Characterizing photothrombotic distal middle cerebral artery occlusion and YAG laser-induced reperfusion model in the Izumo strain of spontaneously hypertensive rats

Abstract

No study has systematically studied the relevance of original Izumo strain of spontaneously hypertensive rats (SHR/Izm) as a stroke model. Furthermore, both SHR/Izm and stroke-prone SHR/Izm (SHRSP/Izm) are commercially available, and recent progress in genetic studies allowed us to use several congenic strains of rats constructed with SHR/Izm and SHRSP/Izm as the genetic background strains. A total of 166 male SHR/Izm and 17 male SHRSP/Izm were subjected to photothrombotic middle cerebral artery (MCA) occlusion with or without YAG laser-induced reperfusion. The pattern of distal MCA was recorded. Infarct volumes were determined with 2,3,5-triphenyltetrazolium chloride. At 24 or 48 h after MCA occlusion, infarct volumes in the permanent occlusion and 2-h occlusion groups (88 ± 22 [SD] and 87 ± 25 mm³, respectively) were significantly larger than that in the 1-h occlusion group (45 ± 14 mm³), indicating the presence of sizeable zone of penumbra. Infarct size in SHRSP/Izm determined at 24 h after MCA occlusion was fairly large (124.0 ± 34.8 mm³, n = 10). Infarct volume in SHR/Izm with simple distal MCA was 76 ± 19 mm³, which was significantly smaller than 95 ± 22 mm³ in the other SHR/Izm with more branching MCA. These data suggest that this stroke model in SHR/Izm is useful in the preclinical testing of stroke therapies and elucidating the pathophysiology of cerebral ischemia/reperfusion.

Fig. 1

a Mean arterial blood pressure (MABP) was increased after the onset of maintenance concentration of halothane anesthesia, which reached a plateau at 30–40 min. The mean value of MABP in SHRSP/Izm was significantly increased from 175 mmHg at 0 min to 203 mmHg at 40 min (paired t test, P = 0.005). In SHR/Izm, MABP increased from 146 mmHg at 0 min to 167 mmHg at 40 min, but the difference was not significant. Bars indicate SD (n = 5 per group). b Regional cerebral blood flow (CBF) was measured at 1 mm posterior and 4 mm lateral to the bregma with laser-Doppler flowmetry. In case of tissue sampling, the brain was cut into a 2-mm-thick coronal block, and four samples (a, b, c, and d) were dissected from the block at −20°C on a cooling plate under a stereomicroscope. R, right. c Changes in CBF after middle cerebral artery (MCA) occlusion and reperfusion (n = 6). Changes in CBF were expressed as a percentage of the average of two or three baseline values. Bars indicate SD. d 2,3,5-Triphenyltetrazolium chloride (TTC) stained sections just anterior and posterior to the 2-mm-thick coronal block shown in b. pMCAO permanent MCA occlusion

Fig. 2

a The experimental set-up of the photothrombotic MCA occlusion and YAG laser-induced reperfusion laser system. b The view of white square on A from the opposite side

Fig. 3

Evolving ischemic injury among the groups of permanent occlusion (PO), reperfusion at 1 h (1hO) or 2 h (2hO) of MCA occlusion. a Infarct volume at 24 or 48 h after MCA occlusion with or without reperfusion. * TTC-defined lesions at 6 h are considered to be ischemic injury not infarction. Open and shaded symbols indicate infarct volume at 24 and 48 h, respectively. ** P < 0.001 vs. 1hO at 24/48 h, and PO at 6 h. ^# P = 0.002 vs. 1hO at 24/48 h, and 2hO at 6 h. b Schematic representation of progression of ischemic injury in the PO and 2hO groups (dark gray area) compared with the 1hO group or lesion sizes at 6 h (outer area). Circles indicate the sites of krypton laser irradiation for MCA occlusion (two-point hit)

Fig. 4

a Infarct volume after permanent distal MCA (dMCA) occlusion between the groups with simple or regular dMCA. ^# P = 0.048 vs. regular dMCA. b Prevalence of simple, regular, and complicated distal MCA in SHR/Izm and SHR/Kyushu. * Historical data from our previous report (Cai et al., Stroke 1988;29:1982–1987)