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. Author manuscript; available in PMC: 2026 Sep 11.
Published in final edited form as: Neurobiol Dis. 2026 Aug 4;228:107563. doi: 10.1016/j.nbd.2026.107563

Chronic hypertension and social isolation disrupt the blood-brain barrier and memory in a sex-dependent manner

Anduela Thaqi a, Gianna M DeMarco a, Emily D Schellinger a, Dinlin Chen a, Jared Luchetta a, Abbie C Chapman a,b,*
PMCID: PMC13559686  NIHMSID: NIHMS2208322  PMID: 42551659

Abstract

Chronic hypertension in mid-life is a leading cause of vascular cognitive impairment (VCI), the second leading cause of age-related cognitive decline. Further, social isolation is another risk factor for dementia, and disproportionately impacts middle-aged adults. However, mechanisms by which social isolation may compound chronic hypertension to damage the cerebrovasculature and contribute to VCI remain unclear. The current study used male and female adult Wistar rats and spontaneously hypertensive rats (SHR) to investigate how social isolation during chronic hypertension disrupted blood-brain barrier (BBB) and memory function. Half of the SHR were housed individually for three months, with remaining SHR and Wistar rats housed in groups and routinely handled (n = 8/group). At six months old, long-term and spatial working memory were assessed using object recognition and continuous y maze tasks, respectively. BBB permeability was measured in-vivo in dorsal hippocampus and prefrontal cortex and serum markers of inflammation assessed. Long-term memory function was impaired only in isolated male SHR, as recognition index was lower compared to socialized male Wistar and SHR (0.59 ± 0.07 vs. 0.70 ± 0.02 and 0.77 ± 0.03; p < 0.05 by 2way ANOVA), which was associated with increased BBB permeability in the hippocampus. In contrast, spatial working memory was only impaired in isolated female SHR that had a decreased alternation index compared to socialized female Wistar rats (0.59 ± 0.04 vs. 0.74 ± 0.02; p < 0.05 by 2way ANOVA). This sex-specific deficit was associated with greater BBB disruption in prefrontal cortex that correlated with elevated levels of circulating oxidative stress marker 8-isoprostane. These findings suggest that social isolation during chronic hypertension differentially impairs memory function in a sex-dependent manner. Overall, this study advances our understanding of cerebrovascular consequences of social isolation in adulthood and indicate brain region/memory domain dysfunction differ by sex during chronic hypertension.

Keywords: Blood-brain barrier, Chronic hypertension, Sex differences, Social isolation, Vascular cognitive impairment

1. Introduction

The incidence of dementia is rising at a devastating rate as the population continues to live longer lives, and is currently estimated to affect at least 1 in 10 people over the age of 65 (Alzheimers Dement., 2022; Cahill, 2020). Vascular contributions to cognitive impairment and dementia are the second leading cause of dementia after Alzheimer's disease (Santisteban and Iadecola, 2025). Vascular cognitive impairment (VCI) involves a spectrum of cognitive dysfunction due to cerebrovascular disease, with its most severe form being vascular dementia (Alzheimers Dement., 2022; Iadecola et al., 2019). Importantly, up to 80% of individuals clinically diagnosed with Alzheimer's disease have cerebrovascular pathology in addition to classic Alzheimer's disease neurodegeneration (e.g., mixed dementia). These pathologies interact synergistically and accelerate cognitive decline (Alzheimers Dement., 2022; Spina et al., 2021; Barnes et al., 2019; Power et al., 2018). Thus, the incidence of VCI is likely underestimated, highlighting the critical need to understand vascular mechanisms of age-related cognitive decline and ease the progression of Alzheimer's disease and related dementias, such as VCI.

Currently, there are no treatments for VCI, and interventions focus solely on preventative strategies including managing modifiable risk factors, such as chronic hypertension. Chronic hypertension is the leading cause of cerebrovascular disease and increases the risk for VCI by up to 60%, particularly when hypertension occurs in mid-life and remains uncontrolled (Whelton et al., 2018; Launer et al., 2010; Fujishima and Kiyohara, 2002; Yamada et al., 1999). This increased risk for VCI is in part due to the prevalence for ischemic stroke in hypertensive patients, resulting in post-stroke cognitive impairment (Iadecola et al., 2019; Kalaria et al., 2016). However, chronic hypertension directly damages the cerebral circulation independently of stroke and accelerates cognitive decline. Specifically, hypertension-induced cerebrovascular dysfunction and atrophy occur in the hippocampus and prefrontal cortex, two brain regions critical to memory, contributing to VCI (Pacholko and Iadecola, 2024; Santisteban et al., 2023; Chapman et al., 2025; Johnson, 2023; Gannon et al., 2024; Lai et al., 2022).

Several aspects of cerebrovascular function are compromised during chronic hypertension, including the highly protective blood-brain barrier (BBB). The BBB is a complex interface between systemically circulating factors and the delicate brain milieu. Cerebral endothelial cells are primary constituents of the BBB that are unique due to specialized high electrical-resistance tight junctions and lack of fenestrations that limit para- and transcellular permeability (Ueno, 2007). BBB disruption can cause neuroinflammation, neuronal dysfunction, and impaired cognition (Pacholko and Iadecola, 2024; Santisteban et al., 2023). In fact, BBB breakdown is considered an early biomarker of human cognitive dysfunction (French et al., 2025). Importantly, the BBB in the hippocampus has been reported to be more susceptible to disruption than other brain regions (Montagne et al., 2015; Johnson et al., 2018; Kuntner et al., 2010; Nation et al., 2019). For example, in patients with mild cognitive impairment, increased BBB permeability in the hippocampus occurred earlier and to a greater extent than in cortical brain regions, as measured via dynamic contrast-enhanced MRI (Montagne et al., 2015; Nation et al., 2019). Therefore, region-specific vulnerability may exist such that chronic hypertension damages the BBB in hippocampus more so than prefrontal cortex. Further, males in general have higher BBB permeability compared to females which is attributed to protective actions of estrogens (Dion-Albert et al., 2022). We have recently reported that circulating proinflammatory mediators that damage the BBB (e.g., intercellular adhesion molecule 1, ICAM-1) are higher in male compared to female chronically hypertensive rats (Chapman et al., 2025), suggesting the BBB of males may be more sensitive to disruption. Whether chronic hypertension disrupts the hippocampal BBB to a greater extent than in other cognition-centric brain regions (e.g., prefrontal cortex), and if this differs by sex, remains unclear but could contribute to mechanisms by which hypertension accelerates age-related memory decline.

