Differential Effects of Murine Stroke Models on Dopaminergic Neurons, Glial Responses, and Neurobehavioral Outcomes

Abstract

Stroke is a leading cause of disability worldwide, often resulting in persistent motor, cognitive, and emotional impairments. While the hippocampus and amygdala play critical roles in post-stroke behavioral changes, specific neuronal alterations and prolonged glial responses within these regions across different stroke types remain unclear. This study investigates the behavioral, neuronal, and glial effects of subarachnoid hemorrhage (SAH), transient middle cerebral artery occlusion (tMCAO), and photothrombotic stimulation (PTS) in mice. SAH and tMCAO models exhibited significant motor deficits, spatial and recognition memory impairments, and increased anxiety- and depressive-like behaviors, whereas the PTS model showed similar motor and cognitive impairments but lacked affective (anxiety- and depressive-like) behavioral changes. Immunohistochemical analysis revealed increased overlap of tyrosine hydroxylase (TH, a dopaminergic marker) process with NeuN (a neuronal marker) in the dentate gyrus (DG) of SAH and tMCAO mice, highlighting region-specific vulnerability to ischemic damage in the hippocampus. In the amygdala, elevated overlap of TH⁺ process with NeuN in SAH and tMCAO mice suggests enhanced dopaminergic involvement in emotional dysregulation. In contrast, the PTS model did not exhibit any changes in overlap of TH⁺ process with NeuN in either the hippocampus or amygdala, consistent with the absence of affective behavioral deficits. Additionally, SAH and tMCAO models exhibited persistent astrocytic and microglial activation in the amygdala, characterized by increased intensity and density without significant morphological changes, indicative of a chronic inflammatory response. The PTS model also showed increased microglial intensity and density without overt morphological changes, suggesting a more moderate, possibly subclinical inflammatory response. These findings highlight the differential effects of stroke models on behavior, neuronal populations, and glial responses in limbic regions. The pronounced dopaminergic and glial alterations in SAH and tMCAO may underlie post-stroke emotional and cognitive disturbances.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12975-025-01381-x.

Introduction

Stroke is the second leading cause of death and a major source of long-term disability globally, affecting millions of individuals each year and posing substantial physical, cognitive, and emotional challenges for survivors [1, 2]. Broadly, strokes are classified into ischemic and hemorrhagic types: ischemic stroke, caused by blood vessel occlusion, accounts for approximately 87% of cases, while hemorrhagic stroke, caused by vessel rupture, comprises around 13% [2]. Hemorrhagic strokes are further divided into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH), each with distinct pathology and clinical outcomes [2]. Despite advancements in acute stroke management, there remains a critical need to understand the long-term neurological sequelae of stroke, especially the persistent cognitive and emotional impairments that profoundly affect the quality of life [3–5]. While motor deficits following stroke have been extensively studied, the neurobiological mechanisms underlying post-stroke mood disorders and cognitive dysfunction remain incompletely understood.

This gap is especially evident when comparing different stroke subtypes, where distinct pathophysiological mechanisms likely contribute to divergent outcomes. The lack of such comparative analyses makes it difficult to identify both shared and unique pathological mechanisms and to develop targeted, subtype-specific therapies. Although models such as subarachnoid hemorrhage (SAH), transient middle cerebral artery occlusion (tMCAO), and photothrombotic stimulation (PTS) are well-established and replicate key features of human stroke subtypes [6, 7], few studies have directly compared their long-term behavioral and neuronal outcomes. This represents a critical gap in preclinical research, limiting our ability to identify shared versus unique neuropathological mechanisms and hindering the development of tailored subtype-specific interventions. Each of these stroke models offers unique translational relevance: SAH mimics aneurysmal bleeding into the subarachnoid space, making it particularly useful for studying hemorrhagic injury mechanisms, such as oxidative stress and blood–brain barrier disruption [8]; tMCAO models arterial occlusion and ischemic damage, commonly seen in human ischemic stroke [9, 10]; and PTS models in situ thrombus formation under photothrombotic conditions, reflecting thromboembolic stroke risk in humans and its vascular implications [11]. Despite their relevance, no prior study has systematically evaluated the long-term behavioral effects spanning motor, cognitive, and emotional domains across all three stroke models, nor has any study linked these behavioral outcomes to specific neuronal and glial changes within the hippocampus and amygdala, two regions critical for memory and emotion. The novelty of this study lies in its integrative, cross-model approach to dissecting stroke-induced dysfunctions. By directly comparing SAH, tMCAO, and PTS models, we identify both converging and diverging effects on behavior and brain structure. This design uniquely allows us to investigate whether distinct stroke etiologies lead to specific behavioral phenotypes and neuropathologies.

The hippocampus and amygdala are essential regions for memory, emotional regulation, and the stress response, making them particularly vulnerable to stroke-related damage [12]. Ischemic injury in the hippocampus disrupts neurogenesis and synaptic plasticity, leading to memory deficits [13], while the amygdala, a region crucial for processing emotions and regulating mood, contributes to post-stroke emotional disorders, such as anxiety and depression [14]. Dopamine plays a pivotal role in both regions, modulating synaptic activity and behavioral outputs. In the hippocampus, dopamine aids memory consolidation through synaptic plasticity and long-term potentiation (LTP) [15]. In the amygdala, dopamine regulates stress and reward responses, fostering emotional resilience through interactions with GABAergic and glutamatergic pathways [16]. Disruption of dopaminergic signaling following stroke can disrupt these pathways, potentially leading to mood disorders, such as post-stroke depression and anxiety [17]. Given that dopamine has a critical role in maintaining cognitive stability in the hippocampus and emotional resilience in the amygdala, dopaminergic dysregulation may partly explain the observed co-occurrence of cognitive and mood disturbances after stroke. Nonetheless, the specific effects of ischemia on dopaminergic neurons in these regions remain inadequately explored.

