Collateral blood flow in different cerebrovascular hierarchy provides endogenous protection in cerebral ischemia

Chuanming Luo; Fengyin Liang; Huixia Ren; Xiaoli Yao; Qiang Liu; Mingyue Li; Dajiang Qin; Ti‐Fei Yuan; Zhong Pei; Huanxing Su

doi:10.1111/bpa.12458

. 2017 Mar 27;27(6):809–821. doi: 10.1111/bpa.12458

Collateral blood flow in different cerebrovascular hierarchy provides endogenous protection in cerebral ischemia

Chuanming Luo ^1,^†, Fengyin Liang ^2,^†, Huixia Ren ^1,^†, Xiaoli Yao ², Qiang Liu ¹, Mingyue Li ², Dajiang Qin ³, Ti‐Fei Yuan ⁴, Zhong Pei ^2,^✉, Huanxing Su ^1,^✉

PMCID: PMC8028906 PMID: 27859886

Abstract

Collateral blood flow as vascular adaptions to focal cerebral ischemia is well recognized. However, few studies directly investigate the dynamics of collateral vessel recruitment in vivo and little is known about the effect of collateral blood flow in different cerebrovascular hierarchy on the neuropathology after focal ischemic stroke. Here, we report that collateral blood flow is critically involved in blood vessel compensations following regional ischemia. We occluded a pial arteriole using femtosecond laser ablating under the intact thinned skull and documented the changes of collateral flow around the surface communication network and between the surface communication network and subsurface microcirculation network using in vivo two photon microscopy imaging. Occlusion of the pial arteriole apparently increased the diameter and collateral blood flow of its leptomeningeal anastomoses, which significantly reduced the cortical infarction size. This result suggests that the collateral flow via surface communicating network connected with leptomeningeal anastomoses could greatly impact on the extent of infarction. We then further occluded the target pial arteriole and all of its leptomeningeal anastomoses. Notably, this type of occlusion led to reversals of blood flow in the penetrating arterioles mainly proximal to the occluded pial arteriole in a direction from the subsurface microcirculation network to surface arterioles. Interesting, the cell death in the area of ischemic penumbra was accelerated when we performed occlusion to cease the reversed blood flow in those penetrating arterioles, suggesting that the collateral blood flow from subsurface microcirculation network exerts protective roles in delaying cell death in the ischemic penumbra. In conclusion, we provide the first experimental evidence that collateral blood vessels at different cerebrovascular hierarchy are endogenously compensatory mechanisms in brain ischemia.

Keywords: collateral blood flow, leptomeningeal anastomoses, microcirculation, focal ischemia, endogenous protection, two‐photon microscopys

Introduction

Focal ischemic stroke is one major cause of disability in adult humans 34. Ischemic injury leads to microvascular dysfunction, oxidative stress injury, inflammation, and cell death 13, 16, 19, 26, 31, 55. Vascular compensation such as collateral flow recruitment has been proposed as an important adaptation following regional ischemia 1, 7, 14, 15, 27, 30, 48, 52. The cerebral angioarchitecture consists of a pial vasculature which forms a communicating network connected with leptomeningeal anastomoses and a three‐dimensional subsurface microvasculature which is supplied by penetrating arterioles that are radially directed from pial arterioles and drained by penetrating venules that return blood to the surface of cortex 10, 11, 25, 38, 51. Anastomotic connections of the cortical surface and interconnections of subsurface microvasculature are the two principal connections underlying the collateral vasculature 20, 25, 38, 48, 51, 52. Previous studies have found that extensive anastomotic connections facilitate partial reperfusion of ischemic territories after focal stroke 1, 2, 4, 7, 8, 9, 10, 15, 27, 30, 33, 44, 45, 48, 51, 52, 58. However, the potential recruitment of subsurface microvessels and their contribution to focal ischemia are yet to be studied because of the limitation of imaging technique and animal models.

Several imaging techniques have been developed to record cortical perfusion after ischemic stroke, including intravital fluorescence imaging 47, laser speckle imaging 5, 22, 57, laser Doppler imaging 3, CT perfusion imaging 7, 17, MR perfusion weighted imaging (PWI) 46 and blood‐oxygenation related functional magnetic resonance imaging (fMRI) 40, 41. Unfortunately, these imaging techniques show their deficiencies in spatiotemporal resolution to the microvasculature and cellular‐level issues. Recently, two‐photon (2P) or multi‐photon microscopy is reported to offer the advantage in real‐time recordings on brain activities of live rodents with high resolution because of reduced photobleaching of dyes 45, 50, 53, suggesting that this technology has promising potential in studying the endogenous protection of collateral blood flow and documenting spatiotemporal cellular and molecular events after focal cortical ischemia.

In the present study, we generated focal cortical infarcts using focused femtosecond laser pulses induced arteriole clot under 2P‐live imaging system through intact thinned skull. The collateral vessels connecting with the target arteriole were then manipulated and recorded to investigate their potential roles during focal ischemia. We documented critical roles of collateral vessel in endogenous neuroprotection, and that subcortical microvessel network is equally important as anastomotic connections in collateral flow recruitment.

Materials and Methods

Animals

Male C57BL/6 mice weighing between 20 and 25 g and aging between 8 and 12 weeks were used in this study. All animal protocols were approved by the ethical committee of the University of Macau and followed the guidelines of animal research in University of Macau.

For surgery, the anesthesia was induced with 5%, and maintained with 2.5% isoflurane in oxygen. Rectal temperature was maintained at 37°C ± 0.5°C using a regulated heating pad with a rectal probe (TR‐200, FST, CA). A pulse oximeter clipped to the hind paw was used to monitor blood oxygen saturation and heart rate (MouseOx; Starr Life Sciences Corp., Oakmont, PA). Glucose in saline (5%) were supplied by subcutaneous injection every 2 h.

