Human umbilical cord blood mononuclear cells ameliorate vascular dementia by modulating microglial myelin debris handling and white matter injury

Abstract

Vascular dementia (VaD), characterized by white matter damage and cognitive decline, currently lacks effective therapeutic options. Human umbilical cord blood mononuclear cells (hUCB-MNCs) have shown neuroprotective and immunomodulatory properties; however, their therapeutic efficacy and underlying mechanisms in VaD remain incompletely understood. In this study, we investigated the effects of hUCB-MNCs treatment in a mouse model of VaD induced by bilateral common carotid artery stenosis (BCAS). Behavioral assessments showed that hUCB-MNCs treatment improved cognitive performance, affective-like behaviors, and motor coordination in BCAS mice. Histopathological analyses demonstrated that hUCB-MNCs treatment attenuated white matter injury, preserved myelin integrity, and mitigated neuronal and synaptic damage. Integrated transcriptomic and proteomic analyses of corpus callosum (CC) tissues revealed enrichment of immune-regulatory, phagocytosis-related, and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT)-associated pathways after hUCB-MNCs treatment. In vivo and in vitro analyses further indicated that hUCB-MNCs helped preserve microglial homeostatic features and improved myelin debris-handling responses. Collectively, these findings suggest that hUCB-MNCs ameliorate VaD-associated pathology, at least in part, by modulating microglial myelin debris-handling responses and PI3K/AKT-related signaling, highlighting hUCB-MNCs as a promising cell-based therapeutic candidate for VaD.

Graphical Abstract

hUCB-MNCs treatment improves microglial myelin debris handling in BCAS-induced VaD. BCAS-induced chronic cerebral hypoperfusion causes white matter injury, myelin debris accumulation, and dysregulated microglial responses. hUCB-MNCs treatment helps restore PI3K/AKT-related activity, improves microglial myelin debris-handling responses, and reduces white matter injury in VaD.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12974-026-03929-6.

Introduction

Vascular dementia (VaD), the second most common form of dementia after Alzheimer’s disease (AD), is characterized by cognitive decline and white matter damage resulting primarily from chronic cerebral hypoperfusion [1]. Sustained hypoperfusion contributes to demyelination, axonal degeneration, and neuronal loss, collectively impairing cognitive domains such as memory, attention, and executive function [2]. Although considerable progress has been made in elucidating the pathophysiology of VaD, effective disease-modifying therapies are still lacking. Thus, there is an urgent need to develop novel therapeutic strategies targeting the underlying pathological mechanisms.

Microglia, the resident immune cells of the central nervous system, play essential roles in maintaining brain homeostasis, sensing tissue injury, removing cellular debris, and coordinating inflammatory responses [3]. In VaD, chronic cerebral hypoperfusion-induced white matter injury is accompanied by myelin disruption and the accumulation of damaged myelin fragments [4]. Inefficient clearance of myelin debris may further aggravate white matter pathology by sustaining neuroinflammation, impairing remyelination, and disrupting neuronal network function [5]. As the major phagocytic cells in the brain, microglia are critically involved in the recognition, engulfment, and processing of myelin debris [6]. However, under chronic hypoperfusion and persistent inflammatory stress, microglial phagocytic and lysosomal functions may become dysregulated, thereby limiting effective debris clearance and tissue repair [7]. Thus, restoring microglial debris-clearing functions may represent a potential therapeutic approach for VaD.

Human umbilical cord blood (hUCB) represents an accessible and ethically acceptable source of hematopoietic and progenitor cells, with well-established immunomodulatory, anti-inflammatory, and neuroprotective properties [8]. Among hUCB-derived therapies, human umbilical cord blood mononuclear cells (hUCB-MNCs) has been extensively studied in both preclinical and clinical settings for a variety of conditions, including hematologic malignancies, ischemic stroke, and traumatic brain injury [9–11]. Their low immunogenicity, abundant availability, and non-invasive collection further support their translational potential [12]. Previous studies of hUCB-based therapies in neurological disorders have mainly focused on neurogenesis, angiogenesis, inflammation, and trophic support [13, 14]. However, whether hUCB-MNCs can ameliorate chronic hypoperfusion-induced cognitive impairment and white matter injury in VaD remains unclear. In particular, the potential effects of hUCB-MNCs on microglial function and myelin debris clearance have not been fully elucidated.

In the present study, we used a bilateral common carotid artery stenosis (BCAS)-induced mouse model of VaD, together with oxygen-glucose deprivation (OGD)-exposed BV2 cells and primary microglia, to evaluate the therapeutic effects of hUCB-MNCs on chronic hypoperfusion-associated cognitive impairment and white matter injury. We further investigated whether hUCB-MNCs modulate microglial myelin debris-handling responses and explored the potential regulatory mechanisms underlying this process. This study aimed to assess the therapeutic potential of hUCB-MNCs in VaD and to determine whether regulation of microglia-mediated myelin debris clearance may contribute to their protective effects on white matter integrity.

Methods

Animals

Male and female C57BL/6J mice (8–9 weeks old, 20–22 g) were obtained from Zhuhai BesTest Bio-Tech Co., Ltd. and housed in a temperature-controlled environment (22 ± 2 °C) with a 12-hour light/dark cycle and free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jinan University and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals.

Total of 102 adult C57BL/6J mice of both sexes were used for the in vivo experiments. According to the requirements of different assays, adult mice were allocated to separate experimental cohorts and randomly assigned to the corresponding treatment groups within each cohort using a random number method. Male mice were used primarily for cognitive behavioral assessments, histopathological analyses, and mechanistic studies, whereas female mice were included in additional validation cohorts for selected behavioral and histopathological endpoints. Detailed animal numbers and sex distribution for each experiment are provided in the corresponding figures and figure legends. In addition, 45 neonatal C57BL/6J mice at postnatal days 2–3 were used exclusively for primary microglia isolation. These neonatal mice were not included in the adult BCAS model, behavioral assessments, or histological analyses. Not all adult animals underwent the same behavioral and histological assessments. To minimize the potential influence of repeated behavioral stress and to meet the requirements of different downstream analyses, mice were assigned to separate experimental cohorts for behavioral testing, frozen sections, paraffin sections, Golgi staining, electron microscopy, and molecular assays.

Animal model preparation

The BCAS model was established as previously described [15]. Briefly, mice were anesthetized with isoflurane (RWD Life Science, China) using an induction concentration of 3% at a flow rate of 1 L/min, followed by maintenance at 1–1.5% at a flow rate of 0.5 L/min. After exposure of one common carotid artery, a microcoil with an inner diameter of 0.18 mm (Sawane Spring Co., Ltd., Japan) was carefully placed around the artery. After 30 min, the contralateral common carotid artery was exposed and another microcoil was placed around it to induce chronic cerebral hypoperfusion. Sham-operated mice underwent the same surgical procedures except for microcoil placement. All surgeries were performed by the same investigator. All animals survived the surgical procedures and completed the planned experimental protocols.

hUCB-MNCs administration

hUCB was collected by the Guangdong Cord Blood Bank and processed in accordance with its standardized protocols [16]. hUCB-MNCs were subsequently isolated and cryopreserved in liquid nitrogen until use.

The hUCB-MNCs used for in vivo treatment were derived from multiple independent donors. To maintain consistency within each animal, all repeated tail-vein injections in the same mouse were performed using hUCB-MNCs from the same donor. Four weeks after BCAS surgery, mice in the hUCB-MNCs treatment group received slow intravenous injections of 200 µL hUCB-MNCs suspension containing 1 × 10⁶ cells via the tail vein once per week for four consecutive weeks. Control mice received an equivalent volume of phosphate-buffered saline (PBS) following the same injection schedule.

Neurological behavioral assessments

To evaluate the effects of hUCB-MNCs intervention on neurological functional recovery following chronic cerebral hypoperfusion, behavioral assessments were initiated four weeks after completion of hUCB-MNCs treatment and completed within a 14-day period. Animals were assigned to separate behavioral cohorts, and not all mice underwent the same behavioral tests.

To minimize the potential influence of behavioral test-related stress on subsequent performance, behavioral assessments were generally arranged from lower to higher stress levels. In one cohort, mice first underwent the fatigue rotarod test, open field test (OFT), followed by Morris water maze (MWM) testing, with appropriate rest intervals between tests. In another cohort, mice sequentially underwent the fatigue rotarod test, OFT, novel object recognition (NOR) test, and tail suspension test (TST), with rest intervals between tests. Because the MWM and TST impose relatively greater stress on mice, both tests were performed at later stages of their respective behavioral testing schedules and were not conducted in the same animals. The experimental procedures were as follows:

NOR test was conducted to assess recognition memory [17]. Mice were first habituated to the test arena for 10 min. After 1 h, they were exposed to two identical objects for 10 min. Another 1 h later, one of the familiar objects was replaced with a novel object, and mice were allowed to explore for 10 min. The recognition index (RI) and discrimination index (DI) were calculated as follows: RI = time spent exploring the novel object / total exploration time. DI = (time spent exploring the novel object − time spent exploring the familiar object) / total exploration time.

