Acute ischemic STROKE – from laboratory to the Patient’s BED (STROKELABED): A translational approach to reperfusion injury. Study Protocol

2.1.1 Clinical setting

All consecutive patients with AIS of the anterior circulation territory who received revascularization therapies (systemic thrombolysis, endovascular treatment) at the Stroke Unit of the Careggi University Hospital from October 1, 2015 to May 31, 2020 are eligible for inclusion (Table 1). After gaining informed consent, individual patient data will be recorded in a dedicated database including demographics and clinical features as well as blood chemistry and neuroradiological parameters. For pathogenesis definition, we will use TOAST classification [10] that denotes five subtypes of ischemic stroke: (1) large-artery atherosclerosis, (2) cardioembolism, (3) small-vessel occlusion, (4) stroke of other determined etiology, and (5) stroke of undetermined etiology. The frequency of various stroke subtypes will be provided in the study population descriptive analyses. Given the retrospective design of the study, it will be possible to evaluate the case-fatality rate, including in-hospital mortality. Blood biomarkers and neuroimaging features will be collected on arrival at the hospital and repeated 24 h following intervention will be included in the analysis. The functional outcome will be assessed 3 months after stroke according to the mRS [9] by outpatient visit or phone interview.

2.1.2 Laboratory protocol

The protocol previously documented [11] will be followed, where blood samples were routinely collected in AIS patients, before recanalization treatment and after 24 h. Whole venous blood is collected in tubes without anticoagulant. Tubes are centrifuged at room temperature at 1,500 g for 15 min, and the supernatants are stored in aliquots at −80°C until measurement of inflammatory markers, Matrix Metalloproteinases (MMPs) and Tissue inhibitors metalloproteinases (TIMPs). Levels of different inflammatory markers (IL-4, IL-6, IL-10, IL-12, alpha2-macroglobulin, Myeloperoxidase, CXCL-8, CXCL10 VEGF, ICAM-1, VCAM-1), zinc-dependent endopeptidases and their inhibitors (MMP-2, -3, -7, -8, -9, TIMP-1, -2, -3, -4 and EMMPRIN) will be determined using Bio-Plex suspension array system and R&D Kits (R&D System, Milan, Italy) following manufacturer’s instructions.

2.1.3 Neuroimaging protocol

A complete neuroimaging assessment, including non-contrast CT (NCCT), CT-angiography (CTA), and CT-perfusion (CTP) is performed at hospital arrival before acute interventions, according to guidelines in place at the time of treatment [12,13]. All patients undergo a 24-h follow-up NCCT. All available neuroimaging will be exported in DICOM format in order to be processed and analyzed and then will be stored in the electronic database.

The presence of HT will be defined according to the European Cooperative Acute Stroke Study (ECASS) II on 24 h NCCT brain scan. HI1: Hemorrhagic infarction type 1; HI2: Hemorrhagic infarction type 2; PH1: Parenchymal hematoma type 1; and PH2: Parenchymal hematoma type 2 [14]. All uncertain cases will be resolved by an experienced neuroradiologist.

Two trained neurologists will independently evaluate each patient’s 24-h NCCT and assess the presence of CE classified according to CE classification used by the Helsinki Stroke Thrombolysis Registry Group [15], where

• CED1 = Focal brain swelling up to one-third of the hemisphere;

• CED2 = Focal brain swelling greater than one-third of the hemisphere;

• CED3 = Brain swelling with midline shift;

• NONE = absence of cerebral edema.

The occurrence of CE and HT after AIS will be also investigated by the means of innovative neuroimaging evaluations to obtain a quantitative and reliable measure, based on the AD concept: both CE and HT imply accumulation of fluid within cerebral tissue creating a brain swelling, which distort normal anatomy of brain itself. Corrected infarct and AD volume will be calculated [16] according to the following outline methodological steps all using FMRIB Software Library v6.0 software (created by the Analysis Group, FMRIB, Oxford, UK):

1. The ischemic lesion on the 24 h NCCT will be masked independently by two neurologists. The Dice similarity index will be calculated for the two masks. For those with a Dice similarity index greater than 0.8, an intersection mask will be generated for subsequent analysis. For those with a Dice similarity index less than 0.8, the mask will be reviewed and finalized by a staff neuroradiologist.

2. The follow-up NCCT scan will be registered to the baseline NCCT scan using FMRIB’s Linear Image Registration Tool, and, FMRIB’s Nonlinear Image Registration Tool [17]. The lesion masks will be resampled on the registered follow-up scans using either the nonlinear warp or the linear matrix generated with a threshold of 0.5 applied. All masks and registrations will be individually validated to ensure optimal registration.

3. Final infarct volume will be defined as the volume of the infarct mask after nonlinear registration. AD will be calculated as the difference in the volume of the infarct masks generated after linear and nonlinear registration to the baseline NCCT.

2.2.1 Months functional outcome

The mRS will be adjudicated in all enrolled patients 3 months following stroke onset [9] either in person from clinic visit or by telephone interview where the patient is unable to attend a clinic in person. The occurrence of CE. Time frame: NCCT brain scans acquired at 24 h after the stroke event (see Neuroimaging protocol). The occurrence of HT. Time frame: NCCT brain scans acquired at 24 h after the stroke event (see Neuroimaging protocol).

