CD36 deficiency reduces chronic BBB dysfunction and scar formation and improves activity, hedonic and memory deficits in ischemic stroke

Abstract

Ameliorating blood–brain barrier disruption and altering scar formation dynamics are potential strategies that may improve post-stroke recovery. CD36 is a class B scavenger receptor that plays a role in innate immunity, inflammation and vascular dysfunction and regulates post-stroke injury, neovascularization, reactive astrogliosis and scar formation. By subjecting WT and CD36KO mice to different MCAo occlusion durations to generate comparable acute lesion sizes, we addressed the role of CD36 in BBB dysfunction, scar formation and recovery. The majority of stroke recovery studies primarily focus on motor function. Here, we employed an extensive behavioral test arsenal to evaluate psychological and cognitive endpoints. While not evident during the acute phase, CD36 deficient mice displayed significantly attenuated BBB leakage and scar formation at three months after stroke compared to wild-type littermates. Assessment of motor (open field, rotarod), anxiety (plus maze, light-dark box), depression (forced swim, sucrose preference) and memory tests (water maze) revealed that CD36 deficiency ameliorated stroke-induced behavioral impairments in activity, hedonic responses and spatial learning and strategy switching. Our findings indicate that CD36 contributes to stroke-induced BBB dysfunction and scar formation in an injury-independent manner, as well as to the chronic motor and neurophysiological deficits in chronic stroke.

Stroke is a major cause of mortality and motor and cognitive impairment in the human population, yet viable therapeutic interventions are limited. Over the past few decades, preclinical research has focused on the development of neuroprotective treatment strategies. Unfortunately, neuroprotective agents that have shown great promise in preclinical studies failed to show significant efficacy in clinical trials. The attention has thus shifted to strategies that aim to augment post-stroke recovery processes. The modification of post-stroke immune responses, neovascularization, scar formation and tissue milieu to ensure an environment that will enhance endogenous recovery processes represents a novel therapeutic approach.^1,2

Post-stroke blood–brain barrier (BBB) impairments, reactive astrogliosis and scar formation have a significant impact on the recovering brain tissue. Disrupted BBB integrity is a key contributor in stroke-induced tissue damage. In experimental stroke, regions with BBB leakage gradually evolve to infarctions. Blocking BBB dysfunction protects parenchyma and improves functional outcome.³ These findings indicate that rather than being solely a consequence of injury, BBB dysfunction also contributes to it. Recent evidence also suggests that post-stroke BBB disruption is not transient but persists to the chronic phases of stroke,^4,5 which negatively influences functional recovery.⁶ Reactive astrogliosis in response to stroke leads to glial scar development that serves to seal and limit the spread of inflammation.^7,8 Attenuation of reactive astrocytes leads to impaired scar formation and increased tissue damage.⁹ Conversely, studies in spinal injury models demonstrate that scar tissue impedes reconstitution and recovery of axonal connections, potentially hampering post-stroke recovery.^10,11 Taken together, interventions that can ameliorate BBB impairment or modulate scar formation have the potential to augment post-stroke spontaneous recovery processes.

CD36 is a class B scavenger receptor that plays a role in innate immunity, inflammation and vascular dysfunction. It is expressed on platelets, endothelial cells, microglia, astrocytes, adipocytes and peripheral monocytes/macrophages. While its expression is low in the healthy brain, it is upregulated after stroke primarily via infiltrating blood-derived monocytes/macrophages.¹² We have previously reported that CD36 contributes to acute ischemic damage by enhancing inflammation and reactive oxygen species.^13–15 In line with these findings, knocking out CD36 reduces acute lesion size in stroke models.¹⁵ Despite its apparent detrimental role in acute inflammatory phase, CD36 may also exert a beneficial role during the resolution phase of inflammation by mediating phagocytosis.¹⁶ As an angiostatic mediator, CD36 regulates post-stroke neovascularization,¹⁷ reactive astrogliosis and scar formation.¹² CD36-deficient mice display reduced scar formation around the infarct core at acute stroke,¹⁸ which may modulate repair/recovery processes. Due to its diverse roles in stroke pathogenesis/repair, understanding the effects of CD36 on long term recovery is important to determine its potential as a therapeutic target.¹²

A vast majority of preclinical studies that assess behavioral endpoints focus solely on motor deficits. Stroke patients, however, display a wide range of cognitive and emotional disturbances such as anxiety, depression, memory impairments, and dementia.¹⁹ The number of studies aiming to model these disturbances and their recovery profiles, as well as investigating whether they can be ameliorated via therapeutic approaches, is very limited.²⁰

The purpose of this study was to define the role of CD36 on BBB integrity, scar formation and motor, neuropsychological and cognitive outcomes in wild type and CD36 deficient mice during acute and recovery phase of stroke. Here, we report that CD36 contributes to stroke-induced BBB permeability and scar formation in the brain, and CD36 deficiency is associated with the improvement of neuropsychological and memory deficits in mice.