MWM test was performed over six consecutive days to assess spatial learning and memory [18]. Mice were trained to locate a hidden platform in a circular water tank, and the escape latency (time to reach the platform) was recorded. On day 7, a probe trial was conducted by removing the platform, during which the time spent in the target quadrant and the number of platform crossings were measured.

OFT was used to assess exploratory behaviour and anxiety levels [19]. Mice were placed in a 40 × 40 × 40 cm open-field box and allowed to explore freely for 15 min. The movement trajectory was recorded, and the latency to enter the central area as well as the time spent in the central area were analyzed.

TST was performed to assess depression-like behaviour [20]. Mice were in a testing chamber using adhesive tape for 7 min, and their behaviour was recorded on video. Total immobility time from 2 to 7 min and longest immobility duration were analyzed using the DBscorer open-source software (https://www.eneuro.org/content/8/6/ENEURO.0305-21.2021).

The rotarod test was performed to evaluate motor coordination and balance, as previously described [21]. The experiment consisted of two phases: In the training phase, mice were placed on a rotating rod for 5 min, followed by a 10-minute rest before the next trial, repeated three times. After 24 h, the testing phase was conducted, where mice underwent three trials, each lasting a maximum of 5 min. During the test, the rotarod speed gradually increased from 4 r/min to 40 r/min. The following parameters were recorded: latency to fall (s), and rotational speed (r/min) at the time of falling.

Immunofluorescence (IF) staining of mouse brain sections

Mice were perfused with ice-cold PBS (Biosharp, China, BL601A), followed by fixation with 4% paraformaldehyde (PFA) (Biosharp, China, BL539A). After gradient sucrose dehydration, brain tissues were embedded in optimal cutting temperature (OCT, Epredia, 6502) compound and coronally sectioned at 10 μm and 30 μm thickness.

Sections were incubated at 37 °C for 15 min and then permeabilized and rehydrated in PBS containing 0.3% Triton X-100 (Sangon Biotech, China, A110694) (PBST) for three washes of 5 min each. Blocking was performed for 1 h at room temperature using blocking buffer containing 3% bovine serum albumin (BSA, BioFroxx, 4240), 10% goat serum (Beyotime, C0265), and 1% Triton X-100 in PBS. Sections were then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-MBP (1:50, Proteintech, China, 10458-1-AP), rabbit anti-dMBP (1:100, Millipore, USA, AB5864), mouse anti-NF200 (1:200, Sigma-Aldrich, USA, N0142), mouse anti-Iba1 (1:100, abcam, USA, ab283319), rabbit anti-Iba1 (1:500, Wako, Japan, 019-19741), rabbit anti-P21(1:200, abcam, USA, ab188224) and rabbit anti-TMEM119 (1:200, Proteintech, China, 27585-1-AP).

After washing in PBST (3 × 5 min), sections were incubated with Alexa Fluor–conjugated secondary antibodies and DAPI (1:1000, stock is 1 mg/mL, Yeasen, 40728ES03) at room temperature for 1 h in the dark. The secondary antibodies included Alexa Fluor 488–conjugated goat anti-rabbit IgG (Invitrogen, A11029), Alexa Fluor 546–conjugated goat anti-rabbit IgG (Invitrogen, A11008), Alexa Fluor 647–conjugated goat anti-rabbit IgG (abcam, ab150079), Alexa Fluor 488–conjugated goat anti-mouse IgG (Invitrogen, USA, A28175), Alexa Fluor 555–conjugated goat anti-mouse IgG (abcam, USA, ab150114), and Alexa Fluor 647–conjugated goat anti-mouse IgG (Invitrogen, USA, A21236). Sections were then washed, mounted with an anti-fade mounting medium (Servicebio, China, G1401) and coverslipped (CITOTEST, China, 80340 − 3610) for imaging. Images were acquired using a leica DMi8 confocal microscope. Fluorescence intensity was quantified using ImageJ 1.49v software, and Imaris 9.0.1 software was used for three-dimensional reconstruction and analysis where indicated.

LFB staining

Luxol Fast Blue (LFB) staining was performed to assess myelin integrity. Mice were perfused with ice-cold PBS (Biosharp, BL601A) followed by 4% PFA (Biosharp, BL539A) fixation, and 3 μm-thick paraffin-embedded coronal brain sections were prepared. After deparaffinization and rehydration through xylene and a graded ethanol series, the sections were incubated overnight at 60 °C in 0.1% LFB solution (Baiqiandu, China, B11003). Following a brief rinse in running tap water and 70% ethanol, the sections were differentiated in 0.05% lithium carbonate solution (APExBIO, USA, K2602) 5 min, then washed in water and counterstained with 0.1% eosin. The sections were then dehydrated in xylene and mounted with neutral resin. Myelin staining was imaged using a StrataFAXS automated slide scanning system (StrataFAXS, Vienna), and the severity of white matter lesions was graded as follows: Grade 0: Normal myelin, Grade 1: Disarrangement of nerve fibres, Grade 2: Formation of marked vacuoles, Grade 3: Disappearance of myelinated fibres [22].

Transmission Electron Microscopy (TEM)

TEM was performed on corpus callosum (CC) tissues, which were dissected and immediately immersed in TEM fixative (Servicebio, China, G1102) overnight at 4 °C. After rinsing in 0.1 M PBS for 3 × 15 min, the samples were post-fixed in 1% osmium tetroxide at room temperature for 2 h. Following another PBS wash (3 × 15 min), the tissues were dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%, 100%; 15 min per step). Samples were then infiltrated overnight with a 1:1 mixture of acetone and EPON 812 embedding resin, followed by full embedding. Ultrathin Sects.  (60–80 nm) were cut and mounted on copper grids, then stained with 2% aqueous uranyl acetate and lead citrate. Images were acquired using a TEM at 5000× and 10,000× magnification. Myelin thickness was quantified by calculating the g-ratio, defined as the ratio of the inner axonal diameter to the total outer diameter of the myelinated fibre. Morphometric analysis and image processing were performed using ImageJ software.

Golgi staining

Fresh mouse brains were fixed in Golgi-Cox fixative solution (Servicebio, China, G1069) for 24 h, then transferred into Golgi-Cox staining solution and incubated for 14 days at room temperature in the dark, with the staining solution replaced every 48 h. After staining, the brains were immersed in tissue processing solution (Servicebio, China, G1069-3) for 3 days at 4 °C in the dark. Brains were then sectioned into 60 μm-thick coronal slices using a vibratome. After rinsing with ultrapure water, slices were incubated with Golgi developer solution (Servicebio, China, G1069-2) for 30 min, washed again, and then coverslipped for imaging. Stained neurons were imaged using a digital slide scanner (Pannoramic DESK, Hungary, HISTECH). The dendritic structure was analyzed using Sholl analysis with the Sholl plugin in Fiji (ImageJ 1.54f). Dendritic spine density was quantified using the Skeletonize plugin, also in Fiji (ImageJ 1.54f).

Nissl staining

Mouse brains were paraffin-embedded and sectioned into 3 μm-thick coronal slices. The sections were heated at 60 °C for 2 h, then deparaffinized in xylene and rehydrated through a graded ethanol series. After rehydration, the sections were stained with Nissl staining solution (Servicebio, China, G1036) for 15 min, followed by rinsing in distilled water. The sections were then dehydrated in absolute ethanol, cleared in fresh xylene for 5 min, and coverslipped using neutral resin. Nissl-stained sections were observed under a light microscope to evaluate neuronal morphology and density in the hippocampal region.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from BV2 cells using TRIzol reagent (Sangon Biotech, China, B511311) according to the manufacturer’s instructions. RNA concentration and purity were determined using a spectrophotometer, and samples with acceptable A260/A280 ratios were used for subsequent analysis. Equal amounts of total RNA were reverse-transcribed into cDNA using a reverse transcription kit (TaKaRa, Japan, RR047A). qRT-PCR was performed using SYBR Green qPCR Master Mix (Applied Biosystems, USA, A25762) on an Applied Biosystems 7500 Real-Time PCR System according to the manufacturer’s protocol.

The relative mRNA expression levels of microglial inflammatory and reparative markers, including Cd86, MRC1, Nos2, Arg1, TNF, TGFB1, IL6, and IL10, were analyzed. Actb was used as the internal control. Relative expression levels were calculated using the 2^−ΔΔCt method. All reactions were performed in triplicate, and primer sequences are provided in Supplementary Table 1.