In this study, we have investigated the sustained behavioral and neuronal impacts of SAH, tMCAO, and PTS stroke models. We assessed a range of motor, memory, and emotional functions using a battery of behavioral assays, including the rotarod, novel object recognition (NOR), Y-maze, Barnes maze, tail suspension (TST), sucrose preference (SPT), and open field tests (OFT). Additionally, we examined neuronal changes in the hippocampus and amygdala, focusing on specific neuronal populations by analyzing NeuN (a neuronal marker) and tyrosine hydroxylase (TH, a dopaminergic marker). We also assessed the quantitative and qualitative characteristics of astrocytes and microglia in the amygdala to explore glial responses in this region. By combining comprehensive behavioral phenotyping with detailed neuroanatomical analysis across multiple stroke models, our study provides a novel framework for understanding the subtype-specific neuropathological basis of post-stroke cognitive and emotional dysfunction. These findings have important implications for identifying mechanistic targets for intervention and improving personalized stroke recovery strategies.

Material and Methods

Animal Models

Male C57BL/J mice (3 months old) were subjected to three distinct stroke models to study the effects of ischemic and hemorrhagic injury. All animal experiments were approved by the Animal Care and Use Committee at The University of Texas Health Science Center at Houston (accredited by the American Association for Laboratory Animal Care) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The reporting of animal experiments followed the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

To ensure reproducibility and transparency, Table 1 summarizes the number of animals used, mortality rates, and final sample sizes. Mice were included only if they survived post-surgery and completed all behavioral and histological assessments. No additional exclusions occurred following stroke modeling.

Table 1.

Summary of animal numbers, mortality rates, and final sample sizes

Experimental Group	Initial Number of Mice	Number Excluded	Number Deceased (within 24 h)	Final Number Included
Sham	30	0	0	30
PTS	16	0	0	16
tMCAO	17	0	2	15
SAH	18	0	3	15

Transient Middle Cerebral Artery Occlusion (tMCAO)

The tMCAO model is an established technique to simulate ischemic stroke by temporarily blocking blood flow to the middle cerebral artery [18]. Focal cerebral ischemia was induced under isoflurane anesthesia according to standard operating procedures [19]. Briefly, a silicone-coated suture (Cat. #702334) (0.23 mm diameter, Doccol Corporation, Massachusetts) was introduced into the right carotid artery and advanced into the internal carotid artery (ICA) to occlude the origin of the middle cerebral artery (MCA), with successful occlusion confirmed by Laser Speckle Imager. During occlusion, body temperature was maintained at 37 ± 0.5℃. After 1 h of occlusion, the monofilament is withdrawn to allow reperfusion with cerebral blood flow monitored by Laser Speckle Imager. The neck incision is then closed, and the animal is monitored on a warm pad until fully awake before being returned to its cage. Postoperatively, bupivacaine (2 mg/kg, s.c.) was used for pain relief and clamps were removed 7–10 days later to complete recovery.

Photothrombotic Occlusion model (PTS)

The PTS model is an experimental method used to induce localized ischemic stroke by administering the photosensitive dye Rose Bengal, followed by targeted light exposure to activate the dye and cause focal vascular occlusion [11]. This approach allows precise control over the stroke location and severity. Briefly, mice were anesthetized with isoflurane and Rose Bengal was administered (a dose of 150 μg/g) through intravenous (i.v.) injection. To induce ischemia, light is applied to the distal middle cerebral artery territory for 15 min. After light exposure, the ferrule is removed, and the incision is closed with nylon sutures or surgical clamps. Postoperatively, bupivacaine (2 mg/kg, s.c.) was used for pain relief and clamps were removed 7–10 days later.

Subarachnoid Hemorrhagic (SAH) Stroke Model

We used the blood injection method for modeling SAH in mice. Briefly, a sterilized Hamilton needle is inserted through the foramen magnum, delivering 0.1 mL of autologous blood (collected from the femoral vein) over 2 min. To prevent backflow, the needle was left in place for an additional 2 min before being withdrawn. Bone wax is applied to seal the burr hole, and the scalp incision is sutured. Isoflurane was discontinued, and the mouse was allowed to recover. Sutures were removed 10–14 days post-surgery for animals undergoing long-term survival.

Sham Group

All sham control mice received isoflurane anesthesia and scalp incision followed by postoperative administration of bupivacaine (2 mg/kg, s.c.) for pain relief. Clamps were removed 7 to 10 days after surgery.

Magnetic Resonance Imaging (MRI)

MRI scans were conducted on a 7 T Bruker Biospec MRI system (Bruker Biospin, Billerica, MA) at the UTHealth Houston preclinical imaging facility. Mice were anesthetized with 1.5–2% isoflurane in a gas mixture of 30% oxygen and 70% medical air throughout the imaging procedure. Body temperature and respiratory rate were continuously monitored using a physiological monitoring system (Small Animal Instruments, Stony Brook, NY) to ensure stable conditions, with temperature maintained at 37 ± 0.5 °C and respiration rate kept between 80 and 100 breaths per minute.

Dynamic Contrast-Enhanced MRI (DCE-MRI) was performed to visualize cerebral infarction. T2-weighted Imaging (T2WI) -MRI acquisition involved a dynamic T2-weighted gradient-echo sequence over 14 min, with six axial slices captured using the following parameters: TR/TE = 50/2.6 ms, flip angle = 15°, resolution = 160 × 160 × 1000 µm³, and temporal resolution = 4 ms. Pre-contrast T1 values were obtained using the same sequence with five flip angles (5°, 10°, 15°, 30°, and 45°). Data processing for DCE imaging was conducted using ROCKETHSIP software.

Behavioral Tests

Two weeks post-surgery, mice underwent a series of behavioral tests to assess motor and cognitive deficits.