In addition to pathological states promoting VCI (e.g., hypertension), prevalent psychosocial factors, such as social isolation, also contribute. In general, social isolation and loneliness are often overlooked cardiovascular risk factors that impact about half of all adults, a statistic that can be much higher based upon factors such as race, ethnicity, and economic status (Guarnera et al., 2023). In fact, we are currently living in a time considered to be an epidemic of adult loneliness and isolation, intensified by the COVID-19 pandemic and is disproportionately impacting middle-aged adults (Richardson et al., 2025; Infurna et al., 2025). Social isolation is associated with cognitive decline and ~ 50% increase in dementia risk that occurs, in part, due to development of cardiovascular disease and cerebrovascular dysfunction. Importantly, chronic social isolation is associated with systemic inflammation and elevated circulating pro-inflammatory factors that damage the BBB (Guarnera et al., 2023; Lao et al., 2024; Zheng et al., 2025). While there is a substantial body of knowledge regarding how social isolation in early life contributes to neurocognitive decline, much less is known about its impact in middle adulthood, including in the setting of comorbidities such as hypertension (Xiong et al., 2023). Therefore, understanding the potential compounding effects of social isolation and chronic hypertension on BBB integrity, and whether one sex or brain region/memory domain is more susceptible is critical to identify novel therapeutic targets to protect neurocognitive health and slow/prevent development of VCI and dementia.

In the current study, we investigated the effects of chronic hypertension and social isolation on long-term and spatial working memory functions in male and female spontaneously hypertensive rats (SHR) in mid adulthood. To understand region-specific susceptibility, BBB permeability was measured in-vivo by quantifying passage of different sized fluorescent tracers into dorsal hippocampus (involved in long-term memory function) and prefrontal cortex (involved in spatial working memory). We hypothesized that memory would be impaired during chronic hypertension that would be exacerbated by social isolation. We further hypothesized that BBB permeability would be higher in males than females, that hypertension and social isolation would increase permeability to a greater extent in the dorsal hippocampus compared to prefrontal cortex, and that corresponding memory functions would be disrupted. To understand potential mechanisms by which chronic hypertension and social isolation may disrupt the BBB, circulating systemic inflammatory mediators that damage BBB tight junctions and play a role in VCI were measured, including ICAM-1, tumor necrosis factor alpha (TNFα) and oxidative stress biomarker 8-isoprostane (Santisteban and Iadecola, 2025; French et al., 2025; Abdoun et al., 2025; Varatharaj and Galea, 2017; Montuschi et al., 2004).

2. Methods

2.1. Animals

Male and female Wistar rats and SHR were purchased from Charles River at 3 months of age (N = 48) and housed in the Binghamton University - SUNY animal facility, an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility. Rats were maintained on a 12-h light/dark cycle with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Euthanasia occurred via exsanguination followed by rapid decapitation under isoflurane anesthesia (3% in oxygen). Male and female SHR were randomly assigned to either isolated or social housing conditions (n = 8/group) until terminal experiments. Isolated rats were single housed and unhandled, whereas socialized Wistar rats and SHR were housed in groups and routinely handled. All rats had a tunnel in their home cage.

2.2. Behavioral assessments

At six months of age, two types of memory function were assessed using behavioral tasks as we have described previously: a novel object recognition (NOR) task to test long-term memory that is largely hippocampal-dependent and a continuous y-maze task to test spatial working memory that is prefrontal cortex-dependent (Chapman et al., 2025; Gannon et al., 2024). Briefly, the NOR task included four phases conducted over the course of two days: 1. habituation – rats were placed in an open field arena for 5 min; 2. acquisition – rats freely explored two identical objects in the arena for 10 min; 3. consolidation – rats were placed back in their home cages and returned to the animal facility where they remained undisturbed for 24 h; and 4. test – rats were placed back in the arena with one novel and one familiar object for 5 min. The time spent exploring each object was quantified using automated tracking software AnyMaze 7.43 (Stoelting Co., Wood Dale, IL, USA). Recognition index was calculated as a measure of long-term memory function using the following formula: TimeNovel / TimeTotal, where TimeNovel is the time spent investigating the novel object, and TimeTotal the total time investigating both objects. In addition, open field data from the NOR habituation phase were used to assess locomotion, including average speed and distance traveled. The following day, rats were allowed to freely explore a y-maze for 8 min. Arm entries were used to quantify an alternation index as a measure of spatial working memory using the formula: # of Alternations / (# of Arm Entries – 2). Rats acclimated to the behavior room for one hour before behavior began and all tasks were completed at the same time of day.

