Impaired motor unit recovery and maintenance in a knock-in mouse model of ALS-associated Kif5a variant.

Kinesin family member 5A (KIF5A) is an essential, neuron-specific microtubule-associated motor protein responsible for the anterograde axonal transport of various cellular cargos. Loss of function variants in the N-terminal, microtubule-binding domain are associated with hereditary spastic paraplegia and hereditary motor neuropathy. These variants result in a loss of the ability of the mutant protein to process along microtubules. Contrastingly, gain of function splice-site variants in the C-terminal, cargo-binding domain of KIF5A are associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving death of upper and lower motor neurons, ultimately leading to degradation of the motor unit (MU; an alpha motor neuron and all the myofibers it innervates) and death. These ALS-associated variants result in loss of autoinhibition, increased procession of the mutant protein along microtubules, and altered cargo binding. To study the molecular and cellular consequences of ALS-associated variants in vivo, we introduced the murine homolog of an ALS-associated KIF5A variant into C57BL/6 mice using CRISPR-Cas9 gene editing which produced mutant Kif5a mRNA and protein in neuronal tissues of heterozygous (Kif5a+/c.3005+1G>A; HET) and homozygous (Kif5ac.3005+1G>A/c.3005+1G>A; HOM) mice. HET and HOM mice appeared normal in behavioral and electrophysiological (compound muscle action potential [CMAP] and MU number estimation [MUNE]) outcome measures at one year of age. When subjected to sciatic nerve injury, HET and HOM mice have delayed and incomplete recovery of the MUNE compared to wildtype (WT) mice suggesting an impairment in MU repair. Moreover, aged mutant Kif5a mice (aged two years) had reduced MUNE independent of injury, and exacerbation of the delayed and incomplete recovery after injury compared to aged WT mice. These data suggest that ALS-associated variants may result in an impairment of the MU to respond to biological challenges such as injury and aging, leading to a failure of MU repair and maintenance. In this report, we present the behavioral, electrophysiological and pathological characterization of mice harboring an ALS-associated Kif5a variant to understand the functional consequences of KIF5A C-terminal variants in vivo.

Robot-assisted mechanical therapy attenuates stroke-induced limb skeletal muscle injury.

The efficacy and optimization of poststroke physical therapy paradigms is challenged in part by a lack of objective tools available to researchers for systematic preclinical testing. This work represents a maiden effort to develop a robot-assisted mechanical therapy (RAMT) device to objectively address the significance of mechanical physiotherapy on poststroke outcomes. Wistar rats were subjected to right hemisphere middle-cerebral artery occlusion and reperfusion. After 24 h, rats were split into control (RAMT-) or RAMT+ groups (30 min daily RAMT over the stroke-affected gastrocnemius) and were followed up to poststroke d 14. RAMT+ increased perfusion 1.5-fold in stroke-affected gastrocnemius as compared to RAMT- controls. Furthermore, RAMT+ rats demonstrated improved poststroke track width (11% wider), stride length (21% longer), and travel distance (61% greater), as objectively measured using software-automated testing platforms. Stroke injury acutely increased myostatin (3-fold) and lowered brain-derived neurotrophic factor (BDNF) expression (0.6-fold) in the stroke-affected gastrocnemius, as compared to the contralateral one. RAMT attenuated the stroke-induced increase in myostatin and increased BDNF expression in skeletal muscle. Additional RAMT-sensitive myokine targets in skeletal muscle (IL-1ra and IP-10/CXCL10) were identified from a cytokine array. Taken together, outcomes suggest stroke acutely influences signal transduction in hindlimb skeletal muscle. Regimens based on mechanical therapy have the clear potential to protect hindlimb function from such adverse influence.-Sen, C. K., Khanna, S., Harris, H., Stewart, R., Balch, M., Heigel, M., Teplitsky, S., Gnyawali, S., Rink, C. Robot-assisted mechanical therapy attenuates stroke-induced limb skeletal muscle injury.

Phytoestrogen isoflavone intervention to engage the neuroprotective effect of glutamate oxaloacetate transaminase against stroke.

In the pathophysiologic setting of cerebral ischemia, excitotoxic levels of glutamate contribute to neuronal cell death. Our previous work demonstrated the ability of glutamate oxaloacetate transaminase (GOT) to metabolize neurotoxic glutamate in the stroke-affected brain. Here, we seek to identify small-molecule inducers of GOT expression to mitigate ischemic stroke injury. From a panel of phytoestrogen isoflavones, biochanin A (BCA) was identified as the most potent inducer of GOT gene expression in neural cells. BCA significantly increased GOT mRNA and protein expression at 24 h and protected against glutamate-induced cell death. Of note, this protection was lost when GOT was knocked down. To validate outcomes in vivo, C57BL/6 mice were intraperitoneally injected with BCA (5 and 10 mg/kg) for 4 wk and subjected to ischemic stroke. BCA levels were significantly increased in plasma and brain of mice. Immunohistochemistry demonstrated increased GOT protein expression in the brain. BCA attenuated stroke lesion volume as measured by 9.4T MRI and improved sensorimotor function-this protection was lost with GOT knockdown. BCA increased luciferase activity in cells that were transfected with the pERRE3tk-LUC plasmid, which demonstrated transactivation of GOT. This increase was lost when estrogen-related receptor response element sites were mutated. Taken together, BCA represents a natural phytoestrogen that mitigates stroke-induced injury by inducing GOT expression.-Khanna, S., Stewart, R., Gnyawali, S., Harris, H., Balch, M., Spieldenner, J., Sen, C. K., Rink, C. Phytoestrogen isoflavone intervention to engage the neuroprotective effect of glutamate oxaloacetate transaminase against stroke.

