Contribution of Matrix Metalloproteinase-9 to Cerebral Edema and Functional Outcome following Experimental Subarachnoid Hemorrhage

Background: Cerebral edema is an important risk factor for death and poor outcome following subarachnoid hemorrhage (SAH). However, underlying mechanisms are still poorly understood. Matrix metalloproteinase (MMP)-9 is held responsible for the degradation of microvascular basal lamina proteins leading to blood-brain barrier dysfunction and, thus, formation of vasogenic cerebral edema. The current study was conducted to clarify the role of MMP-9 for the development of cerebral edema and for functional outcome after SAH. Methods: SAH was induced in FVB/N wild-type (WT) or MMP-9 knockout (MMP-9(-/-)) mice by endovascular puncture. Intracranial pressure (ICP), regional cerebral blood flow (rCBF), and mean arterial blood pressure (MABP) were continuously monitored up to 30 min after SAH. Mortality was quantified for 7 days after SAH. In an additional series neurological function and body weight were assessed for 3 days after SAH. Subsequently, ICP and brain water content were quantified. Results: Acute ICP, rCBF, and MABP did not differ between WT and MMP-9(-/-) mice, while 7 days' mortality was lower in MMP-9(-/-) mice (p = 0.03; 20 vs. 60%). MMP-9(-/-) mice also exhibited better neurological recovery, less brain edema formation, and lower chronic ICP. Conclusions: The results of the current study suggest that MMP-9 contributes to the development of early brain damage after SAH by promoting cerebral edema formation. Hence, MMP-9 may represent a novel molecular target for the treatment of SAH. Copyright (C) 2011 S. Karger AG, Base

Inhaled Nitric Oxide Treatment for Aneurysmal SAH Patients With Delayed Cerebral Ischemia

BACKGROUND: We demonstrated experimentally that inhaled nitric oxide (iNO) dilates hypoperfused arterioles, increases tissue perfusion, and improves neurological outcome following subarachnoid hemorrhage (SAH) in mice. We performed a prospective pilot study to evaluate iNO in patients with delayed cerebral ischemia after SAH. METHODS: SAH patients with delayed cerebral ischemia and hypoperfusion despite conservative treatment were included. iNO was administered at a maximum dose of 40 ppm. The response to iNO was considered positive if: cerebral artery diameter increased by 10% in digital subtraction angiography (DSA), or tissue oxygen partial pressure (PtiO(2)) increased by > 5 mmHg, or transcranial doppler (TCD) values decreased more than 30 cm/sec, or mean transit time (MTT) decreased below 6.5 secs in CT perfusion (CTP). Patient outcome was assessed at 6 months with the modified Rankin Scale (mRS). RESULTS: Seven patients were enrolled between February 2013 and September 2016. Median duration of iNO administration was 23 h. The primary endpoint was reached in all patients (five out of 17 DSA examinations, 19 out of 29 PtiO(2) time points, nine out of 26 TCD examinations, three out of five CTP examinations). No adverse events necessitating the cessation of iNO were observed. At 6 months, three patients presented with a mRS score of 0, one patient each with an mRS score of 2 and 3, and two patients had died. CONCLUSION: Administration of iNO in SAH patients is safe. These results call for a larger prospective evaluation

Experimental subarachnoid hemorrhage causes early and long-lasting microarterial constriction and microthrombosis: an in-vivo microscopy study

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is characterized by a severe, cerebral perfusion pressure (CPP)-independent reduction in cerebral blood flow suggesting alterations on the level of cerebral microvessels. Therefore, we aimed to use in-vivo imaging to investigate the cerebral microcirculation after experimental SAH. Subarachnoid hemorrhage was induced in C57/BL6 mice by endovascular perforation. Pial arterioles and venules (10 to 80 μm diameter) were examined using in-vivo fluorescence microscopy, 3, 6, and 72 hours after SAH. Venular diameter or flow was not affected by SAH, while >70% of arterioles constricted by 22% to 33% up to 3 days after hemorrhage (P<0.05 versus sham). The smaller the investigated arterioles, the more pronounced the constriction (r(2)=0.92, P<0.04). Approximately 30% of constricted arterioles were occluded by microthrombi and the frequency of arteriolar microthrombosis correlated with the degree of constriction (r(2)=0.93, P<0.03). The current study demonstrates that SAH induces microarterial constrictions and microthrombosis in vivo. These findings may explain the early CPP-independent decrease in cerebral blood flow after SAH and may therefore serve as novel targets for the treatment of early perfusion deficits after SAH

The Contractile Apparatus Is Essential for the Integrity of the Blood-Brain Barrier After Experimental Subarachnoid Hemorrhage

