Invest Clin 67(2): 275 - 288, 2026 https://doi.org/10.54817/IC.v67n2a09

Neurotoxic effects of nanoplastics

exposure on depression-like behavior and

cognitive function in mice under chronic

unpredictable mild stress.

Dewei Chang1,#, Miao Xu2,#, Wenning Shi1, Yan He3, Zhe Wu4, Zhifeng Ning4,

Yanling Sun4 and Jianguo Lv5,6

1School of Pharmacy, Department of Medicine, Hubei University of Science

and Technology, Xianning City, Hubei Province, China.

2Outpatient Department, West China Emei Hospital/ Mount Emei Jingchuan Hospital,

Leshan City, Sichuan Province, P. R. China.

3Internal Medicine Department, The Second Affiliated Hospital, Hubei University

of Science and Technology, Xianning City, Hubei Province, P. R. China.

4School of Basic Medical Sciences, Hubei University of Science and Technology,

Xianning City, Hubei Province, P. R. China.

5Psychiatry Department, The Second Affiliated Hospital, Hubei University of Science

and Technology, Xianning City, Hubei province, P. R. China.

6School of Clinical Medical Sciences, Hubei University of Science and Technology,

Xianning City, Hubei Province, P. R. China.

#These authors contributed equally to this work.

Keywords: Microplastics, Nanoparticles; Nanoplastic; BDNF/TrkB; Chronic unpredictable mild

Stress; Depression-like behavior; Disease Models, Animal; Hippocampus.

Abstract. The aim was to investigate the effects of gavage exposure to

nanoplastics (NPs) on cognitive decline and depression-like behavior was in-

vestigated in mice subjected to chronic unpredictable mild stress (CUMS).

BALB/c mice were randomly assigned to four experimental groups: Control

(Ctrl), nanoplastics (NPs), Mod (subjected to CUMS), and NPs+Mod (nano-

plastics + CUMS). We evaluated the role of the brain-derived neurotrophic fac-

tor (BDNF) and its receptor, the tyrosine kinase receptor B (TrkB), signaling

pathway in the hippocampus of mice. Behavioral assessments included the su-

crose preference test, the open field test, the forced swim test, and the Morris

water maze. Nissl staining was used to assess hippocampal neuronal morphol-

ogy. BDNF and TrkB mRNA levels and protein expression in the hippocampus

were measured by qPCR and Western blotting, respectively. Mice in the NPs,

Mod, and NPs+Mod groups showed reduced body weight, lower sucrose pref-

erence, poorer performance in the open field test, and prolonged immobility

in the forced swim test. Additionally, there was a reduction in hippocampal

Corresponding authors: Yanling Sun (Email: yanlingssmed@hotmail.com) and Jianguo Lv (Email: asljgpt@163.com),

Hubei University of Science and Technology, No. 88 Xianning Avenue, Xianning 437100, China.

276 Chang et al.

Investigación Clínica 67(2): 2026

Efectos neurotóxicos de la exposición a nanoplásticos sobre

el comportamiento similar a la depresión y la función cognitiva

en ratones sometidos a estrés crónico leve impredecible.

Invest Clin 2026; 67 (2): 275 – 288

Palabras clave: Nanoplásticos; Microplásticos; Nanopartículas; BDNF/TrkB; Estrés

Crónico; Comportamiento similar a la depresión; Hipocampo.

