Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodacetic acid) and ischemia was examined, using 5-50 mm butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDM's enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.

Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN−/− mice at 14 d. In female VTN−/− mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN−/− mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

Large aspiny neurons are the only non-GABAergic neurons in the striatum. After transient cerebral ischemia, large aspiny neurons survive while medium spiny neurons die. Previous studies have shown that differential changes in the intrinsic membrane properties and excitatory synaptic transmission play a role in this selective vulnerability. However, the role of inhibitory synaptic transmission in this selective vulnerability is still unknown. Since inhibitory tone is very important in the control of neuronal excitability, the present study is aimed at examining if there are any changes in inhibitory synaptic transmission in striatal neurons after ischemia and the possible mechanisms. We also examined if facilitation of inhibitory synaptic transmission by muscimol could attenuate ischemic neuronal injury in the striatum after ischemia. Results from this study will improve the understanding of the mechanisms underlying selective neuronal injury after transient cerebral ischemia. We hope this study could contribute to the translational studies for the stroke patients after cardiac arrest. / Indiana University-Purdue University Indianapolis (IUPUI) / In the striatum, large aspiny (LA) interneurons survive transient cerebral ischemia while medium spiny (MS) neurons die. Excitotoxicity is believed to be the major cause for neuronal death after ischemia. Since inhibitory tone plays an important role in the control of neuronal excitability, the present study is aimed at examining if there are any changes in inhibitory synaptic transmission in striatal neurons after ischemia and the possible mechanisms. Transient forebrain ischemia was induced in male Wistar rats using the four-vessel occlusion method. Inhibitory postsynaptic currents (IPSCs) were evoked intrastriatally and whole-cell voltage-clamp recording was performed on striatal slices. The expression of glutamate decarboxylase65 (GAD65) was analyzed using immunohistochemical studies and Western blotting. Muscimol (a specific GABAA receptor agonist) was injected intraperitoneally to the rats (1 mg/kg) to observe ischemic damage, evaluated by counting the survived cells in the striatum after hematoxylin & eosin (HE) staining. The amplitudes of evoked IPSCs were significantly increased in LA neurons while depressed in MS neurons after ischemia. This enhancement was due to the increase of presynaptic release. Muscimol (1 μM) presynaptically facilitated inhibitory synaptic transmission in LA neurons at 24 h after ischemia. The optical density of GAD65-positive terminals and the number of GAD65-positive puncta was significantly increased in the striatum at both 1 day and 3 days after ischemia. Consistently, data from western blotting suggested an increased expression of GAD65 in the striatum after ischemia. For the rats treated with muscimol, the number of survived cells in the striatum was greatly increased compared to the non-treatment group. The present study demonstrates an enhancement of inhibitory synaptic transmission in LA neurons after ischemia, which is contributed by two mechanisms. One is the increased presynaptic release of GABA mediated by presynaptic GABAA receptors. The other is the increased expression of GAD. Facilitation of inhibitory synaptic transmission by muscimol protects striatal neurons against ischemia. Therefore, the enhancement of inhibitory synaptic transmission might reduce excitotoxicity and contribute to the selective survival of LA neurons after ischemia.

No description available.