Methods

Animals

The use of animals and performance of procedures was approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine. WT and CD36 KO animals were bred and housed at Burke Neurological Institute Animal Facility. All animals were housed under controlled environment conditions (22 ± 3°C, 40% to 70% humidity, 12-h light/dark cycle) with ad libidum access to standard chow and water. CD36 KO mice (>10 times backcrossed with C57BL/6, 99.9% C57BL/6 background) were obtained from homozygote breeding pairs. Animals were genotyped as previously described.²¹ Both male and female mice were used as a biological variable. A total of 78 male and female mice (3–4 months of age) were used: 20 in the acute study and 58 in the behavior baseline, 50 of which were used in the chronic study. Eleven animals were excluded due to surgical complications and/or not meeting criteria for our stroke model. All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and reported in accordance to ARRIVE guidelines. Animals were assigned to sham or MCAo groups via simple randomization (coin flip), and researchers performing the experiments were blind in respect to the groups.

Transient middle cerebral artery occlusion

Middle cerebral artery occlusion was performed as described previously.^22,23 Briefly, mice were anesthetized using a mixture of isoflurane/oxygen/nitrogen. After exposure of the right external carotid artery, a 6-0 Teflon-coated black monofilament surgical suture (Catalog No. 602556PK10, Doccol, Redland, CA, USA) was inserted and advanced into the internal carotid artery and wedged into the circle of Willis to obstruct the origin of the middle cerebral artery. To ensure comparable lesion sizes between WT and CD36 KO animals, the filament was left in place for either 30 (WT) or 40 (CD36 KO) min, then withdrawn.¹⁸ The cerebral blood flow in the center of the ischemic territory was monitored by Laser-Doppler flowmetry (Periflux System 5010; Perimed, Järfälla, Sweden). Animals exhibiting reduced cerebral blood flow >80% during MCAO and restored cerebral blood flow to 80% of baseline by 10 min following reperfusion were included in the study.

Tissue preparation

Brains were removed, frozen, and sectioned using a cryostat (Leica). Because the infarct typically spans ∼6 mm rostrocaudally starting from +2.8 mm from Bregma and extending to −3.8 mm, we used an unbiased stereological sampling strategy to collect tissue in the entire region that included the infarct.^22,24 Tissue sections were collected serially at 600 μm intervals for the analysis of infarct volume and sub-region volume analysis.

Histological assessments

Acute outcome

Infarct volume at three days in the hemisphere and sub-regions was determined as previously described.^24,25 Infarct area was measured on coronal sections serially collected at 600 μm intervals. Infarct volume was calculated by multiplying infarct area (mm²) by the 0.6 mm distance between 13 serial sections. Integrated infarct volume was adjusted for swelling by subtracting the volume difference between hemispheres or subregions. Percent hemispheric swelling was calculated from the hemispheric volume difference divided by the volume of contralateral hemisphere.²⁴

Atrophy

For chronic studies, the extent of atrophy in the hemisphere and sub-regions was determined by integrating the volume from 13 serial sections containing hemisphere or sub-regions (striatum, cortex, hippocampus) from both hemispheres and expressing as a ratio to the contralateral side.

Scar volume and density assessment

Since GFAP staining showed overlap with scar tissue in phase-contrast microscope, scar volume was obtained same way as lesion and atrophy volume using phase-contrast images from serially collected sections. Scar density was assessed by dividing total scar volume by atrophy volume (estimated lesion size) in each individual animal. All measurements were performed by an experimenter blind to the experimental groups.

Assessment of BBB integrity

IgG staining

BBB integrity was assessed using IgG immunohistochemistry as previously described.²⁴ Three sections (+0.98, −0.22 and 0.82 mm from bregma) per animal were collected on slides (Fisherbrand™, Superfrost plus) and fixed with 4% paraformaldehyde (PFA) for 15 min, then incubated for 5 min in 0.3% H₂O₂ in PBS. Sections were blocked in 1% bovine serum albumin (BSA) and 10% normal goat serum (NGS), then labeled overnight at 4°C in goat anti-mouse IgG antibody (1:1000, Vector Lab., Burlingame, CA). Sections were then incubated in avidin/biotinylated peroxidase (ABC reagent, Vector Lab., Burlingame, CA) for 1 h at room temperature. Colorization was done by diaminobenzidine (Sigma, MO) substrate solution. ImageJ software was used to measure IgG stained areas in three separate sections. Since the extend of IgG staining depends on injury size and CD36 KO animals tend to have smaller over all hemispheric volumes, IgG staining was assessed by correcting with lesion areas. IgG density was calculated by dividing the IgG positive area by lesion area (three days) or atrophy (estimated lesion area) (three months) in individual sections.

Dextran extravasation

Animals in the acute survival cohort received 25 mg/kg dextran in 100 µl saline through retro orbital injection at day 3 and sacrificed 6 h after injection. ImageJ software was used to measure dextran intensity in three separate sections and extravasation index was calculated as follows: dextran positive area/lesion area.

