NK1 tachykinin receptor antagonist treatment reduces cerebral edema and intracranial pressure in an ovine model of ischemic stroke.

Following ischemic stroke, substance P (SP)-mediated neurogenic inflammation is associated with profound blood-brain barrier (BBB) dysfunction, cerebral edema, and elevated intracranial pressure (ICP). SP elicits its effects by binding the neurokinin 1 tachykinin receptor (NK1-R), with administration of an NK1-R antagonist shown to ameliorate BBB dysfunction and cerebral edema in rodent and permanent ovine stroke models. Given the importance of reperfusion in clinical stroke, this study examined the efficacy of NK1-R antagonist treatment in reducing cerebral edema and ICP in an ovine model of transient middle cerebral artery occlusion (tMCAo). Anesthetized sheep (n = 24) were subject to 2-hours tMCAo and randomized (n = 6/group) to receive early NK1-R treatment (days 1-3 post-stroke), delayed NK1-R treatment (day 5 post-stroke), or saline vehicle. At 6-days post-stroke animals were re-anaesthetized and ICP measured, followed by MRI to evaluate infarction, edema and BBB dysfunction. Following both early and delayed NK1-R antagonist administration, ICP was significantly reduced on day 6 compared to vehicle animals (p < 0.05), accompanied by a reduction in cerebral edema, midline shift and BBB dysfunction (p < 0.05). This study demonstrates that NK1-R antagonist treatment is an effective novel therapy for cerebral edema and elevated ICP following stroke in an ovine model, warranting future clinical evaluation.

Commentary to Brain-wide continuous functional ultrasound imaging for real-time monitoring of hemodynamics during ischemic stroke.

Functional ultrasound (FUS) has emerged as a novel imaging method to reliably assess relative cerebral blood volume (rCBV) and infer perfusion, with good spatiotemporal resolution. Brunner and colleagues provide what appears to be its first application to characterize peri-infarct spreading depolarizations (SDs) in experimental stroke through recording of transient hyperemic events. They also report incomplete overlap between acute perfusion deficits and subsequent infarct distribution, specifically noting a rostral expansion to involve penumbral territory from which propagating depolarizations had preferentially originated. This observation would not be straightforward using other methodologies. Other strengths and limitations of the study are considered.

Prediction of Stroke Outcome in Mice Based on Noninvasive MRI and Behavioral Testing.

BACKGROUND: Prediction of poststroke outcome using the degree of subacute deficit or magnetic resonance imaging is well studied in humans. While mice are the most commonly used animals in preclinical stroke research, systematic analysis of outcome predictors is lacking. METHODS: We intended to incorporate heterogeneity into our retrospective study to broaden the applicability of our findings and prediction tools. We therefore analyzed the effect of 30, 45, and 60 minutes of arterial occlusion on the variance of stroke volumes. Next, we built a heterogeneous cohort of 215 mice using data from 15 studies that included 45 minutes of middle cerebral artery occlusion and various genotypes. Motor function was measured using a modified protocol for the staircase test of skilled reaching. Phases of subacute and residual deficit were defined. Magnetic resonance images of stroke lesions were coregistered on the Allen Mouse Brain Atlas to characterize stroke topology. Different random forest prediction models that either used motor-functional deficit or imaging parameters were generated for the subacute and residual deficits. RESULTS: Variance of stroke volumes was increased by 45 minutes of arterial occlusion compared with 60 minutes. The inclusion of various genotypes enhanced heterogeneity further. We detected both a subacute and residual motor-functional deficit after stroke in mice and different recovery trajectories could be observed. In mice with small cortical lesions, lesion volume was the best predictor of the subacute deficit. The residual deficit could be predicted most accurately by the degree of the subacute deficit. When using imaging parameters for the prediction of the residual deficit, including information about the lesion topology increased prediction accuracy. A subset of anatomic regions within the ischemic lesion had particular impact on the prediction of long-term outcomes. Prediction accuracy depended on the degree of functional impairment. CONCLUSIONS: For the first time, we developed and validated a robust tool for the prediction of functional outcomes after experimental stroke in mice using a large and genetically heterogeneous cohort. These results are discussed in light of study design and imaging limitations. In the future, using outcome prediction can improve the design of preclinical studies and guide intervention decisions.

Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells.

E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein.

Preserving cognitive function in patients with Alzheimer's disease: The Alzheimer's disease neuroprotection research initiative (ADNRI).