Thin‐skull window preparation

To study the pathophysiological change after distal branch of middle cerebral artery occlusion in vivo, a recent model of femtosecond laser ablation of a single pial artery with minor modifications was used 37, 39. In brief, the distal main branch of MCA between bregma and lambda was selected. After the animal was placed in a stereotaxic apparatus, an incision was made on the middle skin of the skull. A cranial window of 3.5 mm × 3.5 mm size was thinned on the left region over the distal main branch of MCA between bregma and lambda with a microdrill. The skull was cooled with aCSF during the procedure and extensive care was taken to minimize disruption of the intracranial milieu. The skull was thinned to around 30–40 μm until the pial arteries of interest appeared very clear. After completing the operation and/or imaging, the surface of thin‐skull window was covered with agarose dissolved in artificial CSF (pH 7.3, 1.5% at 37°C) and followed with a small square piece (roughly 3.5 × 3.5 mm²) of no.0 glass cover slide.

In vivo two‐photon imaging

For in vivo two‐photon imaging, the mouse was fixed with a custom‐fabricated metal frame by holding the head with a cyanoacrylate and dental cement, and then fixed on the stage of Leica DM6000 CFS. Leica LAS AF 2.5 software was applied to acquire data and control laser scanning. A Leica 0.12 numerical aperture (NA) together with an air objective at 5× magnification was used to locate the target arteriole, and determine the strategy of occlusion with Camera (Leica DFC 365 FX CCD). For high‐resolution imaging, line‐scan measurements, counting of PI‐staining cells, and Femtosecond Laser Ablation, a Leica NA 0.95 and the 25× magnification water‐immersion objectives were used. To evaluate the dynamic evolution of collateral flow and PI‐staining, a Leica NA 0.3 and the 10× magnification water‐immersion objectives were used. All images were acquired by using two‐channel NDD detection with emission filter 525/50 nm and 585/40 nm on TCS SP5 MP System (Leica Microsystems, Mannheim, Germany). The flux of interested vessels was evaluated as previously described 18, 45. Briefly, the diameter of the pial arteriole of interest was determined from at least 20 movie frames. We used line scan to measure centerline red blood cell (RBC) velocity in individual vessels of interest at a line rate of 700 Hz. The centerline RBC velocity was automatically calculated with Leica LAS AF 2.5 software. The average centerline RBC velocity throughout 30 seconds was referred as the RBC speed of interested vessels.

Pial arteriole occlusion by femtosecond laser ablation

To produce pial arteriole occlusions by damaging the endothelium of targeted vessels, intensively focused femtosecond laser pulses by a Ti:Sapphire laser (Coherent Chameleon Ultra II, CA) was employed as a light source tuned at 800 nm with 140 fs pulse width and 80 MHz reputation rate, leading to localized clotting of the vessel. A segment of the arteriole of interest was selected at the appropriate optical zoom setting for femtosecond laser ablation at the two‐photon wavelength of 800 nm. The vascular injury was induced with point bleach mode (the number of point depended on the diameter of target vessel, one second for each point. Normally, 6 points for small vessel of diameter less than 30 μm, 8–10 points for the vessel of diameter between 30 and 50 μm, 12 points for larger vessel of diameter between 50 and 80 μm) in Leica LAS AF 2.5 software. The bleach point was focused on the two edges of the lumen of target vessel in the same plane. The energy was varied according to fluorescence intensity of lumen and the diameter of the target vessel.

To minimize possible collateral damage or avoid bleeding, the endothelium of the target vessel was damaged with 800 nm laser, which intensity (max. power 3.5 W) was controlled by EOM setting at 30% (roughly 1.05 W at the plane). In most cases, this injury triggered the natural clotting cascade, leading to localized clotting of the vessel. If the occlusion did not occur, we gradually increased the intensity by approximately 10% each time until the minimum power was achieved to trigger clot formation in the target vessel. The irradiation was repeated in case of recanalization (within 24 h after the injury). If vascular rupture occurred, the animal was discarded from the study. Overall, the incidence of vascular rupture is 12.5% (totally 240 vessels). Eight mice was discarded (totally 64 mice) in this study. Persistent occlusion was confirmed at 24 h by both visual inspection and two‐photon imaging. The incidence of recanalization is 8.3% (total 240 vessels), and four mice were detected recanalization after 24 h and were excluded in this study. Physiological parameters including temperature, blood oxygen saturation and heart rate did not differ between the various occlusion groups in the course of the experiment. All animals were recovered for 24 h in a recovery cage post‐surgery under a heat lamp and had free access to drinking water and eating food.

Transcranial propidium iodide staining

To evaluate the evolution of ischemic penumbra in the modified mini‐stroke model, propidium iodide (PI) staining was employed and microcirculation network was labeled with fluorescein dextran. PI, as a marker for membrane integrity that binds to DNA/RNA, is reported to detect irreversible cell damage 47, 54. A recent study has shown that PI was able to pass through the intact murine thin‐skull into the meninges and parenchyma to label dead cells 43. Therefore, we detected the cell death by transcranial application of PI in this study. Briefly, the animal was fixed with a custom‐fabricated metal frame by holding the head with a cyanoacrylate and dental cement. The area of thinned skull was exposed through the hole of custom‐fabricated metal frame and incubated with PI (1.5 mM) in aCSF for 30 min. This was followed by a single wash with aCSF and then two‐photon microscopy imaging. Cells labeled by PI with red fluorescence were considered to be dead cells. The region which was predominantly occupied with PI‐positive cells was presumed to be the infarct core and the adjacent region which appeared no‐flow in capillaries or RBC speed in capillaries was more than three times lower than the average RBC speed was considered to be hypoperfused and the ischemic penumbral tissue. To evaluate the effects of collateral flow from subcortical microvasculature on cell death after regional ischemia, we used the 25× magnification water‐immersion objective to select three random views which contained the area of ischemic penumbra in the area of MCA‐ACA. Each view set consisted of four different depths ranging from 50–200 μm below the surface (50‐μm intervals), with four planar images collected at each depth in an XYZ order (1024 × 1024 pixels). The penumbra area in each optical section was measured using ImageJ 2.1.4.7 software (A₅₀, A₁₀₀, A₁₅₀, and A₂₀₀, respectively). The volume of penumbra in each view was calculated by V _t = (A₅₀ + A₁₀₀ + A₁₅₀ + A₂₀₀) × 50 μm. The total volume of penumbra in three views was used for statistical analysis.

