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Popular perception has historically constructed the Highlands and Islands of Scotland to be economically and socially backwards in comparison with the rest of the United Kingdom. As evoked in the phrase the ‘Highland Problem’, the area has been considered by outsiders to be beyond help and destined to remain in a state of underdevelopment and chronic depopulation. Despite the history of economic intervention in the area from the late 18th century onwards from private and government initiatives intended to alleviate poverty and bring wealth to the area, it was not until the 1980s with the implementation of sustained and tailored structural assistance from the European Union that emigration slowed and the population of the Highlands and Islands began to grow significantly. This economic success has largely been the result of not only a significant injection of capital but also the willingness of the EU to use local knowledge and collaborate with local agencies. This remarkable development, which is far from over, is being directly threatened by the Brexit phenomenon.

Ischemic stroke, a leading cause of death and long-term disability, has far-reaching effects across the CNS beyond the region of infarct. As extensions of the brain, neural networks of the eye and spinal cord are affected by cerebral ischemia through the transmission of inflammatory and degenerative changes. Although neuropathological effects of MCAO have been reported in both regions, the contribution of the peripheral inflammatory response to the evolution of injury in these CNS regions is unknown. We evaluated local glial responses, vascular integrity, leukocyte infiltration, and cytokine expression after stroke in the eye and spinal cord. Adult male C57Bl/6 WT or GFP+ bone marrow chimeric mice were subjected to 90-min MCAO or sham surgery (n=3-6). Imaging of the retinal vasculature was performed by ophthalmoscopy. At 8h, 24 and/or 36h, 72h and 7d post-stroke mice were perfused with PBS and sacrificed. Organs were harvested for immunohistochemistry, Evans blue extravasation, flow cytometry, and RT-qPCR. Early (8h) after MCAO, over four-fold increases in TNFα and IL-1β gene expression were observed in the ipsilateral eye relative to sham (4.64±1.21, 1±0.17; 11.03±7.75, 1±0.21). Microglial activation indicated by increased CD11b, CD45, and cell granularity at 36h was accompanied by an influx of peripheral leukocytes predominantly comprised of neutrophils (81%). In contrast to BBB breakdown which was highest at 24h, blood-retinal barrier breakdown peaked in the ipsilateral eye at 36h, shown by a twelve-fold increase in Evans blue concentration (sham, 24h, 36h: 0.16, 0.41, 1.95 ug/mg tissue wet weight). Similarly, major blood-spinal cord barrier breakdown occurred at 36h (0.32 vs 0.052 ug/mg). This was followed by a largely monocytic response with corresponding increases in TNFα, IL-1β, and IFNγ gene expression. Together, our data show robust unilateral peripheral inflammatory responses after stroke in CNS regions outside of the brain. In addition, blood-parenchymal barrier permeability peaks later in the eye and spinal cord than in the brain. Given the distinct time course and inflammatory components associated with each region, these findings may have wide implications for treatments attenuating CNS-wide inflammatory damage after stroke.

Introduction: The impairment of energy metabolism during stroke is a major contributor to ensuing tissue damage and death. The NAD+-dependent histone deacetylase SIRT3 has been identified as a key regulator of mitochondrial energy metabolism. It has also been found to repress the transcription of nuclear genes via histone deacetylation. This study aimed to elucidate the function of nuclear SIRT3 in ischemic stroke. Methods: Focal cerebral ischemia was induced by transient right middle cerebral artery occlusion (MCAO) for 90min followed by 72hrs of reperfusion. Infarct area was quantified with Sigma ScanPro5 on 30μm coronal sections stained with Cresyl Violet. Expression of SIRT3 and acetylated lysine proteins was examined by immunohistochemistry (IHC) and western blotting with anti-SIRT3 and anti-acetylated lysine antibodies. NAD+ levels were measured with a colorimetric kit. Results: SIRT3 expression was increased in the ischemic hemisphere of wild-type (WT) mice at 72hrs. Higher magnification of the IHC image demonstrated localization of SIRT3 to both the nucleus and mitochondria of labeled cells. Subcellular fractionations confirmed this localization, and revealed a dramatic increase in nuclear translocation of SIRT3 with ischemia. With the same duration of MCAO and reperfusion, SIRT3 knockout (KO) mice displayed a reduction in striatal infarct from 60.6±7.2% to 24.9±9.7% (P Conclusions: Our findings suggest that SIRT3 contributes to brain injury during stroke via ischemia-induced upregulation and translocation to the nucleus, where it deacetylates both histone and non-histone proteins and represses nuclear gene transcription. Consistent with the neuroprotection afforded by the protein’s absence in KO mice, the inhibition of SIRT3 may serve as a potential therapeutic strategy for minimizing stroke damage.

