Paul R. Krafft

Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage.

T-lymphocytes promote cerebral inflammation, thus aggravating neuronal injury after stroke. Fingolimod, a sphingosine 1-phosphate receptor analog, prevents the egress of lymphocytes from primary and secondary lymphoid organs. Based on these findings, we hypothesized fingolimod treatment would reduce the number of T-lymphocytes migrating into the brain, thereby ameliorating cerebral inflammation following experimental intracerebral hemorrhage (ICH). We investigated the effects of fingolimod in two well-established murine models of ICH, implementing intrastriatal infusions of either bacterial collagenase (cICH) or autologous blood (bICH). Furthermore, we tested the long term neurological improvements by Fingolimod in a collagenase-induced rat model of ICH. Fingolimod, in contrast to vehicle administration alone, improved neurological functions and reduced brain edema at 24 and 72 h following experimental ICH in CD-1 mice (n=103; p<0.05). Significantly fewer lymphocytes were found in blood and brain samples of treated animals when compared to the vehicle group (p<0.05). Moreover, fingolimod treatment significantly reduced the expression of intercellular adhesion molecule-1 (ICAM-1), interferon-γ (INF-γ), and interleukin-17 (IL-17) in the mouse brain at 72 h post-cICH (p<0.05 compared to vehicle). Long-term neurocognitive performance and histopathological analysis were evaluated in Sprague-Dawley rats between 8 and 10 weeks post-cICH (n=28). Treated rats showed reduced spatial and motor learning deficits, along with significantly reduced brain atrophy and neuronal cell loss within the basal ganglia (p<0.05 compared to vehicle). We conclude that fingolimod treatment ameliorated cerebral inflammation, at least to some extent, by reducing the availability and subsequent brain infiltration of T-lymphocytes, which improved the short and long-term sequelae after experimental ICH in rodents.

P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH.

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1β release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

Isoflurane Attenuates Blood–Brain Barrier Disruption in Ipsilateral Hemisphere After Subarachnoid Hemorrhage in Mice

Background and Purpose— We examined effects of isoflurane, volatile anesthetics, on blood–brain barrier disruption in the endovascular perforation model of subarachnoid hemorrhage (SAH) in mice. Methods— Animals were assigned to sham-operated, SAH+vehicle–air, SAH+1%, or 2% isoflurane groups. Neurobehavioral function, brain water content, Evans blue dye extravasation, and Western blotting for sphingosine kinases, occludin, claudin-5, junctional adhesion molecule, and vascular endothelial cadherin were evaluated at 24 hours post-SAH. Effects of sphingosine kinase (N,N-dimethylsphingosine) or sphingosine-1-phosphate receptor-1/3 (S1P1/3) inhibitors (VPC23019) on isofluranes action were also examined. Results— SAH aggravated neurological scores, brain edema, and blood–brain barrier permeability, which were prevented by 2% but not 1% isoflurane posttreatment. Two percent isoflurane increased sphingosine kinase-1 expression and prevented a post-SAH decrease in expressions of the blood–brain barrier-related proteins. Both N,N-dimethylsphingosine and VPC23019 abolished the beneficial effects of isoflurane. Conclusions— Two percent isoflurane can suppress post-SAH blood–brain barrier disruption, which may be mediated by sphingosine kinase 1 expression and sphingosine-1-phosphate receptor-1/3 activation.

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.

Etiology of stroke and choice of models

Animal models of stroke contribute to the development of better stroke prevention and treatment through studies investigating the pathophysiology of different stroke subtypes and by testing promising treatments before trials in humans. There are two broad types of animal models: those in which stroke is induced through artificial means, modeling the consequences of a vascular insult but not the vascular pathology itself; and those in which strokes occur spontaneously. Most animal models of stroke are in rodents due to cost, ethical considerations, availability of standardized neurobehavioral assessments, and ease of physiological monitoring. While there are similarities in cerebrovascular anatomy and pathophysiology between rodents and humans, there are also important differences, including brain size, length and structure of perforating arteries, and gray to white matter ratio, which is substantially lower in humans. The wide range of rodent models of stroke includes models of global and focal ischemia, and of intracerebral and sub-arachnoid hemorrhage. The most widely studied model of spontaneous stroke is the spontaneously hypertensive stroke-prone rat, in which the predominant lesions are small subcortical infarcts resulting from a vascular pathology similar to human cerebral small vessel disease. Important limitations of animal models of stroke – they generally model only certain aspects of the disease and do not reflect the heterogeneity in severity, pathology and comorbidities of human stroke – and key methodological issues (especially the need for adequate sample size, randomization, and blinding in treatment trials) must be carefully considered for the successful translation of pathophysiological concepts and therapeutics from bench to bedside.

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.

