Introduction: Spontaneously hypertensive stroke-prone rats (SHRSP) are used to model clinically relevant aspects of human cerebral small vessel disease (CSVD). To decipher and understand the underlying disease dynamics, assessment of the temporal progression of CSVD histopathological and neuroimaging correlates is essential. Materials and Methods: Eighty age-matched male SHRSP and control Wistar Kyoto rats (WKY) were randomly divided into four groups that were aged until 7, 16, 24 and 32 weeks. Sensorimotor testing was performed weekly. Brain MRI was acquired at each study time point followed by histological analyses of the brain. Results: Compared to WKY controls, the SHRSP showed significantly higher prevalence of small subcortical hyperintensities on T2w imaging that progressed in size and frequency with aging. Volumetric analysis revealed smaller intracranial and white matter volumes on brain MRI in SHRSP compared to age-matched WKY. Diffusion tensor imaging (DTI) showed significantly higher mean diffusivity in the corpus callosum and external capsule in WKY compared to SHRSP. The SHRSP displayed signs of motor restlessness compared to WKY represented by hyperactivity in sensorimotor testing at the beginning of the experiment which decreased with age. Distinct pathological hallmarks of CSVD, such as enlarged perivascular spaces, microbleeds/red blood cell extravasation, hemosiderin deposits, and lipohyalinosis/vascular wall thickening progressively accumulated with age in SHRSP. Conclusions: Four stages of CSVD severity in SHRSP are described at the study time points. In addition, we find that quantitative analyses of brain MRI enable identification of in vivo markers of CSVD that can serve as endpoints for interventional testing in therapeutic studies.

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.

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.

The purpose of this study was to characterize the effects of tocotrienol form of vitamin E (TCT) on platelet function in patients with stroke or transient ischemic attack (TIA). A double blind, randomized, single center phase II clinical trial was conducted comparing placebo (PBO) and 400 and 800 mg TCT daily for a year in 150 patients with a sentinel ischemic stroke or TIA event in the prior 6 months. Platelet function was measured at baseline and then, at 3 month intervals for a year, using light transmission aggregometry. The incidence of aspirin resistance in aspirin-treated patients or platelet inhibition in patients on clopidogrel alone was compared between the three treatment groups. Results showed that in patients taking aspirin and clopidogrel, the incidence of aspirin resistance was significantly decreased from 40% in PBO-treated patients to 9% in the 400 mg TCT group and 25% in the TCT 800 mg group (P = .03). In conclusion, patients on aspirin and clopidogrel had a higher incidence of aspirin resistance than all patients treated with aspirin alone and TCT decreased the frequency of aspirin resistance in this group.

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.

Introduction: Endothelial dysfunction is an important mediator of post ischemic injury of the heart and brain following ischemia/reperfusion (I/R). We have reported that CD38 activation in heart I/R models leads to NADPH depletion with endothelial nitric oxide synthase (eNOS) impairment, loss of endothelial-mediated coronary dilatation and increased myocardial infarction. While CD38 knockout or inhibition prevents this dysfunction and decreases myocardial infarction, the role of CD38 in ischemic stroke remains uncertain. Hypothesis: We hypothesize that loss of CD38 expression and activity through gene knockout is protective with smaller infarct volume. Methods: 15 week-old male CD38 knock out (KO) n=12 and wild type (WT) mice n=12 underwent middle cerebral artery occlusion (MCAO) for 60 minutes. Stroke volume was calculated using T2 MRI sequences on a 9.4 T MRI system acquired 48 hours post stroke with images analyzed using Osirix software. The ratios of the stroke volume to the affected hemisphere volume and the compensated swelling infarction volume percentage of normal hemisphere were calculated using established methods. Open field test to measure motor impairment was performed at baseline and 48 hours post stroke (KO, n=12, WT n=6). Statistical analysis was completed in STATA using Man-Whitney U test and T-test to compare infarct volumes and cognitive parameters. Values are shown as mean ± SD. P value < 0.05 was considered significant. Results: At 48 hours, brain MRI showed a smaller percentage of cerebral hemisphere affected by stroke in CD38 KO compared to WT (25.9±3.7 vs 41.1±9.4, respectively P=0.0001) and a smaller percentage of compensated swelling infarction volume of normal hemisphere in KO mice compared to WT (19.6±3 vs 33.5±9, respectively P=0.0001). Open field test showed significant post stroke motor impairment in WT compared to CD38KO mice (distance travelled 1.8±1 m vs 4.7±3 m, P=0.04, respectively and average speed 0.006±0.003 m/s vs 0.016±0.01m/s, P=0.04, respectively). Conclusions: Infarct volumes are smaller and motor impairment is decreased in CD38 KO mice compared to WT demonstrating that gene knockout of CD38 confers neuroprotection against acute ischemic brain injury.