Assessment of vessel density

Sections were fixed in 4% PFA for 15 min, washed with PBS three times and incubated in 0.2% Triton X-100 for 15 min. Sections were then blocked in 1% BSA (Sigma) in PBS for 1 h at room temperature, incubated overnight at 4°C with CD31 (1:2000; Millipore Bioscience Research Reagents) for vessel staining, followed by secondary antibodies conjugated with Alexa Fluor 594 (1:2000; Invitrogen) for 1 h at room temperature. Vessel density was measured in accordance with previously described studies.²⁶ Seven to 10 regions of 0.1 mm² areas were randomly selected within the scar tissue at the level of striatum (+1.2 mm from bregma). A 10 × 10 grid in a 0.1 mm² frame was placed within the area and CD31⁺ vessels that cross grid lines were counted. The number of intersections within a frame was counted and averaged for each section. Values were presented as number of intersections per frame area (0.1 mm²).

Behavior assessments

A battery of tests were performed to evaluate motor function (rotarod, open field), anxiety-related behavior (plus maze, dark-light box), depression-related behavior (sucrose preference, Porsolt’s forced swim test) before stroke (behavior baseline) and two and seven weeks after MCAo. Spatial memory was evaluated using a modified Water maze test at week 9. Procedures on behavior tests were adopted from previous publications with minor modifications.^2,27,28

Motor tests

For the rotarod test, animals were placed on a rotating rod (indented rod, 3.5 cm diameter) that accelerates from 4 r/min to 40 r/min over the course of 5 min and evaluated as described previously.^29,30 The latency to fall was used to assess motor performance. To obtain peak performance, animals were trained on the rotarod for five days with three daily trials and compared for baseline differences. At weeks 2 and 7, animals were tested in three consecutive trials and the average was used as the final value. For the open field test, animals were placed into a 40 × 40 cm field surrounded by 40 cm high walls and allowed to explore the setup freely over the course of 10 min. Total distance covered by the animal was tracked by analyzing live video by computer software (ANY-maze, Stoelting Co.) and was used to evaluate gross motor function and activity.²⁷

Anxiety tests

The set up for plus maze consisted of an elevated (60 cm) plus-shaped “maze” with two open (5 × 30 cm) and two closed arms surrounded by walls (5 × 30×15 cm). During the trial, animals explored the setup for 5 min and the entries and time spent in each arm were traced from the live video using ANY-maze software. Entries and time spent in the open arms were used to evaluate anxiety-like behavior.³¹ The light-dark box is a test used to evaluate unconditioned anxiety responses in rodents.³² The apparatus consisted of a dark (20 × 20×40 cm) and a brightly illuminated (40 × 20×40 cm) compartment connected by a door (5 × 7 cm). Animals were released into the dark compartment and transitions between compartments and time spent in each were scored using the ANY-maze software. Entries and time spent in the bright compartment were used to assess anxiety.

Depression tests

Anhedonia – a core symptom of depression – was assessed using the sucrose preference test.^31,33 Animals were first presented with a 1.5% sucrose solution in their home cage (6 h, two consecutive days) to habituate and eliminate any novelty-related aversion. During testing, animals were placed individually in a novel cage and presented with 1.5% sucrose solution and regular drinking water bottles. Bottles were weighed before the test and after 24 h to measure consumption. The final preference score was calculated by dividing total sucrose solution consumption by regular water consumption. The Porsolt’s forced swim test is used for screening potential antidepressant drugs and depression-like behavior in rodents.³⁴ It was performed as previously described.³⁵ Briefly, mice were placed inside a water-filled (20 cm) transparent cylindrical container for 5 min. The latency to stay afloat and total time spent immobile were scored and used to evaluate depressive behavior. One animal was excluded from the test due to drowning risk.

Cognitive tests

For the assessment of spatial learning performance, animals were tested in a Morris water maze task using previously described protocols with minor modifications.^28,36 A circular pool with a diameter of 140 cm was filled with 19°C opacified water (50 cm depth). A Plexiglas platform with a diameter of 10 cm was placed ∼15 cm away from the wall with the top submerged 1 cm beneath the water surface. The experiment consisted of a place learning task, a probe trial and a visible platform task in which animals’ performance (swim speed, latency to reach platform, etc.) was measured using ANY-maze computer tracking system. For the place learning task, animals were released into the pool and allowed to explore for a maximum of 90 s. After reaching the platform, animals were allowed to stay there for 15 s. The test consisted of three trials per days for seven consecutive days. The results of three daily trials were averaged to get the daily means for swim speed and latency to reach the platform. The submerged platform was removed for the probe trial, and animals’ swim path was tracked over 90 s. The time spent in the target quadrant (in which the platform was located) and crosses over the location of the removed platform were recorded. For the visible platform task, a platform marked by a clearly visible marker flag was placed in the opposite quadrant of the previous target quadrant. Animals were tested in three consecutive trials to score their latency to reach the visible platform.

