Dr Lucy Murtha

Introduction The prevalence of heart failure increases in the aging population and following myocardial infarction (MI), yet the extracellular matrix (ECM) remodeling underpinning the development of aging- and MI-associated cardiac fibrosis remains poorly understood. A link between inflammation and fibrosis in the heart has long been appreciated, but has mechanistically remained undefined. We investigated the expression of a novel protein, extracellular matrix protein 1 (ECM1) in the aging and infarcted heart. Methods Young adult (3-month old) and aging (18-month old) C57BL/6 mice were assessed. Young mice were subjected to left anterior descending artery-ligation to induce MI, or transverse aortic constriction (TAC) surgery to induce pressure-overload cardiomyopathy. Left ventricle (LV) tissue was collected early and late post-MI/TAC. Bone marrow cells (BMCs) were isolated from young healthy mice, and subject to flow cytometry. Human cardiac fibroblast (CFb), myocyte, and coronary artery endothelial & smooth muscle cell lines were cultured; human CFbs were treated with recombinant ECM1. Primary mouse CFbs were cultured and treated with recombinant angiotensin-II or TGF-ß1. Immunoblotting, qPCR and mRNA fluorescent in-situ hybridization (mRNA-FISH) were conducted on LV tissue and cells. Results ECM1 expression was upregulated in the aging LV, and in the infarct zone of the LV early post-MI. No significant differences in ECM1 expression were found late post-MI or at any time-point post-TAC. ECM1 was not expressed in any resident cardiac cells, but ECM1 was highly expressed in BMCs, with high ECM1 expression in granulocytes. Flow cytometry of bone marrow revealed ECM1 expression in large granular leucocytes. mRNA-FISH revealed that ECM1 was indeed expressed by inflammatory cells in the infarct zone at day-3 post-MI. ECM1 stimulation of CFbs induced ERK1/2 and AKT activation and collagen-I expression, suggesting a pro-fibrotic role. Conclusions ECM1 expression is increased in ageing and infarcted hearts but is not expressed by resident cardiac cells. Instead it is expressed by bone marrow-derived granulocytes. ECM1 is sufficient to induce cardiac fibroblast stimulation in vitro. Our findings suggest ECM1 is released from infiltrating inflammatory cells, which leads to cardiac fibroblast stimulation and fibrosis in aging and MI. ECM1 may be a novel intermediary between inflammation and fibrosis.

Ischaemia/excitotoxicity produces persistent activation of CaMKII (Ca2+-calmodulin stimulated protein kinase II) that initiates cell death. This study investigated the involvement of CaMKII phosphorylation at T286 and T253 in producing this persistent activation. In T286A-aCaMKII transgenic mice that lack the ability to phosphorylate aCaMKII at T286, transient occlusion of the middle cerebral artery for 90¿min resulted in no significant difference in infarct size compared to normal littermate controls. Overexpression of the phospho-mimic mutant T286D-aCaMKII in differentiated neuroblastoma cell lines did not enhance excitotoxicity-induced cell death compared to overexpression of wild type aCaMKII. By contrast, overexpression of the phospho-mimic mutant T253D-aCaMKII significantly enhanced excitotoxicity-induced cell death whereas overexpression of the phospho-null mutant T253V-aCaMKII produced no enhancement. These results indicate that T286 phosphorylation does not play a significant role in ischaemia/excitotoxicity induced CaMKII-mediated cell death and suggest that T253 phosphorylation is required to produce the persistent activation of CaMKII involved in ischaemia/excitotoxicity induced cell death.

In both the human and animal literature, it has largely been assumed that edema is the primary cause of intracranial pressure (ICP) elevation after stroke and that more edema equates to higher ICP. We recently demonstrated a dramatic ICP elevation 24 hours after small ischemic strokes in rats, with minimal edema. This ICP elevation was completely prevented by short-duration moderate hypothermia soon after stroke. Here, our aims were to determine the importance of edema in ICP elevation after stroke and whether mild hypothermia could prevent the ICP rise. Experimental stroke was performed in rats. ICP was monitored and short-duration mild (35 °C) or moderate (32.5 °C) hypothermia, or normothermia (37 °C) was induced after stroke onset. Edema was measured in three studies, using wet-dry weight calculations, T 2-weighted magnetic resonance imaging, or histology. ICP increased 24 hours after stroke onset in all normothermic animals. Short-duration mild or moderate hypothermia prevented this rise. No correlation was seen between ¿ICP and edema or infarct volumes. Calculated rates of edema growth were orders of magnitude less than normal cerebrospinal fluid production rates. These data challenge current concepts and suggest that factors other than cerebral edema are the primary cause of the ICP elevation 24 hours after stroke onset.

