In Vivo Imaging of the Mouse Neurovascular Unit Under Chronic Cerebral Hypoperfusion

Abstract

T he neurovascular unit (NVU) is a conceptual framework that integrates responses in all cell types, including neuronal, glial, inflammatory, and vascular elements.1-5 This cell-cell integrated response is an indispensable factor used to maintain brain function and homeostasis. In fact, dysfunction of the NVU is the basis for many diseases. [2][3][4] The concept of NVU emphasizes that maintenance of integrated cellular function is more important than just salvaging an individual cell alone. Although the intricate molecular pathway of neuronal death has been dissected in detail, the mechanisms of how the entire NVU responds to cerebral ischemia are not completely understood. Understanding this concept may provide a comprehensive framework for investigating mechanisms and therapies of ischemic brain damage. 5The NVU is a dynamic structure assembled by endothelial cells, basement membranes, perivascular astrocytes, pericytes, and neurons. Therefore, it is difficult to understand the whole structure (including the anatomic relationship between cells) and the dynamic changes that occur within a single specimen. To understand the NVU more easily, an imaging method that can detect the whole NVU component at one time in vivo should be introduced. Thus, the aim of the present experiments was (1) to establish an in vivo imaging method for the NVU, which has extraordinarily spatial and time-dependent features. A spatial feature would require indetail, 3-dimensional (3D) observation of the intricate NVU structure, whereas a time-dependent feature would require repeated longitudinal observation to capture real-time events, such as dynamic microcirculation in capillaries and (2) to clarify the stress response of the NVU in the bilateral common carotid artery stenosis (BCAS) model of chronic cerebral hypoperfusion. Materials and Methods Animal ModelAll procedures were performed in accordance with the guidelines for animal experimentation from the ethical committee of Mie University. Male green fluorescent protein transgenic mice (C57BL/6Background and Purpose-Proper brain function is maintained by an integrated system called the neurovascular unit (NVU) comprised cellular and acellular elements. Although the individual features of specific neurovascular components are understood, it is unknown how they respond to ischemic stress as a functional unit. Therefore, we established an in vivo imaging method and clarified the NVU response to chronic cerebral hypoperfusion. Methods-Green mice (b-act-EGFP) with SR101 plasma labeling were used in this experiment. A closed cranial window was made over the left somatosensory cortex. To mimic chronic cerebral hypoperfusion, mice were subjected to bilateral common carotid artery stenosis operations using microcoils. In vivo real-time imaging was performed using 2-photon laser-scanning microscopy during the preoperative period, and after 1 day and 1 and 2 weeks of bilateral common carotid artery stenosis or sham operations. Results-Our method allowed 3-dimensional observation of most of the ...

Physical exercise promotes astrocyte coverage of microvessels in a model of chronic cerebral hypoperfusion

“…In the 2VO model, it has been observed that endothelial activation markers increase 1 day post-occlusion to 28 days, with a peak at 3 days [37]. Yata et al found increased leukocyte rolling and adhesion in pial surface venules after 24 h, 1, and 2 weeks of cerebral hypoperfusion suggesting that leukocyte activation may be the first step in the whole neurovascular unit stress response to chronic cerebral hypoperfusion [38]. Previous results from our group have shown that physical exercise initiated early following 2VO and maintained for 1 week was able to reduce adherent leukocytes in brain venules and to reduce NADPH-oxidase expression in the brain [14].…”

Increased capillary stalling is associated with endothelial glycocalyx loss in subcortical vascular dementia

“…The underlying mechanism of capillary stalling involves various intravascular components, such as blood clots, red blood cells, and leukocytes. Using a cerebral hypoperfusion model, a previous study demonstrated that capillary stalling is generated by the plugging of leukocytes and not by platelet activation 7 . In another study, leukocyte plugging in stalled capillaries was also identified in an Alzheimer's disease model, demonstrating that it is a plausible mechanism underlying capillary stalling 6,7 .…”

“…Recent studies have found that capillary stalling occurs naturally and not randomly in multiple capillaries 4,5 , while another study reported that increased capillary stalling heavily impacts on cerebral blood flow (CBF) 6 . While previous studies have shown that capillary stalling is induced by leukocyte plugging, the location and mechanism of this phenomenon and its consequences still need to be corroborated 6,7 . The endothelial glycocalyx (EG) is a layer of proteoglycans and glycoproteins at the luminal side of the vessels 8 .…”

“…Since the deficits in SVaD develop over time, a longitudinal investigation is required to properly understand the disease. However, no study has examined microcirculation changes, such as alterations in capillary stalling, in SVaD over several months 7,12 .…”

Interleukin-6 mediates enhanced thrombus development in cerebral arterioles following a brief period of focal brain ischemia

“…While our findings indicate that brain cells account for only one-third of the increased tissue IL-6 concentration detected in ipsilateral brain tissue after MCAo/R, it would appear that either this concentration is sufficient to elicit the thrombosis response or, more likely, that the cells generating the IL-6 lie within (e.g., endothelial cells) and/or in close proximity (e.g., neurons, astrocytes) to cerebral arterioles. For example, astrocytes, which extend their foot processes around cerebral arterioles in mouse brain (Yata et al, 2014), may generate locally high levels of IL-6 that account for the critical role of this cytokine in mediating the accelerated arteriolar thrombosis in the postischemic brain. Additional work is needed to definitively identify the cellular source(s) of the IL-6 that underlies the MCAo/R induced thrombogenesis.…”