Development of vasogenic brain edema is a key event contributing to mortality after subarachnoid hemorrhage (SAH). The precise underlying mechanisms at the neurovascular level that lead to disruption of the blood-brain barrier (BBB) are still unknown. Activation of myosin light chain kinases (MLCK) may result in change of endothelial cell shape and opening of the intercellular gap with subsequent vascular leakage. Male C57Bl6 mice were subjected to endovascular perforation. Brain water content was determined by wet-dry ratio and BBB integrity by Evans-Blue extravasation. The specific MLCK inhibitor ML-7 was administered to the mice to determine the role of the contractile apparatus of the neurovascular unit in determining brain water content, BBB integrity, neurofunctional outcome, brain damage, and survival at 7 days after SAH. Inhibition of MLCK significantly reduced BBB permeability (Evans Blue extravasation - 28%) and significantly decreased edema formation in comparison with controls (- 2%). MLCK-treated mice showed reduced intracranial pressure (- 53%), improved neurological outcome at 24 h and 48 h after SAH, and reduced 7-day mortality. Tight junction proteins claudin-5 and zonula occludens-1 levels were not influenced by ML-7 at 24 h after insult. The effect of ML-7 on pMLC was confirmed in brain endothelial cell culture (bEnd.3 cells) subjected to 4-h oxygen-glucose deprivation. The present study indicates that MLCK contributes to blood-brain barrier dysfunction after SAH by a mechanism that does not involve modulation of tight junction protein levels, but via activation of the contractile apparatus of the endothelial cell skeleton. This underlying mechanism may be a promising target for the treatment of SAH

The Contractile Apparatus Is Essential for the Integrity of the Blood-Brain Barrier After Experimental Subarachnoid Hemorrhage

Development of vasogenic brain edema is a key event contributing to mortality after subarachnoid hemorrhage (SAH). The precise underlying mechanisms at the neurovascular level that lead to disruption of the blood-brain barrier (BBB) are still unknown. Activation of myosin light chain kinases (MLCK) may result in change of endothelial cell shape and opening of the intercellular gap with subsequent vascular leakage. Male C57Bl6 mice were subjected to endovascular perforation. Brain water content was determined by wet-dry ratio and BBB integrity by Evans-Blue extravasation. The specific MLCK inhibitor ML-7 was administered to the mice to determine the role of the contractile apparatus of the neurovascular unit in determining brain water content, BBB integrity, neurofunctional outcome, brain damage, and survival at 7 days after SAH. Inhibition of MLCK significantly reduced BBB permeability (Evans Blue extravasation − 28%) and significantly decreased edema formation in comparison with controls (− 2%). MLCK-treated mice showed reduced intracranial pressure (− 53%), improved neurological outcome at 24 h and 48 h after SAH, and reduced 7-day mortality. Tight junction proteins claudin-5 and zonula occludens-1 levels were not influenced by ML-7 at 24 h after insult. The effect of ML-7 on pMLC was confirmed in brain endothelial cell culture (bEnd.3 cells) subjected to 4-h oxygen-glucose deprivation. The present study indicates that MLCK contributes to blood-brain barrier dysfunction after SAH by a mechanism that does not involve modulation of tight junction protein levels, but via activation of the contractile apparatus of the endothelial cell skeleton. This underlying mechanism may be a promising target for the treatment of SAH. Keywords Subarachnoid hemorrhage Brain edema ML-7 Myosin light chain kinases Intracranial pressure Blood-brain barrie

Nitric oxide inhalation reduces brain damage, prevents mortality, and improves neurological outcome after subarachnoid hemorrhage by resolving early pial microvasospasms.

Subarachnoid hemorrhage is a stroke subtype with particularly bad outcome. Recent findings suggest that constrictions of pial arterioles occurring early after hemorrhage may be responsible for cerebral ischemia and - subsequently - unfavorable outcome after subarachnoid hemorrhage. Since we recently hypothesized that the lack of nitric oxide may cause post-hemorrhagic microvasospasms, our aim was to investigate whether inhaled nitric oxide, a treatment paradigm selectively delivering nitric oxide to ischemic microvessels, is able to dilate post-hemorrhagic microvasospasms; thereby improving outcome after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to experimental SAH. Three hours after subarachnoid hemorrhage pial artery spasms were quantified by intravital microscopy, then mice received inhaled nitric oxide or vehicle. For induction of large artery spasms mice received an intracisternal injection of autologous blood. Inhaled nitric oxide significantly reduced number and severity of subarachnoid hemorrhage-induced post-hemorrhage microvasospasms while only having limited effect on large artery spasms. This resulted in less brain-edema-formation, less hippocampal neuronal loss, lack of mortality, and significantly improved neurological outcome after subarachnoid hemorrhage. This suggests that spasms of pial arterioles play a major role for the outcome after subarachnoid hemorrhage and that lack of nitric oxide is an important mechanism of post-hemorrhagic microvascular dysfunction. Reversing microvascular dysfunction by inhaled nitric oxide might be a promising treatment strategy for subarachnoid hemorrhage