Characterizing the Evolution of Lesion, Penumbra and Blood-Brain Barrier Permeability in a Photothrombotic Juvenile Rat Stroke Model Using MRI and Histology

doi:10.21203/rs.3.rs-4349847/v1

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Characterizing the Evolution of Lesion, Penumbra and Blood-Brain Barrier Permeability in a Photothrombotic Juvenile Rat Stroke Model Using MRI and Histology

https://doi.org/10.21203/rs.3.rs-4349847/v1

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Background: Pediatric stroke is a significant cause of childhood mortality and morbidity. The clinical research in this field bears certain limitations that do not exist in the pre-clinical setting. In pre-clinical research, experimental models of ischemic stroke show differences in lesion evolution and blood-brain barrier (BBB) permeability between adult and neonatal rats. However, little is known about these factors in the juvenile stage.

Aims: To characterize the evolution of the lesion, penumbra and degree of BBB permeability in a photothrombotic ring model of juvenile stroke.

Methods: The design is a mixed longitudinal and cross-sectional study. In 14 Sprague Dawley juvenile rats (weight 130-189 g), lesion, penumbra volume and blood-brain barrier (BBB) leakage were measured longitudinally on days 0, 2, and 7 following photothrombotic stroke. Magnetic resonance imaging (MRI) techniques were conducted to measure lesion and penumbra volumes (T2-weighted imaging [T2] and water restriction (diffusion-weighted imaging [DWI]) and BBB leaking (with dynamic contrast-enhanced imaging [DCE]). Histology was performed to confirm stroke (n=9) with Triphenyltetrazolium chloride staining (TTC); (n=3) for Haemotoxylin and Eosin (H&E) staining; and (n=9) Evans Blue (EB) staining to assess BBB permeability.

Results: We found the penumbra volume to be larger and better delineated on MRI and histology in the acute compared to the subacute and chronic stages, and the lesion to be smaller in volume, increasing over time following same time trajectory. The BBB was most compromised at the hyperacute stage (day 0) and decreasingly, yet persistently, disrupted to day 7.

Conclusions: Our in vivo and ex vivo findings provide insight into the evolution of stroke and could serve as a study model to test blood-brain barrier stabilization agents in the pediatric setting.

Biological sciences/Neuroscience/Blood brain barrier

Health sciences/Neurology/Neurological disorders/Stroke

Health sciences/Health care/Medical imaging/Magnetic resonance imaging

Health sciences/Health care/Paediatrics/Paediatric research

Photothrombosis

stroke

MRI

rats

histology

Blood-brain barrier permeability

Stroke in childhood remains a major cause of significant lifelong neurological impairments,[1–4] and still ranks as one of the top ten causes of death in childhood [5, 6]. Research has revealed childhood stroke incidence, treatment practices, risk factors and neurological outcomes[1–3, 7–9] in the past couple of decades. There remains, however, a critical gap in our understanding of the mechanisms of stroke in the developing brain, especially in stroke that occurs beyond the perinatal or neonatal period (> 28 days of life). In childhood stroke, motor deficits are more common (68%), as are cognitive and language deficits, with the impact lasting many decades[3, 8, 9]. To improve these outcomes, research is needed to understand the basic physiological mechanisms of injury to the developing brain.

A critical physiological mechanism contributing to injury in adult stroke is the blood-brain barrier (BBB) breakdown. Important insights about the mechanisms of injury to the BBB can be revealed in experimental stroke models using advanced neuroimaging. Evidence in adult stroke shows that the barrier becomes disrupted and permeability increases [10] in contrast to rat models of neonatal stroke, where the BBB is more intact [11]. It is not known, however, the response of the BBB is following stroke in childhood and it may influence neurological outcome. This information is necessary to advance targeted neuroprotection in early intervention strategies.

A primary goal of neuroprotective strategies in treating stroke is salvaging viable brain tissue as early as possible. Viable tissue refers to the tissue zone at risk of infringing ischemic damage surrounding the infarct that is hypo-perfused, called the penumbra[12]. A distinctive association has been established between rescuing the penumbra and improving outcomes [13, 14][ Recent evidence also shows an association between the penumbral profile and BBB disruption. In a study by Heidari et al. (2020), diffusion and perfusion-weighted imaging was used to measure core infarct, penumbra, and BBB disruption in 222 adult stroke patients. They found that patients with more significant amounts of preserved ischemic tissue had a favourable penumbral profile and less severe BBB disruption. These patients had less injury to the penumbra and the core infarct[15] A synergistic relationship between the penumbra and the BBB is foreseeable, given the dynamic changes in the pathophysiological environment during acute stroke. However, this relationship has not been studied in childhood acute ischemic stroke.

