Grid Walk Test Research Articles

Delayed Exercise-induced Upregulation of Angiogenic Proteins and Recovery of Motor Function after Photothrombotic Stroke in Mice

Treatments promoting post-stroke functional recovery continue to be an unmet therapeutic problem with physical rehabilitation being the most reproduced intervention in preclinical and clinical studies. Unfortunately, physiotherapy is typically effective at high intensity and early after stroke – requirements that are hardly attainable by stroke survivors. The aim of this study was to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5 h or overnight voluntary wheel running; initiated on post-stroke day 7 and continuing through day 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke and correlate it with angiogenic potential of the brain. Our observations indicate that overnight but not 5 h access to running wheels facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRβ in the peri-infarct and corresponding contralateral motor cortices indicate substantial upregulation of these proteins (≥2-fold) in the infarct core and surrounding cerebral cortex in the overnight running mice on post-stroke day 21. These findings indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins in this expert-recommended mouse ischemic stroke model. Notably, our observations also point out to enhanced angiogenesis and presence of pericytes within the infarct core region during the chronic phase of stroke, suggesting a potential contribution of this tissue area in the mechanisms governing post-stroke functional recovery.

Abstract P750: 3K3A-APC Restores Oligodendrocyte Pools in Models of White Matter Stroke via PAR1 Signaling

A recombinant variant of activated protein C, 3K3A-APC, was recently tested in a successful phase II clinical trial for ischemic stroke (RHAPSODY), which demonstrated that the treatment may reduce hemorrhages and is safe in patients that were treated with tPA and/or thrombectomy. Animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration have shown that the effects of 3K3A-APC are mediated by preserved APC cell signaling activities (anti-apoptotic neuronal protection, protection of blood-brain barrier (BBB) integrity and anti-inflammatory activities). Because recent studies have suggested a role of protease activated receptor 1 (PAR1) in oligodendrocyte recovery after white matter damage, we wanted to test whether biased PAR1 signaling mediated by 3K3A-APC would protect oligodendrocytes in in vivo and ex vivo models of ischemic white matter stroke. First, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results show that treatment with recombinant murine 3K3A-APC (0.2 mg/kg, starting 4h after stroke) protected oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cells (OPCs, Olig2+PDGFRα+) from cell death at 1 and 7 days after stroke. In vitro , 3K3A-APC protected primary mouse oligodendrocytes from apoptosis when exposed to OGD conditions at nanomolar concentrations (EC50 concentration was 5.2 nM). Improved oligodendrocyte viability in vivo was associated by about ~67% reduction in overall cell death (cresyl violet staining, T2w and diffusion tensor MRI) and reduced BBB leakage (fibrin staining and DCE-MRI) as well as improved functional recovery (adhesive removal and grid walking tests). Protective effects of 3K3A-APC were abolished by silencing of PAR1 with small interfering RNA (siRNA) in vivo and in vitro . In conclusion, PAR1 mediated signaling initiated by 3K3A-APC protects oligodendrocyte pools in models of ischemic white matter damage with preservation of neurovascular unit integrity.

The Effect of Histone Deacetylase Inhibitors Panobinostat or Entinostat on Motor Recovery in Mice After Ischemic Stroke.

Using rigorous and clinically relevant experimental design and analysis standards, in this study, we investigated the potential of histone deacetylase (HDAC) inhibitors panobinostat and entinostat to enhance recovery of motor function after photothrombotic stroke in male mice. Panobinostat, a pan-HDAC inhibitor, is a FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor in late stage of clinical investigation. The drugs were administered every other day (panobinostat-3 or 10mg/kg; entinostat-1.7 or 5mg/kg) starting from day 5 to 15 after stroke. To imitate the current standard of care in stroke survivors, i.e., physical rehabilitation, the animals run on wheels (2h daily) from post-stroke day 9 to 41. The predetermined primary end point was motor recovery measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. In addition, we evaluated the running distance and speed throughout the study, and the number of parvalbumin-positive neurons in medial agranular cortex (AGm) and infarct volumes at the end of the study. Both sensorimotor tests revealed that combination of physical exercise with either drug did not substantially affect motor recovery in mice after stroke. This was accompanied by negligible changes of parvalbumin-positive neurons recorded in AGm and comparable infarct volumes among experimental groups, while dose-dependent increase in acetylated histone 3 was observed in peri-infarct cortex of drug-treated animals. Our observations suggest that add-on panobinostat or entinostat therapy coupled with limited physical rehabilitation is unlikely to offer therapeutic modality for stroke survivors who have motor dysfunction.