Resumen. En el presente estudio se evaluó el impacto de la exposición a

nanoplásticos sobre el deterioro cognitivo y el comportamiento depresivo en

ratones sometidos a estrés moderado crónico e impredecible (CUMS). Ratones

BALB/c fueron asignados aleatoriamente a cuatro grupos experimentales: Con-

trol (Ctrl), nanoplásticos (NPs), Mod (sometidos a CUMS) y NPs+Mod (nanoplás-

ticos + CUMS). Se determinó el papel del factor de crecimiento derivado del ce-

rebro (BDNF) y su vía de señalización a través del receptor de la tirosina quinasa

B (TrkB) en el hipocampo. Las pruebas de comportamiento incluyeron la prueba

de preferencia por la sacarosa, la actividad motora en campo abierto, el nado for-

zado y el laberinto de Morris. Se evaluaron la morfología neuronal y la expresión

de BDNF y TrkB mediante técnicas de PCR cuantitativa y Western blot. La expo-

sición a los nanoplásticos, así como el estrés crónico, indujeron una disminución

de la actividad en las pruebas de conducta. Además, se observó una disminución

de las neuronas del hipocampo y de la memoria espacial en ambos tratamientos.

La combinación de estrés crónico y la exposición a los nanoplásticos tuvo un

efecto sumativo sobre las pruebas conductuales y cognitivas, así como sobre la

expresión de BDNF y del receptor Trak B. En conclusión, la exposición a los nano-

plásticos tiene efectos neurotóxicos que pueden potenciar los efectos del estrés

crónico a nivel cognitivo y conductual. Dichos efectos se mediaron mediante la

supresión de la vía de señalización de BDNF y del receptor TrkB en el hipocampo.

Received: 27-11-2025 Accepted: 04-03-2026

neurons and deficits in spatial learning and memory compared with the con-

trol group. BDNF mRNA and TrkB protein levels were decreased. Compared

with the Mod group, the NPs+Mod group exhibited increased depression-like

behaviors and cognitive impairments, greater hippocampal neuronal damage,

and further reductions in BDNF and TrkB mRNA and protein levels. In conclu-

sion, NP exposure has neurotoxic properties that can exacerbate CUMS-induced

depression-like behavior and cognitive deficits, likely by further suppressing the

hippocampal BDNF/TrkB signaling pathway.

INTRODUCTION

Nanoplastics (NPs), an emerging class

of environmental contaminants, are attract-

ing increasing attention due to their poten-

tial health hazards. Owing to their minute

size, NPs can cross biological barriers, dis-

tribute widely throughout the organism,

and adversely affect various physiological

functions, including entry into the nervous

system 1. Accumulating evidence from ex-

perimental studies shows that exposure to

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 277

Vol. 67(2): 275 - 288, 2026

NPs can lead to heightened anxiety and de-

pression-like phenotypes in animals 2. While

direct human epidemiological data linking

NPs to specific mental health disorders re-

main limited, these controlled experimen-

tal models are critical because they provide

a practical tool to elucidate the underlying

neurotoxic mechanisms. For example, they

allow precise dose control, detailed patho-

logical examination, and the isolation of

causal pathways that are not feasible in hu-

man studies. On the other hand, depression

is a debilitating mental disorder character-

ized by low mood and significant impair-

ments in cognition and motivation, repre-

senting a major public health concern 3,4.

Given unavoidable human exposure to plas-

tic pollution, it is imperative to employ such

experimental approaches to comprehensive-

ly investigate the impact of NPs on mental

health, specifically their mechanisms for dis-

rupting emotional regulation and cognitive

function under conditions such as chronic

unpredictable mild stress (CUMS) 5.

The hippocampus is a crucial brain

region that regulates emotional responses

and cognitive functions, and its dysfunction

has been implicated in the pathogenesis of

related disorders 6. The “neuronal damage

hypothesis” of depression posits that an in-

sufficiency of neurotrophic factors plays a

pivotal role in pathological changes within

the hippocampus and in the pathogenesis of

the disorder 7. These neurotrophic factors

are vital for neuronal survival and synaptic

plasticity. Chief among them is brain-derived

neurotrophic factor (BDNF), which, by bind-

ing to its receptor, the tyrosine kinase recep-

tor B (TrkB), activates a critical signaling

pathway for neuronal growth, development,

and maintenance 8. Disruption of the BDNF/

TrkB signaling pathway can impair neuronal

function, thereby adversely affecting emo-

tional regulation and cognitive capabilities.

