Neural stem cell transplantation attenuates cognitive decline and neuroinflammation in a mouse model of Alzheimer’s disease with chronic cerebral hypoperfusion

Abstract

Alzheimer’s disease (AD) and chronic cerebral hypoperfusion (CCH) frequently coexist in aging populations, synergistically aggravating neurodegeneration. To assess the therapeutic potential of stem cell interventions, an AD + CCH mouse model was generated by combining APP/PS1 mice with bilateral common carotid artery stenosis. Neural stem cells (NSCs) or induced pluripotent stem cells (iPSCs) were transplanted into the lateral ventricles at 5 months of age. Behavioral testing, Nissl staining, Western blotting, and immunofluorescence were conducted at 9 and 12 months to evaluate cognition, neuronal survival, cell death pathways (LC3-II, cleaved caspase-3, NLRP3), glial polarization, and neurotrophic/synaptic markers (BDNF, VEGF, VAChT, PSD95). CCH exacerbated AD-related cognitive deficits, neuronal loss, and activation of autophagic, apoptotic, and pyroptotic pathways, accompanied by enhanced M1 microglial polarization, astrogliosis, and downregulation of BDNF and VAChT. NSCs transplantation significantly improved cognitive performance, preserved neuronal integrity, attenuated glial activation, and restored neurotrophic and synaptic protein expression, characterized by increased BDNF, VEGF, and PSD95 levels and partial recovery of VAChT. In contrast, iPSCs transplantation failed to exert comparable effects. These findings demonstrate that NSCs, but not iPSCs, mitigate AD + CCH-induced neuropathology by re-establishing the balance between inflammatory, neurotrophic, and synaptic signaling, supporting NSCs as a promising therapeutic approach for AD with vascular comorbidity.

Keywords

Alzheimer’s disease chronic cerebral hypoperfusion neural stem cells induced pluripotent stem cells microglia polarization

Introduction

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder marked by progressive cognitive impairment and neuropathological hallmarks, including extracellular β-amyloid (Aβ) plaques, intracellular neurofibrillary tangles, and synaptic and neuronal degeneration.¹ Increasing evidence suggests that pure amyloid-driven pathology accounts for only a subset of dementia cases, and that mixed dementia—defined by the coexistence of neurodegenerative and cerebrovascular pathologies—represents the more prevalent clinical phenotype in the elderly population.² Among the vascular insults implicated in the pathogenesis of AD, chronic cerebral hypoperfusion (CCH) has garnered particular attention as a major contributor to cognitive deterioration, neuroinflammation, and glial dysregulation.³

CCH exerts profound effects on brain homeostasis by inducing endothelial dysfunction, blood–brain barrier (BBB) breakdown, white matter demyelination, and mitochondrial energy failure.⁴ These changes disrupt the structural and functional integrity of the neurovascular unit (NVU), reduce cerebral clearance of neurotoxic proteins, and amplify Aβ deposition and tau hyperphosphorylation, thereby promoting a self-reinforcing loop that accelerates AD pathology.⁵ Our previous work demonstrated that CCH exacerbates neurovascular uncoupling and synaptic oxidative stress in transgenic AD models, contributing to the progression of memory impairment and gliosis via NVU disintegration and BBB leakage.⁶ Despite these findings, the precise cellular and molecular cascades through which CCH interacts with AD, particularly concerning innate immune activation, glial polarization, and programmed neuronal death, remain incompletely elucidated.

Recent studies have identified the co-activation of autophagy, apoptosis, and pyroptosis in the AD brain, implicating their convergence as a critical mechanism underlying progressive neuronal loss.⁷ Moreover, dysregulated microglial polarization, shifting from a homeostatic M2 phenotype to a chronically activated M1 profile, has been recognized as a key driver of sustained neuroinflammation in both AD and vascular cognitive impairment.⁸ Compounding these effects, brain-derived neurotrophic factor (BDNF) levels are known to decline in both AD and hypoperfusion, further impairing neuronal survival, synaptic plasticity, and subventricular zone (SVZ) neurogenesis.⁹ Hence, therapeutic strategies that simultaneously target these overlapping neurovascular, neuroinflammatory, and neurotrophic axes are critically needed.

Stem cell-based interventions have emerged as a promising avenue for addressing multifactorial brain injury.¹⁰ Neural stem cells (NSCs), derived from fetal or embryonic sources, have been shown to exert potent neuroprotective effects via paracrine trophic factor release, immune modulation, and support of endogenous repair mechanisms.^11,12 Induced pluripotent stem cells (iPSCs), reprogrammed from adult somatic cells, offer the theoretical advantage of autologous transplantation and unlimited expansion capacity.¹³ However, the therapeutic equivalence and mechanistic divergence between NSCs and iPSCs in models of mixed neurodegenerative–vascular pathology have not been systematically compared.¹⁴ Furthermore, whether these cell types differentially modulate glial polarization, regulated cell death signaling, BDNF expression, and the SVZ stem cell niche in the context of combined AD and CCH remains an open question.

In this study, we established a murine model of mixed dementia by combining APP/PS1 transgenic AD mice with progressive bilateral common carotid artery stenosis to simulate chronic hypoperfusion. This model recapitulates key features of both amyloid-driven and vascular-driven cognitive decline. Using this platform, we performed the first direct comparative evaluation of NSC versus iPSC transplantation via intraventricular delivery. Cognitive function, glial phenotypes, autophagy/apoptosis/pyroptosis pathway activation, neurotrophic signaling, and SVZ niche integrity were systematically assessed in both 9- and 12-month-old animals. Our findings elucidate distinct therapeutic profiles of NSCs and iPSCs in mixed dementia and provide mechanistic insights into stem cell-mediated neurorestoration targeting.