Manganese nanoparticles induce blood-brain barrier disruption, cerebral blood flow reduction, edema formation and brain pathology associated with cognitive and motor dysfunctions.

Nanoparticles affect blood-brain barrier (BBB) and brain edema formation resulting in sensory-motor dysfunction. Exposure of Mn nanoparticles from industrial sources in humans could target basal ganglia resulting in Parkinson's disease. In present investigation, Mn exposure on brain pathology in a rat model was examined. Rats received Mn nanoparticles (30-40nm size) in a dose of 10 or 20mg/kg, i.p. once daily for 7 days and behavioral dysfunctions on Rota Rod performance, inclined plane angle and grid-walking tests as well as gait performances were examined. In addition, BBB breakdown to Evans blue and radioiodine, brain edema formation and neural injuries were also evaluated. Mn nanoparticles treated rats exhibited cognitive and motor dysfunction on the 8th day. At this time, BBB disruption, reduction in cerebral blood flow (CBF), brain edema formation and brain pathology were most marked in the sensory-motor cortex, hippocampus, caudate putamen, cerebellum and thalamus followed by hypothalamus, pons, medulla and spinal cord. In these brain areas, neuronal injuries using Nissl staining was clearly seen. These effects of Mn nanoparticle are dose dependent. These results are the first to demonstrate that Mn nanoparticles induce selective brain pathology resulting in cognitive and motor dysfunction, not reported earlier.

Neuroprotective and Neurorestorative Effects of Holothuria scabra Extract in the MPTP/MPP+-Induced Mouse and Cellular Models of Parkinson’s Disease

Extracts from Holothuria scabra (HS) have been shown to possess anti-inflammation, anti-oxidant and anti-cancer activities. More recently, it was shown to have neuroprotective potential in Caenorhabditis elegans PD model. Here, we assessed whether HS has neuroprotective and neurorestorative effects on dopaminergic neurons in both mouse and cellular models of PD. We found that both pre-treatment and post-treatment with HS improved motor deficits in PD mouse model induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as determined by grid walk test. This was likely mediated by HS protective and restorative effects on maintaining the numbers of dopaminergic neurons and fibers in both substantia nigra pars compacta (SNpc) and striatum. In a cellular model of PD, HS significantly attenuated 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of DAergic-like neurons differentiated from SH-SY5Y cells by enhancing the expression of Bcl-2, suppressing the expression of cleaved Caspase 3 and preventing depolarization of mitochondrial membrane. In addition, HS could stimulate the expression of tyrosine hydroxylase (TH) and suppressed the formation of α-synuclein protein. Taken together, our in vivo and in vitro findings suggested that HS is an attractive candidate for the neuroprotection rather than neurorestoration in PD.

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

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).

Neuroprotection against cerebral ischemia/reperfusion by dietary phytochemical extracts from Tibetan turnip (Brassica rapa L.)

Ethnopharmacological relevanceThe Tibetan turnip (Brassica rapa L.) has a wide array of medicine properties including heat-clearing, detoxifying and anti-hypoxia as listed in the famous centuries-old Tibetan medicine classic “The Four Medical Tantras”. Evidence-based medicine also indicated the anti-hypoxic effect of turnips, suggesting a potential link to neuroprotective effect on ischemic stroke. This thereby enables turnips to serve as a novel nontoxic agent in related treatment. Aim of the studyThis study aimed to investigate the neuroprotective effect and elucidate the mechanism of aqueous extract of turnip (AET) on cerebral ischemia/reperfusion. Materials and methodsThe experimental models of cerebral ischemia included transient middle cerebral artery occlusion/reperfusion (MCAO) in C57BL/6J mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in HT-22 cells. Long-term effect of AET on infarct volume was evaluated by microtubule-associated protein 2 (MAP2) immunofluorescence 28 days after MCAO, and on neurofunctional outcomes determined by rotarod, grid walking, and cylinder tests in the meantime. Efficacy of AET was determined by the cell viability, the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) in neurons. The underlying mechanism of AET rescued OGD/R cells were characterized by PI3K, Akt and mTOR expressions, which were further used to validate AET's role in the pathway. ResultsAET can reduce cerebral infarct volume and ameliorate behavioral deficits of MCAO/R mice dose-dependently. In vitro experiment further demonstrated that suitable concentrations of AET inhibited ROS, LDH production and restored mitochondrial expression induced by OGD/R. AET pretreatment can reverse the OGD/R-induced decreased level of phosphorylation of PI3K, Akt, mTOR, whereas this effect was blocked in the LY294002 (PI3K inhibitor) treatment group. ConclusionsAET improved the survival of OGD/R-injured HT-22 cells by activating the PI3K/Akt/mTOR pathway. Based on the results above, aqueous extract of turnip has a protective effect on focal cerebral ischemic injury.