Introduction: Animal models of human cerebral small vessel disease (CSVD) are important for the study of the disease underlying mechanisms and testing therapeutic interventions. Spontaneusly Hypertensive Rats - Stroke Prone (SHRSP) are used as an animal model of human CSVD. However, there is a lack of data regarding the time course of cognitive and motor impairment. Methods: Male age-matched SHRSP and Wistar Kyoto rats (WKY) were studied. Sensorimotor testing (open field test) and tail-cuff systolic blood pressure (SBP) measurements (Visitech Systems, Inc.) were performed weekly starting from 6 until 24 weeks of age. Brain MRI at 7 and 24 weeks was acquired using a 9.4T MRI system. Brain histology was completed at the same time points. Statistical analysis was performed using a linear mixed model with repeated measurements. P< 0.05 was considered significant. Results: 20 SHRSP and 20 WKY male rats were studied. 10 per group were euthanized following brain MRI at 7 weeks and the rest were followed until 24 weeks. SHRSP weighed on average 30 grams less than WKY throughout the study (P=0.0003). At 7 weeks SBP was not different (WKY 106.6±5.4 vs SHRSP 120.8±5.4, P=0.06). SHRSP started to develop hypertension at 9-12 weeks and maintained hypertension until 24 weeks (average group difference across time P<0.0001; SBP at 21 weeks WKY 134.8±5.4 vs SHRSP 168.9±5.4, P<0.0001). Sensorimotor testing showed higher total distance travelled (TDT) at 7 weeks in SHRSP that trended down with ageing. Both groups became similar at 21 weeks (TDT: at 7 weeks WKY 3.78±1.3 vs SHRSP 7.8±1.3, P =0.037; at 21 weeks: WKY 4±1.3 vs SHRSP 4.4±1.3, P=0.83; average group difference across time P=0.014). Brain MRI was normal at 7 weeks, but small white matter hyperintensities were seen at 24 weeks. Brain Histology showed normal histology on hematoxylin & eosin staining at 7 weeks in both groups, while at 24 weeks SHRSP showed CSVD histopathological changes including microbleed formation, homeostasis and vascular hyalinosis. Conclusions: SHRSP develops hypertension, sensorimotor deficits and CSVD pathology as they age suggesting their utility as human CSVD model. Intervention time points should be selected carefully in future therapeutic drug interventions.

Introduction: While recombinant tissue plasminogen activator (rTPA) is the mainstay of ischemic stroke treatment, recanalization is only achieved in 25-50% of patients. With a significant risk of intracranial hemorrhage, its use has been limited to within 4.5 hours of symptom onset. Previous work has demonstrated that aptamer inhibition of Von Willebrand Factor (VWF) effectively restores reperfusion following murine carotid artery occlusion. Hypothesis: We tested the hypothesis that VWF aptamer would promote recanalization following thrombotic middle cerebral artery (MCA) occlusion, ameliorating stroke burden with greater efficacy than rTPA. Methods: Adult wild-type (C57BL/6J) mice were anesthetized, and the right carotid artery was exposed. A 32-gauge intracranial catheter was advanced within the carotid artery. Murine autologous blood was then mixed with 10 μL 0.9% normal saline and 1 μL murine thrombin and was allowed to stabilize at 37 °C for 15 minutes, after which it was injected through the catheter into the MCA. Laser-doppler flowmetry monitoring measured decreased flow following injection of the embolus. Treatment (vehicle, platelet binding buffer, n=5; VWF aptamer, n=6; rTPA, n=7) was initiated 20 minutes after thrombus injection. An MRI was obtained at 24 hours to assess ischemic stroke volumes. Results: None of the mice receiving rTPA survived to 24 hours, while all mice treated with VWF aptamer and vehicle survived to 24 hours and received an MRI. Ischemic stroke volume was significantly decreased in mice treated with VWF aptamer (5.49 ± 5.01 mm 3 ) compared to vehicle (35.34 ± 9.57 mm 3 , p<0.05)(Figure 1). No evidence of intracranial hemorrhage was identified in either cohort. Conclusions: Treatment with VWF aptamer decreases stroke volume on MRI in a murine model of embolic stroke without the risk of hemorrhagic conversion seen in patients treated rTPA. VWF inhibition represents a promising therapy in stroke treatment.

