Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke.

Animal models and surgery

In total, 57 male Sprague Dawley rats from Charles River were used in this study, ranging in mass from 270 to 310 g. Thirteen animals were imaged during tMCAo using the ‘Koizumi’ method, i.e. , ipsilateral common carotid artery (CCA) ligated during reperfusion ( Koizumi et al. 1986 ), 4 for tMCAO using the ‘Longa’ method, i.e. , ipsilateral CCA intact during reperfusion ( Longa et al. 1989 ), 7 for Sham tMCAo controls, i.e. , ipsilateral CCA ligation and partial filament insertion ( Supplemental Fig. 2 ), 6 for Window only controls, i.e. , cranial window but no vascular manipulation ( Supplemental Fig. 3 ), 4 for hypercapnic treatment to control for large surface arteriole dilation, 12 for pimonidazole studies using the ‘Koizumi’ method, and 11 for LDF studies. Anesthesia was maintained with 1 to 2 % (v/v) isoflurane (Baxter Healthcare) in 30 % oxygen and 70 % nitrous oxide. As isoflurane is a vasodilator that can affect cerebral autoregulation ( Eger 1981 ), the Window only group provided a control for the effects of anesthesia over time ( Supplemental Fig. 3 ). Atropine (American Regent), 0.05 mg per kg body weight intraperitoneal, and lidocaine (Hospira Inc.), 2 % (v/v) subcutaneous, were administered at the start of surgery. Body temperature was maintained at 37°C with a feedback-regulated heat pad (50-7053-F; Harvard). Heart rate and blood oxygen saturation were continuously monitored using a pulse oximeter (8600V; Nonin). Cranial windows, 4 × 4 mm in size and centered at 4.5 mm medial-lateral and −3.0 mm anterior-posterior, were constructed as described previously ( Kleinfeld et al. 2008 ). The left femoral artery was catheterized with polyethylene 50 tubing (Intramedic) connected to a stopcock and 10 mL syringe filled with heparin-saline (20 units per mL, Baxter Healthcare). Arterial blood was sampled from the catheter for blood gas measurement (RapidLab 248; Bayer) during each of three imaging periods, i.e. , baseline, occlusion, and reperfusion. Blood pressure was measured with the tail cuff method (XBP-1000; Kent Scientific) once during each imaging period. Intraperitoneal injections of 5 % (w/v) glucose in 1 mL saline were given every 2 h for re-hydration. Transient middle cerebral artery occlusion was induced using the intraluminal filament method. Ischemia was maintained for 90 min and followed by 90 min of reperfusion. We examined two variations of the model and exploited their differences in post-ischemic blood flow. In the majority of the experiments, the CCA was permanently ligated to generate an incomplete reperfusion ( Koizumi et al. 1986 ). In a control study, we left the CCA intact, which generated a transient hyperemia during the reperfusion period ( Longa et al. 1989 ). In all cases, the filament placement was guided with LDF measurements through the cranial window. Animals that exhibited sub-arachnoid hemorrhage were excluded from the study. The penumbra was defined by the amount of residual blood flow during occlusion, typically ranging within 25-50% of baseline flow values, measured either using LDF or averaging arteriole flux measurements from the cranial window. This level of blood flow in the penumbra is a generally accepted range ( Lipton 1999 ) and is contrasted to flow in the ischemic core, which can be lower than 20% of baseline.

Two-photon microscopy

Images were collected using a two-photon laser scanning microscope of local design ( Tsai et al. 2003 ; Tsai et al. 2002 ) that was controlled by MPScope software ( Nguyen et al. 2006 ). The blood serum was labeled using 0.3 mL of 2 MDa fluorescein-dextran (FD2000S; Sigma) prepared at a concentration of 5% (w/v) in saline, and delivered through the femoral artery catheter, with 0.1 mL supplements as required ( Schaffer et al. 2006 ). A 0.3-numerical aperture (NA), 10-times magnification water-dipping objective (Zeiss) was used to collect a large-scale map to aid navigation through the cortical vasculature, while a 0.8-NA, 40-times magnification water-dipping objective (Olympus) was used to obtain high-resolution line-scan and planar data. The line-scans were collected along the centerline of each vessel over a length of 70 pixels, spanning 7 to 76 μm, at a scan rate of 1.6 kHz/line. RBC velocity was determined from the slope of the line-scan streaks using a method based on singular value decomposition ( Kleinfeld et al. 1998 ). For each vessel, we reported the average velocity over a 1.5 s period. Planar image stacks, 256 by 256 pixels, were acquired to establish the diameter of the vessel. Our analysis was limited to arterioles smaller than 60 μm in diameter as a result of technical constraints. A further limitation was that penetrating arterioles could only be measured if a portion of the vessel was parallel to the cortical surface before diving perpendicularly. In a survey of 154 penetrating arterioles across 11 rats, 17% were not measurable as a result of this limitation. During analysis of surface arterioles, a 23 μm break-point was used to divide the vessels into small and large diameter categories. This number corresponded to the intersection between normalized histograms for penetrating arteriole (n = 215) and surface arteriole (n = 271) diameters at baseline, and by maximum likelihood, was the natural point to divide the data. Further, 23 μm was the median diameter for measured surface arterioles.