RNA sequencing (RNA-seq)

Three freshly isolated CC tissues from each group were immediately snap-frozen in liquid nitrogen and stored until further analysis. Total RNA was extracted using TRIzol (Sangon Biotech, China, B511311), and RNA quality was assessed by Nanodrop spectrophotometry and Agilent 4200 TapeStation for RNA integrity. To isolate mRNA, Oligo (dT) magnetic beads were used to enrich polyadenylated transcripts, leveraging the poly(A) tail feature of most eukaryotic mRNAs. Purified mRNA was fragmented using divalent cations under elevated temperature conditions. The first-strand cDNA was synthesized using random hexamer primers and first-strand synthesis enzyme mix, followed by second-strand cDNA synthesis using dNTPs and second-strand enzyme mix. The resulting double-stranded cDNA was purified, end-repaired, A-tailed, and ligated to sequencing adapters. Fragments of approximately 200–300 bp were selected and enriched by PCR amplification, followed by final purification to obtain sequencing-ready libraries. The quality of the libraries was assessed using Qubit 2.0 Fluorometer for quantification and Agilent 4200 TapeStation for insert size distribution. Libraries passing quality control were sequenced on an Illumina platform using the paired-end 150 bp (PE150) sequencing mode.

Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS)

Three freshly isolated CC tissues were immediately snap-frozen in liquid nitrogen for proteomic analysis. Protein samples were processed via denaturation, reduction, alkylation, trypsin digestion, and peptide desalting, followed by LC-MS/MS. Peptides were reconstituted in 0.1% formic acid (FA) and 200 ng of each sample was separated on an AUR3-15075C18 column (15 cm length, 75 μm inner diameter, 1.7 μm particle size, 120 Å pore size; IonOpticks) using a 30-minute gradient starting from 4% buffer B (80% acetonitrile with 0.1% FA), increasing to 28% in 25 min, then to 90% in 1.5 min, and maintained for 3.5 min. The flow rate was 300 nL/min and the column temperature was set at 50 °C. Data-independent acquisition (DIA) data were acquired in diaPASEF mode. A total of 24 × 25 Th precursor isolation windows were defined from m/z 400 to 1000. The MS1 cycle time was adapted by setting three repetitions in the 8-scan data-independent acquisition parallel accumulation–serial fragmentation (diaPASEF) scheme. During PASEF MS/MS acquisition, collision energy was linearly ramped as a function of ion mobility, from 59 eV at 1/K₀ = 1.6 Vs/cm² to 20 eV at 1/K₀ = 0.6 Vs/cm².

Western blotting

CC tissues were dissected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China, P10013C) supplemented with protease and phosphatase inhibitors (Beyotime, China, P1045). The tissues were homogenized on ice using a mechanical tissue grinder, followed by ultrasonication for 30 min at 4 °C. Lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, twice, and the supernatants were collected. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China, P0010). Equal amounts of protein (50 µg) were mixed with loading buffer (Beyotime, China, P0015), boiled for 10 min, and separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto 0.45 μm transfer membrane (Millipore, USA, IPVH00010). Membranes were blocked in 5% non-fat milk (Sangon Biotech, China, A600669) at room temperature for 1 h, followed by washing in Tris-buffered saline with Tween (TBST) buffer (Servicebio, China, G2150) for 3 × 10 min. Membranes were incubated overnight at 4 °C with the following primary antibodies: Rabbit anti-β-actin (1:1000, CST, USA, 4970), Rabbit anti-PI3K P100α (1:1000, CST, USA, 4249), Rabbit anti-AKT (1:1000, CST, USA, 4691), Rabbit anti-phospho-AKT (p-AKT) (1:1000, CST, USA, 13038). After washing in TBST (3 × 10 min), membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H + L) (1:5000, Yeasen, China, 33101ES60) at room temperature for 1 h. Immunoreactive bands were visualized using a Chemi XX9 imaging system (Syngene, UK), and band intensities were quantified using ImageJ 1.49v software. All target proteins were normalized to β-actin as a loading control.

BV2 cell culture and OGD treatment

BV2 microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA, C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Yeasen, China, 40130ES76) and 1% penicillin-streptomycin (P/S) (Gibco, USA, 15140-122) at 37 °C in a humidified incubator containing 5% CO₂. For OGD treatment, BV2 cells were washed and replaced with glucose-free DMEM (Procell, China, PM150270), followed by incubation in a hypoxic chamber containing 5% CO₂, and 95% N₂ for 4 h.

Primary microglia isolation, culture and OGD treatment

Primary microglia were isolated from neonatal C57BL/6J mice at postnatal days 2–3. Briefly, cerebral cortices were dissected and enzymatically digested with 0.125% trypsin in a 37 °C water bath for 15 min. The digestion was terminated by adding DMEM containing 10% FBS. The tissues were then gently dissociated by pipetting, and the resulting cell suspension was filtered through a 70-µm cell strainer and centrifuged at 1,000 rpm for 10 min. After removal of the supernatant, the cells were resuspended and seeded in DMEM/F12 medium supplemented with 20% FBS and 1% P/S for mixed glial culture. On day 5, the medium was replaced with DMEM/F12 containing 10% FBS and 1% P/S. Between days 12 and 15, primary microglia were collected by gentle shaking at 200 rpm and 37 °C for 2 h. The collected primary microglia were then replated for subsequent experiments.

Primary microglia were maintained in DMEM/F12 containing 10% FBS and 1% P/S at 37 °C in a humidified incubator with 5% CO₂. For OGD treatment, primary microglia were cultured in glucose-free DMEM and incubated in a hypoxic chamber containing 5% CO₂ and 95% N₂ for 4 h.

PI3K inhibitor, cytochalasin D, and annexin V treatments in microglial cells

To investigate the mechanisms underlying hUCB-MNC-mediated regulation of microglial phagocytosis, microglial cells were treated with the PI3K inhibitor LY294002 (Beyotime, China, S1737), the actin polymerization inhibitor Cytochalasin D (CytD) (APEXBIO, USA, B6645), or Annexin V (MCE, USA, HY-P702573). LY294002 was used to assess the involvement of the PI3K/AKT signaling pathway, while CytD was used to inhibit actin-dependent phagocytosis. Annexin V, which binds phosphatidylserine (PS), was used to block PS-mediated recognition and uptake of myelin debris.

Briefly, before OGD exposure, primary microglia were pretreated with 30 µM LY294002 for 1 h, followed by OGD exposure and/or treatment with hUCB-MNC-conditioned medium as indicated. For inhibition of actin-dependent phagocytosis, cells were pretreated with CytD at a final concentration of 10 µM for 1 h before the myelin debris uptake assay. For PS-blocking experiments, Annexin V was used to block PS-mediated recognition of myelin debris. Annexin V was reconstituted to a stock concentration of 100 µg/mL according to the manufacturer’s instructions. Fluorescently labeled myelin debris was prepared at 1 mg/mL and incubated with Annexin V at a final concentration of approximately 9 µg/mL in Annexin V binding buffer at 37 °C for 1 h. The Annexin V-treated myelin debris was then added to microglial cultures for the myelin debris uptake assay.

Myelin isolation, purification, and fluorescent labeling

Myelin was isolated and purified from mouse brain tissue using a modified discontinuous sucrose density-gradient centrifugation method. Briefly, fresh brain tissues were collected from 8-week-old C57BL/6J mice and homogenized in ice-cold 0.32 M sucrose buffer containing 10 mM HEPES (Beyotime, China, C0215) and 5 mM EDTA (Beyotime, China, ST063). The homogenate was then carefully layered over 0.85 M sucrose buffer containing 10 mM HEPES and 5 mM EDTA, followed by ultracentrifugation at 24,600 rpm for 30 min at 4 °C. After centrifugation, the crude myelin fraction at the interface between the two sucrose layers was carefully collected and subjected to washing with sterile distilled water. The crude myelin pellet was subsequently resuspended in 0.32 M sucrose buffer, and the discontinuous sucrose density-gradient centrifugation step was repeated once under the same conditions to further improve myelin purity. The purified myelin fraction was then washed three times with sterile distilled water to remove residual sucrose, soluble proteins, cellular debris, and other contaminants. The purified myelin fraction was finally resuspended in sterile PBS, quantified using a BCA protein assay, adjusted to a final concentration of 1 mg/mL, aliquoted, and stored at − 80 °C until use. Before the phagocytosis assay, purified myelin was adjusted to a stock concentration of 1 mg/mL and fluorescently labeled using a PKH26 labeling kit (Beyotime, China, C2071) according to the manufacturer’s instructions and briefly vortexed before being used in subsequent experiments.

Myelin debris phagocytosis assay

Primary microglia were seeded onto coverslips placed in 24-well plates 24 h before the experiment at a density that resulted in approximately 80% surface coverage, and were then subjected to the indicated treatments according to the experimental design.

For inhibitor experiments, cells were pretreated with the PI3K inhibitor LY294002 or CytD for 1 h before OGD exposure. For PS-blocking experiments, PKH26-labeled myelin debris was preincubated with Annexin V in Annexin V binding buffer at 37 °C for 1 h before being added to microglial cultures.

