Kamil Duris

α7 Nicotinic Acetylcholine Receptor Agonist PNU-282987 Attenuates Early Brain Injury in a Perforation Model of Subarachnoid Hemorrhage in Rats

Background and Purpose— Early brain injury is an important pathological process after subarachnoid hemorrhage (SAH). The goal of this study was to evaluate whether the &agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) agonist PNU-282987 attenuates early brain injury after SAH and whether &agr;7nAChR stimulation is associated with down-regulation of caspase activity via phosphatidylinositol 3-kinase-Akt signaling. Methods— The perforation model of SAH was performed, and neurological score, body weight loss, and brain water content were evaluated 24 and 72 hours after surgery. Western blot and immunohistochemistry were used for quantification and localization of phosphorylated Akt and cleaved caspase 3. Neuronal cell death was quantified with TUNEL staining. &agr;7nAChR antagonist methylcaconitine and phosphatidylinositol 3-kinase inhibitor wortmannin were used to manipulate the proposed pathway, and results were quantified with Western blot. Results— PNU-282987 improved neurological deficits both 24 and 72 hours after surgery and reduced brain water content in left hemispheres 24 hours after surgery. PNU-282987 significantly increased phosphorylated Akt levels and significantly decreased cleaved caspase 3 levels in ipsilateral hemispheres after SAH. Methylcaconitine and wortmannin reversed effects of treatment. Phosphorylated Akt and cleaved caspase 3 were colocalized to neurons in the ipsilateral basal cortex. Phosphorylated Akt was mainly localized in TUNEL-negative cells. PNU-282987 significantly reduced neuronal cell death in the ipsilateral basal cortex. Conclusions— &agr;7nAChR stimulation decreased neuronal cell death and brain edema and improved neurological status in a rat perforation model of SAH. &agr;7nAChR stimulation is associated with increasing phosphorylation of Akt and decreasing cleaved caspase 3 levels in neurons.

α7 Nicotinic Acetylcholine Receptor Agonism Confers Neuroprotection Through GSK-3β Inhibition in a Mouse Model of Intracerebral Hemorrhage

Background and Purpose— Perihematomal edema formation and consequent cell death contribute to the delayed brain injury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of &agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the role of the proapoptotic glycogen synthase kinase-3&bgr; (GSK-3&bgr;) after experimental ICH. Methods— Male CD-1 mice (n=109) were subjected to intracerebral infusion of autologous blood (n=88) or sham surgery (n=21). ICH animals received vehicle administration, 4 or 12 mg/kg of &agr;7nAChR agonist PHA-543613, 12 mg/kg of &agr;7nAChR agonist PNU-282987, 6 mg/kg of &agr;7nAChR antagonist methyllycaconitine (MLA), 15 &mgr;g/kg of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioral deficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluorescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3&bgr; (p-GSK-3&bgr;), and cleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL). Results— &agr;7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery (P<0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3&bgr; and CC3 expressions in the ipsilateral hemisphere (P<0.05, respectively), which was reversed by MLA and wortmannin. P-Akt, p-GSK-3&bgr;, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in the perihematomal area (P<0.05). Conclusions— &agr;7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3&bgr; through the PI3K-Akt signaling pathway.

Nasal Administration of Recombinant Osteopontin Attenuates Early Brain Injury After Subarachnoid Hemorrhage

Background and Purpose— Neuronal apoptosis is a key pathological process in subarachnoid hemorrhage (SAH)–induced early brain injury. Given that recombinant osteopontin (rOPN), a promising neuroprotectant, cannot pass through the blood–brain barrier, we aimed to examine whether nasal administration of rOPN prevents neuronal apoptosis after experimental SAH. Methods— Male Sprague–Dawley rats (n=144) were subjected to the endovascular perforation SAH model. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 and 72 hours after SAH induction. The expressions of cleaved caspase-3, phosphorylated focal adhesion kinase (FAK), and phosphorylated Akt were examined using Western blot analysis. Neuronal cell death was demonstrated with terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling. We also administered FAK inhibitor 14 and phosphatidylinositol 3-kinase inhibitor, Wortmannin, prior to rOPN to establish its neuroprotective mechanism. ELISA was used to measure rOPN delivery into the cerebrospinal fluid. Results— Cerebrospinal fluid level of rOPN increased after its nasal administration. This was associated with improved neurological scores and reduced brain edema at 24 hours after SAH. rOPN increased phosphorylated FAK and phosphorylated Akt expressions and decreased caspase-3 cleavage, resulting in attenuation of neuronal cell death within the cerebral cortex. These effects were abolished by FAK inhibitor 14 and Wortmannin. Conclusions— Nasal administration of rOPN decreased neuronal cell death and brain edema and improved the neurological status in SAH rats, possibly through FAK–phosphatidylinositol 3-kinase–Akt–induced inhibition of capase-3 cleavage.