Rotarod Test

We used the rotarod to evaluate motor coordination and balance by measuring the time a mouse remains on a rotating rod, with progressively increasing speed [20]. Briefly, mice were placed on a rotarod accelerating linearly from 5 to 50 RPM over 3 min. If a mouse fell or completed two passive full rotations, it was removed, and the latency to fall was recorded. The maximum trial duration was 3 min. Mice underwent three consecutive training days with four trials per day and a 10-min inter-trial interval. Latency to fall was used to assess motor function.

Novel Object Recognition (NOR) Test

The NOR test is a commonly used behavioral assay to assess recognition memory in mice [21]. In this experiment, single mice were placed in the open arena with two identical objects in opposite quadrants and were recorded for 20 min. Animals were returned to their home cage after recording the activity. The arena was wiped with 30% ethanol after each trial. Once all animals in a group completed the training phase, the NOR test was conducted following a rest period of at least 20 min. During the test phase, one of the familiar objects (FO) was replaced with a novel object (NO). Each mouse was then individually placed back into the arena for 20 min, and the session was recorded. The percentage of time spent exploring the novel object and the discrimination index was calculated using the following formula:

$$TimespentwithNO=\frac{NOtime}{NOtime+FOtime}\times 100$$

$$Discriminationindex\left(D,I\right)=\frac{NOtime-FOtime}{NOtime+FOtime}\times 100$$

Barnes Maze Test

The Barnes maze is a dry-land-based rodent behavioral paradigm for assessing spatial learning and memory [22]. The Barnes maze consisted of a 60 cm diameter circular table with 20 holes (19 false and 1 escape) each with a 5 cm diameter. The false holes were recessed 1 cm into the table. Visual cues were placed on 4 of the surrounding walls. During the acquisition phase, mice underwent 4 trials per day for 4 consecutive days. Each trial began with the mouse placed inside a cylinder at the center of the dark Barnes maze. The lights were then turned on, the cylinder was lifted, and the mouse was given 5 min to explore the maze. If the mouse failed to locate the escape hole within 5 min, it was gently guided into the hole and allowed to remain there for 1 min before being returned to its home cage. The escape hole remained in the same location throughout the acquisition phase. Between trials, the maze was cleaned with 30% ethanol. On day 5, a single probe trial was conducted, during which the mouse was given 90 s to search the maze. Escape latency was recorded to assess spatial memory performance.

Y-Maze Test

Spatial working memory was assessed in mice through the spontaneous alternation Y-maze test [23]. Spontaneous alternation, a measure of spatial working memory, can be assessed by allowing mice to explore all three arms of the maze and is driven by an innate curiosity of rodents to explore previously unvisited areas. Each mouse was placed in the center of the Y-maze, and the number of entries and arm alterations were recorded over 5 min.

Open Field Test (OFT)

The OFT is a common measure of exploratory behavior, general activity, and anxiety-like behavior in rodents [24], where both the quality and quantity of the activity can be measured. Briefly, mice were placed in a single arena facing the middle of a wall. Mice were allowed to explore the arena for 20 min. Animals were returned to the home cage and the arena was wiped with 30% ethanol. Distance moved and moving velocity was analyzed to assess locomotor and exploratory behavior, while time spent in the center was used to evaluate anxiety-like behavior.

Tail Suspension Test (TST)

The TST is a widely used behavioral assay in mice to assess depressive-like behaviors, particularly behavioral despair, which is thought to model aspects of depression in humans [25]. Each mouse was suspended by the tail 60 cm above the chamber floor using adhesive tape placed less than 1 cm from the tip of the tail. Behavior was recorded using a video camera for 6 min. The behavior was later analyzed to determine the following parameters: number of immobility episodes and total duration of immobility. The total amount of time during which each mouse remained immobile was recorded in seconds and then expressed as a percentage of total time or as a percentage per minute. In this test, the ‘immobile period’ was defined as the period when the animals stopped struggling for ≥ 1 s.

Sucrose Preference Test (SPT)

The SPT in mice is a widely used behavioral assay to assess anhedonia, a core symptom of depression characterized by a reduced ability to experience pleasure [25]. The test evaluates the preference of mice for a sweetened solution (usually sucrose) over plain water. Mice were individually housed in a quiet animal house without any disruptions. Before the test, the mice were trained to drink the sucrose solution. Two bottles containing pure water and 2% sucrose solution were prepared for each mouse. The positions of the two bottles were switched every 24 h. Following two days of habituation, mice were water-deprived overnight, and the water/sucrose consumption test was conducted. A bottle of pure water and a bottle of 2% sucrose solution were weighed in advance and then given to the mice. After 24 h, the two bottles were weighed. The consumption of sucrose solution, pure water, and total water was recorded, and the percentage of sucrose consumed was calculated.

