Sofiyan Saleem

Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats

6-Hydroxydopamine (6-OHDA) is one of the most widely used rat models for Parkinsons disease. There is ample evidence in the literature that 6-OHDA elicits its toxic manifestations through oxidant stress. In the present study, we evaluated the anti-parkinsonian effects of Withania somnifera extract, which has been reported to have potent anti-oxidant, anti-peroxidative and free radical quenching properties in various diseased conditions. Rats were pretreated with 100, 200 and 300 mg/kg b.w. of the W. somnifera extract orally for 3 weeks. On day 21, 2 mL of 6-OHDA (10 mg in 0.1% in ascorbic acidsaline) was infused into the right striatum while sham operated group received 2 mL of the vehicle. Three weeks after 6-OHDA injections, rats were tested for neurobehavioral activity and were killed 5 weeks after lesioning for the estimation of lipidperoxidation, reduced glutathione content, activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase, catecholamine content, dopaminergic D2 receptor binding and tyrosine hydroxylase expression. W. somnifera extract was found to reverse all the parameters significantly in a dose-dependent manner. Thus, the study demonstrates that the extract of W. somnifera may be helpful in protecting the neuronal injury in Parkinsons disease.

Neuroprotection by crocetin in a hemi-parkinsonian rat model

Reactive oxygen species (ROS) are implicated as the leading biochemical cause of neuronal death in various neurologic disorders, including Parkinsons disease. In the present study, neuromodulatory effects of crocetin (active constituent of Crocus sativus) in a 6-hydroxydopamine (6-OHDA) model of rat Parkinsonism were investigated. Male Wistar rats were pre-treated with crocetin (25, 50 and 75 microg/kg body weight) for 7 days and subjected to unilateral intrastriatal injection of 10 microg 6-OHDA on day 8. Locomotion and rotation were observed on day 23 post-injection, and after 4 weeks, striatum and substantia nigra were dissected out by decapitation. Activity of antioxidant enzymes and content of dopamine (DA) and its metabolites were estimated in striatum, whereas glutathione (GSH) content and thiobarbituric acid reactive substance (TBARS) were evaluated in substantia nigra. Levels of GSH and dopamine were protected, while TBARS content was attenuated in crocetin-treated groups. The activity of antioxidant enzymes was decreased in the lesion group, but protected in the crocetin-treated groups. These findings were supported by the histopathologic findings in the substantia nigra that showed that crocetin protects neurons from deleterious effects of 6-OHDA. This study revealed that crocetin, which is an important ingredient of diet in India and also used in various systems of indigenous medicine, is helpful in preventing Parkinsonism and has therapeutic potential in combating this devastating neurologic disorder.

Ginkgo Biloba Extract Neuroprotective Action Is Dependent on Heme Oxygenase 1 in Ischemic Reperfusion Brain Injury

Background and Purpose— Ginkgo biloba extracts are now prescribed in several countries for their reported health benefits, particularly for medicinal properties in the brain. The standardized Ginkgo extract, EGb761, has been reported to protect neurons against oxidative stress, but the underlying mechanisms are not fully understood. Methods— To characterize the oral consumption of EGb761 in transient ischemia, we performed the middle cerebral artery occlusion (MCAO) filament model in wild-type and heme oxygenase 1 (HO-1) knockouts. Mice were pretreated for 7 days before the transient occlusion or posttreated acutely during reperfusion; then neurobehavioral scores and infarct volumes were assessed. Furthermore, primary cortical neuronal cultures were used to investigate the contribution of the antioxidant enzyme HO-1 in the EGb761-associated cytoprotection. Results— Mice that were pretreated with EGb761 had 50.9±5.6% less neurological dysfunction and 48.2±5.3% smaller infarct volumes than vehicle-treated mice; this effect was abolished in HO-1 knockouts. In addition to the prophylactic properties of EGb761, acute posttreatment 5 minutes and 4.5 hours after reperfusion also led to significant reduction in infarct size (P<0.01). After our previous demonstration that EGb761 significantly induced HO-1 levels in a dose- and time-dependent manner in neuronal cultures, here we revealed that this de novo HO-1 induction was required for neuroprotection against free radical damage and excitotoxicity as it was significantly attenuated by the enzyme inhibitor. Conclusion— These results demonstrate that EGb761 could be used as a preventive or therapeutic agent in cerebral ischemia and suggest that HO-1 contributes, at least in part, to EGb761 neuroprotection.

