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

Abstract

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.

Keywords: Cerebral perfusion; early brain injury; microvasospasm; nitric oxide; subarachnoid hemorrhage.

© The Author(s) 2015.

Figures

Figure 1.

NO inhalation improves posthemorrhagic microcirculation. NO inhalation caused a near immediate and long-lasting vasodilation of spastic vessels. (a) Left: a baseline scan after SAH and prior to iNO treatment; right: the same vessel segment at t = 60 min, i. e. after 30 min of iNO treatment. (b) The number of spastic vessel segments (expressed as percent relative to baseline) was significantly decreased by iNO treatment. (c) 60 min after the start of IVM, i.e. 4 h after onset of SAH, spasm severity (i.e., the grade of constriction) was significantly lower in the iNO-treated vessels compared to control-treated vessels; this difference in constriction was largest in the smallest-diameter vessels but was significant in all vessel segments examined.

Figure 2.

iNO treatment does not affect MAP or systemic coagulation. (a) Mean arterial blood pressure (MAP) was measured over time in iNO-treated and control-treated mice. (b) Tail bleeding time was measured in mice treated with iNO for 60 min and control mice (n = 5−7 each, mean +/− SEM).

Figure 3.

NO inhalation reduces basilar arterial spasm. (a) SAH was induced by injecting blood into the cisterna magna; saline was injected as a sham treatment. Representative photomicrographs of a saline-injected brain (left), an SAH-induced brain (middle), and the brain of an animal that received iNO treatment for 12 h following SAH induction (right). (b) Quantification of the diameter of the basilar artery in saline-injected (Sham) mice and SAH-induced mice after 1 and 12 h with or without iNO treatment. The positive effect of iNO observed 12 h after SAH is abated at 24 and 48 h (data not shown).

Figure 4.

iNO treatment reduces SAH-induced brain damage. (a) 72 h after SAH, brain edema (measured as brain water content) was significantly higher in control animals compared with sham (non-SAH) animals (dotted line). iNO treatment significantly reduced brain water content in both hemispheres. (b) 7 days after SAH, neuronal survival (measured as the number of viable cells per ROI) was significantly increased in the CA3 hippocampal region.

Figure 5.

iNO treatment improves outcome following experimental SAH. (a). After SAH (day 0), control animals developed severe neurological deficits measured using a multivariate neurological score (higher scores indicate poorer performance). This impaired neurological performance remained significant through the observation period. In contrast, neurological outcome was significantly better in iNO-treated animals from the third day after SAH, with the iNO-treated mice reaching baseline performance by day 6. (b) Survival plots of mice that were subjected to SAH, then iNO-treated or control-treated for 23 h. The 72-h survival rate in the control-treated mice was 70%, whereas the survival rate in the iNO-treated mice was 100%.