Wet and Dry Biomarkers to Predict Secondary Injury in Stroke: From Bench to Bedside - The NIMBLE Study

Infarct growth (IG), Hemorrhagic Transformation (HT) and Cerebral Edema (CE) can be considered pivotal phenomena of clinical deterioration following an acute ischemic stroke. Innovative techniques applied to neuroimaging allow these phenomena to be identified and measured more adequately than techniques and approaches commonly in use. Some circulating molecules are conceptually usable as biological markers of CE, HT, and IG. The correlation between circulating and neuroimaging biomarkers, and the investigation of neuronal structural remodeling induced by ischemia, may provide fundamental details to prevent or contrast clinical deterioration after ischemic stroke. To achieve this goal, the investigators planned to perform translational research on humans and on a novel mouse model of ischemic stroke. More specifically, the investigators planned a clinical prospective observational study on a consecutive series of patients with acute anterior ischemic stroke either submitted or not submitted to revascularization therapies. Serum levels of several blood biomarkers related to inflammation, blood-brain barrier disruption, and reperfusion injury are analyzed in relation to CE, HT, IG, and final infarct volume, evaluated on CT/MRI images, and to 3 months functional status evaluated by the modified Rankin Scale. In parallel, the investigators employ a newly developed experimental model of stroke and recanalization of the distal branch of the middle cerebral artery in mice to study, with advanced optical imaging techniques, the structural reorganization of neurons at the cellular and subcellular level in relation with the blood vessel extravasation (CE) and with the levels of circulating biomarkers at different time points after stroke. The investigators will verify to what extent the animal model can reliably reproduce significant parameters that are evaluated in stroke patients, i.e. circulating biomarkers levels in relation to lesion volume and edema formation. Once validated, the data on the structural plasticity of mice shall be used to infer the mechanisms that determine the clinical deterioration due to IG, HT, and CE.

BACKGROUND Infarct growth (IG), Hemorrhagic Transformation (HT), and Cerebral Edema (CE) are key factors associated with a negative outcome in ischemic stroke patients, despite the success of recanalization. Futile reperfusion and reperfusion injury are linked to these harmful phenomena but their mechanisms remain largely not understood and consequently they are difficult to be predicted and hindered. Various studies have shown that some circulating biomarkers are associated with the development of IG, HT, and CE. The NIMBLE Study (Integrating novel NeuroImaging Measurements and circulating Biomarkers for the prediction of secondary injury foLlowing strokE: from bench to bedside) is a translational project including clinical research in humans and experimental study in animals. It has the aim to investigate possible factors contributing to IG, HT, and CE after ischemia by studying circulating biomarkers in respect to neuroimaging abnormalities and functional outcome in humans. In mice circulating biomarkers are studied in respect to the neuronal reorganization at the cellular and subcellular level and to experimental evaluation of CE. CLINICAL SETTING Study design: Single-centre, longitudinal, follow-up prospective observational study enrolling a consecutive series of patients, with anterior acute ischemic stroke presenting within 12 hours from symptom onset to the Emergency Department either treated or not treated with revascularization therapies. All patients undergo neuroimaging evaluation, and blood sampling for the dosage of several serum circulating biomarkers (see below for details). Patient enrollment was between 04/24/2021 and 06/30/2023 for a total of 213 patients, median age \[IQR (Interquartile Range)\] 80 \[16\] years, 46% females, median baseline NIHSS (National Institutes of Health Stroke Scale) \[IQR\] 10 \[3\] Work Methodology: Clinical assessment: A full range of clinical data including stroke risk factors, acute stroke treatment, pre-stroke medications are recorded for each patient. Stroke severity is measured using the NIHSS at baseline and at 1, 7 and 90 days follow-up, and post-stroke disability by the modified Rankin Scale (mRS) administered at 3 months by visit or phone interview. Neuroradiological evaluation: Neuroradiological evaluation is blinded to clinical data. Brain imaging includes baseline plain CT, and, when clinically indicated, CT (Computed Tomography) multi-phase angiography and CT perfusion at baseline. CT at 24 h, and non-contrast MRI at 5 days (including diffusion-weighted