Ginkgo biloba affords dose‐dependent protection against 6‐hydroxydopamine‐induced parkinsonism in rats: neurobehavioural, neurochemical and immunohistochemical evidences

Ginkgo biloba extract (EGb), a potent antioxidant and monoamine oxidase B (MAO‐B) inhibitor, was evaluated for its anti‐parkinsonian effects in a 6‐hydroxydopamine (6‐OHDA) rat model of the disease. Rats were treated with 50, 100, and 150 mg/kg EGb for 3 weeks. On day 21, 2 µL 6‐OHDA (10 µg in 0.1% ascorbic acid saline) was injected into the right striatum, while the sham‐operated group received 2 µL of vehicle. Three weeks after 6‐OHDA injection, rats were tested for rotational behaviour, locomotor activity, and muscular coordination. After 6 weeks, they were killed to estimate the generation of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) content, to measure activities of glutathione‐S‐transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD), and to quantify catecholamines, dopamine (DA) D2 receptor binding, and tyrosine hydroxylase‐immunoreactive (TH‐IR) fibre density. The increase in drug‐induced rotations and deficits in locomotor activity and muscular coordination due to 6‐OHDA injections were significantly and dose‐dependently restored by EGb. The lesion was followed by an increased generation of TBARS and significant depletion of GSH content in substantia nigra, which was gradually restored with EGb treatment. EGb also dose‐dependently restored the activities of glutathione‐dependent enzymes, catalase, and SOD in striatum, which had reduced significantly by lesioning. A significant decrease in the level of DA and its metabolites and an increase in the number of dopaminergic D2 receptors in striatum were observed after 6‐OHDA injection, both of which were significantly recovered following EGb treatment. Finally, all of these results were exhibited by an increase in the density of TH‐IR fibers in the ipsilateral substantia nigra of the lesioned group following treatment with EGb; the lesioning had induced almost a complete loss of TH‐IR fibers. Considering our behavioural studies, biochemical analysis, and immunohistochemical observation, we conclude that EGb can be used as a therapeutic approach to check the neuronal loss following parkinsonism.

Histone deacetylase inhibition activates transcription factor Nrf2 and protects against cerebral ischemic damage.

Interest in histone deacetylase (HDAC)-based therapeutics as a potential treatment for stroke has grown dramatically. The neuroprotection of HDAC inhibition may involve multiple mechanisms, including modulation of transcription factor acetylation independent of histones. The transcription factor Nrf2 has been shown to be protective in stroke as a key regulator of antioxidant-responsive genes. Here, we hypothesized that HDAC inhibition might provide neuroprotection against mouse cerebral ischemia by activating the Nrf2 pathway. We determined that the classic HDAC inhibitor trichostatin A increased neuronal cell viability after oxygen-glucose deprivation (from an OD value of 0.10±0.01 to 0.25±0.08) and reduced infarct volume in wild-type mice with stroke (from 49.1±3.8 to 21.3±4.6%). In vitro studies showed that HDAC inhibition reduced Nrf2 suppressor Keap1 expression, induced Keap1/Nrf2 dissociation, Nrf2 nuclear translocation, and Nrf2 binding to antioxidant response elements in heme oxygenase 1 (HO1), and caused HO1 transcription. Furthermore, we demonstrated that HDAC inhibition upregulated proteins downstream of Nrf2, including HO1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit in neuron cultures and brain tissue. Finally, unlike wild-type mice, Nrf2-deficient mice were not protected by pharmacologic inhibition of HDAC after cerebral ischemia. Our studies suggest that activation of Nrf2 might be an important mechanism by which HDAC inhibition provides neuroprotection.

Heme–Hemopexin Complex Attenuates Neuronal Cell Death and Stroke Damage:

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the prooxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme-HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and prooxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme-HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.

Effects of EP1 receptor on cerebral blood flow in the middle cerebral artery occlusion model of stroke in mice.

The lipid mediator prostaglandin E2 (PGE2) exhibits diverse biologic activity in a variety of tissues. Four PGE2 receptor subtypes (EP1–4) are involved in various physiologic and pathophysiologic conditions, but differ in tissue distribution, ligand‐binding affinity, and coupling to intracellular signaling pathways. To characterize the role of the EP1 receptor, physiologic parameters (mean arterial blood pressure, pH, blood gases PaO2 and PaCO2, and body temperature), cerebral blood flow (CBF), and neuronal cell death were studied in a middle cerebral artery occlusion model of ischemic stroke in wild‐type (WT) and EP1 knockout (EP1−/−) mice. The right middle cerebral artery was occluded for 60 min, and absolute CBF was measured by [14C] iodoantipyrine autoradiography. The effect of EP1 receptor on oxidative stress in neuronal cultures was investigated. Although no differences were observed in the physiologic parameters, CBF was significantly (P < 0.01) higher in EP1−/− mice than in WT mice, suggesting a role for this receptor in physiologic and pathophysiologic control of vascular tone. Similarly, neuronal cultures derived from EP1−/− mice were more resistant (90.6 ± 5.8% viability) to tert‐butyl hydroperoxide‐induced oxidative stress than neurons from WT mice (39.6 ± 17.2% viability). The EP1 receptor antagonist SC‐51089 and calcium channel blocker verapamil each attenuated the neuronal cell death induced by PGE2. Thus, the prostanoid EP1 receptor plays a significant role in regulating CBF and neuronal cell death. These findings suggest that pharmacologic modulation of the EP1 receptor might be a means to improve CBF and neuronal survival during ischemic stroke.

