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.

Endothelin B receptor agonist, IRL-1620, has been shown in previous studies, conducted in our lab, to provide significant neuroprotection at both 24h and 1 week following permanent cerebral ischemia. It is possible that IRL-1620 may be neuroprotective due to angiogenesis and neurogenesis. However, the effect of IRL-1620 on neurovascular remodeling following cerebral ischemia has not been established. The present study was conducted to determine the effect of IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)] on astrocytes, neurons, and vascular endothelial cells after induction of cerebral ischemia. Male Sprague-Dawley rats undergoing permanent middle cerebral artery occlusion (MCAO) received three intravenous injections of either vehicle or IRL-1620 at 2, 4, and 6h post occlusion. At 24h post occlusion, IRL-1620 treatment preserved neuronal numbers in the cortex, striatum and subventricular zone (SVZ) of the ischemic rat brain, while simultaneously enhancing the number of blood vessels labeled with vascular endothelial growth factor (VEGF) compared to vehicle treatment. By 1 week following MCAO, VEGF-positive vessels/30 µm brain slice in the IRL-1620 group numbered 11.33±2.13 versus 4.19±0.79 in the vehicle group (P<0.01). Additionally, animals receiving IRL-1620 displayed increased number of proliferating cells (P<0.0001) and cells positively staining for nerve growth factor (NGF; P<0.0001) in the infarcted brain. VEGF and NGF protein expression significantly increased at 1 week post MCAO in the infarcted hemisphere of IRL-1620 treated rats as compared to sham (P<0.01). Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study indicate that IRL-1620, administered on the day of infarct, is neuroprotective and enhances angiogenic and neurogenic remodeling following cerebral ischemia.

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.

Objective: The purpose of this study was to determine the potential neuroprotective effect of endothelin B (ETB) receptor agonist IRL-1620 treatment in a pediatric model of ischemic stroke. Design: A prospective, animal model study. Setting: An experimental laboratory. Subjects: Three-month-old male Wistar Han rats. Interventions: The rats underwent permanent middle cerebral artery occlusion (MCAO). At 2, 4, and 6 h post MCAO, they were treated with saline, IRL-1620 (5 μg/kg, IV), and/or ETB antagonist BQ788 (1 mg/kg, IV). Measurements and Main Results: The rats were evaluated over the course of 7 days for neurological and motor deficit, cerebral blood flow (CBF), and infarct volume. Young rats treated with IRL-1620 following MCAO improved significantly in neurological and motor assessments as compared to the vehicle-treated group, as measured by neurological score (P = 0.00188), grip test (P < 0.0001), and foot-fault error (P = 0.0075). CBF in the infarcted hemisphere decreased by 45-50% in all groups immediately following MCAO. After 7 days, CBF in the infarcted hemisphere of the IRL-1620 group increased significantly (P = 0.0007) when compared to the vehicle-treated group (+2.3 ± 23.3 vs. -45.4 ± 10.2%). Additionally, infarct volume was significantly reduced in IRL-1620-treated rats as compared to vehicle-treated rats (P = 0.0035, 41.4 ± 35.4 vs. 115.4 ± 40.9 mm3). Treatment with BQ788 blocked the effects of IRL-1620. Conclusions: IRL-1620 significantly reduced neurological and motor deficit as well as infarct volume while increasing CBF in a pediatric rat model of cerebral ischemia. These results indicate that selective ETB receptor stimulation may provide a novel therapeutic strategy in the treatment of pediatric ischemic stroke as has been demonstrated in adult ischemic stroke.

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.

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.

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.