Flux quantification

Under the assumption that the flow in the vessels is laminar, RBC velocity and lumen diameter collected from a single vessel can be used to define the average volume flux, F⃗, by: (1) F → = 〈 v → 〉 A = π 8 v → ( 0 ) d 2 , where 〈v⃗〉 is the average RBC velocity, A is the cross-sectional area of the vessel lumen, v⃗(0) is the RBC velocity along the central axis of the vessel, and d is the lumen diameter. Contrawise, the condition of constant flux implies that the change in diameter, Δd, that is required to offset a change in speed, i.e. , Δv⃗(0), is given by (2) Δ d d = 1 1 + [ Δ v → ( 0 ) / v → ( 0 ) ] − 1 → Δ v → ( 0 ) → 0 − 1 2 Δ v → ( 0 ) v → ( 0 ) . A decrease in v⃗(0), i.e. , Δv⃗(0) < 0, yields an increase in d, i.e. , Δd > 0. Vascular casting A fluorescent agarose gel was formulated from 0.42% (w/v) 2 MDa fluorescein-dextran and 1% (w/v) low gelling temperature agarose (A4018; Sigma) in phosphate-buffered saline (PBS), mixed at 60 °C and maintained at this temperature prior to use ( Tsai 2004 ). Cerebral blood vessels were dilated by administration of 5 % CO 2 and 95 % O 2 with 2 % isoflurane through a nose cone for 15 min to facilitate perfusion of the gel. Animals were perfused transcardially through the left ventricle with 100 mL of PBS, 100 mL of 4% paraformaldehyde, another 50 mL of PBS to wash out residual fixative. Fifty mL of the gel was then steadily injected into the ventricle using a syringe at a rate of ∼ 2 mL per second. The gel was rapidly solidified in situ by placing the animal in an ice bath. The brain was carefully extracted to avoid damage to pial vessels. The cortex of the ischemic hemisphere was removed and flattened between two glass slides separated by a distance of 3 mm for wide-field fluorescence microscopy (Axioplan 2; Zeiss). The arteriole network, including the region imaged in vivo , was traced from overlapping images taken with a 0.5-NA 10-times magnification air objective (Zeiss).

Pimonidazole immunohistochemistry

Pimonidazole hydrochloride (Hypoxyprobe™; Hypoxyprobe.com ) is a sensitive method for detecting even small volumes of tissue hypoxia, i.e. , < 10 mm Hg tissue oxygen, compared with approximately 30 mm Hg under normoxic conditions ( Nishimura et al. 2006 ; Takasawa et al. 2008 ). Pimonidazole was injected through the femoral artery catheter at a concentration of 60 mg per kg body weight. For immunostaining, 50 μm frozen sections were treated for 10 min with 3 % (v/v) H 2 O 2 , and incubated for 72 h in anti-Hypoxyprobe™ antibody diluted 10 3 -times in PBS containing 10 % (v/v) normal goat serum (Vector Laboratories), 2 % (v/v) triton X-100 (Sigma), and 0.2 % (v/v) sodium azide (Sigma). Bound antibody was visualized with the Vectastain ABC kit and diaminobenzadine peroxidase substrate kit (both from Vector Laboratories) and brain sections were photographed using a MacroView microscope (MVX10; Zeiss). Pimonidazole staining was quantified from brain sections, originating from Bregma -3.0 mm anterior-posterior, by first determining the normalized intensity histogram for staining in both ischemic (ipsilateral) and non-ischemic (contralateral) cortices. A threshold, set to include the largest 90 % of pixel values from the non-ischemic side, was then used to isolate stained regions on the ischemic side, which was presented as a percentage of the total cortical area.

Laser Doppler flowmetry

Flowmetry measurements were performed with a MoorLab unit (Moor Instruments; λ o = 780 nm and f cut = 15 kHz low-pass filtered, to give a maximum measurable speed of λ o f cut / 2 = 6 mm/s), fitted with a MP1-V2 probe tip that was held in place with a custom adaptor over the cranial window or thinned skull. The LDF “flux” output, a measure of average RBC velocity multiplied by the intensity of the reflected signal, was collected at a sampling rate of 40 Hz using WinEDR software ( http://spider.science.strath.ac.uk/sipbs/software_ses.htm ), and averaged over 60 s intervals.

Statistics

Data are presented as mean ± standard error of the mean (SEM). We used the non-parametric Wilcoxon signed rank test unless otherwise stated since the in vivo imaging data was not always normally distributed. In Fig. 4, differences between experimental means and a theoretical line of conserved flux were tested with a two-tailed one sample t-test. In Fig. 6, differences between pimonidazole stained cortical area was analyzed with an unpaired t-test. In Fig. 7, vasodynamic changes between imaging periods were analyzed using a two-tailed paired t-test.

Supplementary Material Suppl. Figure 1 Suppl. Figure 2 Suppl. Figure 3 Suppl. Methods Suppl. Table