Hi1a Improves Sensorimotor Deficit following Endothelin-1-Induced Stroke in Rats but Does Not Improve Functional Outcomes following Filament-Induced Stroke in Mice

Abstract

Activation of acid-sensing ion channel 1a (ASIC1a) plays a major role in mediating acidosis-induced neuronal injury following a stroke. Therefore, the inhibition of ASIC1a is a potential therapeutic avenue for the treatment of stroke. Venom-peptide Hi1a, a selective and highly potent ASIC1a inhibitor, reduces the infarct size and functional deficits when injected into the brain after stroke in rodents. However, its efficacy when administered using a clinically relevant route of administration remains to be established. Therefore, the current investigation aims to examine the efficacy of systemically administered Hi1a, using two different models of stroke in different species. Mice were subjected to the filament model of middle cerebral artery occlusion (MCAO) and treated with Hi1a systemically using either a single- or multiple-dosing regimen. 24 h poststroke, mice underwent functional testing, and the brain infarct size was assessed. Rats were subjected to endothelin-1 (ET-1)-induced MCAO and treated with Hi1a intravenously 2 h poststroke. Rats underwent functional tests prior to and for 3 days poststroke, when infarct volume was assessed. Mice receiving Hi1a did not show any improvements in functional outcomes, despite a trend toward reduced infarct size. This trend for reduced infarct size in mice was consistent regardless of the dosing regimen. There was also a trend toward lower infarct size in rats treated with Hi1a. More specifically, Hi1a reduced the amount of damage occurring within the somatosensory cortex, which was associated with an improved sensorimotor function in Hi1a-treated rats. Thus, this study suggests that Hi1a or more brain-permeable ASIC1a inhibitors are a potential stroke treatment.

Figure 1

Functional outcomes following ET-1-induced MCAO. Rats were subjected to either sham surgery (n = 6) or ET-1 MCAO. Animals that underwent MCAO received either vehicle (n = 13) or Hi1a 1 mg/kg (n = 12) delivered i.v. at 2 h poststroke. Rats were tested prior to cannula implantation surgery and again prior to stroke. Following stroke, rats were tested on days 1 and 3 poststroke. (A) Time to remove adhesives (s). (B) Percentage error in the ledged beam test expressed as (number of errors/total number of steps) × 100. (C) Neurological score expressed in arbitrary units. (D) Weight change expressed as the percentage change in weight from presurgery baseline. Statistical significance is color coded according to the respective group. Data are presented as mean ± SEM, with ^$P < 0.05 vs vehicle treatment effect, two-way RM ANOVA, *P < 0.05, **P < 0.01 vs prestroke, ^‡P0.05, ^‡‡‡P < 0.001 vs Day 1, ^#P < 0.05 vs sham two-way RM ANOVA followed by Tukey’s multiple comparisons test.

Figure 2

(A) Representative coronal brain sections imaged using the ballistic light method. (B) Infarct volumes (mm³) following ET-1-induced MCAO, for sham-operated nonstroke animals (n = 6), or stroked rats treated with vehicle (n = 13) or Hi1a 1 mg/kg (n = 12) delivered i.v. at 2 h poststroke showing the total infarct volume. (C–E) Infarct volume within the striatum (C), cortex (D), and somatosensory cortex (E). Data are mean ± SEM. Statistical significance measured by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 vs sham. The scale bar in panel (A) represents 2 mm.

Figure 3

Effects of a 30 min occlusion followed by 24 h reperfusion in the filament model of stroke, for sham-operated nonstroke animals (n = 10), or stroked mice treated with vehicle (n = 21) or Hi1a 1 mg/kg (n = 19) delivered i.v. 1 h poststroke (ps), or multiple doses of vehicle (n = 6) or Hi1a 300 μg/kg i.v. 1 h ps +100 μg/kg i.p. 3 and 5 h ps (n = 6). (A) Mean Bederson score expressed in arbitrary units. (B) Latency to fall in hanging wire test. (C) Percentage weight loss compared to prestroke baseline. Data are mean ± SEM. Statistical significance assessed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test for Bederson score, and a one-way ANOVA followed by Tukey’s multiple comparisons test for hanging wire and weight loss. *P < 0.05, **P < 0.01, ***P < 0.001 vs sham.

Figure 4

(A) Representative thionin-stained coronal brain sections. (B) Total infarct volumes (mm³) and (C) infarct volume within the somatosensory cortex after a 30 min occlusion followed by 24 h reperfusion in the filament model of stroke, for sham-operated nonstroke animals (n = 10), or stroked mice treated with vehicle (n = 21) or Hi1a 1 mg/kg (n = 19) delivered i.v. 1 h poststroke (ps), or multiple doses of vehicle (n = 6) or Hi1a 300 μg/kg i.v. 1 h ps +100 μg/kg i.p. 3 and 5 h ps (n = 6). Data are mean ± SEM. Statistical significance assessed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05 vs sham. The scale bar in panel (A) represents 1 mm.