Acute Ischaemic STROKE: From LAboratory to the Patient's BED

The purpose of the project is to evaluate the correlation of circulating biomarkers related to blood brain barrier disruption with the development of cerebral edema, hemorrhagic transformation and the extension of the ischemic area investigated with modern neuroimaging techniques, in order to identify predictors of reperfusion injury in patients undergoing revascularization therapies.

In parallel with the clinical study, the investigators will investigate mechanistic aspects on the correlation between neuronal degeneration and the alteration of vascular permeability. To this end, the investigators will use a combination of advanced microscopy techniques and vital fluorescent markings in a novel mouse model of ischemic stroke.

• Clinical setting: This is a monocentric retrospective observational study of patients with acute ischemic stroke who have undergone revascularization therapies (systemic thrombolysis, endovascular treatment) at the Stroke Unit of the Careggi University Hospital from 1 October 2015 to 31 May 2020. Patients' data will be entered in a dedicated register: demographics and clinical data as well as haematochemical and neuroimaging parameters covered by this study. Available circulating biomarkers and neuroimaging collected at hospital arrival and repeated 24 hours after interventions will be included in the analysis, as well as the clinical/functional outcome assessed 3 months after stroke according to the modified Rankin scale by visit or phone interview.

Laboratory protocol:

In our university hospital, in acute ischemic stroke patients blood samples are routinely collected before and 24 hours after reperfusion treatment (either intravenous thrombolysis or endovascular thrombectomy). The investigators could retrieve blood samples already used for a previous study (Piccardi et al. 2018) and still correctly stored and available in our biological bank: blood is collected in tubes both with anticoagulant and without anticoagulant. Tubes are then centrifuged at room temperature at 1500 x g for 15 minutes, and the supernatant is stored in aliquots at -80° C.

Neuroimaging protocol:

Baseline neuroimaging assessment, including Non-Contrast CT (NCCT), CT-Angiography (CTA) and CT-Perfusion (CTP) has been performed before acute interventions when indicated according to guidelines in place at the time of treatment. A 24 hours follow-up NCCT has been performed in all patients.

After 24 hours, as per international guidelines, all patients undergo a control head CT or brain MRI to determine the presence of hemorrhagic transformation, cerebral edema and the volume of the ischemic area. The re-reading of the images already acquired will be carried out on DICOM format. The data collected will be recorded in a special section of the electronic register.

Two expert neurologists will separately evaluate each patient's baseline and 24-hour CT and record the presence of cerebral edema classified according to the SITS-ISTR 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. The two independent neurologists will also create a manual mapping ("mask") of the ischemic lesion visible on the 24-hour CT scan. Any discrepancies will be resolved by an experienced neuroradiologist. For this process, FSLeyes will be used, a software included in the FSL software (FMRIB 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 ischemic outcomes (already present on baseline CT) will not be included in the masks.

Anatomic distortion (AD) will be assessed by comparing the baseline CT with the 24-hour CT. The steps required to obtain this measurement include delineating the ischemic lesion visible at 24 hours using a "mask" (described before) and registration, a procedure that allows to accurately compare different 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 et al. 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.

3 month outcome: All patients treated with systemic thrombolysis or endovascular treatment are followed-up 3 months after the ictal event with an evaluation of the clinical-functional outcome according to the modified Rankin Scale, directly in the out-patient clinic or by telephone interview.

This data must therefore be acquired from outpatient reports. Only if the data is missing will a telephone interview be carried out.

• Pre-clinical setting: The preclinical research group will characterize a novel mouse model of occlusion/reperfusion of the MCA. All procedures involving mice will be performed in accordance with regulations of the Italian Ministry of Health authorization n. 723/2019.

Mice will be anesthetized with ketamine (50 mg/kg) and xylazine (9 mg/kg). Body temperature will be maintained at 37°C with a heating pad. Mice will be placed on a surgery pad, lying on one side. The mouse hairs between the eye and the ear will be removed and then the skin will be cleaned with betadine and ethanol. Then, local anesthetic lidocaine 2% will be applied. The skin over the squamosal bone will be cut with a blade, the muscle will be detached from the skull and gently pushed down to expose the bone.

