Oxidative stress: A target to treat Alzheimer's disease and stroke.

Oxidative stress has been established as a well-known pathological condition in several neurovascular diseases. It starts with increased production of highly oxidizing free-radicals (e.g. reactive oxygen species; ROS and reactive nitrogen species; RNS) and becomes too high for the endogenous antioxidant system to neutralize them, which results in a significantly disturbed balance between free-radicals and antioxidants levels and causes cellular damage. A number of studies have evidently shown that oxidative stress plays a critical role in activating multiple cell signaling pathways implicated in both progression as well as initiation of neurological diseases. Therefore, oxidative stress continues to remain a key therapeutic target for neurological diseases. This review discusses the mechanisms involved in reactive oxygen species (ROS) generation in the brain, oxidative stress, and pathogenesis of neurological disorders such as stroke and Alzheimer's disease (AD) and the scope of antioxidant therapies for these disorders.

Neuroprotective Effect of Sovateltide (IRL 1620, PMZ 1620) in a Neonatal Rat Model of Hypoxic-Ischemic Encephalopathy.

Therapeutic hypothermia with modest results is the only treatment currently available for neonatal hypoxic ischemic encephalopathy (HIE). Endothelin B (ETB) receptors in the brain are shown to have neural restorative capacity. ETB receptors agonist sovateltide alone or as an adjuvant therapy may enhance neurovascular remodeling in HIE. Sprague-Dawley rat pups were grouped based on treatments into (1) Control; (2) HIE + Vehicle; (3) HIE + Hypothermia; (4) HIE + sovateltide; and (5) HIE + sovateltide + hypothermia. HIE was induced on postnatal day (PND) 7, followed by sovateltide (5 µg/kg) intracerebroventricular injection and/or hypothermia. On PND 10, brains were analyzed for the expression of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), ETB receptors, oxidative stress and cellular damage markers. Vehicle-treated animals had high oxidative stress level as indicated by an increase in lipid peroxidation factor, malondialdehyde, and decreased antioxidants, reduced glutathione and superoxide dismutase, compared to control. These effects were reversed in sovateltide alone (p < 0.001) or in combination with the therapeutic hypothermia (p < 0.001), indicating that ETB receptor activation reduces oxidative stress injury following HIE. Animals receiving sovateltide demonstrated a significant (p < 0.0001) upregulation of ETB receptor, VEGF, and NGF expression in the brain compared to vehicle-treated animals. Additionally, sovateltide alone or in combination with therapeutic hypothermia significantly (p < 0.001) reduced cell death when compared to vehicle or therapeutic hypothermia alone, demonstrating that sovateltide is neuroprotective and attenuates neural damage following HIE. These findings are important and merit additional studies for development of new interventions for improving neurodevelopmental outcomes after HIE.

Centhaquine Restores Renal Blood Flow and Protects Tissue Damage After Hemorrhagic Shock and Renal Ischemia.

Background: Centhaquine (CQ) (Lyfaquin®) is in late stage clinical development as a safe and effective first-in-class resuscitative agent for hemorrhagic shock patients (NCT02408731, NCT04056065, and NCT04045327). Acute kidney injury (AKI) is known to be associated with hemorrhagic shock. Hence, effect of CQ on protection of kidneys from damage due to hemorrhagic shock was investigated. Methods: To assess effect of CQ on AKI in shock, we created a rat model with hemorrhagic shock and AKI. Renal arteries were clamped and de-clamped to induce AKI like ischemia/reperfusion model and hemorrhage was carried out by withdrawing blood for 30 min. Rats were resuscitated with CQ (0.02 mg/kg) for 10 min. MAP, heart rate (HR), and renal blood flow (RBF) were monitored for 120 min. Results: CQ produced a significant improvement in RBF compared to vehicle (p< 0.003) even though MAP and HR was similar in CQ and vehicle groups. Blood lactate level was lower (p = 0.0064) in CQ than vehicle at 120 min post-resuscitation. Histopathological analysis of tissues indicated greater renal damage in vehicle than CQ. Western blots showed higher HIF-1α (p = 0.0152) and lower NGAL (p = 0.01626) levels in CQ vs vehicle. Immunofluorescence in the kidney cortex and medulla showed significantly higher (p< 0.045) expression of HIF-1α and lower expression of Bax (p< 0.044) in CQ. Expression of PHD 3 (p< 0.0001) was higher, while the expression of Cytochrome C (p = 0.01429) was lower in the cortex of CQ than vehicle. Conclusion: Results show CQ (Lyfaquin®) increased renal blood flow, augmented hypoxia response, decreased tissue damage and apoptosis following hemorrhagic shock induced AKI, and may be explored to prevent/treat AKI. Translational Statement: Centhaquine (CQ) is safe for human use and currently in late stage clinical development as a first-in-class resuscitative agent to treat hemorrhagic shock. In the current study, we have explored a novel role of CQ in protection from hemorrhagic shock induced AKI, indicating its potential to treat/prevent AKI.

