Clemastine ameliorates cognitive deficits in an experimental rat model of chronic cerebral hypoperfusion

Abstract

Chronic cerebral hypoperfusion (CCH) profoundly affects patient well-being and has been proposed as a risk factor for cognitive impairment and dementia. However, due to the limited understanding of the pathophysiology of CCH, there are currently no effective preventive or therapeutic approaches for CCH-associated cognitive impairment. Therefore, identifying new targets for CCH is essential to bridge this knowledge gap. In this study, we sought to determine whether enhanced myelination could prevent cognitive impairment associated with CCH. Experimental CCH was induced via bilateral common carotid artery occlusion (BCCAO) in rats, and clemastine, a well-established pro-myelinating agent, was administered to boost myelin renewal. A series of behavioral tests was performed to assess learning and memory. We found that animals exhibited profound cognitive deficits 3 months after BCCAO, accompanied by significant myelin loss, structural disruption at the nodes of Ranvier, and synaptic dysfunction in various brain regions, without notable neuronal loss and oligodendrocyte apoptosis. Importantly, pharmacological enhancement of myelination preserved dendritic spine density and prevented synaptic loss, cognitive deficits, and neurovascular dysfunction following BCCAO. These findings suggest that clemastine may represent a promising therapeutic option for CCH-associated cognitive impairment.

Introduction

Although significant progress has been made in understanding acute cerebral ischemia, our knowledge of the pathophysiology of chronic ischemia remains limited. Chronic cerebral hypoperfusion (CCH), often resulting from cerebral small vessel disease, arteriovenous malformations, or carotid stenosis/occlusion, has been linked to cognitive impairment.^1–3 Additionally, CCH has been proposed as a risk factor for Alzheimer’s disease and other dementias.^4–6 Patients with reduced cerebral perfusion exhibit impaired cognitive function,^7,8 and decreased cerebral blood flow has been linked to more rapid cognitive decline in both vascular cognitive impairment and normal aging. ² Improved management of CCH could, therefore, help reduce the burden of cognitive impairment and dementia. However, there are currently no therapeutic strategies available to prevent or alleviate CCH-associated cognitive deficits. Further investigation into the complex pathophysiology of CCH, in this sense, is essential for identifying new therapeutic targets and may pave the way for the development of targeted therapies.

White matter injury, characterized by oligodendrocyte loss, reduced myelin density, structural disruption at the nodes of Ranvier, and disintegration of white matter tracts, has been observed in both patients with cerebral small vessel disease and in experimental models of CCH.^3,9,10 Myelin pathology and disrupted white matter integrity can be observed as early as 2 weeks following the induction of experimental CCH,^11–13 which is subsequently followed by progressive cognitive deficits.^14,15 Intriguingly, emerging evidence suggests that the myelin sheath, a multi-layered membrane wrapping axons in the nervous system, actively contributes to cognitive processes.^16,17 This raises the possibility that myelin loss may underlie the onset of cognitive deficits following CCH.

High-throughput screening has identified several pro-myelinating compounds, with clemastine being widely used as a pharmacological approach to enhance myelin renewal.^18,19 Administered at a dose of 10 mg/kg body weight over several weeks in experimental animals, clemastine has been shown to effectively promote myelination across various animal models.^19–21 Furthermore, enhancing myelination both pharmacologically and genetically has been demonstrated to alleviate cognitive deficits in different animal models.^22,23 Given the widely reported white matter pathology in CCH, we aimed to determine whether pharmacological enhancement of myelination could prevent cognitive deficits induced by CCH.

Herein, we aim to investigate whether pronounced demyelination and associated cognitive deficits occur in a rat model of CCH and to determine whether pharmacological enhancement of myelin renewal can rescue these deficits. This study will test our hypothesis that prolonged demyelination plays a key role in CCH-induced cognitive impairment and will highlight the potential of pro-myelinating therapies for CCH.

Materials and methods

Animals and experimental design

All experimental procedures followed the ARRIVE guidelines. ²⁴ Three-month-old healthy male Sprague–Dawley rats (280–320 g) were obtained from Charles River Laboratories (Malvern, PA, USA). The animals were housed under standard conditions (12-h light/dark cycles, temperature 23 °C ± 2°C), with ad libitum access to water and food. After a period of acclimation, the rats were randomly assigned to the following three groups: (a) Sham group—no BCCAO, vehicle treatment only; (b) BCCAO group—underwent BCCAO, vehicle treatment only; and (c) BCCAO + clemastine group—underwent BCCAO, received clemastine treatment. Based on previous studies reporting no adverse effects of clemastine in normal animals,^25,26 a sham BCCAO + clemastine group was not included to maintain brevity and clarity. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Louisiana State University Health Science Center and adhered to the Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize both animal distress and the number of animals used.

Bilateral common carotid artery occlusion (BCCAO)

BCCAO was performed on the rats as described by Lana et al. ²⁷ Briefly, the rats were anesthetized with 4% isoflurane in a clear induction chamber, followed by subcutaneous injection of carprofen (5 mg/kg) for analgesia. The animals were then positioned on a surgical platform, and eye ointment was applied to prevent corneal drying. Anesthesia was maintained using a nose cone delivering 2.5% isoflurane. The right common carotid artery was bluntly dissected and permanently ligated with a silk suture. Seven days later, the left common carotid artery was occluded using the same procedure to complete the BCCAO. Throughout the surgery, the rectal temperature was maintained between 36.5 °C and 37.5 °C using a heating pad with an anal probe as a temperature reference (ThermoStar; RWD Life Science). Animals in the sham group underwent bilateral common carotid artery exposure without ligation.

Drug treatment

From days 7 to 28 post-surgery, rats subjected to BCCAO received daily oral gavage of clemastine (Selleck Chemicals, S1847) at a dose of 10 mg/kg in PBS with DMSO. Both the sham and BCCAO control groups were administered an equivalent volume of vehicle (PBS with DMSO) under identical conditions. The clemastine dosage was selected based on previous studies demonstrating its neuroprotective effects.^28,29 In order to examine cell proliferation, BrdU (50 mg/kg body weight) was administered intraperitoneally to each animal every 3 days for 21 days following BCCAO.^30,31 BrdU injections were administrated to all groups of animals.