Neonatal asphyxia is the third largest cause of neonatal mortality in Vietnam, and many of the surviving patients suffer from permanent brain damage. A cooling mattress used for treatment of infants suffering from asphyxia has been shown to have problems with providing a stable body temperature for the patients. The main purpose of this project is to assess potential problems with routines and the use of the most central technical equipment used to treat newborns suffering from asphyxia, as well as constructing a phantom to simulate a newborn infant in the process of cooling. The purpose of the simulation is to investigate new methods of using the cooling mattress to achieve a more stable body temperature when cooling the patient. The project was done at Phu San Hostpial and Vietnam National Children's Hospital in Hanoi, Vietnam. The technical evaluation was conducted with observations, interviews and a question form. The results showed that there were issues with the sensors connected to the ventilators, noise level from alarms and routines concerning sanitation. The phantom was based on a water bottle, an interest heater and a pump to mix the water. It proved not possible to construct a phantom that met the necessary criteria. / Asfyxi hos nyfödda är den tredje största orsaken till neonatal dödlighet i Vietnam, och många av de överlevande får permanenta hjärnskador. En nedkylningsmadrass som används till behandling av nyfödda med asfyxi har visat sig ha problem med att hålla patientens kroppstemperatur stabil. Arbetets huvudsakliga syfte är att undersöka potentiella problem med rutiner och användningen av den viktigaste tekniken vid behandling av nyfödda som lider av asfyxi, samt även konstruera en fantom som ska kunna simulera en nyfödd vid nedkylning. Simuleringen har som syfte att undersöka nya användningsmetoder av nedkylningsmadrassen för att uppnå en mer stabil kroppstemperatur hos patienten. Arbetet gjordes vid Phu San Hospital, och National Children’s Hospital i Hanoi, Vietnam. Utvärderingen av teknikens användning gjordes via observationer, intervjuer och ett frågeformulär. Resultaten sammanställdes för att hitta potentiella förbättringar till teknikens användning. Det framkom problem med sensorerna kopplat till ventilatorerna, ljudnivån från alarmer och rutiner kring sanitering. Fantomet baserades på en vattenflaska, en doppvärmare och en pump som blandade vattnet. Det visade sig inte vara möjligt att konstruera en fantom som uppfyllde alla kriterier med de resurser som var tillgängliga.

Ischemic colitis is a known complication of abdominal aortic aneurysm (AAA) repair. There is still no established consensus regarding the individual significance of factors related to this phenomenon. We detail the hospital course of a patient who unexpectedly developed acute colonic ischemia following open AAA repair. The pathophysiology, diagnostic modalities, potential preventative measures, and effect on patient morbidity and mortality will be presented. Reviewing the current literature, this will focus on the reported statistical importance of various risk factors including the incidence following traditional open repair and endovascular aneurysm repair.

Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Znfinger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure. A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ. Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM's resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4'PBA prevented high glucoseinduced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3'methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.