Materials and methods

Experimental animals

Male C57BL/6J wild-type mice and APP/PS1 double-transgenic mice were utilized in this investigation, with an average postnatal age of 30 ± 1 days (mean ± SD). To minimize confounding factors associated with estrogen fluctuations, only male animals were selected for inclusion in the study.¹⁵ The APP/PS1 mouse line carries mutations in two key genes implicated in the pathogenesis of AD, facilitating the manifestation of AD-like neuropathological hallmarks and cognitive deficits.¹⁶ This transgenic model has been extensively validated and is widely employed in preclinical studies to elucidate disease mechanisms and evaluate potential therapeutic interventions for AD.¹⁷ All mice were maintained under controlled environmental conditions, with regulated temperature and humidity. They were kept in standard laboratory cages under a 12-h light/dark cycle, with ad libitum access to food and water. Animal care and all experimental procedures conformed to the ethical standards outlined in the Guide for the Care and Use of Laboratory Animals (eighth edition, National Research Council, 2011) and received approval from the Institutional Animal Care and Use Committee (IACUC) of Youjiang Medical University for Nationalities (approval no. YYFY-LL-2024-265). Moreover, the study design and reporting adhered strictly to the ARRIVE 2.0 guidelines to ensure methodological rigor and reproducibility, including detailed documentation of housing conditions, experimental interventions, and outcome measures. This animal work constitutes part of a broader research project focusing on the involvement of oxidative stress and autophagic impairment in this AD mouse model.

Experimental design and CCH mouse model

For this study, a total of five experimental groups were delineated: the WT group (C57BL/6J, n = 10), the AD group (APP/PS1, n = 10), the AD + CCH group (APP/PS1 + CCH, n = 10), the NSCs group (APP/PS1 + CCH + NSCs, n = 10), and the iPSCs group (AD + CCH + iPSCs, n = 10). The protocol for establishing the CCH model has been exhaustively documented in our prior investigations.^8,18,19 Briefly, at 4 months of age—prior to the onset of significant amyloid deposition in APP/PS1 mice—animals were anesthetized with 1% isoflurane in a 69%/30% (vol/vol) nitrous oxide/oxygen mixture and placed supine on a temperature-controlled heating pad, and bilateral common carotid arteries (BCCAs) were surgically exposed through neck incisions, where ameroid constrictors (ACs) were carefully placed. These constrictors progressively induced narrowing of the BCCAs, culminating in a reduction of cerebral blood flow (CBF) values to 60%–70% by postoperative day 28. This murine model represents a valuable tool for investigating the impact of CCH on cognitive decline and the pathology of AD.

Intracerebroventricular injection of stem cells

NSCs derived from C57BL/6 mice were obtained from OriCell^® (catalog number: MUBNF-01001), and iPSCs were sourced from the Chinese Academy of Sciences Stem Cell Bank (OSKZ-1, catalog number: SCSP-1204).²⁰ At 5 months of age, mice assigned to the NSCs and iPSCs treatment groups underwent stereotaxic intracerebroventricular (ICV) injection of the respective stem cells (Figure 1(a)). The injection coordinates relative to the Bregma were as follows: 0.3 mm posterior, 1.0 mm lateral to the right, and 2.2 mm below the surface of the skull. A total volume of 2 μL of cell suspension was administered per mouse, resulting in a final stem cell concentration of 5 × 10⁵ cells/μL. The injection was performed at a controlled rate of 1 μL/min using a microsyringe to ensure precision and minimize tissue damage. After delivery of the cell suspension, the needle was left in place for an additional 5 min to allow adequate diffusion and prevent reflux. For the AD + CCH control group, an equivalent volume (2 μL) of sterile physiological saline was injected into the lateral ventricle using the same stereotaxic parameters and injection procedure, serving as the vehicle control.

Behavioral evaluation

T-maze test

The T-maze consists of one start arm and two symmetrical goal arms, forming a T-shaped configuration. At the beginning of each session, mice are allowed to explore the maze freely to acclimate to the testing environment. After this habituation phase, a food incentive is strategically placed at the end of one goal arm to promote goal-directed learning through positive reinforcement. With repeated training trials, the animals gradually learn to identify and navigate toward the rewarded arm with increased efficiency. To assess spatial working memory, a probe test is conducted following the training phase, during which the reward is removed. The arm chosen by the mouse and the latency to reach the decision point are recorded as measures of memory performance. Generally, reduced response time and a higher rate of spontaneous alternation are interpreted as indicators of superior cognitive function, particularly in learning and memory domains.

Novel object recognition (NOR) test

This behavioral paradigm evaluates recognition memory by leveraging rodents’ intrinsic tendency to preferentially investigate unfamiliar stimuli over previously encountered ones. In a standardized open-field environment, two identical objects are placed symmetrically positioned 15 cm from the perimeter walls and separated by 25 cm. Each mouse is initially placed at the center of the arena, equidistant from the objects, and permitted to explore freely for a 10-min habituation session. After a 5-h retention interval, one of the familiar objects is replaced with a novel object differing in color and shape, though matched in material and size. The subject is then reintroduced to the arena from the identical starting location and allowed to explore for another 10 min. All exploratory behavior directed toward familiar and novel objects is recorded using an overhead infrared tracking system. Recognition performance is quantified using the Recognition Index (RI), calculated as: RI = (exploration time of novel object)/(exploration time of novel object + exploration time of familiar object) × 100%. Typically, a reduced RI denotes exacerbated cognitive and memory deficits in mice.

Brain tissue processing

At 9 and 12 months of age—corresponding to the middle and late stages of Alzheimer’s disease progression—mice underwent procedures for brain tissue collection and sectioning. The process of tissue collection commenced with the administration of intraperitoneal pentobarbital sodium (40 mg/kg) to induce deep anesthesia across all experimental groups. Subsequently, mice were subjected to transcardial perfusion with 20 ml of ice-cold phosphate-buffered saline (PBS) to ensure thorough removal of blood from the vasculature. Following perfusion, whole brain specimens were carefully exercised and preserved at −80 °C. Tissue sectioning commenced with the continuation of perfusion, this time with 20 ml of ice-cold 4% paraformaldehyde (PFA, 0.1 mol/L) in PBS. Following PFA perfusion, brains were extracted, fixed overnight in 4% PFA, and subjected to subsequent steps including dehydration, tissue transparency, paraffin embedding, and slicing into thin sections.

