Experimental intracerebral hemorrhage in the mouse: histological, behavioral, and hemodynamic characterization of a double-injection model.

BACKGROUND AND PURPOSE: A major limitation of intracerebral hemorrhage (ICH) research is the lack of reproducible animal models. The present study was conducted to validate in the mouse the double-injection method of ICH initially developed in the rat. We investigated the effect of intrastriatal injection of blood or cerebrospinal fluid (CSF) on cerebral blood flow (CBF), neurological score, hematoma volume, and brain swelling. METHODS: Male C57BL/6 mice were anesthetized with halothane/nitrous oxide delivered by face mask. Rectal and cranial temperatures were regulated at 37 degrees C to 37.5 degrees C. Mice were placed in a stereotactic frame, and a 30-gauge stainless steel cannula was introduced through a burr hole into the left striatum. Each mouse received a 5-microL injection of either whole blood or CSF (over 3 minutes), followed 7 minutes later by 10 microL injected over 5 minutes. The injection cannula was slowly withdrawn 10 minutes after the second injection. Control mice had only cannula insertion. CBF was studied by laser Doppler perfusion imaging. Neurological status was evaluated on days 1 and 2. After 2 days, hematoma volume and brain swelling were calculated. RESULTS: Physiological values were stable. Mice with ICH but not those with CSF or cannula alone had a marked, persistent neurological deficit and a highly reproducible hematoma, whose mean+/-SEM volume was 2.0+/-0.2 mm3 compared with a lesion size of 0.2+/-0.1 mm3 in mice with CSF. Residual swelling of the ipsilateral hemisphere at 48 hours was 5.7% in the hematoma and 1.5% in the CSF groups. Relative CBF in the neocortex ipsilateral to the injection site declined by approximately 45% to 60% during the first 20 minutes after cannula insertion/injection in all groups but began to renormalize at approximately 25 to 30 minutes in the CSF and cannula-only groups; in the hematoma group, cortical hypoperfusion of approximately 35% to 50% persisted during the 90-minute measurement period. CONCLUSIONS: The present ICH model in mice produces a consistent neurological deficit, hypoperfusion, hematoma volume, and brain swelling. This model closely mimics human hypertensive basal ganglionic ICH and should be useful for the evaluation of pharmaceutical therapies. Laser Doppler perfusion imaging is a useful new technique to quantify relative CBF changes and can be used for studies of dynamic changes of CBF in this in vivo model of ICH in mice.

Albumin therapy of transient focal cerebral ischemia: in vivo analysis of dynamic microvascular responses.

BACKGROUND AND PURPOSE: To study whether intravascular or hemodynamic factors contribute to the marked neuroprotective effect of albumin therapy in focal cerebral ischemia, 2 complementary methods were applied: laser-scanning confocal microscopy (LSCM) and laser-Doppler perfusion imaging (LDPI). METHODS: In the LSCM study, Sprague-Dawley rats were anesthetized with halothane/nitrous oxide, and a cranial window was placed over the dorsolateral frontoparietal cortex. Rats received 2-hour middle cerebral artery occlusion (MCAO) by an intraluminal suture and were treated with human albumin (1.25 g/kg; n=4) or saline (n=3) after 30 minutes of recirculation. Video images of cortical vessels were continually acquired and were digitized offline to measure diameters and fluorescent erythrocyte velocities. In the LDPI study, cortical perfusion was measured in anesthetized Sprague-Dawley rats that received 2-hour MCAO and were treated with albumin (2.5 g/kg; n=6) or saline (n=5) at 30 minutes after recirculation. RESULTS: In the LSCM study, MCAO was associated with arteriolar dilation and slowing of capillary and venular erythrocyte perfusion. During the first 15 to 30 minutes of postischemic recirculation, prominent foci of vascular stagnation developed within cortical venules, associated with thrombuslike foci and adherent corpuscular structures consistent in size with neutrophils. Saline administration failed to affect these phenomena, while albumin therapy was followed by significant increases in arteriolar diameter ( approximately 12%; P=0.007) and by a prompt improvement of venular and capillary erythrocyte perfusion and a partial disappearance of adherent thrombotic material. Albumin therapy increased erythrocyte flow velocity in both capillaries (288+/-73% versus 76+/-18% in the saline group; P=0.023) and venules (2.7-fold [P=0.001] versus 1.0-fold in the saline group [P=NS]). In the LDPI study, cortical perfusion declined during MCAO and rose initially with recirculation (to approximately 135% of baseline) in both groups. Mean cortical perfusion improved slightly (approximately 14%; P=NS) in albumin-treated animals. CONCLUSIONS: These results reveal a beneficial effect of albumin therapy in reversing stagnation, thrombosis, and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia and support its utility in the treatment of acute ischemic stroke.

Automated registration of laser Doppler perfusion images by an adaptive correlation approach: application to focal cerebral ischemia in the rat.

Hemodynamic changes are extremely important in analyzing responses from a brain subjected to a stimulus or treatment. The Laser Doppler technique has emerged as an important tool in neuroscience research. This non-invasive method scans a low-power laser beam in a raster pattern over a tissue surface to generate the time course of images in unit of relative flux changes. Laser Doppler imager (LDI) records cerebral perfusion not only in the temporal but also in the spatial domain. The traditional analysis of LD images has been focused on the region-of-interest (ROI) approach, in which the analytical accuracy in an experiment that necessitates a relative repositioning between the LDI and the scanned tissue area will be weakened due to the operator's subjective decision in data collecting. This report describes a robust image registration method designed to obviate this problem, which is based on the adaptive correlation approach. The assumption in mapping corresponding pixels in two images is to correlate the regions in which these pixels are centered. Based on this assumption, correlation coefficients are calculated between two regions by a method in which one region is moved around over the other in all possible combinations. To avoid ambiguity in distinguishing maximum correlation coefficients, an adaptive algorithm is adopted. Correspondences are then used to estimate the transformation by linear regression. We used a pair of phantom LD images to test this algorithm. A reliability test was also performed on each of the 15 sequential LD images derived from an actual experiment by imposing rotation and translation. The result shows that the calculated transformation parameters (rotation: theta =7.7+/-0.5 degrees; translation: Delta x =2.8+/-0.3, Deltay=4.7+/-0.4) are very close to the prior-set parameters (rotation: theta=8 degrees; translation: Delta x=3, Delta y=5). This result indicates that this approach is a valuable adjunct to LD perfusion monitoring. An original sequence of LD images that recorded cerebral perfusion through a cranial window before, during and after middle cerebral artery occlusion (MCAo) is presented, together with the registered image sequence. Cerebral perfusion data acquired in a pixel-based manner from different anatomic locations of the registered LD image sequence are also presented over the whole time-course of the experiment.