According to the experimental grouping, PKH26-labeled myelin debris was added to microglial cultures at a final concentration of 15 µg/mL and co-incubated for 4 h under OGD or normoxic control conditions. For OGD treatment, cells were cultured in glucose-free DMEM and incubated in a hypoxic chamber containing 5% CO₂ and 95% N₂ at 37 °C. After incubation, cells were gently washed three times with PBS to remove non-engulfed or surface-adherent myelin debris and then fixed with pre-cooled 4% paraformaldehyde for subsequent immunofluorescence staining.

Microglia were labeled by Iba1 immunofluorescence staining, myelin components were detected by MBP staining, and nuclei were counterstained with DAPI. Images were acquired using a confocal laser scanning microscope. Quantitative analysis was performed using ImageJ software by calculating the proportion of PKH26-positive area within Iba1-positive microglial regions and the colocalization between PKH26 and Iba1 signals, which were used to evaluate microglial phagocytosis of myelin debris.

Immunofluorescence (IF) staining of cells

Cells were fixed with pre-cooled 4% paraformaldehyde (ECOTOP, ES-8100) at room temperature for 15 min. After three washes with PBS, cells were blocked with blocking buffer containing 0.5% bovine serum albumin (BSA) (BioFroxx, China, 4240GR500), 4% goat serum (Beyotime, China, C0265), and 0.4% Triton X-100 (Biosharp, China, BS084) in 1× PBS for 1 h at room temperature to reduce nonspecific binding.

After blocking, cells were incubated with the indicated primary antibodies, including anti-Iba1 (Rabbit anti-Iba1, 1:1000, Wako, Japan, 019-19741; Guinea pig anti-Iba1, 1:500, Oasis, China, OB-PGP049), anti-MBP (Rabbit anti-MBP, 1:200, Proteintech, China, 10458-1-AP) and anti- Lysosome-Associated Membrane Protein (LAMP) (1:200, CST, USA, 99437) antibodies, overnight at 4 °C. The next day, cells were washed three times with PBS and incubated with the corresponding fluorescent secondary antibodies, including anti-rabbit (Invitrogen, USA, A11008) and anti-guinea pig (Oasis, China, AF647) secondary antibodies, together with DAPI (1:2000; stock concentration 1 mg/mL; Yeasen, 40728ES03) for nuclear counterstaining, for 1 h at room temperature in the dark. For Boron-Dipyrromethene (BODIPY) staining, BODIPY (1:1000, Thermo, USA, D3922) was added during this step. Cells were then washed three times with PBS. Finally, coverslips were mounted with antifade mounting medium, and images were acquired using a confocal laser scanning microscope. Image analysis was performed using ImageJ software.

Statistical analysis

All statistical analyses and graph generation were performed using GraphPad Prism software (Version 10.1.2(342)) (GraphPad Software, Inc., USA). Data from dendritic branching analysis using Golgi-Sholl analysis are presented as mean ± standard error of the mean (SEM), whereas all other data are presented as mean ± standard deviation (SD), unless otherwise indicated. Normality was assessed using the Shapiro–Wilk test before statistical comparisons. For comparisons among multiple groups, normally distributed data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s or Dunnett’s multiple comparisons test, as appropriate. A P-value < 0.05 was considered statistically significant.

Results

hUCB-MNCs treatment improves behavioral deficits in BCAS-induced VaD mice

To evaluate the therapeutic effects of human umbilical cord blood mononuclear cells (hUCB-MNCs) on vascular dementia (VaD)-related behavioral deficits, we established a bilateral common carotid artery stenosis (BCAS)-induced chronic cerebral hypoperfusion mouse model. Intravenous hUCB-MNCs treatment was initiated 1 month after model establishment (Fig. 1A, B). A battery of behavioral tests was then performed to assess recognition memory, spatial memory, exploratory/anxiety-like behavior, depressive-like behavior, and motor coordination.

Fig. 1.

hUCB-MNCs treatment alleviate neurological deficits in BCAS-induced VaD mice. A Schematic illustration of bilateral common carotid artery stenosis (BCAS) surgery used to establish the vascular dementia (VaD) mouse model. B Experimental timeline showing the adaptation period, BCAS or Sham surgery, intravenous administration of human umbilical cord blood mononuclear cells (hUCB-MNCs) or phosphate-buffered saline (PBS), behavioral assessments, and experimental endpoint. Behavioral tests were arranged from lower- to higher-stress procedures; the Morris water maze (MWM) and tail suspension test (TST) were performed in separate animal cohorts to avoid excessive stress-related interference. C–E Novel object recognition (NOR) test: schematic illustration of the habituation, training, and test phases (C), recognition index (RI), calculated as time exploring the novel object/total exploration time × 100% (D), and discrimination index (DI), calculated as (time exploring the novel object − time exploring the familiar object)/total exploration time × 100% (E). n = 12 male mice per group. F–H MWM test: representative swimming trajectories during the probe trial (F), number of platform crossings (G), and time spent in the target quadrant (H). n = 12 male mice per group. I–K Open-field test (OFT): representative movement trajectories (I), time spent in the central area (J), and latency to enter the central area (K). n = 12 male mice per group. L–N TST: representative images of the test (L), maximum immobility duration (M), and total immobility duration during the 5-min test period (N). n = 7, 9, and 6 male mice in the Sham, BCAS, and BCAS + hUCB-MNCs groups, respectively. O Rotarod test assessing motor coordination and fatigue resistance, including latency to fall and drop-off speed. n = 20, 15, and 14 male mice in the Sham, BCAS, and BCAS + hUCB-MNCs groups, respectively. Data are presented as mean ± SD. Each data point represents one mouse. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. * P < 0.05, **P < 0.01, ***P < 0.001

In the novel object recognition (NOR) test, BCAS mice showed markedly reduced recognition index (RI) and discrimination index (DI), indicating impaired recognition memory. hUCB-MNCs treatment significantly restored both indices, suggesting improved object recognition memory in BCAS mice (Fig. 1C–E). Consistently, in the Morris water maze (MWM) probe trial, BCAS mice crossed the former platform location fewer times and spent less time in the target quadrant than Sham mice. These deficits were significantly improved after hUCB-MNCs treatment, as shown by increased platform crossings and longer time spent in the target quadrant (Fig. 1F–H). To determine whether MWM performance was influenced by basic swimming ability, we analyzed total swimming distance and swimming speed during the 60-s probe trial. No significant differences were observed among groups, indicating comparable swimming ability under this testing condition. Therefore, the differences in platform crossings and target quadrant exploration more likely reflected changes in spatial memory rather than being primarily driven by differences in basic motor or swimming performance (Supplementary Fig. 1A, B).

In the open-field test (OFT), BCAS mice spent less time in the central area and showed increased latency to enter the center, suggesting reduced exploratory behavior and increased anxiety-like behavior. These abnormalities were significantly improved after hUCB-MNCs treatment (Fig. 1I–K). In the tail suspension test (TST), BCAS mice exhibited increased maximum immobility duration and total immobility time, whereas hUCB-MNCs treatment reduced immobility, suggesting an improvement in depressive-like behavior (Fig. 1L–N). In addition, the fatigue rotarod test showed that BCAS mice had shorter latency to fall and lower drop-off speed, both of which were improved by hUCB-MNCs treatment, indicating partial recovery of motor coordination and fatigue resistance (Fig. 1O).

To further consider sex as a biological variable, we additionally performed the NOR test in female BCAS mice. Consistent with the findings in male mice, treatment with hUCB-MNCs improved both the RI and DI in female BCAS mice (Supplementary Fig. 1C). Together, these results indicate that hUCB-MNCs treatment alleviates BCAS-induced behavioral deficits across multiple domains, including recognition memory, spatial memory, affective-like behaviors, and motor coordination.

hUCB-MNCs treatment attenuates white matter injury in BCAS-induced VaD mice

Because white matter injury is a major pathological feature of chronic cerebral hypoperfusion, we next evaluated whether hUCB-MNCs treatment could preserve white matter integrity in BCAS-induced VaD mice. The corpus callosum (CC) and external capsule (EC), two major white matter regions in the mouse brain that are vulnerable to chronic hypoperfusion-associated injury, were selected for white matter analysis.

Luxol Fast Blue (LFB) staining showed reduced myelin staining intensity and increased white matter damage scores in the CC of BCAS mice, indicating prominent myelin injury after chronic cerebral hypoperfusion. hUCB-MNCs treatment restored LFB staining intensity and reduced white matter damage scores (Fig. 2A–C). Consistently, MBP immunofluorescence showed a marked reduction in MBP expression in the CC and EC after BCAS, indicating myelin loss, whereas hUCB-MNCs treatment significantly preserved MBP expression in both regions (Fig. 2D–F).

