Loss of AMPK potentiates inflammation by activating the inflammasome after traumatic brain injury in mice.

Traumatic brain injury (TBI) is a significant public health concern characterized by a complex cascade of cellular events. TBI induces adenosine monophosphate-activated protein kinase (AMPK) dysfunction impairs energy balance activates inflammatory cytokines and leads to neuronal damage. AMPK is a key regulator of cellular energy homeostasis during inflammatory responses. Recent research has revealed its key role in modulating the inflammatory process in TBI. Following TBI the activation of AMPK can influence various important pathways and mechanisms including metabolic pathways and inflammatory signaling. Our study investigated the effects of post-TBI loss of AMPK function on functional outcomes inflammasome activation, and inflammatory cytokine production. Male C57BL/6 adult wild-type (WT) and AMPK knockout (AMPK-KO) mice were subjected to a controlled cortical impact (CCI) model of TBI or sham surgery. The mice were tested for behavioral impairment at 24 h post-TBI thereafter, mice were anesthetized, and their brains were quickly removed for histological and biochemical evaluation. In vitro we investigated inflammasome activation in mixed glial cells stimulated with lipopolysaccharides+ Interferon-gamma (LI) (0.1 µg/20 ng/ml LPS/IFNg) for 6 h to induce an inflammatory response. Estimating the nucleotide-binding domain, leucine-rich-containing family pyrin domain containing western blotting ELISA and qRT-PCR performed 3 (NLRP3) inflammasome activation and cytokine production. Our findings suggest that TBI leads to reduced AMPK phosphorylation in WT mice and that the loss of AMPK correlates with worsened behavioral deficits at 24 h post-TBI in AMPK-KO mice as compared to WT mice. Moreover compared with the WT mice AMPK-KO mice exhibit exacerbated NLRP3 inflammasome activation and increased expression of proinflammatory mediators such as IL-1b IL-6 TNF-a iNOS and Cox 2. These results align with the in vitro studies using brain glial cells under inflammatory conditions, demonstrating greater activation of inflammasome components in AMPK-KO mice than in WT mice. Our results highlighted the critical role of AMPK in TBI outcomes. We found that the absence of AMPK worsens behavioral deficits and heightens inflammasome-mediated inflammation thereby exacerbating brain injury after TBI. Restoring AMPK activity after TBI could be a promising therapeutic approach for alleviating TBI-related damage.

Distinctive effect of anesthetics on the effect of limb remote ischemic postconditioning following ischemic stroke.

Limb remote ischemic postconditioning (LRIP) has been reported as an effective method to reduce the induced experimental stroke damage after ischemic reperfusion (IR) injury. Studies suggest that anesthetics used during induction of ischemic stroke can reduce IR injury, which could affect the actual mechanisms of neuroprotection by LRIP. This study focuses on the comparative effects of anesthetics such as isoflurane and ketamine-xylazine on ischemic injury when used during LRIP. Adult C57BL/6 mice were anesthetized by isoflurane or halothane, and transient middle cerebral artery occlusion (MCAO) was induced through insertion of the filament. Under isoflurane or ketamine-xylazine anesthesia, LRIP was performed after 90 min of reperfusion by carrying out three cycles of 5 min ischemia/5 min reperfusion of the bilateral hind limbs for one session per day for a total of 3 days. Results showed that the use of different anesthetics-isoflurane or ketamine-xylazine-during LRIP had no effects on body weight. However, LRIP was able to improve neurological function as observed by the neurological deficit score in ischemic mice. Interestingly, the neurological deficit in the group where ketamine-xylazine was used was better than the group where isoflurane was used during LRIP. Furthermore, the LRIP was able to prolong the period of the ischemic mice on the rotarod and this effect was more significant in the groups where ketamine-xylazine was used during LRIP. Moreover, LRIP significantly attenuated the infarction volume; however, this effect was independent of the anesthetic used during LRIP. From these results, we conclude that ischemic mice that were subjected to LRIP under ketamine-xylazine anesthesia had better neurological deficit outcomes after stroke.