Immunohistochemistry

Following the completion of all behavioral assessments, mice were euthanized under deep anesthesia and transcardially perfused with phosphate-buffered saline (PBS) (0.01 M, pH 7.4) and 10% formalin. The brains were removed and fixed in formalin for 24 h, then dehydrated overnight in a 15% sucrose solution, followed by immersion in 30% sucrose for 48–72 h until they sank. A freezing microtome (RWD, FS800, China) was used to cut serial coronal/sagittal brain Sects. (30 μm thickness). Sections were then preserved with 0.3% Triton-X 100 for membrane permeability before being blocked for 1.5 h at RT with 5% bovine serum albumin (BSA) in 0.01 M PBS, followed by incubation with primary antibody overnight for TH (Novus Biologicals, NB300109; 1:1000), and NeuN (Invitrogen, MA5-33,103; 1:1000), GFAP (Millipore Sigma, MAB3402C3; 1:1000), IBA-1 (FUJIFILM Wako, 019–19741, 1: 1000) at 4 °C. The following day, slices were washed with PBS, incubated with Alexa Fluor 594 donkey anti-rabbit (1:1000; Jackson ImmunoResearch) and Alexa Fluor 488 donkey anti-mouse (1:1000; Jackson ImmunoResearch) for 60 min at room temperature, and then washed three times (10 min each time) in PBS. Following the washing steps, slices were then sequentially coverslipped with Fluoromount-G™ Mounting Medium, with DAPI. Each brain slice was imaged using a fluorescence microscope scanner (VS200 Slide Scanner, Olympus, Tokyo, Japan) under 4 × and 40 × objectives. Images were acquired as Z-stacks with a step size of 1 µm over an 8–10 µm depth, depending on the brain region. Z-stacks were processed using the maximum intensity projection method in ImageJ for figure presentation, and all analyses were performed on the resulting projected images. Glial morphology was quantified using the AnalyzeSkeleton (2D/3D) plugin in ImageJ, which converts fluorescence photomicrographs into skeletonized binary images [26] and enables detailed quantification of glial activation and morphological changes.

Statistical Analysis

Behavioral data were recorded using EthoVision software. All bar graphs were generated in GraphPad Prism 9, with statistical analyses performed using one-way ANOVA followed by post-hoc comparisons to assess differences between experimental groups. Immunostaining quantification was conducted in ImageJ software. Statistical significance was set at p < 0.05, and all values are presented as mean ± SEM.

Results

Stroke-Induced Motor Deficits in Experimental Models: SAH, tMCAO, and PTS

Four groups of mice were included in the study: SAH, tMCAO, PTS, and sham. The experimental timeline is illustrated in Fig. 1A. To capture the subacute to early chronic phase, behavioral assessments were conducted from 2 to 4 weeks post-injury, as shown in Fig. 1A. Following stroke induction, model success was confirmed using MRI (Fig. 1B). Given that motor deficits are commonly observed in stroke patients [27], we first aimed to evaluate similar impairments in mouse models of stroke. Motor performance was assessed using the rotarod test, revealing a significant reduction in latency to fall across all three stroke models (p < 0.0001, sham vs. SAH and tMCAO; p < 0.01 sham vs. PTS; Fig. 1C). In between-group comparisons demonstrated that tMCAO mice exhibited significantly greater motor deficits than the PTS group (p < 0.01, tMCAO vs. PTS). These results indicate substantial motor impairments in SAH, tMCAO, and PTS models, effectively replicating stroke-induced motor deficits reported in clinical settings.

Fig. 1.

Impaired motor function in SAH, tMCAO, and PTS stroke mice. A Schematic illustration of the experimental design. B T2-weighted imaging (T2WI) representative images taken 24 h post-stroke, showing infarct regions in SAH, transient middle cerebral artery occlusion (tMCAO), and photothrombotic stimulation (PTS) models. C Motor deficits were assessed using the rotarod test, showing significantly reduced latency to fall in all three stroke models compared to the sham group, indicating impaired motor function. Data are presented as mean ± SEM, with statistical significance denoted by asterisks (**p < 0.01, ****p < 0.0001). Sample sizes: Sham, n = 30; SAH and tMCAO, n = 15 each; PTS, n = 16

Stroke Impairs Recognition, Working, and Spatial Memory in Experimental Models of SAH, tMCAO, and PTS

Despite motor deficits, stroke is also known to cause cognitive deficits [28]. Building on our earlier findings of motor impairments in stroke models, we extended our assessment to recognition memory using the NOR test. In the NOR test, quantitative analysis of the discrimination index revealed a significant reduction in recognition memory across all stroke models compared to sham, indicating impaired memory function (p < 0.01, sham vs. SAH and tMCAO; p < 0.001, sham vs. PTS; Fig. 2A). Further analysis showed a significant reduction in time spent with the NO in all stroke models compared to sham (p < 0.05, sham vs. SAH and tMCAO; p < 0.01, sham vs. PTS; Fig. 2A). This consistent decrease in the discrimination index and time spent with NO suggests that each stroke model disrupts recognition memory.

Fig. 2.

Impaired recognition, spatial and working memory in SAH, tMCAO, and PTS stroke mice. A Representative images from the novel object recognition (NOR) test showing exploration patterns in all groups. Quantification of discrimination index in the NOR test, showing significant differences in all stroke models compared to the sham group. Percentage of time spent with the novel object significantly reduced in all stroke models. B Representative tracking traces from the Y-maze test, illustrating spatial exploration patterns. Percentage of spontaneous alternation significantly reduced in all stroke models. C Tracking traces from the Barnes maze test, demonstrating spatial navigation. Latency to reach the goal box during training days in the Barnes maze. Latency to reach the goal box on the test day significant reduced in all stroke models compared to the sham group. Data are presented as mean ± SEM, with statistical significance denoted by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Sample sizes: Sham, n = 30; SAH and tMCAO, n = 15 each; PTS, n = 16

To further investigate the impact of stroke on working memory, we employed the Y-maze test and recorded the number of spontaneous alternations. All stroke models exhibited a significant reduction in spontaneous alternation rates compared to sham (p < 0.05, sham vs. SAH; p < 0.0001, sham vs. tMCAO and p < 0.001, sham vs. PTS; Fig. 2B), indicating impaired working memory. This decrease in the alternation rate reflects deficits in short-term information processing and decision-making, which are essential for everyday tasks.

The impact of stroke on spatial learning and memory was further assessed using the Barnes maze. All three stroke models demonstrated a significant increase in escape latency compared to sham (p < 0.001, sham vs. SAH; p < 0.0001, sham vs. tMCAO; p < 0.05, sham vs. PTS; Fig. 2C), indicating deficits in spatial learning and memory. Collectively, our findings demonstrate that the SAH, tMCAO, and PTS models not only induce motor deficits but also significantly disrupt recognition, working and spatial learning, and memory.