Fig. 2.

hUCB-MNCs treatment attenuates white matter injury in BCAS-induced VaD mice. The corpus callosum (CC) and external capsule (EC), two major white matter regions in the mouse brain that are vulnerable to chronic hypoperfusion-induced injury, were selected for white matter analysis. A Representative Luxol Fast Blue (LFB) staining images showing myelin integrity in the CC. B, C Quantification of LFB staining intensity by average optical density (AOD) (B) and white matter damage score (C). n = 6 male mice per group. (D) Representative immunofluorescence images of myelin basic protein (MBP) in the CC and EC. E, F Quantification of MBP fluorescence intensity in the CC (E) and EC (F). n = 6 male mice per group. G Representative immunofluorescence images of degraded myelin basic protein (dMBP) in the CC and striatum. H, I Quantification of dMBP fluorescence intensity in the CC (H) and striatum (I). n = 6 male mice per group. J Representative immunofluorescence images of neurofilament 200 (NF200) and MBP in the EC. K Quantification of NF200 fluorescence intensity. n = 6 male mice per group. L Representative transmission electron microscopy (TEM) images showing myelin ultrastructure in the CC at 5000× and 10,000× magnification. M Scatter plot showing the relationship between inner tongue thickness and axon diameter. Each point represents one myelinated axon. N Quantification of the g-ratio, calculated as the ratio of the inner axonal diameter to the outer diameter of the myelinated fiber. For TEM analysis, n = 3 male mice per group. Six randomly selected TEM images from the CC were analyzed for each mouse. The 18 image-level data points are shown for transparency, and statistical comparisons were performed using mouse-level mean values. Data are presented as mean ± SD. Each data point in the quantitative graphs represents one mouse, except for the image-level data points shown in the TEM analysis as indicated above. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars are indicated in the images

We next examined degraded myelin basic protein (dMBP) to evaluate myelin degradation and myelin debris burden. BCAS mice showed marked dMBP accumulation in the CC and striatum, suggesting increased myelin breakdown after chronic cerebral hypoperfusion. This accumulation was significantly reduced after hUCB-MNCs treatment (Fig. 2G–I). In parallel, NF200 immunofluorescence showed impaired axonal integrity in the EC of BCAS mice, which was partially restored by hUCB-MNCs treatment (Fig. 2J, K).

Transmission electron microscopy (TEM) further confirmed the protective effects of hUCB-MNCs on white matter ultrastructure. BCAS mice exhibited disorganized myelin architecture, abnormal myelin morphology, and increased g-ratio values, indicating myelin thinning and ultrastructural damage. After hUCB-MNCs treatment, myelin ultrastructure was improved, and g-ratio values were reduced compared with BCAS mice and shifted toward Sham levels (Fig. 2L–N). In female BCAS mice, hUCB-MNCs treatment also improved LFB staining, preserved MBP expression, and alleviated myelin ultrastructural abnormalities, supporting a similar white matter-protective effect in female animals (Supplementary Fig. 1D–I).

Together, these results indicate that hUCB-MNCs treatment attenuates chronic hypoperfusion-associated white matter injury in BCAS-induced VaD mice by preserving myelin integrity, reducing myelin debris burden, and improving axonal/myelin ultrastructure.

hUCB-MNCs mitigate synaptic and neuronal injury in BCAS-induced VaD mice

To further evaluate the neuroprotective effects of hUCB-MNCs on hippocampal neuronal integrity and synaptic structure, Golgi staining was conducted to assess dendritic arborization and spine density. Compared with sham-operated controls, BCAS mice displayed a significant reduction in dendritic complexity within the Cornu Ammonis 1 (CA1) and dentate gyrus (DG) regions, together with decreased dendritic spine density in the basal dendrites of CA1 pyramidal neurons. hUCB-MNCs administration substantially reversed these morphological abnormalities (Fig. 3A–E). In parallel, neuronal survival across hippocampal subregions was assessed via NeuN immunostaining and Nissl staining. BCAS mice exhibited pronounced neuronal loss in the CA1, CA2, and CA3 regions, which was significantly mitigated following hUCB-MNCs treatment (Fig. 3F–I). Consistently, in female BCAS mice, hUCB-MNCs treatment also preserved NeuN expression and reduced neuronal loss, as assessed by NeuN immunostaining and Nissl staining (Supplementary Fig. 1J–M).

Fig. 3.

hUCB-MNCs treatment preserves dendritic structure and mitigates neuronal loss in the hippocampus of BCAS-induced VaD mice. A Representative Golgi staining images and reconstructed neuronal morphology in the hippocampal CA1 and dentate gyrus (DG) regions. B, C Sholl analysis showing dendritic complexity in CA1 (B) and DG (C), quantified as the number of dendritic intersections at increasing distances from the soma. Sholl analysis data are presented as mean ± SEM. n = 3 male mice per group. D Representative Golgi staining images showing dendritic spines in the basal dendrites of CA1 pyramidal neurons, with enlarged images showing dendritic spine morphology in each group. E Quantification of dendritic spine density in the basal dendrites of CA1 pyramidal neurons, expressed as the number of spines per 10 μm dendrite. For Golgi staining, Sholl analysis, and dendritic spine analysis, n = 3 male mice per group. Six randomly selected images were analyzed for each mouse. The 18 image-level data points are shown for transparency, whereas statistical comparisons were performed using mouse-level mean values. F Representative immunofluorescence images of NeuN staining in the CA1 regions of hippocampus. G Quantification of NeuN fluorescence intensity. n = 5 male mice per group. H Representative Nissl staining images in the hippocampal CA1, CA2, and CA3 regions. I Quantification of Nissl-positive cells in CA1, CA2, and CA3. n = 5 male mice per group. Data are presented as mean ± SD, except for Sholl analysis data, which are presented as mean ± SEM. Each data point represents one mouse unless otherwise indicated. For dendritic spine analysis in (E), image-level data points are shown, and statistical comparisons were performed using mouse-level mean values. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01

Collectively, these results demonstrate that hUCB-MNCs preserves hippocampal neuronal architecture and synaptic integrity, which may contribute to the observed improvements in cognitive function in VaD mice.

hUCB-MNCs treatment preserve microglial homeostatic features and improve myelin debris handling in BCAS-induced white matter injury

Microglia are key regulators of brain homeostasis, white matter injury responses, and myelin debris handling after chronic cerebral hypoperfusion. Therefore, we next examined whether hUCB-MNCs treatment could modulate microglial responses in the CC of BCAS-induced VaD mice, with a particular focus on microglial homeostatic features and functional states related to myelin debris handling. Because Iba1 can also label some peripheral macrophage-derived cells, we additionally performed Iba1/TMEM119 co-staining. Clear TMEM119 immunoreactivity was observed in Iba1-positive cells in the CC, supporting the interpretation that the Iba1-positive cells analyzed in this study mainly represented resident microglia (Supplementary Fig. 2).

First, we analyzed microglial reactivity and morphological changes in the CC across groups. Iba1 immunofluorescence showed a marked increase in the Iba1-positive area in BCAS mice, suggesting enhanced microglial activation or accumulation in the white matter lesion area. This increase was significantly reduced after hUCB-MNCs treatment (Fig. 4A, B). Further morphological analysis showed that Iba1-positive microglia in BCAS mice exhibited activation-like changes, characterized by enlarged soma area, reduced branch number, and shortened maximum branch length. hUCB-MNCs treatment partially reversed these morphological abnormalities, suggesting attenuation of BCAS-induced microglial activation-like changes (Fig. 4C-F).

Fig. 4.

hUCB-MNCs treatment helps preserve microglial homeostatic features in BCAS-induced white matter injury. A Representative immunofluorescence images of Iba1 staining in the CC. B Quantification of the Iba1-positive cell area in the CC. C Representative high-magnification images of Iba1-positive microglia and corresponding binary images used for morphological analysis. D–F Quantification of microglial soma area (D), number of branches (E), and maximum branch length (F). G Representative immunofluorescence images showing Iba1 and the homeostasis-associated microglial marker P2RY12 co-staining in the CC. H Quantification of P2RY12 fluorescence intensity. I qRT-PCR analysis of the mRNA expression levels of pro-inflammatory markers, including Cd86, TNF, IL6, and Nos2, and anti-inflammatory/immunoregulatory markers, including MRC1, TGFB1, IL10, and Arg1, in BV2 microglial cells under control, oxygen-glucose deprivation (OGD), and OGD + hUCB-MNC-conditioned medium conditions. Statistical comparisons were performed relative to the OGD group. Data are presented as mean ± SD. For in vivo immunofluorescence analyses, n = 6 male mice per group, and each data point represents one mouse. For in vitro qRT-PCR analysis, n = 4 independent experiments, and each data point represents one independent experiment. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars are indicated in the images

To determine whether chronic hypoperfusion affected homeostasis-associated microglial features, we performed co-staining for Iba1 and the homeostasis-associated microglial marker P2RY12. P2RY12 fluorescence intensity was markedly decreased in the CC of BCAS mice, indicating loss of homeostasis-associated microglial characteristics under chronic hypoperfusion. hUCB-MNCs treatment significantly restored P2RY12 expression, suggesting partial preservation or restoration of microglial homeostatic features in BCAS-induced white matter injury (Fig. 4G, H). Meanwhile, to further evaluate the effects of hUCB-MNCs on the inflammatory response state of microglia under hypoxic/ischemic-like stress, we examined the mRNA expression of inflammation-related markers in BV2 microglial cells under OGD conditions. OGD increased the expression of pro-inflammatory markers, including Cd86, TNF, IL6, and Nos2, whereas hUCB-MNC-conditioned medium reduced the expression of these markers and increased the expression of anti-inflammatory or immunoregulatory markers, including MRC1, TGFB1, IL10, and Arg1 (Fig. 4I). These results further support that hUCB-MNCs modulate the microglial response state under hypoxic/ischemic-like stress.