Nissl staining evaluation

Paraffin-embedded brain sections were deparaffinized in xylene, rehydrated through graded ethanol (100%–75%), and stained with 1% toluidine blue (pH 4.0) for 10 min at room temperature. After rinsing with distilled water, sections were briefly differentiated in 95% ethanol, dehydrated, cleared in xylene, and mounted with neutral balsam. Neuronal morphology was examined under a light microscope (Leica DM5000B, Germany). Nissl-positive neurons, identified by intact soma and distinct Nissl bodies, were quantified in three randomly selected non-overlapping fields within layer IV of the motor cortex using ImageJ software (NIH, USA). The mean neuronal count per animal was used for statistical analysis.

Western blot analysis

After animals were euthanized, transcardial perfusion was conducted using ice-cold phosphate-buffered saline (PBS) to eliminate residual blood and preserve tissue integrity. Immediately after perfusion, brains were promptly removed, and both cortical and hippocampal regions were dissected and placed on ice to prevent protein degradation. The excised tissues were rinsed in chilled PBS and subsequently lyzed on ice for 15 min to ensure thorough protein extraction. Lysates were then subjected to centrifugation at 12,000 rpm for 15 min at 4 °C. The resulting supernatants were carefully harvested for subsequent protein analysis. The experiment consisted of five groups, with five mice included in each group (n = 5). Protein concentrations were assessed using a bicinchoninic acid (BCA) assay. For each specimen, 20 µg of total protein was denatured in loading buffer and separated using SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (MERCK) using a standard wet transfer protocol. To block nonspecific antibody binding, membranes were incubated in 5% skim milk prepared in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature.

Following the blocking step, membranes were incubated overnight at 4 °C with appropriate primary antibodies. After thorough rinsing with TBST, the membranes were exposed to horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at ambient temperature. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) reagents, and signal intensity was analyzed using ImageJ software for densitometric quantification. The antibodies applied in this study included: TNF-α (1:1000, 346654; Zen-Bioscience), IL-1β (1:1000, bs-0812R; BOISS), NLRP3 (1:1000, bs-10021R; BOISS), APP (1:1000, ab232136; Abcam), IL-6 (1:1000, 500286; Zen-Bioscience), CD16/32 (1:1000, ab228971; Abcam), β-tubulin (1:10,000, 66240-1-Ig; Proteintech), β-actin (1:10,000, 20536-1-AP; Proteintech), CD31 (1:1000, 347526; Zen-Bioscience), DCX (1:1000, 13925-1-AP; Proteintech), GFAP (1:1000, A19058; ABclonal), Iba1 (1:200, 019-19741; Wako), LC3 (1:1000, 14600-1-AP; Proteintech), cleaved Caspase-3 (1:1000, 9669S; CST), IL-10 (1:1000, bs-0698R; BOISS), IL-4 (1:1000, 66142-1-Ig; Proteintech), CD163 (1:1000, ab182422; Abcam), and BDNF (1:1000, 28205-1-AP; Proteintech), VEGF (1:1000, R389402; Zen-Bioscience), VAChT (1:1000, 190002; Zen-Bioscience), and PSD95 (1:1000, R381001; Zen-Bioscience).

Chemiluminescent signals were recorded using the Bio-Rad ChemiDoc MP Imaging System. To prevent signal oversaturation, the imaging system’s automatic exposure function was employed to identify optimal exposure times. Multiple exposure durations were tested to ensure that signal intensities remained within the linear dynamic range. This methodological control ensured accurate quantification and eliminated the risk of signal saturation compromising data integrity.

Immunofluorescence assessment

Paraffin-embedded brain tissue sections were initially deparaffinized, followed by incubation in 3% hydrogen peroxide for 15 min to quench intrinsic peroxidase activity. After rinsing in phosphate-buffered saline (PBS), antigen retrieval was performed using a 0.1 M sodium citrate buffer solution. Tissue sections were subsequently blocked with normal serum for 1 h at room temperature to prevent nonspecific antibody binding. Thereafter, rabbit and mouse-derived primary antibodies were co-incubated with the sections overnight at 4 °C. On the following day, sections were thoroughly washed in PBS and incubated with fluorescent-conjugated secondary antibodies for 1 h at room temperature. After an additional PBS rinse, nuclear counterstaining was achieved using DAPI for 10 min at room temperature. Following the final wash, sections were imaged using a laser-scanning confocal microscope (LSM-510; Zeiss, Jena, Germany), equipped with argon and HeNe1 lasers. The antibodies applied in the immunostaining procedure included: Ki67 (1:200, 14-5698-80; Invitrogen), CD31 (1:200, 347526; Zen-Bioscience), DCX (1:200, 13925-1-AP; Proteintech), GFAP (1:200, A19058; ABclonal), CD16/32 (1:1000, ab228971; Abcam), CD163 (1:200, ab182422; Abcam), Iba1 (1:200, 019-19741; Wako), Aβ40 (1:200, 014-26923; Wako), NLRP3 (1:200, bs-10021R; BOISS), NeuN (1:200, ab177487; Abcam), as well as the secondary antibodies: goat anti-mouse IgG (H + L), highly cross-adsorbed (1:500, A-11001; Invitrogen) and donkey anti-rabbit IgG (H + L), highly cross-adsorbed (1:500, A-21207; Invitrogen).

Quantitative assessment of fluorescence intensity was performed using ImageJ software (NIH, Bethesda, MD, USA). For each animal, three non-overlapping brain sections were analyzed. Immunoreactive signals for Aβ40, CD16/32, CD163, GFAP, and Iba1 were quantified in both the cortex (CTX) and hippocampus (HI); expression of Ki67, DCX, CD31, GFAP, and Iba1 was examined in the subventricular zone (SVZ); while NLRP3 immunoreactivity was specifically evaluated in layer IV of the motor cortex. Standardized imaging conditions were applied to all sections to ensure reproducibility and comparability. Regions of interest (ROIs) encompassing stained areas were manually delineated, and background fluorescence was subtracted from each ROI to obtain corrected intensity values for accurate quantification.