Balali_Pargol_MSc thesis_Neuroscience department_2023Sep / Background: Cerebral microbleeds are asymptomatic neuroimaging markers of small vessel disease (SVD), visualized as small hypointensities on blood-sensitive magnetic resonance imaging (MRI) sequences. Patients with ischemic stroke and microbleeds are at a higher risk of future ischemic stroke and intracranial hemorrhage. Antithrombotic therapies, the mainstay treatment of secondary stroke prevention, are associated with an increased risk of bleeding. This raises concerns surrounding the net benefit of antithrombotic therapies in these hemorrhage-prone patients. The overarching aim of this thesis is to determine the safety of antithrombotic treatments in patients with hemorrhage-prone SVD marked by microbleeds on MRI or prior intracerebral hemorrhage (ICH). I aimed to characterize the association between baseline microbleeds and the risk of future clinical outcomes in patients with ischemic stroke and whether there exists treatment effect modification of different anticoagulants on clinical outcomes according to microbleeds presence, location, and number. Methods: We performed post hoc analyses on two multicenter previously conducted randomized trials in patients with non-cardioembolic ischemic stroke. For the PACIFIC-STROKE trial, we used multivariable regression models to determine the contribution of microbleeds to the risk of new microbleeds, hemorrhagic transformation (HT), ischemic stroke, intracranial hemorrhage, and death. We assessed the treatment effect of asundexian, a factor XIa inhibitor, vs. placebo on these clinical outcomes, stratified by microbleeds presence, location, and number. I was trained on standardized rating of microbleeds on MRI, achieved excellent interrater reliability, and rated all DATAS-II participant MRIs. I used multivariable logistic regression models to identify the association between microbleeds and HT and 90-day excellent functional outcome. I assessed the interaction between treatment with dabigatran, a direct thrombin inhibitor, vs. aspirin and microbleeds for these outcomes. Separately, I performed a review of the literature and wrote an editorial discussing the optimum timing of antiplatelet re-initiation after ICH. Results: The PACIFIC-STROKE post hoc analyses showed that microbleeds are associated with a 1.6-fold and 4.4-fold higher risk of HT and new microbleeds, respectively. The DATAS-II exploratory analyses demonstrated no association between the risk of outcomes and microbleeds presence. We found no interaction between treatment assignment and microbleed presence for any of the clinical outcomes investigated in either of these studies. Based on the totality of evidence, we concluded that early resumption of antiplatelets in ICH survivors is likely to be safe. Conclusion: Our findings do not support existing concerns surrounding the use of anticoagulants in patients with acute ischemic stroke and microbleeds on MRI, nor for the early resumption of antiplatelets in ICH survivors. / Thesis / Master of Science (MSc) / Diseases of small brain blood vessels can lead to strokes due to blockage or bleeding. Small, asymptomatic brain bleeds on MRIs (microbleeds) are common among affected patients. Patients with clot-induced stroke and microbleeds have a higher risk of both types of strokes. Blood thinners are standard treatments to prevent future clotting events after clot-induced stroke. However, their potential to increase the risk of brain bleeding has raised concerns regarding their use in patients with microbleeds or bleeding-induced stroke. We assessed information from two large, previously completed randomized trials to evaluate the safety of strong blood thinners (anticoagulants) in patients with clot-induced stroke and microbleeds. Additionally, we evaluated the risk vs. benefit of restarting milder blood thinners (antiplatelets) early after bleeding-induced stroke. Bleeding was more prevalent in patients with microbleeds; however, the effect of the anticoagulants tested on bleeding outcomes was not modified by microbleed presence. Overall, our findings suggest that blood thinners are safe in patients with clot-induced stroke and microbleeds, and that early resumption of antiplatelets seems safe in patients with bleeding-induced stroke.

Stroke is a devastating neurological disease with limited treatment opportunities. Recent advances in understanding the underlying pathogenesis of cerebral ischemia support the involvement of multiple biochemical pathways in the development of the ischemic injury. The work reported in this thesis was undertaken to investigate the hypothesis that fenamate NSAIDs have neuroprotective properties against ischemic stroke and to explore the underlying mechanisms for any efficacy. Fenamates are non-selective inhibitors of cyclooxygenases. In addition, fenamates are antagonists of non-selective cation channels, subtype-selective modulators of GABA A receptors, weak inhibitors of glutamate receptors and activators of some potassium channels, all potentially important in the pathogenesis of ischemic stroke. Mefenamic acid, a prototype fenamate, administered by intracerebroventricular (ICV) infusion, reduced the ischemic brain damage and edema volume in the middle cerebral artery occlusion model in male rats. Consistent with these results, systemic administration of mefenamic acid, by multiple intravenous injections, also reduced the ischemic damage and edema volume measured by morphometric analysis and as a function of brain water content. These are the first set of experiments to demonstrate a significant neuroprotective effect of a fenamate against an in vivo model of ischemic stroke. In vitro , mefenamic acid was also shown to reduce glutamate-evoked cell death ( excitotoxicity ) in a concentration-dependent manner in cultured embryonic rat hippocampal neurons. Similarly, selected other fenamates also reduced excitotoxicity in the rank order (from highest): mefenamic acid > flufenamic acid ≥ meclofenamic acid > niflumic acid supporting the idea that this is a drug class action. Three pharmacological properties of fenamates, cyclooxygenase inhibition, GABA A receptor modulation and potassium channel activation were investigated as the potential mechanism(s) for the neuroprotective effects of mefenamic acid against excitotoxicity. The experimental results suggest that these are not the primary mechanisms for neuroprotective effects of mefenamic acid against glutamate-evoked cell death. Collectively, these data support the hypothesis that fenamate NSAIDs are neuroprotective against experimental models of cerebral ischemia and suggest they should be further investigated as potential pharmacological treatments for stroke.