Malignant infarction of the middle cerebral artery in a porcine model. A pilot study

Animal models; Central nervous system; InfarctionModels animals; Sistema nerviós central; InfartModelos animales; Sistema nervioso central; InfartoBackground and purpose Interspecies variability and poor clinical translation from rodent studies indicate that large gyrencephalic animal stroke models are urgently needed. We present a proof-of-principle study describing an alternative animal model of malignant infarction of the middle cerebral artery (MCA) in the common pig and illustrate some of its potential applications. We report on metabolic patterns, ionic profile, brain partial pressure of oxygen (PtiO2), expression of sulfonylurea receptor 1 (SUR1), and the transient receptor potential melastatin 4 (TRPM4). Methods A 5-hour ischemic infarct of the MCA territory was performed in 5 2.5-to-3-month-old female hybrid pigs (Large White x Landrace) using a frontotemporal approach. The core and penumbra areas were intraoperatively monitored to determine the metabolic and ionic profiles. To determine the infarct volume, 2,3,5-triphenyltetrazolium chloride staining and immunohistochemistry analysis was performed to determine SUR1 and TRPM4 expression. Results PtiO2 monitoring showed an abrupt reduction in values close to 0 mmHg after MCA occlusion in the core area. Hourly cerebral microdialysis showed that the infarcted tissue was characterized by reduced concentrations of glucose (0.03 mM) and pyruvate (0.003 mM) and increases in lactate levels (8.87mM), lactate-pyruvate ratio (4202), glycerol levels (588 μM), and potassium concentration (27.9 mmol/L). Immunohistochemical analysis showed increased expression of SUR1-TRPM4 channels. Conclusions The aim of the present proof-of-principle study was to document the feasibility of a large animal model of malignant MCA infarction by performing transcranial occlusion of the MCA in the common pig, as an alternative to lisencephalic animals. This model may be useful for detailed studies of cerebral ischemia mechanisms and the development of neuroprotective strategies.The Neurotraumatology and Neurosurgery Research Unit is supported by a grant from the Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (SGR 2014-844). This work has been supported in part by the Fondo de Investigación Sanitaria (Instituto de Salud Carlos III) with grant FIS PI11/00700, which was co-financed by the European Regional Development Fund (ERDF) and awarded to Dr. J. Sahuquillo. A. Sánchez-Guerrero is the recipient of a personal pre-doctoral grant from the Instituto de Salud Carlos III (grant number grant number FI12/00074)

Preclinical stroke research - advantages and disadvantages of the most common rodent models of focal ischaemia

This review describes the most commonly used rodent models and outcome measures in preclinical stroke research and discusses their strengths and limitations. Most models involve permanent or transient middle cerebral artery occlusion with therapeutic agents tested for their ability to reduce stroke-induced infarcts and improve neurological deficits. Many drugs have demonstrated preclinical efficacy but, other than thrombolytics, which restore blood flow, none have demonstrated efficacy in clinical trials. This failure to translate efficacy from bench to bedside is discussed alongside achievable steps to improve the ability of preclinical research to predict clinical efficacy: (i) Improvements in study quality and reporting. Study design must include randomization, blinding and predefined inclusion/exclusion criteria, and journal editors have the power to ensure statements on these and mortality data are included in preclinical publications. (ii) Negative and neutral studies must be published to enable preclinical meta-analyses and systematic reviews to more accurately predict drug efficacy in man. (iii) Preclinical groups should work within networks and agree on standardized procedures for assessing final infarct and functional outcome. This will improve research quality, timeliness and translational capacity. (iv) Greater uptake and improvements in non-invasive diagnostic imaging to detect and study potentially salvageable penumbral tissue, the target for acute neuroprotection. Drug effects on penumbra lifespan studied serially, followed by assessment of behavioural outcome and infarct within in the same animal group, will increase the power to detect drug efficacy preclinically. Similar progress in detecting drug efficacy clinically will follow from patient recruitment into acute stroke trials based on evidence of remaining penumbra

Development of new pharmacological approaches in ischemic stroke: a systematic review of transient MCAO model in rats

Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de FarmáciaOs modelos in vivo de isquemia cerebral fornecem-nos informações importantes sobre os processos bioquímicos envolvidos nos AVCs, assim como, desempenham um papel fundamental no desenvolvimento de novas abordagens terapêuticas. O modelo de isquemia da artéria cerebral média (ACM) é um dos modelos in vivo amplamente utilizado em estudos experimentais, sendo o adequado para mimetizar a isquemia cerebral, uma vez que é um método robusto, não invasivo e reversível. Esta dissertação tem como objetivo sistematizar os vários métodos de validação do modelo animal de isquemia da ACM para haver uma maior reprodutibilidade em estudos futuros. Para a elaboração desta dissertação foi realizada uma pesquisa na base de dados Medline até março de 2021 para a recolha de todos os artigos. Foi então construída uma expressão de pesquisa recorrendo aos termos MeSH. Foram escolhidos os critérios de inclusão e exclusão, para seleção dos artigos a integrar nesta revisão sistemática da literatura. Subsequentemente, as características do rato, características de indução, anestesia, volume de enfarte e parâmetros estudados foram recolhidos de cada artigo por dois revisores e, quando necessário, com a ajuda de um moderador. Quanto às características dos ratos, as espécies mais utilizadas são os Sprague-Dawley ou Wistar adultos. A anestesia mais adequada para realizar a isquemia da ACM é o isoflurano e os períodos de isquemia variam de 1-2h e reperfusão de 24h a 72h. Foi também averiguado que o modelo de isquemia da ACM induz volumes de enfarte de 40-50%. Os parâmetros avaliados incluíram o comportamento (funções neurológicas, função sensoriomotora e testes cognitivos), testes histológicos (volume de enfarte, edema cerebral, GFAP, Iba1 e NeuN) e marcadores bioquímicos, tais como fatores inflamatórios (iNOS, COX-2, NF-κB, TNF-α, e interleucinas). Com os resultados obtidos neste estudo, será possível realizar estudos mais precisos e reprodutíveis na área da isquemia cerebral.In vivo models of cerebral ischemia provide us with important information regarding the biochemical processes involved behind stroke, as well as playing a central role in the development of new therapeutic options. The transient middle cerebral artery occlusion model (MCAO) is one of the most widely used in vivo models in experimental studies and best suited to mimic cerebral ischemia since it is a robust, non-invasive and reversible method. This study aims to perform a systematic review which follows the PRISMA systematic review-protocol of the MCAO model to systemize the methods of validation of MCAO model so that there is a greater reproducibility in future studies. To perform this study a search in the Medline database was performed until March 2021 to collect all the articles. To find the articles a search expression was built using MeSH terms. The inclusion and exclusion criteria were chosen, to choose the articles to perform the systematic review. Then the rat characteristics, induction characteristics, anesthesia, infarct volume, and parameters studied data were collected from each article by two reviewers and when necessary with the help of one arbitrator. Regarding the rats characteristics the most used rats are male adult Sprague-Dawley or Wistar rats. The preferred anesthesia to perform MCAO is isoflurane and the periods of ischemia vary from 1-2h and reperfusion from 24h to 72h. It was also verified that the MCAO model causes infarct volumes between 40-50%. The parameter studied included behavior (neurological functions, sensorimotor function, and cognitive tests) and histological tests (infarct volume, brain edema, GFAP, Iba1, and NeuN) and biochemical markers, such as inflammatory factors (iNOS, COX-2, NF-κB, TNF-α, and interleukins). With the results obtained in this study, it will be possible to carry out more accurate and reproducible studies of cerebral ischemia

Multiparametric, Longitudinal Optical Coherence Tomography Imaging Reveals Acute Injury and Chronic Recovery in Experimental Ischemic Stroke

Progress in experimental stroke and translational medicine could be accelerated by high-resolution in vivo imaging of disease progression in the mouse cortex. Here, we introduce optical microscopic methods that monitor brain injury progression using intrinsic optical scattering properties of cortical tissue. A multi-parametric Optical Coherence Tomography (OCT) platform for longitudinal imaging of ischemic stroke in mice, through thinned-skull, reinforced cranial window surgical preparations, is described. In the acute stages, the spatiotemporal interplay between hemodynamics and cell viability, a key determinant of pathogenesis, was imaged. In acute stroke, microscopic biomarkers for eventual infarction, including capillary non-perfusion, cerebral blood flow deficiency, altered cellular scattering, and impaired autoregulation of cerebral blood flow, were quantified and correlated with histology. Additionally, longitudinal microscopy revealed remodeling and flow recovery after one week of chronic stroke. Intrinsic scattering properties serve as reporters of acute cellular and vascular injury and recovery in experimental stroke. Multi-parametric OCT represents a robust in vivo imaging platform to comprehensively investigate these properties

Ultrasound-enhanced Fibrinolysis Pro-fibrinolytic and Non-beneficial Effects Of Ultrasound Exposure