Insight into the consequences and mechanisms of acute stroke, such as the viability of the penumbra and the integrity of the BBB, has been challenging to study in childhood stroke. This is partly due to the delay in diagnosis in children since clinical presentations often mimic other diseases[16]. Standardized neuroimaging protocols in pediatric stroke also need to be developed. In addition, ethical considerations of using contrast agents in children to evaluate the BBB are problematic due to reports of concentration-dependent gadolinium deposits found in the brain [17]. Together, these issues limit the knowledge about critical pathophysiological consequences of stroke in the acute setting. The design of treatment studies in children with stroke is hindered by this lack of knowledge, including if and how long there is viability of the penumbra and if, when and how long the BBB is integrity in children following stroke. It is important, therefore, to consider the insights about these processes revealed in experimental models of childhood stroke.

One of these models involves inducing ischemic cortical lesions using photothrombosis, a method using a photoactive dye and light irradiation at a specific wavelength to a defined area on an intact skull[18] that then causes platelet adhesion and aggregation by the release of free radical oxygen. Thrombi then forms and interrupts local cerebral blood flow. This technique produces substantial local vasogenic and cytotoxic edema comparable to that seen in human ischemic stroke [19]. In this model, it is difficult to determine the boundaries of the penumbra. However, in a modified[18] version of this model, a ring-shaped filter provides a central area within the lesioned cortex that is not optically thrombosed, and this area is considered the penumbra. The ‘ring’ in the model facilitates a reproducible study of the penumbra, where the potentially compromised tissue lies within, rather than perifocal, to an ischemic locus. The pathophysiological mechanisms involved in the evolution of ischemic damage are considered characteristic of the evolution of clinical thromboembolic stroke with penumbra. In addition, the size of infarction is easy to establish, and boundaries are delineated, making it highly advantageous for precise lesion/penumbral characterization in controlled brain regions [20]. For these reasons, we used this photothrombotic model in the current study.

The objectives of our study were to 1) quantify the extent of lesion volume and penumbra over time and 2) assess and quantify the degree of BBB disruption over time in a photothrombotic juvenile model of stroke with in vivo MRI and histology using a combined longitudinal and cross-sectional study design.

Study overview

A combined longitudinal and cross-sectional design (summarized in the flow chart, Figure 1.) was used to investigate the effects of photothrombotic ring model of stroke in juvenile rats. In the longitudinal study, the evolution of lesion and penumbra volume, diffusion, and BBB permeability was evaluated on day 0 (d0), day 2 (d2) and day 7 (d7) following stroke induction using in-vivo MRI. A cross-sectional histology study complemented this to corroborate the MRI results.

Figure 1. Experimental Workflow

Figure 1 -For cross-sectional evaluation, H&E staining was conducted on 3 rats (n=1 per time point), EB staining was conducted on 9 rats (n=3 per time point), and TTC staining was conducted on 9 rats (n=3 per time point). *The rats evaluated for TTC on day 7 were used from the day 7 longitudinal MRI subgroup. For longitudinal evaluation of the stroke, T2W and DWI were conducted on n=14 rats on Days 0, 2, and 7. DCE MRI was conducted longitudinally on a subset of seven rats on Days 0, 2, and 7, however, three of the contrast injections failed on day 7 (day 0 n=7, day 2 n=7, day 7 n=4).

Subjects

Thirty-five Sprague Dawley male rats (mean age five weeks, weight range 130-189 g) were used for the combined longitudinal and cross-sectional studies. Before surgery, rats are paired and housed in large cages under a 12-hour light/dark cycle with temperature control (22 °C) and fed ad libitum. All procedures were approved by The Animal Care Committee at the Hospital for Sick Children under the Canadian Council of Animal Care guidelines. Reporting of this work complies with Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines [21].

Photothrombotic Stroke Induction

Prior to induction, anesthesia was induced using 5% isoflurane mixed with a flow rate of 1–2 L/minute of oxygen, maintained at 1.5–2.5%, and followed by intraperitoneal injection of slow-release Buprenorphine (0.03 mg/kg, stock concentration of 0.3 mg/mL diluted into 0.01 mg/mL with 0.9% saline) as an analgesic. The hair on the skull was shaved, and the skin was prepared by disinfecting with 10% betadine (povidone-iodine) followed by 70% isopropyl.

The surgical procedure for photothrombotic stroke was induced in all 35 rats using a ring-shaped lesion. The lesion was created using a hollowed dark polyvinyl chloride mask of 5 mm in diameter with a thickness of 0.5 mm and a second circular mask with a radius of 3mm central to three identifiers (midline, lambda, and bregma) to create an annular ring size of 2mm. The masks were positioned on the skull to restrict the illumination to only the ring-shaped cut-out[18] (Figure 2).