Behavioral assessments

Barnes maze

The Barnes Maze is a widely used behavioral assay for evaluating hippocampus-dependent spatial learning and memory in rodents. ³² The test consisted of a 3-day training phase followed by a probe trial. During the training phase, each animal underwent one session per day. In each session, animals were allowed to explore the maze to locate a hidden escape chamber positioned beneath a designated target hole. The trial was terminated upon successful entry into the chamber or after a maximum exploration time of 3 min. Animal movements were recorded using an overhead video camera connected to the ANY-maze video tracking system (Stoelting, Wood Dale, IL, USA). Key parameters recorded included the latency to locate the escape chamber. During the probe test, the escape chamber was removed and the target hole was blocked with a small black plastic board. Each animal underwent a 90-s probe trial. Average speed, time spent in the target quadrant (where the target hole had been located), and searching errors were quantified using ANY-maze software for subsequent analysis. Searching errors were defined as the percentage of incorrect hole investigations (holes other than the target hole) relative to the total number of hole explorations during the probe trial.

Fear conditioning

The fear conditioning protocol was conducted in soundproof chambers to evaluate associative learning and memory function. Briefly, Chamber A (context A) was equipped with a grid floor designed to deliver electric shocks, controlled by a stimulus isolation unit (SIU-102B; Warner Instruments, Hamden, CT, USA). Chamber B (context B) differed visually and had sawdust bedding to provide a distinct odor. Chambers and equipment were cleaned with 70% ethanol between tests/animals. Fear conditioning consisted of a 2-day protocol, with day 1 for conditioning/training, and day 2 for testing of the conditioning.

Day 1. During the training session, animals were placed in Chamber A for a 90-s acclimation period, followed by three 30-s tones (2.8 kHz sine wave at 85 dB) delivered at 2-min intervals. Each tone co-terminated with a 0.6 mA foot shock lasting 2 s. Freezing behavior, defined as the absence of any detectable movement for at least 1 s, was monitored and analyzed using ANY-maze software. The percentage of time spent freezing during each 2-min interval was recorded and plotted as the conditioning response.

Day 2. Twenty-four hours after the training session, contextual fear memory was assessed by reintroducing the rats to Chamber A for 5 min without any tone presentation. The percentage of time spent freezing during the 1–5 min period was recorded to evaluate contextual memory retention. After a 1-h rest period, cued fear memory was tested in Chamber B. Rats were placed in context B for a 90-s acclimation period, followed by three 30-s tones delivered at 2-min intervals, similar to the training phase but without foot shocks. The percentage of freezing during the initial 90 s was recorded as baseline freezing in the neutral context, and the average percentage of freezing in response to the three tones was used to assess cued memory.

CBF measurement

Laser speckle contrast imaging was utilized for CBF measurements. After anesthesia, the rats were positioned in a stereotaxic frame to stabilize the skull, and eye ointment was applied to prevent corneal drying. Body temperature was maintained at 37 °C. A longitudinal incision (~20 mm) was made along the midline of the head, and the subcutaneous connective tissues were carefully retracted to expose the skull. The exposed skull area was gently scraped with a scalpel to create a dry surface. A 10 mm diameter region over the left frontoparietal cortex, ~1 mm lateral to the bregma, was identified without thinning the skull to avoid variations. A thin layer of transparent gel was evenly applied to the skull surface to improve visibility, as previously described. ³³ Laser speckle contrast imaging was conducted using the RFLSI III Speckle Contrast Imaging System (RWD Life Science). The brain was illuminated with a 110 mW laser to measure cortical blood flow, and the relative blood flow index (a.u.) was calculated using software provided by the vendor (RWD Life Science). Following these procedures, the animals were sacrificed, and brain tissues were collected for further analysis.

Whisker stimulation

Whisker stimulation was performed as previously described by Lecrux et al. ³⁴ Briefly, rats were anesthetized with 4% isoflurane in a mixture of O₂ and N₂O (30:70) using a transparent induction chamber, followed by a subcutaneous injection of carprofen (5 mg/kg) for analgesia. The animals were then positioned on a surgical platform, and ophthalmic ointment was applied to prevent corneal drying. Anesthesia was maintained with 2.5% isoflurane delivered through a nose cone during scalp incision and short skull-thinning procedure and reduced to 2.0% during whisker stimulation. The entire procedure per animal lasted <20 min, and no craniotomy or surgical manipulation was performed. Under these conditions, all animals exhibited stable spontaneous respiration and physiological appearance; thus, mechanical ventilation was not employed. Before testing, the left-side whiskers were fully shaved, while the right-side whiskers were trimmed to ~5 mm. Subsequently, the skull was carefully thinned over the left barrel cortex to create a 3 × 3 mm optical window (bregma coordinates: AP −3.0 mm; L ±7.0 mm) using a high-speed microdrill. During stimulation, the whiskers were activated using an electric toothbrush oscillating side-to-side at a frequency of 8–10 Hz. The stimulation protocol followed a block design consisting of 60 s of baseline, 20 s of stimulation, and 40 s of rest, repeated three times. Changes in regional CBF were averaged and expressed as a percentage increase from baseline values. To assess changes in neuronal activity within the barrel cortex, animals were sacrificed 90 min after whisker stimulation.

Brain sections and protein preparations

Brain collection was performed following the whisker stimulation test. After transcardial perfusion with ice-cold PBS, the rat brains were rapidly removed. The left cerebral hemispheres were post-fixed in 4% paraformaldehyde (Thermo Fisher Scientific™, Inc.) and cryoprotected in 30% sucrose for tissue sectioning. The brains were then embedded in OCT medium, frozen, and coronal sections (25 μm) were prepared using a Leica RM2155 microtome. These sections were stored in a stock solution (FD NeuroTechnologies, Inc., Columbia, MD, USA) for subsequent immunofluorescence and Black-Gold staining. For protein extraction, the hippocampus, mPFC, and corpus callosum were dissected from the right hemispheres. A motor-driven Teflon homogenizer was used to homogenize the tissue in 400 μL of ice-cold homogenization buffer containing protease and phosphatase inhibitors (78420, A32955, Thermo Fisher Scientific™, Inc., Waltham, MA, USA). Protein concentration was measured using the Pierce BCA protein assay kit (23227, Thermo Fisher Scientific™, Inc., Waltham, MA, USA).