The aim of this study was to further clarify the pro-fibrinolytic effects, and to explore the possible non-beneficial effects in ischemic organs, during exposure to pulsed ultrasound. This was accomplished by studies of the effects of different intensities of pulsed ultrasound exposure on the fibrinolytic properties of streptokinase and reteplase. Measurements were performed both following pre-exposure of the drugs and clots to pulsed ultrasound, and following concomitant exposure of clots and drugs. In the exploration of pro-fibrinolytic effects during ultrasound exposure it was shown that exposure of the streptokinase molecule to low-intensity, pulsed high frequency ultrasound modulates its fibrinolytic properties. The effects were present following ultrasound exposure of streptokinase solution and during ultrasound exposure of clots and streptokinase solution concomitantly. Depending on its intensity, modulation was observed as both increased and decreased fibrinolytic effects. Pre-exposure of reteplase solution to low-intensity ultrasound induced changes in the function of the reteplase molecule associated with enhanced fibrinolytic effects. Enhancement effects were also seen when the clots were pre-exposed to high intensities of ultrasound before or concomitantly with exposure to reteplase solution, suggesting that two different intensity-dependent mechanisms are involved in ultrasound-enhanced reteplase fibrinolysis. No decreased fibrinolytic effect of reteplase depending on ultrasound exposure could be seen. In an initial study of the fibrinolytic effects induced by a combination of Sonazoid® microbubbles and ultrasound, the effects of various ultrasound parameters on the microbubble destruction-rate was studied. It was shown that, at the same intensity level, the destruction-rate was faster outside the resonance frequency range. Five pulses were needed to achieve a fast destruction-rate. An ultrasound intensity of 0.5 W/cm2 was shown to be the lowest intensity yielding a fast destruction-rate of Sonazoid® microbubbles. The possible non-beneficial effects in ischemic organs of ultrasound with characteristics used to enhance fibrinolysis in vivo were studied in two different models, one using non-perfused myocardia in pigs, and one using non-perfused brain tissue in rats. It was shown that prolonged exposure of low intensity pulsed ultrasound might increase instantaneous myocardial damage. However, the same frequency, intensity and duration of pulsed ultrasound did not seem to enhance ischemic damage in non-perfused rat brain or induce any damage in the perfused rat brain. In conclusion, ultrasound-enhanced fibrinolysis is induced by multiple mechanisms and drug specific reactions, and this study shows the importance of evaluating the effects of exposure on ischemic tissue, aiming at ultrasound-enhanced fibrinolysis

An examination of ischaemic penumbra in the spontaneously hypertensive stroke-prone rat (SHRSP) using the MRI perfusion-diffusion mismatch model