Behavior study

Beam‐walking task and the adhesive removal test were used to evaluate sensory‐motor behavior in the modified mini‐stroke model according to the methods described previously 6, 12. For a beam‐walking test, we placed a narrow wooden beam which was 120 cm in length and 6 mm in width above a table. The animal was trained to walk from one end of the beam to the other before surgery in three consecutive trials each day for 3 days. The beam‐walk test was carried out on day 1, 3 and 7 after injury. We recorded the hind paw slips on each side of the beam while walking about 80 cm on the beams. Over 50 steps, the number of foot faults of the right hindlimb was counted. Mice with a basal level (<10 faults of totally 50 steps) were selected for the study.

For the Adhesive Removal test, a small adhesive patch (0.3 cm in diameter) was applied to each forepaw. The order of placement alternated between the right and left forepaw in each mice and session. The investigator pressed both forelimbs at the same time to reduce bias after the patches were placed. The animal then returned to its home cage for 120 seconds, and the time to contact and remove each adhesive tape was recorded. This test was repeated on each occasion and the mean score was used in the analysis.

Histological analysis

Mice were perfused transcardially with 50 mL of ice‐cold saline and fixed with 100 mL 4% paraformaldehyde (wt/vol). Brains were post‐fixed overnight at 4°C and then dehydrated by equilibration with 30% sucrose (wt/vol). In order to ascertain the cortical pathological changes after arteriole occlusion, the brain was isolated using the edge of thinned‐skull window as a reference point to mark the region of interest and then cut into 10 μm thick coronal slices. We collected the slices every 200 μm between bregma and lambda and followed with hematoxylin‐eosin staining for histopathological analysis. The volume of the infarct was calculated by measuring infarct areas on each slice using Image J 2.1.4.7 software, and multiplying by the distance between sections.

Statistical analysis

Statistical values were expressed as mean ± SEM. Statistical analysis was performed with SPSS 17.0 (SPSS, Inc., Chicago, IL). The centerline red blood cell (RBC) velocity and the lumen diameter were compared by one‐way Analysis of variance (ANOVA) with a Bonferroni's multiple comparison post‐hoc test; Behavioral changes and penumbral volumes among different occlusion groups were compared by two‐way ANOVA followed by a Newman–Keuls post hoc test. All of the graphs were created by GraphPad Prism 5. Significance was defined as P < 0.05 in all statistical analyses.

Results

Morphology of the target arteriole

To evaluate the effect of collateral blood flow in different cerebrovascular hierarchy on focal ischemic stroke, we first introduced a novel model of focal ischemic lesion in mice with the occlusion of the target arteriole or/and their connected vessels. Anatomical studies on C57BL/6 mice showed that the middle cerebral artery (MCA) travels dorsally toward the top of the brain where it splits into three branches: the rostral branch, the middle branch and the caudal branch (Figure 1A2,C). We intravenously infused fluorescein dextran to the mice and then imaged cortical vasculature with 2P microscopy. The middle branch and the anterior part of the caudal branch of MCA as well as their leptomeningeal anastomoses and penetrating arterioles appeared very clear in the field of the thin‐skull window (Figure 1A–C). The middle branch of the MCA runs from the lateral edge toward the medial axis between bregma and lambda, which mainly supplies the forelimb region and hindlimb region of somatosensory cortex (S1FL and S1HL) and part of the barrel cortex (S1BF) 20. We selected the branch distal to the lenticulostriate branch of the MCA (Coordinates: lateral 3.4 ± 0.1 mm, anterior‐posterior 0.8 ± 0.08 mm) as the target arteriole (named as “the target arteriole” in the following) and traced all of its leptomeningeal anastomoses. Three‐dimensional images under high‐resolution were used to determine the diameter of the target arteriole that lies between the upstream and downstream of the target. The middle section of the target arteriole was used for the diameter measurement. The average diameter of the target arteriole was 56.5 μm (56.5 ± 5.6 μm, n = 8). It contained an average of 5 leptomeningeal anastomoses (mean is 5.25, 42 vessels in eight mice) which displayed variable configurations, including end‐to‐end (52%), end‐to‐side (43%) and communicating arterioles (5%) (Figure 1D). Occlusions of leptomeningeal anastomoses were generated by focusing femtosecond‐duration laser pulses onto the two edges of the lumen in the same plane. The vessel wall was injured by nonlinear absorption of laser energy, which triggered clotting formation as reported 35, 37. Only the occlusion of the anastomoses with this laser method did not cause potential off‐target effects of irradiation and over damage that could be incurred by multiple irradiations (Figure 1E and Supporting Information Video 1).