Background and Purpose: Ischemic stroke is a sexually dimorphic disease. Events that occur during pregnancy can have lasting implications on both the mother and fetus for decades after parturition. In particular, fetomaternal microchimerism, the bidirectional exchange and persistence of cells between a pregnant female and her fetus, has been shown to play a role in various disease pathologies but has not been well studied in the brain. We hypothesized that microchimeric cells (MCs) home to sites of injury as part of the immune response to stroke and display a stem cell phenotype with potential to aid in repair. Methods: C57Bl/6 mice were bred with GFP transgenic mice using specific mating paradigms to generate fetal and maternal microchimeras. Young 10-12 week old maternal-microchimeric mice and 12-14 week old parous fetal-microchimeric mice were subject to a 90 min MCAO or sham surgery and sacrificed at 72hr or 30 day post-reperfusion (n=10). Blood, bone marrow (BM), and brain tissue were analyzed by co-labeling with anti-GFP antibody using immunohistochemistry, flow cytometry, and cytospin preparations. RNA was extracted from GFP+ cells in situ by laser capture microdissection to further characterize MCs. Results: At 72hrs after stroke, clusters of MCs were present in the ischemic area. Flow cytometry of maternal blood and BM identified a rare cell population (0.01-0.001%) of GFP+CD45+CD34+, indicative of umbilical cord stem cells. Engrafted MCs with stem-like phenotypes were also found in cytospin preparations of blood and BM. At 30 day after stroke, MCs in the brain displayed mature endothelial morphology in 60% of animals. This was validated by RT-qPCR, confirming the presence of MCs in the ischemic brain. Conclusions: We have shown that MCs display stem cell phenotypes, are actively recruited to the ischemic brain, and selectively aggregate at sites of injury. Our data show that MCs are mobilized in BM and blood early after stroke and later give rise to endothelial cells, indicating a potential angiogenic role in promoting revascularization. Further characterization of the functional contribution and mechanisms that underlie these responses may prove useful in assessing female responses to brain injury.

Introduction: Early after stroke local inflammatory changes are primarily mediated by microglia, the resident macrophages of the brain. As injury evolves and the vascular integrity becomes compromised, there is a robust infiltration of peripheral myeloid cells into the ischemic area. Given the common lineage ancestry and morphological similarity between bone marrow-derived (BM) macrophages and resident microglia, the two cell types become nearly indistinguishable from each other using standard immunohistological methods. To date, the most sensitive means of discerning between microglia and BM macrophages relies on the relative level of CD45 expression. In this study, ex-vivo analyses were performed to distinguish CD45intCD11b+ microglia and CD45hiCD11b+Ly6C+ BM macrophages. Functional studies were also performed. Materials & Methods: Young male C57Bl/6 mice were subject to sham surgery or middle cerebral artery occlusion for 90min followed by 72hr or 7d of reperfusion. Ipsilateral hemispheres were harvested from PBS perfused mice and fixed for immunohistological preparation or processed by dissociation into single cell suspensions for use in functional flow cytometry assays. Results: Significant microglia cell death occurred by 72hr, and inversely correlated with BM macrophage infiltration. Surviving microglia showed a low rate of proliferation in response to ischemic stress compared to BM macrophages (1% vs. 7%; p Discussion: There is a complex, biphasic macrophage response to stroke injury. The post-acute phase is characterized by widespread microglia activation and dysregulation, whereas the subsequent resolution phase consists of debris clearance in the necrotic core by infiltrating macrophages.