Figure 2

Figure 2 (a) Surgical setup prior to positioning the fibre optic source in the designated area. (b) The 3-mm-diameter circular mask is placed on the skull and is surrounded by another mask with a 5-mm-diameter hollowed circle to create a ring with an annular size of 2mm. The fibre optic will be positioned perpendicularly to the skull surface, with the 3-mm-diameter mask at its centre.

A fibre optic cold light source (SCHOTT MLS, Germany) was then positioned perpendicularly on the entire surface of the exposed area. Rose Bengal dye (Sigma- Aldrich, USA; 2 ml/kg, concentration: 10 mg/ml saline) (34,35) was injected through the tail vein and circulated for 5 minutes, followed by illumination at 100% light intensity for 20 minutes. Following the illumination period, the masks were removed, and the surgical site was sutured. The rats were left to recover under O2 with body temperature monitoring until conscious. They then rested for 2 hours post-op (i.e. the end of light illumination) before use for longitudinal or cross-sectional studies. Three of the 35 rats perished due to complications, leaving 32 for the remaining research.

Longitudinal MRI Assessment

A longitudinal evaluation was conducted with a subset of rats (n=14), with MRI performed on days 0, 2, and 7 post-ischemic stroke induction to evaluate the hyperacute, sub-acute and chronic stages of stroke (see timeline in Figure 3).

Figure 3

Figure 3 - Timeline of longitudinal study following photothrombosis. For animals allocated to Day 0 time point, MRI was conducted 2 hrs post-op ('post-op' determined as the end of light illumination). For animals assigned to Day 2 time point, MRI was conducted 46 hrs post-op, and lastly, for animals allocated to Day 7 time point, MRI was conducted 166 hrs past-op, followed by sacrifice and histology 168 hrs post-op.

Before and during MRI, rats were temporarily anesthetized with isoflurane (2% with oxygen). Breathing was visually monitored between scans to ensure correct isoflurane levels. Body temperature was maintained by positioning a heat-conducting blanket around the rat’s body. Imaging was conducted using a 3.0T MRI system (Philips Achieva 3.0T TX) equipped with an 8-channel wrist coil with rats in a prone position. The MRI protocol consisted of T2-weighted (T2W) and diffusion-weighted imaging (DWI) to characterize the lesion and penumbra evolution and dynamic contrast-enhanced (DCE) imaging sequences to measure BBB permeability. The T2W protocol consisted of a standard 2D turbo spin echo acquisition (TR/TE = 3,000/98 ms, FOV = 100 x 85 mm², Matrix=168x142, pixel size= 0.28x0.28 mm, 5.6 pixels per mm, FA=90˚, number of slices=8, slice thickness=1 mm, number of slices=8). DWI images were obtained with a 2D turbo spin echo acquisition with an additional set of diffusion gradients (TR/TE=801/86 ms, FOV=100x85 mm², Matrix=168x142, pixel size= 0.18x0.18 mm, 3.52 pixels per mm, FA=90˚, slice thickness=2 mm, number of slices=6, b=0, 1000). The DCE MRI sequence consisted of a T1-weighted dynamic 3D gradient echo acquisition was used (TR/TE=6.3/2.2 ms, temporal resolution=6.1 s, FOC=100x85 mm², Matrix=168x142, number of slices=10, number of dynamics per slice=40, slice thickness=1 mm, volumes=36, time=4 min 20 sec). Gadolinium-DTPA was injected through the tail vein at a rate of 60 μL over 10-15 seconds beginning approximately 25 seconds after the start of the DCE acquisition.

Fourteen rats underwent MRI, including T2W and ADC, and a subset (n=7) also had DCE imaging for all time points (see flowchart in Figure 1).

Sacrifice: Following imaging on day 7, all rats were sacrificed using a guillotine where the head was detached from the rest of the body at the neck area. Three out of 14 sacrificed rats were also used for histology (TTC staining) to minimize the number of rats for the cross-sectional study. These are referred to as (+3)* in the cross-sectional study section (see Figure 1 and * below in Cross-Sectional Study Assessment).

MRI Analysis: MRI data were transferred to an independent workstation and analyzed offline. The restriction of water diffusion in the lesion was measured with DWI, BBB permeability in and around the lesion with DCE MRI, and lesion volume was quantified from T2W imaging (as outlined in the next section).

DWI: Apparent diffusion coefficient (ADC) maps were calculated using a least-square fit of the acquired signal [22] at the two b-values (b=0, 1000) for each animal. The ADC maps were created using MATLAB v.7.11 (Mathworks, Natick, MA, USA). ADC values were calculated based on three regions of interest (ROI): the lesion (LN), the penumbra (PN) and the homologous area in the contra-lesional hemisphere (ie. healthy non-lesioned tissue) (CH) (units: x10^-6 mm²/s). The ADC maps were compared to their corresponding high-resolution T2W slices to localize these regions. ADC values were calculated in ROIs drawn on the LN and PN.