Exposure to Morphine and Caffeine Induces Apoptosis and Mitochondrial Dysfunction in a Neonatal Rat Brain.

Background: Preterm infants experience rapid brain growth during early post-natal life making them vulnerable to drugs acting on central nervous system. Morphine is administered to premature neonates for pain control and caffeine for apnea of prematurity. Simultaneous use of morphine and caffeine is common in the neonatal intensive care unit. Prior studies have shown acute neurotoxicity with this combination, however, little information is available on the mechanisms mediating the neurotoxic effects. The objective of this study was to determine the effects of morphine and caffeine, independently and in combination on mitochondrial dysfunction (Drp1 and Mfn2), neural apoptosis (Bcl-2, Bax, and cell damage) and endothelin (ET) receptors (ETA and ETB) in neonatal rat brain. Methods: Male and female rat pups were grouped separately and were divided into four different subgroups on the basis of treatments-saline (Control), morphine (MOR), caffeine (CAFF), and morphine + caffeine (M+C) treatment. Pups in MOR group were injected with 2 mg/kg morphine, CAFF group received 100 mg/kg caffeine, and M+C group received both morphine (2 mg/kg) and caffeine (100 mg/kg), subcutaneously on postnatal days (PND) 3-6. Pups were euthanized at PND 7, 14, or 28. Brains were isolated and analyzed for mitochondrial dysfunction, apoptosis markers, cell damage, and ET receptor expression via immunofluorescence and western blot analyses. Results: M+C showed a significantly higher expression of Bax compared to CAFF or MOR alone at PND 7, 14, 28 in female pups (p < 0.05) and at PND 7, 14 in male pups (p < 0.05). Significantly (p < 0.05) increased expression of Drp1, Bax, and suppressed expression of Mfn2, Bcl-2 at PND 7, 14, 28 in all the treatment groups compared to the control was observed in both genders. No significant difference in the expression of ETA and ETB receptors in male or female pups was seen at PND 7, 14, and 28. Conclusion: Concurrent use of morphine and caffeine during the first week of life increases apoptosis and cell damage in the developing brain compared to individual use of caffeine and morphine.

Sovateltide (IRL-1620) activates neuronal differentiation and prevents mitochondrial dysfunction in adult mammalian brains following stroke.

The development of effective drugs for stroke is urgently required as it is the 2nd largest killer in the world and its incidence is likely to increase in the future. We have demonstrated cerebral endothelin B receptors (ETBR) as a potential target to treat acute cerebral ischemic stroke. However, the mechanism of ETBR mediated neural regeneration and repair remains elusive. In this study, a permanent middle cerebral artery occluded (MCAO) rat model was used. Sovateltide (an ETBR agonist) injected intravenously showed better survival and neurological and motor function improvement than control. Higher neuronal progenitor cells (NPCs) differentiation along with better mitochondrial morphology and biogenesis in the brain of sovateltide rats were noted. Exposure of cultured NPCs to hypoxia and sovateltide also showed higher NPC differentiation and maturation. This study shows a novel role of ETBR in NPCs and mitochondrial fate determination in cerebral ischemia, and in improving neurological deficit after stroke.

Relationship Between Oxidative Stress Markers and Endothelin-1 Levels in Newborns of Different Gestational Ages.