Sample size determination and experimental rigor

A priori power analysis (G*Power 3.1; two-tailed, α = 0.05, power = 0.80) based on pilot effect sizes for T-maze latency (d ≈ 1.2) and NOR index (d ≈ 1.0) determined behavioral sample sizes of n = 10/group. Biochemical and histological analyses were conducted in n = 5/group. Animals were block-randomized by litter and baseline body weight to ensure balanced group allocation. Behavioral testing, immunofluorescence quantification, and Western blot analyses were performed by investigators blinded to group identity. Due to the technical nature of CCH surgery, blinding was not feasible during the procedure; however, surgical interventions and all subsequent outcome assessments were conducted by independent personnel to minimize operator bias. Overall, these measures ensured rigor in experimental design, data collection, and analysis.

Statistical analysis

Statistical analysis of the data was conducted using SPSS 23.0 software. Continuous variables are presented as mean ± standard deviation (x ± s). Within-group differences were analyzed using one-way analysis of variance (ANOVA), followed by post-hoc pairwise comparisons using the LSD test. Between-group differences were analyzed using the t-test, with significance set at p < 0.05.

Results

Cognitive performance and expression of cell death-associated markers in mice

Behavioral assessments using the T-maze and NOR tests demonstrated significant cognitive deficits in both AD and AD + CCH groups compared to the WT group (Figure 1(b)–(d); p < 0.05, p < 0.01, p < 0.001). Mice in the AD + CCH group exhibited longer latency times and reduced correct response rates relative to the AD group at both 9 and 12 months of age (p < 0.05). NSCs transplantation in AD + CCH mice resulted in significantly improved behavioral performance, characterized by decreased latency, increased correct choice rate, and higher recognition index (p < 0.05). iPSCs treatment did not result in statistically significant differences compared to the AD + CCH group. Across all experimental conditions except WT, 12-month-old mice exhibited significantly increased latency and decreased cognitive accuracy and recognition compared to their 9-month-old counterparts (p < 0.05).

Nissl staining demonstrated a significant reduction in Nissl-positive neurons within the motor cortex across all AD-related groups relative to WT mice at both 9 and 12 months (Figure 1(e) and (f); p < 0.05, p < 0.01, p < 0.001). The loss of neurons was most pronounced in the AD + CCH group and was further exacerbated with age. NSCs transplantation partially restored neuronal density (p < 0.05), whereas iPSCs administration produced no significant effect.

Western blot analysis was used to evaluate expression levels of autophagy-related marker LC3-II, apoptosis-related cleaved caspase-3, and pyroptosis-associated protein NLRP3 (Figure 1(g)). Expression of LC3-II (Figure 1(h)), cleaved caspase-3 (Figure 1(i)), and NLRP3 (Figure 1(j)) was significantly upregulated in the AD and AD + CCH groups compared to the WT group at both 9 and 12 months (p < 0.05, p < 0.01, p < 0.001), with greater increases observed in the AD + CCH group (p < 0.05). NSCs treatment significantly reduced the expression of all three markers compared to AD + CCH, while iPSCs treatment showed no significant changes. Protein levels of LC3-II, cleaved caspase-3, and NLRP3 were also significantly higher in 12-month-old mice than in those at 9 months of age (Figure 1(g)–(j); p < 0.05).

Immunofluorescence double staining of NLRP3 and NeuN in the CTX further confirmed the protein-level findings (Figure 1(k)–(m)). Increased co-localization of NLRP3 with NeuN-positive neurons was observed in both AD and AD + CCH groups, with reduced NLRP3 positive cells in the NSCs-treated group.

Expression of M1 microglial polarization-related markers

Western blot analysis demonstrated significant upregulation of pro-inflammatory M1 microglial markers, including CD16/32, IL-1β, IL-6, and TNF-α, in both the AD and AD + CCH groups compared to the WT group (Figure 2(a)–(e); p < 0.05, p < 0.01, p < 0.001). Expression levels of all markers were further elevated in the AD + CCH group relative to the AD group alone (p < 0.05). NSCs transplantation significantly reduced the expression of CD16/32, IL-1β, IL-6, and TNF-α in comparison to the AD + CCH group (p < 0.05). No statistically significant differences in M1 marker expression were observed between the iPSCs-treated and AD + CCH groups.

Age-related analysis revealed that, across all experimental groups, 12-month-old mice exhibited significantly higher expression levels of CD16/32, IL-1β, IL-6, and TNF-α than their 9-month-old counterparts (Figure 2(a)–(e); p < 0.05). Immunofluorescence staining of CD16/32 in the CTX and HI confirmed the Western blot results (Figure 2(f)–(m)). Confocal imaging showed extensive accumulation of CD16/32-positive microglia surrounding Aβ40-positive amyloid plaques, with the highest density observed in the AD + CCH group. The signal intensity and periplaque distribution were more prominent in 12-month-old mice compared to the 9-month-old cohort.

Expression of M2 microglial polarization-associated proteins

Western blot analysis showed that expression levels of M2 microglial polarization markers, including CD163, IL-4, and IL-10, were significantly increased in the AD group compared to the WT group (Figure 3(a)–(d); p < 0.05, p < 0.01, p < 0.001). In the AD + CCH group, expression of CD163, IL-4, and IL-10 was significantly reduced compared to the AD group (p < 0.05). NSCs transplantation resulted in a significant increase in the expression of these proteins compared to the AD + CCH group (p < 0.05). No significant differences in CD163, IL-4, or IL-10 expression were observed between the iPSCs-treated and AD + CCH groups.

Age-dependent analysis revealed that levels of CD163, IL-4, and IL-10 were significantly higher in 12- than in 9-month-old mice across AD-related groups (Figure 3(a)–(d); p < 0.05). Immunofluorescence staining for CD163 in the CTX and HI confirmed the protein-level findings (Figure 3(e)–(k)). CD163⁺ microglial cells were observed in increased numbers surrounding Aβ-positive plaques, with a higher density detected in the NSCs-treated group (Figure 3(e)–(h)) compared to other AD-related groups.