Stroke accounts for 9% of all deaths worldwide and is a major cause of severe disability (Donnan et al, 2008). Following ischaemic stroke, the penumbra represents tissue which is hypoperfused and functionally impaired but is not yet irreversibly damaged. However, the penumbra has a finite lifespan and will proceed to infarction in the absence of swift reperfusion. Therefore, the identification and potential salvage of penumbral tissue in acute ischaemic stroke is the ultimate goal for both clinicians and experimental stroke researchers. Positron emission tomography (PET) is the ‘gold standard’ imaging modality for identifying the penumbra, but the complex logistics of PET limit its widespread use. Magnetic Resonance Imaging (MRI) is widely used for penumbra imaging in both clinical and pre-clinical research. The MRI perfusion-diffusion mismatch model provides an approximation of the penumbra, where diffusion weighted imaging (DWI) identifies the core of ischaemic injury and perfusion weighted imaging (PWI) reveals the perfusion deficit. The mismatch between the DWI and PWI provides a measure of penumbral tissue. However, there is no consensus on the perfusion and diffusion thresholds used to identify mismatch tissue in clinical and preclinical stroke research. Furthermore, in rodent stroke models differences in the evolution of ischaemic injury between strains may limit the use of a single set of threshold values. Therefore, the first aim of this thesis was to establish strain specific perfusion and diffusion thresholds to compare penumbra volume in the clinically relevant spontaneously hypertensive stroke-prone rat (SHRSP) and the normotensive control strain, Wistar-Kyoto (WKY) using 3 different methods. The SHRSP strain is characterised by the progressive development of severe hypertension which is followed by a tendency to spontaneous stroke and an increased sensitivity to experimental stroke. Experimental stroke was induced by permanent middle cerebral artery occlusion (MCAO) by the intraluminal filament method. DWI and PWI were obtained every hour from 1-4 hours post-MCAO. Strain-specific diffusion and perfusion thresholds were established from final infarct at 24 hours post-MCAO, as defined by T2 weighted imaging. The calculated ADC thresholds were comparable between the strains but the absolute perfusion threshold was significantly higher in SHRSP compared to WKY. This may be indicative of an increased sensitivity to ischaemia in the hypertensive strain. Furthermore, application of these thresholds to the acute MRI data revealed that the volume of ischaemic injury and the perfusion deficit were significantly larger in SHRSP compared to WKY and this was also reflected in the significantly larger infarct volume observed in SHRSP at 24 hours post-MCAO. Interestingly, there was evidence of a temporal increase in the volume of the perfusion deficit in SHRSP and WKY. This may indicate that there is a progressive failure of collateral blood supply in both strains following stroke. Penumbra volume was then assessed in SHRSP and WKY rats using the mismatch method and also indirectly by examining the growth of the volume of ADC derived ischaemic injury. Mismatch volume was determined by arithmetic subtraction of the volume of ischaemic injury from the volume of perfusion deficit (volumetric method) and also by manual delineation of mismatch on each of 6 coronal slices (spatial method). There was a limited volume of mismatch tissue in either strain from as early as 1 hour post-MCAO and the volumetric method generated smaller mismatch volumes than the spatial mismatch method. Mismatch volume was comparable in SHRSP and WKY from 1-4 hours post-MCAO. Penumbra was also determined retrospectively by subtracting the volume of ischaemic injury at each time point from final infarct volume. Using this method, penumbra volume was significantly larger in WKY compared to SHRSP at 30 minutes post-MCAO but penumbra volume was comparable at all later time points. This suggests that there is reduced potential for tissue salvage in SHRSP compared to WKY within the first hour following MCAO but from 1 hour onwards, there is limited potential for penumbra salvage in both strains. In addition, there was evidence of ‘negative’ mismatch tissue in SHRSP and WKY rats, where the ADC derived lesion expanded beyond the boundary of the perfusion deficit. The volume of negative mismatch tissue was comparable between the strains and was persistent over the 4 hour time course. This phenomenon may arise from the spread of toxic mediators from the ischaemic core. Oxidative stress is a major mediator of cellular injury following ischaemic stroke and reactive oxygen species, like superoxide, have multiple deleterious effects on the components of the neurovascular unit. It is well established that NADPH oxidase is the principal source of superoxide in acute ischaemic stroke and is therefore a target for potential neuroprotective strategies (Moskowitz et al, 2010). Consequently, the second aim of this thesis was to evaluate the potential neuroprotective effect of NADPH oxidase inhibition with low and high dose apocynin following permanent or transient ischaemia. Rats were administered apocynin at a dose of 5mg/kg or 30mg/kg or vehicle, at 5 minutes post-MCAO. Apocynin treatment had no significant effect on infarct volume or functional outcome at 24 hours following permanent MCAO in WKY rats. However, both low and high dose apocynin treatment significantly reduced infarct volume at 72 hours post-MCAO by 60% following 1 hour of ischaemia in Sprague-Dawley rats. Furthermore, functional outcome was improved in the low dose apocynin treated group, although this did not reach the level of statistical significance. On the basis of these results, low dose apocynin treatment was evaluated in SHRSP rats following 1 hour of ischaemia. However, apocynin treatment had no effect on the acute evolution of ischaemic injury and failed to improve stroke outcome, where the mortality rate was high in both the apocynin treated and the vehicle treated group. The conflicting effects of apocynin may be attributable to a differential expression of NADPH oxidase subunits in normotensive and hypertensive rat strains. These findings may also explain the failure of neuroprotective drugs to translate from bench to bedside, as therapies which are neuroprotective in young healthy animals may not demonstrate the same efficacy in animal models with stroke co-morbidities. Therefore, potential therapeutic strategies should be extensively evaluated in animal models with stroke risk factors before proceeding to clinical trial

Relevance of Porcine Stroke Models to Bridge the Gap from Pre-Clinical Findings to Clinical Implementation

Altres ajuts: This research is supported by grants from the Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III (ISCIII) to A.D. that was susceptible to be co-financed by FEDER funds, and a grant from Agència de Gestió d'Ajuts Universitaris i de Recerca to A.D. and to T.G. The group has received funding from "la Caixa Foundation" CI15-00009, from the European Institute of Innovation and Technology (EIT), which receives support from the European Union's Horizon 2020 research and innovation programme, from the Fundación para la Innovación y la Prospectiva en Salud en España (FIPSE) program 3594-18. M.M.-S. is a recipient of a PFIS contract FI19/00174.In the search of animal stroke models providing translational advantages for biomedical research, pigs are large mammals with interesting brain characteristics and wide social acceptance. Compared to rodents, pigs have human-like highly gyrencephalic brains. In addition, increasingly through phylogeny, animals have more sophisticated white matter connectivity; thus, ratios of white-to-gray matter in humans and pigs are higher than in rodents. Swine models provide the opportunity to study the effect of stroke with emphasis on white matter damage and neuroanatomical changes in connectivity, and their pathophysiological correlate. In addition, the subarachnoid space surrounding the swine brain resembles that of humans. This allows the accumulation of blood and clots in subarachnoid hemorrhage models mimicking the clinical condition. The clot accumulation has been reported to mediate pathological mechanisms known to contribute to infarct progression and final damage in stroke patients. Importantly, swine allows trustworthy tracking of brain damage evolution using the same non-invasive multimodal imaging sequences used in the clinical practice. Moreover, several models of comorbidities and pathologies usually found in stroke patients have recently been established in swine. We review here ischemic and hemorrhagic stroke models reported so far in pigs. The advantages and limitations of each model are also discussed