The global trend toward aging populations has resulted in an increase in the occurrence of Alzheimer's disease (AD) and associated socioeconomic burdens. Abnormal metabolism of amyloid-ß (Aß) has been proposed as a significant pathomechanism in AD, supported by results of recent clinical trials using anti-Aß antibodies. Nonetheless, the cognitive benefits of the current treatments are limited. The etiology of AD is multifactorial, encompassing Aß and tau accumulation, neuroinflammation, demyelination, vascular dysfunction, and comorbidities, which collectively lead to widespread neurodegeneration in the brain and cognitive impairment. Hence, solely removing Aß from the brain may be insufficient to combat neurodegeneration and preserve cognition. To attain effective treatment for AD, it is necessary to (1) conduct extensive research on various mechanisms that cause neurodegeneration, including advances in neuroimaging techniques for earlier detection and a more precise characterization of molecular events at scales ranging from cellular to the full system level; (2) identify neuroprotective intervention targets against different neurodegeneration mechanisms; and (3) discover novel and optimal combinations of neuroprotective intervention strategies to maintain cognitive function in AD patients. The Alzheimer's Disease Neuroprotection Research Initiative's objective is to facilitate coordinated, multidisciplinary efforts to develop systemic neuroprotective strategies to combat AD. The aim is to achieve mitigation of the full spectrum of pathological processes underlying AD, with the goal of halting or even reversing cognitive decline.

Novel targets, treatments, and advanced models for intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is the second most common type of stroke and a major cause of mortality and disability worldwide. Despite advances in surgical interventions and acute ICH management, there is currently no effective therapy to improve functional outcomes in patients. Recently, there has been tremendous progress uncovering new pathophysiological mechanisms underlying ICH that may pave the way for the development of therapeutic interventions. Here, we highlight emerging targets, but also existing gaps in preclinical animal modelling that prevent their exploitation. We particularly focus on (1) ICH aetiology, (2) the haematoma, (3) inflammation, and (4) post-ICH pathology. It is important to recognize that beyond neurons and the brain, other cell types and organs are crucially involved in ICH pathophysiology and successful interventions likely will need to address the entire organism. This review will spur the development of successful therapeutic interventions for ICH and advanced animal models that better reflect its aetiology and pathophysiology.

Long-Term Connectome Analysis Reveals Reshaping of Visual, Spatial Networks in a Model With Vascular Dementia Features.

BACKGROUND: Connectome analysis of neuroimaging data is a rapidly expanding field that offers the potential to diagnose, characterize, and predict neurological disease. Animal models provide insight into biological mechanisms that underpin disease, but connectivity approaches are currently lagging in the rodent. METHODS: We present a pipeline adapted for structural and functional connectivity analysis of the mouse brain, and we tested it in a mouse model of vascular dementia. RESULTS: We observed lacunar infarctions, microbleeds, and progressive white matter change across 6 months. For the first time, we report that default mode network activity is disrupted in the mouse model. We also identified specific functional circuitry that was vulnerable to vascular stress, including perturbations in a sensorimotor, visual resting state network that were accompanied by deficits in visual and spatial memory tasks. CONCLUSIONS: These findings advance our understanding of the mouse connectome and provide insight into how it can be altered by vascular insufficiency.

A pilot of the feasibility and usefulness of an aged obese model for use in stroke research.

Background: Animal models of stroke have been criticised as having poor predictive validity, lacking risk factors prevalent in an aging population. This pilot study examined the development of comorbidities in a combined aged and high-fat diet model, and then examined the feasibility of modelling stroke in such rats. Methods: Twelve-month old male Wistar-Han rats (n=15) were fed a 60% fat diet for 8 months during which monthly serial blood samples were taken to assess the development of metabolic syndrome and pro-inflammatory markers. Following this, to pilot the suitability of these rats for undergoing surgical models of stroke, they underwent 30min of middle cerebral artery occlusion (MCAO) alongside younger controls fed a standard diet (n=10). Survival, weight and functional outcome were monitored, and blood vessels and tissues collected for analysis. Results: A high fat diet in aged rats led to substantial obesity. These rats did not develop type 2 diabetes or hypertension. There was thickening of the thoracic arterial wall and vacuole formation in the liver; but of the cytokines examined changes were not seen. MCAO surgery and behavioural assessment was possible in this model (with some caveats discussed in manuscript). Conclusions: This study shows MCAO is possible in aged, obese rats. However, this model is not ideal for recapitulating the complex comorbidities commonly seen in stroke patients.

Glyceryl trinitrate for the treatment of ischaemic stroke: Determining efficacy in rodent and ovine species for enhanced clinical translation.

