Yutaka Tomita

Vascular fate of adipose tissue-derived adult stromal cells in the ischemic murine brain: A combined imaging-histological study

Increasing evidence indicates that fat tissue can provide a novel source of progenitor cells with therapeutic potential. Here, the fate of adipose tissue-derived stromal cells (ADSCs) transplanted into the mouse ischemic cortex was monitored in the long term using in vivo imaging, and subsequently characterized. The left middle cerebral artery (MCA) was occluded in C57BL/6J mice equipped with a closed cranial window chronically implanted over the left parietal cortex (n = 20). ADSCs expressing the green fluorescent protein (GFP) (approximately 18 x 10(3) cells in 0.5 microl) were transplanted into the ipsilateral cortex, 24 h after MCA occlusion. GFP+-ADSCs were monitored through the window using confocal fluorescence microscopy to assess their single fate in vivo. Co-localization of GFP with vascular, neuronal, glial or proliferation markers was investigated immunohistochemically. Repeated in vivo imaging revealed that GFP+-ADSCs migrated over 1 week toward the lesion, survived for at least 4 weeks, and exhibited a particular tropism for vessels. About 5% of the transplanted GFP+-ADSCs were scattered in the peri-ischemic area on histological sections. Immunohistochemistry evidenced that perivascular GFP+-ADSCs enfolded CD31-labeled endothelial cells, always outside their basal lamina, and occasionally expressed smooth muscle alpha-actin. Less than 1% GFP and BrdU co-labeling indicated a low proliferation rate of ADSCs. These results demonstrate that cerebral ischemia induces ADSCs survival, migration toward the lesion, especially toward microvessels, and occasional differentiation into smooth muscle cells.

Automated Method for Tracking Vast Numbers of FITC-Labeled RBCs in Microvessels of Rat Brain In Vivo Using a High-Speed Confocal Microscope System

High‐speed camera investigation of rapidly moving red blood cells (RBCs) in the microvasculature has been limited by an inability to handle the vast volume of data. We have developed a novel method to analyze large numbers of RBC images captured by a high‐resolution, high‐speed camera fitted on a confocal fluorescence microscope, to determine the velocities of individual RBCs in capillaries in vivo. Fluorescein isothiocyanate (FITC)‐labeled RBCs flowing in the microvasculature of the cerebral cortex of urethane‐anesthetized Wistar rats were recorded through the skull window on video clips during specified periods at high frame rates (500 fps). Sequential frames of moving RBCs in the video clips for a specified period were analyzed offline with in‐house software (KEIO‐IS2). Images of RBCs acquired were numbered automatically in order of appearance and displayed in a two‐dimensional (2‐D) RBC tracking map. The velocities of individual RBCs were automatically computed based on the RBC displacement per frame multiplied by the frame rate (fps), and the results were displayed in a 2‐D velocity map and a 2‐D RBC number map. Single capillaries were identified by staining with FITC‐dextran. The mean capillary velocity of RBCs was evaluated as 2.05 ± 1.59 mm/second in video clips obtained at 500 fps. This method is considered to have wide potential applicability.

In vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse.

The aim of the study was to monitor in vivo and noninvasively the fate of single bone marrow cells (BMCs) transplanted into the ischemic brain of unirradiated mice. In vivo imaging was performed through a closed cranial window, throughout the 2 weeks following cell transplantation, using laser-scanning confocal fluorescence microscopy. The window was chronically implanted above the left parieto-occipital cortex in C57BL/6J adult mice. BMC (3 x 10(5) nucleated cells in 0.5 microL medium) from 5-week-old transgenic mice, ubiquitously expressing green fluorescent protein (GFP), was transplanted into the ipsilateral cortex 24 h after the induction of focal ischemia by coagulation of the left middle cerebral artery (n = 15). Three nonischemic mice served as controls. Repeated in vivo imaging, up to a depth of 200 microm, revealed that BMCs survived within the ischemic and peri-ischemic cortex, migrated significantly towards the lesion, proliferated and adopted a microglia-like morphology over 2 weeks. These results were confirmed using ex vivo imaging after appropriate immunocytochemical treatments. This study indicates that confocal fluorescence microscopy is a reliable and unique tool to repeatedly assess with cellular resolution the in vivo dynamic fate of fluorescent cells transplanted into a mouse brain. These results also provide the first in vivo findings on the fate of single BMCs transplanted into the ischemic brain of unirradiated mice.

A time-variable concentric wave-ring increase in light transparency and associated microflow changes during a potassium-induced spreading depression in the rat cerebral cortex

Abstract The mechanism of coupling between neurons and the microvasculature continues to remain unclear. This was examined during potassium-induced spreading depression (SD), since SD is thought to be a phenomenon of local neuronal depolarization in association with flow changes. In α-chloralose-urethane anesthetized rats, a new optical method was employed by which both the light transparency (LT) changes of the somatosensory cortex as well as its associated capillary-level flow were measured simultaneously in a region of interest (ROI; 2×2 mm) of the sensorimotor cortex. Microinjection of concentrated potassium chloride solution into the cortex produced spreading depression which was observed as a function of time and space, with attention given to the increase and decrease in wave-rings marked by cortical LT variance. The wave-ring was observed to enlarge at a speed of ca. 2.2 mm/min and approximately 1–2 min later, a new wave-ring appeared. This cycle continued repeatedly for more than 30 min. The study was performed on seven rats, with ring propagation in three, an abortive form in another three, and no rings in one rat. In a 3-D microflow map produced by intracarotid injection of diluted carbon black (CB) (Pelikan Werke, Germany) in Ringer solution, a low-flow ring was approximately collocated at the wave-front ΔLT ring, followed by a flow increase. A close correlation between topographical microflow changes and LT changes indicates that local depolarization of the neurons induces an immediate decrease followed rapidly by an increase in microflow, suggesting that the depolarized neurons induced changes in adjacent microvascular (capillary) flow.

Selective Thrombin Inhibitor (Argatroban): Amelioration of Platelet Adhesion to Human Brain Microvascular Endothelial Cells In Vitro: Observation with Video-Enhanced Contrast Microscopy

We examined whether argatroban (a selective thrombin inhibitor) inhibits adhesion of activated platelets to human brain microvascular endothelial cells (HBECs) in vitro using video-enhanced contrast (VEC) microscopy. In the control group (n = 5), HBECs were cultured on a coverglass and put in the observation chamber of VEC microscopy. Platelet-rich plasma (PRP) was then superfused on the HBECs with an infusion pump at a low shear rate (10/s) for 30 min, and platelet adhesion to HBEC was examined. In the ADP group (n = 9), PRP with ADP (2µM) was superfused for 30 min and washed out. In the argatroban group (n = 9), PRP with ADP (2µM) plus argatroan (5µg/ml) was superfused, and platelet adhesion to HBECs was observed. In the control group, platelet adhesion to HBECs was rarely seen. In the ADP group, platelets adhered to HBECs in all experiments, and microaggregates of platelets were seen. In the argatroban group, platelet adhesion to HBECs was clearly suppressed. The average number of platelets adhering and aggregating to HBEC was 0.3 ± 0.6/900µm2 in the control group, 25.5 ± 11.3/900µm2 (P < 0.01, vs. control) in the ADP group, and 1.8 ± 1.8/900µm2 in the argatroban group (P < 0.01, vs. ADP). These results showed that argatroban ameliorated adhesion and pile-up of activated aggregated platelets to HBECs in a low-flow state in vitro. It suggests that thrombin produced by platelet activation makes HBECs a procoagulant and is the most likely candidate subsequently to induce platelet adhesion to HBECs.