Subarachnoid Hemorrhage Mice Research Articles

Abstract T P242: Cognitive Dysfunction in a Pre-chiasmatic Cistern Blood Injection Model of Aneurysmal Subarachnoid Hemorrhage In Mice

Background: Majority of aneurysmal subarachnoid hemorrhage (SAH) survivors develop cognitive dysfunction. To better understand the underlying mechanisms and develop treatments, predictive animal models are required. We carried out a detailed physiological and cognitive characterization of pre-chiasmatic cistern (PC) and cisterna magna (CM) SAH models in mice. Methods: SAH was induced by arterial blood injection into the PC (40 μl) or CM (60 μl) in C57BL6/J mice (male, 25g). Controls received normal saline. Cerebral blood flow (CBF) was imaged using laser speckle flowmetry during and for 60 min after SAH. Intracranial pressure (ICP) and blood pressure (BP) were monitored to calculate cerebral perfusion pressure (CPP). Neurological and cognitive function was assessed 3 weeks after the injection, using pole, novel object recognition, Y maze and Morris water maze tests. Results: Mortality was 10% after PC and 4% after CM SAH. In both groups, CPP decreased from about 65 mmHg to less than 10 mmHg immediately after the injection, and recovered to 40 mmHg within 10 min after PC (n=8) and 7 min after CM (n=8) SAH (Fig A). In both groups, CBF was severely reduced to ~20% of baseline in both hemispheres immediately after SAH. CBF recovered to >40% within 5 min after PC and 2 min after CM SAH (Fig A). In saline controls (n=5 in PC and CM each), CPP and CBF changes were much milder and shorter-lasting. Compared with controls (n=12), PC SAH mice (n=12) performed significantly worse in a subset of sensorimotor and cognitive tests for up to 3 weeks (Fig B). CM SAH did not significantly impact neurological function. Conclusions: Pre-chiasmatic cistern but not cisterna magna SAH model reproduces cognitive dysfunction observed in patients with low mortality and high reproducibility in mice.

Abstract 116: Matrix Metalloproteinase-9 Deletion Attenuates Blood-Brain Barrier Disruption and Consequent Injury in White Matter After Subarachnoid Hemorrhage

Objective: The detailed mechanism of white matter injury after subarachnoid hemorrhage (SAH) remains unclear. We hypothesized that blood-brain barrier (BBB) disruption occurs in white matter after SAH and it leads to consequent injury. In this study, we investigated the potential role of matrix metalloproteinase-9 (MMP9) in SAH-induced white matter injury. Methods: SAH was induced by endovascular perforation in male wild-type (WT) C57BL/6 and MMP9 knockout (MMP9-/-) mice. The following three experiments were devised: 1) to determine BBB disruption and MMP9 activation/expression in white matter, mice underwent MRI at 24 h after SAH. They were then euthanized for immunostaining, transmission electron microscopy and MMP zymography; 2) to investigate the role of MMP9 in BBB disruption, lesion volumes on MRI were compared between WT and MMP9-/- mice at 24 h after SAH; 3) WT and MMP9-/- mice underwent MRI at 1 and 8 days after SAH induction to detect time-dependent changes, and were euthanized at 8 days after SAH. Removed brains were used to investigate myelin integrity and axonal damage in the white matter. Results: In WT mice with SAH, albumin leakage along the white matter was increased (p<0.05; vs. sham) and was consistent with T2-hyperintensity on MRI (r=0.97, p<0.01). In microvessels, tight junction detachment and swollen astrocyte endfeet were demonstrated by electron microscopy. MMP9 activity was elevated in white matter at 24 h after SAH (vs. sham; p<0.05), and MMP9 was mainly expressed in astrocytes and oligodendrocyte precursors. The volume of T2-hyperintensity in the white matter in WT mice with SAH was 6.2 ± 3.0 mm3 (p<0.001; vs. WT sham), while that in MMP9-/- mice with SAH was significantly smaller (1.0 ± 1.4 mm3; p<0.01). At 8 days after SAH, decreased myelin integrity and an increased density of damaged axons were observed in WT with SAH mice compared to normal control mice (p<0.05; for each), while T2-hyperintensity on MRI in the white matter almost disappeared. MMP9-/- mice developed less of those injuries than WT mice at 8 days after SAH (p<0.05; for each). Conclusions: SAH causes BBB disruption and consequent injury in the white matter, and MMP9 plays an important role for those pathologies. MMP9 could be a therapeutic target for SAH-induced white matter injury.

Valproic Acid treatment after experimental subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) can result in significant brain injury. Valproic acid (VPA), a widely-used anti-epileptic drug, was investigated as a possible neuroprotective drug in a prechiasmatic injection model of SAH in mice. Mice were randomized to the following experimental groups: SAH, SAH + VPA, Sham, and Sham + VPA. VPA (400 mg/kg) or saline was administered within 30 min of SAH induction and every 12 h thereafter for 48 h. Neurobehavioral assessments using the modified Garcia Score were conducted at 24 and 48 h. Brain injury was assessed at 48 h with fluoro-jade b and caspase-3/NeuN histo- and immunohistochemistry. Vasospasm was assessed in the MCA branches using hematoxylin & eosin histology. SAH mice treated with VPA appeared to have improved neurobehavioral assessments at both 24 and 48 h compared to untreated SAH mice. VPA treatment in SAH mice also significantly decreased the number of degenerating neurons on fluoro-jade b staining. In VPA-treated SAH mice, there was a trend toward a decrease in the number of apoptotic neurons on caspase-3/NeuN immunohistochemistry. VPA did not significantly affect vasospasm. This study demonstrated that VPA improves neurological outcome and decreases brain injury in a mouse model of SAH.