Effects of stem cell therapy on neuroinflammatory marker expression

Western blot analysis revealed significant differences in the expression of neuroinflammatory markers across experimental groups (Figure 4(a)). Levels of APP, GFAP, and Iba1 were significantly increased in both the AD and AD + CCH groups compared to the WT group (Figure 4(a)–(d); p < 0.05, p < 0.01, p < 0.001). The AD + CCH group exhibited further upregulation of these markers relative to the AD group (p < 0.05). NSCs transplantation significantly reduced APP, GFAP, and Iba1 expression levels compared to the AD + CCH group (p < 0.05), while iPSCs treatment did not produce statistically significant changes. Across all AD-related groups, expression of APP, GFAP, and Iba1 was significantly higher at 12 months than at 9 months (p < 0.05; Figure 4(a)–(d)).

Double immunofluorescence staining confirmed these protein-level findings. In the CTX and HI, increased accumulation of GFAP⁺ astrocytes (Figure 5(a)–(d)) and Iba1⁺ microglia (Figure 5(i)–(l)) was observed surrounding Aβ40-positive plaques in the AD and AD + CCH groups, with more prominent periplaque glial responses in 12-month-old AD + CCH mice. NSCs transplantation resulted in a notable reduction in Aβ plaque burden (Figure 5(e), (f), (m), and (n)) and a decrease in the density of GFAP⁺ (Figure 5(g) and (h)) and Iba1⁺ (Figure 5(o) and (p)) cells in the periplaque regions compared to the iPSCs-treated group.

Effects of stem cell therapy on the NSCs niche and neurotrophic support in the SVZ

Immunofluorescence analysis was conducted to evaluate the cellular composition of the SVZ using markers for proliferating cells (Ki67), immature neurons (DCX), endothelial cells (CD31), astrocytes (GFAP), and microglia (Iba1) in 9- and 12-month-old mice (Figure 6(a)–(h)). All AD-related groups exhibited significantly increased numbers of Ki67⁺, DCX⁺, and CD31⁺ cells in the SVZ compared to the WT group (p < 0.05), with no significant differences detected among the AD, AD + CCH, NSCs-treated, and iPSCs-treated groups. GFAP⁺ and Iba1⁺ cell densities were also elevated in all AD-related groups relative to the WT group.

Quantitative fluorescence intensity measurements confirmed these findings (Figure 6(i)–(m)). Ki-67, DCX, and CD31 fluorescence intensities were significantly higher in all AD-related groups compared to the WT group (p < 0.05). GFAP and Iba1 fluorescence signals were similarly elevated. No statistically significant differences were observed between the 9- and 12-month-old groups for any of the measured markers.

Effects of stem cell therapy on the expression of BDNF, VEGF, VAChT, and PSD95 in the AD + CCH brain

Western blot analysis revealed marked alterations in neurotrophic and synaptic protein expression across experimental groups at 9 and 12 months (Figure 7(a)). BDNF levels were significantly elevated in all AD-related groups compared with WT controls (Figure 7(b); p < 0.01, p < 0.001), with the highest expression observed in the NSCs-treated group (p < 0.05 vs AD + CCH). No significant difference was detected between the iPSCs-treated and AD + CCH groups. Across all conditions, BDNF expression was higher at 12 than at 9 months (p < 0.05).

Similarly, VEGF and PSD95 levels were significantly upregulated in AD, AD + CCH, NSCs-, and iPSCs-treated mice compared with WT (Figure 7(c) and (e); p < 0.05, p < 0.01, p < 0.001). NSCs transplantation further enhanced VEGF and PSD95 expression relative to AD + CCH (p < 0.05), while iPSCs treatment produced no significant change. Both VEGF and PSD95 levels were consistently higher in 12-month-old mice compared to 9-month-old counterparts (p < 0.05).

In contrast, VAChT expression was significantly reduced in all AD-related groups relative to WT at both time points (Figure 7(d); p < 0.05, p < 0.01, p < 0.001), with the most pronounced reduction observed in the AD + CCH group. NSCs transplantation partially restored VAChT expression (p < 0.05), whereas iPSCs administration did not significantly affect its levels. VAChT expression further declined with aging across all AD-related groups (p < 0.05).

Schematic summary of the therapeutic mechanisms of NSCs in the AD + CCH model

A mechanistic summary of the observed cellular and molecular alterations is illustrated in Figure 7(f). CCH acts as a compounding factor in AD, driving excessive autophagy (via LC3-II upregulation), apoptosis (cleaved caspase-3), and pyroptosis (NLRP3), thereby contributing to progressive neuronal loss. Concurrently, CCH exacerbates microglial polarization toward the M1 phenotype, leading to increased production of pro-inflammatory mediators such as CD16/32, IL-1β, IL-6, and TNF-α, which perpetuate neuroinflammation and tissue damage. NSC therapy exerts multifaceted neuroprotective effects by downregulating these cell death pathways, suppressing M1 polarization, and promoting a shift toward the anti-inflammatory M2 phenotype characterized by elevated IL-4, IL-10, and CD163.