Immunohistochemistry staining and confocal microscopy

Immunofluorescence staining of free-floating sections was performed as described in our previous study. ³¹ Briefly, tissue sections were washed in 0.4% Triton X-100 for 3 × 10 min, followed by blocking with 3% BSA for 1 h. Sections were then incubated overnight at 4 °C with primary antibodies, including MBP (1:300, ab62631; Abcam, USA), NF200 (1:300, N4142; Millipore Sigma, USA), MAP2 (1:200, 17490-1-AP; Proteintec, USA), NeuN (1:200, 66836-1-lg; Proteintec, USA), CC1 (1:300, OP80; EMD Millipore, USA), PDGFRα (1:200, 3174S; Cell Signaling Technology, USA), Olig2 (1:100, MABN50; Millipore Sigma, USA or 1:100, AB109186; Abcam, USA), Synaptophysin (1:250, AB8049; Abcam, USA), Spinophilin (1: 100, AB203275; Abcam, USA), Caspr (1:150, 75-001; CiteAb, USA), Nav1.6 (1:200, ASC-009; Alomone Labs, USA), Ki67 (1:200, PA5-19462; Invitrogen, USA), BrdU (1:100, PA5-32256; Invitrogen, USA), and c-fos (1:500, 2250S; Cell Signaling Technology, USA). On the following day, brain slices were washed three times with 0.1% Triton X-100 and then incubated with Alexa Fluor-labeled anti-mouse/rabbit secondary antibodies (488/568; Thermo Fisher Scientific™, Inc., Waltham, MA, USA) for 1 h at room temperature. After washing, the sections were mounted with DAPI fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA).

For staining with BrdU, PFA-fixed brain sections were permeabilized in 1 M HCl for 10 min at 0 °C, followed by sequential treatment with 1 and 2 M HCl for 10 min each at room temperature, and then 2 M HCl for 20 min at 37 °C. The sections were neutralized with 0.1 M sodium tetraborate (pH 8.5) for 20 min, followed by washing, blocking, and overnight incubation at 4 °C with anti-BrdU antibodies.

TUNEL staining was performed on free-floating sections using the Click-iT^® Plus TUNEL assay kit (Thermo Fisher Scientific™, Inc.), following the protocol described in our recent work (PMID: 31515743). Visualization and imaging of double- and triple-stained sections were carried out using an Olympus CSU W1 Spinning Disk microscope (Olympus, Germany) with 40× or 100× oil immersion Neofluor objectives. Fluorescence images were analyzed using Fiji software (ImageJ, NIH, MD, version 1.52q). For each animal, three to five sections were analyzed, and the data from each section were averaged to generate a single value per animal. Group means and standard errors (SE) were calculated, and statistical analyses were performed to compare groups.

ProteinSimple^® Capillary-based immunoassay

Quantification of all protein targets was achieved using simple Western blot analysis in a Jess™ apparatus per the manufacturer’s protocol (ProteinSimple; Bio-techne). Briefly, tissue lysate was diluted to a final concentration of 1 µg/µl for analysis, then added in a 4:1 (lysate: mix) ratio to a mix containing 40 µM fluorescent molecular weight marker and dithiothreitol. Samples were then denatured for 5 min at 95 °C before loading into a microwell plate. Antibody diluent (ProteinSimple; Bio-techne) was used for all antibody dilutions as follows: MBP (1:50, 10458-1-AP; Proteintech), PDGFRα (1:50, 3174S; Cell Signaling Technology), Synaptophysin (1:100, 60191-1-lg; Proteintech). Antibodies were detected using HRP-conjugated secondary anti-rabbit or anti-mouse. Relative protein levels were calculated by measuring the area under the peak of a chemiluminescent chromatogram and normalized to total capillary chemiluminescence for a given sample. All analyses were carried out using Compass for SW software (version 6.0.0, ProteinSimple; Bio-techne). Pseudo-blot images were computer-generated based on peak values derived from Compass for SW software.

Western blotting

Western blotting analysis was conducted as previously described. ³⁰ Briefly, 20 μg of each protein lysate was electrophoresed on 10%–20% gradient gels (Bio-Rad) and transferred to a PVDF membrane. Membranes were blocked for 1 h at room temperature using a blocking buffer with gentle shaking, followed by incubation with primary antibodies at 4 °C overnight. The following primary antibodies were used: CNPase (1:3000, 5664S; Cell Signaling Technology), MBP (1:2500, 10458-1-AP; Proteintech), and β-actin (1:2500, 60008-1-Ig; Proteintech). After washing in PBS-T, membranes were incubated with HRP-conjugated secondary antibodies (31460 or 31430; Invitrogen) for 1 h at room temperature. Chemiluminescent signals were then captured using ECL substrates (34850; Invitrogen) and visualized with a multifunctional imaging system (SCG-W5000; ServiceBio). Fiji (ImageJ) software was used for semi-quantitative analysis. Relative protein expression levels were normalized to β-actin. All uncropped western blot images are provided in Supplementary Material (Figure S11).

Black-Gold staining

Black-Gold II stain (#TR-100-BG; Biosensis, USA) was applied according to the manufacturer’s instructions. Briefly, dried frozen brain sections were incubated in pre-warmed Black-Gold^® II sodium solution and sodium thiosulfate solution for 12 min at 65 °C in the dark. The sections were then rinsed in distilled water and fixed in 1% sodium thiosulfate for 3 min at 65 °C. After three washes, the sections were dehydrated through a graded ethanol series, cleared in xylene, and cover-slipped. Images were captured in the hippocampal CA1 region, medial prefrontal cortex (mPFC), and corpus callosum using an Olympus BX43 microscope equipped with a 10× objective. For analysis, three sections per animal were processed using ImageJ software.

Spine density analysis

Golgi staining was performed following the manufacturer’s protocol (PK401; FD NeuroTechnologies). Fresh brain samples were collected and incubated in an impregnation solution at room temperature for 10 days in the dark. The samples were then transferred to Solution C and stored at room temperature for 3 days, also in the dark. Coronal sections, 200 μm thick, were prepared using a Vibrating Microtome (VT1000E; Leica) and mounted onto gelatin-coated slides (PO101; FD NeuroTechnologies). After rinsing with Milli-Q water, the sections were stained with the provided staining solution for 10 min at room temperature, followed by another Milli-Q water rinse. The sections were then dehydrated through an ethanol series, cleared in xylene, and mounted with Permount™ mounting medium (SP15-100; Fisher Scientific, USA). Images were captured using an Olympus CSU W1 Spinning Disk. ImageJ software was used for image processing and analysis. Spine density was quantified from five animals per group, with five sections analyzed per brain.