Transplantation of Induced Neural Stem Cells Derived from Human Fibroblasts Promotes Functional Recovery of Spinal Cord Injury in Rats

Background: Traumatic injury to the spinal cord causes cell death, demyelination, axonal degeneration, and cavitation, resulting in functional motor and sensory loss. Stem cell therapy as a promising approach for spinal cord injury (SCI) has some challenges, such as immunological responses to grafted cells. Transplantation of autologous trans-differentiated cells can be a useful strategy to overcome this problem. Materials and Methods: In this research, we transplanted human-induced neural stem cells (hiNSCs), which were trans-differentiated from adult human fibroblasts into the injured spinal cord of adult rats on day seven post-injury. Before transplantation, hiNSCs were explored for expressing NSCs general protein and genes and also, their normal karyotype was examined. After hiNSCs transplantation, behavioural tests (BBB score and grid walk tests) were performed weekly and finally histological assessment was done for exploring the cell fates and migration. Results: Our results showed the cell viability, differentiation, and migration of transplanted hiNSCs was significantly improved in the injured site of the spinal cord up to seven weeks after the SCI. Also, the behavioural analysis revealed the enhanced locomotor functions of the animals that underwent transplantation after seven weeks. Our data provide strong evidence in support of the feasibility of hiNSCs for cell-based therapy in SCI rats.

Blood glutamate scavengers and exercises as an effective neuroprotective treatment in mice with spinal cord injury.

Excitotoxicity due to neuronal damage and glutamate release is one of the first events that leads to the progression of neuronal degeneration and functional impairment. This study is based on a paradigm shift in the therapeutic approach for treating spinal cord injury (SCI). The authors tested a new treatment targeting removal of CNS glutamate into the blood circulation by injection of the blood glutamate scavengers (BGSs) recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its cosubstrate oxaloacetic acid (OxAc). Their primary objective was to investigate whether BGS treatment, followed by treadmill exercises in mice with SCI, could attenuate excitotoxicity, inflammation, scarring, and axonal degeneration and, at a later time point, improve functional recovery. A pharmacokinetic experiment was done in C57BL/6 naive mice to verify rGOT1/OxAc blood activity and to characterize the time curve of glutamate reduction in the blood up to 24 hours. The reduction of glutamate in CSF after BGS administration in mice with SCI was confirmed by high-performance liquid chromatography. Next, SCI (left hemisection) was induced in the mice, and the mice were randomly assigned to one of the following groups at 1 hour postinjury: control (underwent SCI and received PBS), treadmill exercises, rGOT1/OxAc treatment, or rGOT1/OxAc treatment followed by treadmill exercises. Treatment started 1 hour postinjury with an injection of rGOT1/OxAc and continued for 5 consecutive days. Starting 1 week after SCI, the exercises and the combined treatment groups recommenced the treadmill exercise regimen 5 days a week for 3 months. Locomotor function was assessed for 3 months using the horizontal grid walking test and CatWalk. Axonal anterograde and wallerian degenerations were evaluated using tetramethylrhodamine dextran. Tissue sections were immunofluorescently stained for Iba1, GFAP, GAP-43, synaptophysin, and NeuN. BGS treatment decreased the CSF glutamate level up to 50%, reduced axonal wallerian degeneration, and increased axonal survival and GAP-43 expression in neuronal cells. Combined treatment reduced inflammation, scarring, and lesion size. Additionally, the combination of BGS treatment and exercises increased synapses around motor neurons and enhanced axonal regeneration through the lesion site. This resulted in motor function improvement 3 months post-SCI. As shown by biochemical, immunohistochemical, and functional analysis, BGSs exhibit a substantial neuroprotective effect by reducing excitotoxicity and secondary damage after SCI. Furthermore, in combination with exercises, they reduced axonal degeneration and scarring and resulted in improved functional recovery.