2.3. BBB permeability in vivo

To understand how social isolation may compound hypertension-related BBB dysfunction and impair memory, BBB permeability to three different sized tracers was measured in-vivo: 0.3 kDa sodium fluorescein (NaFl, 5 mg/mL), 3 kDa Texas red dextran (1 mg/mL) and 10 kDa cascade blue dextran (1 mg/mL) (Johnson et al., 2014). Under isoflurane anesthesia, blood was collected from the tail vein, centrifuged, and serum stored at −80 °C. Rats were injected via tail vein with the cocktail of tracers (1.5 mL at 5 mL/min) which circulated for 5 min. The circulation was then flushed via transcardiac perfusion of cold 0.1 M PBS (~ 80 mL, 10 mL/min). All tracer stocks and cocktails were protected from light. Under red light, brains were extracted and prefrontal cortex and dorsal hippocampus dissected. Tissue was weighed and homogenized using a bead homogenizer for two minutes (2 m/s in 800 μL of 0.1 M PBS; VWR Bead Mill Homogenizer 4). Tissue homogenates were deproteinated by adding 800 μL of 50% TCA (trichloroacetic acid) then vortexed for one minute. Samples were then centrifuged for 10 min at 4500 rpm and 4 °C, supernatants collected and centrifuged again at the same parameters. Samples were plated into a black 96-well plate in triplicate. TCA/PBS wells, consisting of an equal mixture of 50% TCA and PBS were also plated to be analyzed as a blank control. Pilot studies were conducted to determine appropriate concentrations for all tracers and circulation time was sufficient to detect passage from blood to tissue. Additionally, a portion of kidney was processed as a positive control to confirm that tail vein injections were successful. Fluorescence was quantified as relative fluorescence unit (RFU) using a plate reader (Tecan Infinite 200 Pro). Background fluorescence from TCA/PBS wells was subtracted and values were normalized to tissue weight. Triplicate values were averaged per sample.

2.4. Assessment of estrous stage

To understand whether hormonal status impacted BBB permeability, estrous stage was assessed in all female rats on the day of terminal BBB permeability experiments via vaginal cytology. Vaginal lavages were completed using ~200 μL 0.1 M PBS and cell type identified using an inverted microscope at 10× magnification. Based upon cells present in the vaginal smear, estrous stage was assigned (Goldman et al., 2007; Cora et al., 2015).

2.5. Measurement of circulating factors

To determine circulating levels of inflammatory factors that may disrupt BBB permeability, various enzyme-linked immunosorbent assays (ELISAs) were performed. Commercially available ELISA kits were used to quantify serum levels of TNF-α (Product RTA00; samples undiluted), and soluble ICAM-1 (Product RIC100; sample run with 50-fold dilution), which were obtained from R&D Systems (Minneapolis, MN, USA). Oxidative stress was assessed using an 8-Isoprostane ELISA kit (Item No. 516351; samples run with 2-fold dilution) from Cayman Chemicals (Ann Arbor, MI, USA). All samples were randomized across plates and ran in duplicate according to manufacturer instructions. Due to limited blood volume from tail vein withdrawal and space constraints of 96-well plates, one sample/group was excluded for the 8-isoprostane ELISA to accommodate additional wells needed for the assay, and serum from only four normotensive controls of both sexes were assayed (chosen at random).

2.6. Drugs and solutions

All tracers used in this study were diluted in sterile Lactated Ringer's solution (Baxter, Deerfield, IL, USA) and prepared fresh daily. Stock tracer solutions were stored according to manufacturer recommendations and protected from light. 0.1 M PBS was prepared weekly and stored at 4 °C. A 50% TCA (Sigma-Aldrich, St. Louis, MO, USA) solution was prepared daily. Working solutions were made fresh as needed for protein precipitation during tissue processing. All buffer solutions were prepared weekly and stored at 4 °C until use.

2.7. Statistical analysis

A statistical power calculation was done to determine the number of animals needed using a two-sided 95% confidence interval for a single mean and 1 – β of 0.80 based on our prior studies using similar methodology (Gannon et al., 2024; Johnson et al., 2014; Johnson et al., 2021). We determined that n = 6/group was sufficient to detect differences in memory function of SHR and passage of fluorescent tracers into the brain. Based on an 80% success rate in pilot BBB permeability studies, we added n = 2/group. Statistical comparisons were made to determine main effects and the interaction between sex and hypertension/housing condition using a two-way ANOVA with a Šídák's test to correct for multiple comparisons. To determine effects of brain region on BBB permeability within sex, a two-way mixed-effects model with a nested design and Šídák's multiple comparison test was used. Linear regression analyses were generated to understand the relationships between circulating factors and BBB permeability. Results are reported as mean ± SEM, with p < 0.05 considered significant. All analyses were performed in GraphPad Prism 10.3. The presence or absence of cage mates prevented behavioral testing from being completed by investigators who were completely blinded to group. Tissue processing and BBB permeability analyses were completed by investigators who were blinded to group.

3. Results

To understand how chronic hypertension and social isolation impact memory function, we assessed long-term memory and spatial working memory in male and female Wistar rats and compared to SHR without and with social isolation. There were main effects of sex, condition and a significant interaction between the two for body weight (F1, 42 = 1373, p < 0.01; F2, 42 = 163.3, p < 0.01 F2, 42 = 24.03, p < 0.01 by two-way ANOVA, respectively; Fig. 1). In general, males weighed more than females, and normotensive Wistar rats weighed more than SHR. In females, SHR that were housed in isolation had higher body weight than those housed in groups whereas social isolation did not affect body weight in male SHR (Fig. 1). There were main effects of sex, condition, and a significant interaction between the two for locomotion, including average speed (F1, 42 = 15.09, p < 0.01; F2, 42 = 42.77, p < 0.01 F2, 42 = 9.33, p < 0.01 by two-way ANOVA, respectively) and distance traveled (F1, 42 = 14.94, p < 0.01; F2, 42 = 42.38, p < 0.01; F2, 42 = 9.17, p < 0.01 by two-way ANOVA, respectively). Female Wistar rats had higher locomotion than all other groups, with greater average speed and distance traveled during the NOR habituation phase in the open field arena (Fig. 2A and B; p < 0.01 by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons). Chronic hypertension had no effect on locomotion in males but decreased locomotion in females; however, both male and female SHR that were socially isolated had substantially reduced locomotion compared to the socialized groups (Fig. 2A and B; p < 0.05 by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons).

