MDA level was found to be elevated significantly (P>.001) after BCCAO followed by reperfusion in group II animals (vehicle control) compared with group I (sham-operated control). Dose-dependent and significant (P>.001) decreases were observed in animals treated with cow colostrum in doses of 125, 250, and 500 mg/kg, as compared to the vehicle control group. Similar results were observed for the animals treated with TMP in doses 12.5, 25, and 50 mg/kg [Table 3].
Table 3: Effect of treatment on lipid peroxidation (MDA) level

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Effect on catalase activity

Catalase activity was found to be decreased significantly (P>.001) after BCCAO followed by reperfusion in group II animals (vehicle control) compared with group I (sham-operated control). Dose-dependent and significant (P>.001) increases were observed in animals treated with cow colostrum in doses of 125, 250 and 500 mg/kg, compared to the vehicle control group. Similar results were observed for the animals treated with TMP in doses of 12.5, 25, and 50 mg/kg [Table 4].
Table 4: Effect of treatment on catalase activity

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Effect on reduced glutathione (GSH) level

GSH level was found to be decreased significantly (P>.001) after BCCAO followed by reperfusion in group II (vehicle control) compared with group I (sham-operated control). In the groups treated with cow colostrum, i.e., group III, IV, and V, the decrease in GSH was dose dependently recovered. The change was statistically significant (P>.001) compared to vehicle control group. Similarly, GSH level was dose-dependently and significantly increased (P>.001) in the animals treated with TMP in doses of 12.5, 25, and 50 mg/kg, as compared to the vehicle control group [Table 5].
Table 5: Effect of treatment on reduced glutathione (GSH) level


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   Discussion Top


In the present study, a preclinical model of BCCAO for 30 minutes followed by reperfusion for 7 days was used for global cerebral ischemia. Adult male rats were used because they have an intracranial circulation similar to that of humans. Infarction can be induced consistently and invariably and a large amount of neurochemical data is available on rats. [15] Female rats show a lesser susceptibility to postischemic brain injury in experimental models of stroke, and research suggests that this is likely due to circulating estrogen. [40]

BCCAO causes moderate and, most likely, permanent reduction of cortical and cerebral blood flow in diverse areas of the brain. [41] Previous research suggests that sensorimotor and neurological deficits take place in the animals following BCCAO and reperfusion injury. [42] BCCAO and reperfusion-induced damage in the hippocampal CA1 region is associated with behavioral alterations that are similar to the clinical symptoms observed in stroke patients. [43],[44] Hence, the model of BCCAO and reperfusion was used to induce global cerebral ischemia.

The relationship between stroke and functional impairment is complex. Approximately 80% of patients with acute stroke present with focal weakness or paralysis. Consistent with previous studies, the present study found that after BCCAO and reperfusion injury in the vehicle control group food intake decreased markedly and animals experienced severe weakness throughout the experiment period. In the other groups, treatment with TMP and cow colostrumrestored the food intake of animals to normal and appeared to protect against the weakness.

Dementia is frequently observed after stroke. About one-third of stroke survivors assessed during hospitalization were found to meet the criteria for dementia at 4-year follow-up assessment. [45] Global cerebral ischemia impairs memory function because of its influence on hippocampal neurons. [46]

In the present investigation, the elevated plus-maze task was used to evaluate memory impairment after BCCAO and reperfusion injury. To perform the retention task correctly, it is necessary for the rats to use two different memories. One of these is the 'reference memory' that includes extra-maze cues. The day-to-day performance of a task essentially depends on retrieval from this memory. The other is the 'working memory,' the memory of the arms of the maze that the rat has already visited during the trial. In the present study the pretrained rats, after BCCAO and reperfusion, were found to have impaired reference memory but unimpaired working memory. Thus, the results of the present study corroborate the hypothesis that global cerebral ischemia impaires memory function. [46] From the results of our study it can be observed that the sham-operated control group has an RTL on the second and seventh days that is the same as the ITL. However, in the vehicle control group, the RTL on the second and seventh days was significantly increased. This suggests that induction of ischemia impaired the reference memory in the vehicle control group. Treatment with cow colostrum and TMP recovered the impaired reference memory in a dose-dependent manner. With the dose of 500 mg/kg of cow colostrum and the doses of 25 mg/kg and 50 mg/kg of TMP there was marked recovery of memory. This suggests the therapeutic potential of TMP at very low doses. Cow colostrum also produced an beneficial effect on memory impairments, but at higher doses. This suggests that TMP and cow colostrum have an effect on such behavioral alterations after ischemic-reperfusion injury.

Oxidative insults, whether over-excitation or excessive release of glutamate or ATP caused by stroke, ischemia, or inflammation may initiate various signaling cascades, leading to apoptotic cell death and neuronal damage. [34],[47],[48] It has been well documented that cerebral hypoperfusion induces oxidative stress. [49],[50] Oxidative reperfusion injury could be one of the possible cellular cascades affecting all organs and tissue[es during ischemia. However, the mechanisms that trigger and modulate the vicious cellular cascades are still only partially understood. [51] Since reperfusion injury is associated with an imbalance of oxidant and antioxidant defense system, prevention of cerebral ischemia-induced oxidative damage could be a possible therapeutic strategy to manage the problem, [52] which is why we have studied the effect of treatments on the oxidative parameters.

