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J Cereb Blood Flow Metab. 2010 August; 30(8): 1432–1436.
Published online 2010 June 2. doi:  10.1038/jcbfm.2010.73
PMCID: PMC2949243

Laser speckle contrast imaging of collateral blood flow during acute ischemic stroke

Abstract

Collateral vasculature may provide an alternative route for blood flow to reach the ischemic tissue and partially maintain oxygen and nutrient support during ischemic stroke. However, much about the dynamics of stroke-induced collateralization remains unknown. In this study, we used laser speckle contrast imaging to map dynamic changes in collateral blood flow after middle cerebral artery occlusion in rats. We identified extensive anastomatic connections between the anterior and middle cerebral arteries that develop after vessel occlusion and persist for 24 hours. Augmenting blood flow through these persistent yet dynamic anastomatic connections may be an important but relatively unexplored avenue in stroke therapy.

Keywords: animal models, cerebral blood flow, embolic stroke, focal ischemia, neuroprotection, pial vessels

Introduction

Death and disability after focal stroke results from neuronal death induced by impaired blood flow to a particular region of the brain. As cell death evolves over hours and days after ischemic onset (Hossmann, 2006), neuroprotective strategies that restore cerebral blood flow to the ischemic region or that halt the cell death cascade in injured neurons may save the brain tissue from death and improve recovery (Green, 2008). It has been suggested that augmenting collateral circulation may be neuroprotective during stroke.

Collateral circulation refers to subsidiary vascular networks in the brain, the pathophysiological recruitment of which can partially maintain blood flow when primary vascular routes are blocked (Liebeskind, 2003). In particular, clinical data suggest that collateral blood flow through the circle of Willis and anastomatic connections between the distal segments of cerebral arteries may facilitate partial reperfusion of ischemic territories after focal stroke (Liebeskind, 2003). Treatments that increase cerebral blood flow to ischemic regions through this collateral vasculature may therefore be neuroprotective (Liebeskind, 2005). However, the development of collateral therapeutics is limited in part by a paucity of research on the extent and duration of the changes in collateral circulation elicited by stroke. In this study, we define dynamic changes in collateral blood supply to ischemic territories using repeated laser speckle contrast imaging (LSCI) over a 24-hour period after ischemic onset in a clinically relevant thromboembolic model of middle cerebral artery occlusion (MCAo) in rats (Wang et al, 2001).

Materials and methods

A total of 15 male Sprague–Dawley rats (weighing 300 to 350 g) were used in this study. Experimental protocols conform to the guidelines established by the Canadian Council on Animal Care. Briefly, anesthetized rats implanted with a chronic imaging window underwent LSCI immediately before and after MCAo and 24 hours after ischemic onset. For the duration of surgical and imaging procedures (~3 hours for initial sessions incorporating imaging window implantation, MCAo, and image acquisition; <1 hour for 24 hours imaging sessions), rats were anesthetized with 1.5% to 2.0% isoflourane (30:70, O2:N20). Temperature was maintained at 36.7°C, and heart rate, oxygen saturation, and breath rate were monitored using a MouseOx pulse oximeter (STARR Life Sciences, Oakmont, PA, USA). Animals were housed in standard cages in a 12-hour light/dark cycle with free access to food and water before and after surgical procedures.

Imaging Window

A 5 × 10 mm2 craniotomy was performed over the right sensorimotor cortex (~1 to 5.5 mm lateral of the midline and ±5 mm from the bregma, Figure 1A; Paxinos and Watson, 2007), leaving the dura intact. A thin layer of 1.3% low-melt agarose (in artificial cerebrospinal fluid) was placed on the exposed dura, sealed with a coverslip, and anchored in place with a perimeter of dental cement.

Figure 1
Surgical preparation, histology, and blood flow mapping in sham animals. (A) Imaging windows were placed 1 mm lateral from the bregma directly over the primary motor and somatosensory cortex for the forelimb and hindlimb. (B) Representative hematoxylin ...

Middle Cerebral Artery Occlusion

Embolic MCAo was induced as reported previously (Wang et al, 2001). In brief, a 1.5-cm longitudinal incision was made along the midline of the ventral cervical skin. The distal portion of the external carotid artery was ligated and cut. A modified PE-10 catheter filled with bovine thrombin was inserted into the right external carotid artery and blood was withdrawn and allowed to clot for 15 minutes. Once the clot formed, the catheter was advanced 17 mm into the internal carotid artery until it reached the origin of the MCA. The clot was injected into the MCA, the catheter removed, and the wound closed. Sham animals underwent identical surgery without blood clot injection.