Hypertension is a leading risk factor for death and dependency after ischaemic stroke. However, administering anti-hypertensive medications post-stroke remains contentious with concerns regarding deleterious effects on cerebral blood flow and infarct expansion. This study sought to determine the effect of glyceryl trinitrate (GTN) treatment in both lissencephalic and gyrencephalic pre-clinical stroke models. Merino sheep underwent middle cerebral artery occlusion (MCAO) followed by GTN or control patch administration (0.2 mg/h). Monitoring of numerous physiologically relevant measures over 24 h showed that GTN administration was associated with decreased intracranial pressure, infarct volume, cerebral oedema and midline shift compared to vehicle treatment (p < 0.05). No significant changes in blood pressure or cerebral perfusion pressure were observed. Using optical imaging spectroscopy and laser speckle imaging, the effect of varying doses of GTN (0.69-50 µg/h) on cerebral blood flow and tissue oxygenation was examined in mice. No consistent effect was found. Additional mice undergoing MCAO followed by GTN administration (doses varying from 0-60 µg/h) also showed no improvement in infarct volume or neurological score within 24 h post-stroke. GTN administration significantly improved numerous stroke-related physiological outcomes in sheep but was ineffective in mice. This suggests that, whilst GTN administration could potentially benefit patients, further research into mechanisms of action are required.

UK consensus on pre-clinical vascular cognitive impairment functional outcomes assessment: Questionnaire and workshop proceedings.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

Transfemoral Approach to Induce Transient Middle Cerebral Artery Occlusion in Rats: The Use of Commercially Available Endovascular Wires.

BACKGROUND: Animal models of stroke play a crucial role in determining the pathophysiology of stroke progression and assessment of any new therapeutic approaches. Transient middle cerebral artery occlusion (tMCAo) in rodent models are the most common site-specific type of ischemia because of their relevance to the clinical setting. Compared with the intraluminal filament technique for inducing tMCAo, the transfemoral approach using endovascular wires is relatively a new technique METHODS: Here we present the use of commercially available wires used for neuro-endovascular surgical procedures to induce tMCAo in rats via a transfemoral approach. We used male Wistar rats in four groups to assess the effect of occlusion time (1 vs. 2 hours) and the wire type (PT2 TM 0.014″ vs. TransendTM EX, 0.014″, Boston Scientific, MA, USA). Infarct volume, edema, neurological deficits, and pro-inflammatory/anti-inflammatory blood biomarkers were used as outcome measures. RESULTS: We observed a significant effect of the wire type on the infarct volume (p value = 0.0096) where infarcts were slightly larger in the PT2 wiregroups. However, the occlusion time had no significant effect on infarct volume, even though the interaction between wire-type * occlusion-time was significant (p value = 0.024). Also, the amount of edema and blood pro-inflammatory/anti-inflammatory biomarkers were not statistically different among the wire-type and occlusion-time groups. CONCLUSIONS: The choice of appropriate endovascular wire should probably be the focus of the study design instead of the occlusion time when planning an experiment. The transfemoral approach using endovascular wires for inducing tMCAo in rats provides a more consistent outcome with fewer complications compared with suture filament models.

Recommendations for measuring whisker movements and locomotion in mice with sensory, motor and cognitive deficits.

BACKGROUND: Previous studies have measured whisker movements and locomotion to characterise mouse models of neurodegenerative disease. However, these studies have always been completed in isolation, and do not involve standardized procedures for comparisons across multiple mouse models and background strains. NEW METHOD: We present a standard method for conducting whisker movement and locomotion studies, by carrying out qualitative scoring and quantitative measurement of whisker movements from high-speed video footage of mouse models of Amyotrophic Lateral Sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, Cerebellar Ataxia, Somatosensory Cortex Development and Ischemic stroke. RESULTS: Sex, background strain, source breeder and genotype all affected whisker movements. All mouse models, apart from Parkinson's disease, revealed differences in whisker movements during locomotion. R6/2 CAG250 Huntington's disease mice had the strongest behavioural phenotype. Robo3R3-5-CKO and RIM-DKOSert mouse models have abnormal somatosensory cortex development and revealed significant changes in whisker movements during object exploration. COMPARISON WITH EXISTING METHOD(S): Our results have good agreement with past studies, which indicates the robustness and reliability of measuring whisking. We recommend that differences in whisker movements of mice with motor deficits can be captured in open field arenas, but that mice with impairments to sensory or cognitive functioning should also be filmed investigating objects. Scoring clips qualitatively before tracking will help to structure later analyses. CONCLUSIONS: Studying whisker movements provides a quantitative measure of sensing, motor control and exploration. However, the effect of background strain, sex and age on whisker movements needs to be better understood.