Oxidative stress results from excessive reactive oxygen species formation and/or inadequate antioxidant defense. Premature and critically ill infants are especially susceptible due to an immature intrinsic antioxidant system that cannot fully compensate for a free radical load. Oxidative stress is also associated with endothelial dysfunction and alterations in Endothelin-1 (ET-1) signaling pathways. However, the effects of the complex interaction between oxidative stress and ET-1 in newborns are not well-understood. The objective of this pilot study was to determine the relationship between levels of common oxidative stress biomarkers [glutathione (GSH), malondialdehyde (MDA)] and ET-1 in newborns of different gestational ages. In a level IV NICU, 63 neonates were prospectively enrolled and divided into groups based on gestational age at birth: Early Preterm (24 0/7-30 6/7 weeks), Late Preterm (31 0/7-36 6/7 weeks), and Term (37 0/7-42 weeks). Umbilical cord (1.5 mL) and 24(±4) h of life (24 h) (1 mL) blood samples were collected for GSH, MDA, and ET-1 analyses. GSH, MDA, and ET-1 were determined using established methodology. Mean cord MDA levels for all age groups, Early Preterm (2.93 ± 0.08 pg/ml), Late Preterm (2.73 ± 0.15 pg/ml), and Term (2.92 ± 0.13 pg/ml), were significantly higher than those at 24 h of life (p < 0.001). Mean cord ET-1 levels were significantly higher than 24 h samples in both Early and Late Preterm groups (p < 0.05). Cord and 24 h ET-1 levels did not correlate with MDA and GSH levels at birth (r2 = 0.03, p > 0.05 and r2 = 0.001, p > 0.05, respectively) or 24 h of life (r2 = 0.001, p > 0.05 and r2 = 0.03, p > 0.05, respectively). Preterm neonates exposed to prenatal corticosteroids (1.87 ± 0.31 pg/ml) had lower cord MDA levels than non-exposed neonates (2.85 ± 0.12 pg/ml) (p < 0.05). Both cord and 24 h OS markers were significantly higher in neonates treated with oxygen therapy (p < 0.005 and p < 0.05, respectively) than those who did not receive supplemental oxygen. Oxidative stress markers (MDA and GSH) and ET-1 levels act independently. MDA is higher in cord blood than at 24 h of life regardless of gestational age. In preterm neonates, ET-1 levels are higher in umbilical cord blood compared to 24 h of life.

Sovateltide (IRL-1620) affects neuronal progenitors and prevents cerebral tissue damage after ischemic stroke.

Stimulation of endothelin B receptors by its agonist IRL-1620 (INN, sovateltide) provides neuroprotection and neurological and motor function improvement following cerebral ischemia. We investigated the effect of sovateltide on stem and progenitor cells mediated neural regeneration and its effect on the cerebral tissue repair and restoration of neurological and motor function. Sovateltide (5 µg/kg) was injected intravenously in permanent middle cerebral artery occluded (MCAO) rats at 4, 6, and 8 h at days 0, 3, and 6. Neurological and motor function tests were carried out pre-MCAO and at day 7 post-MCAO. At day 7, significantly reduced expression of neuronal differentiation markers HuC/HuD and NeuroD1 was seen in MCAO + vehicle than sham rats. Sovateltide treatment upregulated HuC/HuD and NeuroD1 compared to MCAO + vehicle and their expression was similar to sham. Expression of stem cell markers Oct 4 and Sox 2 was similar in rats of all of the groups. Significantly reduced infarct volume and DNA damage with recovery of neurological and motor function was observed in sovateltide-treated MCAO rats. These results indicate that sovateltide initiates a regenerative response by promoting differentiation of neuronal progenitors and maintaining stem cells in an equilibrium following cerebral ischemic stroke.

Anti-apoptotic and Immunomodulatory Effect of CB2 Agonist, JWH133, in a Neonatal Rat Model of Hypoxic-Ischemic Encephalopathy.