Role of the L-PGDS-PGD2-DP1 receptor axis in sleep regulation and neurologic outcomes.

To meet the new challenges of modern lifestyles, we often compromise a good night's sleep. In preclinical models as well as in humans, a chronic lack of sleep is reported to be among the leading causes of various physiologic, psychologic, and neurocognitive deficits. Thus far, various endogenous mediators have been implicated in inter-regulatory networks that collectively influence the sleep-wake cycle. One such mediator is the lipocalin-type prostaglandin D2 synthase (L-PGDS)-Prostaglandin D2 (PGD2)-DP1 receptor (L-PGDS-PGD2-DP1R) axis. Findings in preclinical models confirm that DP1R are predominantly expressed in the sleep-regulating centers. This finding led to the discovery that the L-PGDS-PGD2-DP1R axis is involved in sleep regulation. Furthermore, we showed that the L-PGDS-PGD2-DP1R axis is beneficial in protecting the brain from ischemic stroke. Protein sequence homology was also performed, and it was found that L-PGDS and DP1R share a high degree of homology between humans and rodents. Based on the preclinical and clinical data thus far pertaining to the role of the L-PGDS-PGD2-DP1R axis in sleep regulation and neurologic conditions, there is optimism that this axis may have a high translational potential in human therapeutics. Therefore, here the focus is to review the regulation of the homeostatic component of the sleep process with a special focus on the L-PGDS-PGD2-DP1R axis and the consequences of sleep deprivation on health outcomes. Furthermore, we discuss whether the pharmacological regulation of this axis could represent a tool to prevent sleep disturbances and potentially improve outcomes, especially in patients with acute brain injuries.

Unique Properties Associated with the Brain Penetrant Iron Chelator HBED Reveal Remarkable Beneficial Effects after Brain Trauma.

Iron is postulated to contribute to secondary injury after brain trauma through various pathways including oxidative stress and inflammation. Therefore, one goal is to limit iron toxicity by either directly limiting iron activity, or limiting the secondary cascade mediated by iron, therefore rescuing the brain from damage after trauma. The N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid monohydrochloride (HBED) is a unique iron chelator that has the ability to cross the intact blood-brain barrier; it has a higher affinity to iron, and it has a longer half-life than most commonly used chelators. A controlled-cortical impact model of traumatic brain injury (TBI) was induced in mice. Mice were subcutaneously injected with HBED immediately after TBI, then at 12 h after, followed by a twice-a-day regimen until an end-point of 3 days. Neurobehavioral tests were performed daily. Cortical injury volume, hemispheric enlargement, and hippocampal swelling were quantified. Perls' iron immunostaining along with markers of gliosis, oxidative stress, and aquaporin (AQP) 4 were also performed. Data revealed that HBED treatment significantly decreases motor deficits and improves recovery after TBI. It also reduces cortical injury volume by 36.6 ± 6.8% (p < 0.001), hippocampal swelling by 23.4 ± 3.8% (p < 0.05), and total hemispheric volume by 13.3 ± 2.7% (p < 0.01). These effects are related to a reduction in microgliosis and oxidiative stress markers in the impacted corpus callosum area by 39.8 ± 7.3%, and by 80.5 ± 0.8% (p < 0.05), respectively. AQP4 staining is also attenuated in the hippocampus of HBED-treated mice. Therefore, our results suggest that HBED should be considered as a therapeutic tool to facilitate the recovery process following brain trauma.