Recent human imaging studies indicate that reduced blood flow through pial collateral vessels ('collateral failure') is associated with late infarct expansion despite stable arterial occlusion. The cause for 'collateral failure' is unknown. We recently showed that intracranial pressure (ICP) rises dramatically but transiently 24 hours after even minor experimental stroke. We hypothesized that ICP elevation would reduce collateral blood flow. First, we investigated the regulation of flow through collateral vessels and the penetrating arterioles arising from them during stroke reperfusion. Wistar rats were subjected to intraluminal middle cerebral artery (MCA) occlusion (MCAo). Individual pial collateral and associated penetrating arteriole blood flow was quantified using fluorescent microspheres. Baseline bidirectional flow changed to MCA-directed flow and increased by >450% immediately after MCAo. Collateral diameter changed minimally. Second, we determined the effect of ICP elevation on collateral and watershed penetrating arteriole flow. Intracranial pressure was artificially raised in stepwise increments during MCAo. The ICP increase was strongly correlated with collateral and penetrating arteriole flow reductions. Changes in collateral flow post-stroke appear to be primarily driven by the pressure drop across the collateral vessel, not vessel diameter. The ICP elevation reduces cerebral perfusion pressure and collateral flow, and is the possible explanation for 'collateral failure' in stroke-in-progression.

Background: Perfusion computed tomography is becoming more widely used as a clinical imaging tool to predict potentially salvageable tissue (ischemic penumbra) after ischemic stroke and guide reperfusion therapies. Aims: The study aims to determine whether there are important changes in perfusion computed tomography thresholds defining ischemic penumbra and infarct core over time following stroke. Methods: Permanent middle cerebral artery occlusion was performed in adult outbred Wistar rats (n=6) and serial perfusion computed tomography scans were taken every 30 mins for 2h. To define infarction thresholds at 1h and 2h post-stroke, separate groups of rats underwent 1h (n=6) and 2h (n=6) of middle cerebral artery occlusion followed by reperfusion. Infarct volumes were defined by histology at 24h. Co-registration with perfusion computed tomography maps (cerebral blood flow, cerebral blood volume, and mean transit time) permitted pixel-based analysis of thresholds defining infarction, using receiver operating characteristic curves. Results: Relative cerebral blood flow was the perfusion computed tomography parameter that most accurately predicted penumbra (area under the curve=0·698) and also infarct core (area under the curve=0·750). A relative cerebral blood flow threshold of <75% of mean contralateral cerebral blood flow most accurately predicted penumbral tissue at 0·5h (area under the curve=0·660), 1h (area under the curve=0·659), 1·5h (area under the curve=0·636), and 2h (area under the curve=0·664) after stroke onset. A relative cerebral blood flow threshold of <55% of mean contralateral most accurately predicted infarct core at 1h (area under the curve=0·765) and at 2h (area under the curve=0·689) after middle cerebral artery occlusion. Conclusions: The data provide perfusion computed tomography defined relative cerebral blood flow thresholds for infarct core and ischemic penumbra within the first two hours after experimental stroke in rats. These thresholds were shown to be stable to define the volume of infarct core and penumbra within this time window.

Background: Early recanalization of occluded vessels in stroke is closely associated with improved clinical outcome. Microbubble-enhanced sonothrombolysis is a promising therapy to improve recanalization rates and reduce the time to recanalization. Testing any thrombolytic therapy requires a model of thromboembolic stroke, but to date these models have been highly variable with regards to clot stability. Here, we developed a model of thromboembolic stroke in rats with site-specific delivery of platelet-rich clots (PRC) to the main stem of the middle cerebral artery (MCA). This model was used in a subsequent study to test microbubble-enhanced sonothrombolysis. Methods: In Study 1 we investigated spontaneous recanalization rates of PRC in vivo over 4 hours and measured infarct volumes at 24 hours. In Study 2 we investigated tPA-mediated thrombolysis and microbubble-enhanced sonothrombolysis in this model. Results: Study 1 demonstrated stable occlusion out to 4 hours in 5 of 7 rats. Two rats spontaneously recanalized at 40 and 70 minutes post-embolism. Infarct volumes were not significantly different in recanalized rats, 43.93 ± 15.44% of the ischemic hemisphere, compared to 48.93 ± 3.9% in non-recanalized animals (p = 0.7). In Study 2, recanalization was not observed in any of the groups post-treatment. Conclusions: Site specific delivery of platelet rich clots to the MCA origin resulted in high rates of MCA occlusion, low rates of spontaneous clot lysis and large infarction. These platelet rich clots were highly resistant to tPA with or without microbubble-enhanced sonothrombolysis. This resistance of platelet rich clots to enhanced thrombolysis may explain recanalization failures clinically and should be an impetus to better clot-type identification and alternative recanalization methods.

