Mouse Model of Microembolic Stroke and Reperfusion

Adult C57BL/6 mice (WT) and tPA^−/− mice (Jackson Laboratory, Bar Harbor, Maine) weighing 20 to 30 g were used. All experiments were approved by the institutional animal care and use committees at University of Pennsylvania and Massachusetts General Hospital.

Microemboli were prepared by adding thrombin (0.2 U/mL) to a fibrinogen solution (10 mg/mL).^6,7 After overnight maturation at 4°C, clots were dissected and homogenized. Radiolabeled microemboli were prepared by adding 20 μg of ¹²⁵I-labeled human fibrinogen before clot formation. Analysis with the use of a ZM Coulter Counter (Coulter Electronic, Ltd) showed that 1 μL of standard ¹²⁵I-labeled microemboli suspension contained 8100±750 microparticles, with an average diameter of 2.5 to 3 μm and an average total volume of microparticles of 5 to 10 μm³. To determine tissue distribution, a bolus of radiolabeled microemboli was injected into the tail vein or the carotid artery. Animals were killed 10 minutes after injection to measure percent distribution of radioactivity in the blood and organs. In separate experiments, a bolus of radiolabeled microemboli was injected into the carotid artery to determine distribution within the brain. The brain was dissected into regions as shown in Figure 1B: anterior, temporal, cerebellum, and brain stem. Mice were killed at defined time points (10, 60, 180, and 300 minutes) after injection, and data were calculated as residual percentage of radioactivity recovered at the first time point.

Animals were anesthetized with 2% isoflurane and maintained with 1.5% isoflurane in 70% N₂O and 30% O₂ by facemask. Rectal temperature was maintained at 36.5±0.5°C with the use of a feedback-regulated heating system (FHC Inc). The left common carotid artery (CCA), external carotid artery (ECA), and ICA were exposed via a midline incision. A 6-0 silk suture was tied to the distal end of the ECA, and the left CCA, ICA, and pterygopalatine artery (PPA) were temporarily tied. A polyethylene catheter with inner diameter 0.02 mm was attached to a 1-mL syringe, introduced into the ECA, and secured with the 6-0 silk suture. The ligature around the ICA was removed, and the catheter was advanced into the lumen of the ICA. The microemboli solution was injected as a bolus into the ICA. The catheter was then withdrawn, and the ligature was removed from the CCA and PPA. To evaluate the effect of thrombotic burden on perfusion and brain injury, we used 3 doses of microemboli: small (4.0×10⁵ particles), moderate (8.0×10⁵ particles), and large (1.4×10⁶ particles).

Permanent (24 hours) or transient (1 hour of ischemia, 24 hours of reperfusion) middle cerebral artery (MCA) occlusion (MCAO) was caused by inserting an 8-0 nylon filament covered by silicon into the ICA and advancing it to the origin of the MCA.⁸

CBF was determined by laser-Doppler flowmetry (LDF) with a fiberoptic probe (Perimed) attached to the skull 2 mm posterior and 6 to 7 mm lateral to bregma, measured along the surface of the skull (MCA territory). rCBF was measured before ICA ligation (100% baseline) and after injection of microemboli.

For laser speckle imaging (LSI), mice were placed in a stereotaxic frame, paralyzed with pancuronium bromide (0.4 mg/kg IV every 45 minutes), and ventilated (SAR-830 ventilator, Charles Ward Equipment) with monitoring of end-tidal CO₂. After reflection of the scalp, the skull surface was covered with a thin layer of mineral oil and diffusely illuminated with the use of a laser diode at 780 nm. The 5×4-mm imaging field of the charge-coupled device camera (Cohu) was positioned over the left hemisphere. Speckle contrast images were converted to images of correlation time values, which were assumed to be inversely proportional to rCBF.⁶ Relative blood flow images were color coded for percentage of baseline, as described.⁹

Neurological deficit measurements were performed at 24 hours after injection of microemboli, on a 5-point scale¹⁰: normal motor function was scored as 0, flexion of the contralateral torso and forelimb on lifting the animal by the tail as 1, circling to the contralateral side but normal posture at rest as 2, leaning to the contralateral side at rest as 3, and no spontaneous motor activity as 4.

