Robert Ayer

A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model

The endovascular perforation rodent model for experimental subarachnoid hemorrhage (SAH) studies is criticized for lack of control over bleeding. Presently, there is no practical grading system to categorize the severity of SAH depending on the amount of blood. We outline a simple and objective novel SAH grading system by examining the subarachnoid blood clots in the basal cisterns, and evaluate for correlation with neurological status and cerebral vasospasm. Effects of simvastatin, known to reduce vasospasm, were examined using this grading system. Seventy-seven adult male Sprague-Dawley rats were divided randomly into three groups: sham-operated (n=24), SAH (n=32), and SAH+simvastatin (n=25). High-resolution brain pictures were used to grade the severity of SAH and categorize animals into mild, moderate and severe groups. The SAH grades were compared with neurological scores and internal carotid artery parameters such as diameter, perimeter and wall thickness at 24h. Two investigators verified the grading system independently. The SAH grade showed linear correlation functionally with neurological status (r=0.42, p<0.01) and morphometrically with the degree of vasospasm (|r|>0.7, p<0.01), and also between two independent investigators (r=0.937, p<0.001). Simvastatin improved neurological score in moderate and severe (p<0.05) but not mild SAH groups (p=0.28). This grading system has the potential to be adopted for SAH experimental rodent models.

Oxidative stress in subarachnoid haemorrhage: significance in acute brain injury and vasospasm

Aneurismal subarachnoid haemorrhage (SAH) is a devastating disease that is associated with significant morbidity and mortality. The mortality is approximately 50%, with 30% of survivors having significant morbidity. There is substantial evidence to suggest that oxidative stress is significant in the development of acute brain injury and cerebral vasospasm following SAH. There are several sources for the excessive generation of free radicals following SAH, including disrupted mitochondrial respiration and extracellular hemoglobin. There is also the upregulation of free radical producing enzymes such as inducible nitric oxide synthase (iNOS), xanthine oxidase, NADPH oxidase (NOX), as well as enzymes involved in the metabolism of arachidonic acid. Additionally, intrinsic antioxidant systems such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are inhibited. Experiments have linked free radicals to the apoptosis of neurons and endothelial cells, BBB breakdown and the altered contractile response of cerebral vessels following SAH. Antioxidant therapy has provided neuroprotection and antispasmotic effects in experimental SAH and some therapies have demonstrated improved outcomes in clinical trials. These studies have laid a foundation for the use of antioxidants in the treatment of aneurismal SAH.

Matrix metalloproteinases inhibition provides neuroprotection against hypoxia-ischemia in the developing brain

The present study was designed to investigate the role of matrix metalloproteinases (MMPs) in the immature brain and the long term effects of early MMPs inhibition after hypoxic‐ischemic (HI) injury. HI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O2 for 2 h) in P7 rat pups. GM6001, a broad spectrum MMPs inhibitor, was injected (50 mg/kg or 100 mg/kg) intraperitoneally at 2 h and 24 h after HI injury. Blood‐brain barrier (BBB) integrity, brain edema, MMP‐2/‐9 activity, TIMP‐1/‐2 and tight junction protein (TJP) level were evaluated using IgG staining, Evan’s blue extravasation, brain water content, zymography and western blot. Doxycycline, another MMPs inhibitor, was injected (10 mg/kg or 30 mg/kg) intraperitoneally at 2 h after HI, then BBB integrity and brain edema were measured at 48 h post‐HI using brain water content measurement and IgG staining. The long‐term effects of early MMPs inhibition (GM6001, 100 mg/kg) were evaluated by neurobehavioral tests, body weight, and brain atrophy measurement. GM6001 attenuated brain edema and BBB disruption at the dosage of 100 mg/kg. MMP‐2 activity increased at 24 h and peaked at 48 h after HI, whereas MMP‐9 activity peaked at 24 h and tapered by 48 h after HI. MMP‐9/‐2 activities were significantly attenuated by GM6001 at 24 h and 48 h after HI. The degradation of TJPs (ZO‐1 and occludin) at 48 h after HI was reversed by GM6001 treatment. Early MMPs inhibition had long‐term effects that attenuated ipsilateral brain tissue loss, and improved neurobehavioral outcomes after HI. These results suggest that early MMPs inhibition with a broad‐spectrum inhibitor provides both acute and long‐term neuroprotection in the developing brain by reducing TJPs degradation, preserving BBB integrity, and ameliorating brain edema after neonatal HI injury.