Dynamic changes in collateral flow of leptomeningeal anastomoses after occlusion of the target arteriole

The baseline of RBC speed and vessel diameter (pre‐occlusion) was measured in all of leptomeningeal anastomoses connected to the target arteriole. Then, occlusion of the target arteriole was generated by Femtosecond Laser Ablation (Figure 2A). Briefly, the occlusion of target arteriole was induced by focusing femtosecond laser pulses on 12 points at the two edges of the lumen of the target vessel at the same plane with bleach mode controlled by Leica LAS AF 2.5 software. At 30 min, 3 h and 24 h after the clot formation, the lumen diameter and RBC speed of the leptomeningeal anastomoses were continuously recorded. In the first 3 min after vascular occlusion, brain perfusion was found to decrease rapidly and dramatically in the area located downstream of the occlusion site in all animals. Subsequently, the flow direction was reversed and the RBC speed gradually accelerated in a large proportion of leptomeningeal anastomoses (Figure 2B; Supporting Information Video 2).

At 30 min after a clot, the diameter of leptomeningeal anastomoses slightly dilated when compared with the baseline (Figure 2B,D; P < 0.05, n = 42). Correspondingly, the RBC speed apparently accelerated in comparison with the baseline (Figure 2B, C, E, and F; P < 0.01, n = 42). At 3 h after a clot, the diameter of leptomeningeal anastomoses became apparently dilated (Figure 2B,D; P < 0.001, n = 42). Meanwhile, the RBC speed further accelerated in a 95% of leptomeningeal anastomoses (Figure 2C, E, and F; P < 0.01, n = 42). At 24 h after a clot, the vessel dilation became more prominent and the RBC speed obviously accelerated compared with the baseline (Figure 2B–F; P < 0.0001, n = 42).

Collateral blood flow via leptomeningeal anastomoses reduces brain infarction volume

To evaluate whether the collateral blood flow of leptomeningeal anastomoses affected focal cortical ischemia, we used a 5× magnification air objective to obtain images of the target arteriole as well as leptomeningeal anastomoses and penetrating arterioles, and then outlined the area supplied by the target arteriole with Camera (Leica DFC 365 FX CCD). We further modified the area supplied by the target arteriole to an approximate 2.0 × 2.0 mm area containing its leptomeningeal anastomoses (Figure 3A). Five types of occlusion have been performed: Type I is occlusion of the target arteriole without collateral vessel clot; Type II is selective occlusion of leptomeningeal anastomoses (with 1–2 leptomeningeal anastomoses of MCA–ACA remaining intact) without clotting the target arteriole; Type III is occlusion of all of its leptomeningeal anastomoses without clotting the target arteriole; Type IV is occlusion of the target arteriole as well as selectively clotting leptomeningeal anastomoses (with 1–2 leptomeningeal anastomoses of MCA–ACA remaining intact); Type V is occlusion of the target arteriole and all of its leptomeningeal anastomoses (Figure 3A).

Three days after the clot, HE staining showed that there was a huge variation in cortical infarct volume among the five groups (Figure 3A–C). Ischemia lesion was almost invisible in type I and completely invisible in type II and III. Ischemia lesion could be observed but with a great variety in the infarction volume in type IV (The mean infarction volume was 2.138 ± 0.378 mm³ with the maximal volume being 3.68 mm³ and the minimal volume being 0.32 mm³; Coefficient of variation was 17.68%; n = 8).

In type V animals, the infarction volume exhibited some degree of variation but predominantly centered around the mean number (3.85 ± 0.186 mm³) in that the maximal infarct volume was found to be 4.22 mm³ and the minimal volume 3.22 mm³ (Coefficient of variation is 3.08%; n = 8) (Figure 3B,C). We then further performed the behavioral evaluation using adhesive removal test and the beam‐walking test to display any neurological deficits in the five groups. Consistent with the histological studies, the adhesive removal abilities of clotted animals were significantly impaired on day 1, day 3 and day 7 in type V animals, but not in type I, II, III and IV (Figure 3D; two‐way ANOVA followed by Newman–Keuls post hoc test; F = 23.96, df = 4, P < 0.0001; n = 8 per group). No significant difference has been found with beam‐walking test in each type animals (Figure 3E; two‐way ANOVA followed by Newman–Keuls post hoc test; F = 0.87, df = 4, P = 0.484; n = 8 per group).

Penetrating arterioles reverse blood flow after occlusion of the target arteriole and all its connected vessels

After occlusion of the target pial arteriole and all its connected vessels to cease the collateral blood flow from anastomotic connections of the cortical surface, we further monitored the collateral blood flow from the subsurface microvasculature through documented the change of blood flow in penetrating arterioles using live imaging. The targeted region in our clotting experiment contained an average of 16 penetrating arterioles (48 vessels in three mice) in an approximate 2.0 × 2.0 mm area. In the first 3 min after the clot, we found that the RBC speed of surface vessels and penetrating arterioles decreased dramatically and even completely ceased (Supporting Information Videos 3 and 4). Subsequently, some penetrating arterioles, mainly proximal to the target arteriole and the edge of ischemic lesions, reversed the flow direction to return the blood to surface pial arterioles from subcortical circulatory networks (Figure 4A–C; Supporting Information Videos 3 and 4). Around 22.9%, 20.8% and 14.6% penetrating arterioles at 30 min, 3 h and 24 h after the clot respectively was found to reverse the blood flow direction. We also found that there was still blood flow with a very slow speed in the normal direction in most penetrating arterioles distal to the target arteriole (Figure 4C). Since we occluded all the surface collateral vessels of the target pial arteriole, we proposed that the reversed blood in the penetrating arterioles has come from the subsurface microvasculature. The averaged absolute values of RBC speed in penetrating arterioles were extremely slow compared with the baseline at each time point after occlusion (Figure 4E; P < 0.001, n = 48) and the diameter of penetrating arterioles was found to contract significantly compared with the baseline (Figure 4D; P < 0.05, n = 48). The number of penetrating arterioles with reversed blood at 24 h was decreased compared with that at 30 min, 3 h and 24 h after the clot.

