John W. Calvert

Neurovascular Protection Reduces Early Brain Injury After Subarachnoid Hemorrhage

Background and Purpose— Cell death, especially apoptosis, occurred in brain tissues after subarachnoid hemorrhage (SAH). We examined the relationships between apoptosis and the disruption of blood–brain barrier (BBB), brain edema, and mortality in an established endovascular perforation model in male Sprague-Dawley rats. Methods— A pan–caspase inhibitor (z-VAD-FMK) was administered intraperitoneally at 1 hour before and 6 hours after SAH. Expression of caspase-3 and positive TUNEL was examined as markers for apoptosis. Results— Apoptosis occurred mostly in cerebral endothelial cells, partially in neurons in the hippocampus, and to a lesser degree in the cerebral cortex. Accordingly, increased BBB permeability and brain water content were observed, accompanied by neurological deficit and a high mortality at 24 hours after SAH. z-VAD-FMK suppressed TUNEL and caspase-3 staining in endothelial cells, decreased caspase-3 activation, reduced BBB permeability, relieved vasospasm, abolished brain edema, and improved neurological outcome. Conclusions— The major effect of z-VAD-FMK on early brain injury after SAH was probably neurovascular protection of cerebral endothelial cells, which results in less damage on BBB.

Neonatal Hypoxia/Ischemia Is Associated With Decreased Inflammatory Mediators After Erythropoietin Administration

Background and Purpose— Erythropoietin (EPO), a hematopoietic growth factor, has been shown to be neuroprotective when administered as either a pretreatment or posttreatment. This study tested the hypothesis that one of the mechanisms of protection afforded by posttreatment with recombinant human EPO (rh-EPO) is an anti-inflammatory effect via inhibition of interleukin (IL)-1&bgr;. Methods— Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 90 minutes of hypoxia (8% O2 at 37°C). Pups were divided into the following groups: control, hypoxia/ischemia, and hypoxia/ischemia plus rh-EPO. In the rh-EPO–treated pups, rh-EPO (5 U/g body weight IP) was administered starting 24 hours after the insult and then for 2 additional days. Samples were collected at 3, 7, 14, and 21 days after the insult. IL-1&bgr; mRNA and protein levels were determined by quantitative real-time reverse transcription–polymerase chain reaction and ELISA. Tumor necrosis factor (TNF)-&agr; mRNA levels were determined by colorimetric microplate assay. Results— rhEPO attenuated brain injury, as assessed by brain weight, and attenuated both the hypoxia/ischemia–induced increases in IL-1&bgr; mRNA and protein levels. TNF-&agr; mRNA levels did not increase at 3 to 14 days after the hypoxic/ischemic insult. Conclusions— Administration of exogenous rh-EPO starting 24 hours after a hypoxic/ischemic insult is neuroprotective in the neonatal rat. This neuroprotective activity prevented the secondary, delayed rise in IL-1&bgr; and attenuated the infiltration of leukocytes into the ipsilateral hemisphere.

Inhibition of Apoptosis by Hyperbaric Oxygen in a Rat Focal Cerebral Ischemic Model

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.

A possible role of RhoA/Rho-kinase in experimental spinal cord injury in rat.

Secondary injury following traumatic spinal cord injury is induced by the activation of a number of cellular and molecular changes. RhoA, a small GTPase, regulates the organization of the actin cytoskeleton, gene expression, cell proliferation, and has been implicated in the regenerative process. This study was undertaken to investigate the involvement of the RhoA signaling pathway in the secondary injury that follows traumatic spinal cord injury in rats. RhoA mRNA and protein expressions were enhanced significantly in the injured spinal cord 1 week after surgery (P<0.05, ANOVA). C3 exozyme (RhoA inhibitor), Y-27632 (selective Rho kinase inhibitor), and Fasudil (non-selective protein kinase inhibitor) were administered after spinal cord injury, and the subjects were evaluated for 5 weeks as per BBB locomotor score. Poor rat response interrupted the C3 experiment. Y-27632 slightly, but significantly (P<0.05, ANOVA), delayed the recovery. Fasudil significantly improved the BBB score (P<0.05, ANOVA). In conclusion, spinal cord injury activates the RhoA/Rho-kinase alpha, beta associated pathway. However, their role in secondary injury or in the improvement of functional recovery remains unclear. Fasudil might exert a cytoprotective effect by mechanisms other than inhibiting Rho-kinase alpha, beta.