Introduction: Neonatal HIE is associated with high morbidity and mortality. Current research, is focused on developing alternative treatments to therapeutic hypothermia for treatment of HIE. The endocannabinoid system is known to be influential in neuronal protection. Activation of brain CB2 receptors, has been shown to reduce inflammatory markers and decrease infarct volume in adult cerebral ischemic models. Methods: Rat pups were divided into six groups: 1-Placebo; 2-JWH133; 3-HIE + Placebo; 4-HIE + JWH133; 5-HIE + Hypothermia + Placebo; and 6-HIE + Hypothermia + JWH133. HIE was induced in in groups 3-6 by right carotid ligation on postnatal day 7 followed by placement in a hypoxic chamber. Pups in groups 5 and 6 were treated with hypothermia. Western blot analysis was used to analyze brain tissue for acute inflammatory markers (IL-6, TNFα, MIP1α, and RANTES), immunoregulatory cytokines (TGFß and IL-10), and CB2 receptor expression. DNA fragmentation in the brains of pups was determined via TUNEL staining post HIE. Results: The combination of JWH133 and hypothermia significantly reduced tumor necrosis factor α (TNFα) (-57.7%, P = 0.0072) and macrophage inflammatory protein 1α (MIP1α) (-50.0%, P = 0.0211) as compared to placebo. DNA fragmentation was also significantly reduced, with 6.9 ± 1.4% TUNEL+ cells in HIE+JWH133 and 12.9 ± 2.2% in HIE+Hypothermia + JWH133 vs. 16.6 ± 1.9% in HIE alone. No significant difference was noted between groups for the expression of interleukins 6 and 10, RANTES, or TGFß. After 8 h, CB2 receptor expression increased nearly 2-fold in the HIE and HIE + JWH133 groups (+214%, P = 0.0102 and +198%, P = 0.0209, respectively) over placebo with no significant change in the hypothermia groups. By 24 h post HIE, CB2 receptor expression was elevated over five times that of placebo in the HIE (P < 0.0001) and HIE + JWH133 (P = 0.0002) groups, whereas hypothermia treatment maintained expression similar to that of placebo animals. Conclusion: These results indicate that the combination of CB2 agonist and hypothermia may be neuroprotective in treating HIE, opening the door for further studies to examine alternative or adjuvant therapies to hypothermia.

Author Correction: Anti-apoptotic activity of ETB receptor agonist, IRL-1620, protects neural cells in rats with cerebral ischemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anti-apoptotic activity of ETB receptor agonist, IRL-1620, protects neural cells in rats with cerebral ischemia.

Endothelin-B receptor agonist, IRL-1620, provides significant neuroprotection following cerebral ischemia in rats. Whether this neuroprotection is due to inhibition of apoptosis is unknown. IRL-1620-treated rats following permanent middle cerebral artery occlusion (MCAO) showed significant improvement in neurological and motor functions along with a decrease in infarct volume at 24 h (-81.3%) and day 7 (-73.0%) compared to vehicle group. Cerebral blood flow (CBF) significantly improved in IRL-1620-treated animals compared to vehicle by day 7 post MCAO. IRL-1620-treated rats showed an increase in phospho-Akt and decrease in Bad level 7 h post-occlusion compared to vehicle, while Akt and Bad expression was similar in cerebral hemispheres at 24 h post-MCAO. The phospho-Bad level was lower in vehicle- but not in IRL-1620-treated rats at 24 h. Anti-apoptotic Bcl-2 expression decreased, while pro-apoptotic Bax expression increased in vehicle-treated MCAO rats, these changes were attenuated (P < 0.01) by IRL-1620. Mitochondrial membrane-bound Bax intensity significantly decreased in IRL-1620 compared to vehicle-treated MCAO rats. IRL-1620 treatment reduced (P < 0.001) the number of TUNEL-positive cells compared to vehicle at 24 h and day 7 post MCAO. The results demonstrate that IRL-1620 is neuroprotective and attenuates neural damage following cerebral ischemia in rats by increasing CBF and reducing apoptosis.

24-Hour Pharmacokinetic Relationships for Vancomycin and Novel Urinary Biomarkers of Acute Kidney Injury.

