Fatima A. Sehba

Mechanisms of acute brain injury after subarachnoid hemorrhage

Abstract Brain injury after subarachnoid hemorrhage (SAH) is a biphasic event with an acute ischemic insult at the time of the initial bleed and secondary events such as cerebral vasospasm 3 to 7 days later. Although much has been learned about the delayed effects of SAH, less is known about the mechanisms of acute SAH-induced injury. Distribution of blood in the subarachnoid space, elevation of intracranial pressure, reduced cerebral perfusion and cerebral blood flow (CBF) initiates the acute injury cascade. Together they lead to direct microvascular injury, plugging of vessels and release of vasoactive substances by platelet aggregates, alterations in the nitric oxide (NO)/nitric oxide synthase (NOS) pathways and lipid peroxidation. This review will summarize some of these mechanisms that contribute to acute cerebral injury after SAH.

Acute Decrease in Cerebral Nitric Oxide Levels After Subarachnoid Hemorrhage

Disturbances in the nitric oxide (NO) vasodilatory pathway have been implicated in acute vasoconstriction and ischemia after subarachnoid hemorrhage (SAH). The authors hypothesize that blood released during SAH leads to vasoconstriction by scavenging NO and limiting its availability. This was tested by measuring the major NO metabolites nitrite and nitrate in five different brain regions before and after experimental SAH. The basal NO metabolites levels were as follows (mean ± SD, μmol/mg wet weight): brain stem, 0.14 ± 0.07; cerebellum, 0.12 ± 0.08; ventral convexity cortex, 0.22 ± 0.15; dorsal convexity cortex, 0.16 ± 0.11; and hippocampus, 0.26 ± 0.17. In sham-operated animals, no effect of the nitric oxide synthase (NOS) inhibitor lG-nitro-L-arginine-methyl-ester (30 mg/kg) was found on NO metabolites 40 minutes after administration, but a significant decrease was seen after 120 minutes. The NO metabolites decreased significantly 10 minutes after SAH in all brain regions except for hippocampus, and recovered to control levels in cerebellum at 60 minutes and in brain stem and dorsal cerebral cortex 180 minutes after SAH, while remaining low in ventral convexity cortex. Nitrite recovered completely in all brain regions at 180 minutes after SAH, whereas nitrate remained decreased in brain stem and ventral convexity cortex. Our results indicate that SAH causes acute decreases in cerebral NO levels by a mechanism other than NOS inhibition and provide further support for the hypothesis that alterations in the NO vasodilatory pathway contribute directly to the ischemic insult after SAH.

Experimental models of subarachnoid hemorrhage in the rat: a refinement of the endovascular filament model

The rat endovascular filament model has been utilized to study subarachnoid hemorrhage (SAH). Because the severity of the hemorrhage with this model has proven difficult to modulate, we attempted to vary the hemorrhage by modifying filament size, and compared this model to the blood injection method with regards to acute physiological responses and hemorrhage size. SAH was achieved using either a 3-0 or 4-0 filament, or by injecting 0.3 cc of autologous blood into the cisterna magna. Peak ICP elevations were lowest in the 4-0 filament group. CBF decreased acutely and rose from its nadir in all three models with the injection model demonstrating the earliest recovery. In the injection group, mean arterial blood pressure rose acutely and remained elevated, whereas in the 3-0 group, MABP rose transiently and in the 4-0 group it did not rise significantly. Histologically, there was less subarachnoid blood in the 4-0 group vs. the injection or 3-0 groups and a different distribution of blood in the two experimental models. Varying filament size provides a method to modulate the severity of SAH in the filament model. In addition, the rat endovascular filament and blood injection models produce different distribution of blood and physiological responses.

Effects of S-Nitrosoglutathione on Acute Vasoconstriction and Glutamate Release After Subarachnoid Hemorrhage

BACKGROUND AND PURPOSE Subarachnoid hemorrhage (SAH) causes acute vasoconstriction that contributes to ischemic brain injury shortly after the initial bleed. It has been theorized that decreased availability of nitric oxide (NO) may contribute to acute vasoconstriction. Therefore we examined the effect of the NO donor N-nitroso glutathione (GSNO) on acute vasoconstriction and early ischemic glutamate release after experimental SAH. METHODS SAH was induced by the endovascular suture method in anesthetized rats. GSNO (1 micromol/L/kg, n=31) or saline (n=21) was injected 5 minutes after SAH. Sham-operated rats received GSNO (1 micromol/L/kg, n=5) 5 minutes after sham surgery. Arterial and intracranial pressures, cerebral blood flow (CBF), and extracellular glutamate release were measured serially for 60 minutes after SAH. SAH size was determined, and vascular measurements were made histologically. RESULTS GSNO had no effect on resting blood pressure, intracranial pressure, cerebral perfusion pressure, or CBF in sham-operated animals. However, administration of GSNO after SAH was associated with significantly increased CBF (161.6+/-26.6% versus saline 37.1+/-5.5%, 60 minutes after SAH, P<0.05), increased blood vessel diameter (internal carotid artery [ICA] 285.0+/-16.5 microm versus saline 149.2+/-14.1 microm, P<0.01), decreased vessel wall thickness (ICA12.9+/-0.7 microm versus saline 25.1+/-1.6 microm, P<0.01), and decreased extracellular glutamate levels (3315.6+/-1048.3% versus saline469. 7+/-134.3%, P<0.05). Blood pressure decreased transiently, whereas intracranial pressure, cerebral perfusion pressure, and SAH size were not affected. CONCLUSIONS These results suggest that GSNO can reverse acute vasoconstriction and prevent ischemic brain injury after SAH. This further implies that acute vasoconstriction contributes significantly to ischemic brain injury after SAH and is mediated in part by decreased availability of NO.