Pathophysiology of an hypoxic-ischemic insult during the perinatal period.

Abstract Hypoxia–ischemia is a leading cause of morbidity and mortality in the perinatal period with an incidence of ~1/4000 live births. Biochemical events such as energy failure, membrane depolarization, brain edema, an increase of neurotransmitter release and inhibition of uptake, an increase of intracellular Ca2+, production of oxygen–free radicals, lipid peroxidation, and a decrease of blood flow are triggered by hypoxia–ischemia and may lead to brain dysfunction and neuronal death. These abnormalities can result in mental impairments, seizures, and permanent motor deficits, such as cerebral palsy. The physical and emotional strain that is placed on the children affected and their families is enormous. The care that these individuals need is not only confined to childhood, but rather extends throughout their entire life span, so it is very important to understand the pathophysiology that follows a hypoxic–ischemic insult. This review will highlight many of the mechanisms that lead to neuronal death and include the emerging area of white matter injury as well as the role of inflammation and will provide a summary of therapeutic strategies. Hypothermia and oxygen will also be discussed as treatments that currently lack a specific target in the hypoxic/ischemic cascade.

Hyperbaric oxygenation prevented brain injury induced by hypoxia-ischemia in a neonatal rat model.

The occurrence of hypoxia-ischemia (HI) during early fetal or neonatal stages of an individual leads to the damaging of immature neurons resulting in behavioral and psychological dysfunctions, such as motor or learning disabilities, cerebral palsy, epilepsy or even death. No effective treatment is currently available and this study is the first to use hyperbaric oxygen (HBO) as a treatment for neonatal HI. Herein, we sought out to determine if HBO is able to offer neuroprotectivity against an HI insult. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O(2) at 37 degrees C). HBO treatment was administered by placing pups in a chamber (3 ATA for 1 h) 1 h after hypoxia exposure. Brain injury was assessed based on ipsilateral hemispheric weight divided by contralateral hemispheric weight, light microscopy, and EM. Sensorimotor functional tests were administered at 5 weeks after hypoxia exposure. After HI, the ipsilateral hemisphere was 52.65 and 57.64% (P<0.001) of the contralateral hemisphere at 2 and 6 weeks, respectively. In HBO treated groups, the ipsilateral hemisphere was 77.77 and 84.19% (P<0.001) at 2 and 6 weeks. There was much less atrophy and apoptosis in HBO treated animals under light or electron microscopy. Sensorimotor function was also improved by HBO at 5 weeks after hypoxia exposure (Chi-square, P<0.050). The results suggest that HBO is able to attenuate the effects of HI on the neonatal brain by reducing the progression of neuronal injury and increasing sensorimotor function.

Multiple effects of hyperbaric oxygen on the expression of HIF-1α and apoptotic genes in a global ischemia–hypotension rat model

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor specifically activated by hypoxia. Activation of proapoptotic caspase-9 and caspase-3 pathways, by binding with tumor suppressor p53, HIF-1alpha could lead to harmful actions such as apoptosis. We examined whether increasing oxygen levels by hyperbaric oxygen (HBO) offers neuroprotection, at least partially by suppression of HIF-1alpha and apoptotic genes. Male SD rats (n = 78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia-hypotension (GI), and 6 groups of HBO treatment after global ischemia-hypotension (GI + HBO). HBO (3 ATA for 2 h) was applied at 1 h after global ischemia-hypotension. Rats were sacrificed at 6, 12, 24, 48, and 96 h and 7 days. Global ischemia-hypotension (10 min ischemia, 30-35 mm Hg) produced a marked increase of HIF-1alpha expressions in the hippocampus and cortex at 6 h and peaked at 48-96 h. The expressions of p53, caspase-9, and caspase-3 were all increased in a similar time course. These molecular changes were accompanied by massive cell loss in the hippocampal regions and to a lesser degree in the cortex, with features of apoptosis. HBO treatment reduced expressions of HIF-1alpha, p53, caspase-9, and caspase-3 and decreased cell death. The protein levels of proapoptotic caspase-8 and antiapoptotic bcl-2 were increased after global ischemia-hypotension and HBO potentiated the expression of caspase-8 and decreased expression of bcl-2. These results indicate that HBO has multiple actions on apoptotic genes even though the overall effect of HBO was decreased HIF-1alpha expression and reduced apoptosis after global ischemia-hypotension.