DCE MRI: Permeability maps were generated on a pixel-by-pixel basis (expressed as permeability surface area product (KPS) in units: mL/100 g/min) in MATLAB v.7.11 (Mathworks, Natick, MA, USA) using the Patlak model [23]. The sagittal sinus generated the input function [24]. After creating the KPS map, permeability values within the lesion's high-intensity areas were outlined, calculated, and averaged on a single coronal slice. The location of the lesion was determined by comparing the DCE scans with their corresponding high-resolution T2W image (20). The permeability value of a homologous area in the CH was also calculated to control for variability between scans. The ratio between the KPS values (LN and CH) was then determined. Two independent investigators (A.K. and F.H.) conducted inter-rater reliability for DCE MRI analysis.

T2W Imaging: To calculate stroke lesion volumes, 3D Slicer image analysis platform (3D slicer, USA) using a GrowCut algorithm [25,26] was used. Two independent investigators calculated the total lesion volume, including the penumbra and lesion, across all slices. The ratio between the total lesion volume and brain volume was calculated for each rat to measure total brain volume. Each rat's average relative lesion volume was then calculated per time point.

Cross-Sectional Assessment

Cross-sectional evaluation was conducted in a subset of rats (n=18 + 3*). In this cohort, triphenyl tetrazolium chloride (TTC) staining was performed on 9 (6 + 3*) rats on days 0, 2 and 7 (n=3 for each time point) post-ischemic stroke induction to verify the presence and size of the lesion and penumbra. H&E staining was performed on three rats on days 0, 2 and 7 (n=1 for each time point) to examine further stroke characteristics, including the pathophysiological differences between the lesion and penumbra. Histological staining was conducted using EB on nine rats on days 0, 2 and 7 (n=3 for each time point) following stroke to investigate BBB permeability.

Following photothrombotic surgery, rats undergoing histology procedures (TTC, H&E and EB) were sacrificed via decapitation using a guillotine. The brains were extracted within 10 minutes by cutting through the skull posterior to anteriorly. The skull was then carefully removed, and the brain extracted, washed in iced phosphate buffer saline (PBS), placed in a brain mould and a sheet of weighing paper and put in a -20℃ freezer for 20 minutes to maintain structure. This procedure was also performed on the +3* (see Figure 1) in the TTC Staining group in the longitudinal study after sacrifice.

TTC Assay: Three rats at each time point, days 0, 2, and 7 after initiation of photothrombotic stroke, were sacrificed. The three rats in the d7 group were carried over from the d7 longitudinal study for 7 TTC analysis. The brains were extracted, and following 20 minutes of freezing time, the cerebrum was cut into 1 mm thick coronal slices and placed in 2 % TTC solution (0.3 g of TTC powder dissolved in 15 mL of 0.9% saline) for 15 min in a 37 °C incubator for the macroscopic determination of tissue viability. All slices were photographed for delineation of the infarct. TTC slides were inspected visually, where negative regions represent the necrotic and non-viable tissue (i.e. white tissue) and TTC-positive, viable tissue (pink-coloured tissue or gradient).

Haematoxylin and Eosin (H&E) Staining: After initiating stroke, three rats at each time point, days 0, 2, and 7, were sacrificed, and the brains were extracted. After 20 min of freezing, extracted brains were placed in a 1:10 diluted, buffered 10 % formalin solution (Fisher-brand, USA) and stored in a 4˚C freezer for 22 hours to dehydrate and harden the brain tissue. Following this, the slice representing the center of the infarct and penumbra was removed and processed with H&E staining.

Evan’s Blue (EB) Extravasation: Rats were fully anesthetized using 5% isoflurane with an oxygen flow rate of 1-2 L/minute. EB dye (Sigma, 10 mg/ml in saline, 2.5 ml/kg rat weight) was injected into the tail vein on days 0, 2, and 7 (n=3 at each time point) following photothrombotic lesion induction. Two hours after EB injection, the rats were anesthetized again and sacrificed, and the brains were then removed from skulls immediately after death. The brains were then sliced coronally into 1 mm sections and labelled as lesioned and contra-lesional hemispheres. The slices were then stored in -80 ˚C freezers and thawed for measurement.

To quantify EB, each slice was placed in a 3.5% formaldehyde solution, and the tissue was then pelleted with centrifugation. The concentration of EB absorbance was measured with a spectrophotometer at 620 nm against a set of EB standards. The values were then normalized against the weight of the tissue sample to generate a value of EB (µg)/brain tissue (mg). EB leakage was expressed as a ratio of the average lesion value (LN) to contralesional homologous tissue (CH).