This study aims to investigate the com-

bined neurotoxic effects of NPs and CUMS

in mice, with a specific focus on depression-

like behaviors and cognitive deficits. The

effects were evaluated using a battery of

behavioral tests, histopathological examina-

tions, and molecular biological techniques.

Furthermore, the study analyzes the involve-

ment of the BDNF/TrkB signaling pathway

in these processes, thereby providing a sci-

entific basis for understanding the mecha-

nisms underlying NPs-induced neurotoxicity

and informing the development of potential

intervention strategies.

MATERIALS AND METHODS

Experimental animals

We purchased a total of thirty-two fe-

male BALB/c mice. They were SPF-grade and

6 to 8 weeks old. The overall body weight of

the mice was approximately 20-25 grams.

The mice were obtained from the Hubei

Provincial Research Center for Laboratory

Animals (animal qualification certificate no.

42000600056335). The animal room’s envi-

ronmental parameters were set to maintain

a temperature of 24-26°C and a humidity of

45-50%. Additionally, a 12-hour light/dark cy-

cle was implemented. Mice had unrestricted

access to food and water. This study was ap-

proved by the Ethics Committee of Hubei Uni-

versity of Science and Technology (Approval

Certificate ID: HBUST-IACUC-2024-11-010),

which is available upon request.

Reagents and kits

Polystyrene nanoparticles (PS-NPs)

with a diameter of 25 nm were obtained

from Zhongke Leiming Technology Co., Ltd.

(Product No. PS000025). The Nissl stain-

ing solution was obtained from Shanghai

Biyuntian Biotechnology Co., Ltd. (Product

No. C0117). For RNA extraction, we used a

kit from Shenzhen Dakewei Biotechnology

Co., Ltd. (Product No. 8034111). The quan-

titative PCR (qPCR) kit was obtained from

Beijing Quanshijin Biotechnology Co., Ltd.

(Product No. Q601-02). Furthermore, anti-

bodies against BDNF and TrKB were obtained

from Abcam, USA (Product Nos. ab108319

and ab187041).

278 Chang et al.

Investigación Clínica 67(2): 2026

Fig. 1. Neurobiological effects of NPs on stressed mice. A: Scientific hypothesis of the study. B: Experimental

groups and treatment protocol.

The equipment used in this research in-

cluded the following: EthoVision XT Version

12.0 by Noldus (Netherlands) for record-

ing neurobehavioral data in experimental

animals; an Olympus dp73 biological mi-

croscope (Japan); a Leica RM2245 paraffin

microtome (Germany); a Sartorius BAS124S

electronic balance (Germany); a Sanyo MDF-

U53V ultra-low-temperature freezer operat-

ing at -80 °C (Japan); an Eppendorf 5415R

low-temperature centrifuge (Germany); a

BioTek Epoch microplate reader (USA); a

Bio-Rad CFX Connect real-time PCR system

(USA); and a Bio-Rad PowerPac Basic system

for Western blot electrophoresis (USA).

Animal grouping and modeling

After a week of adaptive feeding, 32

BALB/c mice were randomly assigned to

four groups (n=8 per group): the control

group (Ctrl), the nanoplastics group (NPs),

the model group (Mod), and the combined

nanoplastics and model group (NPs+Mod).

The NPs and NPs+Mod groups received oral

gavage with NPs (5 mg/kg/d, 10 μL/g) 9,

whereas the Mod and NPs+Mod groups un-

derwent CUMS to establish a depression

model. Mice in the Ctrl group received an

equivalent volume of 0.9% sodium chloride

solution via gavage.

