Many stroke survivors suffer long‐term functional disabilities with no effective treatment option available. For that reason, we tested the potential effects of enhancing noradrenergic and serotoninergic systems in combination with delayed voluntary running on recovery after stroke in mice. To accomplish this, we used adult male mice (n=12/group) and divided them into sham, stroke, vehicle, or drug‐treated groups with either selective norepinephrine reuptake inhibitor atomoxetine (0.3, 1 mg/kg, once a day i.p.) or selective serotonin reuptake inhibitor fluoxetine (3, 10 mg/kg, once a day i.p.) starting from day 5 after stroke and continued for 12 days. For evaluation of motor function, we used grid walking and cylinder tests 3 days prior and 3, 7, 14, 28, 42 days after using ischemic stroke model (photothrombosis). At 42 days after stroke, mice brains were collected either after fixing the brain through cardiac perfusion (for infarct size measurements and immunohistochemistry experiments after brain sectioning) or as a fresh tissue from the motor cortex area (for immunoblotting experiments).The results of both motor function tests showed that all stroke‐subjected groups had comparable and substantial motor impairment on day 3 after stroke when compared to their baseline and that voluntary running group did not significantly improve throughout the days after stroke. However, administrating either atomoxetine or fluoxetine along with exercise promoted motor recovery at 42 days after stroke dose‐dependently with no significant changes seen in infarct size among groups (0.5 ± 0.1 mm 3 ). Further, immunoblotting experiment showed no significant changes amongst groups in synaptophysin, an integral membrane glycoprotein of neuronal synaptic vesicles, nor with PSD‐95, an excitatory postsynaptic density scaffolding protein, after normalization to beta actin in the ischemic hemisphere. However, the immunohistochemistry data showed that atomoxetine and fluoxetine treatment significantly decreased the expression of parvalbumin (PV), a calcium binding albumin protein associated with depressing synapses, in the boundaries of medial agranular cortex and medial frontal cortex areas of the ischemic hemisphere when compared to their corresponding area in the healthy hemisphere. Whereas no significant relative changes seen with the expression of PV showed between the healthy and ischemic hemispheres of sham, stroke, and vehicle groups. Our data showed that low duration voluntary running does not facilitate motor recovery independently after ischemic stroke in mice. But it promoted recovery of motor function only when combined with atomoxetine or fluoxetine in a dose‐dependent manner. Our data showed a significant decrease of the inhibitory interneurons PV concomitant with the recovery seen with atomoxetine and fluoxetine treatment. Our ongoing experiments include the evaluation of growth associated protein (GAP‐43) and glutamate receptors expression. Support or Funding Information Research in the Karamyan laboratory is supported by R01NS106879 Enhanced motor recovery of mice treated with atomoxetine or fluoxetine after an experimental stroke in the grid walking test (A) and the cylinder test (B). Figure 1 Atomoxetine and fluoxetine treatment significantly decreased parvalbumin expression in the ischemic hemisphere compared to the healthy hemisphere on 42 days after stroke. Figure 2
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).
The aim of this study was to evaluate the potential of pharmacological modulation of adrenergic and serotoninergic systems in post-stroke motor recovery in mice. For this purpose, the effects of selective norepinephrine reuptake inhibitor atomoxetine (1 mg/kg, once a day, i.p.) and selective serotonin reuptake inhibitor fluoxetine (10 mg/kg, once a day, i.p.) were studied on recovery of motor function in adult male mice after photothrombotic stroke. Vehicle or drug treatments were initiated on day 5 after photothrombosis and lasted until post-stroke day 16. In addition to pharmacological therapy, mice were allowed to voluntarily run on a wheel in their home cage starting from day 9 after stroke. Motor function was assessed in grid walking and cylinder tests 3 days before and 3, 7, 14, 28 and 42 days after stroke. On post-stroke day 42, mouse brains were fixed by cardiac perfusion and collected for sectioning and evaluation of infarct volume (cresyl violet staining) and molecular markers (immunofluorescence). The results of both sensorimotor tests indicated that all groups had substantial and comparable motor impairment on post-stroke day 3 (p < 0.01 compared to respective baselines), and that voluntary running started 9 days post-stroke did not significantly facilitate motor recovery after stroke (p > 0.05, vehicle-treated vs. stroke alone). However, treatment of mice with atomoxetine or fluoxetine significantly potentiated motor recovery by day 42 after stroke (p < 0.05 compared to vehicle-treated and stroke alone groups). The average total distance run by mice receiving vehicle or either of the drugs was comparable (1653 ± 110 meters). Infarct volumes measured at day 42 post-stroke were also comparable in experimental groups (1.7 ± 0.2 mm 3 ). Our data indicate that moderate voluntary running starting 9 days after stroke does not facilitate motor recovery after stroke in mice. However, pharmacological modulation of adrenergic or serotoninergic systems, in addition to moderate voluntary running, enhances motor recovery in mice. Our ongoing studies focus on evaluation of the dose-dependence of atomoxetine and fluoxetine effects, and elucidation of molecular signaling pathways responsible for the observed effects of these drugs.