Vancomycin has been associated with acute kidney injury in preclinical and clinical settings; however, the precise exposure profiles associated with vancomycin-induced acute kidney injury have not been defined. We sought to determine pharmacokinetic/pharmacodynamics indices associated with the development of acute kidney injury using sensitive urinary biomarkers. Male Sprague-Dawley rats received clinical-grade vancomycin or normal saline as an intraperitoneal injection. Total daily doses between 0 and 400 mg/kg of body weight were administered as a single dose or 2 divided doses over a 24-h period. At least five rats were utilized for each dosing protocol. A maximum of 8 plasma samples per rat were obtained, and urine was collected over the 24-h period. Kidney injury molecule-1 (KIM-1), clusterin, osteopontin, cystatin C, and neutrophil gelatinase-associated lipocalin levels were determined using Milliplex multianalyte profiling rat kidney panels. Vancomycin plasma concentrations were determined via a validated high-performance liquid chromatography methodology. Pharmacokinetic analyses were conducted using the Pmetrics package for R. Bayesian maximal a posteriori concentrations were generated and utilized to calculate the 24-h area under the concentration-time curve (AUC), the maximum concentration (Cmax), and the minimum concentration. Spearman's rank correlation coefficient (rs ) was used to assess the correlations between exposure parameters, biomarkers, and histopathological damage. Forty-seven rats contributed pharmacokinetic and toxicodynamic data. KIM-1 was the only urinary biomarker that correlated with both composite histopathological damage (rs = 0.348, P = 0.017) and proximal tubule damage (rs = 0.342, P = 0.019). The vancomycin AUC and Cmax were most predictive of increases in KIM-1 levels (rs = 0.438 and P = 0.002 for AUC and rs = 0.451 and P = 0.002 for Cmax). Novel urinary biomarkers demonstrate that kidney injury can occur within 24 h of vancomycin exposure as a function of either AUC or Cmax.

Attenuation of opioid tolerance by ETB receptor agonist, IRL-1620, is independent of an accompanied decrease in nerve growth factor in mice.

AIM: ETA receptor antagonists reverse opioid tolerance but the involvement of ETB receptors is unknown. In morphine or oxycodone tolerant mice we investigated (1) the effect of ETB receptor agonist, IRL-1620, on analgesic tolerance; (2) changes in expression of the brain ETA and ETB receptors; and (3) alterations in the brain VEGF, NGF, PI3K and notch-1 expression. MAIN METHODS: Body weight, body temperature, and tail-flick latency were assessed before and after a challenge dose of morphine or oxycodone in vehicle or IRL-1620 treated mice. Expression studies were carried out using Western blots. KEY FINDINGS: Tail flick latency to a challenge dose of opioid was significantly increased by IRL-1620 from 39% to 100% in morphine tolerant and from 8% to 83% in oxycodone tolerant mice. Morphine or oxycodone did not alter ETA or ETB receptor expression. IRL-1620 had no effect on ETA however it increased (61%) expression of ETB receptors. IRL-1620-induced increase in ETB receptor expression was attenuated by morphine (39.8%) and oxycodone (51.8%). VEGF expression was not affected by morphine or oxycodone and was unaltered by IRL-1620. However, NGF and PI3K expression was decreased (P < 0.001) by morphine and oxycodone and was unaffected by IRL-1620. Notch-1 expression was not altered by morphine, oxycodone or IRL-1620. SIGNIFICANCE: ETB receptor agonist, IRL-1620, restored analgesic tolerance to morphine and oxycodone, but it did not affect morphine and oxycodone induced decrease in NGF/PI3K expression. It is concluded that IRL-1620 attenuates opioid tolerance without the involvement of NGF/PI3K pathway.

Ontogeny of endothelin receptors in the brain, heart, and kidneys of neonatal rats.

BACKGROUND: Endothelin (ET) plays an important role in many physiological functions. It has been demonstrated that endogenous ET-1 concentration in the central nervous system (CNS) changes with age; however the ontogeny of ETA and ETB receptors in the brain, heart, and kidneys during postnatal development has not been studied. METHODS: Brains, hearts and kidneys of rats at postnatal days 1, 7, 14 and 28 were evaluated for the expression of ETA and ETB receptors via Western blot. ETB receptors within the developing brain were further accessed via immunofluorescence. RESULTS: The mean organ and body weights increased proportionally with advancing age demonstrating normal growth. The expression of ETA receptors in the brain, heart, and kidneys and ETB receptor expression in the heart and kidneys was similar in these rats at postnatal ages 1, 7, 14 and 28days. However, brain ETB receptor expression significantly (P<0.001) decreased by 72% on day 28 compared to the levels on postnatal day 1. Upon immunofluorescent analysis, the intensity of ETB staining in the cerebral cortex and subventricular zones of the developing rat brain decreased significantly from day 1 to day 7 (P<0.001) and from day 7 to day 14 (P<0.0001). There was no further decrease in ETB intensity noted in the cerebral cortex and subventricular zones between day 14 and day 28 of postnatal age. The intensity of ETB receptor staining within the cerebrovasculature, on the other hand, increased significantly (P<0.05) from days 1 and 7 to day 14. CONCLUSIONS: These results demonstrate that expression of ETA receptors does not change with postnatal development. On the other hand ETB receptors in the cerebral cortex and subventricular zones of the brain decrease with age, while ETB receptors in the cerebrovasculature increase with age, implicating ETB receptor involvement in the structural maturity and development of the CNS.

