Istvan Schiszler

Initial oligemia with capillary flow stop followed by hyperemia during K+-induced cortical spreading depression in rats

Local cerebral blood volume (CBV) and capillary flow changes in regions of depolarizing neurons during K+-induced cortical spreading depression (CSD) in the cerebral cortex of α-chloralose-urethane-anesthetized rats were examined employing a transillumination (550 nm) video system. Capillary flow was calculated as the reciprocal of mean transit times of blood in pixels of 40 μm × 40 μm, each of which contains a few capillaries. Potassium microinjection into the cortex evoked repetitive wave-ring spreads of oligemia at a speed of ca. 2.33±0.48 mm/min. During the spread of CSD, tracer (either saline or carbon black) was injected into the internal carotid artery. Colocated with the oligemic wave, we detected capillary flow stop as evidenced by disappearance of the hemodilution curves. At any location in the region of interest within the cerebral cortex, we observed cyclic changes of capillary flow stop/hyperperfusion in synchrony with oligemia/hyperemia fluctuations. The initial flow stop and oligemia were ascribed to capillary compression by astroglial cell swelling, presumably at the pericapillary endfeet, since the oligemia occurred before larger vessel changes. We conclude that local depolarizing neurons can decrease adjacent capillary flow directly and immediately, most likely via astroglial cell swelling, and that the flow stop triggers upstream arteriolar dilatation for capillary hyperperfusion.

Repetitive concentric wave-ring spread of oligemia/hyperemia in the sensorimotor cortex accompanying K+-induced spreading depression in rats and cats

Vascular changes accompanying spreading depression (SD) remain controversial. We examined dynamic alterations of local cerebral blood volume (CBV) during SD by observing light transmission at an isosbestic point of hemoglobin (550 nm) in seven rats and five cats under alpha-chloralose/urethane anesthesia. The two species were used for comparison between the lissencephalic and gyrencephalic brains. We found that a concentrated K(+) solution microinjected into the sensorimotor cortex provoked CBV changes that appeared as a repetitive propagation of concentric wave-rings of ischemia followed by hyperemia expanding peripherally from the injection site at speeds of 1.9-3.2 mm/min. The dynamic CBV changes continued repeatedly every 1-5 min for more than 30 min in three rats, ceased within 30 min in three rats and remained at the site of K(+) injection in one rat. Similar repeated CBV changes occurred in two out of five cats.

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.

Platelet adhesion to human brain microvascular endothelial cells in vitro. Observation with video-enhanced contrast microscopy

Employing video-enhanced contrast (VEC) microscopy and perfusion systems, we examined whether platelets adhere directly to human brain microvascular endothelial cells (HBEC) in vitro after thrombin treatment and whether adenosine diphosphate (ADP) or thromboxane A2-stimulated platelets adhere directly to HBEC at a low flow state in vitro. HBECs were cultured on a coverglass and put in the observation chamber of VEC microscopy. Following pretreatment with human alpha-thrombin 1.0 units/ml (n = 8) for 20 min, thrombin was thoroughly washed out. Platelet rich plasma (PRP) was perfused over HBEC at a low shear rate of 10 s(-1) for 30 min. Platelets adhered directly to thrombin-treated HBEC. Activated platelets by ADP (2 microM, n = 8) or thromboxane A2 (U-46619 10 microM, n = 5) were perfused over HBEC for 30 min and washed out. Platelets also adhered directly to HBEC. However, platelets did not adhere to HBEC when PRP only (n = 6) was perfused over HBEC for 30 min and washed out. Platelet adhesion directly to HBEC following thrombin treatment or platelet activation may play a pivotal role in secondary thrombus formation and microcirculatory disturbance in the ischemic brain.

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.

Oxygen-Induced Cytoskeleton Rearrangement of Cultured Human Brain Microvascular Endothelial Cells

We conclude that the cytoskeleton of HBECs rapidly and reproducibly remodels after successive contraction cycles in response to multiple oxygen exposures. The appearance of a mesh network and adhesion plaques suggests that HBECs are mobile cells.

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.