Protective Effects of Leukadherin1 in a Rat Model of Targeted Experimental Autoimmune Encephalomyelitis (EAE): Possible Role of P47phox and MDA Downregulation.

BackgroundReactive oxygen and nitrogen species (ROS and RNS) are involved in pathologic mechanisms underlying demyelination and exacerbation in multiple sclerosis (MS) lesions. P47phox is the most important subunit of an ROS-producing enzyme (NADPH oxidase) which is reportedly upregulated in MS plaques due to the intense activity of infiltrated immune cells and resident microglia. Leukadherin1 is a specific CD11b/CD18 agonist that inhibits signaling and transmigration of inflammatory cells to sites of injury. Based on this mechanism, we evaluated therapeutic effects of leukadherin1 in an animal model of targeted experimental autoimmune encephalomyelitis (EAE) through focal injection of inflammatory cytokines to the spinal cord.MethodsFor model induction, Lewis rats were first immunized with 15µg MOG 1–125 emulsion. Twenty days later, animals were subjected to stereotaxic injection of IFNγ and TNFα to the specific spinal area (T8). One day after injection, all animals presented EAE clinical signs, and their behaviors were monitored for eight days through open-field locomotion and grid-walking tests. Leukadherin1-treated animals received daily intraperitoneal injections of 1mg/kg of the drug. The specific spinal tissues were extracted on day 5 in order to measure nitric oxide (NO), malon di-aldehyde (MDA), and TNFα concentrations alongside P47phox real-time PCR analysis. In addition, spinal sections were prepared for immunohistochemical (IHC) observation of infiltrated leukocytes and activated microglia.ResultsLeukadherin1 exhibited promising improvements in EAE clinical scores and behavioral tests. Demyelination, CD45+ leukocyte infiltration, and Iba1+ microglia activation were reduced in spinal tissues of leukadherin1-treated animals. Furthermore, P47phox expression levels, MDA, and NO amounts were decreased in treated animals. However, TNFα concentrations did not differ following treatment.ConclusionBased on our results, we suggest that leukadherin1 may be used as a novel therapeutic agent in tackling the clinical challenge of multiple sclerosis, especially during the acute phase of the disease. This effect was possibly mediated through decreased leukocyte infiltration and oxidative stress.

New PAR1 Agonist Peptide Demonstrates Protective Action in a Mouse Model of Photothrombosis-Induced Brain Ischemia.

Protease-activated receptors (PARs) are involved not only in hemostasis but also in the development of ischemic brain injury. In the present work, we examined in vivo effects of a new peptide (AP9) composing Asn47-Phen55 of PAR1 “tethered ligand” generated by activated protein C. We chose a mouse model of photothrombosis (PT)-induced ischemia to assess AP9 effects in vivo. To reveal the molecular mechanism of AP9 action, mice lacking β-arrestin-2 were used. AP9 was injected intravenously once 10 min before PT at doses of 0.2, 2, or 20 mg/kg, or twice, that is, 10 min before and 1 h after PT at a dose of 20 mg/kg. Lesion volume was measured by magnetic resonance imaging and staining of brain sections with tetrazolium salt. Neurologic deficit was estimated using the cylinder and the grid-walk tests. Blood–brain barrier (BBB) disruption was assessed by Evans blue dye extraction. Eosin-hematoxylin staining and immunohistochemical staining were applied to evaluate the number of undamaged neurons and activated glial cells in the penumbra. A single administration of AP9 (20 mg/kg), as well as its two injections (20 mg/kg), decreased brain lesion volume. A double administration of AP9 also reduced BBB disruption and neurological deficit in mice. We did not observe the protective effect of AP9 in mice lacking β-arrestin-2 after PT. Thus, we demonstrated for the first time protective properties of a PAR1 agonist peptide, AP9, in vivo. β-Arrestin-2 was required for the protective action of AP9 in PT-induced brain ischemia.