Laser Speckle Contrast Imaging and Analysis

Rats were placed in a custom stereotaxic plate under a Dalsa 1M60 Pantera camera (Dalsa Corporation, Waterloo, ON, Canada) mounted on a video macroscope. A total of 300 to 500 image frames were collected at 10 Hz (with 15 milliseconds exposure time) during illumination with a 784 nm (32 mW) laser (StockerYale, Inc., Salem, NH, USA). Analysis of laser speckle images was performed using ImageJ software (NIH, Bethesda, MD, USA). The speckle contrast factor K is a measure of the local spatial contrast of the laser speckle pattern and is defined as the ratio of the s.d. to the mean intensity (K=σs/I) in a small (5 × 5 pixels) region of the speckle image. K has a minimal value of 0 when the scattering particles are moving quickly, a maximal value of 1 with no movement, and is inversely related to blood flow velocity (Ayata et al, 2004; Li and Murphy, 2008; Shin et al, 2008). Maps of the speckle contrast value at each pixel of the image show the pattern of blood flow on the cortical surface during imaging, with darker vessels showing relatively faster blood flow than lighter vessels. The mean speckle contrast values in large-diameter surface veins (labeled V in Figure 1D) and branches of the MCA (labeled MCA in Figure 1D) were calculated by measuring the average speckle contrast in 2 to 8 equidistant regions of interest (ROIs) within the vessel borders defined by LSCI along the medial-lateral extent of these large veins (average of 3.84 ROIs per vein, mean area of 565±86 pixels2 per ROI) and MCA branches (average of 4.42 ROIs per artery, mean area of 143±13 pixels2 per ROI).

Stroke Size and Location

After the final imaging session, anesthetized animals were killed and their brains removed. Stroke size and location was assessed on cryostat-sectioned 20 μm coronal brain sections stained with hematoxylin and eosin or fresh 2 mm brain sections stained with 2,3,5-triphenyltetrazolium chloride.

Results

Histology

Infarct size and location was assessed 24 hours after MCAo onset (n=10). Nine rats had infarcts incorporating the striatum as well as the primary and secondary somatosensory cortices. In three animals, the infarct extended to the primary motor cortex, with two infarcts also incorporating the secondary motor cortex. In one rat, the infarct was restricted to the cortex (no striatal damage was observed). Sham animals (n=5) did not have infarcts. Figures 1B and 1C show representative hematoxylin and eosin staining and the largest and smallest infarcts produced by MCAo, respectively.

Laser Speckle Contrast Imaging

Maps of blood flow on the surface of the frontoparietal cortex incorporating the sensorimotor cortex for the forelimb and hindlimb were made using LSCI immediately before and after MCAo or sham surgery and again at 24 hours after ischemic onset (or sham). As shown in Figure 1D, the map of blood flow attained through LSCI in sham animals was highly consistent over time. Importantly, sham animals never exhibited anastomatic connections between the distal segments of the anterior cerebral artery (ACA) and MCA. As speckle contrast values are inversely related to blood flow velocity, a relative measure of blood flow changes in vessels within the imaging window could be made by comparing prestroke and poststroke speckle contrast values in rats after MCAo. By measuring speckle contrast in multiple ROIs along the path of large surface veins or branches of the MCA, the relative change in venous or arterial flow downstream of the MCAo could be evaluated. Importantly, a comparison of presham and postsham speckle contrast images showed no significant change in the LSCI-derived map of cortical blood flow (Figure 1D) or the mean speckle values obtained from surface veins (paired t-test, t(4)=0.157, P=0.883) or branches of the MCA (paired t-test, t(4)=0.315, P=0.769). Conversely, LSCI in rats after MCAo identified reduced venous and arterial blood flow and new patterns of collateral blood flow in the sensorimotor cortex. Immediately after vessel occlusion, MCAo animals exhibited reduced venous flow from ischemic territories (Figure 2A, it is noteworthy that veins are lighter gray after MCAo), as indicated by significantly elevated speckle contrast values (1.90-fold increase in speckle contrast, paired t-test, t(7)=2.456, P=0.0437) in large surface veins. Similarly, decreased blood flow in branches of the MCA downstream of the occlusion was confirmed by increased speckle contrast (1.63-fold increase in speckle contrast, paired t-test, t(7)=2.366, P=0.0499) within these vessels. Strikingly, our LSCI data suggested that partial blood flow in branches of the MCA was maintained through anastomatic connections between distal segments of the ACA and MCA (Figure 2A, arrows) that developed immediately after MCAo.

Figure 2
Mapping blood flow after focal stroke using laser speckle contrast imaging (LSCI). (A) LSCI image showing blood flow in the surface veins (V) and branches of the middle cerebral artery (MCA) over the hindlimb and forelimb sensorimotor cortex. It is noteworthy ...