Background: Intracranial pressure elevation, peaking three to seven post-stroke is well recognized following large strokes. Data following small-moderate stroke are limited. Therapeutic hypothermia improves outcome after cardiac arrest, is strongly neuroprotective in experimental stroke, and is under clinical trial in stroke. Hypothermia lowers elevated intracranial pressure; however, rebound intracranial pressure elevation and neurological deterioration may occur during rewarming. Hypotheses: (1) Intracranial pressure increases 24h after moderate and small strokes. (2) Short-duration hypothermia-rewarming, instituted before intracranial pressure elevation, prevents this 24h intracranial pressure elevation. Methods: Long-Evans rats with two hour middle cerebral artery occlusion or outbred Wistar rats with three hour middle cerebral artery occlusion had intracranial pressure measured at baseline and 24h. Wistars were randomized to 2·5h hypothermia (32·5°C) or normothermia, commencing 1h after stroke. Results: In Long-Evans rats (n=5), intracranial pressure increased from 10·9±4·6mmHg at baseline to 32·4±11·4mmHg at 24h, infarct volume was 84·3±15·9mm3. In normothermic Wistars (n=10), intracranial pressure increased from 6·7±2·3mmHg to 31·6±9·3mmHg, infarct volume was 31·3±18·4mm3. In hypothermia-treated Wistars (n=10), 24h intracranial pressure did not increase (7·0±2·8mmHg, P<0·001 vs. normothermia), and infarct volume was smaller (15·4±11·8mm3, P<0·05). Conclusions: We saw major intracranial pressure elevation 24h after stroke in two rat strains, even after small strokes. Short-duration hypothermia prevented the intracranial pressure rise, an effect sustained for at least 18h after rewarming. The findings have potentially important implications for design of future clinical trials. © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization.

Background: Many aspects of CSF dynamics are poorly understood due to the difficulties involved in quantification and visualization. In particular, there is debate surrounding the route of CSF drainage. Our aim was to quantify CSF flow, volume, and drainage route dynamics in vivo in young and aged spontaneously hypertensive rats (SHR) using a novel contrast-enhanced computed tomography (CT) method.Methods: ICP was recorded in young (2-5 months) and aged (16 months) SHR. Contrast was administered into the lateral ventricles bilaterally and sequential CT imaging was used to visualize the entire intracranial CSF system and CSF drainage routes. A customized contrast decay software module was used to quantify CSF flow at multiple locations.Results: ICP was significantly higher in aged rats than in young rats (11.52 ± 2.36 mmHg, versus 7.04 ± 2.89 mmHg, p = 0.03). Contrast was observed throughout the entire intracranial CSF system and was seen to enter the spinal canal and cross the cribriform plate into the olfactory mucosa within 9.1 ± 6.1 and 22.2 ± 7.1 minutes, respectively. No contrast was observed adjacent to the sagittal sinus. There were no significant differences between young and aged rats in either contrast distribution times or CSF flow rates. Mean flow rates (combined young and aged) were 3.0 ± 1.5 µL/min at the cerebral aqueduct; 3.5 ± 1.4 µL/min at the 3rd ventric= and 2.8 ± 0.9 µL/min at the 4th ventricle. Intracranial CSF volumes (and as percentage total brain volume) were 204 ± 97 µL (8.8 ± 4.3%) in the young and 275 ± 35 µL (10.8 ± 1.9%) in the aged animals (NS).Conclusions: We have demonstrated a contrast-enhanced CT technique for measuring and visualising CSF dynamics in vivo. These results indicate substantial drainage of CSF via spinal and olfactory routes, but there was little evidence of drainage via sagittal sinus arachnoid granulations in either young or aged animals. The data suggests that spinal and olfactory routes are the primary routes of CSF drainage and that sagittal sinus arachnoid granulations play a minor role, even in aged rats with higher ICP. © 2014 Murtha et al.; licensee BioMed Central Ltd.