Late Atomoxetine or Fluoxetine Treatment with Low Duration Voluntary Running Facilitate Recovery of the Motor Function after Photothrombotic Stroke in Mice

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 mm3). 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 InformationResearch in the Karamyan laboratory is supported by R01NS106879Enhanced 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 1Atomoxetine and fluoxetine treatment significantly decreased parvalbumin expression in the ischemic hemisphere compared to the healthy hemisphere on 42 days after stroke.Figure 2

P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis.

IntroductionClearance of damaged cells and debris is beneficial for the functional recovery after ischemic brain injury. However, the specific phagocytic receptor that mediates microglial phagocytosis after ischemic stroke is unknown.AimTo investigate whether P2Y6 receptor‐mediated microglial phagocytosis is beneficial for the debris clearance and functional recovery after ischemic stroke.ResultsThe expression of the P2Y6 receptor in microglia increased within 3 days after transient middle cerebral artery occlusion. Inhibition of microglial phagocytosis by the selective inhibitor MRS2578 enlarged the brain atrophy and edema volume after ischemic stroke, subsequently aggravated neurological function as measured by modified neurological severity scores and Grid walking test. MRS2578 treatment had no effect on the expression of IL‐1α, IL‐1β, IL‐6, IL‐10, TNF‐α, TGF‐β, and MPO after ischemic stroke. Finally, we found that the expression of myosin light chain kinase decreased after microglial phagocytosis inhibition in the ischemic mouse brain, which suggested that myosin light chain kinase was involved in P2Y6 receptor‐mediated phagocytosis.ConclusionOur results indicate that P2Y6 receptor‐mediated microglial phagocytosis plays a beneficial role during the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.

Abstract TMP27: Par1 Mediates Protective Effect of 3K3K-APC After White Matter Stroke in Mice

3K3A-APC is a recombinant variant of activated protein C (APC) with > 90% reduced anticoagulant activity but preserved cell signaling activity. Benefical effects are mediated by its antiapoptotic direct neuronal protective and vasculoprotective effects including protection of blood-brain barrier (BBB) integrity as well as anti-inflammatiory activity as shown in multiple animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration. These preclinical studies led to a successfully completed phase II clinical trial of 3K3A-APC for ischemic stroke (RHAPSODY). RHAPSODY trial showed that 3K3A-APC is safe and may reduce hemorrhage in patients that were treated with tPA and/or thrombectomy standard of care therapy for stroke. Even though the protective effects of 3K3A-APC are well known to be mediated by biased signaling via protease activated receptor 1 (PAR1) activation, the role of PAR1 has not been studied in ischemic white matter stroke. In the present study, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results suggest that 4 hrs post-treatment with 3K3A-APC (0.2 mg/kg) reduced lesion volumes by about ~67% (cresyl violet staining and T2w MRI), which was associated with reduced BBB leakage (fibrinogen staining and DCE-MRI), and improved function of oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cell (OPCs, Olig2+PDGFRα+) at 1 and 7 days after stroke. Beneficial effects of 3K3A-APC were associated with reduced axonal damage on diffusion tensor MRI, and improved behavioral performance on adhesive removal and grid walking tests. We next showed that PAR1 is expressed in all major cell types in the white matter (e.g., OLS, OPCs, microglia and astrocytes) and that silencing PAR1 with small interfering RNA (siRNA) abolishes the protective effect of 3K3A-APC in this model. Taken together, our data suggest that 3K3A-APC is protective in ischemic white matter disease via PAR1 mediated signaling, which improves oligovascular integrity and neuronal survival.

