Delayed atomoxetine or fluoxetine treatment coupled with limited voluntary running promotes motor recovery in mice after ischemic stroke

Abstract

Currently, there is an unmet need for treatments promoting post-stroke functional recovery. The aim of this study was to evaluate and compare the dose-dependent effect of delayed atomoxetine or fluoxetine therapy (starting on post-stroke day 5), coupled with limited physical exercise (2 hours daily voluntary wheel running; post-stroke days 9 to 42), on motor recovery of adult male mice after photothrombotic stroke. These drugs are selective norepinephrine or serotonin reuptake inhibitors indicated for disorders unrelated to stroke. The predetermined primary end-point for this study was motor function measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. Additionally, we quantified the running distance and speed throughout the study, the number of parvalbumin-positive neurons in the medial agranular cortex and infarct volumes. Both sensorimotor tests revealed that neither limited physical exercise nor a drug treatment alone significantly facilitated motor recovery in mice after stroke. However, combination of physical exercise with either of the drugs promoted restoration of motor function by day 42 post-stroke, with atomoxetine being a more potent drug. This was accompanied by a significant decrease in parvalbumin-positive inhibitory interneurons in the ipsilateral medial agranular cortex of mice with recovering motor function, while infarct volumes were comparable among experimental groups. If further validated in larger studies, our observations suggest that add-on atomoxetine or fluoxetine therapy coupled with limited, structured physical rehabilitation could offer therapeutic modality for stroke survivors who have difficulty to engage in early, high-intensity physiotherapy. Furthermore, in light of the recently completed Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) and Efficacy oF Fluoxetine-a randomisEd Controlled Trial in Stroke (EFFECTS) trials, our observations call for newly designed studies where fluoxetine or atomoxetine pharmacotherapy is evaluated in combination with structured physical rehabilitation rather than alone. This study was approved by the Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee (protocol # 16019).

Keywords: FLAME trial; drug repurposing; neural repair; physical exercise; physiotherapy; post-stroke recovery; pre-clinical trial; selective norepinephrine reuptake inhibitor; selective serotonin reuptake inhibitor; stroke pharmacotherapy.

Figure 1

Schematic representation of the study design. Atomo 0.3: 0.3 mg/kg atomoxetine; Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 3.0: 3 mg/kg fluoxetine; Fluox 10.0: 10 mg/kg fluoxetine; R: randomization.

Figure 2

Motor recovery augmented by atomoxetine or fluoxetine is associated with decreased number of inhibitory interneurons. (A) Fluoxetine or atomoxetine-treated mice have statistically significantly decreased number of parvalbumin positive (PV⁺) neurons in ipsilateral (I) medial premotor area (AGm, medial agranular cortex) compared to that of the contralateral (C) hemisphere (n = 6 per group; unpaired t-test, ***P < 0.001). Values are expressed as mean ± standard error. (B) A representative immunofluorescence confocal microscopy image of a mouse brain section on day 42 after stroke. PV signal is depicted in green and cell nuclei (stained with DAPI) in blue. Note, the representative Cresyl violet-stained brain section is shown to illustrate the imaged medial premotor area (red boxes). Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 10.0: 10 mg/kg fluoxetine.

Figure 3

Voluntary running distance and speed. Daily running distance (panel A) and total average running (B) of mice in experimental groups. Mice treated with atomoxetine and fluoxetine covered shorter distance of running compared to vehicle-treated animals (one - (B) or two- (A) way repeated measures ANOVA followed by Dunnett’s multiple comparison, *P < 0.05, ***P < 0.001, vs. vehicle-treated group). (C) Speed of running in experimental groups did not differ significantly during the last three weeks of the study (one-way ANOVA followed by Dunnett’s multiple comparison). (D) Compared to their baseline body weights sham and stroke (^sP < 0.001) and 10 mg/kg fluoxetine-treated animals (^fP = 0.014) gained substantial weight by completion of the study (paired t-test). Values are expressed as mean ± standard error. n = 12/group in all panels. ANOVA: Analysis of variance, Atomo 0.3: 0.3 mg/kg atomoxetine; Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 3.0: 3 mg/kg fluoxetine; Fluox 10.0: 10 mg/kg fluoxetine.

Figure 4

Enhanced motor recovery with atomoxetine or fluoxetine treatment in grid-walking test. (A, B) Following photothrombotic stroke mice treated with atomoxetine or fluoxetine exhibited dose-dependent improvement in affected forelimb motor function (i.e., decreased number of footfaults; n = 12 per group). Compared to the vehicle-treated group, mice treated with 1 mg/kg atomoxetine (^aP = 0.007) or 10 mg/kg fluoxetine (^fP = 0.011) showed statistically significantly improved motor function on day 42 after stroke (two-way repeated measures ANOVA followed by Dunnett’s multiple comparison). (C) No functional deficit was observed in the unaffected (i.e., ipsilateral) forepaw of mice in experimental groups (two-way repeated measures ANOVA followed by Dunnett’s multiple comparison, P > 0.05 for all within group and within day comparisons). Values are expressed as mean ± standard error. ANOVA: Analysis of variance. Atomo 0.3: 0.3 mg/kg atomoxetine; Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 3.0: 3 mg/kg fluoxetine; Fluox 10.0: 10 mg/kg fluoxetine.

Figure 5

Enhanced motor recovery with atomoxetine or fluoxetine treatment in cylinder test. (A, B) Following photothrombotic stroke mice treated with atomoxetine or fluoxetine exhibited dose-dependent improvement in affected forelimb motor function (n = 12 per group). Compared to vehicle-treated group, mice treated with 1 mg/kg atomoxetine (^aP < 0.001) or 10 mg/kg fluoxetine (^fP = 0.04) showed statistically significantly improved motor function on day 42 after stroke (two-way repeated measures analysis of variance followed by Dunnett’s multiple comparison). Values are expressed as mean ± standard error. Atomo 0.3: 0.3 mg/kg atomoxetine; Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 3.0: 3 mg/kg fluoxetine; Fluox 10.0: 10 mg/kg fluoxetine.

Figure 6

Infarct location and volume. (A) Volumetric measurements of brain infarction did not reveal statistically significant differences among experimental groups on day 42 after stroke (n = 6 per group; one-way analysis of variance followed by Dunnett’s multiple comparison, P > 0.05). Values are expressed as mean ± standard error. (B) A representative Cresyl violet-stained mouse brain on day 42 after stroke, indicating location of infarction (outlined in red dotted line) in the primary motor cortex. Atomo 0.3: 0.3 mg/kg atomoxetine; Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 3.0: 3 mg/kg fluoxetine; Fluox 10.0: 10 mg/kg fluoxetine. Similar results were observed in the second cohort of experimental animals which received high dose drug treatments but no physical rehabilitation.

Figure 7

Lack of motor recovery with atomoxetine or fluoxetine in the absence of physical rehabilitation. (A, B) Following photothrombotic stroke mice treated with fluoxetine (10 mg/kg) or atomoxetine (1 mg/kg) did not show improvement in the affected forelimb motor function in comparison to vehicle-treated mice in grid-walking test (n = 7 per group, two-way repeated measures analysis of variance followed by Dunnett’s multiple comparison, P > 0.05). (C) No improvement was observed in the affected forelimb motor function of the same animals in cylinder test (n = 7 per group; two-way repeated measures analysis of variance followed by Dunnett’s multiple comparison, *P < 0.05 for vehicle vs. Atomo 1.0 on post-stroke day 14). Values are expressed as mean ± standard error. Atomo 1.0: 1.0 mg/kg atomoxetine; Fluox 10.0: 10 mg/kg fluoxetine.