Endothelin receptor type B agonist, IRL-1620, prevents beta amyloid (Aß) induced oxidative stress and cognitive impairment in normal and diabetic rats.

Alzheimer's disease (AD) is a progressive brain disorder leading to impairment of learning and memory. Amyloid ß (Aß) induced oxidative stress has been implicated in the initiation and progression of AD. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies indicate that stimulation of ETB receptors may provide neuroprotection. The purpose of this study was to determine the preventative effect of selectively stimulating ETB receptors on cognitive impairment and oxidative stress in Aß treated non-diabetic and diabetic (induced by streptozotocin) rats. Rats were concurrently treated with Aß1-40 (day 1, 7 and 14) and either saline, IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) daily for 14 days in the lateral cerebral ventricles using sterotaxically implanted cannula; experiments were performed on day 15. Aß treatment produced a significant (p<0.0001) increase of 360% and 365% in malondialdehyde levels (a marker of lipid peroxidation) in non-diabetic and diabetic rats, respectively, compared to sham group. Antioxidants (superoxide dismutase and reduced glutathione) decreased following Aß treatment compared to sham group. Treatment with IRL-1620 reversed these effects, indicating that ETB receptor stimulation reduces oxidative stress injury following Aß treatment. In Morris swim task, Aß treated rats showed impairment in spatial memory. Rats treated with IRL-1620 significantly reduced the cognitive impairment induced by Aß. BQ788 treatment completely blocked IRL-1620 induced reduction in oxidative stress and cognitive impairment. Results of the present study demonstrate that IRL-1620 improved both acquisition (learning) and retention (memory) on water maze task and reduced oxidative stress parameters. It can be speculated that ETB receptor stimulation prevents cognitive impairment and may be useful in neurodegenerative diseases.

Endothelin B receptor agonist, IRL-1620, provides long-term neuroprotection in cerebral ischemia in rats.

We have earlier shown that stimulation of endothelin B receptors by IRL-1620 provides significant neuroprotection at 24h following cerebral ischemia. However, the effect of IRL-1620 is not known in the subacute phase of cerebral ischemia, where development of cerebral edema further contributes towards brain damage. This study was designed to determine the effect of IRL-1620 on neurological functions, infarct volume, oxidative stress, and endothelin receptors following permanent middle cerebral artery occlusion for 7 days. Rats received three intravenous injections of either vehicle or IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)] at 2, 4, and 6h post occlusion. Treatment with IRL-1620 reduced infarct volume (54.06 ± 14.12 mm(3) vs. 177.06 ± 13.21 mm(3)), prevented cerebral edema and significantly improved all neurological and motor function parameters when compared to the vehicle-treated group. Vehicle-treated middle cerebral artery occluded rats demonstrated high levels of malondialdehyde and low levels of reduced glutathione and superoxide dismutase; these effects were reversed in IRL-1620 treated rats. No change in expression of endothelin A receptor was observed 7 days after induction of cerebral ischemia in vehicle or IRL-1620 treated rats. Rats receiving IRL-1620 demonstrated an upregulation of endothelin B receptor only in the infarcted hemisphere 7 days following occlusion. All effects of IRL-1620 were blocked by endothelin B receptor antagonist, BQ788. Results of the present study demonstrate that IRL-1620, administered on day 1, provides significant neuroprotection till 7 days after the induction of cerebral ischemia in rats. Selective endothelin B receptor activation may prove to be a novel therapeutic target in the treatment of cerebral ischemia.

Repeated administration of exendin-4 reduces focal cerebral ischemia-induced infarction in rats.