Stroke Induces Distinct Profiles of Anxiety and Depression-like Behaviors in Experimental Models of SAH, tMCAO, and PTS

To assess anxiety and depressive-like behaviors in stroke models, we used the OFT, SPT, and TST, which are standard for evaluating post-stroke emotional behaviors.

In the OFT, we measured the total distance moved and moving velocity to assess general locomotor activity and exploratory behavior. No significant differences were observed across the groups, suggesting that overall activity levels were comparable between stroke models and sham animals (Fig. 3A-C). These findings suggest that any differences identified in further evaluations would likely reflect specific emotional and behavioral disturbances associated with stroke, rather than being attributable to general hypoactivity or fatigue. Despite the comparable locomotor activity, tMCAO and SAH mice exhibited a significant reduction in the time spent in the center of the open field compared to sham (p < 0.01, sham vs. SAH; p < 0.0001, sham vs. tMCAO; Fig. 3D), suggesting heightened anxiety-like behavior in SAH and tMCAO mice. In contrast, the PTS model showed no significant difference in center time relative to sham, indicating less pronounced anxiety-related behavior. In pairwise comparisons, tMCAO mice also showed a difference in center time compared to SAH (p < 0.05) and PTS (p < 0.0001), while SAH mice also differed significantly from PTS mice (p < 0.05). These results highlight the distinct emotional responses across the stroke models and emphasize the variability in anxiety-related behaviors following different types of cerebrovascular injury.

Fig. 3.

Impaired anxiety and depression-like behavior in SAH and tMCAO models, but not in the PTS mice. A Representative images from the open field test showing exploration. B Total distance moved in the open field test is similar among groups. (C) Moving velocity across groups, with no significant changes observed. D Time spent in the center zone of the open field is significantly altered in the SAH and tMCAO groups but not in the PTS group. E Percentage of sucrose preference is significantly reduced in the SAH and tMCAO groups but not in the PTS group. F Immobility time in the tail suspension test is significantly increased in the SAH and tMCAO groups but not in the PTS group. Data are presented as mean ± SEM, with statistical significance denoted by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Sample sizes: Sham, n = 30; SAH and tMCAO, n = 15 each; PTS, n = 16

In the SPT, SAH and tMCAO mice exhibited a significant reduction in sucrose preference (p < 0.0001 for sham vs. SAH; p < 0.001 for sham vs. tMCAO; Fig. 3E), indicating anhedonia, while the PTS model showed no significant change. Pairwise comparisons revealed that sucrose preference was significantly higher in the PTS group compared to the SAH group (p < 0.01). Similarly, TST analysis showed a significant increase in immobility time in SAH and tMCAO mice, suggesting depressive-like behavior (p < 0.0001 sham vs. SAH; p < 0.05 sham vs. tMCAO; Fig. 3F). In contrast, the PTS model did not show significant changes in immobility time, further indicating an absence of depressive-like symptoms. These findings suggest that the SAH and tMCAO models more closely represent depression-related symptoms, while the PTS model does not fully capture this aspect of mood dysfunction.

Stroke-Induced Alterations in Dopaminergic Populations in the Dentate Gyrus Across Multiple Models

Following behavioral assessments, immunohistochemical analyses were performed at 30 days post-injury to reflect chronic-stage pathology (Fig. 1A). Given the significant cognitive deficits observed across all stroke models, and considering the critical role of the dentate gyrus (DG) in memory formation, learning, and emotional regulation [29], as well as the influence of dopaminergic signaling on neuronal plasticity and function [30], we hypothesized that stroke-induced alterations in NeuN-positive neurons and TH-positive processes within the DG may contribute to the behavioral impairments observed. Analysis of TH-positive process overlap with NeuN revealed a significant increase in the SAH and tMCAO groups compared to sham (p < 0.01, sham vs. SAH and tMCAO; Fig. 4A-B), suggesting enhanced dopaminergic input to neurons in these models. Pairwise comparisons showed significantly higher overlap in the tMCAO group compared to the PTS group (p < 0.05; Fig. 4B). NeuN-positive neuronal density was significantly reduced in all stroke models (p < 0.001, sham vs. SAH; p < 0.01, sham vs. PTS and tMCAO; Fig. 4C). Furthermore, TH-positive area fraction (Fig. 4D) was significantly increased in the SAH (p < 0.05, sham vs. SAH) and tMCAO (p < 0.01, sham vs. tMCAO) groups but remained unchanged in the PTS group. These findings highlight distinct alterations in dopaminergic processes and neuronal populations across stroke models, underscoring the complexity of stroke-induced neurochemical remodeling and its potential role in cognitive and emotional dysfunction.

Fig. 4.

Altered TH-positive process overlap with NeuN, NeuN density, and TH area fraction in the dentate gyrus across stroke models. A Representative immunofluorescence images of NeuN (neuronal marker, green) and TH (tyrosine hydroxylase, red) in the DG across all experimental groups. Nuclei are counterstained with DAPI (blue). Scale bar: 1 mm and 20 µm B Quantification of TH-positive process overlap with NeuN reveals significantly increased colocalization in the SAH and tMCAO groups compared to the Sham group, with no significant change in the PTS group. C NeuN-positive neuronal density is significantly decreased in the SAH, tMCAO, and PTS groups compared to the Sham group. D TH-positive area fraction in the DG is significantly increased in the SAH and tMCAO group but remains unchanged in the PTS groups. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham group (one-way ANOVA with post hoc Tukey's test); n = 5 per group

Stroke-Induced Alterations in Dopaminergic Populations in the Amygdala Across Multiple Models