Fig. 1.

Fig. 1.

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Body weights of male and female rats. Socialized normotensive Wistar rats and spontaneously hypertensive rats (SHR) that were either socially housed or housed in isolation (+Isol.) had body weights measured prior to terminal blood-brain barrier permeability experiments. **p < 0.01; n = 8/group. Data are mean ± SEM and analyzed by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons.

Fig. 2.

Fig. 2.

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Locomotion and exploratory behavior. A. Average speed and B. total distance traveled in an open field arena during the habituation phase of the novel object recognition task. Socialized male and female normotensive Wistar rats and spontaneously hypertension rats (SHR) that were either socialized or socially isolated (SHR + Isol.) freely explored the arena for five minutes. *p < 0.05, **p < 0.01; n = 8/group. Data are mean ± SEM and analyzed by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons.

Fig. 3A shows the schematic of the NOR paradigm used to assess long-term memory function, which is largely hippocampal-dependent (Broadbent et al., 2010). The NOR task is based upon a rodent's innate preference for novelty and compares the exploration time of a familiar object and a novel object. When given the option to explore both an identical and novel object, a rat with intact memory function will spend the majority of its time with the novel object (recognition index >0.50). If memory is impaired, then time will be split more evenly between “novel” and “familiar” objects, as it does not remember the object as familiar. Despite differences in locomotion, all rats spent >35 s investigating the identical objects during the acquisition phase of NOR, which is sufficient for memory encoding. There were no main effects of sex or condition on long-term memory function (F1, 42 = 0.951, p = 0.335; F2, 42 = 1.776, p = 0.182 by two-way ANOVA, respectively). There was, however, a significant interaction between sex and condition (F2, 42 = 3.61, p = 0.036 by two-way ANOVA). Object recognition memory was similar between Wistar rats and SHR in both males and females (Fig. 3B), however, social isolation of male SHR impaired memory function (p = 0.011 by two-way ANOVA with post-hoc Šídák's test for multiple comparisons) but had no effect in female SHR (Fig. 3B).

Fig. 3.

Fig. 3.

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The combination of social isolation during chronic hypertension selectively disrupted memory function in a sex- and domain-dependent manner. A. Schematic of the novel object recognition paradigm used to calculate a recognition index and assess long-term memory function in socialized male and female normotensive Wistar rats and spontaneously hypertensive rats (SHR) that were either socialized or isolated (SHR + Isol.). B. Recognition index was decreased in male SHR that were socially isolated compared to socialized SHR. Recognition index was unaffected in females. C. Schematic of continuous 8 min y-maze task used to calculate alternation index as a measure of spatial working memory function. D. Alternation index was unaffected by chronic hypertension without and with social isolation in males, but decreased in female SHR that were socially isolated compared to Wistar rats. *p < 0.05; n = 8/group. Data are mean ± SEM and analyzed by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons.

Spatial working memory largely involves prefrontal cortex signaling and was tested using a continuous y-maze task (Liu et al., 2018; Spellman et al., 2015). If a rat has intact working memory, the rat will alternate from arm A to B to C without making a reversal entry, which is considered an error (Fig. 3C). An alternation index was calculated as a measure of spatial working memory, with a higher index indicating better working memory performance. There was no main effect of sex or interaction between sex and condition on spatial working memory (F1, 42 = 1.417, p = 0.2405; F2, 42 = 1.311, p = 0.2803, respectively, by two-way ANOVA). However, there was a trending main effect of condition (F2, 42 = 3.039, p = 0.0585 by two-way ANOVA). In males, neither chronic hypertension alone nor in combination with social isolation impaired spatial working memory, as alternation indices were similar between groups (Fig. 3D). In females, however, alternation index was lower in isolated SHR compared to socialized Wistar rats that trended towards significance compared to socialized SHR (Fig. 3C; p = 0.0363 and p = 0.0536 by two-way ANOVA with post-hoc Šídák's test). Together, these data suggest that social isolation impaired memory function during chronic hypertension in a sex-dependent manner, such that hippocampal-dependent memory was disrupted in males, whereas prefrontal-dependent memory was disrupted in females.

To investigate cerebrovascular mechanisms by which chronic hypertension and social isolation may impair memory function, in-vivo BBB permeability to NaFl and 3 kDa Texas red was measured in dorsal hippocampus (Fig. 4) and prefrontal cortex (Fig. 5). There was no permeability to 10 kDa cascade blue in either brain region or any group. In the dorsal hippocampus, there were main effects of sex, condition, and a significant interaction between the two on BBB permeability to NaFl (F1,37 = 4.058, p = 0.0513; F2,37 = 15.62, p < 0.001; F2,37 = 5.263, p = 0.010, by two-way ANOVA, respectively). Further, there was a main effect of condition on permeability to 3 kDa Texas red (F2,35 = 14.64, p < 0.0001 by two-way ANOVA), but no main effect of sex or an interaction between the two (F1,35 = 0.002, p = 0.964; F2,35 = 1.165, p = 0.3239 by two-way ANOVA, respectively). Social isolation in male SHR increased BBB permeability to NaFl (Fig. 4A; p < 0.01) and 3 kDa Texas red (Fig. 4B; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). However, there was no effect of chronic hypertension alone. BBB permeability to NaFl in female SHR was higher compared to male SHR (Fig. 4A; p < 0.01 by two-way ANOVA with post-hoc Šídák's test), suggesting chronic hypertension disrupted the BBB in dorsal hippocampus specifically in the female sex. In contrast to males, chronic hypertension increased permeability to NaFl and 3 kDa Texas red in females that was not further increased by social isolation (Fig. 4A and B; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). Taken together, these findings suggest differential effects of chronic hypertension and social isolation on permeability occur in a sex-dependent manner in dorsal hippocampus: chronic hypertension alone disrupted the BBB in females with no impact of isolation, whereas in males, it was the combination of hypertension and social isolation that disrupted the BBB.