Objective: To compare the functional effects of von Willebrand Factor (VWF) inhibition by RNA aptamer (T79) vs rTPA utilizing in vivo arterial thrombosis in mice. Approach and Results: We previously demonstrated that inhibition of VWF by a targeted RNA aptamer (T79) both prevents thrombosis and thrombolyses stabilized clots in a murine model of ferric chloride (FeCl 3 )-induced vascular injury suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesized that T79 treatment, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA, would result in improved behavioral outcome with 7 day survival after vascular injury. Baseline locomotor testing in an open field was performed on both male and female, 8-16 week old, wild-type C57BL/6J mice. Occlusive arterial thrombus formation was induced by a 3 minute exposure to 10% FeCl 3 on the right common carotid monitored by Doppler flow and time to occlusion (blood flow of 0 ml/min) was measured. Twenty minutes after thrombus stabilization either T79 (0.1 mg/kg or 0.5 mg/kg bolus), rTPA (10 mg/kg 45 min infusion) or saline vehicle (45 min infusion) was introduced via a saphenous catheter. Animals were recovered and locomotor testing was repeated 48 hours and 7 days after injury with baseline set as 100%. All groups including vehicle control resulted in a statistically significant decrease in distance traveled (meters) and speed (meters/sec) at 48 hours after injury. At 7 days, 0.1 mg/kg T79 increased 29.89% from 48 hours, 0.5 mg/kg T79 increased 22.50% from 48 hours but rTPA treatment showed no significant improvement (2.79% increase) (n=4, p<0.001). In addition, 0.1 mg/kg and 0.5 mg/kg T79 resulted in a 28.46% and 22.44% increase in speed, respectively, at 7 days compared to 48 hours whereas rTPA did not (2.94% increase) (n=4, p<0.001). Interestingly, males scored lower on most behavioral parameters at 48 hours, but recovered to the same level as females by 7 days. No significant differences in time to occlusion or baseline locomotor testing were observed in any group. Conclusion: Inhibition of VWF by T79 aptamer markedly improves behavioral outcomes compared to rTPA after survival carotid artery occlusion following ferric chloride-induced injury in mice.

Ischemic stroke results in excessive release of glutamate, which contributes to neuronal cell death. Here, we test the hypothesis that otherwise neurotoxic glutamate can be productively metabolized by glutamate oxaloacetate transaminase (GOT) to maintain cellular energetics and protect the brain from ischemic stroke injury. The GOT-dependent metabolism of glutamate was studied in primary neural cells and in stroke-affected C57-BL6 mice using magnetic resonance spectroscopy and GC-MS. Extracellular Glu sustained cell viability under hypoglycemic conditions and increased GOT-mediated metabolism in vitro Correction of stroke-induced hypoxia using supplemental oxygen in vivo lowered Glu levels as measured by 1H magnetic resonance spectroscopy. GOT knockdown abrogated this effect and caused ATP loss in the stroke-affected brain. GOT overexpression increased anaplerotic refilling of tricarboxylic acid cycle intermediates in mouse brain during ischemic stroke. Furthermore, GOT overexpression not only reduced ischemic stroke lesion volume but also attenuated neurodegeneration and improved poststroke sensorimotor function. Taken together, our results support a new paradigm that GOT enables metabolism of otherwise neurotoxic extracellular Glu through a truncated tricarboxylic acid cycle under hypoglycemic conditions.-Rink, C., Gnyawali, S., Stewart, R., Teplitsky, S., Harris, H., Roy, S., Sen, C. K., Khanna, S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain.