The amygdala is a brain region integral to emotion and memory processing [31]. It plays a critical role in regulating emotional responses [32], and disruptions in its neuronal populations may contribute to the behavioral deficits associated with stroke. Given the observed impairments in motor function and cognition across the SAH, tMCAO, and PTS stroke models, along with differential anxiety- and depression-like behaviors, we aimed to investigate potential neuronal changes in the amygdala. Specifically, we aimed to analyze changes in neuronal populations by assessing NeuN and TH expression levels. Quantification of TH-positive process overlap with NeuN revealed a significant increase in the amygdala of the SAH and tMCAO models compared to sham (p < 0.0001 for sham vs. SAH; p < 0.01 for sham vs. tMCAO; Fig. 5A-B). In contrast, the PTS model showed no significant change in the TH-positive process and NeuN overlap. Pairwise comparisons demonstrated a significant increase in TH-positive process overlap in the SAH group compared to the PTS group (p < 0.01; Fig. 5B). NeuN-positive neuronal density was significantly increased in the SAH and tMCAO groups (p < 0.0001 for sham vs. SAH and sham vs. tMCAO; Fig. 5C). Pairwise comparisons revealed significantly higher numbers of NeuN-positive cells in the SAH and tMCAO groups compared to the PTS group (p < 0.0001 for SAH vs. PTS and tMCAO vs. PTS). Furthermore, TH-positive area fraction (Fig. 5D) was significantly elevated in SAH (p < 0.05, sham vs. SAH) and tMCAO (p < 0.001, sham vs. tMCAO) compared to sham but remained unchanged in the PTS group. Pairwise comparisons revealed an additional increase in tMCAO compared to PTS (p < 0.05; Fig. 5D). These findings indicate that stroke induces model-specific alterations in dopaminergic processes and neuronal populations within the amygdala, with SAH and tMCAO showing more pronounced changes than PTS.

Fig. 5.

Increased TH-positive process overlap with NeuN, NeuN density, and TH area fraction in the amygdala of SAH and tMCAO Mice, but not PTS mice A Representative immunofluorescence images of NeuN (neuronal marker, green) and TH (tyrosine hydroxylase, red) in the amygdala across all experimental groups. Nuclei are counterstained with DAPI (blue). Scale bar: 1 mm and 20 µm. B Quantification of TH-positive process overlap with NeuN shows significantly increased overlap in the SAH and tMCAO groups compared to sham, with no significant change in the PTS group. C NeuN-positive neuronal density is significantly increased in the SAH and tMCAO groups compared to the sham group, while the PTS group shows no change. D TH-positive area fraction in the amygdala is elevated in SAH and tMCAO mice but remains unchanged in the PTS group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus sham group (one-way ANOVA with post hoc Tukey's test); n = 5 per group

Stroke-induced Glial Activation and Morphological Changes in the Amygdala Across SAH, tMCAO, and PTS Mouse Models

Astrocytes and microglia are key components of the brain’s immune system, playing essential roles in maintaining neural homeostasis and mediating inflammatory responses following stroke [33]. The activation of these glial cells has been increasingly recognized as a key factor contributing to post-stroke pathophysiology, particularly in the context of cognitive and emotional deficits observed in stroke survivors [34]. Specifically, astrocytes, via their GFAP expression, and microglia, identified by IBA-1 positivity, are involved in neuroinflammation, neuronal survival, and synaptic plasticity, processes that are disrupted in stroke models. The amygdala, a brain region intimately involved in emotional regulation, has been shown to undergo significant glial and neuronal changes after stroke, further influencing the development of mood disorders such as depression and anxiety [35]. Despite the well-established role of glial activation in stroke recovery, the relationship between these glial responses and specific behavioral outcomes, especially in emotional domains, remains incompletely understood. Therefore, we investigated GFAP-positive astrocytes and IBA-1-positive microglia in the amygdala of SAH, tMCAO, and PTS mouse models to better understand how glial activation may contribute to the behavioral deficits observed in these stroke models. Our results demonstrate increased GFAP-positive astrocyte density in the SAH and tMCAO groups compared to sham (p < 0.01, sham vs. SAH and p < 0.0001, sham vs. tMCAO; Fig. 6A-B), with higher density in both compared to PTS (p < 0.01 for SAH vs. PTS; p < 0.0001 for tMCAO vs. PTS). GFAP fluorescence intensity also increased in SAH and tMCAO groups (p < 0.01 for sham vs. SAH and tMCAO; Fig. 6C), while no significant changes were observed in PTS. Interestingly, astrocyte branch length was significantly increased in the PTS group compared to other groups (p < 0.01 for sham vs. PTS and sham vs SAH; p < 0.001 for PTS vs. tMCAO; Fig. 6D), indicating structural remodeling without robust activation. The number of endpoint voxels remained unchanged across groups (Fig. 6E). Additionally, IBA-1-positive microglial density was significantly elevated in all stroke models (p < 0.05 for sham vs. SAH; p < 0.01 for sham vs. tMCAO and PTS; Fig. 6F-G), with increased fluorescence intensity (p < 0.05 for sham vs. SAH and PTS; p < 0.01 for sham vs. tMCAO; Fig. 6H). However, no significant differences were observed in microglial branch length (Fig. 6I) or endpoint voxel number (Fig. 6J). These findings demonstrate distinct glial responses in the amygdala across stroke models, characterized by pronounced astrogliosis in SAH and tMCAO, and astrocyte remodeling in PTS. While microglial activation was evident in all models, it occurred without notable morphological changes.

Fig. 6.