Fig. 4.

Fig. 4.

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Chronic hypertension and social isolation differentially increased blood-brain barrier (BBB) permeability in the dorsal hippocampus based upon sex. BBB permeability to A. small solute sodium fluorescein (NaFl) and B. 3 kDa Texas red dextran was increased in socially isolated male spontaneously hypertensive rats (SHR + Isol.) compared to socialized Wistar and SHR. Permeability was higher in female SHR compared to normotensive Wistar rats and was unaffected by social isolation. *p < 0.05, **p < 0.01; n = 6–8/group. Data are mean ± SEM and analyzed by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons.

Fig. 5.

Fig. 5.

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Increased blood-brain barrier (BBB) permeability occurred in prefrontal cortex during chronic hypertension to a greater extent in females than males. A. BBB permeability to small solute sodium fluorescein (NaFl) was increased in socialized spontaneously hypertensive rats (SHR) of both sexes compared to socialized Wistar rats and unaffected by social isolation (SHR + Isol.). B. In males, there were no differences in BBB permeability to 3 kDa Texas red dextran. Permeability was increased in female SHR and SHR + Isol. compared to female Wistar rats. *p < 0.05, **p < 0.01; n = 6–8/group. Data are mean ± SEM and analyzed by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons.

In prefrontal cortex, there was a main effect of sex and condition on BBB permeability to NaFl, but no interaction between the two (F1, 38 = 18.07, p < 0.001; F2, 38 = 8.874, p = 0.001; F2, 38 = 0.1319, p = 0.8768, respectively, by two-way ANOVA). Female rats had higher permeability compared to males and chronic hypertension increased permeability to NaFl in both sexes, but there was no additional effect of social isolation in SHR of either sex (Fig. 5A; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). In regards to BBB permeability to 3 kDa Texas red (Fig. 5B), there was a main effect of condition (F2,35 = 14.64, p < 0.001), but not sex (F1,35 = 0.002, p = 0.964), and no interaction between the two (F2,35 = 1.165, p = 0.3239 by two-way ANOVA). There were no increases in permeability to 3 kDa Texas red during hypertension or after social isolation in males, however, chronic hypertension increased permeability in females that was unaffected by social isolation (Fig. 5B; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). These findings suggest greater hypertension-induced BBB disruption occurred in prefrontal cortex of females. The majority of female rats were in met/diestrus (17/24, 70.8%), with few rats being in estrus (4/24, 16.7%) and proestrus (3/24, 12.5%). There were no detectable differences in permeability to any tracer in any brain region between estrous stages (data not shown). Two isolated male SHR and one female Wistar were excluded from all BBB studies due to technical difficulties. One male SHR and one isolated female SHR were excluded from hippocampal measurements and 3 kDa Texas red measurements, respectively, due to being a statistical outlier (>2 SDs away from the mean).

To understand potential regional differences in the susceptibility of the BBB to disruption, and whether this differed by sex, we compared permeability to NaFl and 3 kDa Texas red between dorsal hippocampus and prefrontal cortex in both male and female rats. In females, there was a main effect of brain region and condition, but no interaction between the two on BBB permeability to NaFl (F1,18 = 63.80, p < 0.001; F2,21 = 8.486, p = 0.002; F2,18 = 0.5193, p = 0.6036, respectively, by two-way mixed-effects analysis). Prefrontal cortex had greater NaFl permeability compared to dorsal hippocampus in all groups (p < 0.01 by two-way mixed-effects analysis with post-hoc Šídák's test). There was a main effect of condition, but no effect of brain region and no interaction between the two on BBB permeability to 3 kDa Texas red (F2,38 = 11.45, p < 0.001; F1,38 = 2.142, p = 0.1515; F2,38 = 1.062, p = 0.3558, respectively, by two-way mixed-effects analysis). In males, there was a main effect of brain region and condition, but no interaction between the two on BBB permeability to NaFl (F1,16 = 11.33, p = 0.004; F2,21 = 11.00, p = 0.001; F2,16 = 3.010, p = 0.0777, respectively, by two-way mixed-effects analysis). This brain region effect was largely driven by greater permeability to NaFl in prefrontal cortex compared to hippocampus in male SHR (p < 0.001), with no differences between brain region in normotensive rats (p = 0.1584) or isolated SHR (p = 0.6088 by two-way mixed-effects analysis with post-hoc Šídák's test). Similarly to females, there was a main effect of condition, but not brain region and no interaction between the two on BBB permeability to 3 kDa Texas red (F2,37 = 6.029, p = 0.005; F1,37 = 3.397, p = 0.0733; F2,37 = 1.234, p = 0.3030, respectively, by two-way mixed-effects analysis). Thus, basal permeability to small solute NaFl differed by brain region in a sex-dependent manner, potentially increasing general vulnerability of females to BBB disruption in the prefrontal cortex.

