Indian-Ink Perfusion Based Method for Reconstructing Continuous Vascular Networks in Whole Mouse Brain

Abstract

The topology of the cerebral vasculature, which is the energy transport corridor of the brain, can be used to study cerebral circulatory pathways. Limited by the restrictions of the vascular markers and imaging methods, studies on cerebral vascular structure now mainly focus on either observation of the macro vessels in a whole brain or imaging of the micro vessels in a small region. Simultaneous vascular studies of arteries, veins and capillaries have not been achieved in the whole brain of mammals. Here, we have combined the improved gelatin-Indian ink vessel perfusion process with Micro-Optical Sectioning Tomography for imaging the vessel network of an entire mouse brain. With 17 days of work, an integral dataset for the entire cerebral vessels was acquired. The voxel resolution is 0.35×0.4×2.0 µm3 for the whole brain. Besides the observations of fine and complex vascular networks in the reconstructed slices and entire brain views, a representative continuous vascular tracking has been demonstrated in the deep thalamus. This study provided an effective method for studying the entire macro and micro vascular networks of mouse brain simultaneously.

Stroke and the Connectome: How Connectivity Guides Therapeutic Intervention

“…In contrast to the brain's electrical circuit diagram, which is far from complete in any mammalian model organism, recent breakthroughs in imaging and sectioning technology have resulted in high-resolution capillary-level reconstructions of the rodent brain angiome Mayerich et al, 2011;Pathak et al, 2011;Tsai et al, 2009;Xue et al, 2014). Tracing blood vessels is (A) At the microscale level, serial blockface scanning electron microscopy has been used to reconstruct a volume of rodent brain tissue and manually trace a segment of spiny dendrite (shown in red) to visualize synaptic connections.…”

“…Optimization of histological and sectioning techniques in combination with classic methods such as India ink perfusion has resulted in remarkably complete brain-wide capillary-level data sets for mice (Mayerich et al, 2011;Xue et al, 2014). Such data may be used to predict local vulnerability to vascular disruptions, but insight from the data with respect to stroke is still in progress.…”

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

“…[1][2][3] . Although structural and molecular analyses of cerebral vasculature are promising for providing fundamental insights into cerebral circulation and cerebrovascular diseases, which are major causes of death and disability 4 , few studies have captured detailed structural information of molecularly identified vascular networks due to technical limitations (Supplementary Tables 1 and 2) [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] .…”

Chronic pain alters microvascular architectural organization of somatosensory cortex

“…After the loss of any reflex, before the euthanasia by the barbiturate overdose administered intraperitoneally (Pentobarbital solution, Sigma-Aldrich, 300mg/kg), the animals were intracardiacally perfused according to a common protocol [4]. The procedure was performed in four stages: heparinized saline (150-180 ml), 10% formalin (150 ml), air (ten minutes of air insufflation through the same route of fluids and, eventually, undiluted Indian-ink (100 ml), this last used in accordance with the modified protocol by Xue et al [64]. The unusual step of injecting air into the circulating system after the vessel fixation was adopted to empty the vascular system before the ink injection.…”