Statistical analysis

GraphPad Prism and SigmaStat Software were used to perform statistical analyses. For data that met the normality assumptions of the statistical test, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was conducted to analyze all dependent variables. Kruskal–Wallis test followed by Dunn’s post-hoc test was used for data that did not pass the test for normality. The escape latency during the training phase in the Barnes maze and the cued memory test were analyzed using two-way ANOVA, followed by Tukey’s post-hoc test. CBF at various time points was analyzed using repeated measures one-way ANOVA, followed by Tukey’s multiple comparisons. All data were presented as mean ± SD. A value of p < 0.05 was considered significant for all statistical tests. Statistical details are provided in Supplementary Table 1.

Results

BCCAO leads to chronic cerebral hypoperfusion without affecting neuronal survival

To examine the effects of CCH, the BCCAO model, a well-established approach, was used (Figure S1(A, a)). Laser speckle contrast imaging was performed at baseline, and on days 7 and 29 post-BCCAO, to monitor changes in CBF in the anterior and posterior regions of the cortex (Figure S1(A, b) and (B, a)). As shown in Figure S1(B, b), significant reductions in CBF were observed on both days 7 and 29 in the anterior and posterior cortex. No differences in CBF were detected between the left and right hemispheres (data not shown). To assess whether enhanced myelination could improve cognitive outcomes following CCH, clemastine, a pro-myelinating compound, was administered daily starting on day 7 post-BCCAO for three consecutive weeks. Behavioral tests were conducted from days 90 to 111, and tissues were collected on day 112 (Figure S1(C)).

Immunofluorescence staining was subsequently performed to assess whether BCCAO induces neuronal loss. Given the critical role of the hippocampal CA1 region and medial prefrontal cortex (mPFC) in cognitive function, these two key brain regions were chosen for analysis (Figure S2(B)). Quantification of NeuN⁺ neurons and MAP2⁺ neuronal cell bodies/dendrites revealed no significant neuronal loss on day 112 post-BCCAO (Figure S2(C, a)–(C, d)), and clemastine treatment did not have a significant effect on neuronal survival in this BCCAO model.

Clemastine promotes axonal myelination after BCCAO

Clemastine, known for its pro-myelinating properties, has been widely recognized for promoting myelin regeneration in various neurological conditions.^19,20,35 Given that CCH can lead to significant myelin degradation,^11,12 we explored whether clemastine could promote axonal myelination following BCCAO. On day 112 post-BCCAO, immunostaining for NF200 and MBP (Figures 1(A, a) and S3(A)) was used to assess axons and myelin. No significant differences were observed in NF200⁺ axonal fluorescence intensity in either the hippocampus or mPFC among the groups (Figure 1(A, b) and (A, d)), indicating that CCH does not appear to induce substantial axonal degeneration in these regions at 112 days post-BCCAO. Nevertheless, a reduction in NF200 fluorescence intensity was detected in the corpus callosum, which was reversed by clemastine treatment (Figure S3(A)).

Figure 1.

Clemastine promotes axonal myelination after BCCAO. (A, a) Representative confocal immunofluorescence images showing MBP-positive myelin (green), NF200-positive axon (red), and DAPI (blue) in the hippocampal CA1 region and mPFC 112 days after BCCAO or sham surgery. Scale bar = 500 and 20 μm, respectively. The NF200 intensity and the MBP/NF200 ratio in the hippocampus (b, c) and mPFC (d, e) were quantitatively analyzed. N = 6/group. (B, a) Representative synthetic protein bands and quantification analyses of MBP in the hippocampus (b) and mPFC (c) 112 days after BCCAO or sham surgery. MBP was obtained using the ProteinSimple^® Capillary-based immunoassay, and corresponding peak areas were normalized to total protein levels (n = 4–6/group). (C, a) Representative images showing Black-Gold II myelin staining of the hippocampal CA1 region, and mPFC 112 days after BCCAO or sham surgery. Scale bar = 100 μm. (b, c) Quantitative analysis of the average intensity in the hippocampus and mPFC. N = 5/group. All data are presented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex; NS: no significant differences.

To evaluate axonal myelination, we analyzed the proportion of MBP⁺ myelin relative to NF200⁺ axons. As expected, BCCAO led to a marked reduction in myelinated axons across the hippocampus, mPFC, and corpus callosum (Figures 1(A, c) and (A, e) and S3(A)). These findings were further validated by ProteinSimple^® Capillary-based immunoassay (Figure 1(B, a)–(B, c)). In line with these observations, Black-Gold myelin staining revealed fewer myelin sheaths in the hippocampus, mPFC, and corpus callosum. This myelin loss was partially rescued by clemastine (Figures 1(C, a)–(C, c) and S3(B)). These results indicate that CCH induces persistent myelin loss, and clemastine, as a pharmacological strategy, can significantly enhance myelination after BCCAO.

To further investigate whether BCCAO induces demyelination at an earlier time point, additional experimental groups were included in which tissues were collected 29 days post-BCCAO. Although no obvious axonal loss was observed at this stage, substantial myelin loss was evident in the corpus callosum (Figure S4(A)), as indicated by a reduced ratio of MBP⁺ myelin to NF200⁺ axons. Notably, this myelin loss was rescued by clemastine treatment. Western blot analysis showed that the expression of CNPase and MBP—two well-established myelin markers—was significantly reduced in the corpus callosum following BCCAO, while clemastine treatment restored their expression (Figure S4(B)).

