Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

BackgroundLeukocytes contribute to tissue damage after cerebral ischemia;however, the mechanisms underlying this process are still unclear. This study investigates the temporal and spatial relationship between vascular leukocyte recruitment and tissue damage and aims to uncover which step of the leukocyte recruitment cascade is involved in ischemic brain injury. MethodsMale wild-type, ICAM-1-deficient, anti-CD18 antibody treated, or selectin-deficient [fucusyltransferase (FucT IV/VII-/-)] mice were subjected to 60 min of middle cerebral artery occlusion (MCAo). The interaction between leukocytes and the cerebrovascular endothelium was quantified by in vivo fluorescence microscopy up to 15 h thereafter. Temporal dynamics of neuronal cell death and leukocyte migration were assessed at the same time points and in the same tissue volume by histology. ResultsIn wild-type mice, leukocytes started to firmly adhere to the wall of pial postcapillary venules two hours after reperfusion. Three hours later, neuronal loss started and 13 h later, leukocytes transmigrated into brain tissue. Loss of selectin function did not influence this process. Application of an anti-CD18 antibody or genetic deletion of ICAM-1, however, significantly reduced tight adhesion of leukocytes to the cerebrovascular endothelium (-60%;p < 0.01) and increased the number of viable neurons in the ischemic penumbra by 5-fold (p < 0.01);the number of intraparenchymal leukocytes was not affected. ConclusionsOur findings suggest that ischemia triggers only a transient adhesion of leukocytes to the venous endothelium and that inhibition of this process is sufficient to partly prevent ischemic tissue damage

Neurovascular Reactivity in the Aging Mouse Brain Assessed by Laser Speckle Contrast Imaging and 2-Photon Microscopy: Quantification by an Investigator-Independent Analysis Tool

The brain has a high energy demand but little to no energy stores. Therefore, proper brain function relies on the delivery of glucose and oxygen by the cerebral vasculature. The regulation of cerebral blood flow (CBF) occurs at the level of the cerebral capillaries and is driven by a fast and efficient crosstalk between neurons and vessels, a process termed neurovascular coupling (NVC). Experimentally NVC is mainly triggered by sensory stimulation and assessed by measuring either CBF by laser Doppler fluxmetry, laser speckle contrast imaging (LSCI), intrinsic optical imaging, BOLD fMRI, near infrared spectroscopy (NIRS) or functional ultrasound imaging (fUS). Since these techniques have relatively low spatial resolution, diameters of cerebral vessels are mainly assessed by 2-photon microscopy (2-PM). Results of studies on NVC rely on stable animal physiology, high-quality data acquisition, and unbiased data analysis, criteria, which are not easy to achieve. In the current study, we assessed NVC using two different imaging modalities, i.e., LSCI and 2-PM, and analyzed our data using an investigator-independent Matlab-based analysis tool, after manually defining the area of analysis in LSCI and vessels to measure in 2-PM. By investigating NVC in 6–8 weeks, 1-, and 2-year-old mice, we found that NVC was maximal in 1-year old mice and was significantly reduced in aged mice. These findings suggest that NVC is differently affected during the aging process. Most interestingly, specifically pial arterioles, seem to be distinctly affected by the aging. The main finding of our study is that the automated analysis tool works very efficiently in terms of time and accuracy. In fact, the tool reduces the analysis time of one animal from approximately 23 h to about 2 s while basically making no mistakes. In summary, we developed an experimental workflow, which allows us to reliably measure NVC with high spatial and temporal resolution in young and aged mice and to analyze these data in an investigator-independent manner

Inhalation of nitric oxide following experimental ischemic stroke reduces neurovascular inflammation

Brain injury following ischemic stroke is driven to a large extent by a robust inflammatory response involving the brain parenchyma and cerebral vessels. Thus, anti-inflammatory treatment strategies may have the potential to reduce this damage and improve outcome in patients following a stroke. Here, we investigated whether inhaled NO (iNO) can reduce post-ischemic vascular inflammation. Male C57BL/6 mice underwent middle cerebral artery occlusion (MCAo) for one hour and then received NO (50 ppm) by inhalation upon reperfusion; systemic delivery of NO was confirmed by a significant increase in the plasma levels of NO metabolites. Leukocyte-endothelium interactions were then imaged in vivo using 2-photon microscopy, and plasma and tissue samples were collected five hours after reperfusion and analyzed for pro-inflammatory cytokines, NO metabolites, NO synthases, and adhesion molecules. We found that iNO normalized cGMP levels, virtually eliminated the adhesion of leukocytes to the cerebrovascular endothelium, and reduced the expression of parenchymal inflammatory cytokines and endothelial adhesion molecules. Together, our findings show that iNO increases systemic NO, thereby potently inhibiting ischemia-induced vascular inflammation in the brain. Thus, iNO, which is clinically approved for the treatment of several pulmonary conditions, may represent a novel therapeutic strategy for ischemic stroke and other neuroinflammatory disorders