Next, we assessed the spatial association between microglia and damaged myelin debris using dMBP and Iba1 immunofluorescence co-staining combined with three-dimensional reconstruction. The volume of degraded myelin basic protein (dMBP) was markedly increased in the CC of BCAS mice, whereas total dMBP accumulation was significantly reduced after hUCB-MNCs treatment (Fig. 5A, B). In parallel, BCAS mice showed increased Iba1-positive cell volume, elevated dMBP signal within Iba1-positive cells, and an increased proportion of dMBP-positive signal within Iba1-positive cells; these changes were significantly reversed by hUCB-MNCs treatment (Fig. 5C–E). These findings suggest prominent myelin debris accumulation in the CC after BCAS, accompanied by increased spatial association between microglia and damaged myelin debris, whereas hUCB-MNCs treatment reduced myelin debris burden and improved the state of microglia involved in myelin debris handling. Notably, increased dMBP signal associated with Iba1-positive microglia in vivo does not necessarily indicate enhanced microglial phagocytic capacity, because this signal may also be influenced by the overall extent of myelin injury and debris burden. Therefore, we further evaluated the effects of hUCB-MNCs on microglial myelin debris uptake under controlled in vitro conditions using equal amounts of myelin debris substrate in subsequent experiments.

Fig. 5.

hUCB-MNCs treatment reduces myelin debris burden and improves microglial debris-handling responses. A Representative immunofluorescence images showing degraded myelin basic protein (dMBP), Iba1, DAPI, and dMBP signals within Iba1-positive cells in the CC. B–E Quantification of total dMBP volume (B), Iba1 volume (C), dMBP volume within Iba1-positive cells (D), and the percentage of dMBP-positive staining within Iba1-positive cells (E). F, G Representative immunofluorescence images of Iba1, BODIPY, and DAPI staining in BV2 microglial cells incubated with myelin debris under control, OGD, and OGD plus hUCB-MNC-conditioned medium conditions (F), with quantification of BODIPY fluorescence intensity (G). H, I Representative immunofluorescence images of Iba1, LAMP, and DAPI staining in BV2 microglial cells incubated with myelin debris under control, OGD, and OGD plus hUCB-MNC-conditioned medium conditions (H), with quantification of LAMP fluorescence intensity (I). J, K Representative immunofluorescence images of Iba1, p21, and DAPI co-staining in the CC of Sham, BCAS, and BCAS plus hUCB-MNCs-treated mice (J), with quantification of p21 fluorescence intensity and the percentage of p21^⁺Iba1^⁺ overlapping area relative to total Iba1^⁺ area (K). Data are presented as mean ± SD. For in vivo dMBP/Iba1 three-dimensional reconstruction analysis, n = 6 male mice per group, with each data point representing one mouse. For in vitro Boron-Dipyrromethene (BODIPY) and Lysosome-Associated Membrane Protein (LAMP) immunofluorescence analyses, n = 4 independent experiments, with each data point representing one independent experiment. For in vivo p21 staining analysis, n = 5 male mice per group, with each data point representing one mouse. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars are indicated in the images

Considering that myelin debris is lipid-rich and requires lysosome-associated degradation and metabolism after uptake, we further added myelin debris to BV2 microglial cells under OGD conditions and assessed Boron-Dipyrromethene (BODIPY) and Lysosome-Associated Membrane Protein (LAMP) expression. OGD increased BODIPY fluorescence intensity, indicating increased intracellular lipid burden after exposure to myelin debris. hUCB-MNC-conditioned medium reduced BODIPY signals, suggesting attenuation of myelin debris-associated lipid accumulation (Fig. 5F, G). In addition, OGD reduced LAMP fluorescence intensity, whereas hUCB-MNC-conditioned medium partially restored LAMP expression, suggesting improved lysosome-associated responses during myelin debris processing (Fig. 5H, I).

Furthermore, to assess whether the white matter lesion area was accompanied by stress- or senescence-like changes under chronic hypoperfusion, we examined p21 expression. p21 staining showed increased p21 fluorescence intensity in the CC of BCAS mice, together with an increased percentage of p21⁺Iba1⁺ overlapping area relative to the total Iba1^⁺ area. After hUCB-MNCs treatment, both p21 fluorescence intensity and the p21^⁺ Iba1^⁺ overlapping area ratio were reduced (Fig. 5J, K). These findings suggest enhanced p21-associated stress or senescence-like responses in the white matter lesion area after BCAS, which were attenuated by hUCB-MNCs treatment.

Together, these results indicate that BCAS induces a dysregulated microglial response characterized by increased Iba1 reactivity, activation-like morphological changes, reduced P2RY12 expression, abnormal inflammatory responses, increased myelin debris burden, and impaired lipid/lysosome-associated processing. hUCB-MNCs treatment helps preserve or restore microglial homeostatic features, reduces myelin debris burden, and improves the functional state of microglia involved in myelin debris handling during chronic hypoperfusion-associated white matter injury.

PI3K/AKT signaling contributes to hUCB-MNC-mediated improvement of microglial myelin debris uptake

To further elucidate the molecular mechanisms by which hUCB-MNCs regulate microglial function and promote white matter repair, we performed integrated transcriptomic and proteomic analyses using freshly isolated CC tissues from the BCAS and BCAS + hUCB-MNCs-treated groups (Fig. 6A). Proteomic GSEA showed that hUCB-MNCs treatment regulated pathways associated with inflammatory responses, tissue repair, and PI3K/AKT-related processes (Fig. 6B). Differential expression analysis further identified a series of genes altered by hUCB-MNCs treatment compared with the BCAS group (Fig. 6C). KEGG enrichment analysis showed that PI3K/AKT-related signaling was markedly enriched among the differentially expressed genes. GO biological process analysis further revealed enrichment of biological processes related to phagocytosis, cell adhesion, migration, and immune regulation (Fig. 6D, E). Consistently, transcriptomic GSEA also indicated activation of the PI3K/AKT/mTOR signaling pathway in the hUCB-MNCs-treated group (Fig. 6F).

Fig. 6.

PI3K/AKT signaling contributes to hUCB-MNC-mediated improvement of microglial myelin debris uptake under OGD-induced stress. A Schematic illustration of CC tissue collection for RNA sequencing and proteomic analysis, followed by differentially expressed gene/protein, KEGG, GO, and GSEA analyses. B GSEA of proteomic data showing enriched pathways in the comparison between the BCAS and BCAS + hUCB-MNCs groups. C Volcano plot showing differentially expressed genes between the BCAS and BCAS + hUCB-MNCs groups. D KEGG pathway enrichment analysis of differentially expressed genes after hUCB-MNCs treatment. E GO biological process enrichment analysis of differentially expressed genes after hUCB-MNCs treatment. F Transcriptomic GSEA plot showing enrichment of the PI3K/AKT/mTOR signaling pathway after hUCB-MNCs treatment. G Representative Western blot images of PI3K, phosphorylated AKT (p-AKT), total AKT, and β-actin in CC tissue. H Quantification of PI3K protein expression and the p-AKT/AKT ratio. For Western blot analysis, n = 4 male mice per group; each data point represents one mouse. I Representative immunofluorescence images showing myelin debris uptake by Iba1-positive primary microglia under control or oxygen-glucose deprivation (OGD) conditions, with or without hUCB-MNC-conditioned medium treatment. Where indicated, cells were pretreated with the PI3K inhibitor LY294002 (30 µM, 1 h) or Cytochalasin D (CytD, 10 µM, 1 h) before the myelin debris uptake assay. For phosphatidylserine (PS)-blocking experiments, fluorescently labeled myelin debris was preincubated with Annexin V (approximately 9 µg/mL, 37 °C, 1 h) in Annexin V binding buffer before being added to microglial cultures. J, K Quantification of the percentage of myelin debris-positive signal within Iba1-positive cells (J) and myelin debris/Iba1 colocalization normalized to the control group (K). For in vitro microglial uptake assays, n = 3 independent experiments. Six randomly selected images were analyzed per independent experiment. The 18 image-level data points are shown for transparency, whereas statistical comparisons were performed using independent experiment-level mean values. Data are presented as mean ± SD. Normality was assessed using the Shapiro–Wilk test. For panel H, statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. For panels J–K, statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. ns = no significance. Scale bars are indicated in the images

To validate the omics findings, we examined PI3K/AKT-related signaling in CC tissues by western blotting. BCAS mice showed reduced PI3K expression and a decreased phosphorylated AKT/total AKT ratio in the CC, whereas hUCB-MNCs treatment restored PI3K expression and increased AKT phosphorylation (Fig. 6G, H). These results suggest that hUCB-MNCs treatment reactivates PI3K/AKT-related signaling in BCAS-induced white matter injury.