Statistics

Comparisons between two groups (i.e. infarct size, swelling, atrophy) were performed using Student’s t-tests. Statistical analysis of multiple comparisons was evaluated using analysis of variance (ANOVA). In post hoc analyses that involve more than one factor (i.e. effect of stroke (sham vs. stroke) and effect of genotype (WT vs. CD36 KO), two-way ANOVA was used followed by a post hoc Bonferroni’s correction. For tests that were repeated over several days or required trials (i.e. Rotarod, Water maze), repeated measures ANOVA followed by Fisher’s LSD test was utilized. All data in figures are presented as mean ± 95% confidence interval (CI). Normality of data was analyzed using D’Agostino-Pearson normality test. In cases where data did not show normal distribution, Mann–Whitney U test was utilized. We used the term trend when defining statistical values that fall within the range 0.05 ≤ p < 0.1.

Results

CD36 deficiency does not alter BBB permeability or vessel density at acute phase of stroke

We have previously reported that CD36 KO animals have smaller lesions compared to WT mice.¹⁵ To exclude possible effect of infarct size, we subjected each genotype of animals to different durations of MCA occlusion to normalize lesion size between genotypes. As previously described,¹⁸ CD36 KO mice (n = 10) subjected to 40 min displayed a similar infarct size and swelling as compared to WT mice (n = 11) subjected to 30 min MCAO, assessed at three days post-ischemia (Figure 1(b) and (c)). Further analyses of sub-regions also showed no difference in the extent of lesion in the cortex, striatum and hippocampus (Figure 1(b)). Vascular integrity, assessed by IgG staining at three days, showed a trend (p = 0.061) towards reduced and less diffuse IgG immunoreactivity without reaching significance in CD36 KO mice compared to WT mice (Figure 1(d)). However, there was no difference between groups in dextran extravasation (Figure 1(e)). Vessel densities in the penumbra (WT = 27.1 ± 4.3, KO = 25.5 ± 6.6) and infarct regions (WT = 20.5 ± 3.1, KO = 20.7 ± 6.3) were also not different among groups (n = 15, WT = 7, CD36 KO = 8).

Figure 1.

Experimental timeline, histological outcome, IgG staining and dextran extravasation three days post-stroke. (a) Experimental timeline: Mice were subjected to 30 (WT) and 40 min (CD36 KO) MCAo to generate comparable injury size between genotypes. A cohort of animals was sacrificed at day 3 for histological analyses. Another cohort of animals was assessed in various behavioral tests and sacrificed at ∼3 months. (b) Infarct sizes in whole hemisphere and subregions (three days post stroke). All results are presented as percentage (%) of contralateral region volume. (c) Ipsilateral hemisphere swelling, calculated by subtracting contralateral hemisphere volume from ipsilateral hemisphere volume; presented as percentage of contralateral hemisphere volume. (d) IgG density in three separate sections at the level +0.98, −0.22 and 0.82 mm from bregma. IgG area was obtained from individual sections, then normalized by dividing by infarct area, thereby giving density values. (e) Dextran extravasation was assessed in three separate sections at the level +0.98, −0.22 and 0.82 mm from bregma. Dextran positive area was measured in the ipsilateral hemisphere and normalized by dividing by infarct area to calculate “dextran density”. Statistical tests: t-test for hemispheric, striatal and hippocampal lesion size and swelling; Mann–Whitney U test for cortical lesion size; two-way ANOVA for IgG and dextran density. Values are expressed as mean ± 95% confidence interval.

CD36 deficiency attenuates BBB disruption and scar formation at chronic phase of stroke

For the chronic outcome study, the extent of brain atrophy was assessed to estimate infarct size. WT and CD36 KO mice subjected to different occlusion times (WT 30 min, CD36 KO 40 min) showed similar atrophy (WT vs. CD36 KO, 24.6 ± 10.9, 20.0 ± 8.6, ns, n = 13/group) (Figure 2(a)). Further analyses of sub-regions also showed similar extent of atrophy in the cortex, striatum and hippocampus.

Figure 2.

Reduced IgG extravasation and scar tissue in CD36 KO animals at three months post stroke. (a) Estimated infarct volumes (atrophy) in ipsilateral hemisphere and subregions. All results presented as percentage (%) of contralateral region volume. (b) IgG density in three separate sections (level +0.98, −0.22 and 0.82 mm from bregma) and representative images. IgG area was obtained from individual sections, then normalized by dividing by infarct area, thereby giving density values. (c) Scar volume corrected to estimated lesion (atrophy) volume. (d) Correlation between scar volume and estimated lesion volume (atrophy) for KO and WT animals. (e) CD31 immunohistochemistry vessel density measurements (*p < 0.05, mean ± 95% confidence interval, n = 8 per group). Statistical tests: t-test for striatal and cortical atrophy, scar and vessel density; Mann–Whitney U test for hemispheric and hippocampal atrophy; Two-way ANOVA for IgG density. Values are expressed as mean ± 95% confidence interval.

With the confirmation of comparable injury size between the genotypes, we next assessed BBB integrity at three months after stroke. The cortex (including the entorhinal cortex) and striatum were the primary regions showing strong IgG staining in 90% of animals. The hippocampus was weakly IgG positive in ∼44% of WT and ∼30% of CD36 KO mice. Compared to WT mice, CD36 KO mice showed a significant reduction of IgG density (Figure 2(b), two-way ANOVA, significant effect of genotype, F (1, 51) = 6.165, p = 0.0164, n = 9/group). Sub-regions analyses of IgG staining in the cortex, striatum, hippocampus and thalamus did not show significant differences between genotypes (data not shown).