Background: MKP-1, a negative modulator of several MAP kinases including JNK and p38, is a critical regulator of inflammation and apoptosis in the CNS. Inhibition of MKP-1 worsens stroke outcome in mice, suggesting that MKP-1 is an endogenous protective molecule, although the underlying mechanisms remain unclear. Here we investigated the downstream mediators of MKP-1’s effects in stroke. Methods: Stroke was induced by 60 min transient middle cerebral artery occlusion (tMCAO) in male mice. Levels of p-JNK and p-p38 in MKP-1 KO mice and WT littermates 6 hours after stroke were assessed by Western blots. In a second cohort, the selective JNK inhibitor (SP600125) was administered intraperitoneally to both MKP-1 KO and WT mice 30 min before stroke then infarcts were analyzed at 72 hours post ischemia. In a third cohort, WT mice were intraperitoneally administrated the inhibitor or vehicle 30 min before stroke and sacrificed at 6 hours for protein analysis. Results: MKP-1 deletion produced significantly higher levels of p-JNK 6 hours after stroke (0.71±0.12 WT vs. 1.32± 0.14 KO, p0.05, n=3 p/g). There was no additive deleterious effects of pharmacological JNK inhibition in MKP-1 KO mice as measured by infarcts at 72 hours (cortex: 25.84±2.44%WT vs. 22.32±1.91%KO, striatum: 33.10±3.61% WT vs. 26.52±1.92% KO, total: 27.85±2.95% WT vs. 26.75±1.61% KO, p>0.05, n=6 p/g). In WT mice, JNK inhibition significantly decreased p-JNK (0.99 ± 0.14 vehicle vs. 0.44 ± 0.13 drug, p0.05, n=3 p/g). Conclusions: JNK is an important downstream mediator of MKP-1’s effects in stroke. In the ischemic brain, activating MKP-1 may restrain JNK activity, thereby producing neuroprotective effects.

Introduction: Evidence suggests that post-stroke inflammation and outcome is influenced by sex, yet the mechanistic processes behind these disparities remain largely unknown. Studies have shown that ischemic damage following experimental stroke is reduced in young female mice when compared to their male counterparts and that this protective effect can be diminished by ovariectomy (OVX). TGF-B activated kinase (TAK-1) is an important mediator in inflammatory signaling. TAK decreases following cerebral ischemia and inhibition of TAK reduces infarct size following experimental stroke. TAB2 is an activating binding partner that binds to TAK-1 following stimulation, allowing TAK-1 auto-phosphorylation and activation of the p38/MAPK and JNK inflammatory pathways. Post-stroke regulation of TAK levels and activation may contribute to the ischemic resistance seen in females. Hypothesis: We hypothesized that female mice would have a greater decrease in TAK1 and activated p-TAK1 than their male counterparts following experimental stroke. Methods: Male, intact female and ovariectomized female (OVX) mice were subjected to 90-minute middle cerebral artery occlusion (MCAO) and sacrificed at 24 hours post-stroke. Protein levels of TAK-1, TAB2, pTAK and JNK were examined by Western Blot. Results: TAK and pTAK levels were reduced following stroke in female, male and OVX mice. Baseline levels of both TAK1 and pTAK were lower in female mice and the loss of TAK, pTAK and TAB2 following stroke was more dramatic. JNK levels were lower in female mice than in male mice at baseline (p Conclusions: Female mice exhibit lower baseline JNK levels and do not demonstrate a stroke-induced increase in JNK, indicating a protective resistance against stroke-induced inflammation. The dramatic loss of TAB2 in females results in less binding and activation of TAK, reducing inflammation and conferring neuro-protection. OVX mice showed an intermediate phenotype, indicating that estrogen may play a role in TAK regulation following stroke.