Collateral blood flow from subcortical microvasculature delays cell death in ischemic penumbra

To evaluate the effect of collateral blood flow from subcortical microvasculature on ischemic lesions, we further introduced the PI‐staining to indicate the cell damage. PI, as a marker for membrane integrity that binds to DNA/RNA, has been reported to detect irreversible cell damage 47, 54. A recent study has shown that PI is able to pass through the intact murine thin‐skull into the meninges and parenchyma to label dead cells 43. Therefore, we detected the cell death by transcranial application of PI in this study. We firstly performed the transcranial PI‐labeling in vivo and then NeuN‐staining at 24 h after occlusion of the target arteriole and all its connected vessels. NeuN staining showed that the PI‐occupied region was devoid of NeuN‐positive cells, suggesting that there was no neuronal survival in the PI‐occupied region (Figure 5A).

Collateral blood flow from subcortical microvasculature delays cell death in the ischemic penumbra. (A) NeuN staining and transcranial PI‐labeling showed that the PI‐occupied region was devoid of NeuN‐positive cells. (B) Representative images showing the decrease of the penumbral zone over time after occlusion of the target arteriole as well as its all leptomeningeal anastomoses. Infarction core: above the blue dotted line; the region of hypoperfusion: between the blue dotted line and the white dotted line. (C) Representative images under high‐solution illustrated the evolution of the cell death labeled by PI and the region of ischemic penumbra in Type V animals and Type VI animals. (D) Quantification showing the penumbral volume in type VI animals was apparently decreased compared with type V animals (Two‐way ANOVA followed by Newman–Keuls *post hoc* test; F = 172.86, df = 1, P < 0.001; n = 8 per group). Scale bar: 150 μm for A, 100 μm for B and 60 μm for C.

To document the evolution of ischemic penumbra, we imaged the cell death in the target area at 100 μm below the cortical surface, at 0 min, 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after occlusion of the target arteriole and all its connected vessels. The region of hypoperfusion appeared immediately after the clot (Figure 5A). The number of PI‐positive cells increased gradually and the PI‐positive cells‐occupied region expanded over time, suggesting a temporal increase in the infarct core (Figure 5A,B). Accordingly, the penumbral region decreased gradually over time (Figure 5B). PI‐positive cells were found to be filled with the penumbral region at 72 h after the clot (Figure 5B; Supporting Information Video 5).

In order to evaluate the effects of collateral flow from subcortical microvasculature on cell death after regional ischemia, another experimental group was used in which a further clot was performed on the superficial section of penetrating arterioles by using focusing femtosecond‐duration laser pulses to cease all of reversed blood flow from the subsurface microcirculation after occlusion of the target arteriole and all its connected vessels (named as “type VI”). Our data showed that the volume of ischemia penumbra in type V animals was 0.132 ± 0.042 mm³, 0.092 ± 0.025 mm³, 0.075 ± 0.013 mm³ and 0.019 ± 0.0060 mm³ at 12 h, 24 h, 48 h and 72 h after the clot, respectively, and the volume of ischemia penumbra in type VI animals was 0.025 ± 0.012 mm³, 0.012 ± 0.008 mm³, 0.007 ± 0.002 mm³ and 0.001 ± 0.000 mm³ at 12 h, 24 h, 48 h and 72 h after the clot, respectively. The volume of ischemia penumbra decreased quickly and PI‐positive cells were found to be filled with the penumbral region much earlier in type VI animals compared with type V animals (Figure 5C,D; two‐way ANOVA followed by Newman–Keuls post hoc test; F = 172.86, df = 1, p < 0.001; n = 8 per group), indicating that collateral blood flow from subcortical microvasculature has delayed cell death in ischemic penumbra.

Discussions

Collateral flow reduces the infarction volume after focal stroke 52. Different cerebrovascular hierarchy may exert different contribution to the effectiveness of collateral flow 10, 11, 25, 29, 37, 38, 51, 52. However, little is known about their effect on focal stroke because of the limitation of imaging technique and animal models. Here, we report a focal stroke in the somatosensory cortex induced by focused femtosecond laser pulses through an intact thinned‐skull window. In this model, we have firstly in vivo documented the dynamic change of collateral blood flow via surface communicating networks or subsurface microvasculature networks and proved its importance as endogenous protection mechanisms following focal stroke.

The pial network of arterioles in the neocortex exhibits similar structure in both rodents and humans and neighboring cerebral arteries form a communicating network connected with leptomeningeal anastomoses 10, 11, 38, 48, 51. Those surface arterioles send out penetrating arterioles which plunge perpendicularly into the brain to supply subsurface microvascular bed and connected to an underlying, three‐dimensional microcirculation network 10, 11, 38, 51. Such a network is an ideal platform to study the ischemic lesion and cerebral blood flow redistribution in response to vascular occlusions.

In this study, we have developed a focal stroke model which is different from classical dMCAO model induced by ligation or electrocoagulation of the MCA distal to the lenticulostriate branch. We selected the trunk of the middle branch of MCA, the downstream branch of lenticulostriate branch, as the target arteriole, which runs from the lateral edge toward the medial axis between bregma and lambda mainly supplying somatosensory cortex. This target arteriole is more suitable for obtaining the spatiotemporal high‐resolution imaging of collateral flow events from direct observation through an intact thin‐skulled window using 2‐photon microscopy. The target arteriole contains abundant leptomeningeal anastomoses (an average of five leptomeningeal anastomoses) in C57BL/6 mice.