Efficacy of Laropiprant in Minimizing Brain Injury Following Experimental Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most devastating and disabling forms of stroke, yet effective treatments are still lacking. Prostaglandins and their receptors have been implicated in playing vital roles in ICH outcomes. Recently, laropiprant, a DP1 receptor antagonist, has been used in combination with niacin to abolish the prostaglandin D2-(PGD2)-induced flushing. Here, we test the hypothesis that laropiprant limits bleeding and rescues the brain from ICH. Wildtype (WT) and DP1-/- mice were subjected ICH and neurologic deficits and hemorrhagic lesion outcomes were evaluated at 72 hours after the ICH. To test the therapeutic potential of laropiprant, WT mice subjected to ICH were treated with laropiprant at 1 hour after the ICH. The putative effect of laropiprant on limiting hematoma expansion was tested by an in vivo tail bleeding cessation method and an ex vivo coagulation method. Finally, the roles of laropiprant on gliosis and iron accumulation were also investigated. A significant decrease in the injury volume was observed in DP1-/- as well as laropiprant-treated WT mice. The tail bleeding time was significantly lower in laropiprant group as compared with the vehicle group. Significantly lower Iba-1 and Perls' iron staining in DP1-/- and laropiprant-treated WT groups were observed. Altogether, the data suggest that laropiprant treatment post-ICH attenuates brain damage by targeting primary as well as secondary injuries.

Protective Role of Arginase II in Cerebral Ischemia and Excitotoxicity.

BACKGROUND: Arginase (Arg), one of the enzymes involved in the urea cycle, provides an essential route for the disposal of excess nitrogen resulting from amino acid and nucleotide metabolism. Two reported subtypes of Arg (ArgI and II) compete with nitric oxide synthase (NOS) to use L-arginine as a substrate, and subsequently regulate NOS activity. It has been reported that Arg has significant effects on circulation that suggest the potential role of this enzyme in regulating vascular function. However, the role of Arg following brain damage has not been elucidated. In this study, we hypothesize that the deletion of ArgII will lead to aggravated brain injury following cerebral ischemia and excitotoxicity. METHODS AND FINDINGS: To test our hypothesis, male C57BL/6 wildtype (WT) and ArgII-/- mice were subjected to permanent distal middle cerebral artery occlusion and survived for 7 d. Cerebral blood flow (CBF) data revealed a statistically non-significant decrease in CBF in ArgII-/- mice. However, ArgII-/- mice had significantly higher neurologic deficit scores and brain infarctions. The hypothesis was further tested in a more specific N-methyl-D-aspartate (NMDA)-induced acute excitotoxic model. WT and ArgII-/- mice were given a single intrastriatal injection of 15 nmol NMDA. Forty-eight hours later, the excitotoxic brain damage was significantly worse in ArgII-/- mice. The data from both models confirm the neuroprotective effect of ArgII. CONCLUSION: Targeting ArgII could be considered an integrative part of a multi-modal approach to fight acute brain damage excitotoxicity, ischemic brain injury, and other forms of brain trauma.

Considerations for the Optimization of Induced White Matter Injury Preclinical Models.

White matter (WM) injury in relation to acute neurologic conditions, especially stroke, has remained obscure until recently. Current advances in imaging technologies in the field of stroke have confirmed that WM injury plays an important role in the prognosis of stroke and suggest that WM protection is essential for functional recovery and post-stroke rehabilitation. However, due to the lack of a reproducible animal model of WM injury, the pathophysiology and mechanisms of this injury are not well studied. Moreover, producing selective WM injury in animals, especially in rodents, has proven to be challenging. Problems associated with inducing selective WM ischemic injury in the rodent derive from differences in the architecture of the brain, most particularly, the ratio of WM to gray matter in rodents compared to humans, the agents used to induce the injury, and the location of the injury. Aging, gender differences, and comorbidities further add to this complexity. This review provides a brief account of the techniques commonly used to induce general WM injury in animal models (stroke and non-stroke related) and highlights relevance, optimization issues, and translational potentials associated with this particular form of injury.

Validity of Laser Doppler Flowmetry in Predicting Outcome in Murine Intraluminal Middle Cerebral Artery Occlusion Stroke.