Inhibition of Rho kinase by Hydroxyfasudil Attenuates Brain Edema after Subarachnoid Hemorrhage in Rats

The blood-brain barrier (BBB) disruption and brain edema are important pathophysiologies of early brain injury after subarachnoid hemorrhage (SAH). This study is to evaluate whether Rho kinase (Rock) enhances BBB permeability via disruption of tight junction proteins during early brain injury. Adult male rats were assigned to five groups; Sham-operated, SAH treated with saline, a Rock inhibitor hydroxyfasudil (HF) (10 mg/kg) treatment at 0.5 h after SAH, HF treatment at 0.5 and 6 h (10 mg/kg, each) after SAH, and another Rock inhibitor Y27632 (10 mg/kg) treatment at 0.5 h after SAH. The perforation model of SAH was performed and neurological score and brain water content were evaluated 24 and 72 h after surgery. Evans blue extravasation, Rock activity assay, and western blotting analyses were evaluated 24 h after surgery. Treatment of HF significantly improved neurological scores 24 h after SAH. Single treatment with HF and Y27632, and two treatments with HF reduced brain water content in the ipsilateral hemisphere. HF reduced Evans blue extravasation in the ipsilateral hemisphere after SAH. Rock activity increased 24 h after SAH, and HF reversed the activity. SAH significantly decreased the levels of tight junction proteins, occludin and zonula occludens-1 (ZO-1), and HF preserved the levels of occluding and ZO-1 in ipsilateral hemisphere. In conclusion, HF attenuated BBB permeability after SAH, possibly by protection of tight junction proteins.

Minocycline improves functional outcomes, memory deficits, and histopathology after endovascular perforation-induced subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) results in significant long-lasting cognitive dysfunction. Therefore, evaluating acute and long-term outcomes after therapeutic intervention is important for clinical translation. The aim of this study was to use minocycline, a known neuroprotectant agent, to evaluate the long-term benefits in terms of neurobehavior and neuropathology after experimental SAH in rats, and to determine which neurobehavioral test would be effective for long-term evaluation. SAH was induced by endovascular perforation in adult male Sprague-Dawley rats (n=118). The animals were treated with intraperitoneal injection of minocycline (45 mg/kg or 135 mg/kg) or vehicle 1 h after SAH induction. In the short-term, animals were euthanized at 24 and 72 h for evaluation of neurobehavior, brain water content, and matrix metalloproteinase (MMP) activity. In the long-term, neurobehavior was evaluated at days 21-28 post-SAH, and histopathological analysis was done at day 28. High-dose but not low-dose minocycline reduced brain water content at 24 h, and therefore only the high-dose regimen was used for further evaluation, which reduced MMP-9 activity at 24 h. Further, high-dose minocycline improved spatial memory and attenuated neuronal loss in the hippocampus and cortex. The rotarod, T-maze, and water maze tests, but not the inclined plane test, detected neurobehavioral deficits in SAH rats at days 21-28. This study demonstrates that minocycline attenuates long-term functional and morphological outcomes after endovascular perforation-induced SAH. Long-term neurobehavioral assessments using the rotarod, T-maze, and water maze tests could be useful to evaluate the efficacy of therapeutic intervention after experimental SAH.

Role of the pituitary–adrenal axis in granulocyte-colony stimulating factor-induced neuroprotection against hypoxia–ischemia in neonatal rats.