Vasospasm and p53-Induced Apoptosis in an Experimental Model of Subarachnoid Hemorrhage

Background and Purpose— Despite intensive research efforts, the etiology of vasospasm (sustained constriction of the cerebral vessels) remains unknown. In this study, we investigated the role of p53-induced apoptosis in the vasculature at 24 and 72 hours. To completely examine the apoptotic cascades, key proteins of the caspase-dependent, -independent and mitochondrial pathways were examined. Methods— In this study, adult rats were divided into 3 groups: sham (n=21), nontreatment (subarachnoid hemorrhage [SAH]+dimethyl sulfoxide; n=42), and treatment (SAH+pifithrin-α) (n=42) groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the basilar artery was harvested at 24 and 72 hours for analysis. Results— We found severe vasospasm at the 24-hour time point, which persisted to 72 hours. Furthermore, we found that the markers of the apoptotic cascades rose significantly at the 24-hour time point but had dissipated by 72 hours. However, the neurological outcome and mortality scores improved at the 72-hour time point. Conclusions— Apoptosis, and in particular p53, may play an important role in the etiology of vasospasm with relation to SAH, and in this model, vasospasm persisted to 72 hours, despite the fact that apoptosis does not.

Inhibition of Integrin αvβ3 Ameliorates Focal Cerebral Ischemic Damage in the Rat Middle Cerebral Artery Occlusion Model

Background and Purpose— Recent studies have shown that selective inhibition of specific subsets of intercellular adhesion molecules protects the brain during ischemia. We studied selective inhibition of integrin αvβ3 with cyclo [Arg-Gly-Asp-d-Phe-Val] (cRGDfV) in the rat middle cerebral artery occlusion model (MCAO). Methods— Rats were treated before and after MCAO with cRGDfV. Physiological parameters, expression of integrin αvβ3, infarction volume, brain water content, Evans Blue exudation, IgG exudation, histology, immunohistochemistry, and western blotting were studied in 4 groups of animals: sham operation (n=13), untreated (n=18), nonfunctioning peptide treatment (n=19), and cRGDfV treatment (n=27). Results— Treatment with cRGDfV reduced infarction, reduced brain edema, reduced exudation of Evans blue and IgG, and prevented fibrinogen deposition. Western blotting showed reduction of phosphorylated Flk-1 (a vascular endothelial growth factor [VEGF] receptor), reduction of phosphorylated FAK (an intracellular kinase phosphorylated in the presence of VEGF), reduction of VEGF, and reduction of fibrinogen in the cRGDfV treatment group. Conclusions— The selective integrin αvβ3 inhibitor cRGDfV improves outcomes in the MCAO model by preserving the blood-brain barrier, which mechanistically may occur in a VEGF- and VEGF-receptor–dependent manner.

Role of c-Jun N-Terminal Kinase in Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Background and Purpose— Inflammation could play a role in cerebral vasospasm after subarachnoid hemorrhage (SAH). SP600125 a c-Jun N-terminal kinase (JNK) inhibitor reduces inflammation. The present study examined if SP600125 could reduce cerebral vasospasm. Methods— Twenty-seven dogs were assigned to 5 groups: control, SAH, SAH plus dimethyl sulfoxide (DMSO), SAH plus SP600125 (10 &mgr;mol/L), and SAH plus SP600125 (30 &mgr;mol/L). SAH was induced by the injection of autologous blood into the cisterna magna on day 0 and day 2. Angiograms were evaluated on day 0 and day 7. The behavior of the dogs was evaluated daily. The activation of the JNK pathway, the infiltration of leukocytes, and the production of cytokines were also evaluated. Results— Severe vasospasm was observed in the basilar artery of SAH and DMSO dogs. The JNK signaling pathway was activated in the basilar artery after SAH and SP600125 reduced angiographic and morphological vasospasm and improved behavior scores with a concomitant reduction of infiltrated leukocytes and IL-6 production. Conclusions— These results demonstrate that SP600125 attenuated cerebral vasospasm through a suppressed inflammatory response, which may provide a novel therapeutic target for cerebral vasospasm.