BACKGROUND: Laser Doppler flowmetry (LDF) can reliably reflect brain perfusion in experimental stroke by monitoring both the degree and the duration of relative regional cerebral blood flow (rCBF). Variation in rCBF was continuously monitored in 68 mice undergoing middle cerebral artery occlusion (MCAO) and 25 mice undergoing sham-operation and documented as LDF (%). Transcranial LDF changes in the territory of right middle cerebral artery during MCAO procedure were correlated with corrected infarct volume (CIV) and neurological deficit score (NDS). METHODS: Ninety-three C57BL/6 mice (Harlan Laboratories, Indianapolis, IN) between 9 and 11 weeks old were randomly selected and assigned to either MCAO for 45 minutes (n = 68) or sham group (n = 25). Ischemia was induced using the transient intraluminal filament model of MCAO based on Koizumi's method and transcranial LDF was used to measure CBF during the procedure. Neurological deficits were measured at 2 and 23 hours after MCA reperfusion with NDS and 2% triphenyltetrazolium chloride (TTC) staining of carefully dissected brains was performed at 23 hours after reperfusion to determine infarct area. RESULTS: After common carotid artery occlusion (CCAO), there was a negative association between LDF drop from base line and NDS at 2 hours (r = -0.43, P = 0.038) and 23 hours (r = -0.61, P = 0.003). Also, a negative correlation was noted between MCA reperfusion LDF and NDS at 23 hours (r = -0.53, P = 0.001). Moreover, post-MCA reperfusion LDF had a positive association with initial CCAO LDF (r = 0.761, P = 0.000) and MCA occlusion LDF (r = 0.31, P = 0.036) in predicting neurological outcome. NDS at 23 hours corresponded well with the infarct volume (r = 0.31, P = 0.005). CONCLUSIONS: Greater augmentation of rCBF after MCA reperfusion was associated with improved neurological deficit scoring. Interestingly, greater reduction of regional cerebral blood flow after CCAO was also associated with improved neurological outcomes. The favorable neurological outcome is possibly due to interplay of factors such as vascular reserve, collaterals, and autoregulation mechanisms. We propose LDF changes as an additional noninvasive prognosticator of stroke outcome in the setting of experimental brain ischemia.

Role of PGE2 EP1 receptor in intracerebral hemorrhage-induced brain injury.

Prostaglandin E2 (PGE2) has been described to exert beneficial and detrimental effects in various neurologic disorders. These conflicting roles of PGE2 could be attributed to its diverse receptor subtypes, EP1-EP4. At present, the precise role of EP1 in intracerebral hemorrhage (ICH) is unknown. Therefore, to elucidate its possible role in ICH, intrastriatal injection of collagenase was given in randomized groups of adult male wildtype (WT) and EP1 receptor knockout (EP1⁻/⁻)C57BL/6 mice. Functional outcomes including neurologic deficits, rotarod performance, open field activity, and adhesive removal performance were evaluated at 24, 48, and 72 h post-ICH. Lesion volume, cell survival and death, were assessed using Cresyl Violet, and Fluoro-Jade staining, respectively. Microglial activation and phagocytosis were estimated using Iba1 immunoreactivity and fluorescently-labeled microspheres. Following 72 h post-ICH, EP1⁻/⁻ mice showed deteriorated outcomes compared to the WT control mice. These outcomes were demonstrated by elevated neurological deficits, exacerbated lesion volume, and significantly worsened sensorimotor functions. Fluoro-Jade staining showed significantly increased numbers of degenerating neurons and reduced neuronal survival in EP1⁻/⁻ compared to WT mice. To assess in vivo phagocytosis, the number of microspheres phagocytosed by Iba1-positive cells was 145.4 ± 15.4 % greater in WT compared to EP1⁻/⁻ mice. These data demonstrate that EP1 deletion exacerbates neuro-behavioral impairments following ICH, potentially by slowing down/impairing microglial phagocytosis. A better understanding of this EP1 mechanism could lead to improved intervention strategies for hemorrhagic stroke.

PGE2 EP1 receptor deletion attenuates 6-OHDA-induced Parkinsonism in mice: old switch, new target.

