CBF and red blood cell kinetics in perfused capillaries after MCAO

Typical light microscope images of the rat cortical vasculature (a) before and (b) 10 minutes after middle cerebral occlusion (MCAO) in a rat. Black arrows indicate small arterial branches and cortical arterioles whose diameter decrease dramatically 10 minutes after occlusion. Within the white circles, the black lines indicate the outer arterial circumference before occlusion to visualize the diameter change. The scale bar indicates 200 μm. (c) Laser speckle contrast (LSC) image after 30 min of filament occlusion. Red colors correspond to high image intensity and high blood flow, whereas blue colors represent low LSC image intensity and low perfusion. (d) The same image thresholder so only tissue with image intensity between 25-50% of the contralateral hemisphere average is shown (the white dotted line indicates midline). Penumbral tissue was identified as tissue with LSC image intensity between 25 and 50% of contralateral tissue (pink) and infarct core (grey) as tissue with image intensity less than 25% LSC image intensity compared to a contralateral tissue. The core area was further verified by 2,3,5-Triphenyltetrazolium chloride (TTC) staining of the intact brain (e), where the white tissue shows the core 4h following MCA occlusion. The contralateral region of interest (turquoise) encircles the mirror-regions of the two, relative to the mid line (dotted while line). (f) LSC image intensity signal as a function of time for the core, penumbra, and contralateral mirror region. Asterisk indicates statistical significance between the three areas. (g) Comparing MACO and control animals, red blood cell (RBC) kinetics measured by two photon microscopy (TPM) line scanning of 530 individual penumbral capillaries (270 in control animals, 260 in MCAo, n= 8 for each group) every 30 min after filament occlusion provided estimates of capillary diameter, (h) RBC flux, and (i) RBC velocity. Asterisk indicates statistical significance (p < 0.0001) by two-way ANOVA test and Tukey multiple pairwise comparisons test.

TPM line scans reveal increasingly chaotic RBC kinetics after MCAO.

Using two-photon line scanning, 530 capillaries were scanned (270 in control animals, 260 in penumbral tissue of MCAo in the depth range between 100-250μm. Twenty-five capillaries were excluded from further analysis as the analysis yielded unrealistic cell densities (in excess of 300 cells/mm. (a) Typical line scanning trajectory. Injected FITC makes plasma appear bright while individual red blood cells (RBCs) can be observed as dark shadows within the vessel lumen. Two scan paths were prescribed for each capillary; one along the axis for RBC velocity estimation, and one transversal scan for RBC flux and capillary diameter assessment. (b) Typical line scan data for cortical capillaries in control animal and penumbral tissue of an MCAO animal, respectively. The corresponding, 30-second velocity profiles are shown for the control animal capillary (top) and a capillary within penumbral tissue (bottom). Note that RBC flow direction changes during the 30 second observation period (red dotted line indicates zero velocity). (c) shows the percentage of penumbral capillaries with such ‘chaotic’ behaviour as a function of the duration of ischemia. Note how the percentage of capillaries with reversing flows remains constant in normal tissue but increases dramatically in penumbral capillaries. (d) shows (left) the line scan of a capillary with stalled flow (flux=0) and (right) the percentage such capillaries represented of the total number of scanned capillaries for control and penumbral tissue over time. Asterisk denotes statistically significant difference (p<0.05) by two-way ANOVA test and Tukey multiple pairwise comparisons test. See Supplementary Material for further information about the 60 min data point.

TPM bolus tracking technique in the penumbral area.

A) Arterial input function (AIF) and venous output function (VOF) for quantifications of mean transit time (MTT) and capillary transit time heterogenity (CTH) in the capillary network. B). A total measurement of 303 bolus pairs were performed with 104 in controls and 199 in MCAo animals at a depth of 80-100 μm from the selected 0-position at the z-axe identified by a 20 sec spiral scan (512x512 pixels, time per pixel = 1.2 μsec.). The vessels were scanned as defined by the scanning route (grey line) crossing all AIF and VOF in the field of view (FOV). Red line marks the main AIF and blue the main VOF. The time series of the bolus injection is shown in C. Based on the scans, the 60 sec line scanned by time and length is shown in D), where the red and blue mark corresponds to the vessels marked in B. From the AIF and VOF intensity curves as a function on the 60 sec scan time, a 5 sec curve matching region was selected and curves were adjusted according to this including a baseline region and a post baseline. Scale bars = 200μm.

The transit time was disturbed across the penumbral capillary bed.