Clemastine increases the proportion of mature oligodendrocytes after BCCAO

The mechanism by which clemastine exerts its pro-myelinating effect is thought to involve the modulation of the M1 muscarinic receptor, leading to enhanced differentiation of oligodendrocyte precursor cells (OPCs) and an increase in mature oligodendrocyte populations. ¹⁸ To further investigate this mechanism, we performed immunostaining for PDGFRα, Olig2, and CC1, which are markers representing different stages of the oligodendrocyte lineage (Figure 2(A)). As shown in Figures 2(B) and (D) and S6(A), BCCAO induced a marked reduction in the proportion of CC1⁺/Olig2⁺ mature oligodendrocytes across the hippocampus, mPFC, and corpus callosum. In contrast, clemastine treatment significantly increased the proportion of CC1⁺/Olig2⁺ mature oligodendrocytes. In addition, no significant changes were observed in the number of Olig2⁺ oligodendrocytes or PDGFRα⁺ Olig2⁺ cells (Figures 2(C) and (D) and S6(A)), nor the protein expression levels of PDGFRα (Figures 2(E, a) and (E, b) and S6(B)).

Figure 2.

Clemastine increases the number of mature oligodendrocytes in the rat BCCAO model. (A) A graph displaying the various stages of oligodendrocyte lineage and the corresponding markers utilized in this study. (B) Representative confocal immunofluorescence images showing Olig2⁺ oligodendrocytes (red), and CC1-positive mature oligodendrocytes (green) in the hippocampal CA1 region and mPFC 112 days post-BCCAO or sham surgery. Scale bar = 100 μm. (C) Representative confocal immunofluorescence images showing Olig2⁺ oligodendrocytes (red), PDGFRα⁺ oligodendrocyte precursor cells (green), and DAPI (blue) in hippocampal CA1 region and mPFC 112 days post-BCCAO or sham surgery. Scale bar = 100 μm. (D) Quantification of Olig2⁺ cells, the percentage of CC1⁺ Olig2⁺ cells and PDGFRα⁺ Olig2⁺ cells among total Olig2⁺ cells in the hippocampal CA1 region and mPFC. (E, a) Representative synthetic protein bands of PDGFRα in the hippocampus and mPFC 112 days after BCCAO or sham surgery. (b) Protein quantification results for PDGFRα in the hippocampus and mPFC. The corresponding peak areas were normalized to total protein levels (n = 4–6/group). All data are presented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex; NS: no significant differences.

To further examine changes in oligodendrocyte lineage cells at an earlier time point, immunofluorescence staining was performed at 29 days post-BCCAO to assess mature oligodendrocytes and OPCs (Figure S5(A) and (B)). Although the total number of Olig2⁺ oligodendrocytes did not differ among groups, the proportion of CC1⁺/Olig2⁺ mature oligodendrocytes was also significantly reduced across multiple brain regions, and this reduction was partially reversed by clemastine treatment. In contrast, there were no significant differences in the proportion of PDGFRα⁺/Olig2⁺ OPCs or PDGFRα protein expression among groups at this time point (Figure S5(B) and (C)).

We next investigated whether clemastine influences the proliferation of oligodendrocyte lineage cells. Since OPCs can originate from neural progenitor cells, which are abundant in the subgranular zone (SGZ) of the hippocampus (Figure S7(A)), we performed immunofluorescence for Olig2/Ki67 and BrdU/Olig2 (Figure S7(B) and (C)) in this brain area. As quantified in Figure S7(E), no significant differences were observed among the groups. To assess whether BCCAO or clemastine impacts oligodendrocyte apoptosis, TUNEL assay was conducted. As shown in Figure S7(D) and (E), double staining of TUNEL/Olig2 and data quantification revealed no significant differences in oligodendrocyte apoptosis across the groups. Taken together, our data indicate that clemastine promotes OPC differentiation and oligodendrocyte maturation rather than increasing the overall OPC or oligodendrocyte lineage cell population. Next, we sought to determine whether oligodendrocyte apoptosis occurs at an earlier time point. Given that myelin loss is evident in the corpus callosum at 29 days post-BCCAO, TUNEL assay and immunofluorescence staining for Olig2 were performed to evaluate oligodendrocyte death in this region (Figure S8(A)). However, no obvious oligodendrocyte death was observed at this time point (Figure S8(A)). These findings suggest that BCCAO induces selective loss of mature oligodendrocytes, and that early clemastine treatment may confer neuroprotection.

Treatment of clemastine preserves the structural integrity of the node of Ranvier

The node of Ranvier is a small gap between adjacent myelin sheaths along myelinated axons, characterized by the clustering of Na⁺ channels. ³⁶ The structural integrity of the node of Ranvier is essential for the rapid and efficient propagation of action potentials. ³⁷ The disrupted structural integrity of the node of Ranvier is widely recognized as an indicator of myelin dysfunction or impaired axon—oligodendrocyte communication. Lengthening of the node of Ranvier, typically due to demyelination, can weaken the electrically resistive seal and impair the speed of action potential propagation.^36,38

Next, we sought to determine if BCCAO disrupts the integrity of the node of Ranvier and whether enhanced myelination can preserve its structure. Figure 3(A) presents a schematic diagram of myelinated axons, illustrating the nodes of Ranvier and the paranodal regions. We performed immunofluorescence staining for Caspr, a paranodal marker, and Nav1.6, a nodal marker (Figures 3(B) and S9(A)). The node of Ranvier length was calculated as the distance between two Caspr-positive regions, and nodal density was evaluated by measuring the density of Nav1.6. Quantification in Figures 3(B, b) and (B, c) and S9(A, b) reveals a significant reduction in nodal intensity within the hippocampus, mPFC, and corpus callosum following BCCAO, a disruption that was prevented by enhanced myelination with the treatment of clemastine. Additionally, nodal length was increased in these brain areas after BCCAO, but this alteration was reversed following enhanced myelination (Figures 3(B, d) and (B, e) and S9(A, b)).

Figure 3.

Treatment of clemastine preserves the nodes of Ranvier in the rat BCCAO model. (A, a) Schematic diagram of myelinated axons, illustrating the nodes of Ranvier and the paranode. (b) A representative node from the sham group is shown at higher magnification in the right panel, and the node length was measured as the distance between the two Caspr-positive paranodes (red). (B, a) Representative confocal immunofluorescence images showing Nav1.6-positive nodes (green) and Caspr-positive paranode regions (red) in the mPFC and hippocampal CA1 region 112 days after BCCAO or sham surgery. Scale bar = 10 μm. (b, c) The density of nodes in the hippocampal CA1 area and mPFC were quantified. Nodes were counted only if both Nav1.6 and Caspr staining were positive. Five brain slices from each animal were used for the node counts (n = 25 from five animals per group). (d, e) The average nodal gap length in the mPFC and hippocampal CA1 region was compared (n = 50 nodes from five animals per group). All data are presented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex.