Dynamics of Cerebral Anastomoses After Middle Cerebral Artery Occlusion

Blood flow through anastomatic connections between the ACA and MCA was dynamic and dependent on the reperfusion status of the animal. As MCAo involves injection of a blood clot that can displace or degenerate, spontaneous reperfusion can occur in the thromboembolic model of MCAo. In this study, two MCAo animals exhibited spontaneous reperfusion. Representative data illustrating the pattern of blood flow before and after reperfusion are shown in Figure 2B. Immediately after MCAo onset, blood flow through anastomatic connections between the ACA and MCA is observed (Figure 2B, arrows). However, data acquired 24 hours after ischemic onset showed that spontaneous reperfusion occurred between imaging sessions, closing anastomatic connections (right panel, thin arrows), and increasing blood flow in veins draining the territory of the MCA (asterisks).

In MCAo rats that did not spontaneously reperfuse, collateral circulation through anastomatic connections between the ACA and MCA identified using LSCI persisted over the 24-hour recovery period. Figure 2C shows LSCI blood flow maps from a rat before, after, and at 24 hours after MCAo (left, middle, and right panels, respectively). Large arrows indicate blood flow through anastomatic connections between the ACA and MCA initially identified immediately after MCAo that persisted for 24 hours. A small arrow indicates blood flow through an anastomatic connection identified 24 hours after MCAo that was not apparent immediately after ischemic onset. In MCAo animals imaged at all three time points that did not reperfuse (n=5), blood flow through 35 anastomatic connections between the distal segments of the ACA and MCA was detected immediately after MCAo. Blood flow through 27 (77%) of these anastomatic connections persisted for 24 hours, whereas five anastomoses (in four rats) were observed 24 hours after but not immediately after MCAo.

Discussion

Persistence and Dynamics of Anastomatic Connections Between the Anterior Cerebral Artery and Middle Cerebral Artery

Improving blood flow to the ischemic tissue is one of the primary mechanisms by which the tissue can be saved and permanent disability reduced after focal stroke. It has been suggested that collateral circulation may partially restore blood flow to ischemic territories during focal stroke. Pathophysiological recruitment of anastomatic connections between the ACA and MCA may contribute to collateral flow in human stroke patients (Liebeskind, 2003), and it has been shown in patients that good collateral blood flow is associated with decreased infarct growth and improved tissue survival (Bang et al, 2008).

In this study, we used repeated LSCI before and after thromboembolic MCAo to map the pattern of blood flow through collateral vasculature after stroke. Laser speckle contrast imaging can be used to create high-resolution maps of blood flow on the cortical surface based on the blurring of the laser speckle patterns by the motion of blood cells (Dunn et al, 2001). As such, LSCI allowed us to map the blood flow reaching ischemic territories through collateral circulation. In particular, we showed that the pathophysiological recruitment of anastomatic connections between distal segments of the ACA and MCA provides immediate and persistent collateral blood flow to ischemic territories, although these connections are dynamic and disappear after spontaneous reperfusion. Our data are consistent with those of previous studies in mice that identified anastomatic connections between the ACA and MCA during transient (60 minutes) occlusion of the proximal or distal MCA (Li and Murphy, 2008; Shin et al, 2008). As was the case in our model, anastomoses closed after reperfusion.

Laser Speckle Contrast Imaging and Blood Flow Velocity

In this study, measures of the mean speckle contrast in surface veins and branches of the MCA confirmed reduced blood flow downstream of the MCAo. Although speckle contrast is inversely related to blood flow velocity, the exact quantitative relationship between speckle contrast and blood flow velocity remains undefined (Duncan and Kirkpatrick, 2008), and the absolute values of the speckle contrast are susceptible to experimental artifacts that may vary between animals and imaging sessions (Ayata et al, 2004; Parthasarathy et al, 2008). For this reason, LSCI is best used to describe relative changes in blood flow (Ayata et al, 2004; Parthasarathy et al, 2008). In this study, LSCI permitted high-resolution mapping of MCAo-induced changes in collateral blood flow through anastomatic connections between the ACA and MCA.

Collateral Therapeutics

On the basis of stroke-induced collateralization, it has been suggested that therapies that can enhance collateral blood flow to ischemic territories may benefit stroke patients. Although clinical data and animal studies suggest that collateral therapeutics such as blood pressure augmentation (Shin et al, 2008) and partial occlusion of the descending aorta (Uflacker et al, 2008; Noor et al, 2009) may reduce infarct size and improve recovery after focal stroke, collateral therapeutics remain relatively unexplored (Liebeskind, 2003). It has also been proposed that collateral blood supply could also augment the effectiveness of neuroprotective drug therapies by providing a more effective route of drug delivery to ischemic territories (Liebeskind, 2005). Given the high failure rate of the current neuroprotective therapies, a better understanding of the dynamics and mechanisms of stroke-induced collateralization may facilitate the implementation of collateral therapeutics of acute focal stroke.

Acknowledgments

We thank Tim Murphy and Albrecht Sigler for previous technical assistance with laser speckle contrast imaging.

Notes

The authors declare no conflict of interest.

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