Rodent neonatal germinal matrix hemorrhage mimics the human brain injury, neurological consequences, and post-hemorrhagic hydrocephalus

Germinal matrix hemorrhage (GMH) is the most common neurological disease of premature newborns. GMH causes neurological sequelae such as cerebral palsy, post-hemorrhagic hydrocephalus, and mental retardation. Despite this, there is no standardized animal model of spontaneous GMH using newborn rats to depict the condition. We asked whether stereotactic injection of collagenase type VII (0.3 U) into the ganglionic eminence of neonatal rats would reproduce the acute brain injury, gliosis, hydrocephalus, periventricular leukomalacia, and attendant neurological consequences found in humans. To test this hypothesis, we used our neonatal rat model of collagenase-induced GMH in P7 pups, and found that the levels of free-radical adducts (nitrotyrosine and 4-hyroxynonenal), proliferation (mammalian target of rapamycin), inflammation (COX-2), blood components (hemoglobin and thrombin), and gliosis (vitronectin and GFAP) were higher in the forebrain of GMH pups, than in controls. Neurobehavioral testing showed that pups with GMH had developmental delay, and the juvenile animals had significant cognitive and motor disability, suggesting clinical relevance of the model. There was also evidence of white-matter reduction, ventricular dilation, and brain atrophy in the GMH animals. This study highlights an instructive animal model of the neurological consequences after germinal matrix hemorrhage, with evidence of brain injuries that can be used to evaluate strategies in the prevention and treatment of post-hemorrhagic complications.

Hyperbaric oxygen preconditioning attenuates hyperglycemia-enhanced hemorrhagic transformation by inhibiting matrix metalloproteinases in focal cerebral ischemia in rats

Hyperglycemia dramatically aggravates brain infarct and hemorrhagic transformation (HT) after ischemic stroke. Oxidative stress and matrix metalloproteinases (MMPs) play an important role in the pathophysiology of HT. Hyperbaric oxygen preconditioning (HBO-PC) has been proved to decrease oxidative stress and has been demonstrated to be neuroprotective in experimental stroke models. The present study determined whether HBO-PC would ameliorate HT by a pre-ischemic increase of reactive oxygen species (ROS) generation, and a suppression of MMP-2 and MMP-9 in hyperglycemic middle cerebral artery occlusion (MCAO) rats. Rats were pretreated with HBO (100% O₂, 2.5 atmosphere absolutes) 1 h daily for 5 days before MCAO. Acute hyperglycemia was induced by an injection of 50% dextrose. Neurological deficits, infarction volume and hemorrhagic volume were assessed 24 h and 7 days after ischemia. ROS scavenger n-acetyl cysteine (NAC), hypoxia-inducible factor-1α (HIF-1α), inhibitor 2-methoxyestradiol (2ME2) and activator cobalt chloride (CoCl₂), and MMP inhibitor SB-3CT were administrated for mechanism study. The activity of MMP-2 and MMP-9, and the expression HIF-1α were measured. HBO-PC improved neurological deficits, and reduced hemorrhagic volume; the expression of HIF-1α was significantly decreased, and the activity of MMP-2 and MMP-9 was reduced by HBO-PC compared with vehicle group. Our results suggested that HBO-PC attenuated HT via decreasing HIF-1α and its downstream MMP-2 and MMP-9 in hyperglycemic MCAO rats.

Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice.

Fibroblast growth factors (FGFs) maintain and promote vascular integrity; however whether FGFs protect the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unexplored. In this present study, we hypothesized that exogenous FGF administration attenuates brain injury after ICH, specifically by preserving endothelial adherens junctions, therefore reducing vasogenic brain edema and attenuating neurofunctional deficits in mice subjected to experimental ICH. Acid fibroblast growth factor (FGF1) or basic fibroblast growth factor (FGF2) was administered intracerebroventricularly (ICV) at 0.5 h after intrastriatal injection of bacterial collagenase (cICH) or autologous whole blood (bICH). Fibroblast growth factor receptor (FGFR) inhibitor PD173074 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were additionally administered with FGF2. The selective Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor Y27632 was independently administered at 0.5 h after cICH. Brain water content and neurofunctional deficits were evaluated at 24 and 72h after ICH induction. Evans blue extravasation as well as Western blot analysis for the quantification of activated FGFR, Akt, Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and adherens junction proteins (p120-catenin, β-catenin and VE-cadherin) were conducted at 72 h post-cICH. FGF treatment reduced perihematomal brain edema and improved neurofunctional deficits at 72 h after experimental ICH (p<0.05, compared to vehicle); however, FGFR and PI3K inhibition reversed these neuroprotective effects. Exogenous FGF2 increased activated FGFR, Akt, and Rac1 but reduced activated RhoA protein expression at 72 h after cICH (p<0.05, compared to vehicle), which was reversed by FGFR and PI3K inhibition. Y27632 treatment reduced brain injury at 72 h after cICH (p<0.05, compared to vehicle) and increased the expression of catenins (p120-catenin, β-catenin). In conclusion, our findings suggest that exogenous FGF treatment reduced RhoA activity via FGFR-induced activation of the PI3K-Akt-Rac1 signaling pathway, thus preserving BBB integrity, and therefore attenuating secondary brain injury after experimental ICH in mice.