Abstract TP114: Histone Deacetylase Inhibitors Panobinostat and Entinostat for Motor Recovery After Ischemic Stroke in Mice

In this study, we investigated the potential of two histone deacetylase (HDAC) inhibitors, panobinostat and entinostat, to enhance recovery of motor function after ischemic stroke in CD-1 male mice. Panobinostat, which is a pan-HDAC inhibitor, is an FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor and is widely expected to get approval for clinical use in near future. Stroke was induced by photothrombosis and the drugs were administered intraperitoneally every other day (panobinostat at 10 mg/kg or 30 mg/kg; entinostat at 5 mg/kg or 15 mg/kg) starting from day 5 to day 15 after stroke. The control group received only vehicle following the same regimen. In addition, all drug or vehicle-treated mice exercised for 2 hours (voluntary wheel running) starting from day 9 to day 41 after stroke. Additional control groups included sham-operated animals, and mice which had stroke but were not drug/vehicle-treated or allowed to run. Motor function of the mice was evaluated by blinded investigators using gridwalk and cylinder tests before and after stroke. Acetylation of histone 3 in the peri-infarct region of the brain samples was measured by immunoblotting. Our results indicate that lower dose of both panobinostat and entinostat marginally improved motor function in mice by day 42 after stroke, although it did not reach statistical significance. Notably, this improvement trend was lost with the higher dose of both drugs which also showed some toxicity. No statistical difference was observed in running distance of mice among experimental groups. Likewise, we did not observe statistically significant difference in stroke volumes among the experimental groups. Immunoblotting experiments indicated that both panobinostat and entinostat dose-dependently increased the level of acetylated histone in the cortical, peri-infarct region of drug-treated animals compared to the non-treated groups. In summary, our results indicate that both panobinostat and entinostat do not facilitate improvement of motor function after stroke at the tested doses. However, it is likely that lower doses of these drugs may enhance recovery of motor function after stroke.

Abnormal Pyramidal Decussation and Bilateral Projection of the Corticospinal Tract Axons in Mice Lacking the Heparan Sulfate Endosulfatases, Sulf1 and Sulf2.

The corticospinal tract (CST) plays an important role in controlling voluntary movement. Because the CST has a long trajectory throughout the brain toward the spinal cord, many axon guidance molecules are required to navigate the axons correctly during development. Previously, we found that double-knockout (DKO) mouse embryos lacking the heparan sulfate endosulfatases, Sulf1 and Sulf2, showed axon guidance defects of the CST owing to the abnormal accumulation of Slit2 protein on the brain surface. However, postnatal development of the CST, especially the pyramidal decussation and spinal cord projection, could not be assessed because DKO mice on a C57BL/6 background died soon after birth. We recently found that Sulf1/2 DKO mice on a mixed C57BL/6 and CD-1/ICR background can survive into adulthood and therefore investigated the anatomy and function of the CST in the adult DKO mice. In Sulf1/2 DKO mice, abnormal dorsal deviation of the CST fibers on the midbrain surface persisted after maturation of the CST. At the pyramidal decussation, some CST fibers located near the midline crossed the midline, whereas others located more laterally extended ipsilaterally. In the spinal cord, the crossed CST fibers descended in the dorsal funiculus on the contralateral side and entered the contralateral gray matter normally, whereas the uncrossed fibers descended in the lateral funiculus on the ipsilateral side and entered the ipsilateral gray matter. As a result, the CST fibers that originated from 1 side of the brain projected bilaterally in the DKO spinal cord. Consistently, microstimulation of 1 side of the motor cortex evoked electromyogram responses only in the contralateral forelimb muscles of the wild-type mice, whereas the same stimulation evoked bilateral responses in the DKO mice. The functional consequences of the CST defects in the Sulf1/2 DKO mice were examined using the grid-walking, staircase, and single pellet-reaching tests, which have been used to evaluate motor function in mice. Compared with the wild-type mice, the Sulf1/2 DKO mice showed impaired performance in these tests, indicating deficits in motor function. These findings suggest that disruption of Sulf1/2 genes leads to both anatomical and functional defects of the CST.

Growth Hormone Promotes Motor Function after Experimental Stroke and Enhances Recovery-Promoting Mechanisms within the Peri-Infarct Area.