Cognition in Stroke Rehabilitation and Recovery Research: Consensus-Based Core Recommendations From the Second Stroke Recovery and Rehabilitation Roundtable.

Cognitive impairment is an important target for rehabilitation as it is common following stroke, is associated with reduced quality of life and interferes with motor and other types of recovery interventions. Cognitive function following stroke was identified as an important, but relatively neglected area during the first Stroke Recovery and Rehabilitation Roundtable (SRRR I), leading to a Cognition Working Group being convened as part of SRRR II. There is currently insufficient evidence to build consensus on specific approaches to cognitive rehabilitation. However, we present recommendations on the integration of cognitive assessments into stroke recovery studies generally and define priorities for ongoing and future research for stroke recovery and rehabilitation. A number of promising interventions are ready to be taken forward to trials to tackle the gap in evidence for cognitive rehabilitation. However, to accelerate progress requires that we coordinate efforts to tackle multiple gaps along the whole translational pathway.

Cognition in stroke rehabilitation and recovery research: Consensus-based core recommendations from the second Stroke Recovery and Rehabilitation Roundtable.

Cognitive impairment is an important target for rehabilitation as it is common following stroke, is associated with reduced quality of life and interferes with motor and other types of recovery interventions. Cognitive function following stroke was identified as an important, but relatively neglected area during the first Stroke Recovery and Rehabilitation Roundtable (SRRR I), leading to a Cognition Working Group being convened as part of SRRR II. There is currently insufficient evidence to build consensus on specific approaches to cognitive rehabilitation. However, we present recommendations on the integration of cognitive assessments into stroke recovery studies generally and define priorities for ongoing and future research for stroke recovery and rehabilitation. A number of promising interventions are ready to be taken forward to trials to tackle the gap in evidence for cognitive rehabilitation. However, to accelerate progress requires that we coordinate efforts to tackle multiple gaps along the whole translational pathway.

A Brief Review of Edema-Adjusted Infarct Volume Measurement Techniques for Rodent Focal Cerebral Ischemia Models with Practical Recommendations.

BACKGROUND: Determining cerebral infarction volume is an important part of preclinical studies to determine the benefit of potential therapies on stroke outcome. A well-known problem in determining the actual infarction volume of rodent models is the presence of edema. Because of this, algorithms must be utilized to obtain the edema-adjusted (E A)-infarct volume. Different methods based on 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining have been published describing algorithms to determine the E A-infarct volume. MATERIALS AND METHODS: Simulated models of infarction and corresponding swelling were employed to determine which absolute method of calculation (Lin et al., Reglodi et al., or Belayev et al.) is the most accurate in calculating the absolute E A-infarct volume. RESULTS: The Reglodi and Belayev methods were statistically more accurate in measuring E A-infarct volume than Lin's method, p = 0.0078. Though there was no significant difference between Reglodi's and Belayev's methods for the E A-infarction volume calculation, Reglodi's approach was closer to the ground-truth infarct volume while also being simpler and more straightforward to use. CONCLUSION: We recommend that Reglodi's method, that is E A-infarct volume = infarct volume × (contralateral hemisphere/ipsilateral hemisphere), to be used in calculating E A-infarct volume in TTC stained rodent brains. Further, factors such as inhomogeneous infarction distribution in a given brain slice can also contribute to the error in volume calculation. Therefore, the average of the infarct area obtained from anterior and posterior views of a given slice should be used to account for the variation. Considering different factors, we have provided a summary recommendation for calculating the infarction volume.

Atlas registration for edema-corrected MRI lesion volume in mouse stroke models.

Lesion volume measurements with magnetic resonance imaging are widely used to assess outcome in rodent models of stroke. In this study, we improved a mathematical framework to correct lesion size for edema which is based on manual delineation of the lesion and hemispheres. Furthermore, a novel MATLAB toolbox to register mouse brain MR images to the Allen brain atlas is presented. Its capability to calculate edema-corrected lesion size was compared to the manual approach. Automated image registration performed equally well in in a mouse middle cerebral artery occlusion model (Pearson r = 0.976, p = 2.265e-11). Information encapsulated in the registration was used to generate maps of edema induced tissue volume changes. These showed discrepancies to simplified tissue models underlying the manual approach. The presented techniques provide biologically more meaningful, voxel-wise biomarkers of vasogenic edema after stroke.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).