Differential Astrogliosis and Microgliosis in the Amygdala Across SAH, tMCAO, and PTS Mice. A Representative immunofluorescence images of GFAP (astrocytes, green) in the amygdala across all experimental groups. Nuclei are counterstained with DAPI (blue). Scale bar: 1 mm and 20 µm. B Quantification of GFAP-positive cell density shows significantly increased astrocyte density in the SAH and tMCAO groups compared to the sham group. C Fluorescence intensity of GFAP reveals a significant increase in the SAH and tMCAO groups compared to the sham group, with no significant change in the PTS group. D Morphological analysis of astrocytes indicates increased branch length in the PTS group compared to the other groups. E The number of endpoint voxels of astrocyte branches is unchanged across all groups. F Representative immunofluorescence images of IBA-1 (microglia, red) in the amygdala across all experimental groups. G Density of IBA-1-positive microglia is significantly increased in SAH, tMCAO, and PTS groups compared to the sham group. H Fluorescence intensity of IBA-1 is significantly elevated in all experimental groups (SAH, tMCAO, and PTS) compared to the sham group. I-J Microglial morphology analysis shows no significant changes in branch length or the number of endpoint voxels (J) across groups. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham group (one-way ANOVA with post hoc Tukey's test); n = 5 per group

Discussion

This study provides a detailed comparison of behavioral, neuronal, and glial outcomes across three mechanistically distinct mouse models of stroke: SAH, tMCAO, and PTS. By examining motor, cognitive, and emotional outcomes alongside neuronal and glial changes, we delineate model-specific pathophysiological signatures relevant to translational stroke research.

Our findings reveal significant impairments in motor coordination and spatial memory across all three stroke models (Figs. 1 and 2), consistent with the well-established cognitive and motor deficits experienced by human stroke survivors [27, 36–38]. In addition to physical impairments, psychological disturbances such as depression and anxiety are common post-stroke complications, affecting up to one-third of patients and substantially diminishing quality of life [39, 40]. In our study, we observed anxiety- and depression-like behaviors in the SAH and tMCAO models (Fig. 3), which align with the emotional consequences observed in human patients following hemorrhagic and ischemic strokes [41, 42]. These findings suggest that both models can be used to explore the neurobiological mechanisms driving emotional dysregulation following stroke. In contrast, the PTS model, which simulates focal cortical infarction and is less invasive and more localized compared to the tMCAO and SAH model [43], predominantly induced motor and cognitive deficits without accompanying anxiety- or depression-like behaviors. This likely reflects the relatively limited and superficial nature of the injury in PTS, which spares subcortical limbic structures such as the hippocampus and amygdala that are critically involved in mood regulation [44, 45]. Thus, the selection of stroke models should be guided by the specific behavioral domains of interest: SAH and tMCAO models are particularly suited for studying emotional dysfunctions, whereas PTS may be more appropriate for investigating localized cortical pathology and associated motor and memory deficits. For ease of comparison, we have included a summarized table (Table 2) outlining the behavioral tasks and the corresponding outcomes observed across all three stroke models.

Table 2.

Behavioral tasks and outcomes across stroke models

Domain	Behavioral Test	Function Assessed	Outcome in SAH	Outcome in tMCAO	Outcome in PTS
Motor Function	Rotarod	Motor coordination, balance	Impaired	Severely impaired	Mildly impaired
Cognition	Novel Object Recognition (NOR)	Recognition memory	Impaired	Impaired	Strongly impaired
	Y-maze	Working memory (spontaneous alternation)	Impaired	Strongly impaired	Moderately impaired
	Barnes Maze	Spatial learning and memory	Moderately impaired	Strongly impaired	Mildly impaired
Affective Behavior	Open Field Test (OFT)	Anxiety-like behavior (center time)	Increased anxiety	Strongly increased anxiety	No significant change
	Sucrose Preference Test (SPT)	Anhedonia (depression-like behavior)	Strongly reduced preference	Moderately reduced preference	No significant change
	Tail Suspension Test (TST)	Behavioral despair (depression-like behavior)	Increased immobility	Mildly increased immobility	No significant change

Affective behaviors are tightly regulated by limbic circuits, particularly the reciprocal connections between the hippocampus and amygdala [45, 46]. Under pathophysiological conditions, these circuits become hyperactive and contribute to the development of anxiety and depressive-like phenotypes [47]. Dopaminergic neurons, particularly those projecting to the amygdala and hippocampus, play a central role in modulating affective states [47], and dysregulation of dopaminergic signaling in these regions has been closely linked to heightened anxiety and depressive symptoms [48]. Our data indicate region- and model-specific dopaminergic alterations that may underlie the observed behavioral outcomes. We observed increased TH-positive process overlap with NeuN in the DG (Fig. 4) and amygdala (Fig. 5) of SAH and tMCAO mice, indicating enhanced dopaminergic projections to neurons in these regions. NeuN expression was significantly reduced in the DG across SAH, tMCAO, and PTS models, whereas it was increased in the amygdala of SAH and tMCAO mice. This divergent pattern suggests injury-induced neuronal loss in the hippocampus but possible compensatory neuroplasticity in the amygdala. Notably, although MRI conducted at 24 h post-injury (Fig. 1B) did not reveal hippocampal infarction in the PTS model, Fig. 4C shows significant NeuN loss in the hippocampus at 30 days. This discrepancy likely reflects the timing of assessments. It is well-established that regions like the hippocampus can undergo secondary degeneration undetectable by acute imaging. A longitudinal clinical MRI study reported delayed atrophy in limbic structures, including the hippocampus, several weeks post-stroke [49]. Such chronic changes are thought to arise from excitotoxicity, inflammation, and cortico-hippocampal disconnection [50, 51], which may evade early MRI detection but become apparent with delayed histological analysis.

