Brain injury begins early after aneurysmal subarachnoid hemorrhage (SAH). Although cell death via apoptosis and necrosis is known to be present in brain 24 hours after SAH, it is not known how soon after SAH cell death begins. We have previously described structural changes in rat brain microvessels 10 minutes after induction of SAH by endovascular puncture. This study examined brain for evidence of cell death beginning 10 minutes after induction of SAH. Cleaved caspase-3 (cl-caspase-3) staining was evident in vascular and parenchymal cells at 10 minutes 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 hour after SAH. TUNEL assay revealed apoptotic cells present at 10 minutes, with substantially more at 24 hours after SAH. Scattered Fluoro-Jade positive neurons appeared at 1 hour after SAH and their number increased with time. At 1 hour Fluoro-Jade positive neurons were present in cortical and subcortical regions but not in hippocampus; at 24 hours 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.

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.

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 10 seconds before sacrifice, and parenchymal escape of tracer was rare. At ten minutes and 3 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 24 hours posthemorrhage, indicating that vascular perfusion had recovered. Parenchymal escape of intravascular tracer was detected at 10 minutes 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 minutes after SAH and identify focal microvascular constriction and local accumulation of luminal platelet aggregates as potential initiators of that compromise.

Platelet aggregates are present in parenchymal vessels as early as 10 minutes 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 topographical 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 minutes after SAH. High-resolution three-dimensional image analysis placed many platelets at the ab-luminal (basal) side of endothelium at 10 minutes, and others either within the vascular basal lamina or in nearby parenchyma. By 24 hours 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.

Object-The role of adenosine A 2A receptors (A 2A R) in the early vascular response after subarachnoid hemorrhage (SAH) is not known. In other forms of cerebral ischemia both activation and inhibition of A 2A R is reported to be beneficial. However, these studies mainly used pharmacological receptor modulation and most of the agents available exhibit low specificity. We used adenosine A 2A receptor knockout mice to study the role of A 2A R in the early vascular response to SAH.Method-SAH was induced in the wild type (WT; C57BL/6) and A 2A R knockout mice (A 2A R-KO) by endovascular puncture. Cerebral blood flow (CBF), intracranial pressure (ICP) and blood pressure (BP) were recorded, cerebral perfusion pressure (CPP) was deduced. Animals were sacrificed at 1, 3 and 6 hours after SAH or sham surgery. Coronal brain sections were immunostained for collagen-IV; major protein of basal lamina. The internal diameter of major cerebral arteries and the area fraction of collagen-IV positive microvessels (<100μm) were determined.Results-Initial ICP rise and CPP fall at SAH induction was similar but CBF fall was significantly smaller in A 2A R-KO as compared to WT cohorts. The internal diameter of major cerebral vessels decreased progressively after SAH. The extent of diameter reduction was significantly less in A 2A R-KO than in WT mice. Collagen-IV immunostaining decreased progressively after SAH. The decrease was significantly less in A 2A R-KO than in WT.Conclusion-Our results demonstrate that global inactivation of A 2A R decreases the intensity of the early vascular response to SAH. Early inhibition of A 2A R after SAH might reduce cerebral injury.

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