Damian McLeod

Intracranial pressure elevation reduces flow through collateral vessels and the penetrating arterioles they supply. A possible explanation for ‘collateral failure' and infarct expansion after ischemic stroke

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 4450% 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.

Intracranial pressure elevation after ischemic stroke in rats: cerebral edema is not the only cause, and short-duration mild hypothermia is a highly effective preventive therapy

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, T2-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 ΔICP 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.

Establishing a Rodent Stroke Perfusion Computed Tomography Model

Brain computed tomography perfusion imaging in acute stroke may help guide therapy. However, the perfusion thresholds defining potentially salvageable (penumbra) and irreversibly injured (infarct core) tissue require further validation. The aim of this study was to validate infarct core and penumbra perfusion thresholds in a rodent stroke model by developing and optimising perfusion computed tomography imaging, performing serial scanning and correlating scans with final histology. Stroke was induced in male Wistar rats (n = 17) using the middle cerebral artery thread-occlusion method. Perfusion computed tomography scans were obtained immediately pre- and postocclusion, and every 30 min for 2·5 h. Histological changes of infarction were assessed after 24 h. High-quality maps of cerebral blood flow and cerebral blood volume were generated at multiple coronal planes after optimisation of contrast injection and scanning parameters. The prestroke absolute cerebral blood flow and cerebral blood volume values (mean ± SD) were 158·2 ± 49·94 ml/min per 100 g and 5·6 ± 1·13 ml per 100 g, respectively. Cerebral blood flow was significantly lower in the infarct region of interest than the contralateral hemisphere region of interest at all time points, except the 0·5 h postocclusion time point. However, cerebral blood volume was only significantly lower in the infarct region of interest than the contralateral hemisphere region of interest at the 1 h and the 1·5 h time points (postocclusion). This study has demonstrated for the first time the feasibility of performing perfusion computed tomography in the most commonly used animal model of stroke. The model will allow definitive studies to determine optimal thresholds and the reliability of perfusion computed tomography measures for infarct core and penumbra.

Short-Duration Hypothermia after Ischemic Stroke Prevents Delayed Intracranial Pressure Rise:

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 24 h after moderate and small strokes. (2) Short-duration hypothermia-rewarming, instituted before intracranial pressure elevation, prevents this 24 h 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 24 h. Wistars were randomized to 2·5 h hypothermia (32·5°C) or normothermia, commencing 1 h after stroke. Results In Long-Evans rats (n = 5), intracranial pressure increased from 10·9 ± 4·6 mmHg at baseline to 32·4 ± 11·4 mmHg at 24 h, infarct volume was 84·3 ± 15·9 mm3. In normothermic Wistars (n = 10), intracranial pressure increased from 6·7 ± 2·3 mmHg to 31·6 ± 9·3 mmHg, infarct volume was 31·3 ± 18·4 mm3. In hypothermia-treated Wistars (n = 10), 24 h intracranial pressure did not increase (7·0 ± 2·8 mmHg, P < 0·001 vs. normothermia), and infarct volume was smaller (15·4 ± 11·8 mm3, P < 0·05). Conclusions We saw major intracranial pressure elevation 24 h after stroke in two rat strains, even after small strokes. Short-duration hypothermia prevented the intracranial pressure rise, an effect sustained for at least 18 h after rewarming. The findings have potentially important implications for design of future clinical trials.

Inadvertent Occlusion of the Anterior Choroidal Artery Explains Infarct Variability in the Middle Cerebral Artery Thread Occlusion Stroke Model

Intraluminal occlusion of the middle cerebral artery (MCAo) in rodents is perhaps the most widely used model of stroke, however variability of infarct volume and the ramifications of this on sample sizes remains a problem, particularly for preclinical testing of potential therapeutics. Our data and that of others, has shown a dichotomous distribution of infarct volumes for which there had previously been no clear explanation. When studying perfusion computed tomography cerebral blood volume (CBV) maps obtained during intraluminal MCAo in rats, we observed inadvertent occlusion of the anterior choroidal artery (AChAo) in a subset of animals. We hypothesized that the combined occlusion of the MCA and AChA may be a predictor of larger infarct volume following stroke. Thus, we aimed to determine the correlation between AChAo and final infarct volume in rats with either temporary or permanent MCA occlusion (1 h, 2 h, or permanent MCAo). Outbred Wistar rats (n = 28) were imaged prior to and immediately following temporary or permanent middle cerebral artery occlusion. Presence of AChAo on CBV maps was shown to be a strong independent predictor of 24 h infarct volume (β = 0.732, p <0.001). This provides an explanation for the previously observed dichotomous distribution of infarct volumes. Interestingly, cortical infarct volumes were also larger in rats with AChAo, although the artery does not supply cortex. This suggests an important role for perfusion of the MCA territory beyond the proximal occlusion through AChA-MCA anastomotic collateral vessels in animals with a patent AChAo. Identification of combined MCAo and AChAo will allow other investigators to tailor their stroke model to reduce variability in infarct volumes, improve statistical power and reduce sample sizes in preclinical stroke research.