Recent experimental data on Parkinson's disease (PD) predicts the critical role of inflammation in the progression of neurodegeneration and the promising preventive effects of nonsteroidal anti-inflammatory drugs (NSAIDs). Previous studies suggest that NSAIDs minimize cyclooxygenase-2 (COX-2) activity and thereby attenuate free radical generation. Prostaglandin E2 (PGE2) is an important product of COX activity and plays an important role in various physiologic and pathophysiologic conditions through its EP receptors (EP1-EP4). Part of the toxic effect of PGE2 in the central nervous system has been reported to be through the EP1 receptor; however, the effect of the EP1 receptor in PD remains elusive. Therefore, in our pursuit to determine if deletion of the PGE2 EP1 receptor will attenuate 6-hydroxy dopamine (6-OHDA)-induced Parkinsonism, mice were given a unilateral 6-OHDA injection into the medial forebrain bundle. We found that apomorphine-induced contralateral rotations were significantly attenuated in the 6-OHDA-lesioned EP1(-/-) mice compared with the 6-OHDA-lesioned WT mice. Quantitative analysis showed significant protection of dopaminergic neurons in the substantia nigra pars compacta of the 6-OHDA-lesioned EP1(-/-) mice. To the best of our knowledge, this is the first in vivo study to implicate the PGE2 EP1 receptor in toxin-induced Parkinsonism. We propose the PGE2 EP1 receptor as a new target to better understand some of the mechanisms leading to PD.

Prostaglandin D2 DP1 receptor is beneficial in ischemic stroke and in acute exicitotoxicity in young and old mice.

The cardiovascular complications reported to be associated with cyclooxygenase inhibitor use have shifted our focus toward prostaglandins and their respective receptors. Prostaglandin D(2) and its DP1 receptor have been implicated in various normal and pathologic conditions, but their role in stroke is still poorly defined. Here, we tested whether DP1 deletion aggravates N-methyl-D: -aspartic acid (NMDA)-induced acute toxicity and whether DP1 pharmacologic activation protects mice from acute excitotoxicity and transient cerebral ischemia. Moreover, since the elderly are more vulnerable to stroke-related damage than are younger patients, we tested the susceptibility of aged DP1 knockout (DP1(-/-)) mice to brain damage. We found that intrastriatal injection of 15 nmol NMDA caused significantly larger lesion volumes (27.2 +/- 6.4%) in young adult DP1(-/-) mice than in their wild-type counterparts. Additionally, intracerebroventricular pretreatment of wild-type mice with 10, 25, and 50 nmol of the DP1-selective agonist BW245C significantly attenuated the NMDA-induced lesion size by 19.5 +/- 5.0%, 39.6 +/- 7.7%, and 28.9 +/- 7.0%, respectively. The lowest tested dose of BW245C also was able to reduce middle cerebral artery occlusion-induced brain infarction size significantly (21.0 +/- 5.7%). Interestingly, the aggravated NMDA-induced brain damage was persistent in older DP1(-/-) mice as well. We conclude that the DP1 receptor plays an important role in attenuating brain damage and that selective targeting of this receptor could be considered as an adjunct therapeutic tool to minimize stroke damage.

GOSPEL: a neuroprotective protein that binds to GAPDH upon S-nitrosylation.

We recently reported a cell death cascade whereby cellular stressors activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The nuclear GAPDH/Siah complex augments p300/CBP-associated acetylation of nuclear proteins, including p53, which mediate cell death. We report a 52 kDa cytosolic protein, GOSPEL, which physiologically binds GAPDH, in competition with Siah, retaining GAPDH in the cytosol and preventing its nuclear translocation. GOSPEL is neuroprotective, as its overexpression prevents NMDA-glutamate excitotoxicity while its depletion enhances death in primary neuron cultures. S-nitrosylation of GOSPEL at cysteine 47 enhances GAPDH-GOSPEL binding and the neuroprotective actions of GOSPEL. In intact mice, virally delivered GOSPEL selectively diminishes NMDA neurotoxicity. Thus, GOSPEL may physiologically regulate the viability of neurons and other cells.

PGF(2alpha) FP receptor contributes to brain damage following transient focal brain ischemia.