Discussion

This study delineated the exacerbating effects of CCH on AD pathology and, for the first time, provided a direct comparison of NSCs and iPSCs transplantation in a combined AD + CCH mouse model. CCH markedly aggravated cognitive dysfunction and neuronal loss in AD mice (Figure 1), accompanied by enhanced autophagic, apoptotic, and pyroptotic activity, as reflected by increased LC3-II, cleaved caspase-3, and NLRP3 expression. Concurrently, microglial activation shifted toward a pro-inflammatory M1 phenotype (Figure 2), with concomitant suppression of M2 markers (CD163, IL-4, IL-10; Figure 3) and neurotrophic factors, including BDNF and VEGF (Figure 7). Astroglial reactivity and Aβ-associated gliosis were also intensified (Figures 4 and 5), and SVZ niche organization was altered (Figure 6). NSCs transplantation effectively mitigated these pathological alterations by improving behavioral performance, reducing neuronal stress and glial activation, restoring M2 polarization, and upregulating neurotrophic and synaptic proteins such as BDNF, VEGF, and PSD95. In contrast, iPSCs transplantation failed to induce comparable neuroprotective or reparative effects. Collectively, these findings demonstrate that NSCs, but not iPSCs, confer multifaceted protection against AD + CCH-induced neurodegeneration by modulating inflammatory, neurotrophic, and synaptic pathways, providing new experimental evidence for stem cell-based interventions targeting mixed dementia pathology.

AD and CCH are two converging pathophysiological processes that frequently coexist in aging populations, compounding cognitive decline through shared molecular and cellular mechanisms.²¹ CCH has been implicated as a critical contributor to mixed dementia, not merely as a comorbidity, but as an active driver that accelerates the onset and progression of AD pathology.²² Accumulating evidence supports that chronic hypoperfusion can amplify amyloidogenic processes, impair clearance of misfolded proteins, and disrupt neurovascular coupling, thereby promoting synaptic failure and neuronal loss. In our previous work, we demonstrated that CCH leads to structural disintegration of the NVU, BBB leakage, mitochondrial impaired energy metabolism,^6,23 which in turn compromise cerebral homeostasis and neuronal viability in AD-prone models. These findings are consistent with the concept that CCH acts as a sensitizer for neurodegeneration, weakening the intrinsic neuroprotective architecture and creating a permissive environment for toxic protein accumulation and inflammatory priming.²⁴ In the current study, combining APP/PS1 double-transgenic mice with gradual bilateral common carotid artery stenosis produced more severe cognitive deficits than AD pathology alone, as measured by T-maze and novel object recognition performance (Figure 1). The deterioration was observed at both 9 and 12 months, with further worsening over time, highlighting not only the additive burden of vascular insufficiency but also the temporal vulnerability of the aged brain. This aligns with prior studies demonstrating that hypoperfusion-induced white matter rarefaction, oxidative stress, and reduced interstitial fluid clearance can accelerate amyloid deposition and tau phosphorylation.²⁵ Importantly, these changes form a self-perpetuating cycle in which vascular dysfunction aggravates amyloid pathology, which in turn exacerbates endothelial and glial reactivity, further compromising perfusion and barrier function.²⁶

At the mechanistic level, this study provides direct evidence of the simultaneous engagement of autophagy (LC3-II), apoptosis (cleaved caspase-3), and pyroptosis (NLRP3) in the AD + CCH mice (Figure 1), reflecting a multifaceted and interlinked neuronal stress response. While autophagy is initially neuroprotective, chronic activation without effective clearance may result in autophagic flux blockage, leading to cellular collapse and transition to apoptotic or pyroptotic pathways.²⁷ Recent studies have demonstrated that NLRP3 inflammasome activation is not only downstream of lysosomal stress but can also inhibit autophagic degradation by sequestering key autophagy regulators.²⁸ Furthermore, caspase-3-mediated apoptosis is frequently observed in neurons with mitochondrial dysfunction, which is a hallmark of both AD and hypoperfusion models.²⁹ The co-occurrence of these death pathways, particularly in aged animals, supports the notion that compensatory survival mechanisms become exhausted in the face of chronic metabolic and inflammatory insults. In sum, our findings highlight a pathological convergence between neurodegenerative and vascular mechanisms, where CCH serves as both a trigger and amplifier of AD-relevant cascades.³⁰ This model recapitulates key clinical features of mixed dementia and provides a relevant platform for evaluating therapies that target overlapping neurovascular and neuroimmune axes.

Microglial activation represents a central feature in the pathogenesis of both AD and CCH, with polarization toward pro-inflammatory or anti-inflammatory phenotypes dictating the trajectory of neuroimmune responses.³¹ In the present study, a pronounced shift toward the M1 phenotype was observed in both AD and AD + CCH mice, as evidenced by increased expression of CD16/32, IL-1β, IL-6, and TNF-α (Figure 2). These pro-inflammatory markers were further elevated in the AD + CCH group, highlighting the potentiating effect of vascular insufficiency on microglial-mediated neuroinflammation.³² Conversely, the expression of M2-associated markers, including CD163, IL-4, and IL-10 (Figure 3), was significantly reduced in AD + CCH mice compared to AD alone, indicating impaired compensatory or repair-oriented microglial functions.³³ These findings are in line with recent reports showing that chronic hypoperfusion enhances M1 polarization while suppressing anti-inflammatory microglial signaling, thereby creating a persistently neurotoxic milieu.³⁴ In AD models, Aβ accumulation has been shown to activate pattern recognition receptors on microglia, including TLR4 and NLRP3, leading to cytokine production and inflammasome assembly.^35,36 Our immunofluorescence data further revealed that M1 marker CD16/32⁺ microglia were localized around Aβ40 plaques in the cortex and hippocampus, with higher densities in the AD + CCH group, suggesting enhanced plaque-associated microglial recruitment under vascular stress. This spatial association has been linked to impaired Aβ clearance, excessive synaptic pruning, and neurotoxic cytokine release.

In parallel with microglial activation, astrogliosis was observed in all AD-related groups and was particularly prominent in the AD + CCH condition. Increased GFAP expression and accumulation of reactive astrocytes around amyloid plaques suggest a coordinated glial response to chronic neuroinflammation and neuronal injury.³⁷ Astrocytes, while playing essential roles in blood–brain barrier maintenance and metabolic support, can also adopt a reactive phenotype under pathological conditions, secreting inflammatory mediators and forming glial scars that restrict tissue repair.³⁸ The synergistic activation of both astrocytes and microglia likely contributes to the amplification of cytokine signaling, ROS production, and excitotoxicity, particularly in aged animals where glial priming is more pronounced.³⁹ Together, these results illustrate that the inflammatory landscape in the AD + CCH brain is characterized by a skewed microglial M1/M2 balance, sustained astroglial activation, and reinforced glial cross-talk, all of which converge to exacerbate neurodegeneration.⁸ Therapeutic strategies aimed at rebalancing microglial phenotypes and attenuating glial reactivity may therefore hold promise in the context of mixed neurodegenerative-vascular dementia.