Previously, we reported that CD36-deficient mice have attenuated GFAP expression and scar formation in acute phase of stroke.¹⁸ We thus addressed the involvement of CD36 in chronic scar formation. Since scar formation depends on injury size and CD36 KO animals tend to have smaller over all hemispheric volumes, the extent of scar formation was assessed relative to estimated lesion size (i.e. atrophy volume) at 3 m. Compared to WT animals, CD36 KO animals had significantly attenuated scar formation (Figure 2(c), p = 0.0482). There was a significant correlation between scar volume and hemispheric atrophy volume in WT mice (p = 0.0375), while such correlation was not present in CD36 KO mice (Figure 2(d)), confirming involvement of CD36 in glial scar formation. Vessel density within the glial scar, however, did not show difference between genotypes (Figure 2(e)), suggesting that previously reported increases in neoangiogenesis in CD36 KO mice during the sub-acute phase of stroke²⁶ do not persist during the chronic phases of stroke.

CD36 KO mice do not display abnormal anxiety or depression-related behaviors but do exhibit reduced motor fitness at pre-stroke baseline

WT and CD36 KO animals were trained and compared at baseline before sham and MCAo operations to identify genotype-related differences in behavioral outcomes. In Rotarod, which is used to evaluate motor learning and performance, there was a significant effect of genotype (Figure 3(a), Rotarod) with CD36 KO animals spending less time on the rod compared to WT (repeated measures ANOVA, effect of genotype, F (1,55) =12.87, p = 0.0007, N WT = 28, KO = 29). In the open field test, which assessed gross motor function and activity, CD36 KO animals covered significantly less distance within the 10-min test duration, indicating a minor hypoactive phenotype. There were no differences in open center entries or time (data not shown). In tests for anxiety-related behavior (Figure 3(b)), there was no difference among groups in bright compartment time in light-dark box and open arm time in plus maze tests, showing that the groups do not differ in their anxiety profile. Similarly, in test for depressive behavior, total immobile time in Porsolt’s forced swim test and sucrose preference tests CD36 KO and WT mice was not significantly different (Figure 3(c)). These results indicate a minor reduction in motor fitness and activity in CD36 KO animals. Anxiety and depression profiles were not different in any behavior test suggesting that minor hypoactivity observed in CD36 deficiency animals did not affect these evaluations.

Figure 3.

Baseline behaviors prior to stroke in WT and CD36 KO mice. (a) motor tests: rotarod performance through five days of baseline training and distance traveled in open field test. (b) Test for anxiety-related behavior. The light-dark box test showing time spent in the bright compartment of the box. Plus maze test showing the time spent in the open arms of the elevated plus maze. (c) Test for depression-related behavior. In the Porsolt’s forced swim test, total time spent immobile is indicated. Ratio of sucrose to water consumption in sucrose preference test. Statistical tests: t-test for open field, light-dark box, forced swim and sucrose preference; Mann–Whitney U test for plus maze; RM ANOVA for rotarod. Values are expressed as mean ± 95% confidence interval. All values are expressed with mean ± 95% confidence interval.

CD36 deficiency ameliorates stroke-induced hyperactivity in the subacute phase

Animals were tested at two weeks post-stroke. Repeated measures ANOVA analysis revealed a highly significant effect of stroke on rotarod performance (F (1, 39) = 2.746 p < 0.0001, n = 13 per group) without any effect of genotype, showing that stroke impairs motor performance and that CD36 deficiency did not have any significant effect on outcome (Figure 4(a) – rotarod).

Figure 4.

CD36 deficiency abolishes stroke-induced hyperactivity in the subacute phase. (a) Motor tests: Latency to drop from the accelerating rotarod and distance travelled in open field test. (b) Tests for anxiety-related behavior: Time spent in the bright compartment of the light-dark box, time spent in the open arms of the plus maze test. (c) Tests for depression-related behavior: Total time spent immobile in the Porsolt’s forced swim test, ratio of sucrose to water consumption in sucrose preference test. Statistical tests: Two-way ANOVA for all (*p < 0.05, **p < 0.01, ***p< 0.001 ****p< 0.0001, mean ± 95% confidence interval, N = WT Sham 7, WT stroke 13, CD36 KO Sham 9, CD36 KO Stroke 13).

In the open field test, both stroke (F (1, 39) = 6.552, p = 0.0145) and genotype (F (1, 39) = 7.282, p = 0.0102) had significant effect on open field activity without a significant interaction. Post hoc analysis revealed a significant difference between stroke and sham animals only in the WT group. In addition, WT stroke animals covered significantly more distance compared to CD36 KO stroke animals (Figure 4(a) – open field). These findings indicate that in the subacute phase, stroke induced a hyperactive phenotype that is ameliorated by CD36 deficiency.

