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Stroke, a life-threatening condition, primarily resulting from ischemic events often caused by occlusion of the middle cerebral artery (MCA). Pre-existing leptomeningeal collateral (LMC) vessels connect MCA branches to anterior or posterior arteries, situated along the brain's cortical surface or meninges, under healthy conditions these vessels remain dormant due to their small diameters and relatively low flow velocity. LMCs serve as vascular redundancies that retrogradely re-supply blood to help salvage the penumbra following cerebral vascular occlusion. Their outward growth or remodeling (arteriogenesis) is essential for promoting cerebral reperfusion and preventing tissue damage after ischemic stroke. Increased fluid shear stress on collateral vessel wall activates arteriogenesis result in the activation of the endothelium and subsequent recruitment of peripheral-derived immune cells (PDICs), which have been shown to aid this unique adaptive process in other organ systems, however their role and mechanism(s) involved in LMC remodeling in stroke has not previously been evaluated. Initial findings suggest the EphA4, a well-established axonal growth and guidance receptors, plays a novel role in LMC arteriogenesis. This dissertation examined PDIC-specific functions of EphA4 using GFP labeled bone marrow chimeric mice subjected to permanent middle cerebral artery occlusion (pMCAO). We assessed immune cell population changes, infarct volume, functional recovery, characterized subtypes of infiltrated immune cell, and measured collateral vessel diameters. Additionally, we explored the Tie2-mediated PI3K signaling pathway in peripheral-derived monocyte/macrophages (PDM) treated with soluble Tie2-Fc and a PI3K p110α inhibitor. The results from this dissertation show that loss of PDIC-specific EphA4 led to increased collateral remodeling, associated with decreased infarct volume, improved cerebral blood flow, and functional recovery within 24 hours post-pMCAO. The crosstalk between EphA4-Tie2 signaling in PDMs, regulated through PI3K/Akt axis, inhibited pial collateral remodeling. In conclusion, our findings highlight the negative regulatory role of PDM-specific EphA4 in collateral growth and remodeling by inhibiting Tie2 function via the PI3K regulated pathway. Peripheral myeloid-derived EphA4 emerges as a new regulator of cerebral vascular injury and neuroinflammation following acute ischemic stroke. / Doctor of Philosophy / Stroke, a life-threatening condition, occurs when blood flow to part of the brain is disrupted due to the vascular occlusion of a major brain artery, such as the MCA. Within protective layers of our brain, there are pre-existing pial collateral vessels that act as backup connections. These vessels play an important role in increasing cerebral reperfusion and preventing tissue damage after stroke. One fascinating aspect of stroke recovery involves PDICs. These immune cells migrate into the blood hypo-perfused region of the brain and regulate the growth of collateral vessels. However, the specific functions of PDICs, particularly a receptor called EphA4, has remained unclear. Our research delved into the immune response following ischemic stroke using genetically modified mice. We examined immune cell populations, infarct volume (the damaged brain tissue), functional recovery, and collateral vessel diameters. Notably, we discovered that deletion of PDIC-specific EphA4 enhanced collateral vessel remodeling. This led to decreased infarct volume, better blood flow, and improved functional recovery within 24 hours after stroke. Furthermore, we explored a signaling pathway involving Tie2 and PI3K in PDM. This crosstalk between EphA4 and Tie2, mediated through PI3K regulation, played a critical role in suppressing collateral vessel remodeling. In summary, understanding how immune cells contribute to stroke recovery may pave the way for novel therapeutic approaches to enhance outcomes for stroke patients.

Tyrimo tikslas. Palyginti eisenos atsigavimą asmenims, patyrusiems trauminį galvos smegenų sužalojimą (TGSS) ar galvos smegenų infarktą (GSI), pirmajame reabilitacijos etape taikant kineziterapiją. Tyrimo uždaviniai.1) Įvertinti asmenų, patyrusių TGSS ar GSI, protinę būklę ir mobilumą prieš ir po kineziterapijos; 2) Įvertinti asmenų, patyrusių TGSS, eiseną taikant kineziterapiją pirmojo reabilitacijos etapo pradžioje ir pabaigoje; 3) Įvertinti asmenų, patyrusių GSI, eiseną taikant kineziterapiją pirmojo reabilitacijos etapo pradžioje ir pabaigoje; 4) Įvertinti asmenų, patyrusių TGSS ar GSI, proto būklės ir mobilumo atsigavimo įtaką eisenos atsigavimui, taikant kineziterapiją. Tyrimo metodai. Tyrime dalyvavo 60 asmenų: 30 asmenų, patyrusių TGSS, ir 30 asmenų, patyrusių GSI. Visiems tiriamiesiems buvo sutrikusi eisena. Vertinome tiriamųjų proto būklę pagal Trumpą Proto Būklės Tyrimo Testą, (TPBVT), mobilumą pagal Rivermead’o mobilumo indeksą (Rivermead Mobility Index) ir eiseną pagal Dinaminį Eisenos Indeksą (Dynamic Gait Index). Rezultatai skaičiuoti atliekant matematinę statistinę analizę. Visiems tiriamiesiems buvo taikoma kineziterapija siekiant eisenos atsigavimo. Tyrimo rezultatai. Tyrimo rezultatai parodė, kad taikant kineziterapiją pagerėjo proto būklė, mobilumas ir eisena abiejose tiriamųjų grupėse. Atlikus asmenų po TGSS ar GSI proto būklės, mobilumo ir eisenos palyginamąją analizę, gavome, kad kineziterapija turi teigiamą įtaką abiejų grupių proto būklės, mobilumo... [toliau žr. visą tekstą] / The aim. To compare gait recovery of individuals after Traumatic Brain Injury (TBI) or Ischemic Brain Stroke (IBS) using Physical Therapy during first rehabilitation stage. The tasks were: 1) To evaluate individuals after TBI or IBS mental state and mobility before and after Physical Therapy. 2) To evaluate individuals gait after TBI before and after Physical Therapy during first rehabilitation state. 3) To evaluate individuals gait after IBS before and after Physical Therapy during first rehabilitation state. 4) To find correlation between mental state, mobility and gait recovery of individuals after TBI or IBS using Physical Therapy. Methods and Material. In this study there were 60 persons participated: 30 after TBI and 30 after IBS. All patients had gait disorders. We evaluated mental state using Mini Mental State Examine (MMSE), mobility using Rivermead Mobility Index (RMI) and gait using Dynamic Gait Index. The results were calculated using mathematical statistical analysis. All patients received Physical Therapy to improve their gait. Results. The results showed significant mental state, mobility and gait recovery after Physical Therapy in both groups. Comparative analysis of persons after TBI or IBS mental state, mobility and gait showed that Physical Therapy has a statistically significant influence on mental state, mobility and gait recovery in both groups. We found correlation between mental state, mobility and gait recovery for patients after TBI or IBS. Gait... [to full text]