2.3.1 Mouse model of MCA occlusion and recanalization

In this project, we will apply a newly developed mouse model of stroke targeted to the para-orbital branch of the MCA [18]. Mice will be anesthetized with ketamine (50 mg/kg) and xylazine (9 mg/kg), and body temperature will be monitored and maintained at 37°C with a heating pad (ThermoStar Temperature Controller, RWD, USA). To induce the occlusion of the blood vessel, we will perform a small craniotomy on the squamosal bone and then, after an intraperitoneal injection of the photosensitive dye Rose Bengal, we will illuminate the MCA with a green laser. The dye activation will induce the formation of oxygen free radicals, causing endothelial damage that triggers platelet aggregation and clot formation. After defined periods of stable occlusion (30, 60, and 90 min), we will illuminate the occlusion site with a UV LED capable of disrupting the fibrine bounds inside the clot resulting in the recanalization of the blood vessel.

2.3.2 Behavioral evaluation

Functional impairment induced by the occlusion of the distal MCA with and without recanalization will be assessed according to a panel of different motor tests to assess strength, balance, adduction, and flexion capability modifying the protocol applied by Balbi and collaborators [19], based on a standardized qualitative assessment for measuring the degree of motor impairment [20]. The experimental protocol will include three-time points: pre-stroke, 24 h, and 5 days after the damage. This will allow the longitudinal comparison of motor performance in the pre-stroke, acute, and subacute phases.

2.3.3 Characterization of BBB leakage

We will assess the presence of alterations in BBB permeability 24 h after the insult through ex vivo experiments. To this aim, we will inject the Evans Blue dye in the mouse tail vein, characterized by a very high affinity for serum albumin. In physiological conditions, serum albumin cannot pass through the BBB. Where BBB integrity is lost, the Evans Blue dye will cross into the brain parenchyma and stain the brain tissue.

2.3.4 Investigation of tissue-damaged extension

We will evaluate the presence of necrotic tissue 7 days after ischemia through ex vivo experiments, as previously described by Conti et al., [21]. After anesthetizing the animals with an overdose of ketamine (50 mg/kg) and xylazine (9 mg/kg), mice will be transcardially perfused with 4% paraformaldehyde to allow tissue fixation. The brain will then be cut into 100 µm thick coronal sections with a vibrating-blade vibratome (Leica, Germany) to perform immunostaining of the Neuronal marker, NeuN (1:1,000, anti-NeuN chicken, Millipore, Germany). The stroke volume for each animal will be calculated by summing up all damaged areas, characterized by the absence of immunostaining, 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 value ± standard error of all analyzed animals.

2.3.5 Reactive astrogliosis characterization

The emergence of the acute inflammatory response associated with the upregulation of the glial fibrillary acidic protein (GFAP) in astrocytes will be assessed. The number of 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). We will focus our investigation on four regions of interest, i.e., the peri-infarct area (ischemic border zone), a region in the ipsilesional hemisphere distant to the stroke core (remote zone in the ipsilesional hemisphere), a region in the contralateral hemisphere homologous to the ischemic core, and a region in the contralateral hemisphere homologous to the periinfarct area.

2.4.1 Clinical section

We will divide the study cohort into two groups according to the outcome to be explored: functional outcome (mRS0-2 vs mRS 3-6), CE occurrence (CE yes vs CE no), relevant HT occurrence (HI-2, PH-1 or PH-2 vs HI-1 or no HT). Data will be presented as mean value (standard deviation, SD), median (interquartile range, IQR), and number (percentage) as appropriate. The levels of all circulating biomarkers will be recorded as continuous variables. We will describe the general characteristics of the population and use Student’s t-test, the Mann–Whitney U-test, and Pearson’s chi-squared test, as appropriate, to test the differences between groups. Moreover, logistic regression analysis will be used to calculate odds ratio (OR) for each outcome under investigation with 95% confidence intervals (CI) adjusting for age, gender, stroke severity (according to the National Institutes of Health Stroke Scale, NIHSS), AD, BBB leakage (according to CTP K trans value > 0.63), history of hypertension, diabetes, atrial fibrillation, taking anticoagulants or antiplatelets, type of treatment (Thrombolysis rt-PA, endovascular, and combined). The presence or absence of systemic inflammation will be assessed by a proxy measure: current adherence to corticosteroid therapy. Analyses will be restricted to patients with complete data. The study design is retrospective and the data collection process is embedded in the usual clinical routine for AIS patients at the single center of Careggi University Hospital of Florence, Italy; therefore, it is likely that missing data could be few and completely at random, resulting in unbiased estimates. For all statistical analyses, a two-sided p-value < 0.05 will be considered as statistically significant. Analyses are performed using IBM SPSS Statistics for Windows, Version 23.0 (IBM CO. Armonk, NY).

2.4.2 Preclinical section

All the analyses of both in vivo and ex vivo experiments will be blinded. Moreover, all the data will be independently evaluated by the two researchers who perform the experiments and the analysis. Results will be considered statistically significant if their corresponding P value will be less or equal to 0.05. OriginPro software (OriginLab Corporation) will be used for all other statistical analyses. For all ANOVAs that prove statistically significant, multiple comparisons among time points and different regions of the cortex will be assessed using the ANOVA Repeated Measures followed by a post hoc Tukey HSD test.

Mice will be housed in clear plastic cages under a 12 h light/dark cycle and given ad libitum access to water and food. We will use a transgenic mouse line, C57BL/6J-Tg(Thy1-EGFP)MJrs/J, from Jackson Laboratories (Bar Harbor, Maine USA).

1. Ethics statement clinical research: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee. The study has been approved by the local Ethics Committee (ethics committee registration number: Comitato Etico Area Vasta Centro [CEAVC] 16923_oss).

2. Informed consent statement: Informed consent has been obtained from all individuals included in this study.

3. Ethical approval: The research related to animals’ will be complied with all the relevant national regulations and institutional policies for the care and use of animals. All procedures involving mice will be performed in accordance with regulations of the Italian Ministry of Health authorization n. 723/2019.