Treatment of clemastine preserves dendritic spine density and prevents synaptic loss after BCCAO

Impaired synaptic plasticity is a prominent consequence of CCH.^39,40 To investigate whether enhanced myelination could mitigate synaptic dysfunction following BCCAO, Golgi staining was conducted (Figure 4(A) and (B)). Quantification revealed a significant decrease in the density of total dendritic spines and mushroom (mature) spines in both the hippocampus and mPFC after BCCAO (Figure 4(C, a) and (C, b)). Remarkably, treatment of clemastine effectively preserved dendritic spine density in these brain regions (Figure 4(C, a) and (C, b)).

Figure 4.

Treatment of clemastine preserves dendritic spine density 112 days after BCCAO in the mPFC and hippocampal CA1 region. (A) The left panel shows a representative image of the dendritic branching pattern from Golgi staining in the mPFC (blue rectangle) and hippocampal CA1 region (red rectangle; 10× magnification). The middle panel provides a higher magnification of neurons from the left rectangle. The right panel exhibits apical dendrites selected for analysis at 100× magnification. Scale bar = 20 μm. (B) Selected dendritic segments in the hippocampal CA1 region and mPFC from each group. (C) The total number of spines and mushroom-like spines per 40 μm apical dendrites in the hippocampal CA1 region (a) and mPFC (b) were manually counted and summarized. All data are presented as mean ± SD. Five brain slices from each animal were used for the dendritic spine counting (n = 20 from five animals per group). *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex.

Next, we sought to examine whether BCCAO can lead to synaptic deficits. To this end, immunofluorescence staining for synaptophysin and spinophilin was performed (Figure 5(A, a)). Synaptic density was measured by quantifying synaptophysin, a presynaptic protein marker. As shown in Figure 5(A, b) and (A, c), the density of spinophilin⁺ dendritic spines was significantly reduced after BCCAO, consistent with the Golgi staining results. Additionally, synaptophysin⁺ synapses and spinophilin⁺ synaptophysin⁺ puncta were diminished post-BCCAO. Treatment of clemastine, notably, rescued these synaptic deficits (Figure 5(A, b) and (A, c)). These findings were further supported by ProteinSimple^® Capillary-based immunoassay, which showed elevated synaptophysin protein expression with enhanced myelination (Figure 5(B)). To further determine whether clemastine treatment reverses or prevents synaptic dysfunction, the same analysis was conducted at 29 days post-BCCAO in the corpus callosum (Figure S10(A) and (B)). Intriguingly, no apparent synaptic deficits were observed at this time point, suggesting that clemastine prevents, rather than reverses, synaptic dysfunction in the rat BCCAO model.

Figure 5.

Treatment of clemastine inhibits BCCAO-induced synaptic dysfunction. (A, a) Representative confocal immunofluorescence images of the dendritic spine marker, spinophilin (green), the presynaptic marker, synaptophysin (red), and the colocalized puncta in the hippocampal CA1 region (scale bar = 10 μm) and mPFC (scale bar = 50 μm) 112 days after BCCAO or sham surgery. (b, c) Quantification of synaptophysin and spinophilin granule density, as well as their colocalization, in the hippocampal CA1 region and mPFC (n = 6/group). (B) Representative synthetic protein bands of synaptophysin in the hippocampal CA1 region 112 days after BCCAO or sham surgery. Corresponding peak areas were normalized to total protein levels. (n = 5/group). All data are presented as mean ± SD. *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex.

Treatment of clemastine prevents cognitive deficits after BCCAO

To evaluate the therapeutic potential of enhancing myelination in CCH, several cognition-associated behavioral tests were conducted. First, the Barnes maze test was performed to assess spatial learning and memory (Figure 6(A, a) and (A, b)). The results indicated impaired spatial learning performance following BCCAO, as demonstrated by longer escape latencies during the 3-day training phase. However, treatment of clemastine rescued this deficit (Figure 6(A, c)). Similarly, during the probe test, animals subjected to BCCAO displayed poor performance (Figure 6(A, e) and (A, f)), indicating impaired spatial memory, which was reversed by enhancing myelination. Additionally, there was no difference in average speed among the groups (Figure 6(A, d)), suggesting that BCCAO does not significantly affect locomotor activity, eliminating it as a potential confounding factor in the behavioral results.

Figure 6.

Treatment of clemastine preserves spatial learning and memory performance following BCCAO. (A, a, b) Representative escaping traces (training phase) and heat maps (probe test) of animals from each group recorded during the Barnes maze tests, 3 months after BCCAO or sham surgery. (c) Analysis of escape latencies during the training phase. (d–f) Analysis of average speed, quadrant occupancy in the TQ, and errors in finding the target box during the probe test. Errors were defined as searches of any hole without the hidden escape chamber beneath it. Quadrant occupancy was assessed during the probe test on day 4, following 3 days of training. (B, a, b) Paradigms for the contextual and cued memory tests. (c) Occupancy plot of the rats’ center point during the contextual memory test. Freezing time in response to the conditioned room context (Context-A) in the contextual memory test is shown in (d). (e) Occupancy plot of the rats’ center point during cued memory test. (f) Freezing response to the unconditioned room context (Context-B). No significant difference was found for baseline among groups. (g) Analysis of the freezing response to the conditioned stimulus tone during the cued memory test. All data are presented as mean ± SD (n = 10–11). *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex; NS: no significant differences; TQ: target quadrant.

The fear conditioning test was also performed to evaluate associative learning and memory function (Figure 6(B, a) and (B, b)). Animals subjected to BCCAO showed less freezing time during the contextual memory test, suggesting compromised fear memory (Figure 6(B, c) and (B, d)). As expected, enhanced myelination prevented this deficit, as evidenced by increased freezing time (Figure 6(B, c) and (B, d)). However, no significant differences were observed during the cued memory test (Figure 6(B, e)–(B, g)). One possible explanation is that the BCCAO model did not affect the brain regions or neural circuits involved in cued fear memory retrieval, allowing animals to exhibit intact cued memory.