To further determine the functional relevance of this pathway in microglial myelin debris handling, we performed an in vitro myelin debris uptake assay using primary microglia under controlled substrate conditions. Equal amounts of fluorescently labeled myelin debris were added to each group to minimize the influence of differences in myelin debris burden. OGD exposure reduced myelin debris uptake by Iba1-positive primary microglia, suggesting that hypoxic/ischemic-like stress impairs microglial debris-handling capacity. hUCB-MNC-conditioned medium significantly enhanced myelin debris uptake by microglia under OGD conditions (Fig. 6I-K). We next examined whether PI3K/AKT signaling was involved in hUCB-MNC-mediated improvement of myelin debris uptake. Pretreatment with the PI3K inhibitor LY294002 attenuated the hUCB-MNC-conditioned medium-mediated increase in myelin debris uptake, suggesting that PI3K/AKT signaling participates in the hUCB-MNCs-induced improvement of microglial myelin debris handling (Fig. 6I-K). In parallel, Cytochalasin D (CytD), an inhibitor of actin-dependent phagocytosis, markedly attenuated the hUCB-MNC-conditioned medium-mediated increase in myelin debris uptake. This finding supports the interpretation that the increased intracellular myelin debris signal after hUCB-MNC-conditioned medium treatment primarily reflects active, actin-dependent phagocytic uptake rather than nonspecific debris adhesion, residual extracellular signal, or passive accumulation. We also used Annexin V to block phosphatidylserine (PS)-mediated recognition of myelin debris. Annexin V partially reduced myelin debris uptake under normal conditions (Supplementary Fig. 3A-B), suggesting that PS-mediated recognition contributes to basal myelin debris uptake. However, under OGD conditions, Annexin V did not markedly block the enhancing effect of hUCB-MNC-conditioned medium on myelin debris uptake, suggesting that hUCB-MNC-mediated improvement of myelin debris uptake may not mainly depend on PS-mediated recognition (Fig. 6I–K).

Together, these results indicate that hUCB-MNCs improve microglial uptake of myelin debris under hypoxic/ischemic-like stress, at least partly through PI3K/AKT-related signaling mechanisms. This mechanism may help enhance microglial myelin debris-handling function and contribute to the protective effects of hUCB-MNCs against chronic hypoperfusion-associated white matter injury in vascular dementia.

Discussion

VaD, primarily driven by chronic cerebral hypoperfusion, remains a major clinical challenge due to the lack of effective therapeutic strategies [23]. In this study, we demonstrated that hUCB-MNCs alleviated cognitive impairment and white matter injury in a BCAS-induced mouse model of VaD. These protective effects were accompanied by reduced myelin loss and axonal damage, as well as improved microglial myelin debris-handling responses. Mechanistically, our findings suggest that PI3K/AKT-related signaling pathway may participate in hUCB-MNC-mediated regulation of microglial debris processing. Together, these results support the possibility that hUCB-MNCs protect against chronic hypoperfusion-associated white matter injury, at least in part, by improving microglial debris-clearing function. These findings provide preclinical evidence supporting hUCB-MNCs as a potential cell-based therapeutic strategy targeting microglial dysfunction in VaD.

White matter injury is a major pathological feature of VaD and is closely associated with cognitive decline [1]. Chronic cerebral hypoperfusion can lead to oligodendrocyte injury, demyelination, axonal degeneration, and disruption of neural network connectivity, thereby impairing learning, memory, and executive function [24]. Consistent with these pathological features, BCAS mice in the present study exhibited cognitive behavioral deficits accompanied by white matter disruption, myelin loss, axonal injury, and reduced neuronal integrity [4]. hUCB-MNCs treatment improved these abnormalities, preserved neuronal integrity, and alleviated white matter damage, suggesting its potential protective effects against chronic hypoperfusion-induced cognitive impairment and white matter pathology. Because motor deficits may confound the interpretation of cognitive behavioral tests in chronic cerebral hypoperfusion models, we further considered the contribution of motor function to behavioral performance. In the present study, motor-related behavioral parameters did not indicate that the cognitive improvement after hUCB-MNCs treatment was primarily attributable to differences in motor ability (Supplementary Fig. 1A–B). Therefore, the observed improvement in cognitive performance more likely reflects cognitive benefits rather than a secondary consequence of altered motor function.

An important finding of this study is that hUCB-MNCs treatment may improve microglial myelin debris-handling responses and contribute to reduced myelin debris burden. Myelin debris is not merely a passive consequence of demyelination; if not efficiently removed, it can sustain local inflammatory responses, impair remyelination, and further aggravate white matter pathology [18, 25, 26]. Microglia are the principal immune cells responsible for recognizing, engulfing, and processing damaged myelin in the central nervous system [27]. During the early phase of acute injury, microglial activation and phagocytic responses contribute to debris removal and tissue repair [28]. However, under chronic cerebral hypoperfusion and persistent inflammatory stress, microglia may enter a maladaptive or functionally dysregulated state in which inflammatory/morphological activation coexists with insufficient debris-processing capacity. Therefore, our findings should not be interpreted as indicating that microglial activation per se suppresses phagocytosis. Rather, they suggest that BCAS-associated microglia may be activated but inefficient in completing debris processing and clearance [4]. This activated but functionally dysregulated phenotype may help explain the coexistence of increased Iba1-positive microglial activation, loss of homeostasis-associated features, and persistent myelin debris accumulation in BCAS-induced white matter lesions [18].

In this study, BCAS mice exhibited pronounced microglial activation, morphological abnormalities, and downregulation of the homeostasis-associated marker P2RY12 [29], whereas these alterations were partially improved after hUCB-MNCs treatment. In parallel, hUCB-MNCs-based intervention enhanced the uptake of myelin debris by microglia under OGD conditions in vitro, further supporting the modulatory effect of hUCB-MNCs on microglial phagocytic function. These findings suggest that hUCB-MNCs may not simply suppress microglial activation, but may instead modulate microglial function and improve their capacity to process myelin debris under chronic hypoperfusion-related stress. The possibility that BCAS-associated microglia acquire senescence-like or exhaustion-like features is also important. In the present study, we added p21 staining and observed increased p21-associated signals in the white matter lesion area after BCAS, which were reduced by hUCB-MNCs treatment. However, other senescence-related markers, such as p16, ferritin light chain, and SASP-related cytokines, were not systematically examined, and future studies assessing these markers will be useful to determine whether microglial senescence-like dysfunction contributes to impaired debris handling in chronic hypoperfusion. Notably, the reduced myelin debris burden observed after hUCB-MNCs treatment in vivo may reflect two non-mutually exclusive mechanisms. On the one hand, hUCB-MNCs may enhance microglial clearance of pre-existing myelin debris. On the other hand, hUCB-MNCs may reduce the generation of new myelin debris by attenuating white matter injury. Therefore, the protective effects of hUCB-MNCs on white matter integrity are likely multifactorial, involving both improved pathological debris clearance and reduced tissue damage.

The role of microglial phagocytosis in CNS diseases should be interpreted in a disease- and stage-specific manner. In chronic neurodegenerative diseases or chronic brain injury conditions, such as AD, VaD, and chronic white matter injury [30–32], microglia often exhibit sustained inflammatory activation together with impaired homeostatic and debris-clearing functions. In this context, restoring microglial clearance capacity may contribute to inflammation resolution and tissue repair. However, in CNS disorders with acute onset, such as epilepsy, intracerebral hemorrhage, ischemic stroke, and traumatic brain injury [33–37], microglial responses evolve dynamically across the acute, subacute, and chronic phases. The consequences of enhancing microglial phagocytosis may therefore differ depending on the disease stage and the functional state of microglia. In some settings, excessive or dysregulated phagocytic activity may aggravate tissue injury, whereas appropriately regulated phagocytosis may facilitate debris clearance and recovery [38]. Thus, our findings should not be interpreted as supporting nonspecific enhancement of microglial phagocytosis, but rather as suggesting that hUCB-MNCs enhance the capacity of microglia to process myelin debris in the context of chronic hypoperfusion-associated white matter injury.