Animals were euthanized for infarct volume measurements 24 hours after microemboli injection or MCAO, immediately after scoring for neurological deficit. The volume of cerebral infarcts was measured with the use of Image analysis software (M4). With the use of an RMB-200C mouse brain matrix (Activation Systems), each brain was cut into 2-mm-thick coronal blocks, for a total of 6 blocks per animal. The tissue was immersed in 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) for 12 hours, which provides better contrast between normal and infarct zones than staining for 1 hour. The areas of infarction and each hemisphere were traced, and volumes were determined by integrating the appropriate area with the section thickness. The area of infarction in each section was expressed as a fraction of the nonischemic part of ipsilateral hemisphere (indirect volume of infarct). Measurements of infarct volumes and neurological deficits were performed in randomized-blind fashion.

All data, except for neurological deficit measurements, are expressed as mean±SEM, and statistical analysis was performed with the use of t test comparisons. Differences of P<0.05 were considered significant. Neurological deficit data as seen in Figure 3F were analyzed with the Mann-Whitney test.

We compared the deposition of ¹²⁵I-labeled microemboli injected via the tail vein and the carotid artery. As seen in Figure 1A, intravenous injection deposited microemboli primarily in the lung, in agreement with previous data.⁷ In contrast, intracarotid injection resulted in the major fraction of ¹²⁵I-labeled microemboli being lodged in the brain. After carotid injection, the major fraction of ¹²⁵I-labeled microemboli was localized in the temporal part in the ipsilateral hemisphere, although detectable amounts were found in the contralateral temporal hemisphere and other regions bilaterally (Figure 1B).

The extent and duration of thrombotic occlusion and pathological consequences of microemboli depended on the dose of microemboli. Moderate and large doses of microemboli caused marked (≈80%) reductions of rCBF measured by LDF in the ipsilateral hemisphere by 5 minutes after injection, while small doses had little effect (Figure 2 A, 2B, 2C). After a moderate dose, rCBF recovered to >60% by 1 hour, while it remained <20% with a large dose of microemboli.

We used LSI⁹ to study the spatial distribution of ischemia induced by injection of microemboli. Small doses of microemboli caused a transient and variable rCBF reduction within the ipsilateral MCA territory (data not shown). Moderate doses of microemboli caused a more reproducible and severe reduction in rCBF in the ipsilateral MCA and, to a lesser extent, the PCA and ACA territories (Figure 2D).

We compared the effects of intracarotid microemboli injection and MCAO by filament, a commonly used mouse model of stroke. A small dose of microemboli caused no significant injury. A moderate microemboli dose provided an intermediate extent of injury and caused a clearly detectable neurological deficit manifested by circling and depression of spontaneous activity. A large dose of microemboli led to a severe neurological deficit manifested by side positioning. Figure 3 shows the effects of microemboli dose on infarct size measured 24 hours later, images of representative brain sections, and neurological deficit scoring. Generally, the microembolic model of cerebral ischemia caused damage in the zone supplied by the MCA, but the affected areas also included other territories. The effects of the large dose of microemboli were similar to those of permanent MCAO by a filament.

The rate of postsurgical stroke-related death was paralleled by the size of infarcts. No animals died after injection of a small dose of microemboli, 10% died after a moderate dose, and 70% died after a large dose. In comparison, 13% died after transient filament MCAO, and 37% died after permanent MCAO by filament.

Previous studies showed that microemboli lodged in the pulmonary vasculature spontaneously dissolve within a few hours.^6,7 In contrast, radiolabeled tracing revealed that after initial 40% to 50% dissolution within the first hour, a large dose of ¹²⁵I-labeled microemboli lodged in the cerebral vasculature failed to resolve completely within 3 hours (Figure 4). These data agree with measurements of blood flow: rCBF remained markedly reduced even several hours after injection of a large dose of microemboli (Figure 2A). This dose caused permanent reduction of ipsilateral rCBF and later reduction of contralateral rCBF (Figure 2A).

Five minutes after a moderate dose of microemboli, rCBF was reduced to similar levels in WT and tPA^−/− mice (Figure 5A). However, tPA^−/− mice demonstrated a delay of reperfusion rate measured by LDF within the first 30 minutes compared with WT mice (27.4±5.6 in tPA^−/− mice, 56.9±9.6 in WT mice [% baseline]; n=7 each group). By 60 minutes after injection, rCBF returned to normal in WT mice, while it remained significantly depressed in tPA^−/− mice. ¹²⁵I-labeled microemboli tracing revealed that genetic ablation of tPA markedly retarded the rate of thrombi dissolution in the brain (Figure 5B). Infarct volume was significantly greater in tPA^−/− mice than in WT mice (Figure 5C), and neurological score revealed a trend to more severe injury (2.0±0.6 in tPA^−/− mice [n=4]; 0.97±0.15 in WT mice [n=16]). Mean arterial blood pressure and temperature did not differ between WT and tPA^−/− mice.