Decreased nitric oxide availability contributes to acute cerebral ischemia after subarachnoid hemorrhage.

OBJECTIVE Disturbances of the L-arginine-nitric oxide (NO) vasodilatory pathway have been implicated as a cause of acute vasoconstriction and ischemia after subarachnoid hemorrhage (SAH). Because NO-dependent vasodilatory mechanisms are still intact in this setting, acute vasoconstriction may be the result of limited NO availability after SAH. The present study examines this hypothesis by administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). METHODS SAH was induced by the endovascular suture method in anesthetized rats. L-NAME (30 mg/kg intravenously) was injected 20 minutes before or 15, 30, or 60 minutes after SAH. Control rats received normal saline. Arterial and intracranial pressure and cerebral blood flow (CBF) were measured continuously for 60 minutes after SAH. RESULTS L-NAME administration 20 minutes before SAH produced a significant decrease in resting CBF (29.4 +/- 3.4%; P < 0.05), but it had no effect on the acute decrease in CBF after SAH or on its early recovery up to 30 minutes after SAH. However, a significant decrease in CBF recovery was found in animals receiving L-NAME injections (28.7 +/- 9.4%; P < 0.05 versus controls) 60 minutes after SAH. Administration of L-NAME 15 or 30 minutes after SAH had no effect on CBF recovery, as compared with controls. However, when administered 60 minutes after SAH, L-NAME decreased CBF significantly (45.4 +/- 8.8%; P < 0.05 versus controls). CONCLUSION These results indicate a biphasic pattern of NO availability after SAH. NO-mediated vasodilation is limited during the first 30 minutes of SAH and is restored 60 minutes after SAH.

Reduction of neutrophil activity decreases early microvascular injury after subarachnoid haemorrhage

BackgroundSubarachnoid haemorrhage (SAH) elicits rapid pathological changes in the structure and function of parenchymal vessels (≤ 100 μm). The role of neutrophils in these changes has not been determined. This study investigates the role of neutrophils in early microvascular changes after SAHMethodRats were either untreated, treated with vinblastine or anti-polymorphonuclear (PMN) serum, which depletes neutrophils, or treated with pyrrolidine dithiocarbamate (PDTC), which limits neutrophil activity. SAH was induced by endovascular perforation. Neutrophil infiltration and the integrity of vascular endothelium and basement membrane were assessed immunohistochemically. Vascular collagenase activity was assessed by in situ zymography.ResultsVinblastine and anti-PMN serum reduced post-SAH accumulation of neutrophils in cerebral vessels and in brain parenchyma. PDTC increased the neutrophil accumulation in cerebral vessels and decreased accumulation in brain parenchyma. In addition, each of the three agents decreased vascular collagenase activity and post-SAH loss of vascular endothelial and basement membrane immunostaining.ConclusionsOur results implicate neutrophils in early microvascular injury after SAH and indicate that treatments which reduce neutrophil activity can be beneficial in limiting microvascular injury and increasing survival after SAH.

Cell death starts early after subarachnoid hemorrhage.

Brain injury begins early after aneurysmal subarachnoid hemorrhage (SAH). Although cell death via apoptosis and necrosis is known to be present in brain 24 h after SAH, it is not known how soon after SAH cell death begins. We have previously described structural changes in rat brain microvessels 10 min after induction of SAH by endovascular puncture. This study examined brain for evidence of cell death beginning 10 min after induction of SAH. Cleaved caspase-3 (cl-caspase-3) staining was evident in vascular and parenchymal cells at 10 min after SAH and was significantly greater than in time-matched, sham-operated controls. The number of cl-caspase-3 positive cells was increased further at 24 h after SAH. TUNEL assay revealed apoptotic cells present at 10 min, with substantially more at 24 h after SAH. Scattered Fluoro-Jade positive neurons appeared at 1h after SAH and their number increased with time. At 1 h Fluoro-Jade positive neurons were present in cortical and subcortical regions but not in hippocampus; at 24h they were also present in hippocampus and were significantly greater in the hemisphere ipsilateral to the vascular puncture. No Fluoro-Jade staining was present in shams. These data demonstrate an early activation of endothelial and parenchymal cells apoptosis and neuronal necrosis after SAH and identifies endpoints that can be targeted to reduce early brain injury after SAH.