Mechanistically, our results indicate that the PI3K/AKT signaling pathway may be involved in hUCB-MNC-mediated regulation of microglial phagocytosis. PI3K/AKT signaling is widely implicated in cell survival, cytoskeletal remodeling, membrane trafficking, and phagocytic responses [39]. In microglia, activation of this pathway may facilitate phagocytic uptake and contribute to adaptive responses after tissue injury [40]. Integrated transcriptomic and proteomic analyses of CC tissue revealed significant enrichment of PI3K/AKT-related signaling after hUCB-MNCs treatment, and the increased levels of PI3K and phosphorylated AKT in western blot analyses further supported this finding. In vitro, pharmacological inhibition of PI3K with LY294002 attenuated the hUCB-MNCs-induced enhancement of microglial myelin phagocytosis, suggesting that PI3K/AKT signaling contributes to hUCB-MNC-mediated regulation of microglial debris clearance. However, PI3K/AKT is a broad signaling pathway with multiple downstream targets, and its activation may also influence inflammatory responses, cellular metabolism, and cell survival [39]. Therefore, further studies are needed to identify the specific upstream factors and downstream effectors linking hUCB-MNCs-derived signals to microglial phagocytic function.

The mechanism by which intravenously administered hUCB-MNCs affect brain pathology remains an important question. In chronic cerebral hypoperfusion, blood-brain barrier disruption may occur to varying degrees, potentially allowing circulating cells or their secreted factors to influence the brain microenvironment [41]. However, unlike the pronounced blood-brain barrier disruption observed in acute ischemic stroke and other acute brain injuries, blood-brain barrier impairment associated with chronic cerebral hypoperfusion may be relatively mild [42, 43]. Therefore, whether intravenously administered hUCB-MNCs can directly enter the brain and exert sustained effects within the central nervous system remains to be clarified. Previous studies have detected human-specific gene signals in brain tissue from disease-model mice treated with hUCB-MNCs [44], suggesting that exogenous human cells or their components may reach the brain. However, direct visual evidence demonstrating long-term engraftment of hUCB-MNCs within the central nervous system under chronic cerebral hypoperfusion conditions remains limited. Thus, the effects of hUCB-MNCs may not fully depend on their long-term persistence in the brain. Accumulating evidence suggests that hUCB-MNCs may exert therapeutic effects mainly through paracrine and immunomodulatory mechanisms [45]. hUCB-MNCs may release trophic and immune-regulatory factors that indirectly reshape microglial responses and improve the brain microenvironment [46]. Our in vitro data further support this possibility, showing that soluble factors derived from hUCB-MNCs can enhance microglial phagocytic activity under OGD-induced stress. Future studies using cell tracking, biodistribution analysis, and secretome profiling are needed to further clarify whether the effects of hUCB-MNCs depend on their entry into the central nervous system or are primarily mediated by paracrine factors and immune regulation.

Several limitations should be acknowledged. First, although we observed that hUCB-MNCs also improved cognitive behavioral abnormalities and white matter injury in female BCAS mice, future studies should include both male and female animals in mechanistic investigations to determine whether sex-specific differences exist in hUCB-MNC-mediated regulation of microglial function. Second, the contribution of infiltrating monocyte-derived macrophages cannot be fully excluded. These cells may share overlapping markers and functions with resident microglia in white matter lesions, and future studies using multiple microglia-specific markers, flow cytometry, or lineage-tracing approaches would help distinguish their respective contributions. Third, because human cells were used in a mouse model in this study, cross-species immune recognition may have influenced host immune responses. This xenogeneic context differs from potential human-to-human therapeutic application and should be considered when interpreting the translational implications. Future studies using more clinically relevant models or human-compatible systems will be needed to further evaluate the therapeutic mechanisms and safety of hUCB-MNCs. Fourth, we did not directly track the biodistribution or persistence of intravenously administered hUCB-MNCs. Therefore, whether their effects depend on direct entry into the central nervous system or secreted factors remains to be further clarified. Fifth, although hUCB-MNCs used for in vivo administration were derived from multiple independent donors, the present study was not designed to systematically compare donor-dependent differences in therapeutic efficacy. Given that donor-to-donor variability may involve differences in cellular composition, activation status, and secreted factor profiles, future studies using a donor-stratified experimental design will be important to determine whether hUCB-MNCs from different donors differ in therapeutic efficacy and to clarify the underlying mechanisms.

In conclusion, our study demonstrates that hUCB-MNCs treatment alleviates chronic hypoperfusion-associated cognitive impairment and white matter injury in a mouse model of VaD. The protective effects of hUCB-MNCs may involve modulation of microglial function and improved myelin debris-handling responses, potentially through PI3K/AKT-related signaling. These findings suggest that targeting the pathological debris-clearing capacity of microglia may represent a potential therapeutic strategy for VaD and support further investigation of hUCB-MNCs as a cell-based intervention for chronic cerebral hypoperfusion-related cognitive impairment.

Conclusion

In summary, this study demonstrates that hUCB-MNCs ameliorate cognitive dysfunction and white matter injury in a mouse model of VaD induced by chronic cerebral hypoperfusion. Mechanistically, hUCB-MNCs may improve microglial myelin debris-handling responses, at least partly through PI3K/AKT-related signaling. These findings suggest that modulating microglial debris-processing capacity may contribute to the therapeutic effects of hUCB-MNCs and support further investigation of hUCB-MNCs as a potential cell-based intervention for chronic hypoperfusion-associated cognitive impairment.

Abbreviations

AD

Alzheimer’s Disease

AnnV

Annexin V

AKT

Protein Kinase B

ANOVA

Analysis of Variance

AOD

Average Optical Density

BCA

Bicinchoninic Acid

BCAS

Bilateral Common Carotid Artery Stenosis

BSA

Bovine Serum Albumin

BODIPY

Boron-Dipyrromethene

CA1

Cornu Ammonis 1

CC

Corpus Callosum

CNS

Central Nervous System

CytD

Cytochalasin D

DAPI

4’,6-DiAmidino-2-PhenylIndole

DEGs

Differentially Expressed Genes

DI

Discrimination Index

DIA

Data-independent acquisition

DMEM

Dulbecco’s modified Eagle’s medium

DG

Dentate Gyrus

dMBP

Degraded Myelin Basic Protein

EC

External Capsule

GO

Gene Ontology

GSEA

Gene Set Enrichment Analysis

hUCB-MNCs

Human Umbilical Cord Blood Mononuclear Cells

KEGG

Kyoto Encyclopedia of Genes and Genomes

LAMP

Lysosome-Associated Membrane Protein

LC-MS/MS

Liquid Chromatography-Tandem Mass Spectrometry

LFB

Luxol Fast Blue

MBP

Myelin Basic Protein

MWM

Morris Water Maze

NeuN

Neuronal Nuclear Antigen

NF200

Neurofilament 200

NOR

Novel Object Recognition

OCT

Optimal Cutting Temperature Compound

OGD

Oxygen-Glucose Deprivation

OFT

Open Field Test

PBS

Phosphate Buffered Saline

PBST

Phosphate Buffered Saline with Triton

PFA

Paraformaldehyde

PI3K

Phosphoinositide 3-Kinase

PKH26

Paul Karl Horan 26 fluorescent dye

PS

Phosphatidylserine

P2RY12

Purinergic Receptor P2Y12

RI

Recognition Index

RIPA

Radio-Immunoprecipitation Assay

SEM

standard error of the mean

SD

Standard Deviation

SDS-PAGE

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

TBST

Tris Buffered Saline with Tween

TEM

Transmission Electron Microscopy

TST

Tail Suspension Test

IACUC

Institutional Animal Care and Use Committee

Iba1

Ionized Calcium-Binding Adaptor Molecule 1

VaD

Vascular Dementia

Authors’ contributions

Conceptualization: XY.W., YS. Z., ZG. Y. and KS. L; Methodology, experiments, and data analysis: XY.W., KJ. H., C. H. and CY. S.; Preparation of hUCB-MNCs: CY. S. and W. W.; Data discussions: XY.W., KJ. H., C. H., CY. S. and W. W.; Writing—original draft: XY.W., KJ. H. and CY. S.; Writing—review and editing: XY.W. C. H. YS. Z., ZG. Y. and KS. L; Funding acquisition and resources: XY.W., ZG. Y. and KS. L. All authors reviewed the manuscript.

Funding

This work was supported by grants from the Guangzhou Technology Program of Agriculture and Social Development of Key Research and Development Scheme (Grant No. 2023B03J1351), National Natural Science Foundation of China (Grant No. 82171344), the Science and Technology Projects in Guangzhou (Grant No. 2025A03J4169), the China Postdoctoral Science Foundation (Grant No. 2024M751134 and 2025M782186), and the Scientific Research Foundation for Young Doctors of the Second Affiliated Hospital, AMU (Grant No. 2024YQB003).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Jinan University (Approval No. IACUC-20240408-06).

Consent for publication

All authors have read and approved the final manuscript for publication.

Competing interests

The authors declare no competing interests.