Introduction: While recombinant tissue plasminogen activator (rTPA) is the mainstay of ischemic stroke treatment, few patients are eligible for treatment, and recanalization is only seen in 25-50%. Von Willebrand Factor (VWF) inhibition may play a role in thrombolysis. Hypothesis: VWF inhibition with an RNA aptamer lyses arterial thrombus and decreases ischemic injury. Furthermore, aptamer reversal with an antidote oligonucleotide ameliorates intracranial hemorrhage (ICH). Methods: Adult wild-type (C57BL/6J) mice were anesthetized, and the right carotid artery was exposed. Baseline carotid flow was obtained using a Doppler flow probe, and thrombotic occlusion was induced with a ferric chloride patch. After clot stabilization, mice were administered vehicle (platelet binding buffer, n=11), no infusion (n=8), rTPA (n=5) or VWF aptamer (n=5). Carotid flow was monitored for an additional 100 minutes. In a second cohort of mice, a 6-0 nylon suture was advanced within the carotid artery to generate vascular injury and ICH. Mice were given vehicle (n=16), rTPA (n=11), VWF aptamer (n=9) or aptamer/antidote (n=8). An MRI was obtained after 90 minutes to assess stroke and ICH volumes. Results: VWF aptamer successfully restored carotid blood flow 45 minutes following carotid occlusion (Figure 1) compared to controls (p<0.01*) and rTPA (p<0.05 + ). Stroke volume was significantly decreased in mice treated with VWF aptamer (23.03 ± 6.81 mm 3 ) and aptamer/antidote (12.48 ± 5.68 mm 3 ) compared to vehicle (45.25 ± 4.14 mm 3 , p<0.01). ICH volumes in mice treated with rTPA (2.64 ± 0.84 mm 3 ) were trending higher than vehicle (1.51 ± 0.17 mm 3 ), VWF aptamer (1.92 ± 0.22 mm 3 ) or aptamer/antidote (1.31 ± 0.35 mm 3 ). Conclusions: Aptamer inhibition of VWF is a potent thrombolytic agent with greater efficacy compared to rTPA. VWF inhibition appears safe with a trend toward lower ICH volumes in animals treated with aptamer and aptamer/antidote compared to rTPA.

Objective: (1) Develop a standardized approach to quantitatively measure residual limb skin health. (2) Report reference residual limb skin health values in people with transtibial and transfemoral amputation. Approach: Residual limb health outcomes in individuals with transtibial (n=5) and transfemoral (n=5) amputation were compared to able-limb controls (n=4) using noninvasive imaging (hyperspectral imaging and laser speckle flowmetry) and probe-based approaches (laser doppler flowmetry, transcutaneous oxygen, transepidermal water loss, surface electrical capacitance). Results: A standardized methodology that employs noninvasive imaging and probe-based approaches to measure residual limb skin health are described. Compared to able-limb controls, individuals with transtibial and transfemoral amputation have significantly lower transcutaneous oxygen tension, higher transepidermal water loss, and higher surface electrical capacitance in the residual limb. Innovation: Residual limb health as a critical component of prosthesis rehabilitation for individuals with lower limb amputation is understudied in part due to a lack of clinical measures. Here, we present a standardized approach to measure residual limb health in people with transtibial and transfemoral amputation. Conclusion: Technology advances in noninvasive imaging and probe-based measures are leveraged to develop a standardized approach to quantitatively measure residual limb health in individuals with lower limb loss. Compared to able-limb controls, resting residual limb physiology in people that have had transfemoral or transtibial amputation is characterized by lower transcutaneous oxygen tension and poorer skin barrier function.

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 pERRE(3)tk-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.