Then, a dental drill (Silfradent, Italia) will be used to create a small craniotomy to expose the distal branch of the middle cerebral artery. Once removed the flap bone, a photosensitive dye, Rosebengal (0.2 ml, 10 mg/ml solution in Saline Solution; Sigma Aldrich, USA) will be intraperitoneally injected. Five minutes after the injection, a 532 nm laser (Laser Diode CPS532, Thorlabs, Germany) will be focused upstream of the MCA branching for twenty-five minutes to induce the formation of a stable clot. Thirty minutes after the occlusion of the distal branch of the MCA, in order to promote the disruption of the clot and the consequent recanalization of the blood vessel, the investigators will illuminate the same region with a 365 nm UV LED (High-Power LED SOLIS-365C, Thorlabs, Germany) for fifteen minutes. After the UV irradiation period, the distal branch of the MCA result recanalized. At the end of the procedure, the muscle will be replaced over the bone and the skin sutured.

During the experimental time window the investigators will characterize the functional impairment induced by the occlusion of the distal branch of the MCA with and without recanalization.

To this aim the investigators will use the neurodeficit score modifying the protocol applied by Balbi and collaborators in 2019, based on a standardized qualitative assessment for measuring the degree of motor impairment (Bederson et al. 1986) employing a panel of five motor tests to evaluate strength, balance, and adduction and flexion capability.

In a subgroup of mice the investigators will investigate the emergence of blood-brain barrier alterations 24 hours after distal MCA occlusion.

To this aim, the investigators will inject in the mouse tail vein 0.20 mL of Evans Blue dye (0.20 mg/mL), at the end of the surgery to occlude the distal branch of the MCA. 24 hours after the injection the animal will be anesthetized with an overdose of ketamine (50 mg/kg) and xylazine (9 mg/kg) and then perfused with 100 mL of PBS in order to remove the blood from the brain tissue. The brain will be then extracted and incubated in PFA 4% for one hour. Then the brain will be sectioned with a brain matrix producing approximately 15 slices 1 mm thick.

In another subgroup of mice the investigators will characterize lesion extension and astrocytes recruitment 7 days after stroke induction.

To this aim, animals will be anesthetized with an overdose of ketamine (50 mg/kg) and xylazine (9 mg/kg) and then transcardially perfused with 4% paraformaldehyde. Brains were cut using a vibrating-blade vibratome (Leica, Germany) to obtain 100 µm thick coronal sections for immunostaining of Neuronal marker, NeuN (1:1000, anti-NeuN chicken, Millipore, Germany), and Glial Fibrillary Acidic Protein, GFAP (1:1000, anti-GFAP rabbit, Abcam, United Kingdom).

The NeuN immunostaining will be performed to quantify stroke lesion volume 7 days after photothrombosis. The stroke volume for each animal will be calculated by summing up all damaged areas and multiplying the number by section thickness and by 4 (the spacing factor). Images will be acquired with a (Stemi 508, Carl Zeiss). The total volume in mm3 will be given as the mean ± standard error of all analyzed animals.

The number of Glial Fibrillary Acid Protein (GFAP) positive neurons will be analyzed using a confocal fluorescence microscope (Nikon Eclipse TE 300, Tokyo, Japan) with a Nikon Plan EPO 60X objective (NA 1.4, oil immersion Nikon, Tokyo, Japan). The investigators will focus our investigation on 4 regions of interest, i.e. the peri-infarct area (ischemic border zone), a region in the ipsilesional hemisphere distant to the stroke core (ipsilesional remote zone), a region contralateral to the peri-infarct area (contralateral ischemic border zone), and a region in the healthy hemisphere contralateral to the ischemic core (contralateral ischemic core).