Systemically circulating factors are known to have damaging effects on the BBB and are elevated during chronic hypertension (Chapman et al., 2025). Therefore, we measured serum levels of 8-isoprostane, ICAM-1, and TNFα to assess oxidative stress, endothelial dysfunction and inflammation. There were main effects of sex, condition and a trend towards significant interaction between the two on serum levels of 8-isoprostane (F1,30 = 26.49, p < 0.001; F2,30 = 19.22, p < 0.001; F2,30 = 3.144, p = 0.0576, respectively, by two-way ANOVA). Females had higher levels of 8-isoprostane compared to males (Fig. 6A). Regardless of social isolation, chronic hypertension increased 8-isoprostane levels in male and female SHR compared to normotensive control rats (Fig. 6A; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). Female SHR were in a state of greater oxidative stress as indicated by higher levels of 8-isoprostane compared to male SHR (Fig. 6A; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). There was a significant correlation between 8-isoprostane levels and permeability to both NaFl and 3 kDa Texas red in dorsal hippocampus (Fig. 6B) and prefrontal cortex (Fig. 6C). When stratified by sex, 8-isoprostane levels in females were correlated with higher permeability to both tracers in both brain regions, with no statistical correlations present in males (Table 1). There was a main effect of sex and condition but no interaction between two on serum levels of ICAM-1 (F1,34 = 17.14, p < 0.001; F2,34 = 16.30, p < 0.001; F2,34 = 0.3133, p = 0.7331, respectively, by two-way ANOVA). Males, in general, had higher levels of circulating ICAM-1 compared to females (Fig. 6D). Chronic hypertension in males did not affect ICAM-1 levels, however, isolated male SHR had increased circulating ICAM-1 compared to normotensive controls (Fig. 6D; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). In females, chronic hypertension alone increased ICAM-1 levels that were not further increased by social isolation (Fig. 6D; p < 0.05 by two-way ANOVA with post-hoc Šídák's test). Serum levels of ICAM-1 correlated significantly with BBB permeability to both NaFl and 3 kDa Texas red in dorsal hippocampus (Fig. 6E) but not prefrontal cortex (Fig. 6F). When stratified by sex, ICAM-1 levels correlated with permeability to both tracers in dorsal hippocampus but not prefrontal cortex of females and to a lesser extent in males (Table 1). There were no main effects of sex or condition, and no interaction between the two on serum levels of TNFα (F1,33 = 0.7430, p = 0.3949; F2,33 = 2.144, p = 0.1333; F2,33 = 0.9440, p = 0.3993, respectively, by two-way ANOVA). Circulating TNFα did not differ between group and were not correlated with permeability to NaFl or 3 kDa Texas red in either brain region or sex (Fig. 6G-5I, Table 1). Taken together, these findings suggest a prominent role of oxidative damage of the BBB in both brain regions, and that the BBB in dorsal hippocampus may be more susceptible to disruption in response to high ICAM-1 levels, specifically in females.

Fig. 6.

Fig. 6.

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Serum levels of markers of oxidative stress, endothelial dysfunction and inflammation and the relationship with blood-brain barrier (BBB) permeability. A-C. 8-isoprostane; D-F. intercellular adhesion molecule-1 (ICAM-1); G-I. tumor necrosis factor-alpha (TNFα) serum levels and correlations with BBB permeability to sodium fluorescein (NaFl) and 3 kDa Texas red dextran (TxR) in dorsal hippocampus and prefrontal cortex of male and female normotensive Wistar rats and spontaneously hypertensive rats (SHR) that were either socialized or isolated (SHR + Isol.). Data are mean ± SEM; n = 4–8/group. Panels A, D and G *p < 0.05, **p < 0.01 by two-way ANOVA with a post-hoc Šídák's test for multiple comparisons. Panels B-C, E-F, and H-I *p < 0.05, **p < 0.01 by linear regression analyses to detect if the slope is significantly non-zero.

Table 1.

Correlations between serum ICAM-1, 8-isoprostane (8-Iso) and TNFα levels and BBB permeability in male and female rats.

8-Iso Region/Tracer F DF r2 p-value significance
Male dHipp/NaFl 0.846 1,14 0.0569 0.3733 ns
dHipp/TxR 3.904 1,14 0.2181 0.0682 trending
PFC/NaFl 0.000 1,14 0.0000 0.9863 ns
PFC/TxR 1.510 1,15 0.0915 0.2380 ns
Female dHipp/NaFl 19.45 1,16 0.5487 0.0004 **
dHipp/TxR 4.141 1,16 0.2056 0.0588 trending
PFC/NaFl 10.29 1,15 0.4068 0.0059 **
PFC/TxR 8.830 1,16 0.3556 0.0090 **
ICAM-1
Male dHipp/NaFl 4.651 1,15 0.2367 0.0477 *
dHipp/TxR 2.427 1,15 0.1393 0.1401 ns
PFC/NaFl 0.499 1,16 0.0302 0.4902 ns
PFC/TxR 6.327 1,16 0.2834 0.0230 *
Female dHipp/NaFl 4.489 1,18 0.1996 0.0483 *
dHipp/TxR 8.638 1,17 0.3369 0.0092 **
PFC/NaFl 3.414 1,17 0.1672 0.0821 ns
PFC/TxR 1.150 1,18 0.0600 0.2978 ns
TNFα
Male dHipp/NaFl 0.438 1,14 0.0303 0.5191 ns
dHipp/TxR 0.037 1,14 0.0026 0.8504 ns
PFC/NaFl 0.523 1,15 0.0337 0.4809 ns
PFC/TxR 1.822 1,15 0.1083 0.1970 ns
Female dHipp/NaFl 0.166 1,18 0.0091 0.6885 ns
dHipp/TxR 1.683 1,17 0.0901 0.2119 ns
PFC/NaFl 1.085 1,17 0.0600 0.3121 ns
PFC/TxR 0.037 1,18 0.0020 0.8494 ns
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*

p < 0.05

**

p < 0.01, “trending” is p < 0.08, ns is p > 0.08 by linear regression analyses to detect if slope is significantly non-zero. dHipp, dorsal hippocampus; NaFl, sodium fluorescein; PFC, prefrontal cortex; TxR, 3 kDa Texas red dextran.