Previous studies have reported conflicting findings in anxiety and depression-related behaviors after stroke.²⁰ In our study, there were no significant differences between groups in time spent in the bright compartment of the light-dark box apparatus and open arm time in the elevated plus maze test (Figure 4(b)), showing that our stroke model did not alter anxiety-like behavior at the subacute phase of stroke. Two-way ANOVA analysis of Porsolt’s forced swim test shows that stroke significantly reduced immobility time (F (1, 38) = 7.166, p < 0.05) with no effect of genotype or interaction. Post hoc analysis revealed significant differences only among the WT groups. These findings suggest a reduced depressive phenotype induced by stroke. However, immobility time in PSFT is also influenced by hyperactivity (e.g. induced by psychostimulants); therefore, reduced immobility time may also reflect the increased activity observed primarily in WT stroked animals. Conversely, stroke led to a significant reduction in sucrose preference at two weeks (F (1, 37) = 21.50, p < 0.0001) with post hoc analysis revealing significant difference in both genotypes. However, no effect of genotype or interaction was observed (Figure 4(c)). Taken together, in the subacute phase, stroke induced a significant hyperactivity and hedonic deficit along with alterations in immobility duration in the forced swim test. CD36 deficiency ameliorated hyperactivity.

Stroke induced hedonic deficit resolves in CD36 KO mice in the chronic phase

Motor performance on the rotarod was still impaired in both stroke groups at the chronic phase. Two-way ANOVA analysis showed a significant effect of stroke (F (1, 38) = 17.29, p = 0.0002) without genotype or interaction effect. Post hoc analysis shows the effect of stroke on both genotypes as well (Figure 5(a) – rotarod). These findings indicate that stroke-induced motor deficit persists and is not significantly altered by CD36 deficiency at subacute or chronic phases of stroke.

Figure 5.

Stroke-induced hedonic deficit resolves in CD36 KO mice in chronic phase. (a) Motor tests: Latency to drop from the accelerating rotarod and distance travelled in open field test. (b) Anxiety-related behaviors: Time spent in the bright compartment of the light-dark box, time spent in the open arms of the plus maze Test. (c) Depression-related behaviors: total time spent immobile in the Porsolt’s forced swim test, ratio of sucrose to water consumption in sucrose preference test. Statistical tests: two-way ANOVA for all (*p < 0.05, **p < 0.01, mean ± 95% confidence interval, N = WT Sham 7, WT stroke 13, CD36 KO Sham 9, CD36 KO Stroke 13).

Two-way ANOVA analysis of open field activity in the chronic phase shows a significant effect of stroke (F (1, 38) = 13.40, p = 0.0008) without genotype effect or interaction (Figure 5(a)). Post hoc analysis, however, indicates a significant difference between sham and stroke animals only in the WT groups. Taken together with acute phase finding, the ameliorating effects of CD36 deficiency on stroke-induced hyperactivity appear to persist into the chronic phases of stroke.

Anxiety-related behaviors were not altered as assessed in light-dark box and elevated plus maze (Figure 5(b)) in the chronic phase as well. Reductions in immobility time in stroke animals observed at week two persisted into chronic phases after stroke. Two-way ANOVA analysis revealed a significant effect of stroke (F (1, 37) = 7.484, p = 0.0095) with no genotype effect or interaction. Analysis of sucrose preference test (Figure 5(c)) revealed no effect of stroke or genotype but an interaction effect (F (1, 37) = 6.007, p = 0.0191). Post hoc analysis shows a significantly reduced sucrose preference only between the WT sham and stroke groups. Our results indicate that CD36 deficiency ameliorates hedonic deficit at the chronic stages of stroke.

Knocking out CD36 drastically alleviates stroke-induced learning deficits

During the seven-day learning period (place task) of the MWM test (Figure 6), mice from all groups improved in their time to find the hidden platform. There was no difference among groups in swim speed. Repeated measures ANOVA revealed a significant effect of time (F (6, 216) = 74.65 p < 0.0001), group (F (3, 36) = 6.534 p = 0.0012) and group × time interaction F (18, 216) = 2.146 p = 0.0055, n = 13 per group). Post hoc analysis reveals a significant difference between WT stroke animals and WT sham (days 2, 3, 4 and 6) and CD36 KO stroke animals (days 4, 5 and 6). WT stroke animals performed significantly worse compared to all other groups (Figure 6(a)), whereas CD36 KO stroke group performance was comparable to sham groups, indicating a stroke-induced learning impairment that is mitigated by knocking out CD36. Conversely, during the probe trial, parameters such as the time spent in the target quadrant (data not shown), mean distance to target (data not shown) or number of crosses through the removed visible platform location were not different between groups (Figure 6(b)). These findings demonstrate that stroke impaired learning performance during the classical water maze task, but the reference memory performance was not drastically impaired.

Figure 6.