Statistical Analysis

A one-way repeated measures Analysis of Variance (ANOVA) was used to evaluate the changes in T2W and a two-way repeated measures ANOVA was used to compare ADC changes over time. Paired t-tests were used for both DCE and EB to compare the dye extravasation levels between the lesion and contra-lesional homologous tissue. Differences in DCE and EB extravasation ratios at different time points were tested using one-way ANOVA. Post-hoc pairwise comparisons were conducted using the Tukey-Kramer test. Continuous variables were presented as average ± standard deviation (SD).

Inter-rater reliability was evaluated for the T2W and DCE measurements obtained from two independent raters (AK and FH), and intraclass correlation coefficients (ICC) were calculated. ICC values were classified as <0.41, “poor”; ≥0.41 and <0.59, “fair”; ≥0.59 and <0.74, “good,” and ≥0.74, “excellent” (43). A p-value of <0.05 was deemed statistically significant. Analyses were conducted using SAS University Edition (SAS Institute, Inc., Cary, NC).

The effects of photothrombotic ring stroke in a juvenile rat model were investigated using a combined longitudinal and cross-sectional design (summarized in the flow chart, Figure 1.) In the longitudinal study, the evolution of diffusion, BBB permeability and lesion volume was evaluated on d0, d2 and d7 following stroke induction with MRI. A cross-sectional study with histology complemented this to corroborate the MRI findings.

To monitor the evolution of lesion and penumbra with longitudinal MRI, we measured differences in diffusion (ADC), lesion volumes (T2W), and BBB permeability (DCE) on d0, d2 and d7 in the LN. We compared these values to the CH within subjects.

The mean ADC ± standard deviation values in the lesion increased from 520.01±40.07 [x10^-6mm²/s], 783.84±72.23 [x10^-6mm²/s] to 917.70±140.12 [x10^-6mm²/s], p<0.0001, respectively. In the penumbra, the mean ADC values also increased from days 0 to 2 from 503.09±66.87 [x10^-6mm²/s] to 1139.32±109.32 [x10^-6mm²/s] (p<0.001) respectively, and on d7 to 1214.39±122.41 [x10^-6mm²/s], (p=0.11). In the CH, mean ADC scores on d0 increased from mean=797.24±54.38 to mean 822.75±58.68 on d2, then decreased on d7 (775.71±79.76, p<0.0001) (see Figure 4).

Figure 4. Average ADC Values in the Lesion, Penumbra and Contra-lesional Homologous Tissue

Figure 4 Average longitudinal ADC quantification results for three different ROI on three time points. Paired t-test indicated significant difference between Day 0 and Day 2 average penumbral area (p<0.001) and no significant differences between Day 2 and Day 7 average penumbral area (p=0.11). Paired t-test indicated significant differences between Day 0 and Day 2 average lesion area (p<0.001) as well as between Day 2 and Day 7 average lesion area (p<0.01). Paired t-test indicated no significant differences between any pair of days for average ADC of contralateral areas. * Indicates p<0.01 and ** indicates p<0.001 for student’s t-test between average lesion (or penumbral area) and contralateral area.

Lesion volume ratios measured on T2W imaging differed across d0, d2 and d7 (mean=0.090±0.017, mean=0.123±0.023, and mean=0.085±0.029, respectively, p<0.001) (see Figure 5). Post-hoc pairwise comparisons indicated that lesion volumes between days 0 and 2 (p<0.05) and days 2 and 7 (p<0.05) differed significantly. However, differences between d0 and d7 did not reach significance (p=0.57). Lesion volumes were measured with excellent inter-rater reliability (ICC =0.86 [95% CI: 0.66-0.94] p<0.001).

Figure 5

Figure 5 T2W average total relative lesion volume (ratio between total lesion volume and brain volume). No significant difference in paired t-tests between Day 0 and Day 7 (p=0.57). Significant difference between Day 2 and both Days 0 and 7 (p<0.01 (*) and p<0.001 (**), respectively).

BBB permeability showed evidence of gadolinium extravasation at all three time points in each rat (see Figure 6 a and b). The average gadolinium extravasation ratios (LH: CH) on days 0, 2, and 7 were 3.25±0.15, 2.02±0.33, and 2.44±0.49, respectively (see Figure 6b). Significant differences were detected between d0, d2, and d0, d7 (p=0.0022, p=0.0243, respectively); however, no further significant differences were seen in the later time points d2 and d7 (p=0.28).