Although some of the COX-2 metabolites and prostaglandins have been implicated in stroke and excitotoxicity, the role of prostaglandin F(2alpha) (PGF(2alpha)) and its FP receptor have not been elucidated in the pathogenesis of ischemic-reperfusion (I/R) brain injury. Here we investigated the FP receptor's contribution in a unilateral middle cerebral artery (MCA) occlusion model of focal cerebral ischemia in mice. The MCA in wild type (WT) and FP knockout (FP(-/-)) C57BL/6 male mice was transiently occluded with a monofilament for 90 min. After 96 h of reperfusion, the FP(-/-) mice had 25.3% less neurological deficit (P < 0.05) and 34.4% smaller infarct volumes (P < 0.05) than those of the WT mice. In a separate cohort, physiological parameters were monitored before, during, and after ischemia, and the results revealed no differences between the groups. Because excitotoxicity is an acute mediator of stroke outcome, the effect of acute NMDA-induced neurotoxicity was also tested. Forty-eight hours after unilateral intrastriatal NMDA injection, excitotoxic brain damage was 20.8% less extensive in the FP(-/-) mice (P < 0.05) than in the WT counterparts, further supporting the toxic contribution of the FP receptor in I/R injury. Additionally, we investigated the effect of post-treatment with the FP agonist latanoprost in mice subjected to MCA occlusion; such treatment resulted in an increase in neurological deficit and infarct size in WT mice (P < 0.05), though no effects were observed in the latanoprost-treated FP(-/-) mice. Together, the results suggest that the PGF(2alpha) FP receptor significantly enhances cerebral ischemic and excitotoxic brain injury and that these results are of importance when planning for potential development of therapeutic drugs to treat stroke and its acute and/or long term consequences.

Selective blockade of PGE2 EP1 receptor protects brain against experimental ischemia and excitotoxicity, and hippocampal slice cultures against oxygen-glucose deprivation.

Cyclooxygenase-2 (COX-2) enzyme increases abnormally during excitotoxicity and cerebral ischemia and promotes neurotoxicity. Although COX-2 inhibitors could be beneficial, they have significant side effects. We and others have shown that the EP1 receptor is important in mediating PGE2 toxicity. Here, we tested the hypothesis that pretreatment with a highly selectiveEP1 receptor antagonist, ONO-8713, would improve stroke outcome and that post-treatment would attenuate NMDA-induced acute excitotoxicity and protect organotypic brain slices from oxygen-glucose deprivation (OGD)-induced toxicity. Male C57BL/6 mice were injected intracerebroventricularly with ONO-8713 before being subjected to 90-min middle cerebral artery occlusion (MCAO) and 96-h reperfusion.Significant reduction in infarct size was observed in groups given 0.1 (25.9 +/- 4.7%) and 1.0 nmol(27.7 +/- 2.8%) ONO-8713 as compared with the vehicle-treated control group. To determine the effects of ONO-8713 post-treatment on NMDA induced excitotoxicity, mice were given a unilateral intrastriatal NMDA injection followed by one intraperitoneal injection of 10 microg/kg ONO-8713, 1 and 6 h later. Significant attenuation of brain damage (26.6 +/-4.9%) was observed at 48 hin the ONO-8713-treated group. Finally, brain slice cultures were protected (25.5 +/- 2.9%) by the addition of ONO-8713 to the medium after OGD.These findings support the notion that the EP1receptor propagates neurotoxicity and that selective blockade could be considered as a potential preventive and/or therapeutic tool against ischemic/hypoxic neurological conditions.

Oral supplementation of Majun Baladar ameliorates antioxidant enzyme activities in cerebral ischaemic damage.