Simvastatin Attenuation of Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats Via Increased Phosphorylation of Akt and Endothelial Nitric Oxide Synthase

The mechanisms involved in simvastatin‐mediated attenuation of cerebral vasospasm after subarachnoid hemorrhage (SAH) are unclear. We investigated the role of the phosphatidylinositol 3‐kinase/Akt (PI3K/Akt) pathway and endothelial nitric oxide synthase (eNOS) in the cerebral vasculature in statin‐mediated attenuation of cerebral vasospasm using wortmannin, an irreversible pharmacological PI3K inhibitor, and a rat SAH endovascular perforation model. Simvastatin was administered intraperitoneally in two dosages (1 mg/kg and 20 mg/kg) at 0.5, 24, and 48 hr after SAH and histological parameters of ipsilateral intracranial carotid artery (ICA) were assessed at 24 and 72 hr. SAH significantly decreased ICA diameter and perimeter while increasing wall thickness at both 24 and 72 hr. High‐dosage simvastatin prevented the reduction of ICA diameter and perimeter following SAH, whereas both high and low dosages reduced wall thickness significantly at 24 and 72 hr. The effects of simvastatin were significantly reversed by wortmannin. High‐dosage simvastatin increased pAkt and peNOS (phosphorylated forms) levels without increasing Akt and eNOS expression compared with the SAH group and also improved neurological deficits at 24 and 72 hr. Simvastatin did not affect protein levels by itself compared with untreated sham group. The present study elucidates the critical role of the PI3K activation leading to phosphorylation of Akt and eNOS in simvastatin‐mediated attenuation of cerebral vasospasm after SAH.

Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats

Objective: Accumulated evidence suggests that the primary cause of poor outcome after subarachnoid hemorrhage is not only cerebral arterial narrowing but also early brain injury. Our objective was to determine the effect of recombinant osteopontin, a pleiotropic extracellular matrix glycoprotein, on early brain injury after subarachnoid hemorrhage in rats. Design: Controlled in vivo laboratory study. Setting: Animal research laboratory. Subjects: One hundred seventy-seven male adult Sprague-Dawley rats weighing 300 to 370 g. Interventions: The endovascular perforation model of subarachnoid hemorrhage was produced. Subarachnoid hemorrhage or sham-operated rats were treated with an equal volume (1 &mgr;L) of pre-subarachnoid hemorrhage intracerebroventricular administration of two dosages (0.02 and 0.1 &mgr;g) of recombinant osteopontin, albumin, or vehicle. Body weight, neurologic scores, brain edema, and blood–brain barrier disruption were evaluated, and Western blot analyses were performed to determine the effect of recombinant osteopontin on matrix metalloproteinase-9, substrates of matrix metalloproteinase-9 (zona occludens-1, laminin), tissue inhibitor of matrix metalloproteinase-1, inflammation (interleukin-1&bgr;), and nuclear factor-&kgr;B signaling pathways. Measurements and Main Results: Treatment with recombinant osteopontin prevented a significant loss in body weight, neurologic impairment, brain edema, and blood–brain barrier disruption after subarachnoid hemorrhage. These effects were associated with the deactivation of nuclear factor-&kgr;B activity, inhibition of matrix metalloproteinase-9 induction, the maintenance of tissue inhibitor of matrix metalloproteinase-1, the consequent preservation of the cerebral microvessel basal lamina protein laminin, and the tight junction protein zona occludens-1. Conclusions: These results demonstrate that recombinant osteopontin treatment is effective for early brain injury after subarachnoid hemorrhage.

Protective Effect of Melatonin upon Neuropathology, Striatal Function, and Memory Ability after Intracerebral Hemorrhage in Rats

Since free radicals play a role in the mechanisms of brain injury after hemorrhagic stroke, the effect of melatonin (a potent antioxidant and free-radical scavenger) on outcomes was investigated after intracerebral hemorrhage (ICH) in rats. ICH was induced by clostridial collagenase infusion into the right caudate putamen, and several time points and doses of melatonin were studied. Brain edema and neurological function at 24 h were unchanged in comparison with vehicle-treated groups, in spite of oxidative stress reductions. Repeated treatment with the lower dose of melatonin (5 mg/kg) given at 1 h and every 24 h thereafter for 3 days after ICH, led to normalization of striatal function and memory ability over the course of 8 weeks, and less brain atrophy 2 weeks later. These results suggest that melatonin is safe for use after ICH, reduces oxidative stress, provides brain protection, and could be used for future investigations of free radical mechanisms after cerebral hemorrhage.