Exendin-4 is a GLP-1 agonist that is clinically used for the treatment of diabetes mellitus and may also have neuroprotective effect. We explored the effect of repeated administration of exendin-4 (0.5 µg/kg, intraperitoneal twice a day for 7 days) on infarct volume, neurological deficit (neurological score, grip test, foot fault and rota rod tests), oxidative stress parameters (malondialdehyde, reduced glutathione, and superoxide dismutase) and expression of endothelin (ET) ET(A) and ET(B) receptors following cerebral ischemia produced in rats by permanent middle cerebral artery occlusion (MCAO). Since ET(A) receptors in the central nervous system (CNS) are involved in cerebral ischemia, we determined the effect of a specific ET(A) receptor antagonist, BQ123 (1mg/kg, intravenously administered thrice: 30 min, 2h and 4h after MCAO for a total dose of 3 mg/kg) on cerebral ischemia in control and exendin-4 treated rats. Results indicate that exendin-4 treated rats had significant protection following MCAO induced cerebral ischemia. The infarct volume was 27% less compared to vehicle treated rats. The neurological deficit following MCAO was lower and oxidative stress parameters were improved in exendin-4 treated rats compared to control. BQ123 significantly improved infarct volume, oxidative stress parameters and neurological deficit in ischemic rats treated with vehicle or exendin-4. BQ123 induced protection from cerebral ischemia was similar in vehicle or exendin-4 treated rats. Expression of ET(A) receptors was significantly increased following cerebral ischemia which was not affected by exendin-4 treatment or by BQ123 administration. No change in expression of ET(B) receptors was observed following cerebral ischemia or any treatment. It is concluded that exendin-4 protects the CNS from damage due to cerebral ischemia by reducing oxidative stress and is independent of ET receptor involvement.

Endothelin B receptor agonist, IRL-1620, reduces neurological damage following permanent middle cerebral artery occlusion in rats.

Endothelin and its receptors have long been considered therapeutic targets in the treatment of ischemic stroke. Recent studies indicate that ET(B) receptors may provide both vasodilatation and neuroprotection. The purpose of this study was to determine the effect of selectively activating the ET(B) receptors following permanent middle cerebral artery occlusion in rats. IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)], a highly selective ET(B) agonist, was used alone and in conjunction with BQ788, an ET(B) antagonist, to determine the role of ET(B) receptors in cerebral ischemia. Rats were assessed for neurological deficit and motor function, and their brains were evaluated to determine infarct area, oxidative stress parameters, and ET receptor protein levels. Animals treated with IRL-1620 showed significant improvement in all neurological and motor function tests when compared with both vehicle-treated and BQ788-treated middle cerebral artery occluded groups. In addition, there was a significant decrease in infarct volume 24h after occlusion in animals treated with IRL-1620 (24.47±4.37mm(3)) versus the vehicle-treated group (153.23±32.18mm(3)). Blockade of ET(B) receptors by BQ788 followed by either vehicle or IRL-1620 treatment resulted in infarct volumes similar to those of rats treated with vehicle alone (163.51±25.41 and 139.21±15.20mm(3), respectively). Lipid peroxidation, as measured by malondialdehyde, increased and antioxidants (superoxide dismutase and reduced glutathione) decreased following infarct. Treatment with IRL-1620 reversed these effects, indicating that ET(B) receptor activation reduces oxidative stress injury following ischemic stroke. Animals pretreated with BQ788 showed similar oxidative stress damage as those in the vehicle-treated group. No significant difference was observed in ET(B) receptor levels in any of the groups. The present study demonstrates that ET(B) receptor activation may be a novel neuroprotective therapy in the treatment of focal ischemic stroke.

Endothelin-A receptor antagonists prevent amyloid-ß-induced increase in ETA receptor expression, oxidative stress, and cognitive impairment.