Motor impairment is the most common and widely recognised clinical outcome after stroke. Current clinical practice in stroke rehabilitation focuses mainly on physical therapy, with no pharmacological intervention approved to facilitate functional recovery. Several studies have documented positive effects of growth hormone (GH) on cognitive function after stroke, but surprisingly, the effects on motor function remain unclear. In this study, photothrombotic occlusion targeting the motor and sensory cortex was induced in adult male mice. Two days post-stroke, mice were administered with recombinant human GH or saline, continuing for 28 days, followed by evaluation of motor function. Three days after initiation of the treatment, bromodeoxyuridine was administered for subsequent assessment of cell proliferation. Known neurorestorative processes within the peri-infarct area were evaluated by histological and biochemical analyses at 30 days post-stroke. This study demonstrated that GH treatment improves motor function after stroke by 50%–60%, as assessed using the cylinder and grid walk tests. Furthermore, the observed functional improvements occurred in parallel with a reduction in brain tissue loss, as well as increased cell proliferation, neurogenesis, increased synaptic plasticity and angiogenesis within the peri-infarct area. These findings provide new evidence about the potential therapeutic effects of GH in stroke recovery.

Motor deficit in the mouse ferric chloride-induced distal middle cerebral artery occlusion model of stroke

Ferric chloride-induced distal middle cerebral artery occlusion (MCAO) model of stroke was described in mice several years ago, however it lacked in-depth evaluation of the post-stroke functional outcomes in the animals. In this study, we reproduced the recently developed model and expanded its characterization by thorough evaluation of blood supply, cerebral infarction, and motor function in adult male and female mice up to 14 days after stroke. Our observations indicate near complete interruption of blood flow in the distal MCA shortly after application of 20 % ferric chloride over the artery through a cranial window, which remained occluded for at least 4 h. As expected, infarction of the brain tissue, documented by TTC and hematoxylin stains, was restricted to the cerebral cortex. We also systematically evaluated motor impairment of the animals in this model. For this, a series of studies were carried out in male and female mice up to 14 days after stroke, and motor function was assessed in cylinder and grid-walking tests in blinded manner. Contrary to our expectations, the results of both motor tests indicated minor, transient motor deficit in mice after stroke. Based on these observations, we conclude that the mouse ferric chloride-induced distal MCAO model is likely not suitable for proof-of-concept and preclinical studies where motor function is an important outcome measure.

Progranulin deficiency exacerbates spinal cord injury by promoting neuroinflammation and cell apoptosis in mice

PurposeSpinal cord injury (SCI) often results in significant and catastrophic dysfunction and disability and imposes a huge economic burden on society. This study aimed to determine whether progranulin (PGRN) plays a role in the progressive damage following SCI and evaluate the potential for development of a PGRN derivative as a new therapeutic target in SCI.MethodsPGRN-deficient (Gr−/−) and wild-type (WT) littermate mice were subjected to SCI using a weight-drop technique. Local PGRN expression following injury was evaluated by Western blotting and immunofluorescence. Basso Mouse Scale (BMS), inclined grid walking test, and inclined plane test were conducted at indicated time points to assess neurological recovery. Inflammation and apoptosis were examined by histology (Hematoxylin and Eosin (H&E) staining and Nissl staining, TUNEL assays, and immunofluorescence), Western blotting (from whole tissue protein for iNOS/p-p65/Bax/Bcl-2), and ex vivo ELISA (for TNFα/IL-1β/IL-6/IL-10). To identify the prophylactic and therapeutic potential of targeting PGRN, a PGRN derived small protein, Atsttrin, was conjugated to PLGA-PEG-PLGA thermosensitive hydrogel and injected into intrathecal space prior to SCI. BMS was recorded for neurological recovery and Western blotting was applied to detect the inflammatory and apoptotic proteins.ResultsAfter SCI, PGRN was highly expressed in activated macrophage/microglia and peaked at day 7 post-injury. Grn−/− mice showed a delayed neurological recovery after SCI at day 21, 28, 35, and 42 post-injury relative to WT controls. Histology, TUNEL assay, immunofluorescence, Western blotting, and ELISA all indicated that Grn−/− mice manifested uncontrolled and expanded inflammation and apoptosis. Administration of control-released Atsttrin could improve the neurological recovery and the pro-inflammatory/pro-apoptotic effect of PGRN deficiency.ConclusionPGRN deficiency exacerbates SCI by promoting neuroinflammation and cellular apoptosis, which can be alleviated by Atsttrin. Collectively, our data provide novel evidence of using PGRN derivatives as a promising therapeutic approach to improve the functional recovery for patients with spinal cord injury.

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260-330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.