Endovascular perforation subarachnoid hemorrhage fails to cause Morris water maze deficits in the mouse.

Cognitive dysfunction is the primary driver of poor long-term outcome in aneurysmal subarachnoid hemorrhage (SAH) survivors; modeling such deficits preclinically is thus key for mechanistic and translational investigation. Although rat SAH causes long-term deficits in learning and memory, it remains unknown whether similar deficits are seen in the mouse, a species particularly amenable to powerful, targeted genetic manipulation. We thus subjected mice to endovascular perforation SAH and assessed long-term cognitive outcome via the Morris water maze (MWM), the most commonly used metric for rodent neurocognition. No significant differences in MWM performance (by either of two protocols) were seen in SAH versus sham mice. Moreover, SAH caused negligible hippocampal CA1 injury. These results undercut the potential of commonly used methods (of SAH induction and assessment of long-term neurocognitive outcome) for use in targeted molecular studies of SAH-induced cognitive deficits in the mouse.

In vivo and ex vivo dysfunction of neurovascular coupling in a mouse model of subarachnoid hemorrhage (676.3)

Neurovascular coupling (NVC) represents activity‐dependent focal increases in cerebral blood flow (CBF) crucial for the enhanced delivery of nutrients to maintain brain function in regions of high metabolic demand. We have previously reported inversion of NVC, with neuronal activation causing vasoconstriction rather than vasodilation, in brain slices from subarachnoid hemorrhage (SAH) model rats (Koide et al, 2012). Here, we examined ex vivo NVC, in vivo functional hyperemia and sensory motor function using a mouse endovascular perforation SAH model. In brain slices, astroctyic endfoot Ca2+ and adjoining parenchymal arteriolar diameter were measured using two‐photon and infrared‐differential interference contrast microscopy. Neuronal activation caused increased endfoot Ca2+ that was followed by an inverted neurovascular response (i.e. vasoconstriction rather than vasodilation) in ~ 80% of brain slices from day 1 and day 2 SAH mice and ~ 30% of slices from SAH day 4 mice. In vivo functional hyperemia (whisker stimulation‐induced CBF increases) measured by laser Doppler flowmetry in SAH day 1 mice was also significantly attenuated. Consistent with impaired NVC, SAH mice showed a decreased ability to perform sensory motor tasks. These data demonstrate dysfunction of neurovascular coupling occurs both ex vivo and in vivo following SAH, which may contribute to the development of neuronal deficits. Grant Funding Source: Supported by Totman Research Trust, P. Martin Endowment, NIH P01HL095488, P30RR032135 & P30GM103498.

Simvastatin increases endothelial nitric oxide synthase and ameliorates cerebral vasospasm resulting from subarachnoid hemorrhage.

Endothelial nitric oxide synthase (eNOS) activity is decreased after subarachnoid hemorrhage (SAH). Simvastatin increases eNOS activity. We hypothesized that simvastatin would increase eNOS protein and ameliorate SAH-induced cerebral vasospasm. Mice were treated with subcutaneous simvastatin or vehicle for 14 days and then subjected to endovascular perforation of the right anterior cerebral artery or sham surgery. Three days later, neurological deficits were scored (5 to 27; 27=normal), and middle cerebral artery diameter and eNOS protein were measured. The study was repeated, but simvastatin treatment was started after SAH or sham surgery. In SAH mice, simvastatin pretreatment increased middle cerebral artery diameter (SAH-simvastatin=74+/-22 micro m, SAH-vehicle=52+/-18 micro m, P=0.03; sham-simvastatin=102+/-8 micro m, sham-vehicle=105+/-6 micro m). Pretreatment reduced neurological deficits (SAH-simvastatin=25+/-2, SAH-vehicle=20+/-2, P=0.005; sham-simvastatin and sham-vehicle=27+/-0). Simvastatin pretreatment also increased eNOS protein. Simvastatin posttreatment caused a modest increase in middle cerebral artery diameter in SAH mice (SAH-simvastatin=56+/-12 micro m, SAH-vehicle=45+/-4 micro m, P=0.03; sham-simvastatin=92+/-13 micro m, sham-vehicle=99+/-10 micro m) and reduced neurological deficits (SAH-simvastatin=21+/-1, SAH-vehicle=19+/-2, P=0.009). Simvastatin posttreatment did not significantly increase eNOS protein. Simvastatin treatment before or after SAH attenuated cerebral vasospasm and neurological deficits in mice. The mechanism may be attributable in part to eNOS upregulation.