Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

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Intracerebral hemorrhage (ICH) is a severe form of stroke, with a high mortality rate and often resulting in irreversible neurological deterioration. Although animal studies have provided insight into the etiology of the disease, many of the causative genes and mechanisms implicated in cerebral-vascular malformations are unknown. Treatment options remain ineffective. With the present models, the pathophysiological consequences of ICH can only be assessed in situ and after histological analysis. Furthermore, common deficiencies of the current models include the heterogeneity, low expression and low reproducibility of the desired phenotype. Hence, there is a requirement for novel approaches to model ICH pathogenesis. Zebrafish (Danio rerio) has gained recognition as a vertebrate model for stroke research. Through a combination of pharmacological blockers, metabolite rescue, genetic approaches, and confocal imaging analysis, I demonstrate a requirement for the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway in regulating developmental cerebral-vascular stabilization. A transient loss in HMGCR function induces ICH, characterised by progressive dilation of blood vessels, vascular permeability and vessel rupture. These effects are likely due to reduced prenylation of Rho GTPases, evidenced by morpholino-mediated blocking of the prenylation pathway and in vivo assessment of endothelial-specific localization of cdc42, a Rho GTPase family protein. These results are in conformity with recent clinical and experimental evidence. I have further shown that this model consistently replicates common pathoghysiological processes associated with ICH. The hemorrhages are associated with the disruption of the blood-brain barrier, vessel disintegration, hematoma expansion and edema into the adjacent brain regions. Also, enhanced apoptosis, activation of inflammatory mediators in the periphery of the hematoma, enriched heme oxygenase 1 (HO-1) expression and localised thrombosis were observed in these embryos. I show that the patterning and distribution of catecholaminergic neurons, response to sensory stimulus and swimming speed were impaired as a consequence of ICH. These results suggest that HMGCR contributes to cerebral-vascular stabilisation through Rho GTPase mediated-signalling and that zebrafish can serve as a powerful paradigm for the systemic analysis of the etiological and pathophysiological underpinnings of ICH and can help establish the basis for future studies into screening for putative therapeutics and elucidating mechanisms aiding functional recovery.

<strong>Aims:</strong> The purpose of the research presented in this thesis is to elucidate the mechanism of the stimulation of oxidative metabolism and contractile function that occurs in vascular smooth muscle during cerebral vasospasm (CV) after subarachnoid haemorrhage (SAH). The biochemical mechanisms leading to CV were investigated using an in vitro model of CV developed for this research. CSF (cerebrospinal fluid) from SAH patients at risk of vasospasm which stimulated oxygen consumption (CSF S ) was used to model vasospasm. The hypothesis is CSF_S contains a substance which stimulates tension generation over that of CSF_N ,(non-stimulatory cerebrospinal fluid) and also inhibits the myosin light chain phosphatase. <strong>Methods:</strong> The porcine carotid artery was used as a model for the human basilar artery. The rate of oxygen consumption (JO₂) was measured in response to CSF_S and tension generation was also examined. Various agents were used to treat or pretreat the tissue such as magnesium and andalpha;₁-adrenergic receptor agonists. Their effects on the CSF_S-induced stimulation were measured to study the mechanism of vasospasm. A myosin light chain phosphatase (MLCP) assay was developed to study the mechanisms leading to CV. <strong>Results and conclusion:</strong> Addition of CSF_S to the porcine carotid artery is a reliable and reproducible in vitro model of CV. Using this model, it was found that Mg⁺⁺ loading and andalpha;₁-adrenergic receptor agonists attenuated the vasospasm, but a non-specific endothelin antagonist had no effect. Acute addition of 12mM Mg⁺⁺ relaxed the tissue from a CSF_S induced contraction significantly and rendered the contraction rinsible. Okadaic acid (InM), a phosphatase inhibitor, had very similar effects to CSF_S because it stimulated JO₂ and slowed relaxation after a stretch. There was also significant inhibition of phosphatase caused by the CSF_S. Vasospasm appears to be caused by a combination of a contractile stimulus, and inhibition of MLCP activity.

Purpose: The principal aim of this project was to investigate the influence of clinical indices of injury severity and polymorphism of the apolipoprotein E gene upon the long-term physical, cognitive and emotional sequelae of traumatic brain injury and spontaneous subarachnoid haemorrhage. It was also intended to determine the extent to which changes occur in these sequelae beyond the initial six months post injury. Method: Sixty-two brain injury patients who had previously taken part in a neuropsychological assessment at six months post injury were traced and participated in a follow-up assessmens some 6-9 years subsequent to their injury. Separately, a group of 70 subarachnoid patients drawn from a consecutive series of neurosurgical admissions participated in a neuropsychological assessment at 14 months subsequent to their haemorrhage. In both studies, the assessment comprised a semi-structured interview and a battery of cognitive measures focusing principally upon memory and executive function tasks. A questionnaire including a range of standardised measures of anxiety, depression and quality of life was left with patients to be returned by post. Results: The ApoE e4 allele did not appear to influence recovery amongst these brain injury survivors, though there are suggestions that it may have an influence upon subgroups of patients. Amongst traumatic brain injury survivors, post-traumatic amnesia was a better predictor of functional or emotional outcome than consciousness based measures. However, consciousness based measures were more predictive of cognitive sequelae and low admission Glasgow Coma Scale was associated with continued improvement on information processing tasks. Other than on these tasks, there was little evidence of change between 6 months and 6-9 years post injury. Amongst the subarachnoid haemorrhage patients, Fisher Grade was found to be more predictive of subsequent Glasgow Outcome Scale and cognitive function than WFNS Grade or other clinical indices. Surviving aneurysmal patients had comparable levels of recovery to patients who had a negative angiogram. In both studies emotional sequelae, in particular anxiety-related difficulties, were found to be a principal factor in the functional outcome of some 40% of patients. Conclusions: Greater emphasis should be placed upon measures of post-traumatic amnesia as predictors of functional recovery in surviving patients. The use of an amnesia measure may also be warranted in studies of outcome following subarachnoid haemorrhage or other stroke. The ApoE e4 allele does not appear to have a strong influence upon functional recovery after brain injury across all patients, though it is possible that it interacts with other factors to influence recovery in subgroups. Greater emphasis should be placed upon the prevention and/or detection and treatment of mood disorders following brain injury. In the absence of intensive rehabilitative interventions, survivors of serious brain injury are more likely to deteriorate than to continue to recover beyond six months post injury.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Hyperthermia worsens outcome after ischemia. While it seems reasonable that hyperthermia would also worsen outcome after intracerebral hemorrhage (ICH), clinical studies attempting to find a causative relationship between hyperthermia and outcome have been inconclusive. We induced ICH with an injection of autologous whole blood (100 l) immediately followed by 3 hours of hyperthermia (HYPER; 39C) or normothermia (NORMO; 37C). Surprisingly, hyperthermia reduced edema at 72 hours, and improved outcome on day 3 post-ICH. There were no behavioural differences at later time points (day 11 and 32 post-ICH) and no difference in lesion volume (NORMO 14.0 mm3, HYPER: 14.5 mm3). Overall, this study does not support the hypothesis that mild, transient hyperthermia worsens outcome after ICH. Further research is needed to determine if more severe or prolonged hyperthermia worsens outcome, or if the cause of hyperthermia (e.g. infection) is important.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.