imaging (DWI), apparent diffusion coefficient (ADC), fluid- attenuated inversion recovery (FLAIR) and fast field echo (FFE) sequences) are performed. Early ischemic signs (Alberta Stroke Programme Early CT \[ASPECTS\] score), presence and severity of small vessel disease, presence and grading of CE and HT are assessed by two neuroradiologists at baseline and follow-up CT scans. CE is classified according to the SITS-ISTR (Safe Implementation of Treatments in Stroke - International Stroke Thrombolysis Register) criteria: COED1 = focal brain swelling up to one third of the hemisphere, COED2 = focal brain swelling greater than one third of the hemisphere, COED3 = brain swelling with midline shift, NONE = absence of cerebral edema. HT is assessed according to the ECASS (European Cooperative Acute Stroke Study) radiographic classification: HI-1: Haemorrhagic infarction type 1 = small petechiae HI-2: Haemorrhagic infarction type 2 = more confluent petechiae PH-1: Parenchymal hematoma type 1 = \<30% of infarcted area with mild space-occupying effect PH-2: Parenchymal hematoma type 2 =. \>30% of infarcted area with significant space-occupying effect Perfusion data derived following local clinical protocol are evaluated by an expert neuroradiologist to detect infarct core and hypoperfusion volumes. Furthermore, an MRI characterization of the edema is performed to differentiate vasogenic edema from cytotoxic edema, analyzing the signal characteristics and the lesional morphology. A new method to quantify brain swelling (induced by CE and HT) and to define lesion growth and final infarct volume, called Anatomical Distortion (AD) is also applied. Two expert operators will independently create a manual mapping ("mask") of the ischemic lesion visible on the 24-hour CT scan. A lesion "mask" will be defined by one operator also on the 5 days MRI (Magnetic Resonance Imaging) scan (using DWI sequence). Any discrepancies will be resolved by a separate neuroradiologist. For this process, FSLeyes will be used, a software included in the FSL software (FMRIB \[Functional Magnetic Resonance Imaging of the Brain\] Software Library v6.0, Created by the Analysis Group, FMRIB, Oxford, UK) which offers the possibility to generate a three-dimensional lesion mask for each patient, covering the infarct tissue on all CT sections. Previous/non-acute ischemic lesions (already present on baseline CT) will not be included in the masks. AD will be assessed by comparing the baseline CT with the 24-hour CT and 5 days MRI scans. The steps required to obtain this measurement include delineating the ischemic lesion visible on follow-up scans using a "mask" (described before) and registration, a procedure that allows accurate comparison betweendifferent images of the brain, even at different times and individuals. AD can be calculated by comparing the two possible registration approaches and in particular by quantifying the difference between the lesion masks processed through Linear and Non-Linear registration according to the method described by Harston in 2018. All image processing and analysis will be carried out using FSL software (FMRIB Software Library v6.0, Created by the Analysis Group, FMRIB, Oxford, UK), a free access software developed by a research group of the University of Oxford. Subtracting AD from total lesion volume, the investigators can obtain the corrected infarct volume. Laboratory protocol: Biomarker measurements is blinded to clinical data. In all patients the following serum levels of biomarkers linked to inflammation, blood-brain barrier disruption, and reperfusion injury, are measured on arrival and after 24 hours. They can be divided into three groups according to the type and speed of the measurement technique: * rapidly available molecules: alpha 2 macroglobuline (A2M); Serum Amyloid A protein (SAA); Haptoglobin (Hp); high sensitivity C Reactive Protein (hsCRP); Neuron-specific enolase (NSE); S100Beta (S100B) * biomarkers requiring longer times to be measured: pro-and anti-inflammatory cytokines and chemokines, matrix metalloproteases (MMPs) and their inhibitors (TIMPs), von Willebrand factor (vWF), endothelial dysfunction markers and tight junction proteins. * Metabolomic and lipoproteomic biomarkers All the data are reported in a dedicated Case report form (CRF). Outcome measure Primary Outcome Measure: 1. Infarct growth, Hemorrhagic Transformation and Cerebral Edema Secondary Outcome Measure: 2. Functional outcome Functional outcome will be assessed by measuring the modified Rankin Scale 3 months after acute ischaemic stroke onset. Statistical analysis plan: Univariate linear regression analyses will be conducted to evaluate the association between each primary radiological outcome and selected baseline variables (including