PGD2 DP1 receptor protects brain from ischemia-reperfusion injury

Prostaglandin D2 is the most abundant prostaglandin in the brain. It has long been described as a modulator of the neuroinflammatory process, but little is known regarding the role of its Gαs‐coupled receptor, DP1. Therefore, in this study, the effect of the DP1 receptor on the outcome of cerebral ischemia in wildtype (WT) and DP1 knockout (DP1–/–) C57Bl/6 mice was investigated. Ischemia‐reperfusion injury was produced by a 90‐min occlusion of the right middle cerebral artery followed by a 4‐day reperfusion. Infarct size was 49.0 ± 11.0% larger in DP1–/– mice (n = 11; P < 0.01) than in WT mice (n = 9 per group). However, no differences were detected in the relative cerebral blood flow (CBF) or any of the physiological parameters measured (n = 5 per group) or in the large blood vessel anatomy (n = 3 per group). To further address whether the DP1 protective role in the brain could be extended to neurons, mouse primary corticostriatal neuronal cultures were exposed to the DP1‐selective agonist, BW245C, which provided dose‐dependent protection against excitotoxicity induced by glutamate. Protection was significant at a dose as low as 0.05 µm. The results indicate that the DP1 receptor is neuroprotective in both in vivo and in vitro paradigms. Development of drugs to stimulate the DP1 receptor in brain could provide a new therapeutic strategy against cerebral ischemia and potentially other neurological conditions.

Effects of Pycnogenol and vitamin E on cognitive deficits and oxidative damage induced by intracerebroventricular streptozotocin in rats.

Oxidative stress plays a crucial role in the progression of cognitive decline in Alzheimers disease (AD). Considerable attention has been focused on increasing the internal antioxidant defenses in response to AD. This study was designed to examine and compare the pretreatment effects of Pycnogenol (PYC) and vitamin E (Vit E) on cognitive deficits and oxidative damage in the hippocampus and cerebral cortex of intracerebroventricular streptozotocin (ICV-STZ)-infused rats. Rats pretreated with PYC (10 mg/kg), Vit E (100 mg/kg), and vehicle (intraperitoneal; once daily for 3 weeks) were bilaterally injected with ICV-STZ (3 mg/kg), whereas sham rats received the same volume of vehicle. After 2 weeks of ICV-STZ infusion, rats were tested for cognitive performance using passive avoidance and water maze tasks, and then killed for biochemical assays. ICV-STZ induced significant declines in cognitive performance and choline acetyltransferase activity in the hippocampus, which were significantly attenuated with PYC and Vit E. Pretreatment with PYC and Vit E produced a significantly enhanced glutathione level and Na+/K+-ATPase activity and decreased thiobarbituric acid reactive substances and protein carbonyl. These findings suggest that PYC and Vit E may provide a promising approach for the treatment of oxidative stress-related neurodegeneration in conditions such as AD.

1-HydroxyPGE1 reduces infarction volume in mouse transient cerebral ischemia

Differential neurological outcomes due to prostaglandin E2 activating G‐protein‐coupled prostaglandin E (EP) receptors have been observed. Here, we investigated the action of the EP4/EP3 agonist 1‐hydroxyPGE1 (1‐OHPGE1) in modulating transient ischemic brain damage. C57BL/6 mice were pretreated 50 min before transient occlusion of the middle cerebral artery with an intraventricular injection of 1‐OHPGE1 (0.1, 0.2, 2.0 nmol/0.2 µL). Brain damage 4 days after reperfusion, as estimated by infarct volume, was significantly reduced by more than 19% with 1‐OHPGE1 in the two higher‐dose groups (P < 0.05). To further address whether protection also was extended to neurons, primary mouse cultured neuronal cells were exposed to N‐methyl‐d‐aspartate. Co‐treatment with 1‐OHPGE1 resulted in significant neuroprotection (P < 0.05). To better understand potential mechanisms of action and to test whether changes in cyclic adenosine monophosphate (cAMP) levels and downstream signaling would be neuroprotective, we measured cAMP levels in primary neuronal cells. Brief exposure to 1‐OHPGE1 increased cAMP levels more than twofold and increased the phosphorylation of extracellular‐regulated kinases at positions Thr‐202/Tyr‐204. In a separate cohort of animals, 1‐OHPGE1 at all doses tested produced no significant effect on the physiological parameters of core body temperature, mean arterial pressure and relative cerebral blood flow observed following drug treatment. Together, these results suggest that modulation of PGE2 receptors that increase cAMP levels and activate extracellular‐regulated kinases 1/2 caused by treatment with 1‐OHPGE1 can be protective against neuronal injury induced by focal ischemia.