Treatment of clemastine preserves neurovascular coupling

Neurovascular coupling reflects the intricate interaction between neuronal activity and CBF and is essential for proper brain function. ⁴¹ Impaired neurovascular responses have been observed in patients with severe brain trauma, ⁴² but whether CCH also affects neurovascular coupling remains unclear. To investigate this, whisker stimulation was performed 29 days after BCCAO or sham surgery. This test operates on the principle that tactile stimulation of rodent whiskers activates sensory neurons, leading to increased neuronal activity and CBF in the barrel cortex.^34,43

The region for stimulation and the brain area monitored for CBF changes are illustrated in Figure 7(A). Figure 7(B, a) shows the barrel cortex monitored for CBF changes during the whisker stimulation test using laser speckle imaging. Representative images of CBF in the barrel cortex before and after whisker stimulation are shown in Figure 7(B, b) and (B, c), respectively. Subtracting the baseline image from the stimulation image revealed CBF changes localized to the barrel cortex (Figure 7(B, d)). Initially, no significant differences in barrel cortex CBF were observed among the three experimental groups before stimulation (Figure 7(C, a)). However, following whisker stimulation, animals subjected to BCCAO exhibited a significantly blunted CBF increase in the barrel cortex compared to the sham group, indicating impaired neurovascular coupling in CCH (Figure 7(C, b) and (C, c)). Importantly, enhanced myelination by clemastine preserved neurovascular coupling, as evidenced by the improved regional CBF response to stimulation (Figure 7(C, b) and (C, c)).

Figure 7.

Clemastine improves neurovascular coupling after BCCAO. (A) Schematic diagram depicting the experimental timeline, the whisker stimulation paradigm, and the corresponding barrels in the primary somatosensory barrel cortex (S1). (B, a) Representative images of CBF in the barrel cortex obtained using laser speckle imaging, shown before (b) and after (c) whisker stimulation in different groups. (d) CBF changes were visualized by subtracting baseline images from stimulation images using ImageJ software. After separating into RGB channels, only the red channel is shown. (C, a) Baseline CBF in three groups before whisker stimulation. (b) Quantification of the CBF increase relative to baseline in response to whisker stimulation in the S1 barrel cortex. (c) Typical couple CBF response during whisker stimulation across three sessions among different groups. (D, a) Representative confocal immunofluorescence images showing c-fos (red) and MBP (green) in the Barrel cortex and (b, c) quantification of c-fos⁺ cells and MBP intensity 29 days post-BCCAO. Scale bar = 100 μm. All data are presented as mean ± SD (n = 4–5). *p < 0.05 versus sham, ^#p < 0.05 versus BCCAO group.

BCCAO: bilateral common carotid artery occlusion; Cle: clemastine; mPFC: medial prefrontal cortex; NS: no significant differences.

To further determine whether the enhanced regional CBF response was attributable to increased local neuronal activity, animals were sacrificed 90 min after whisker stimulation, and c-Fos immunofluorescence staining was performed in the barrel cortex (Figure 7(D, a)). As quantified in Figure 7(D, b), rats that underwent BCCAO exhibited a marked reduction in the number of c-Fos⁺ cells in the barrel cortex, indicating diminished neuronal activity in response to stimulation. Notably, animals treated with clemastine following BCCAO showed an increased number of c-Fos⁺ cells, supporting the interpretation that the enhanced regional CBF response is driven by increased neuronal activity in response to stimulation. In line with this, rats subjected to BCCAO exhibited reduced MBP intensity in the barrel cortex, which was reversed by clemastine (Figure 7(D, c)). These findings further support that improved local neuronal activity and neurovascular coupling responses may be attributed to enhanced myelination.

Discussion

CCH, a severe condition caused by various risk factors, has been associated with neurocognitive disorders.^5,44 However, effective therapies to prevent the devastating consequences of CCH remain limited. As an insulating layer, myelin supports axons and ensures the efficient transmission of electrical signals in the nervous system. ⁴⁵ Recently, myelin has been demonstrated to be involved in cognitive processes and memory.^16,17 Myelin pathology has been implicated in various neurological disorders, and enhancing myelin renewal, either pharmacologically or genetically, has been shown to rescue memory deficits in experimental models.^22,23,35,46 Given the widespread presence of myelin pathology in CCH, we sought to examine whether enhancing myelination could rescue cognitive deficits resulting from CCH. To this end, clemastine—a widely used pro-myelinating drug—was used in this study.

Clemastine fumarate is an FDA-approved antihistamine with antimuscarinic properties. Both in vitro and in vivo studies have demonstrated that clemastine enhances OPC differentiation through its antagonism of the M1 muscarinic acetylcholine receptor, a known negative regulator of OPC maturation.^18,21 Furthermore, clemastine has been shown to promote remyelination in various experimental models when administered at a dose of 10 mg/kg.^19,22,28,47 Based on this evidence, the same dosing regimen was employed in the present study. Our findings showed that clemastine treatment promoted myelination following BCCAO and prevented BCCAO-induced cognitive deficits. Given that this dosage has consistently been shown to facilitate remyelination, our results support the idea that myelin repair may represent a promising therapeutic strategy for preventing neurocognitive symptoms associated with CCH. These findings also highlight the therapeutic potential of pro-myelinating compounds in the context of CCH. Indeed, clemastine has attracted growing research interest, and multiple clinical trials are currently ongoing or recruiting to evaluate its efficacy in demyelinating conditions (e.g. NCT05359653, NCT05338450, NCT06087757, and NCT06315699). The findings of these trials are expected to help bridge the gap between preclinical findings and clinical practice, further evaluating the therapeutic potential and safety of clemastine in demyelinating diseases.

Although clemastine is well established as a pro-myelinating compound, potential off-target effects beyond its myelination-promoting properties cannot be ruled out and may also contribute to the neuroprotection observed in the present study. A feasible approach to determine whether the therapeutic effects of clemastine are specifically attributable to enhanced myelination would involve the use of a tamoxifen-inducible Cre/loxP system to selectively delete Myrf, a key transcription factor required for oligodendrocyte differentiation, in OPCs.^48,49 This genetic manipulation is expected to impede myelination upon Cre-mediated recombination during clemastine treatment. However, such inducible Cre/loxP systems are currently only available in mice, which prevents us from directly validating whether the neuroprotective effects of clemastine in the BCCAO rat model used in this study are exclusively mediated through enhanced myelination.