Alzheimer's disease is a neurodegenerative disorder associated with abnormal accumulation of amyloid-ß (Aß) which can release endothelin (ET). The present study was conducted to investigate the effect of ET antagonists on Aß-induced changes in ETA and ETB receptor expression, oxidative stress, and cognitive impairment. Male Sprague-Dawley rats were treated with Aß1-40 in the lateral cerebral ventricles and were administered vehicle or ET antagonists for 14 days. Aß treatment produced an increase in ETA receptor expression in the cerebral cortex, hippocampus, and brain stem by 72%, 85%, and 90%, respectively. No change in ETB receptor expression was observed. There was an increase in malondialdehyde (MDA) and decrease in reduced glutathione (GSH) and superoxide dismutase (SOD) levels in Aß-treated rats. In the Morris swim task, Aß treated rats showed a significant impairment in spatial memory. ET receptor antagonists, BQ123, BMS182874, and TAK-044, significantly decreased Aß-induced increase in ETA expression in the cortex, hippocampus, and brain stem. Rats treated with ET antagonists showed significant attenuation of Aß-induced changes in the brain MDA, GSH, and SOD levels. Rats treated with specific ETA receptor antagonists, BQ123 and BMS182874, significantly reduced the cognitive impairment induced by Aß. However, nonspecific ETA/ETB receptor antagonist TAK-044 did not show any improvement in the learning and memory parameter. This study demonstrates that ETA receptor antagonists are effective in preventing cognitive impairment, changes in ETA expression and oxidative stress induced by Aß. It is concluded that ETA receptor antagonists may be useful in improving cognitive impairment due to Alzheimer's disease.

Therapeutic potential of herbal drugs in cerebral ischemia.

Stroke is one of the most important causes of mortality and morbidity in the world. Prevention and effective treatment of stroke is of the utmost importance. Cerebral ischemia causes disturbances in a variety of cellular and molecular mechanisms, including oxidative phosphorylation, membrane function, neurotransmitter release, and free radical generation. It has been years since tissue-type plasminogen activator (t-PA) became the first medication approved by the FDA for the management of stroke, with limited success. Thrombolytic therapy is the most effective therapeutic strategy for the prevention of brain injury and reduction of mortality in patients with cerebral infarction. However, a combination of established thrombolytic therapy and effective neuronal protection therapy may have more beneficial effects for patients with cerebral infarction. Because clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned towards approaches which include herbal drugs that can be used in limiting the neurological damage associated with stroke. Herbal drugs may be used as prophylactic treatment in patients with high risk of stroke. Herbals drugs have been described in ancient systems of medicine for the treatment of various ailments associated with stroke and have more recently been reported to be beneficial in treating stroke. However, the strength of evidence to support the use of these herbal drugs is unclear. This review focuses on putative mechanisms underlying the beneficial effects of herbal drugs in patients with stroke and on the possibility of herbal drugs to increase the therapeutic time window in patients with cerebral ischemia.

Endothelin modulates the cardiovascular effects of clonidine in the rat.

Clonidine decreases mean arterial pressure (MAP) by acting as an α(2)-adrenergic receptor (AR) agonist in the central nervous system; it also acts on peripheral α-ARs to produce vasoconstriction. Endothelin (ET) has been shown to modulate the action of ARs. The present study was conducted to determine the involvement of ET in cardiovascular effects of clonidine. Intravenous administration of clonidine (10, 30 and 90µgkg(-1)) produced a dose-dependent decrease in MAP and heart rate (HR). Treatment with ET-1 (100, 300 and 900ngkg(-1)) significantly attenuated clonidine (10µgkg(-1)) induced fall in MAP and HR. Rats treated with ET-1 (900ngkg(-1)) showed an increase in MAP and HR after clonidine administration compared to untreated rats, while ET(A/B) antagonist, TAK-044 (1mgkg(-1)) and ET(A) antagonist, BMS-182874 (9mgkg(-1)) potentiated the hypotensive effect of clonidine. ET(B) receptor agonist, IRL-1620 (5µgkg(-1)) produced significant attenuation of clonidine induced fall in MAP and HR, while ET(B) receptor antagonist, BQ-788 (0.3mgkg(-1)), potentiated the hypotensive effect of clonidine. Prazosin (0.1mgkg(-1)) completely blocked ET-1 induced changes in cardiovascular effects of clonidine. Clonidine-induced contraction of rat abdominal aortic ring was potentiated by ET-1, which was completely blocked by prazosin. Clonidine produced an increase in ET(A) receptor expression in the brain and abdominal aorta while ET(B) receptors were not affected. It is concluded that ET enhances the responsiveness of vascular ARs to the constrictor effect of clonidine and ET antagonists potentiate the hypotensive effect of clonidine suggesting that a combination of ET antagonist with clonidine may be a useful option to treat hypertension.