Lipids are the macromolecules that are most susceptible to oxidative stress. The results of the current study suggest that lipid peroxides, measured in terms of MDA level, was significantly increased during ischemia-reperfusion. This finding is in accordance with that of Bromont et al. [53] who showed that reperfusion of the ischemic brain raises lipid peroxidation and nitrite levels. Treatment with cow colostrum and TMP ameliorated MDA levels in the brains of ischemic rats. In the present study, a dose-dependent decrease in MDA level after treatment was observed.

BCCAO followed by 7 days of reperfusion significantly decreased the level of catalase in the vehicle control group. Treatment with cow colostrum and TMP significantly restored the level of catalase dose dependently. Literature suggests that catalase and superoxide dismutase are enzymes present endogenously in the brain for antioxidant defense against free radical action.

The doses of 500 mg/kg of cow colostrum and 50 mg/ kg of TMP ameliorated the level of MDA and also restored the level of catalase after ischemic injury. This suggests that cow colostrum and TMP have strong antioxidant potential in ischemic injury.

Glutathione, which is considered as a central component of the antioxidant defenses of cells, acts both to directly detoxify reactive oxygen species and as a substrate for various peroxidases. Dysfunction of the glutathione system has been implicated in a number of neurodegenerative diseases [54],[55] and is a potential contributor to oxidative damage following temporary ischemia. The role of glutathione in cognitive function and synaptic plasticity processes as well as its involvement in neurotrophic and neurodegenerative events in rodents has been documented. [56] Redox ratio or disruption of the glutathione system is a hallmark of cerebral ischemic damage. In the present study, ischemia-reperfusion injury significantly decreased GSH in brain, indicating weak antioxidant defense. Treatment of cow colostrum and TMP markedly recovered the GSH level in brain dose dependently.

Overall, the results of the present study show that TMP in doses of 25 mg/kg and 50 mg/kg have marked effect on cognitive impairments induced by global cerebral ischemia. Cow colostrum also has an effect on cognitive impairments, but at high doses, i.e. 500 mg/kg. The results further show that cow colostrum and TMP have strong antioxidant potential as they increase catalase and GSH levels and decrease MDA levels.

The study shows that TMP produces marked improvements in memory impairments and demonstrates significant neuroprotective activity after ischemic-reperfusion injury. These actions may be due to its ability to improve cardiac and cerebral reperfusion, [57],[58] its property of calcium antagonism in vascular tissues, [59] its ROS scavenging activity, [60] and its ability to inhibit inflammation by modulating secretion of specific cytokines and nitric oxide-related pathways. [61],[62] Systemic administration of TMP also decreased the impairment of learning and improved memory performance and shows that TMP may have potent therapeutic efficacy Alzheimer disease. [63],[64]

Our study suggests that cow colostrum mainly acts by decreasing oxidative damage and may protect neurons against ischemic injury. This activity may due to its many constituents, such as immune factors and growth factors. It is reported that immune factors such as immunoglobulins, lactoferrins, and lymphokines have immunomodulatory activity; [65],[66] lactoferrin and cytokines have anti-inflammatory activity; [67] and lactalbumin has mood-elevating properties by increasing serotonin level and decreasing cortisol level. [68],[69] Also, growth factors such as epithelial growth factors have a role in the regulation of blood pressure and cholesterol levels, and platelet-derived growth factor helps in neuronal survival and regeneration. [70],[71] All these actions may contribute to cow colostrum's neuroprotective action in cerebral ischemia.

Taken together, the data from our study suggest that cow colostrum and TMP have antioxidant and free radical scavenging properties and the ability to modulate neurodegeneration-induced oxidative impairments in the brain. They can be effectively employed as neuroprotective adjuvants to reduce oxidative stress during ischemic injury in vivo. They can also be used in memory impairments. Both of these drugs are traditional medicines. Whereas there was good safety and tolerance with cow colostrum, TMP showed a narrow safety margin. Although, cow colostrum and TMP showed promising results in the BCCAO and reperfusion model of cerebral ischemia, further studies are required to unravel their mechanism of action. Once this is known, it may become possible to combine these two drugs to get a synergistic effect, which will allow lowering of the dose of TMP to avoid its toxicity.


   Conclusion Top


In conclusion, TMP offered significant neuroprotection in the BCCAO model of global cerebral ischemia in rats. The neuroprotective effect of TMP was associated with memory improvements. Cow colostrum also offered neuroprotective activity in the BCCAO model of global cerebral ischemia. Further studies on cow colostrum and TMP are warranted to unravel the molecular basis of their pharmacological efficacy.


   Acknowledgment Top


We are thankful to Dhanwantari Distributers Pvt. Ltd. for providing financial support and giving the gift sample of cow colostrum and to Mohanish Chemicals Pvt., Ltd. for providing the gift sample of tetramethylpyrazine. The authors are also thankful to PDEA's Seth Govind Raghunath Sable College of Pharmacy, Saswad, for providing the necessary facilities for this research work.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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