The injured brain typically exhibits both demyelination and a limited degree of endogenous remyelination; however, this intrinsic remyelination is generally insufficient for functional recovery.^20,22 Differentiating between changes in demyelination and remyelination in response to treatment in demyelinating disorders can provide critical insight into the underlying mechanisms of therapeutic efficacy. To achieve this, multiple inducible Cre/loxP systems are often required, including the use of fluorescent reporter mice and distinct Cre drivers to visualize pre-existing myelin or to trace the fate of OPCs contributing to newly formed myelin.^22,49 While previous studies using inducible Cre/loxP systems to trace OPC lineage have demonstrated that clemastine promotes new myelin formation,^20,22 whether it also attenuates ongoing demyelination—albeit potentially to a lesser extent—remains to be determined. In the present study, we observed early loss of mature oligodendrocytes following BCCAO, and early clemastine treatment remarkably increased the proportion of mature oligodendrocytes, which was accompanied by an increase in myelin levels. Therefore, the therapeutic effects of clemastine in our BCCAO model may be primarily attributable to enhanced myelination.

Recent studies highlight a strong link between myelination and synaptic plasticity, suggesting that myelination may be an underappreciated mechanism of activity-dependent plasticity.^19,50 Synaptic dysfunction is widely observed following central nervous system (CNS) insults and has been implicated in cognitive impairment.^51,52 To gain further insights into how enhanced myelin renewal could prevent CCH-associated cognitive deficits, we performed Golgi staining and observed a marked reduction in both total and mature dendritic spine density in the hippocampal CA1 region and the mPFC. This deficit was alleviated by clemastine treatment. Next, we examined the expression of synaptophysin, a presynaptic vesicle marker, in these brain regions and found that their expression was reduced following BCCAO. As expected, clemastine treatment significantly increased synaptophysin expression. To gain further insight into whether clemastine prevents or rescues synaptic dysfunction resulting from CCH, immunofluorescence staining for synaptophysin and spinophilin, as well as synaptophysin protein expression, were assessed at 29 days post-BCCAO. No evidence of synaptic dysfunction was detected at this time point, suggesting that clemastine prevents, rather than reverses, synaptic impairment in CCH. The maintenance of synaptic function may further counteract cognitive deficits following CCH. Since synaptogenesis marks the final stage of axonal pathfinding, the increased proliferative capacity of OPCs and enhanced myelination may play a role in this process. ⁵³ Further research is needed to clarify the impact of myelination on synaptic plasticity. Interestingly, studies have revealed that synaptic vesicles may also play a role in axonal myelination in the CNS. In zebrafish, blocking synaptic vesicle release leads to a significant reduction in the number of myelin sheaths. ⁵⁴ This raises the possibility that enhanced myelination may promote synaptic vesicle release following CNS insults, which could, in turn, contribute to further axonal myelination. This could form a positive feedback loop, where enhanced myelination supports increased synaptic activity, which in turn drives additional myelination, ultimately fostering neuronal plasticity and recovery following brain injury. However, this remains to be confirmed by future studies.

Normal neurovascular coupling is thought to be critical for cognitive health and compromised neurovascular coupling has been proposed as a key contributor to cognitive impairment in aging and brain disorders. ⁵⁵ Our results demonstrated that CCH induced significant neurovascular dysfunction, as evidenced by a reduced regional CBF response during the whisker stimulation test—a well-established measure of neurovascular function. Notably, clemastine treatment significantly improved the regional CBF response to whisker stimulation. Animals treated with clemastine also exhibited a marked increase in neuronal activation within the barrel cortex in response to stimulation. We speculate that the restoration of neurovascular coupling following clemastine treatment may result from global remyelination in the brain. Neurovascular coupling relies on the complex communication among multiple cell types. Enhanced myelination can increase the speed and efficiency of action potential propagation along axons, potentially strengthening communication between neurons and other cells, such as astrocytes and endothelial cells. In the context of CCH, this improved intercellular communication may contribute to the restoration of neurovascular coupling. Nevertheless, these observations might be interpreted with caution. Although clemastine, at the dosages used in the present study, has primarily been shown to promote myelin renewal without affecting other cell types, such as astrocytes and microglia, ¹⁹ it might still have unrecognized effects on neurovascular function independent of myelination. At this point, genetic manipulation of axonal myelination may represent a feasible approach to determine whether enhanced myelination can directly improve neurovascular coupling. One limitation of this study is the lack of continuous monitoring of arterial blood gases and blood pressure during isoflurane anesthesia. Although all animals were maintained under comparable anesthetic conditions with similar durations and showed stable spontaneous respiration throughout the procedures, the absence of CO₂ challenge and physiological monitoring may preclude a more precise assessment of cerebrovascular reactivity.

Overall, this study provides the first evidence that the pro-myelinating compound clemastine can prevent synaptic dysfunction, impaired neurovascular coupling, and cognitive deficits following CCH. These findings are promising, as drug repurposing may accelerate clinical translation and offer a practical therapeutic approach for CCH. Moreover, in light of the well-established pro-myelinating properties of clemastine, our results suggest that myelin repair could represent a novel therapeutic target for preventing CCH-associated neurocognitive disorders. This may also open new avenues for exploring the efficacy of other remyelinating therapies in the context of CCH. In addition to its common use at high doses to promote myelin renewal in animal models, clemastine has also demonstrated beneficial effects at lower doses in patients with multiple sclerosis, a demyelinating disease. ⁵⁶ Further research is needed to determine the therapeutic dosage ranges of clemastine, as well as its optimal timing and routes of administration. Moreover, given the identification of various pro-myelinating compounds and growing research interest in the field, it would be appealing to examine whether other pro-myelinating agents with distinct pharmacological properties exert similar therapeutic effects. These efforts could potentially lead to effective treatment options for patients with CCH.

Conclusions

As a risk factor for cognitive impairment and dementia, CCH imposes a significant burden on society. However, the mechanisms by which CCH affects cognitive processes remain incompletely understood. Our results suggest that myelin loss may be a key contributor to CCH-associated cognitive impairment. Enhancing myelination with clemastine, a well-established pro-myelinating agent, significantly prevented cognitive deficits and preserved neurovascular function, as well as synaptic function in an experimental model of CCH. These results suggest that enhancing myelination might offer a promising therapeutic strategy for CCH.