Stem cell-based therapy has emerged as a promising approach for neurodegenerative diseases, not only by replacing lost neurons but more importantly by exerting paracrine, immunomodulatory, and trophic effects that reshape the neuroinflammatory microenvironment.⁴⁰ In this study, transplantation of NSCs into the lateral ventricles of AD + CCH mice significantly ameliorated cognitive deficits, reduced the expression of cell death-associated markers (LC3-II, cleaved caspase-3, and NLRP3; Figure 1), suppressed glial activation, and rebalanced microglial polarization toward an anti-inflammatory M2 phenotype (Figure 3). In contrast, transplantation of iPSCs did not confer comparable neuroprotective or anti-inflammatory benefits. These findings are consistent with prior reports demonstrating that NSCs can modulate host immune responses, attenuate glial reactivity, and enhance neuronal survival through the release of anti-inflammatory cytokines, neurotrophic factors, and extracellular vesicles.⁴¹ In particular, NSCs-derived secretomes contain bioactive molecules such as BDNF, GDNF, IGF-1, and IL-10, which can act synergistically to reduce microglial activation, restore synaptic function, and inhibit apoptosis and inflammasome activation.⁴² Our results showing simultaneous downregulation of LC3-II, cleaved caspase-3, and NLRP3 suggest that NSCs transplantation modulates multiple regulated cell death pathways—autophagy, apoptosis, and pyroptosis—through a coordinated cellular stress-sensing mechanism. These effects are especially relevant in the context of AD + CCH, where chronic hypoxia, metabolic insufficiency, and neuroinflammation converge to drive neuronal loss.

The ineffectiveness of iPSC transplantation in this model may be attributed to several factors, including limited immunoregulatory competence, absence of lineage specification toward neural/glial fates in vivo, or insufficient secretion of reparative factors.⁴³ Previous studies have reported that undifferentiated iPSCs may trigger host immune activation or tumorigenic transformation when not appropriately preconditioned.⁴⁴ Additionally, NSCs possess innate homing capabilities toward injured brain regions and can integrate transiently into the neurovascular niche, thereby enhancing local trophic support and immune quiescence.^45,46 Importantly, the beneficial effects of NSCs were evident at both behavioral and molecular levels, with reductions in glial activation and periplaque inflammatory cell accumulation, and increases in M2 markers and neurotrophic signaling.⁴⁷ These results provide compelling evidence that NSC transplantation promotes a shift from a neurotoxic to a reparative brain microenvironment, supporting functional recovery in the context of combined neurodegenerative and vascular pathology.

Neurotrophic factor deficiency is a well-established feature of AD, contributing to synaptic dysfunction, impaired neurogenesis, and cognitive decline.^48,49 BDNF, in particular, plays a crucial role in neuronal survival, dendritic plasticity, and the formation of long-term memory.⁵⁰ In the present study, the transplantation of NSCs into the lateral ventricle resulted in a marked increase in BDNF expression (Figure 7). which was in accordance with previous studies reporting that NSCs grafts can upregulate endogenous BDNF levels through both direct secretion and indirect stimulation of host cells.⁵¹ Notably, BDNF has been shown to facilitate migration and differentiation of neural progenitors, promote synaptic integration, and counteract Aβ-mediated toxicity.⁵² It also modulates astrocytic and microglial responses by acting on TrkB and p75NTR receptors, thereby integrating neurotrophic and immune pathways.⁵³ Importantly, the failure of iPSC transplantation to enhance BDNF expression further underscores the differential therapeutic capacity of stem cell types in this mixed pathology model.¹⁴ Taken together, these results suggest that NSCs promote neurological recovery in the AD + CCH brain, likely through a combination of trophic factor support, anti-inflammatory effects, and niche-specific microenvironmental modulation.

In addition, we assessed synaptic integrity, cholinergic neurotransmission, and angiogenic signaling by analyzing the expression of PSD95, VAChT, and VEGF, respectively (Figure 7). Western blot analysis demonstrated that VEGF and PSD95 levels were significantly elevated in all AD-related groups compared with WT controls at both 9 and 12 months, reflecting a potential compensatory upregulation of synaptic and vascular plasticity in response to chronic neurodegenerative stress. Notably, NSCs transplantation further augmented VEGF and PSD95 expression relative to the AD + CCH group, indicating that NSCs therapy promotes synaptic stabilization and angiogenic support beyond the intrinsic compensatory mechanisms. VAChT expression, a marker of cholinergic neurotransmission, was similarly upregulated following NSCs transplantation, suggesting restoration of cholinergic function, which is critically impaired in AD + CCH pathology. These neurorestorative effects likely involve paracrine mechanisms, including the release of neurotrophic and angiogenic factors such as BDNF and VEGF, which facilitate synaptic repair and neurovascular remodeling.^54
–56 In contrast, iPSCs treatment elicited minimal changes in these markers, highlighting the limited reparative potential of undifferentiated pluripotent stem cells within the pathological brain microenvironment. Collectively, NSCs transplantation exerts multifaceted neuroprotective and neurorestorative actions in the AD + CCH brain, encompassing synaptic preservation, cholinergic system enhancement, and angiogenic support.