Arrayed nanochannels can be used to controllably transfect and reprogram tissues in vivo for applications in regenerative medicine and cell-based therapies. Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency)1,2,3,4,5,6. In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods11,12. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Introduction: A growing body of literature supports acute changes to skeletal muscle physiology in response to stroke-induced central nervous system injury. While stroke survivors depend on rehabilitation to facilitate functional recovery, study of post-stroke rehabilitation and mechanisms of recovery remains limited. The current work addresses development of a Robot-Assisted Mechanical Therapy (RAMT) device and facilitation of reproducible, objective analysis of post-stroke rehabilitation. We hypothesize that RAMT permits systematic study of post-stroke mechano-physiotherapy and restores hindlimb function after stroke by protecting against skeletal muscle injury. Methods: Wistar rats (male, N=26) were subjected to middle cerebral artery occlusion (MCAO), after which they received daily RAMT (RAMT+; 0.5N force, 1Hz frequency, 10mm linear motion over medial stroke-affected gastrocnemius) or none (RAMT-; anesthesia only) for 14 days. Assessment of gait, sensorimotor behavior, and muscle perfusion quantified effects of RAMT on post-stroke function, while skeletal muscle analysis (RT-PCR, immunohistochemistry) evaluated expression of myostatin, a molecular target of stroke. Results: Compared to RAMT- controls, RAMT+ rats benefited from higher perfusion in stroke-affected gastrocnemius (47.7%, p<0.05). RAMT+ improved post-stroke gait and sensorimotor behavior, evidenced by better track width (11.9%, p<0.05), less time in quad support (54.4%, p<0.05), greater travel distance (60.5%, p<0.05), and more time mobile (41.7%, p<0.05). Additionally, RAMT+ protected from post-stroke induction of myostatin, decreasing mRNA and protein expression (39.1% and 88.6% respectively, p<0.05). Conclusion: RAMT facilitates reproducible, objective, pre-clinical study of post-stroke mechano-physiotherapy. RAMT successfully improves muscle perfusion, rescues gait deficits, preserves sensorimotor behavior, and attenuates the stroke-induced rise in myostatin. Ongoing efforts focus to characterize the mechano-sensitive miRNA transcriptome in skeletal muscle and employ electrophysiology to quantitatively map post-stroke neuroplasticity in response to RAMT.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated bymodification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to similar to 9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viralmethods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage.From the Clinical Editor: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs. (c) 2016 Elsevier Inc. All rights reserved.

Abstract Individuals living outside the tropics need to adjust their behavioral and physiological repertoires throughout the year to adapt to the changing seasons. White-footed mice (Peromyscus leucopus) reduce hippocampal volumes, hippocampal-dependent memory function, long-term potentiation, and alter neurogenesis in response to short (winter-like) day lengths (photoperiods). During winter, these mice putatively shunt energy away from the brain to maximize peripheral thermogenesis, immune function, and survival. We hypothesized that these changes in brain function are accompanied by alterations in brain vasculature. We maintained white-footed mice in short (8 h light/16 h dark) or long (16 h light/8 h dark) photoperiods for 8–9 weeks. Mice were then perfused with fluorescein isothiocyanate (FITC)-conjugated tomato (Lycopersicon esculentum) lectin to visualize the perfused cerebrovasculature. Short-day mice reduced hippocampal and cortical capillary density (FITC+ area); vessels isolated from short day-exposed mice expressed higher mRNA levels of the gelatinase matrix metalloproteinase 2 (MMP2). Additionally, short-day mice reduced cerebral blood flow ∼15% compared with their long-day counterparts, as assessed by laser speckle flowmetry. Immunohistochemistry revealed higher levels of MMP2 in the hippocampus of mice maintained in short days compared with long days, potentially contributing to the observed vascular remodeling. These data demonstrate that a discrete environmental signal (i.e., day length) can substantially alter cerebral blood flow in adult mammals.

A growing number of clinical trials and case reports support qualitative claims that use of an elevated vacuum suspension (EVS) prosthesis improves residual-limb health on the basis of self-reported questionnaires, clinical outcomes scales, and wound closure studies. Here, we report first efforts to quantitatively assess residual-limb circulation in response to EVS. Residual-limb skin health and perfusion of people with lower-limb amputation (N = 10) were assessed during a randomized crossover study comparing EVS with nonelevated vacuum suspension (control) over a 32 wk period using noninvasive probes (transepidermal water loss, laser speckle imaging, transcutaneous oxygen measurement) and functional hyperspectral imaging approaches. Regardless of the suspension system, prosthesis donning decreased perfusion in the residual limb under resting conditions. After 16 wk of use, EVS improved residual-limb oxygenation during treadmill walking. Likewise, prosthesis-induced reactive hyperemia was attenuated with EVS following 16 wk of use. Skin barrier function was preserved with EVS but disrupted after control socket use. Taken together, outcomes suggest chronic EVS use improves perfusion and preserves skin barrier function in people with lower-limb amputation.CLINICAL TRIAL REGISTRATIONClinicalTrials.gov; "Evaluation of limb health associated with a prosthetic vacuum socket system": NCT01839123; https://clinicaltrials.gov/ct2/show/NCT01839123?term=NCT01839123&rank=1.

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.