4. Discussion

There are currently no treatments for VCI. Therefore, understanding mechanisms by which modifiable risk factors damage the cerebrovasculature in cognition-centric brain regions is critical to determine novel therapeutic targets needed for preventing and/or slowing vascular contributions to cognitive impairment and dementia. Further, understanding how such mechanisms may differ by biological sex is of utmost importance. Not only to bolster treatment efficacy, but also to expand our knowledge of sex-specific changes in neurocognition, as females have historically not been included in most basic cardiovascular and cerebrovascular research studies. In the current study, we investigated for the first time that we are aware of how the combination of two major dementia risk factors – chronic hypertension and social isolation – damage the cerebrovasculature to contribute to VCI and whether one sex, brain region or memory domain may be more vulnerable to disruption. Our findings showed that social isolation during chronic hypertension selectively impaired memory function in a sex- and memory domain-dependent manner that was associated with region-specific BBB disruption. We further showed that chronic hypertension was the driver of BBB damage in females that correlated with biomarkers of inflammation and oxidative stress. In males, however, it was the combination of hypertension and social isolation that disrupted the BBB and a lesser role for damaging circulating factors. These findings advance our understanding of sex-specific effects of hypertension and social isolation on the cerebrovasculature. This not only highlights the importance of blood pressure control and maintenance of social connections to combat VCI, but also that efficacy of interventions may depend upon sex.

Chronic hypertension accelerates age-related cognitive decline, particularly when it occurs mid-life (Whelton et al., 2018; Launer et al., 2010; Fujishima and Kiyohara, 2002; Yamada et al., 1999). We have previously shown that age-related long-term memory decline began in adult male SHR between ~4–8 months of age (Gannon et al., 2024). In the current study, socialized male and female SHR at 6 months of age did not demonstrate impaired long-term memory compared to normotensive controls, which was in contrast to our hypothesis. Interestingly, unlike long-term memory, spatial working memory has been shown to be impaired in male SHR as early as 4 months old (Gannon et al., 2024). This was not replicated in the current study, as chronic hypertension did not impact alternation indices in either sex (Fig. 2). However, part of the socialization paradigm was routine handling by investigators that could account for the sustained function of both memory domains during chronic hypertension. In fact, we recently reported that cognitive stimulation with environmental enrichment factors including stimulated learning and routine handling protected memory function in aged (16 month old) male and female SHR (Chapman et al., 2025). Regardless, the current findings of BBB disruption in chronically hypertensive rats with intact memory supports a causal role for BBB disruption in hypertension-induced memory decline.

Social isolation, particularly during adolescence, is known to have a profound impact on neurocognitive function later in life, but much less is known about the effects in adulthood (Xiong et al., 2023). Further, chronic stress associated with social isolation damages the cerebral circulation, including the BBB, but there is little known about how isolation may exacerbate underlying cerebrovascular disease (Guarnera et al., 2023; Lao et al., 2024; Zheng et al., 2025). In the current study, chronic hypertension and social isolation differentially disrupted BBB integrity in the dorsal hippocampus in a sex-dependent manner. Males had increased BBB permeability only with combined hypertension and social isolation, whereas in females this occurred in response to chronic hypertension with no additional impact of isolation. Regardless, BBB permeability was similar between isolated male SHR and isolated female SHR, however, it was only the males that had hippocampal-dependent memory dysfunction. Male SHR are in a higher proinflammatory state than female SHR and have more robust neuroimmune responses to brain injury (i.e., cerebral ischemia) (Gillis and Sullivan, 2016; Dinh et al., 2025; Tariq et al., 2023). Therefore, there may be a greater neuroinflammatory cascade occurring in response to increased BBB permeability in the hippocampus of males, thereby compromising memory function despite similar levels of BBB disruption between sexes. These findings suggest that males may be more susceptible to hippocampal dysfunction under stressful and/or socially isolated conditions. Future studies are needed to investigate neuroinflammatory consequences of chronic hypertension and social isolation, and whether these differ by sex or brain region.

Here, we report that chronic hypertension increased BBB permeability in females to a greater extent in prefrontal cortex compared to hippocampus, and to a higher degree than in males. Further, and in contrast to our hypothesis, there was no damaging impact of social isolation in females. Despite this BBB disruption, only isolated female SHR had impaired prefrontal-dependent memory. This may be due to a heightened neuroinflammatory state in response to the stress of social isolation, isolation-induced neuronal damage, or both (Xiong et al., 2023; Al Omran et al., 2022; Adzic et al., 2009). In the current study, we focused on the potential compounding effects of adult social isolation in the setting of chronic hypertension as primary modifiable risk factors for cognitive decline and dementia. Therefore, we cannot distinguish between the potential damaging effects of social isolation and isolation-induced neuronal dysfunction. In addition, social isolation could further increase blood pressure in SHR that may contribute to changes in BBB permeability. While blood pressures can be measured non-invasively via the tail cuff method (Gannon et al., 2024), this was not performed as the training period involves repeated handling that would interfere with the isolation paradigm. Further, the acute stress of being restrained for 10 consecutive days could also impact BBB permeability. Future studies are needed to determine the mechanisms of sex- and region-specific BBB damage and memory dysfunction, and whether there is a social isolation-related increased in blood pressure. Regardless, the BBB was more permeable in the prefrontal cortex during chronic hypertension in females, suggesting that brain region is more vulnerable to additional insults than in males. This may be particularly important later in life once females enter reproductive senescence (e.g., post-menopausal), when protective effects of estrogens on neurons, glia and the vasculature, including the BBB, are lost (Maggioli et al., 2016; Mervosh and Devi, 2025).