Majun Baladar (MB), a traditional herbal formulation of the Unani system of medicine, was studied for its efficacy against cerebral ischaemia-induced oxidative damage in hippocampus and associated neurobehavioural deficits. Adult male Wistar rats were divided into four groups. The first group was sham, the second group was ischaemic (MCAO: middle cerebral artery occluded) and the third group was a MB pre-treated ischaemic group (MCAO + MB). The fourth group was given MB (1.05 g/kg) orally for 15 days as a drug control. The middle cerebral artery was occluded for 2 hr and reperfused for 22 hr in the ischaemic as well as the drug pre-treated group. The activity of the various enzymatic antioxidants like glutathione peroxidase, glutathione reductase, glutathione S-transferase and non-enzymatic antioxidants, glutathione along with levels of lipid peroxidation were evaluated. Cerebral ischaemic rats showed elevated level of lipid peroxidation and decreased levels of various antioxidants significantly over sham values. As a result of MB pre-treatment, the level of lipid peroxidation was found to be significantly depleted as compared to the ischaemic group. Furthermore, depleted levels of glutathione and the activity of glutathione peroxidase, glutathione S-transferase and glutathione reductase were restored significantly in MB treated group. Majun Baladar exhibited a significant improvement in neurobehavioural activities in the drug pre-treated animals as compared to the ischaemic group as evidenced by the grip strength test, Rota-Rod and video path analysis. The results of the present study provide baseline information regarding the neuroprotective efficacy of MB and also open a window for a potent therapeutic use of this traditional herbal Unani medicine.

Stimulation of prostaglandin E2-EP3 receptors exacerbates stroke and excitotoxic injury.

The effect of PGE(2) EP3 receptors on injury size was investigated following cerebral ischemia and induced excitotoxicity in mice. Treatment with the selective EP3 agonist ONO-AE-248 significantly and dose-dependently increased infarct size in the middle cerebral artery occlusion model. In a separate experiment, pretreatment with ONO-AE-248 exacerbated the lesion caused by N-methyl-d-aspartic acid-induced acute excitotoxicity. Conversely, genetic deletion of EP3 provided protection against N-methyl-d-aspartic acid-induced toxicity. The results suggest that PGE(2), by stimulating EP3 receptors, can contribute to the toxicity associated with cyclooxygenase and that antagonizing this receptor could be used therapeutically to protect against stroke- and excitotoxicity-induced brain damage.

Stimulation of prostaglandin EP2 receptors prevents NMDA-induced excitotoxicity.

Prostaglandin E(2) (PGE(2)) plays an important role in inflammation and neurologic disorders. The neuromodulatory effects of PGE(2) are mediated through regulation of four G-protein-coupled receptors known as EP1, EP2, EP3, and EP4. The goal of the current study was to determine whether EP2 receptor activation protects neurons from acute NMDA-mediated excitotoxicity. To examine the effects of EP2 activation, mice were given an injection of the EP2 receptor-selective agonist butaprost (K (i) = 110 nM for EP2 receptor; K (i) > 10,000 for other prostaglandin receptors) in the cerebral ventricle and then an injection of NMDA in the right striatum. After 48 h, a significant reduction in NMDA-induced lesion volume was observed in groups pretreated with butaprost (1-300 nmol/L), with maximal protection at 100 nmol/L (p < 0.001). To determine if EP2-activated protection was specific to neurons, mouse neuronal cultures were treated with butaprost, and cell viability was analyzed after 24 h of NMDA excitotoxicity. The results showed that butaprost significantly increased neuron survival in a dose-dependent fashion. Furthermore, treatment of primary neurons with butaprost significantly increased cAMP levels (p < 0.001). Together, these data reveal that EP2 receptor stimulation mediates neuroprotection against NMDA excitotoxicity both in vivo and in vitro and that butaprost can limit acute brain damage. Development and testing of specific PGE(2) receptor mimetics could lead to a decrease in side effects associated with anti-inflammatory drugs and could help to fight acute and/or chronic neurologic disorders.

Protective effects of ethanolic extract of Delphinium denudatum in a rat model of Parkinson's disease.