Occluding the target arteriole has not only induced the reversal in blood flow from the downstream but also led to the dynamic change in the diameter and the RBC speed in leptomeningeal anastomoses apparently. Correspondingly, very minimal infarction has been observed in this model after clotting the target arteriole only, suggests that collateral flow from leptomeningeal anastomoses may be sufficient to rescue the territory of a distal MCA branch. In contrast, the infarction volume was greatly variable when we occluded the target arteriole as well as selective leptomeningeal anastomoses. These findings have suggested that the collateral flow via surface communicating network connected with leptomeningeal anastomoses could greatly impact on the extent of infarction, and were highly consistent with previous studies which reported a robust redistribution in blood flow after vascular occlusion of cortical surface vessels 1, 4, 8, 25, 30, 33, 44, 45, 48, 52.

Previous studies have found that the subsurface microvasculature forms interconnected loops with a topology which limit collateral flow from neighboring penetrating arterioles 10, 11, 35, 37, 38, 49. Surprisingly, we were unable to obtain a very consistent infarction (Coefficient of variation is 3.08%; n = 8) even if we occluded the target pial arteriole and all its connected vessels in an outlined area. Meanwhile, we were also unable to make the blood flow completely cease although we performed a clot of the target arteriole and all its connected vessels to limit the collateral blood flow from leptomeningeal anastomoses. We observed the blood flow reversal in some of the penetrating arterioles mainly proximal to the target arteriole and the edge of ischemic lesions when the collateral reflow from leptomeningeal anastomoses was absent. Compared with those changes in the surface anastomoses, the RBC speed of reversed blood in penetrating arterioles was extremely slow. Subsequently, we further investigated the effect of collateral blood flow from subcortical microvasculature on ischemic lesions using PI‐staining to indicate the cell death. Interesting, the cell death in the area of ischemic penumbra was apparently accelerated when we performed a clot to cease all of blood flow reversals from penetrating arterioles after occlusion of the target arteriole and all its connected vessels. Therefore, our study suggested that the collateral blood flow from subsurface microcirculation network may exert protective roles in delaying the cell death in ischemic penumbra in the absence of the collateral flow from the surface communicating network.

Rapid reperfusion of the penumbra using thrombolytic therapy or endovascular treatment should be at the core of all acute ischemic stroke interventions 23, 24. However, a relatively narrow reperfusion time window limits the application of these therapeutic strategies 23, 24, 32. How to protect the ischemic penumbra hence to prevent the infarct core growing may be a potential interesting therapeutic target. The augmentation or maintenance of collateral flow is considered to be an effective strategy for protecting the ischemic penumbra after acute stroke. Several experimental techniques such as partial aortic occlusion, drug‐induced volume expansion, external pressure cuffs, and stimulation of the sphenopalatine ganglion could be used to increase circulation or opening of the collaterals 36, 52. However, the effectiveness of these techniques needs to be validated with randomized controlled trials in patients, and the protective mechanism of the collateral blood flow at the cellular level and molecular level needs to be further investigated in the future. In this study, we have developed a new focal stroke model induced by focused femtosecond laser pulses with 2P‐live imaging system through the intact skull, which is different from classical MCAO model induced by ligation or electrocoagulation of the MCA proximal to the lenticulostriate branch 21, 28, 39, 42, 56. Our model shows its advantages in intravital study of pathophysiological event of ischemic stroke including collateral flow, ischemia penumbra and cell death. In contrast to the cortical stroke model induced by photothrombosis, this focal stroke model is induced by the occlusion of arterial system only rather than the venous system and microcirculation. Meanwhile, we could investigate the role of the collateral blood flow from subsurface microcirculation network on cellular level through controlling the collateral reflow from leptomeningeal anastomoses using focused femtosecond laser pulses with 2P microscopy.

In summary, we have firstly documented the phenomenon of collateral flow within subsurface microcirculation network, which cooperates with surface arterioles to function as endogenous vascular compensation after ischemic stroke. Targeting these vasculatures therefore might promote the endogenous protective mechanism in brain ischemia.

Author Contributions

HS and ZP designed the study; CL, FL, HR, QL, and ML performed the experiment; CL, XY, TY and DQ analyzed the data; CL, TY, and HS wrote the manuscript together.

Competing Financial Interests

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web‐site:

Video 1. Occlusion of leptomeningeal anastomoses by nonlinear absorption of ultrashort laser pulses to generate highly focused irradiation without affecting adjacent tissues. The bloodstream in anastomoses in the video was shown by FITC‐d2000 labeling through tail intravenous injection. Only the occlusion of the anastomoses with this laser method did not appear over damage incurred by multiple irradiations and potential off‐target effects of irradiation.

Click here for additional data file.^{(2MB, wmv)}

Video 2. Dynamic changes in collateral flow of leptomeningeal anastomoses after occlusion of the target arteriole. The bloodstream in the video was shown by FITC‐d2000 labeling through tail intravenous injection. The video firstly showed the baseline of RBC speed and diameter of anastomoses connected to the target arteriole. Then, occlusions of the target arteriole were generated as described above. At 30 min after a clot, the diameter of anastomoses slightly dilated when compared with the baseline. Correspondingly, the RBC speed apparently accelerated in comparison with the baseline. At 3 h after a clot, the diameter of anastomoses became remarkably dilated. Meanwhile, the RBC speed further accelerated. At 24 h after a clot, the vessel dilation became more prominent and the RBC speed obviously accelerated compared with the baseline.

Click here for additional data file.^{(4.5MB, wmv)}

Video 3. The reversal of blood flow in penetrating arterioles after occlusion of the target arteriole and all its connected vessels. The bloodstream in the video was shown by FITC‐d2000 labeling through tail intravenous injection. The video firstly showed the baseline of RBC speed and diameter of penetrating arteriole. Then, the target arteriole and all its connected vessels were occluded as described above. Penetrating arterioles were found to reverse the blood flow direction at 30 min, 3 h and 24 h after the clot and the diameter of penetrating arterioles significantly contracted compared with the baseline.