Study limitations and future directions

Despite the comprehensive nature of the current investigation, several limitations should be acknowledged. First, although the study demonstrated functional and molecular benefits of NSCs transplantation, the fate, integration, and longevity of the grafted cells were not tracked. As NSCs may exert effects via transient paracrine signaling or longer-term structural integration, future studies employing lineage tracing, reporter labeling, or single-cell transcriptomics are warranted to delineate the dynamics and cellular targets of NSC action in vivo. Second, the iPSCs treatment group received undifferentiated iPSCs without preconditioning. Given the risk of teratoma formation and limited differentiation efficiency in vivo, future studies should consider the use of lineage-committed iPSC-derived neural progenitor cells (iPSC–NPCs) or astrocyte-restricted precursors, which may exhibit improved safety and efficacy profiles. Third, this study focused exclusively on male animals to control for sex hormone effects. However, both AD and vascular dementia exhibit sex-specific differences in incidence, progression, and treatment response. Future experiments incorporating female cohorts will be critical for validating the generalizability and translational relevance of stem cell therapies in mixed dementia. Lastly, long-term outcomes beyond 12 months were not assessed, limiting the understanding of whether the observed effects are sustained or transient. Extending the follow-up period, combining cell therapy with pharmacological modulation of inflammatory or neurotrophic pathways, and applying advanced neuroimaging to monitor in vivo changes would provide deeper mechanistic insights and inform clinical translation.

Conclusions

This study delineates an integrated pathological and therapeutic framework for AD with CCH, a condition that closely mirrors the neurovascular comorbidity observed in clinical dementia. The coexistence of AD pathology and CCH synergistically aggravated cognitive decline, neuronal loss, M1 microglial polarization, astroglial activation, and dysregulation of autophagic, apoptotic, and pyroptotic signaling, together with suppression of neurotrophic and synaptic proteins such as BDNF, VEGF, VAChT, and PSD95. These pathological alterations were further intensified with aging, emphasizing the progressive nature of neurovascular-driven neurodegeneration.

For the first time, this study provides a direct comparison of NSCs and iPSCs in a mixed AD + CCH model. NSC transplantation markedly ameliorated behavioral deficits, enhanced neuronal survival, attenuated glial reactivity, rebalanced microglial polarization, and restored neurotrophic and synaptic protein expression, whereas iPSC transplantation failed to produce comparable effects. Collectively, these findings demonstrate that NSCs exert broad-spectrum neuroprotective and neurorestorative actions across inflammatory, trophic, and synaptic pathways. NSC-based therapy therefore represents a promising candidate for mitigating mixed Alzheimer’s and vascular cognitive impairment. Future investigations should optimize cell characterization, delivery precision, and long-term integration to advance translational applicability in neurovascular dementia.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X251400239 – Supplemental material for Neural stem cell transplantation attenuates cognitive decline and neuroinflammation in a mouse model of Alzheimer’s disease with chronic cerebral hypoperfusion

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X251400239 for Neural stem cell transplantation attenuates cognitive decline and neuroinflammation in a mouse model of Alzheimer’s disease with chronic cerebral hypoperfusion by Zhicheng Lu, Hong Zhou, Haibo Tang, Shiyu Luo, Jie Wang, Xingwu Zheng, Yuanhong Hu, Qiuyan Qin, Chengmin Yang, Shenglong Mo, Xiaorui Huang, Lina Tang, Bing Huang, Weishan Xu, Jingtang Nong, Guangqing Gan, Donghui Qin, Zhao Peng, Chongdong Jian, Xia Liu, Xuebin Li and Jingwei Shang in Journal of Cerebral Blood Flow & Metabolism

Footnotes

Acknowledgements

The authors gratefully acknowledge Professor Jinsong Li of the State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences for kindly providing the induced pluripotent stem cells (iPSCs) used in this study.

Abbreviations used

Alzheimer’s disease (AD), ameroid constrictors (ACs), β-amyloid (Aβ), autophagy-related protein LC3-II (LC3-II), blood–brain barrier (BBB), brain-derived neurotrophic factor (BDNF), chronic cerebral hypoperfusion (CCH), common carotid artery (CCA), cortex (CTX), doublecortin (DCX), glial fibrillary acidic protein (GFAP), hippocampus (HI), induced pluripotent stem cells (iPSCs), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), microtubule-associated protein β-tubulin (β-tubulin), neural stem cells (NSCs), neurovascular unit (NVU), novel object recognition (NOR), NOD-like receptor family pyrin domain-containing 3 (NLRP3), programmed cell death protein 3 (caspase-3), subventricular zone (SVZ), T-maze test (T-maze), tumor necrosis factor-α (TNF-α), wild-type (WT).

Author contributions

All authors had full access to the data in the study and take responsibility for the integrity and accuracy of the data analysis. Contributions are listed according to the CRediT taxonomy: conceptualization: Jingwei Shang, Xuebin Li; methodology: Jingwei Shang, Xuebin Li, Zhicheng Lu, Chengmin Yang; investigation: Zhicheng Lu, Hong Zhou, Haibo Tang, Shiyu Luo, Chengmin Yang, Xingwu Zheng, Yuanhong Hu, Lina Tang, Xiaorui Huang; formal analysis: Zhicheng Lu, Chengmin Yang, Shenglong Mo, Bing Huang, Weishan Xu, Jingtang Nong, Guangqing Gan, Donghui Qin, Zhao Peng, Lina Tang; visualization: Zhicheng Lu, Chengmin Yang, Lina Tang, Xia Liu; resources: Hong Zhou, Haibo Tang, Xingwu Zheng, Yuanhong Hu, Xiaorui Huang; writing—original draft: Jingwei Shang, Chongdong Jian; writing—review and editing: Jingwei Shang, Jie Wang; supervision: Jingwei Shang, Xuebin Li; funding acquisition: Jingwei Shang, Zhicheng Lu.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research received support from the National Natural Science Foundation of China (grant no. 82160254), the Baise City Scientific Research and Technology Development Program (grant no. Baike 20224117), the High-level Talent Scientific Research Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities (grant no. R20213001), and Innovation Project of Guangxi Graduate Education (grant no. YCSW2024527).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ORCID iD

Jingwei Shang

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. All data generated or analyzed during this study, including raw behavioral scores, Western blot images, and immunofluorescence quantifications, are stored in secure institutional repositories and will be shared in compliance with ethical and institutional guidelines.

Supplemental material

Supplemental material for this article is available online.

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