Figure 6. Evolution of Gadolinium Extravasation on DCE MRI

Figure 6 Sample longitudinal KPS maps of a rat subject. (a) Evidence of Gadolinium extravasation exists on all three time points. The average ratios of ipsilateral lesion areas to contralateral areas on Days 0, 2, and 7 were 3.25, 2.02, and 2.44, respectively. (b) Plotting the values on a graph indicated similar pattern in Gadolinium extravasation existing in subjects over the 7-day period.

Histology

TTC-negative regions were present both in the lesion and penumbra on day 0 (see Figure 7a). On day 2, TTC-negative regions persisted in both the lesion and the penumbra and were indistinguishable from one another (Figure 7b). This pattern continued to day 7 (Figure 7c).

Figure 7

Figure 7. Representative coronal TTC slice from three representative rats (one rat per endpoint). (a) Day 0 (4 hrs post-op). There is evidence of both TTC positive and TTC negative in the lesioned hemisphere. (b) Day 2 (48 hrs post-op). No evidence of TTC positive tissue, or viable tissue representing the penumbra. (c) The lesion and penumbra have further combined as TTC negative tissue in the lesioned hemisphere and is smaller in size than Day 0 and Day 2.

The EB results revealed that the highest leakage occurred on d0, followed by a reduction on d2 and persistence of leakage at similar levels on d7 (Figure 8). Leakage was significant between d0 and d2 (p<0.05).

Figure 8

Figure 8. Coronal images of EB-stained rat whole brains and slices (3 brains per time point). Three rats sacrificed on Day 0 (2-3 hrs post-op). Vast amount of EB extravasation present on the ipsilateral hemisphere in the area of direct illumination. Three rats sacrificed on Day 2 (48 hrs post-op). Eb extravasation still present, but less than that seen-on Day 0. Three rats sacrificed on Day 7. EB extravasation present at similar levels to that seen on Day 2. * Indicates the number of hours after the end of illumination.

The H&E stain results indicated evidence of neuronal hypoxia in the lesion on d0 (see Figure 9), with the penumbra still containing healthy cells (Figure 9a). Neuronal loss was visible in the lesion, with no evidence of glial proliferation. On d2, the pathology was more marked (Figure 9b) with evidence of neuronal anoxia in the lesion, an increased loss of neurons, and the presence of macrophages. There was also evidence of cytoplasmic hypereosinophilia, a condition where the number of eosinophils in the cytoplasm increases. On d7, the pathology (Figure 9c) showed a clear indication of infarction, with complete cell death in both the lesion and penumbra and a significant presence of both glial cells and macrophages in the peri-infarct areas.

Figure 9

Figure 9Representative coronal H&E-stained slice from three rats (one rat per time point). (a) Day 0 (4 hrs post-op). There is evidence of healthy cells in the penumbral area and neuronal hypoxia as well as loss of neurons in the ring lesion. (b) Day 2 (48 hrs post-op). Neuronal anoxia present in both penumbral area and ring lesion. More loss of neurons detected. Some evidence of macrophages in the peri-infarct areas. (c) Complete non-hemorrhagic infarction in both penumbral area and ring lesion. Significant presence of glial cells and macrophages.

Corroborative Findings in MRI and Histological Measures

The MRI findings (i.e. ADC and T2W measures) corroborated TTC findings, indicating characteristics of the lesion and the penumbra on d0. However, on d2 and 7d, TTC showed no evidence of the penumbra (see Figure 10). DCE measures of BBB permeability over time corroborated the corresponding EB measures (Figure 11) with no significant difference. The average DCE values on day 0 were 3.25±0.15 compared to 2.42±0.16 in EB (p=0.07). Similarly, differences were not significant on d2 (average DCE: 2.02±0.33, average EB: 1.52±0.28, p=0.22) and d7 (average DCE: 2.44±0.49, average EB: 2.22±0.39, p=0.61).

Figure 10 - MRI T2, T2W and TTC Results on Days 0, 2 and 7

Figure 10 Visual comparison of structural MRI data with their corresponding histological TTC-stained slice. Penumbral area was distinguishable from lesion area in DWI, T2, and T2W images (all three days). Penumbral area not different from lesion area on TTC slices (Days 2 and 7). * Indicates number of hours after the end of light illumination. All images have been configured to represent actual right and left.

Figure 11

Our study uses MRI to characterize the evolution of the BBB, penumbra and lesion volume in a photothrombotic stroke model in juvenile rats. Our findings show BBB permeability increases in the acute stage, followed by a successive decrease at the chronic stage following stroke. We found the penumbra volume to be larger and better delineated on MRI and histology in the acute stage than the subacute and chronic stages, and the lesion to be smaller in volume, increasing over time in the same time trajectory.