The BBB is critical to protect the brain from unregulated entry of what is circulating systemically, and its disruption is an early biomarker of cognitive decline (Nation et al., 2019). ICAM-1 on endothelial cells has a critical and protective role in immune cell infiltration into the brain during systemic inflammation. However, disease states including chronic hypertension upregulate expression of ICAM-1, resulting in tight junction disruption (e.g., Claudin-5), increased BBB permeability, and increased serum levels of its cleaved, soluble form (Dietrich, 2002; Liao et al., 2025). Importantly, recent longitudinal data from the HeartSCORE Study reported that midlife circulating levels of ICAM-1 are a predictor of both future cardiovascular disease and cognitive decline (Abdoun et al., 2025). Circulating 8-isoprostane is a reliable biomarker of systemic oxidative stress that, through interactions with prostaglandins causes cytotoxicity of cerebral endothelial cells, and targeting ICAM-1 and 8-isoprostane have been shown to be protective after acute brain injury (Montuschi et al., 2004; Brault et al., 2003; Glushakov et al., 2013; Saikia et al., 2024). In the current study, serum levels of 8-isoprostane were higher in female SHR compared to males that was correlated with increased BBB permeability in both dorsal hippocampus and prefrontal cortex. This suggests increased permeability in prefrontal cortex of females may be due to oxidative damage. Circulating ICAM-1 levels were increased in a similar pattern as BBB permeability. The combination of hypertension and social isolation increased ICAM-1 levels in males, whereas chronic hypertension alone increased levels in females. ICAM-1 levels correlated with increased BBB permeability of both sized tracers in the dorsal hippocampus only, suggesting potential regional susceptibility of the BBB to inflammation-mediated damage.

In addition to the structural barrier formed largely by tight junctions, there is a critical biochemical/selective component of the BBB comprised of specific transport proteins known as efflux transporters (Miller et al., 2008). Efflux transporters contained within the BBB are powerful ATP-driven pumps that actively efflux ligands (e.g., cytokines, steroids, xenobiotics) back into the circulation, thereby preventing passage into the brain (Begley, 2004; Hartz et al., 2010; Hartz et al., 2006). Proinflammatory conditions can impair activity of efflux transporters at the BBB (Miller et al., 2008; Hartz et al., 2006). In fact, NaFl is a ligand of BBB efflux transporters (Schoknecht and Eilers, 2024). Therefore, decreases in efflux transporter activity and/or expression during hypertension and social isolation could contribute to increased passage of tracers into the brain. Future studies are needed to determine the contributions of structural and functional aspects of the BBB that are compromised in a sex- and region-specific manner, including in other cell types (e.g., astrocytic endfeet, pericytes).

There are several strengths and limitations of the current study. Primary hypertension (i.e., “essential hypertension”) accounts for the vast majority (~ 90%) of adult cases (Palaniappan et al., 2026). Adult SHR are a widely used model of primary hypertension, and undergo similar neuropathology of VCI that occurs in humans (Yang et al., 2016). Therefore, the current study was completed using a highly relevant rat model to understand how social isolation in adulthood may exacerbate hypertension-related cerebrovascular dysfunction and memory decline. Chronic hypertension damages the cerebral circulation differently based upon sex, yet females remain rarely included in basic science studies of hypertension and ischemic stroke (Kremer et al., 2022). The use of both sexes in the current study is not only a strength from an inclusivity standpoint, but the data suggest sex differences in the vulnerability of the BBB to social isolation. The combined behavioral assessments with in vivo BBB permeability and serum levels of inflammation/oxidative markers allows for the integration of memory function with BBB integrity to better understand vascular contributions to cognitive impairment and dementia. However, while the current study demonstrates strong associations, it remains largely correlative, as a mechanistic, causal pathway between social isolation, hypertension, BBB disruption and memory impairment could not be established. Further, the current study assessed BBB permeability after three months of social isolation and therefore is unable to determine the duration of social isolation that initiates BBB disruption. Future studies are needed to determine causal mechanisms by which social isolation damages the BBB during chronic hypertension, assess temporal aspects of isolation-related decreases in BBB integrity and if these differ by brain region and/or sex.

In conclusion, the findings from the current study advance our understanding of cerebrovascular consequences of social isolation in adulthood and indicate that brain region/memory domain dysfunction differ by sex during chronic hypertension. That oxidative and inflammatory mediators correlated with BBB disruption to a greater extent in females than males suggests that antioxidant and anti-inflammatory agents have higher therapeutic potential in hypertensive females. In contrast, socialization was more effective at protecting BBB integrity in hypertensive males. The compounding effects of social isolation during chronic hypertension on BBB integrity in males are particularly important, as living alone is known to cause greater loneliness in men than women (van den Broek, 2017; Barreto et al., 2021; Greenfield and Russell, 2010). Overall, females may be more receptive to pharmacological intervention that target inflammation/oxidative stress to protect from VCI, whereas psychosocial interventions may be more effective in curbing VCI and dementia risk in males.

Funding sources

This work was supported by the NIH National Institute of Neurological Disorders and Stroke and National Institute on Aging 1R01 NS127284 (ACC), Binghamton University External Scholarships and Undergraduate Research Center (AT) and Binghamton University – State University of New York (ACC). Funding sources were not involved in study design, collection, analysis or interpretation of data, in the writing of the manuscript, or in the decision to submit the article for publication.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Abbie Chapman reports financial support was provided by Binghamton University. Abbie Chapman reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

CRediT authorship contribution statement

Anduela Thaqi: Writing – review & editing, Writing – original draft, Visualization, Investigation, Funding acquisition, Formal analysis, Data curation. Gianna M. DeMarco: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Emily D. Schellinger: Writing – review & editing, Investigation, Formal analysis, Data curation. Dinlin Chen: Writing – review & editing, Investigation, Formal analysis, Data curation. Jared Luchetta: Writing – review & editing, Methodology, Formal analysis, Data curation. Abbie C. Chapman: Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Data availability

Data will be made available on request.

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