Several reports indicate that the activity of the hypothalamic–pituitary–adrenal axis (HPA) is increased after a brain insult and that its down-regulation can improve detrimental outcomes associated with ischemic brain injuries.Granulocyte-colony stimulating factor (G-CSF) is a neuroprotective drug shown in the naïve rat to regulate hormones of the HPA axis. In this study we investigate whether G-CSF confers its neuroprotective properties by influencing the HPA response after neonatal hypoxia–ischemia (HI). Following the Rice–Vannucci model, seven day old rats (P7)were subjected to unilateral carotid ligation followed by 2.5 h of hypoxia. To test our hypothesis,metyrapone was administered to inhibit the release of rodent specific glucocorticoid, corticosterone, at the adrenal level. Dexamethasone, a synthetic glucocorticoid, was administered to agonize the effects of corticosterone.Our results show that both G-CSF and metyrapone significantly reduced infarct volume while dexamethasone treatment did not reduce infarct size even when combined with G-CSF. The protective effects of G-CSF do not include blood brain barrier preservation as suggested by the brain edema results. G-CSF did not affect the pituitary released adrenocorticotropic hormone (ACTH) levels in the blood plasma at 4 h, but suppressed the increase of corticosterone in the blood. The administration of G-CSF and metyrapone increased weight gain, and significantly reduced the Bax/Bcl-2 ratio in the brain while dexamethasone reversed the effects of G-CSF. The combination of G-CSF and metyrapone significantly decreased caspase-3 protein levels in the brain, and the effect was antagonized by dexamethasone.We report that G-CSF is neuroprotective in neonatal HI by reducing infarct volume, by suppressing the HI-induced increase of the Bax/Bcl-2 ratio, and by decreasing corticosterone in the blood. Metyrapone was able to confer similar neuroprotection as G-CSF while dexamethasone reversed the effects of G-CSF. In conclusion, we show that decreasing HPA axis activity is neuroprotective after neonatal HI, which can be conferred by administering G-CSF.

Erythropoietin inhibits HIF-1α expression via upregulation of PHD-2 transcription and translation in an in vitro model of hypoxia-ischemia.

Hypoxia inducible factor (HIF)-1α is the central transcriptional factor for the regulation of oxygen-associated genes in response to hypoxia. Erythropoietin (EPO), a hematopoietic growth factor, increases oxygen availability during hypoxia/ischemia and is associated with neuroprotection following hypoxia–ischemia in laboratory models of stroke. However, EPO has failed to translate in a clinical setting. Thus, it is critical to elucidate the key players in EPO-induced neuroprotection. Our preliminary studies have shown that EPO, as a downstream gene of HIF, inhibits HIF-1α in a dose-dependent manner in an in vitro model of hypoxia–ischemia. This study is designed to elucidate the primary mediator of EPO-induced HIF-1α inhibition and subsequent cell survival/neuroprotection. Oxygen and glucose deprivation (OGD) of nerve growth factor-differentiated rat pheochromocytoma (PC-12) cells were used to model hypoxia–ischemia in an in vitro environment. The profile of HIF-1α, HIF-2α and prolyl hydroxylase domain 2 (PHD-2) expression; HIF-1α and prolyl hydroxylase (PHD-2) mRNA levels; matrix metalloproteinase (MMP)-9; and cell death was evaluated in the presence and absence of either EPO or PHD-2 inhibitor during OGD. Our findings showed that EPO treatment resulted in an increase in PHD-2 transcription and translation, inhibition of HIF-1α expression, reactive oxygen species formation, and MMP-9 activity, resulting in increased cell survival after OGD. We also observed that EPO-induced cell survival/neuroprotection was reversed by siRNA silencing of PHD-2. This led to the conclusion that PHD-2 is a key mediator of EPO-induced HIF-1α inhibition and subsequent neuroprotection in an in vitro model of hypoxia–ischemia.

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date(1,2). Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies(3). Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase(4). The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours(5). Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood(6) as well as an ICH injection model of bacterial collagenase(7), both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 μl) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p<0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Sampling of CSF via the Cisterna Magna and Blood Collection via the Heart Affects Brain Water Content in a Rat SAH Model

The aim of this study was to evaluate whether sampling of cerebrospinal fluid (CSF) via the cisterna magna and of blood via the heart affects brain water content in a rat subarachnoid hemorrhage (SAH) model. Twenty-nine animals were divided into four groups: sham-operated group with sampling of CSF and blood (Sham S+), sham-operated group without sampling of CSF and blood (Sham S−), SAH group with sampling of CSF and blood (SAH S+), and SAH without sampling of CSF and blood (SAH S−). SAH was induced via endovascular perforation of the left internal carotid artery bifurcation. Cerebrospinal fluid via the cisterna magna and blood via cardiac puncture was collected in the Sham S+ and SAH S+ groups before killing the animals for brain water content measurements. Left hemisphere brain water content was significantly higher in the SAH S− group compared with the Sham S− group (p < 0.05) and in Sham S+ group compared with the Sham S− group (p < 0.05). There was no significant difference in brain water content of the left hemisphere between the SAH S+ and Sham S+ groups (p = NS). There was no significant difference in brain water content in other parts of brains. Sampling of CSF and blood affected brain water content in Sham animals and therefore it is not accurate to use these values from Sham animals for comparison with SAH animals.