In the penumbra and lesion, diffusion restriction was significant acutely (likely due to clot formation), followed by increased diffusion in the lesion and abnormal hyper-diffusion in the penumbra. This finding likely represents vasogenic edema in the acute phase (day 0), also visible on T2W. In the subacute stage (day 2) and chronic stage (day 7), hyper-diffusivity was noted in the penumbra and the lesion, likely reflecting the further loss of neurons and necrosis in the lesion, allowing for more free movement of water within the tissue[27, 28] The increased diffusivity in the penumbra over time was also found in a study by Hilger et al. (2004) using the ring stroke model of photothrombosis[29]. However, the relative change in signal intensity was higher in our study. This may be due to the differences in age with younger rats in the Hilger et al. (2004) study, and the laser light source instead of fibre optic cold light source that differ in light intensity. We also found the lesion volume increased significantly from day 0 to day 2, representing the subacute inflammatory process post-hypoxia, including vasogenic edema. This manifested as an increase in T2 delayed signalling[29] that persisted to d7 [30]characterizing gliosis.

We found a significant increase of BBB permeability in the lesion across all time points using DCE MRI. This was corroborated in vivo with histology findings showing the persistence of BBB leakage in the stroke hemisphere across the seven days from the acute to chronic stage of stroke. However, our sample size for each time point was small. Therefore, these findings require corroboration in a larger sample size to confirm reliability.

The histology measures, TTC and H&E, used to characterize the lesion and penumbra indicated 1) the presence of both the penumbra and ring lesion on Day 0 post-ischemia and 2) the persistence of the ring lesion and complete transformation of the penumbra into necrotic tissue by Day 7 post-ischemia. This suggests that at the hyper-acute stage of ischemia, the penumbra in our model is initially a metabolically viable region characterized by impaired neuronal activity without irreversible neuronal damage due to significant acidosis [31]. This condition puts the penumbra at risk of transforming into necrotic tissue without timely therapeutic intervention.

In our model, the penumbra did not remain metabolically stable over time due to the progressive metabolic failure resulting from the ring lesion blocking the transportation of oxygen and other nutrients via the vessels to the penumbra [32][33]. Our TTC and H&E results indicated that this process was apparent by Day 2 post-ischemia, suggesting that in this model, the time to intervene therapeutically is within two days to salvage as much tissue as possible before recruitment into the infarct core.

The H&E results of our study, the appearance of phagocytes (such as macrophages) as early as Day 2 post-ischemia, followed by a substantial increase in the presence of glial cells and macrophages by Day 7, is in line with earlier photothrombotic studies using different light sources. [34, 35]. The detection of microglia/macrophages on subsequent days was expected. Previous reports have shown that astrocytes produce pro-inflammatory cytokines and chemokines in response to severe lesion insult, which recruits immune cells to clear dead tissue [36, 37].

The BBB permeability characterization using EB indicated a defined pattern of BBB permeability over a 7-day period following ischemia. The high EB extravasation on Day 0 was not surprising, as photothrombosis is known to induce a high degree of vasogenic edema at the acute stage of stroke [38], and vasogenic edema is linked with the occurrence of BBB disruption. [27, 30, 35]. The decrease in EB leakage on Day 1 and Day 2 can be attributed to the reduction in oxygen radicals that trigger inflammation in the hyperacute stage and cause vasogenic edema [39]. Our findings that persistence of BBB disruption can occur up to Day 7 is shown in previous studies [30, 40]. This can be partially attributed to the recruitment of inflammatory cells that contribute to maintaining edema [35]. Another reason for continuous leakage is that the damage caused by the lesion is substantial enough that the BBB cannot recover its function for at least up to the 7-day endpoint. Further studies are needed to study the evolution of the BBB beyond seven days following stroke in the juvenile stage of development.

Our MRI and histology findings were corroborated, indicating that these in vivo techniques complement the measures in the longitudinal study of BBB integrity following stroke. Our structural MRI measures were comparable to TTC findings of the lesion over seven days in that both indicated an evident lesion and the persistence of the lesion over time. This is like earlier reports that show the comparability of DWI and T2W imaging techniques with TTC stains regarding infarct size[40]. The penumbra, however, on MRI and TTC was only detected on Day 0. We suspect that TTC results did not differentiate the lesion and penumbra by Day 2 because inflammatory cell infiltration harboured potentially intact mitochondria at the penumbra within 24 hours of ischemia, keeping that area also unstained[41]. Given this limitation in TTC staining, MRI was effective in confirming the contrast between the lesion and penumbra throughout the seven days.