<p>Cerebrovascular disease (stroke), especially ischemic stroke, is a major cause of death and neurological disability in adults. Because of its clinical heterogeneity, stroke is considered as a multi-factorial and polygenic disorder. Most current genetic studies of ischemic stroke focus on genetic susceptibility rather than factors determining stroke outcome. The genetic components of ischemic stroke outcome are difficult to study in humans due to environmental factors and medical intervention. Thus, we proposed to use a surgically induced, permanent, focal cerebral ischemic stroke mouse model to investigate genetic factors of ischemic stroke outcome measured by infarct volume. This model is the middle cerebral artery occlusion (MCAO) model. First, we screened infarct volumes across 32 inbred mouse strains. The infarct volume varies between strains, and this strongly suggests that infarct volume is genetically determined. To identify these genetic factors, we used genome-wide association study [Efficient Mixed-Model Association (EMMA) analysis] on infarct volume from 32 inbred mouse strains. Using the EMMA analysis, we identified 11 infarct volume-associated loci; however, most loci were mapped with missing alleles. This suggests that these loci might be false positives. Thus, we used specifically designed scripts of EMMA analysis with updated mouse SNP database to correct for potential false positives. The loci identified by the updated EMMA analyses will led us to the identification of genes involved in ischemic stroke outcome. </p><p> There are two major mechanisms were proposed to be determinants of infarct volume, the extent of native collateral circulation and neuroprotection. Using the infarct volume screening panel from 32 inbred strains, we observed that infarct volume is inversely correlated with the native collateral vessel number. However, among these inbred strains, we also observed several strains differ significantly in infarct volumes but harbor similar collateral numbers. In order to identify genetic factors determining infarct volume in a collateral-independent manner (neuroprotection), we used quantitative trait locus (QTL) mapping on mouse strains that exhibit the most difference in infarct volumes but the least difference in collateral numbers (C57BL/6J and C3H/HeJ). From the F2 B6 x C3H cross, we mapped 4 loci determining infarct volume (cerebral infarct volume QTL 4 to 7, Civq4 to Civq7). The Civq4 locus is the strongest locus (LOD 9.8) that contributes 21% of phenotypic variance in infarct volume. We also used a parallel F2 B6 x C3H cross to perform a QTL mapping on collateral vessel traits to further verify these collateral-independent loci. Among these 4 loci, the Civq4 and Civq7 loci appear to be truly collateral-independent. Based on strain-specific sequence variants and mRNA expression differences, we proposed Msr1 and Mtmr7 are the potential candidate genes of the Civq4 locus. Identification of the collateral-independent genetic factors will help to understand the genetic architecture, disease pathophysiology and potential therapeutic targets for of ischemic stroke</p> / Dissertation

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant association between the common c.454-397T/T genotype in ESR1 and ICH, remaining after adjustments for conventional stroke risk factors. The c.454-397T/T genotype also associated with increased systolic (SBP) and diastolic blood pressure (DBP). The combination of c.454- 397T/T and either hypertension, increased SBP, or increased DBP boosted this association substantially and significant synergistic effects on ICH risk between this genotype and increased blood pressure were demonstrated. In a second study, we found a similar association between the common OPG-1181C/C genotype and ICH. Cognitive impairments, including spatial memory and learning deficiencies, are common after stroke. Estrogens improve cognitive functions, including memory and learning processes, in postmenopausal women and ovariectomized rodents. Post-ischemic housing of rats in an enriched environment (EE) improves recovery of spatial memory and learning impairments. Both estrogen and EE induce neuroplasticity in the hippocampus. We hypothesized that 17β- estradiol combined with EE would accelerate recovery after experimental focal brain ischemia in ovariectomized rats and that such improvements could be related to expression of nerve growth factor-induced gene A (NGFI-A) in the hippocampus. Five to six weeks after middle cerebral artery occlusion, 17β-estradiol–treated rats housed in an EE showed significant improvements in cognitive function (i.e., shorter latency and path in the Morris water maze task) and significantly higher NGFI-A mRNA expression in bilateral cornu ammonis 1 (CA1) and ipsilateral dentate gyrus (DG) compared to placebo-treated animals in EE. In conclusion, we present evidence for the association between polymorphic variants in the ESR1 and TNFRS11B genes and ICH and show that 17β-estradiol in combination with EE accelerates cognitive functions in a rat stroke model, putatively through upregulation of NGFI-A in hippocampal subregions. These findings may contribute to an increased understanding of the underlying genetic etiology of ICH and may be informative for the primary prevention of this disease. They also provide hope for 17β-estradiol combined with early environmental enrichment as a novel therapeutic option following ischemic stroke.

Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.