Thrombin Inhibition by Argatroban Ameliorates Early Brain Injury and Improves Neurological Outcomes After Experimental Subarachnoid Hemorrhage in Rats

Background and Purpose— We investigated the role of thrombin in early brain injury after subarachnoid hemorrhage (SAH). Methods— The standard intravascular perforation model was used to produce experimental SAH in Sprague Dawley rats. Low-dose (0.3 mg/h) and high-dose (0.9 mg/h) argatroban, a direct thrombin inhibitor, were evaluated for effects on brain edema, blood–brain barrier (BBB) disruption, apoptotic cell death, inflammatory marker, and neurological outcomes after SAH. Results— Both doses of argatroban attenuated BBB disruption; however, only high-dose was effective in lowering edema in all brain regions, reducing cell death, and inflammatory marker expression, and improving neurological outcomes. Conclusions— Thrombin inhibition by argatroban improves neurological outcomes and provides neuroprotection against acute events after SAH such as BBB disruption, brain edema, and cell death.

Melatonin decreases mortality following severe subarachnoid hemorrhage

Abstract:  Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease that is associated with significant morbidity and mortality. There is substantial evidence to suggest that oxidative stress is significant in the development of acute brain injury following SAH. Melatonin is a strong antioxidant that has low toxicity and easily passes through the blood–brain barrier. Previous studies have shown that melatonin provides neuroprotection in animal models of ischemic stroke. This study hypothesizes that melatonin will provide neuroprotection when administered 2 hr after SAH. The filament perforation model of SAH was performed in male Sprague–Dawley rats weighing between 300 and 380 g. Melatonin (15 or 150 mg/kg), or vehicle was given via intraperitoneal injection 2 hr after SAH. Mortality and neurologic deficits were assessed 24 hr after SAH. A significant reduction in 24‐hr mortality was seen following treatment with high dose melatonin. There was no improvement in neurologic scores with treatment. Brain water content and lipid peroxidation were measured following the administration of high dose melatonin to identify a mechanism for the increased survival. High dose melatonin tended to reduce brain water content following SAH, but had no effect on the lipid peroxidation of brain samples. Large doses of melatonin significantly reduces mortality and brain water content in rats following SAH through a mechanism unrelated to oxidative stress.

The clinical significance of acute brain injury in subarachnoid hemorrhage and opportunity for intervention

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating neurological event that accounts for 3-7% of all strokes and carries a mortality rate as high as 40%. Delayed cerebral vasospasm has traditionally been recognized as the most treatable cause of morbidity and mortality from SAH. However, evidence is mounting that the physiological and cellular events of acute brain injury, which occur during the 24-72 h following aneurysm rupture, make significant contributions to patient outcomes, and may even be a more significant factor than delayed cerebral vasospasm. Acute brain injury in aneurysmal SAH is the result of physiological derangements such as increased intracranial pressure and decreased cerebral blood flow that result in global cerebral ischemia, and lead to the acute development of edema, oxidative stress, inflammation, apoptosis, and infarction. The consequence of these events is often death or significant neurological disability. In this study of acute brain injury, we elucidate some of the complex molecular signaling pathways responsible for these poor outcomes. Continued research in this area and the development of therapies to interrupt these cascades should be a major focus in the future as we continue to seek effective therapies for aneurysmal SAH.

The Neuroprotective Effects of Cyclooxygenase-2 Inhibition in a Mouse Model of Aneurysmal Subarachnoid Hemorrhage

The CNS inflammatory reaction occurring after aneurysmal subarachnoid hemorrhage (SAH) involves the upregulation of numerous cytokines and prostaglandins. Cyclooxygenase (COX) inhibition is a well-established pharmacological anti-inflammatory agent. Previous studies have shown marked increases in COX-2 expression in neurons, astrocytes, microglia, and endothelial cells following brain injury. COX-2 inhibition has been shown to be beneficial following various types of brain injury. This experiment investigates the role of COX-2 activity in early brain injury following SAH. CD-1 mice were subjected to an endovascular perforation model of SAH or SHAM surgery. Following experimental SAH animals were treated with the specific COX-2 inhibitor, NS398, in dosages of either 10 or 30 mg/kg. Neurological performance and brain edema were evaluated 24 and 72 h after SAH. NS398 at 30 mg/kg significantly reduced SAH-induced neurological deterioration. NS 398 at 30 mg/kg resulted in a trend toward the reduction of SAH-induced cerebral edema. Treatment had no effect on mortality. This experiment provides preliminary evidence that COX-2 inhibition is an effective pharmacological intervention for the prevention of brain edema and the preservation of neurological function following SAH.