CD36 deficiency enhances cognitive function at three months after stroke. (a) Submerged platform learning task. Time to locate the submerged platform over seven days, showing learning curves. (b) Number of crossovers in the region of the removed submerged platform during the probe trial. (c) Time elapsed before the animals can reach the visible platform in three consecutive trials of the visible platform task. (d) Time spent by the animals in the quadrant of the removed submerged platform during the visible platform task trials. (e) Representative drawings of the path taken by different groups of animals in visible platform task trials. Statistical tests: two-way ANOVA for platform crosses; RM ANOVA for other parameters (*p < 0.05, **p < 0.01, ***p < 0.001, sham vs. stroke (effect of stroke), ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001, WT stroke vs. CD36 KO stroke (effect of genotype), mean ± 95% confidence interval).

A visible platform task was used in this experiment as a form of reversal learning in which the animal is required to adapt and learn the change in escape platform location. Despite the relative ease of the task, we observed that WT stroke animals took significantly more time to reach the visible platform that is particularly significant during the third trial of the protocol (Figure 6(c)) and were significantly worse compared to Sham and CD36 KO stroke animals. Closer inspection revealed that WT stroke animals were persistently searching for the removed hidden platform (Figure 6(e)) and spent significantly more time compared to WT Sham and CD36 KO stroke animals. Importantly, stroked CD36 KO animals were able to adapt and navigate to the visible platform and did not differ significantly from sham CD36 KO mice. Our results demonstrate that stroke induces a learning deficit that manifests as a problem in strategy switching and relearning. Importantly, lack of CD36 ameliorates the observed learning deficits both during the learning and relearning (visible platform task) phases of the water maze.

Discussion

Genetically modified animals are powerful tools in identifying the role of the gene in question in stroke pathophysiology. A significant number of stroke preclinical studies utilized KO or knock-in models that alter lesion size, which confounds interpretation of changes in pathophysiological cascades and behavioral outcome in acute to chronic phases of stroke. As CD36 KO mice display smaller infarct size,¹³ the current study investigated the role of CD36 in stroke pathophysiology and behavior outcome by subjecting WT and CD36 KO animals to different durations of MCA occlusions to achieve comparable infarcts. We demonstrated that CD36 contributes to BBB disruption, scar formation, as well as clinically relevant neuropsychological and cognitive behavior impairments in the chronic phase of stroke, independent of stroke severity.

BBB breakdown is one of the hallmarks of ischemic stroke, and leads to vasogenic edema, hemorrhagic transformation and progression of stroke.^37,38 Both in stroke patients and in preclinical models, interventions that preserve BBB integrity reduce lesion size and improve functional outcome. BBB disruption is also predictive of outcome irrespective of lesion size.^39,40 BBB dysfunction, as evaluated by IgG staining in our study, was significantly attenuated in CD36 KO animals only in the chronic, but not acute, phase of stroke. Since the lesions were comparable in size between genotypes, reduced BBB disruption in the chronic stage indicates CD36 is an important mediator of its integrity. The mechanisms by which CD36 deficiency ameliorates BBB dysfunction may involve multiple factors. Studies have revealed the pro-inflammatory nature of CD36 in mediating ROS generation and NF-kB activation in acute stroke.^13,41 In addition, it has been shown that in sterile inflammation, CD36 mediates assembly of the NLRP3 inflammasome complex, leading to the release of active IL-1β which is known to impair BBB integrity.⁴² Thus, reduced inflammatory state and oxidative stress maybe an underling event preserving BBB integrity in CD36 KO animals.

Stroke-induced glial scar formation may be beneficial, by blocking the spread of toxic mediators to adjacent tissue, or detrimental, by creating a physical barrier that prevents regrowth and reconstitution of neuronal connections.⁴³ We have previously reported the involvement of CD36 in glial proliferation and scar formation in acute post-ischemia.¹⁸ The observations in the current study showing reduced scar formation at the chronic stage of stroke suggest a role of CD36 in scar formation. Importantly, glial scars have been shown to be permeable to the neurotoxic environment of infarcts and show IgG staining seven weeks post stroke.⁴ As such, reduced scar formation in CD36 KO may lead to a more permissive milieu that promotes the observed functional recovery. Despite of our earlier findings that suggest increased neovascularization in the absence of CD36 during sub-acute (seven days) stroke,¹⁸ the current study shows little evidence of CD36 involvement in angiogenesis in acute and chronic stroke, as we have not observed differences in vessel density between genotypes.

A vast majority of studies on stroke recovery primarily focuses on motor function.²⁰ However, stroke survivors also suffer from a wide range of cognitive and neuropsychological problems ranging from attention deficit, memory problems, and anxiety or depression.^44,45 which evolve over time in stroke patients.⁴⁵ In our long-term behavioral assessments, we observed that stroke leads to hyperactivity, reduced despair behavior, anhedonia and learning and strategy switching deficits, which were attenuated in mice with CD36 deficiency. The behavioral improvements were associated with reduced BBB disruption in CD36 KO mice in the chronic phase of stroke. Literature indicates that psychological⁴⁶ and cognitive disturbances are linked to post-stroke inflammatory⁴⁷ status and BBB leakage and that altering BBB permeability may influence these disturbances and their recovery.⁴⁸ BBB impairment persists for months after stroke, leading to a sustained toxic environment that has been proposed to contribute to delayed brain atrophy and dementia in certain stroke patients.^4,49 In animal models, BBB dysfunction can lead to deficits in various learning and memory tasks including Morris water maze.⁵⁰ Furthermore, the reported implicated role of CD36 in Alzheimer’s disease and mild cognitive impairment, vascular dementia^51–53 and anxiety⁵⁴ supports an involvement of CD36 in stroke-induced cognitive deficit and its link to BBB disruption.