Violin plots of mean transit time (MTT, a), capillary transit time heterogenity (CTH, b) and coefficient of variance (CoV, c) as a function of time every 30 min during the 4 h permanent occlusion period for both control (grey) and stroke (yellow) animals. d) CTH as a function of MTT for both control (grey circles) and stroke (yellow circles) visualized in an arrow diagram with the time of the bolus shown. Asterisk indicates statistical significance between groups (stroke, control) by mixed linear models. The data points from 60 min after occlusion (2nd bolus) are faded out -see supplementary figure 1 for further information. The difference between groups at specific time points were tested by a t-test, p<0.01 Bar plots showing the estimated cerebral metabolic rate of oxygen (CMRO2, e) and tissue oxygen tension (PtO2, f) for a given MTT and CTH at each time point based on the models from {Angleys:2015iv}. Black bars indicate control animals, whereas yellow are MCAo.

Capillary pericytes do not affect penumbral RBC hemodynamics in vivo.

Two-photon image of a line scan of an individual capillary defined as a vessel showing single cell passages (cell’ shadows) within the vessel lumen. Pericytes in the penumbral region were stained by topical injections of Nissl bodies and were visible down to a depth of 350μm in the in both control and MCAo animals. They lined capillaries with multiple processes extending along vessel branches. The labelling was bright and concentrated at both cell soma and throughout the processes, where it displayed a punctate pattern. Two scan paths were performed on each capillary, along the axis for RBCv estimation, and a transversal scan for RBCflux and diameter assessment. Green colours denote plasma, whereas red colours represent a capillary pericyte cell body and arms. Line scans were made on both side of the pericyte and diameter scans were made next to the pericyte body and at a far distance, where 1 denotes upstream pericytes and 2 downstream (a). Data is shown in bar charts, where diameter at the pericyte cell body (dp) and at a far distance (p, b), linear density (c), RBC flux (d), variance of the line scan velocity profile (e) and RBC velocity (f). Black bars are control and yellow stroke. Asterisk indicates significance difference between upstream and downstream the pericyte in the specific type, i.e. control or stroke, p<0.05.

Contour plots reveals low penumbral tissue oxygen tension.

To access the effects of capillary flow pattern changes on oxygen availability during stroke, cerebral metabolic rate of oxygen (CMRO2), tissue oxygen tension (PtO2) and oxygen extraction fraction (OEF) were predicted based on the MTT and CTH values in a biophysical model for oxygen extraction32 assuming all vessels stay open. The effect of increased CTH on tissue oxygen uptake i based on the theory by Jespersen and Østergaard31. According to the model, residual metabolism results in a low tissue oxygen tension (PtO2), and thus an elevated oxygen extraction fraction (OEF) until the network collapses, keeping in mind that the model assumes that vascular volume is preserved (all vessels are open). CTH as a function of MTT for both control (white boxes) and stroke (red/blue triangles) visualized in an arrow diagram with the time of the bolus shown.

Laser speckle imaging reveals the fate of the penumbral tissue during the 4h experimental period.

Following filament occlusion of the middle cerebral artery (MCAo) in Sprague-Dawley rats, a thin scull was made over the cortical area, where CBF was measured by the Laser Speckle Contrast Imaging (LSCI) (Fig.1a). Fig a. shows the LS signal in a sham animal, during acute MCAo, and 4 and 24h following reperfusion. We used the extent of LSCI signal reduction to subdivide hypoperfused cortex into penumbral calculated penumbral tissue21-22,26-28 after 4h of MACo and subsequent 24h of reperfusion. Row b shows the raw LS signal in a representative MACo animal during MCA occlusion at the tome points from 0-210 min at 30 min interval as where the 2PM measurements where performed. Row c shows the corresponding calculated penumbral tissue as it is affected by time after occlusion start. The fate of penumbral and core tissue was verified by TTC staining (row d). Thirty minutes after occlusion, an unexpected increase in blood flow in both ischemic and control animals was observed, resulting in elevated MTT and CTH values in both animal groups (Fig. 4a,b, Supplementary material Fig. 1b,c). These data are shown with transparent images, as it occurred in both MCAo animal and controls and seems to be a feature of our animal preparation, rather than of the stroke model.

Penumbral RBC variance and linear density in MACo and control animals.

TPM measurements of a) the variance of RBC velocities in the penumbral area, and b) linear density. Both measured parameters were not affected of MCAo or the fate of the penumbral tissue over time.