Motor deficit in the mouse ferric chloride-induced distal middle cerebral artery occlusion model of stroke

Abstract

Ferric chloride-induced distal middle cerebral artery occlusion (MCAO) model of stroke was described in mice several years ago, however it lacked in-depth evaluation of the post-stroke functional outcomes in the animals. In this study, we reproduced the recently developed model and expanded its characterization by thorough evaluation of blood supply, cerebral infarction, and motor function in adult male and female mice up to 14 days after stroke. Our observations indicate near complete interruption of blood flow in the distal MCA shortly after application of 20 % ferric chloride over the artery through a cranial window, which remained occluded for at least 4 h. As expected, infarction of the brain tissue, documented by TTC and hematoxylin stains, was restricted to the cerebral cortex. We also systematically evaluated motor impairment of the animals in this model. For this, a series of studies were carried out in male and female mice up to 14 days after stroke, and motor function was assessed in cylinder and grid-walking tests in blinded manner. Contrary to our expectations, the results of both motor tests indicated minor, transient motor deficit in mice after stroke. Based on these observations, we conclude that the mouse ferric chloride-induced distal MCAO model is likely not suitable for proof-of-concept and preclinical studies where motor function is an important outcome measure.

Keywords: Cylinder test; FeCl(3)-induced distal MCAO model of stroke; Grid-walking test; Motor impairment; Mouse stroke model.

Figure 1.. Blood flow monitoring in the distal MCA by laser Doppler flowmetry.

Panel a, representative tracing of the blood flow in a mouse distal MCA during occlusion by ferric chloride. Note, ~5 min of tracing is not presented for the period immediately after removal of the filter paper (due to distortion of the recording during removal of the filter paper and drying of the surface by gentle aspiration). Panel b, average tracing of blood flow in the distal MCA for up to 4 hours after stroke (n = 5).

Figure 2.. Representative TTC-stained coronal brain sections of sham and distal MCAO-operated CD-1 mice on days 1 and 3 after stroke.

Calculated infarct volume is 23.2 ± 1.68 mm³ on day 1 post-stroke (1d PS) and 23.5 ± 5.3 mm³ on day 3 post-stroke (3d PS).

Figure 3.. Hematoxylin-stained coronal brain sections of sham and distal MCAO-operated B6;129 mice.

Top panel, representative brains 4 hours and 1, 3, 7 and 14 days after stroke. Bottom panel, calculated infarct volumes (n = 3 per post-stroke time-point): 3.52 ± 0.24 mm² (4 h), 18.78 ± 2.56 mm² (1 d), 18.93 ± 4.37 mm² (3 d), 16.56 ± 1.59 mm² (7 d), 5.34 ± 0.30 mm² (14 d).

Figure 3.. Hematoxylin-stained coronal brain sections of sham and distal MCAO-operated B6;129 mice.

Top panel, representative brains 4 hours and 1, 3, 7 and 14 days after stroke. Bottom panel, calculated infarct volumes (n = 3 per post-stroke time-point): 3.52 ± 0.24 mm² (4 h), 18.78 ± 2.56 mm² (1 d), 18.93 ± 4.37 mm² (3 d), 16.56 ± 1.59 mm² (7 d), 5.34 ± 0.30 mm² (14 d).

Figure 4.. Grid-walking test.

Following ferric chloride-induced distal MCAO mice exhibited mild to moderate but transient functional deficit in the affected forepaw (i.e., increased number of footfaults; panels a and c) on day 1 after stroke (n =8 for sham and stroke female mice, n = 6 for sham and n = 8 for stroke male mice; *, p < 0.05 in comparison to the stroke group baseline; #, p < 0.05 in comparison to the same day of the sham group). As expected, no functional deficit was observed in the un-affected (i.e., ipsilateral, panels b and d) forepaw of stroke and either forepaw of sham-operated animals.

Figure 5.. Cylinder test.

Mice did not show functional deficit in the affected forelimb (i.e., decreased use of the affected forelimb upon rears) after ferric chloride-induced distal MCAO. As expected, sham-operated animals also lacked functional deficit (n =8 for sham and stroke female mice, n = 6 for sham and n = 8 for stroke male mice; p > 0.05 for all within group and within day comparisons).

Figure 6.. Grid-walking and cylinder tests (14 day-long study; experimental set #3).

Panel a, grid-walking test, mice showed mild, transient functional deficit in the affected forepaw on day 3 after stroke when compared to its baseline performance (p < 0.05). However, no statistically significant difference was observed in performance of the affected forepaw between sham and stroke-operated mice on day 3 after stroke (p > 0.05). Panel b, grid-walking test, no functional deficit was observed in the un-affected forepaw of sham and stroke-operated animals. Panel c, cylinder test, neither sham nor stroke-operated mice showed functional deficit after ferric chloride-induced distal MCAO on any of the evaluation days (n =5 for sham and n = 12 for stroke groups; *, p < 0.05 in comparison to the stroke group baseline).