TH-positive area fraction was also significantly elevated in both regions in SAH and tMCAO mice, whereas no notable changes were detected in the CA1 and CA3 subfields (Supplementary Figs. 1 and 2), further emphasizing the spatial specificity of these effects. These findings are consistent with previous studies demonstrating that ischemic injury involving limbic structures, including the hippocampus and amygdala can lead to long-term motor, cognitive, and emotional impairments, including mood disorders [52–56]. Notably, our results also revealed persistent motor deficits, cognitive dysfunction, and anxiety- and depression-like behaviors in SAH and tMCAO mice, supporting the link between region-specific dopaminergic alterations and long-term neurobehavioral outcomes. In contrast, the PTS model did not produce significant changes in TH-positive area fraction within these regions (Figs. 4 & 5), suggesting a more limited effect on affective circuitry. This differential response can likely be attributed to the extent and distribution of injury and inflammation among the models. Both SAH and tMCAO are associated with widespread blood–brain barrier (BBB) disruption, extensive neurovascular injury, and robust inflammatory responses that can extend into subcortical and limbic regions [57–59]. These conditions promote dopaminergic dysregulation, either through neurotoxicity or compensatory plasticity. However, the PTS model induces a localized cortical infarct with relatively preserved BBB integrity and attenuated microglial activation [60, 61], limiting the propagation of inflammatory signals to distant brain regions. Additionally, the restricted nature of PTS lesions spares subcortical and limbic areas, which may further explain the absence of dopaminergic alterations in the hippocampus and amygdala or the focal injury may also facilitate faster recovery and restoration of dopaminergic homeostasis. Although PTS mice exhibited cognitive and motor deficits, they did not display significant anxiety-like behavior, consistent with the lack of dopaminergic alterations in limbic regions. This suggests that while focal cortical injury in the PTS model is sufficient to impair sensorimotor and memory functions, it may not strongly engage the dopaminergic pathways that regulate emotional behaviors.

Glial responses further support the model-dependent nature of neuroinflammation following stroke. Astrocytes and microglia play central roles in mediating post-injury inflammation and neuroimmune signaling [62, 63]. Our results show persistent astrocytic and microglial activation in the amygdala of SAH and tMCAO models (Fig. 6), consistent with chronic neuroinflammatory signaling even in regions not directly impacted by the initial insult, such as the amygdala [47]. Although we didn’t observe significant morphological changes (Fig. 6), this suggests a functional rather than structural adaptation. The greater glial activation in SAH and tMCAO may again reflect the greater injury burden, deeper tissue penetration, and more widespread BBB leakage [57–59], all of which can promote glial priming and reactivity in both local and remote regions. Microglial activation in the PTS model appeared primarily functional, occurring without significant morphological changes, suggesting involvement in cytokine production and phagocytic activity rather than structural remodeling [64]. These differences in astrocytic and microglial responses across models likely reflect the varying extent, location, and nature of injury. While SAH and tMCAO trigger diffuse neuroinflammatory cascades due to global or subcortical involvement and BBB breakdown, the PTS model, with its localized cortical infarct, may engage glial plasticity in a more targeted and structurally adaptive manner.

Conclusion

This study provides new insights into the pathophysiological mechanisms underlying emotional and cognitive impairments in stroke by comparing the SAH, tMCAO, and PTS models. Unlike previous studies that have predominantly focused on motor outcomes or general neuroinflammation, our study uniquely highlights chronic dopaminergic dysregulation and sustained glial activation in the hippocampus and amygdala, areas crucial for mood regulation and cognitive function. These findings are particularly novel in their demonstration of how these stroke models recapitulate key behavioral and neuropathological features seen in stroke patients, including the emotional consequences of stroke, which are often underexplored in preclinical research. Although translating emotional and cognitive deficits from rodents to humans remains complex, our study advances the understanding of model-specific neurobiological mechanisms that drive affective and cognitive disturbances following stroke. In particular, by contrasting the more localized pathology of the PTS model, which exhibited limited limbic dopaminergic and glial changes, with the broader neurovascular impact of SAH and tMCAO models, our findings underscore the importance of selecting stroke models based on the neurobehavioral domains under investigation.

Despite these strengths, several important considerations should be acknowledged. First, only male mice of a single age group were used to minimize hormonal variability and establish baseline comparisons across models. However, both sex and age are critical biological variables that influence stroke pathology and recovery; therefore, future studies will incorporate female cohorts and multiple age groups to better capture the clinical heterogeneity observed in stroke patients. Second, our neuroanatomical analysis was limited to the hippocampal subfields (DG, CA1, CA3) and the amygdala, regions closely associated with emotional and cognitive functions. While this focus aligned with our behavioral endpoints, dopaminergic and glial alterations in cortical areas directly affected by ischemic injury were not examined. Expanding analyses to include cortical structures will be essential for a more comprehensive understanding of stroke-induced spatial pathophysiology. Third, glial responses were assessed only in the amygdala, selected for its strong association with mood regulation. We recognize that investigating glial reactivity in other brain regions, such as the hippocampus and cortex, may yield additional insights and plan to address this in future studies.

In summary, our study identifies dopaminergic signaling and glial responses as potential therapeutic targets for post-stroke emotional and cognitive impairments. These findings not only enhance the translational relevance of commonly used stroke models but also pave the way for mechanistic studies aimed at improving neuropsychiatric outcomes in stroke survivors.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors Contribution

ST: Writing- original draft, behavior and immunostaining experiments, Figures. HH: Animal stroke models. SW: Data analysis, Writing – review & editing. SH: MRI. BSKY: Data analysis, Writing – review & editing. CHL: Writing – review & editing. AG: Writing – review & editing, Funding acquisition. XR: Conceptualization, Supervision, Writing – review & editing, Funding acquisition.

Funding

The authors gratefully acknowledge support from the National Institute of Neurological Disorders and Stroke (NINDS; 1R01NS117606, K23NS121628), the National Institute on Aging (NIA; 1R01AG073659), the Joe Niekro Foundation, and new faculty start-up funds from the University of Texas Health Science Center at Houston.

Data Availability

The research data supporting the results of this manuscript will be made available by the corresponding author upon reasonable request.

Declarations

Competing interest

The authors declare no competing interests.