Click here for additional data file.^{(4.4MB, wmv)}

Video 4. Dynamic change of blood flow in penetrating arterioles of the distal branch of the MCA after occlusion of the target arteriole and all its connected vessels. The bloodstream in the video was shown by FITC‐d2000 labeling through tail intravenous injection. The video firstly showed the baseline of RBC speed and diameter of penetrating arteriole of distal MCA. Then, the target arteriole and all its connected vessels were occluded as described above. Blood flow at a very slow speed in the normal direction was found in the penetrating arterioles distal to the target arteriole at 30 min, 3 h and 24 h after the clot.

Click here for additional data file.^{(5MB, wmv)}

Video 5. Dynamic change of the ischemic penumbra after occlusion of the target arteriole and all its connected vessels. As shown in the video, the region which appeared no‐flow in blood was considered to be the ischemic penumbral tissue. The number of PI‐positive cells increased gradually and the PI‐positive cells‐occupied region expanded at 12 h, 24 h and 48 h after a clot. PI‐positive cells were found to be filled with the penumbral region at 72 h after the clot (Green fluorescent was labeled by FITC‐d2000 through tail intravenous injection; Red fluorescent was labeled by transcranial PI staining).

Click here for additional data file.^{(15.6MB, wmv)}

Cartoon. Using femtosecond laser ablation, the distal main branch of middle cerebral artery (MCA) between bregma and lambda was occluded (green arrow). The collateral blood flow of its leptomeningeal anastomoses (yellow arrows) was initiated immediately after occlusion. After further occlusion of all of its leptomeningeal anastomoses, a reversal of blood flow in the penetrating arterioles (red arrows) mainly proximal to the occluded pial arteriole was observed.

Click here for additional data file.^{(3.2MB, avi)}

Acknowledgments

This study was supported by Macao Science and Technology Development Fund (018/2013/A1), Ministry of Science and Technology 973 program of China (2012CB966800), and MYRG2016‐00184‐ICMS‐QRCM, Science and Technology Planning Project of Guangdong Province (2013B051000018), and the National Key Clinical Department, National Key Discipline, and Guangdong Key Laboratory for diagnosis and treatment of major neurological disease.

Contributor Information

Zhong Pei, Email: peizhong@mail.sysu.edu.cn.

Huanxing Su, Email: huanxingsu@umac.mo.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's web‐site:

Click here for additional data file.^{(2MB, wmv)}

Click here for additional data file.^{(4.5MB, wmv)}

Click here for additional data file.^{(4.4MB, wmv)}

Click here for additional data file.^{(5MB, wmv)}

Click here for additional data file.^{(15.6MB, wmv)}

Click here for additional data file.^{(3.2MB, avi)}

[bpa12458-bib-0001] 1. Akamatsu Y, Nishijima Y, Lee CC, Yang SY, Shi L, An L et al (2015) Impaired leptomeningeal collateral flow contributes to the poor outcome following experimental stroke in the Type 2 diabetic mice. J Neurosci 35:3851–3864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0002] 2. Alves HC, Pacheco FT, Rocha AJ (2016) Collateral blood vessels in acute ischemic stroke: a physiological window to predict future outcomes. Arq Neuropsiquiatr 74:662–670. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0003] 3. Ances BM, Greenberg JH, Detre JA (1999) Laser doppler imaging of activation‐flow coupling in the rat somatosensory cortex. Neuroimage 10:716–723. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0004] 4. Aoki J, Tateishi Y, Cummings CL, Cheng‐Ching E, Ruggieri P, Hussain MS, Uchino K (2014) Collateral flow and brain changes on computed tomography angiography predict infarct volume on early diffusion‐weighted imaging. J Stroke Cerebrovasc Dis 23:2845–2850. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0005] 5. Armitage GA, Todd KG, Shuaib A, Winship IR (2010) Laser speckle contrast imaging of collateral blood flow during acute ischemic stroke. J Cereb Blood Flow Metab 30:1432–1436. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0006] 6. Balkaya M, Krober J, Gertz K, Peruzzaro S, Endres M (2013) Characterization of long‐term functional outcome in a murine model of mild brain ischemia. J Neurosci Methods 213:179–187. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0007] 7. Bang OY, Goyal M, Liebeskind DS (2015) Collateral circulation in ischemic stroke: assessment tools and therapeutic strategies. Stroke 46:3302–3309. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0008] 8. Beretta S, Cuccione E, Versace A, Carone D, Riva M, Padovano G et al (2015) Cerebral collateral flow defines topography and evolution of molecular penumbra in experimental ischemic stroke. Neurobiol Dis 74:305–313. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0009] 9. Beyer SE, von Baumgarten L, Thierfelder KM, Rottenkolber M, Janssen H, Dichgans M et al, (2015) Predictive value of the velocity of collateral filling in patients with acute ischemic stroke. J Cereb Blood Flow Metab 35:206–212. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0010] 10. Blinder P, Shih AY, Rafie C, Kleinfeld D (2010) Topological basis for the robust distribution of blood to rodent neocortex. Proc Natl Acad Sci U S A 107:12670–12675. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0011] 11. Blinder P, Tsai PS, Kaufhold JP, Knutsen PM, Suhl H, Kleinfeld D (2013) The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow. Nat Neurosci 16:889–897. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0012] 12. Bouet V, Boulouard M, Toutain J, Divoux D, Bernaudin M, Schumann‐Bard P, Freret T (2009) The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice. Nat Protoc 4:1560–1564. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0013] 13. Broughton BR, Reutens DC, Sobey CG (2009) Apoptotic mechanisms after cerebral ischemia. Stroke 40:e331–e339. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0014] 14. Brunner F, Tomandl B, Hanken K, Hildebrandt H, Kastrup A (2014) Impact of collateral circulation on early outcome and risk of hemorrhagic complications after systemic thrombolysis. Int J Stroke 9:992–998. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0015] 15. Campbell BC, Christensen S, Tress BM, Churilov L, Desmond PM, Parsons MW et al (2013) Failure of collateral blood flow is associated with infarct growth in ischemic stroke. J Cereb Blood Flow Metab 33:1168–1172. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0016] 16. Chamorro A, Meisel A, Planas AM, Urra X, van de Beek D, Veltkamp R (2012) The immunology of acute stroke. Nat Rev Neurol 8:401–410. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0017] 17. Chen H, Wu B, Liu N, Wintermark M, Su Z, Li Y et al (2015) Using standard first‐pass perfusion computed tomographic data to evaluate collateral flow in acute ischemic stroke. Stroke 46:961–967. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0018] 18. Cho EE, Drazic J, Ganguly M, Stefanovic B, Hynynen K (2011) Two‐photon fluorescence microscopy study of cerebrovascular dynamics in ultrasound‐induced blood‐brain barrier opening. J Cereb Blood Flow Metab 31:1852–1862. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0019] 19. Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391–397. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0020] 20. Dorr A, Sled JG, Kabani N (2007) Three‐dimensional cerebral vasculature of the CBA mouse brain: a magnetic resonance imaging and micro computed tomography study. Neuroimage 35:1409–1423. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0021] 21. Doyle KP, Buckwalter MS (2014) A mouse model of permanent focal ischemia: distal middle cerebral artery occlusion. Methods Mol Biol 1135:103–110. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0022] 22. Dunn AK, Bolay H, Moskowitz MA, Boas DA (2001) Dynamic imaging of cerebral blood flow using laser speckle. J Cereb Blood Flow Metab 21:195–201. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0023] 23. Eltzschig HK, Eckle T (2011) Ischemia and reperfusion–from mechanism to translation. Nat Med 17:1391–1401. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0024] 24. Goyal M, Demchuk AM, Menon BK, Eesa M, Rempel JL, Thornton J et al (2015) Randomized assessment of rapid endovascular treatment of ischemic stroke. N Engl J Med 372:1019–1030. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0025] 25. Hirsch S, Reichold J, Schneider M, Szekely G, Weber B (2012) Topology and hemodynamics of the cortical cerebrovascular system. J Cereb Blood Flow Metab 32:952–967. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0026] 26. Iadecola C, Anrather J (2011) The immunology of stroke: from mechanisms to translation. Nat Med 17:796–808. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0027] 27. Jung S, Gilgen M, Slotboom J, El‐Koussy M, Zubler C, Kiefer C et al (2013) Factors that determine penumbral tissue loss in acute ischaemic stroke. Brain 136(Pt 12):3554–3560. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0028] 28. Kuraoka M, Furuta T, Matsuwaki T, Omatsu T, Ishii Y, Kyuwa S, Yoshikawa Y (2009) Direct experimental occlusion of the distal middle cerebral artery induces high reproducibility of brain ischemia in mice. Exp Anim 58:19–29. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0029] 29. Lau AY, Wong EH, Wong A, Mok VC, Leung TW, Wong KS (2012) Significance of good collateral compensation in symptomatic intracranial atherosclerosis. Cerebrovasc Dis 33:517–524. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0030] 30. Leger PL, Bonnin P, Moretti R, Tanaka S, Duranteau J, Renolleau S et al (2016) Early recruitment of cerebral microcirculation by neuronal nitric oxide synthase inhibition in a juvenile ischemic rat model. Cerebrovasc Dis 41:40–49. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0031] 31. Love S (1999) Oxidative stress in brain ischemia. Brain Pathol 9:119–131. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bpa12458-bib-0032] 32. Manning NW, Campbell BC, Oxley TJ, Chapot R (2014) Acute ischemic stroke: time, penumbra, and reperfusion. Stroke 45:640–644. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0033] 33. Martinon E, Lefevre PH, Thouant P, Osseby GV, Ricolfi F, Chavent A (2014) Collateral circulation in acute stroke: assessing methods and impact: a literature review. J Neuroradiol 41:97–107. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0034] 34. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M et al, (2015) Heart disease and stroke statistics–2015 update: a report from the American Heart Association. Circulation 131:e29–322. [DOI] [PubMed] [Google Scholar]

[bpa12458-bib-0035] 35. Nguyen J, Nishimura N, Fetcho RN, Iadecola C, Schaffer CB (2011) Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries. J Cereb Blood Flow Metab 31:2243–2254. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Collateral blood flow in different cerebrovascular hierarchy provides endogenous protection in cerebral ischemia

Chuanming Luo

Fengyin Liang

Huixia Ren

Xiaoli Yao

Qiang Liu

Mingyue Li

Dajiang Qin

Ti‐Fei Yuan

Zhong Pei

Huanxing Su

Abstract

Introduction

Materials and Methods

Animals

Thin‐skull window preparation

In vivo two‐photon imaging

Pial arteriole occlusion by femtosecond laser ablation

Transcranial propidium iodide staining

Behavior study

Histological analysis

Statistical analysis

Results

Morphology of the target arteriole

Figure 1.

Figure 2.

Dynamic changes in collateral flow of leptomeningeal anastomoses after occlusion of the target arteriole

Collateral blood flow via leptomeningeal anastomoses reduces brain infarction volume

Figure 3.

Penetrating arterioles reverse blood flow after occlusion of the target arteriole and all its connected vessels

Figure 4.

Collateral blood flow from subcortical microvasculature delays cell death in ischemic penumbra

Figure 5.

Discussions

Author Contributions

Competing Financial Interests

Ethical Approval

Supporting information

Acknowledgments

Contributor Information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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