Transient Middle Cerebral Artery Occlusion with an Intraluminal Suture Enables Reproducible Induction of Ischemic Stroke in Mice.

Ischemic stroke is a leading cause of mortality and chronic disability worldwide, underscoring the need for reliable and accurate animal models to study this disease's pathology, molecular mechanisms of injury, and treatment approaches. As most clinical strokes occur in regions supplied by the middle cerebral artery (MCA), several experimental models have been developed to simulate an MCA occlusion (MCAO), including transcranial MCAO, micro- or macro-sphere embolism, thromboembolisation, photothrombosis, Endothelin-1 injection, and - the most common method for ischemic stroke induction in murine models - intraluminal MCAO. In the intraluminal MCAO model, the external carotid artery (ECA) is permanently ligated, after which a partially-coated monofilament is inserted and advanced proximally to the common carotid artery (CCA) bifurcation, before being introduced into the internal carotid artery (ICA). The coated tip of the monofilament is then advanced to the origin of the MCA and secured for the duration of occlusion. With respect to other MCAO models, this model offers enhanced reproducibility regarding infarct volume and cognitive/functional deficits, and does not require a craniotomy. Here, we provide a detailed protocol for the surgical induction of unilateral transient ischemic stroke in mice, using the intraluminal MCAO model. Graphic abstract: Overview of the intraluminal monofilament method for transient middle cerebral artery occlusion (MCAO) in mouse.

Ischemic stroke-induced polyaxonal innervation at the neuromuscular junction is attenuated by robot-assisted mechanical therapy.

Ischemic stroke is a leading cause of disability world-wide. Mounting evidence supports neuromuscular pathology following stroke, yet mechanisms of dysfunction and therapeutic action remain undefined. The objectives of our study were to investigate neuromuscular pathophysiology following ischemic stroke and to evaluate the therapeutic effect of Robot-Assisted Mechanical massage Therapy (RAMT) on neuromuscular junction (NMJ) morphology. Using an ischemic stroke model in male rats, we demonstrated longitudinal losses of muscle contractility and electrophysiological estimates of motor unit number in paretic hindlimb muscles within 21 days of stroke. Histological characterization demonstrated striking pre- and postsynaptic alterations at the NMJ. Stroke prompted enlargement of motor axon terminals, acetylcholine receptor (AChR) area, and motor endplate size. Paretic muscle AChRs were also more homogenously distributed across motor endplates, exhibiting fewer clusters and less fragmentation. Most interestingly, NMJs in paretic muscle exhibited increased frequency of polyaxonal innervation. This finding of increased polyaxonal innervation in stroke-affected skeletal muscle suggests that reduction of motor unit number following stroke may be a spurious artifact due to overlapping of motor units rather than losses. Furthermore, we tested the effects of RAMT - which we recently showed to improve motor function and protect against subacute myokine disturbance - and found significant attenuation of stroke-induced NMJ alterations. RAMT not only normalized the post-stroke presentation of polyaxonal innervation but also mitigated postsynaptic expansion. These findings confirm complex neuromuscular pathophysiology after stroke, provide mechanistic direction for ongoing research, and inform development of future therapeutic strategies. SIGNIFICANCE: Ischemic stroke is a leading contributor to chronic disability, and there is growing evidence that neuromuscular pathology may contribute to the impact of stroke on physical function. Following ischemic stroke in a rat model, there are progressive declines of motor unit number estimates and muscle contractility. These changes are paralleled by striking pre- and postsynaptic maladaptive changes at the neuromuscular junction, including polyaxonal innervation. When administered to paretic hindlimb muscle, Robot-Assisted Mechanical massage Therapy - previously shown to improve motor function and protect against subacute myokine disturbance - prevents stroke-induced neuromuscular junction alterations. These novel observations provide insight into the neuromuscular response to cerebral ischemia, identify peripheral mechanisms of functional disability, and present a therapeutic rehabilitation strategy with clinical relevance.

Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke.

Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.

Beyond the Brain: The Systemic Pathophysiological Response to Acute Ischemic Stroke.

Stroke research has traditionally focused on the cerebral processes following ischemic brain injury, where oxygen and glucose deprivation incite prolonged activation of excitatory neurotransmitter receptors, intracellular calcium accumulation, inflammation, reactive oxygen species proliferation, and ultimately neuronal death. A recent growing body of evidence, however, points to far-reaching pathophysiological consequences of acute ischemic stroke. Shortly after stroke onset, peripheral immunodepression in conjunction with hyperstimulation of autonomic and neuroendocrine pathways and motor pathway impairment result in dysfunction of the respiratory, urinary, cardiovascular, gastrointestinal, musculoskeletal, and endocrine systems. These end organ abnormalities play a major role in the morbidity and mortality of acute ischemic stroke. Using a pathophysiology-based approach, this current review discusses the pathophysiological mechanisms following ischemic brain insult that result in end organ dysfunction. By characterizing stroke as a systemic disease, future research must consider bidirectional interactions between the brain and peripheral organs to inform treatment paradigms and develop effective, comprehensive therapeutics for acute ischemic stroke.