‘Salvaged’ Stroke Ischaemic Penumbra Shows Significant Injury: Studies with the Hypoxia Tracer FMISO

The degree of cellular injury within the stroke ischaemic penumbra is controversial. Clinical and experimental studies using the hypoxia tracer fluoromisonidazole (FMISO) have shown retention of this tracer in the penumbra, but cellular outcome has not been well characterised. We hypothesised that macroscopically intact FMISO-retaining penumbral tissues would show evidence of microscopic injury, and that no FMISO retention would be seen in the infarct core. To determine the distribution of FMISO retention, a tritium-labelled tracer (hydrogen-3 FMISO ([3H]FMISO)) was administered 5 minutes after induction of 2-hour temporary middle cerebral artery occlusion. Coregistered brain histology and autoradiography at 24 hours revealed marked retention of FMISO within the infarct. However, 48% of the FMISO-retaining tissue was not infarcted. Within this noninfarcted tissue, only 27% (17 of 64) of sampled regions showed no evidence of neuronal loss, whereas 44% (28 of 64) showed injury to >50% of neurons within the sample. To determine whether FMISO retention occurred after the tissue was already committed to infarction, FMISO was administered 4 to 6 hours after the onset of permanent vessel occlusion. Intense FMISO retention was consistently seen throughout the infarct core. In conclusion, FMISO retention occurs both within the ischaemic penumbra and within the early infarct core. Most penumbral tissues show evidence of selective cellular injury.

Perfusion computed tomography thresholds defining ischemic penumbra and infarct core: studies in a rat stroke model.

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 2 h. To define infarction thresholds at 1 h and 2 h post-stroke, separate groups of rats underwent 1 h (n = 6) and 2 h (n = 6) of middle cerebral artery occlusion followed by reperfusion. Infarct volumes were defined by histology at 24 h. 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 0·5 h (area under the curve = 0·660), 1 h (area under the curve = 0·659), 1·5 h (area under the curve = 0·636), and 2 h (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 1 h (area under the curve = 0·765) and at 2 h (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.

Immunosuppression for in vivo research: state-of-the-art protocols and experimental approaches

Almost every experimental treatment strategy using non-autologous cell, tissue or organ transplantation is tested in small and large animal models before clinical translation. Because these strategies require immunosuppression in most cases, immunosuppressive protocols are a key element in transplantation experiments. However, standard immunosuppressive protocols are often applied without detailed knowledge regarding their efficacy within the particular experimental setting and in the chosen model species. Optimization of such protocols is pertinent to the translation of experimental results to human patients and thus warrants further investigation. This review summarizes current knowledge regarding immunosuppressive drug classes as well as their dosages and application regimens with consideration of species-specific drug metabolization and side effects. It also summarizes contemporary knowledge of novel immunomodulatory strategies, such as the use of mesenchymal stem cells or antibodies. Thus, this review is intended to serve as a state-of-the-art compendium for researchers to refine applied experimental immunosuppression and immunomodulation strategies to enhance the predictive value of preclinical transplantation studies.

Neural control of the bronchial circulation

A distinction may be drawn between studies that define potential mechanisms of control, those that define components of control systems, and those that determine the priority of controls in the integrated system. It is the priority controls that determine survival in the environment. These different kinds of studies are necessary and complementary. In the case of the bronchial circulation, we continue with an era of defining potential mechanisms and component systems. These have been excellently reviewed by Godden (1990) and by Coleridge & Coleridge (1994). However, we have a long way to go before we establish priority neural controls in the integrated system. Cogent examples in the bronchial circulation might be the interaction at rest between neural controls and tonic NO released from vascular endothelium in determining basal autonomic tone. Another might be the autonomic controllers of bronchial resistance vessels reflexly evoked at the onset of exercise, and how these controls are modified by central temperature regulation as exercise continues. These kinds of data are necessary to understand evolutionary mechanisms that protect the organism from untoward environmental influence, and that point to support therapies for systems breakdown.

Intracranial Pressure and Collateral Blood Flow

Leptomeningeal collateral vessels, linking the 3 major arterial territories over the surface of the brain, have been recognized for >140 years.1 More widespread use of advanced clinical imaging in the past decade has led to increasing recognition of the key importance of collaterals in ischemic stroke outcome.2 However, recent studies from several groups indicate that failure of initially good collateral supply is a key feature of patients with delayed infarct expansion.3,4 This clinically challenging problem typically occurs in the first 1 to 2 days after hospital admission in patients with initially mild stroke symptoms. Rethrombosis of reperfused vessels was previously thought to be the likely cause of delayed infarct expansion in most patients. However, this theory is not supported by more recent evidence from imaging studies. Despite the important recent observations, there is limited understanding of the dynamic control of the collateral circulation, in particular, the cause of collateral blood flow failure. In this article, we will discuss recent observations from our experimental stroke model, indicating a dramatic increase in intracranial pressure (ICP) occurring around 24 hours after onset of even small stroke.5,6 We have also shown a significant linear reduction of collateral blood flow in response to progressive ICP elevation.7 We believe that a similar transient ICP elevation occurring during the first 1 to 2 days post stroke is a likely mechanism to explain delayed infarct expansion in patients with minor stroke. Perhaps surprisingly, we can find no published data on ICP at 24 hours in patients with minor stroke. The preclinical findings suggest that gathering such data should be a priority. ### Human Stroke There is a strong association between the extent of leptomeningeal collaterals and clinical stroke outcome. Initial studies using digital subtraction angiography permitted direct visualization of collateral vessels and …