Escape of intraluminal platelets into brain parenchyma after subarachnoid hemorrhage.

Platelet aggregates are present in parenchymal vessels as early as 10 min after experimental subarachnoid hemorrhage (SAH). Structural injury to parenchymal vessel walls and depletion of collagen-IV (the major protein of basal lamina) occur in a similar time frame. Since platelets upon activation release enzymes which can digest collagen-IV, we investigated the topographic relationship between platelet aggregates, endothelium, and basal lamina after SAH produced by endovascular perforation, using triple immunofluorescence and confocal microscopy with deconvolution. The location of platelet aggregates in relation to zymography-detected active collagenase was also examined. As reported previously, most cerebral vessels profiles contained platelets aggregates at 10 min after SAH. High-resolution three-dimensional image analysis placed many platelets at the ab-luminal (basal) side of endothelium at 10 min, and others either within the vascular basal lamina or in nearby parenchyma. By 24 h post hemorrhage, large numbers of platelets had entered the brain parenchyma. The vascular sites of platelet movement were devoid of endothelium and collagen-IV. Collagenase activity colocalized with vascular platelet aggregates. Our data demonstrate that parenchymal entry of platelets into brain parenchyma begins within minutes after hemorrhage. Three-dimensional analysis suggests that platelet aggregates initiate or stimulate local disruption of endothelium and destruction of adjacent basal lamina after SAH.

Acute ischaemia after subarachnoid haemorrhage, relationship with early brain injury and impact on outcome: a prospective quantitative MRI study

Objective To determine if ischaemia is a mechanism of early brain injury at the time of aneurysm rupture in subarachnoid haemorrhage (SAH) and if early MRI ischaemia correlates with admission clinical status and functional outcome. Methods In a prospective, hypothesis-driven study patients with SAH underwent MRI within 0–3 days of ictus (prior to vasospasm) and a repeat MRI (median 7 days). The volume and number of diffusion weighted imaging (DWI) positive/apparent diffusion coefficient (ADC) dark lesions on acute MRI were quantitatively assessed. The association of early ischaemia, admission clinical status, risk factors and 3-month outcome were analysed. Results In 61 patients with SAH, 131 MRI were performed. Early ischaemia occurred in 40 (66%) with a mean DWI/ADC volume 8.6 mL (0–198 mL) and lesion number 4.3 (0–25). The presence of any early DWI/ADC lesion and increasing lesion volume were associated with worse Hunt-Hess grade, Glasgow Coma Scale score and Acute Physiology and Chronic Health Evaluation II physiological subscores (all p<0.05). Early DWI/ADC lesions significantly predicted increased number and volume of infarcts on follow-up MRI (p<0.005). At 3 months, early DWI/ADC lesion volume was significantly associated with higher rates of death (21% vs 3%, p=0.031), death/severe disability (modified Rankin Scale 4–6; 53% vs 15%, p=0.003) and worse Barthel Index (70 vs 100, p=0.004). After adjusting for age, Hunt-Hess grade and aneurysm size, early infarct volume correlated with death/severe disability (adjusted OR 1.7, 95% CI 1.0 to 3.2, p=0.066). Conclusions Early ischaemia is related to poor acute neurological status after SAH and predicts future ischaemia and worse functional outcomes. Treatments addressing acute ischaemia should be evaluated for their effect on outcome.

Luminal platelet aggregates in functional deficits in parenchymal vessels after subarachnoid hemorrhage

The pathophysiology of early ischemic injury after aneurysmal subarachnoid hemorrhage (SAH) is not understood. This study examined the acute effect of endovascular puncture-induced SAH on parenchymal vessel function in rat, using intravascular fluorescent tracers to assess flow and vascular permeability and immunostaining to assess structural integrity and to visualize platelet aggregates. In sham-operated animals, vessels were well filled with tracer administered 10s before sacrifice, and parenchymal escape of tracer was rare. At ten minutes and three hours after hemorrhage, patches of poor vascular filling were distributed throughout the forebrain. Close examination of these regions revealed short segments of narrowed diameter along many profiles. Most vascular profiles with reduced perfusion contained platelet aggregates and in addition showed focal loss of collagen IV, a principal component of basal lamina. In contrast, vessels were well filled at 24h post-hemorrhage, indicating that vascular perfusion had recovered. Parenchymal escape of intravascular tracer was detected at 10 min post-hemorrhage and later as plumes of fluorescence emanating into parenchyma from restricted microvascular foci. These data demonstrate that parenchymal microvessels are compromised in function by 10 min after SAH and identify focal microvascular constriction and local accumulation of luminal platelet aggregates as potential initiators of that compromise.