The visual comparison between the DCE KPS maps and their corresponding EB-stained slices showed similarities in the location of contrast dye extravasation. The areas that differed visually in dye extravasation could be attributed to the duration of time allowed for the extravasation to occur. For EB staining, the dye was allowed to circulate in the body for 2 hours prior to sacrifice. On the other hand, to acquire DCE scans, gadolinium circulates for less than 5 minutes. Studies suggest that an EB circulation of at least 1 hour (ideally 2 hours) is optimal [40, 42]. Therefore, it was not possible to match the optimal EB circulation time to the Gadolinium circulation time of 5 minutes. Despite the differences in circulation times, the visual patterns between the two techniques remained similar for all three time points. Furthermore, the quantified cross-sectional comparisons of the two techniques also indicated a similar pattern of BBB disruption over time, with no significant differences between any pair of respective time points. Thus, we showed that the two techniques are independently comparable. A minor issue with this comparison is that because both techniques were not performed on the same rats, our DCE data could not be statistically validated by our EB data. However, previous literature has validated DCE results with EB results in adult ischemic stroke rats [43] [10], and we do not suspect this to be any different in our younger rats. By demonstrating the persistence of BBB disruption in the juvenile stage of development using in vivo and ex vivo techniques, we have characterized BBB disruption in a developmental stage where knowledge of BBB post-ischemia is lacking. This suggests that DCE MRI can be integrated into future pre-clinical and clinical studies to examine the therapeutic effects of drugs in stabilizing BBB integrity after ischemic stroke.

Another important consideration revealed in experimental studies of stroke is the changes in BBB permeability from the acute, sub-acute and chronic phases following stroke ictus. Several longitudinal animal studies have shown an ‘open-close-open’ biphasic pattern in which the BBB has increased permeability at the first stage, followed by a return to normal values and a second permeability increase [44–46]. More recent literature points to a more continuous opening of the BBB with biphasic permeability peaks but without total closing [47–49]. Very few human studies have focused on studying this evolution[11, 50, 51] and do not include the pediatric population. Furthermore, the existing studies do not offer clear and collective evidence on the magnitude of this opening in the different phases. A quantitative assessment of the evolution of BBB disruption could add valuable information to the evaluation of children with AIS, such as extending the time window for treatment interventions or using different strategies at various stages.

To our knowledge, this is the first time BBB disruption has been characterized in juvenile rats in a photothrombotic ischemic stroke model. We found decreased but persistent BBB leakage up to Day 7. Our results are meaningful in many ways. The photothrombosis model used for stroke induction has many advantages, as it can be easily manipulated, is highly reproducible, allows for variable lesion/penumbra sizes and has a very low mortality rate [30, 38]. There are, however, differences between the mechanisms causing stroke in this model and those in human arterial ischemic stroke. In photothrombotically-induced stroke, all vessels within the illuminated area are occluded by thrombi that are formed from oxygen intermediates, inducing endothelial cell membrane peroxidation. This differs from human arterial ischemic stroke, where usually, one or two large arteries are blocked by thrombi[52]. Another difference in mechanisms is that at the hyperacute stage, human arterial ischemic stroke primarily involves the occurrence of cytotoxic edema followed by vasogenic edema after several hours, not simultaneously as it does in the photothrombotic model[38]. There are known inherent differences in animal models compared to human models of stroke. The advantages of this photothrombotic surgical approach are the low complication rate, the high survival rate of the animals (over 90%), and the high reproducibility of the ring lesion in an anatomically predefined region at risk.

We characterized the evolution of ischemic stroke in a ring model of photothrombosis in juvenile rats. Since stroke evolution has not been well studied in this stage of development, our study is among the first pre-clinical studies examining the evolution of the lesion and penumbra and BBB permeability in juvenile rats after ischemic stroke. Our results showed that the penumbra eventually transforms into infarct by Day 7 post-ischemia. Using the photothrombotic model, we found that the BBB is highly permeable at the hyper-acute stage, with the permeability reducing but persisting over time up to Day 7. Since this model is reasonably straightforward and results in high reproducibility, co-opting the different types of available in vivo (MRI) and ex vivo techniques can effectively examine the longitudinal effects of therapeutic drugs in reducing lesion size and stabilizing the BBB.

Animal Ethics: The following Animal Use Protocol was obtained at Sickkids for the work presented in our manuscript:

AUP#: 1000038848

AUP Title: Investigating blood-brain barrier permeability in Sprague Dawley rats as an experimental model of childhood stroke using advanced MR imaging.

Funding: This work was supported by a training grant from Brain Canada (6200300068), funding from NSERC (6280100041) and the Stroke Imaging Lab for Children at Sickkids.

Data Availability: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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No competing interests reported.

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Characterizing the Evolution of Lesion, Penumbra and Blood-Brain Barrier Permeability in a Photothrombotic Juvenile Rat Stroke Model Using MRI and Histology

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