In long-term behavioral assessments, a major observation in our study was stroke-induced hyperactivity. Various studies in rodents have reported chronic hyperactivity,^31,35,55 while others reported no effect.⁵⁶ In human patients, hyperactive motor behavior may present itself in cases of mania, which is associated with abnormally elevated mood in which energy and activity levels are increased. Mania manifests in less than 2% of stroke patients and is associated with right infarctions.^57,58 In our study, we did not observe a significant effect in anxiety behavior. Despair behavior was significantly reduced after stroke, which is in line with another report.⁵⁹ It can be argued that hyperactivity and reduced despair observed after stroke in our model capture the manic phenotype observed in a subset of patients. Alternatively, stroke-induced changes in despair behavior may be an “experimental artifact” due to the sensitivity of Porsolt’s FST to increases in activity.^60,61

Stroke induced a significant hedonic deficit in our study, in accordance with several other studies.^31,62 Given the limited number of studies investigating post-stroke depression (PSD)-like phenomenon in animal studies, our findings are of importance in underlining the translational value of MCAo models. While various antidepressant drugs are used with relative success in the treatment of PSD, further research is crucial in understanding its pathophysiology and establishing better treatments. In animal models of stress and depression, it has been suggested that reduction in sucrose preference and increased despair is associated with cytokine levels.^63,64 Accumulating evidence indicates that BBB dysfunction is involved in the pathogenesis of various psychiatric disorders including bipolar disorder⁶⁵ and major depression.⁶⁶ Importantly, chronic stress models of depression that induce robust anhedonia lead to BBB leakage. Menard et al.⁶⁷ have demonstrated that chronic social stress leads to BBB permeability particularly in the nucleus accumbens. In our study, the nucleus accumbens region stained positive for IgG, indicating BBB dysfunction in the region. Better BBB function and reduced inflammatory state may explain the resolution of hedonic deficit in CD36 KO animals.

Cognitive decline affecting memory, alertness, attention and concentration are a common sequelae after stroke.^19,68 In our study, we evaluated learning and memory performance using the MWM and observed learning and memory deficit along with a drastic impairment in strategy switching and cognitive flexibility. These observations closely resemble the dysexecutive syndrome observed in stroke patients. MWM learning is complex and numerous brain regions are implicated including cerebral cortex, striatum, basal forebrain and cerebellum.⁶⁹ Hippocampal lesions lead to impairments in hidden (but not in visible) platform MWM learning in rodents.^70,71 Striatal lesions are shown to impair hidden platform learning.⁷² Lesions of the dorsomedial striatum are shown to delay spatial learning, and mice displayed inflexible search patterns and repeated visits to towards the previously reinforced cue in cued-based navigation tasks.⁷³ Other studies also reported perseverance and cognitive inflexibility in MWM reversal learning tasks after lesions of the medial striatum.⁷⁴ These Findings are quite similar to the strategy switching problem and perseverance in searching for the removed submerged platform we observed in our WT stroked animals. In accordance with our findings, Winter et al.²⁸ have shown that visible platform task is useful in detecting differences in mild strokes and that striatal lesion volume correlates with the time animal spends exploring the former submerged platform quadrant. These findings indicate the importance of the integrity of the striatum, the primary structure damaged in our model, in this task. In our study, we did not observe significant difference in hippocampal or striatal atrophy between genotypes, indicating that the beneficial effects of CD36 deficiency are not due to reductions in gross atrophy of those structures. Taken together, sustained BBB integrity by CD36 deficiency may be responsible for the observed improvements in MWM performance in our study.

A major shortcoming of our study is that the genetic model used is a full-body knock out. Due to the diverse function of CD36 in various cell types ranging from astrocytes, endothelial cells to infiltrating monocyte and macrophages, identifying the detailed mechanisms by which CD36 deficiency exerts its effect on BBB function, scar tissue formation and behavioral improvements have been limited in our current experimental design. Future studies with conditional deletion of CD36 in monocytes or endothelial cells would define the tissue-specific role of CD36.

In summary, our findings demonstrate that CD36 KO mice displayed improved stroke-induced BBB dysfunction, reduced scar formation and enhanced behavioral impairments in activity, hedonic responses, spatial learning and strategy switching. The improvements of chronic histological and behavior outcomes in CD36 KO mice show a contributing role of CD36 to pathology and long-term functional deficits in stroke. Our findings indicate that various neuropsychological and cognitive deficits observed in the clinical populations can reliably modeled via the MCAo stroke model and the inhibition of CD36 may serve as a potential target to enhance functional recovery in chronic stroke.