Comprehensive Study on the Potential Therapeutic Use of Brevetoxin‐2 to Aid Post‐stroke Recovery

OBJECTIVE Ischemic stroke is a leading cause of long‐term disability that triggers structural and physiological changes in neuronal circuits adjacent to the infarct region. This region, peri‐infarct area, plays a key role in recovery and represents a potential therapeutic target. Brevetoxin‐2 (PbTx‐2) interacts with site 5 of the α‐subunit of the voltage gated sodium channels (VGSC) and augments Na + influx and Ca 2+ dependent downstream signaling pathways. Previous studies in murine neocortical neurons found that PbTx‐2 influences the activation of key signaling proteins that alter physiologic events involved in survival of neurons as well as synaptic plasticity. We therefore assessed the effects of PbTx‐2 on parameters such as motor function, cerebralcortical neuron dendritic arborization and synapse density. HYPOTHESES We hypothesized that PbTx‐2 would aid in functional recovery post‐stroke by increasing dendritic arbor complexity of the layer V pyramidal neurons and excitatory synapse density in the peri‐infarct region METHODS A photothrombotic stroke was produced by administration of an intraperitoneal injection of Rose Bengal solution followed by a 12.5 min light illumination (150 mW) of the motor cortex region resulting in clotting in 2 to 4 months old YFP mice. Epicortical application of hydrogel (controls) or hydrogel + PbTx‐2 was applied over the infarct area on day 5 post‐stroke using a Hamilton syringe. A pasta matrix reach task (PMRT) was used to assess the ability of food restricted mice to grasp pasta pieces so as to determine the number of pieces of pasta retrieved post‐stroke and post‐hydrogel treatment from a 5x5 matrix of pasta. A second motor test, the foot fault test (FFT), was performed where the mouse could freely walk on an elevated wire grid for five minutes, was recorded by video camera, and manually scored for foot faults. Mice were sacrificed by 24 hours on day 6 and the brains were collected to obtain 100 μm sections. These sections were imaged using a Leica confocal microscope which were then analyzed with Imaris software to measure dendritic arbor complexity by Sholl analysis. Immunohistochemistry (IHC) was performed using VGlut1 and PSD95 as synaptic markers to measure excitatory synapse density that was analyzed by Volocity Software. RESULTS The number of pasta pieces retrieved was significantly impaired on day 1 post‐stroke in all animals with confirmed evidence of stroke. There was subsequent improvement of this task following treatment with PbTx‐2 as compared to the controls on day 6. In addition, the PbTx‐2 treated mice had a decrease in the number of foot faults in comparison to the controls on day 6. PbTx‐2 treated layer V pyramidal neurons had a two‐fold increase in dendritic arbor complexity as compared to controls. Preliminary IHC data suggest that the excitatory synapse density in the peri‐infarct region was increased in PbTx‐2 treated mice compared to the control mice. CONCLUSION Preclinical research in a murine stroke model provided behavioral assessments of neural insult‐induced functional deficits and PbTx‐2 induced motor recovery. The PMRT and FFT proved to be sensitive for the study of motor impairment and recovery. Increased dendritic arbor complexity and excitatory synapse density indicates that PbTx‐2 can potentially augment neuroplasticity in the peri‐infarct region. Identifying the appropriate dosage range of PbTx‐2 in future studies will establish the dose‐response relationships for these effects. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .