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Purpose: Approximately one-fourth of stroke survivors are aphasic. Speech therapy is the main treatment approach but leaves most patients with chronic disability. Attempts to improve this situation are hampered by a lack of mechanistic understanding of the disability and treatments, reflecting the neglect of this impairment modality in pre-clinical research. Accordingly, we devised a novel murine model of speech-related impairment after stroke to investigate the role of language- and plasticity-associated molecules. Rodents communicate socially with ultrasonic vocalizations (USVs), conveying semantic and semiotic information with complex frequency modulated "songs" and alarm calls., Methods: Transient focal cerebral ischemia was induced in male C57BL6 mice via either 30 or 45 minutes of reversible right MCAO using the intraluminal filament technique. Nine days post-operatively brains are stained with TTC and analyzed for infarct volume. For behavioral measures health scores are taken (days 1-4), cylinder tests and USV recordings performed at days 3 and 7 post operatively. Real time PCR was performed at 24 and 48 hour and 7 day time points to quantify mRNA expression of communication-related genes (Foxp2, Foxp1, Srpx2, Cntnap2 and Gapdh). Immunohistochemistry was performed to localize FOXP2 protein., Results: After middle cerebral artery occlusion of either 30 or 45 minutes duration, mice demonstrate profoundly impaired socially evoked USVs. In addition, there is suppression of the language-associated transcription factor, Forkhead box protein 2 (Foxp2), and its downstream binding partner, contactin-associated protein 2 (Cntnap2)., Conclusion: These findings set a foundation for further studies of mechanisms and novel treatment strategies for post-stroke vocalization impairments.

Background: Stroke survivors suffer from persistent disability, as well as severe sensorimotor and cognitive deficits. The preclinical assessment of such deficits is important for the development of novel interventions and therapeutics., New Method: The aim of this study was to develop a quantitative behavioral measure of hindlimb functionality in rodents, which could be used to assess deficits after a neural injury, such as stroke. Here we introduce a test to measure long jump behavior in mice., Results: Using this test we first showed that while male and female mice exhibited no differences in jump success rate, the female mice showed lower baseline jumping latencies. Next we demonstrated that the induction of a cerebral stroke via middle cerebral artery occlusion (MCAO) for 45min did not affect the jump success rate in either group; however, it did significantly increase jump latencies in both male and female mice. Finally, we used therapeutic interventions to explore mechanisms that may be involved in producing this increase in jump latency by administering the anti-depressant fluoxetine prior to the long jump assay, and also tested for potential changes in anxiety levels after stroke., Comparison With Existing Methods: Other methods to assess hindlimb functionality are not specific, because they measure behaviors that rely not only on hindlimbs, but also on forelimbs and tail., Conclusions: This study introduces a novel assay that can be used to measure a stroke induced behavioral deficit with great sensitivity, and raises interesting questions about potential mechanisms regulating this effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Transmigration of peripheral leukocytes to the brain is a major contributor to cerebral ischemic cell death mechanisms. Humanized partial major histocompatibility complex class II constructs (pMHC), covalently linked to myelin peptides, are effective for treating experimental stroke in males, but new evidence suggests that some inflammatory cell death mechanisms after brain injury are sex-specific. We here demonstrate that treatment with pMHC constructs also improves outcomes in female mice with middle cerebral artery occlusion (MCAO). HLA-DR2 transgenic female mice with MCAO were treated with RTL1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide), HLA-DRa1-MOG-35-55, or vehicle (VEH) at 3, 24, 48, and 72 h after reperfusion and were recovered for 96 h or 2 weeks post-injury for measurement of histology (TTC staining) or behavioral testing. RTL1000- and DRa1-MOG-treated mice had profoundly reduced infarct volumes as compared to the VEH group, although higher doses of DRa1-MOG were needed for females vs. males evaluated previously. RTL1000-treated females also exhibited strongly improved functional recovery in a standard cylinder test. In novel studies of post-ischemic ultrasonic vocalization (USV), as measured by animal calls to their cage mates, we modeled in mice the post-stroke speech deficits common in human stroke survivors. The number of calls was reduced in injured animals relative to pre-MCAO baseline regardless of RTL1000 treatment status. However, call duration was significantly improved by RTL1000 treatment, suggesting benefit to the animal's recovery of vocalization capability. We conclude that both the parent RTL1000 molecule and the novel non-polymorphic DRα1-MOG-35-55 construct were highly effective immunotherapies for treatment of transient cerebral ischemia in females.

Cerebrovascular disease is a leading cause of death-from-disease and of disability worldwide, affecting some 15 million people. The incidence of stroke or "brain attack" is unlikely to recede for a decade at minimum by most predictions, despite large public health initiatives in stroke prevention. It has been well established that stroke is also one of the most strikingly sex-specific diseases in its epidemiology, and in some cases, in patient outcomes. For example, women sustain lower rates of ischemic stroke relative to men, even beyond their menopausal years. In contrast, outcomes are worse in women in many clinical studies. The biological basis for this sexual dimorphism is a compelling story, and both hormone-dependent and hormone-independent factors are involved, the latter of which is the subject of this brief review. Understanding the molecular and cell-based mechanisms underlying sex differences in ischemic brain injury is an important step toward personalized medicine and effective therapeutic interventions in patients of both sexes.

In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca(2+)-activated K(+) channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg(2+) block that reduces Ca(2+) influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.

Until recently, supplementation with 100% oxygen was standard therapy for newborns who required resuscitation at birth or suffered later hypoxic-ischemic events. Exposure to high concentrations of oxygen, however, may worsen oxidative stress induced by ischemic injury. In this study we investigated the short- and long-term behavioral outcomes in rats that had undergone hypoxic-ischemic brain injury on postnatal day 7, followed by 2h exposure to 21%, 40%, or 100% oxygen, compared to normal controls. There were no differences in the development of walking, head lifting and righting reflexes from postnatal days 9 to 15. Cliff avoidance showed some abnormal responses in the H21 animals. From postnatal days 28 to 56, three tests of sensorimotor coordination were performed weekly: ledged tapered beam, cylinder, and bilateral tactile stimulation. The ledged tapered beam test without prior training of animals was sensitive to injury, but did not distinguish between treatment groups. The cylinder test showed a greater use of the unimpaired limb in female 21% and 40% oxygen groups compared to controls. Performance in both cylinder and the beam tests showed a correlation with the degree of brain injury. The bilateral tactile stimulation test showed that the male 21% oxygen groups had worse sensory asymmetry than male 40% or 100% oxygen groups, but was not statistically significantly different from controls. We thus found a minor benefit to post-hypoxia-ischemic treatment with 100% and 40% oxygen compared to 21% in one test of early motor skills. Our results for long-term sensorimotor behavior, however, showed conflicting results, however, as males treated with 40% or 100% oxygen had less sensory asymmetry (better performance) in the bilateral tactile stimulation test than males treated with 21% oxygen, while females had impaired motor performance in the cylinder test with both 21% and 40% oxygen., (Copyright © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.)

A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a therapeutically relevant timing after middle cerebral artery occlusion (MCAO) and verify functional benefit to complement histological outcome measures. We observed that the administration of mouse-specific RTL551 reduced infarct size and improved sensorimotor outcome when administered within a 3 h post-ischemic therapeutic window. RTL551 treatment reduced cortical, caudate putamen, and total infarct volume as compared to vehicle-treated mice. Using a standard behavioral testing repertoire, we observed that RTL551 reduced sensorimotor impairment 3 days after MCAO. Humanized RTL1000 (HLA-DR2 moiety linked to hMOG-35-55 peptide) also reduced infarct size in HLA-DR2 transgenic mice. These data indicate that this neuroantigen-specific immunomodulatory agent reduces damage when administered in a therapeutically relevant reperfusion timeframe.

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7-13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male+/-dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke., (Copyright (c)10 IBRO. Published by Elsevier Ltd. All rights reserved.)