Parkinson's disease (PD) is one of the major neurodegenerative disorders, and oxidative stress has been implicated in playing an important role in the pathogenesis of the disease. In the present study, we investigated if Delphinium denudatum extract can slow down the neuronal injury in 6-hydroxydopamine (6-OHDA) rat model of Parkinsonism. Rats were treated with 200, 400 and 600 mg/kg body weight (b.w.) of D. denudatum extract for 3 weeks. On day 22, 2 microL of 6-OHDA (10 microg in 0.1% ascorbic acid-saline) or vehicle was infused into the right striatum of the animals. Three weeks after the 6-OHDA injections, the rats were killed for estimation of lipid peroxidation (LPO), reduced glutathione (GSH) content, superoxide dismutase (SOD) and catalase (CAT) activities, catecholamines, dopaminergic D2 receptor binding and tyrosine hydroxylase (TH) expression. Increased LPO and significant depletion of reduced GSH content in the substantia nigra resulting from the lesion were appreciably prevented with Delphinium treatment. Delphinium extract also dose-dependently attenuated the activities of SOD and CAT in striatum, which had been reduced significantly by lesioning. A significant decrease in the level of dopamine (DA) and its metabolites and an increase in the number of dopaminergic D2 receptors in striatum were observed after 6-OHDA injection, both parameters were significantly recovered with treatment of the extract. Finally, all these results were confirmed by an increase in expression of TH in the ipsilateral striatum of the lesioned groups following treatment with Delphinium extract. Thus, the study indicates that D. denudatum extract may be helpful in checking neuronal injury in Parkinsonism.

Effect of Saffron (Crocus sativus) on neurobehavioral and neurochemical changes in cerebral ischemia in rats.

The modifying effects of Crocus sativus (CS) stigma extract on neurobehavioral activities, malondialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase (SOD), catalase (CAT), and Na(+),K(+)-ATPase activities, and glutamate (Glu) and aspartate (Asp) content were examined in the middle cerebral artery (MCA) occlusion (MCAO) model of acute cerebral ischemia in rats. The right MCA of male Wistar rats was occluded for 2 hours using intraluminal 4-0 monofilament, and reperfusion was allowed for 22 hours. MCAO caused significant depletion in the contents of GSH and its dependent enzymes while significant elevation of MDA, Glu, and Asp. The activities of Na(+),K(+)-ATPase, SOD, and CAT were decreased significantly by MCAO. The neurobehavioral activities (grip strength, spontaneous motor activity, and motor coordination) were also decreased significantly in the MCAO group. All the alterations induced by ischemia were significantly attenuated by pretreatment of CS (100 mg/kg of body weight, p.o.) 7 days before the induction of MCAO and correlated well with histopathology by decreasing the neuronal cell death following MCAO and reperfusion. The present results may suggest the effectiveness of CS in focal ischemia most probably by virtue of its antioxidant property.

Prevention of cognitive impairments and neurodegeneration by Khamira Abresham Hakim Arshad Wala.

Khamira Abresham Hakim Arshad Wala (KAHAW) is an effective and potent cardiac tonic with well-known antioxidant properties. The extensive use of this preparation in Indian system of Unani medicine led us to hypothesize that the pretreatment of this drug to male Wistar rats would prevent cognitive and neurobehavioral impairments. The cognitive impairment was developed by giving single intracerebroventricular injection of 1.5 mg/kg body weight of streptozotocin (STZ) bilaterally. An increased latency and path length was observed in cognitive, i.e. STZ group as compared to sham group and these were restored significantly in STZ group pretreated with KAHAW (700 mg/kg body weight for 15 days). The activity of antioxidant enzymes, viz. glutathione reductase, glutathione S-transferase, glutathione peroxidase, and superoxide dismutase was decreased in STZ group as compared to sham group and pretreatment of STZ group with KAHAW has protected their activities significantly. Moreover, the significantly depleted content of reduced glutathione and significantly elevated level of thiobarbituric acid reactive substances (TBARS) in STZ group were protected significantly with KAHAW. The study concludes that the therapeutic intervention of KAHAW may be used to prevent or to decrease the deterioration of cognitive function and neurobehavioral activities, often associated with the generation of free radicals.