Characterization of a thromboembolic photochemical model of repeated stroke in mice

Abstract

Many stroke research groups utilize the model of middle cerebral artery occlusion induced by insertion of an intraluminal thread, owing to its pragmatism and reliability of cerebral infarct generation. However, 75% of stroke cases result from a thromboembolic event and 10% from occlusive atherothrombosis in situ. Here, we characterize a mouse model of repeated thromboembolic stroke, which closely mimics the intravascular pathophysiology of arterial thrombus generation from an atherosclerotic plaque, and subsequent release of a thrombus into the cerebral circulation as an embolus. Common carotid artery thrombosis (CCAT) was induced photochemically leading to non-occlusive platelet aggregation in C57/BL6 male mice (n=35), and was followed by mechanical assistance to facilitate release of the thrombus (MRT) and thus promote embolism. Six experimental groups, differing by changes in the surgical protocol, were used for the purpose of determining which such procedure yielded the most reliable and consistent brain infarct volumes with the lowest mortality at 3 days after surgery. The group which best satisfied these conditions was a double insult group which consisted of animals that underwent CCAT for 2 min by means of argon laser irradiation (514.5 nm) at an intensity of ca. 130 W/cm(2), with concomitant injection of erythrosin B (EB) (35 mg/kg infused over those same 2 min), followed by MRT 1 min later; the entire procedure was repeated 24h later. This group showed a percent of brain lesion volume of 15+/-4% (mean+/-S.D.) with no associated 3-day mortality. Compared to a single insult group which sustained a percent brain lesion volume of 7+/-3%, there was a statistically significant (p<0.05) increase in the volume of infarction in the double-insult group.

Figure 1. Doppler blood flow recordings of the common carotid artery (CCA)

*₁ = Pre-irradiation period (laser is off); *₂ = period of irradiation (laser is on with concomitant 2 minute infusion of EB); *₃ = Post-irradiation period (laser is off). (A) Typical blood flow recording of common carotid artery thrombosis (CCAT) until occlusion. Total occlusion of the right CCA of this animal was attained after approximately four minutes and fifteen seconds of irradiation. Note that the laser is shut off once this occlusion is evidenced and that, in this case, there is a brief period of reperfusion before reocclusion. This phenomenon was seen in most of the animals subjected to occlusive CCAT. (B) Typical right CCA blood flow recording of an animal subjected to two minutes of laser irradiation. Changes in blood flow are subtle; there is slight hyperemia during the post-irradiation period.

Figure 2. Hematoxylin-Eosin (H & E)-stained tissue from an injured mouse from group 3

Insets magnify regions of interest. Arrows within insets highlight evidence of infarction and associated tissue breakdown. (A) Bregma level (BL) +1.5. (B) BL +0.14. (C) BL −1.22. (D) BL −2.7. Hippocampal infarcts are seen in (C) and (D).

Figure 3. H & E staining of different areas from an injured brain

(A) Cortical infarct at Bregma Level (BL) +1.42 showing area of non-necrosed tissue but with neuronal dropout (*). (B) Cortical infarct at BL −1.9; the transition from injured (superior aspect of picture) to normal (inferior aspect) tissue is shown. (C) Infarct in CA1, subiculum, and dentate gyrus at BL −2.7. (D) Thalamic infarct in subventricular area at BL −0.94. 3v = Third ventricle.

Figure 4. Vascular consequences of CCAT

H&E staining of brain from a mouse subjected to CCAT. (A) Cortex at Bregma level −2.6; arrow points towards area of focal hemorrhage (dark spots). White streaks in cortex correspond to tissue breakdown. (B) Higher magnification of a cortical infarct. Arrowheads point to endothelial cells which line a thrombosed vessel wall.

Figure 5. Percent brain lesion volume (%BLV), expressed as the percentage of infarcted tissue with respect to total brain volume (±SD)

* = p < 0.05, denotes statistically significant difference in %BLV between single common carotid artery thrombosis followed by mechanical assistance in the release of the thrombus (CCAT+MRT) (group 5) and double CCAT+MRT (group 6) animals. CCAT among these groups of animals was sustained for 2 minutes followed by MRT a minute later (if applied to surgical protocol).