Diving head-first into brain intravital microscopy.

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.