circulating biomarkers). Independent variables associated with the outcome at univariate analysis will be included in multivariate models. In another analysis, univariate logistic regression models will be created to assess the association between baseline circulating and radiological biomarkers and clinical variables and the dichotomized mRS score recorded at three months. The risk factors found to be significant on a univariate analysis will enter into a multiple logistic regression model with a backward selection criterion. PRECLINICAL SETTING Ethic statement preclinical research: All procedures involving mice are performed in accordance with regulations of the Italian Ministry of Health authorization n. 723/2019. Mice are housed in clear plastic cages under a 12 h light/dark cycle and are given ad libitum access to water and food. The investigators use a transgenic mouse line, C57BL/6J-Tg(Thy1-EGFP)MJrs/J, from Jackson Laboratories (Bar Harbor, Maine USA). Mouse model of Middle Cerebral Artery (MCA) occlusion and recanalization: In this project, a novel method of transient photothrombotic distal MCA occlusion developed by our group is applied. Briefly, photothrombotic MCA occlusion is performed by irradiating the distal MCA with a focused green laser, after an intraperitoneal injection of the photosensitive dye Rose Bengal. At selected times after MCA occlusion (30 min, 60 min, 90 min), recanalization is performed by irradiating the occluded distal MCA with a Ultraviolet (UV) LED capable of disrupting the fibrine bonds inside the clot. In vivo two-photon imaging of dendrites and vasculature: The structural plasticity of axons and dendrites in the acute phase after stroke compared to pre-stroke condition is investigated. In Thy1-GFP mice a cranial window on the peri-infarct area to allow permanent optical access to the mouse cortex is performed. The superficial blood vessels of the cranial window is used as a landmark to retrace the same region of the cortex in different imaging sessions performing a longitudinal investigation of synaptic terminals turnover and neuronal processes orientation. Moreover, the vascular permeability at the capillary level is characterized. To this aim, a systemic injection of a fluorescent dye (e.g. Texas Red Dextran, Thermo Fisher Scientific, USA) in the caudal vein of mice right before the imaging session is performed. This dye is vital for several hours after the injection and diffuses into the parenchyma only if and where extravasation occurs. Then, imaging stacks of the labeled vasculature every 5 minutes from immediately to 30 minutes after injection is acquired. To evaluate the permeability of blood vessels in vivo, the average fluorescence values within a square Region of Interest centered either inside or right outside a blood vessel and repeat the measure on the same location at different time points after injection is performed. The permeability ⍺(t) will be calculated according to the following formula described by Nhan in 2013): \[⍺(t) = (dIe/dt)/\[(Ii(t)/0.55)-(Ie(t)/(Ve/Vi)\]. Edema: 24 hours after stroke induction, the brain water content, as an indirect measure of cerebral edema is calculated in sacrificed mice, according to Kenne and collaborators. The brain is divided along the midline and the contralateral and ipsilateral tissues are weighed right after removal to obtain wet weight (WW). The tissues are dried at 60°C for 72 hours and weighed to obtain dry weight (DW). Water content is calculated as a percentage of wet weight; % water content = \[(WW-DW)/(WW)\]\*100. Circulating biomarkers investigation: In order to test at different time points (pre-stroke, baseline, and 24 hours after the occlusion) the same blood biomarkers evaluated in patients, blood is drawn through the retro-orbital sinus in anesthetized mice. Statistical analyses: The sample will be divided into three experimental groups (Sham, No-Recanalized, and Recanalized). For the sample sizing, the investigators will use the ANOVA test (fixed effects, omnibus, one-way) and the following data: 3 experimental groups, effect size f = 0.5, α = 0.05, Power = 0.8 (software used G \* Power, version 3.1.9.2, Franz Faul, University of Kiel, Germany). Data of mice of the three groups will be compared at different time points (pre-stroke, baseline, and 24 after stroke). A one-way analysis of variance (ANOVA) repeated measures will be performed on two-photon imaging data in order to assess the synaptic turnover and blood-brain barrier permeability of cortical blood vessels at different time points. Data will be given as mean ± standard error of the mean. Fundings: * Tuscany Region "Bando Salute 2018"; * #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) - A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022).