The CUMS protocol for the Mod and

NPs+Mod groups consisted of a variety of

unpredictable mild stressors applied over 4

weeks 10. The stressors included tail clamp-

ing for 10 minutes, a 24-hour fast, a 24-hour

water deprivation, inversion of the circadian

rhythm, overnight light exposure, place-

ment in an empty cage for 12 hours, contact

with damp bedding for 12 hours, cage shak-

ing for 15 minutes, foot shocks for 15 min-

utes, stroboscopic lighting for 10 minutes,

refrigeration for 10 minutes, and swimming

in cold water at 10°C for 10 minutes. Mice

experienced 1-2 stressors each day, with the

type of stressor varying daily to avoid repeti-

tion and ensure variability over a three-day

period. One or two different stressors were

applied daily in a random order to prevent

habituation. The experimental design is

shown in Fig. 1.

General condition inspection

The eating patterns, fur quality, and

body weight of mice were monitored before

the first exposure and after the final expo-

sure to the experimental protocol.

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 279

Vol. 67(2): 275 - 288, 2026

Behavioral testing

Sucrose preference test experiment

Before this experiment commenced,

mice underwent a 3-day training phase that

included sucrose water. Each cage contained

two identical bottles in appearance and vol-

ume, which were rotated every 12 hours to

prevent the formation of a routine memory.

On the first day, each bottle was filled with a

1% sucrose solution. The next day, one bottle

was replaced with an equivalent amount of

distilled water. By the third day, the mice

were denied access to any food or water. The

evaluation took place on the fourth day, when

each cage received one bottle of distilled wa-

ter and another containing the 1% sucrose

solution. After 24 hours, the weights of the

bottles (in grams) were recorded. Sucrose

solution and water consumption were deter-

mined by measuring the weight loss of each

bottle. The sucrose preference rate (%) was

calculated as: sucrose solution consumption

/ (sucrose solution consumption + water

consumption) × 100%, which indicated the

proportion of sucrose solution consumed by

each group of mice prior to and following

the final exposure.

Open field test

After the final NP administration, mice

were placed at the center of an open-field re-

action chamber measuring 100 cm in length,

100 cm in width, and 40 cm in height. At

the same time, video recording and timing

were started, and behavior was recorded for

5 minutes. The experimental data were col-

lected and analyzed using EthoVision XT

Version 13.0 software.

Forced swim test

Mice were placed in a clear acrylic cylin-

der, 50 cm high and 30 cm in diameter, filled

with water to a depth of 30 cm at a tempera-

ture of (24±2) °C. Each mouse was carefully

positioned upright in the water, ensuring

that its limbs did not touch the bottom of the

cylinder as it struggled and swam. The first 2

minutes were not timed, and the duration of

immobility during the subsequent 4 minutes

was measured. Immobility was defined as the

state in which mice ceased struggling and

made only minimal movements necessary to

keep their heads above water. Following the

swimming session, the animal was swiftly re-

moved from the water, dried, and placed in a

warm setting.

Morphological detection

After the final NPs administration, mice

were anesthetized intraperitoneally with

1% sodium pentobarbital (40 mg/kg) and

placed in a supine position on the surgical

table. An incision was made in the chest cav-

ity to expose the heart, and an infusion nee-

dle was carefully inserted 5-6 mm into the

cardiac apex, aligned with the heart’s axis.

Once perfusion commenced, the right atri-

um was cut. The blood vessels were quickly

flushed with 50 mL of warm physiological sa-

line, followed by fixation perfusion with 450

mL of pre-cooled 4% paraformaldehyde. The

whole brain was then removed and immersed

in the same fixative for an additional 6 to 12

hours. Following standard paraffin embed-

ding procedures, sections approximately 5

μm thick were prepared. These sections were

stained with Nissl for 10 minutes to 1 hour,

rinsed with double-distilled water, differenti-

ated with 70% alcohol, dehydrated through

a graded alcohol series, cleared with xylene,

and finally mounted. Morphological altera-

tions in hippocampal neurons were exam-

ined under an optical microscope and docu-

mented for archival records.

Cognitive function assessment

This study used the Morris water maze,

a widely recognized method for assessing

spatial learning and memory in rodents. The

apparatus consisted of a cylindrical water

tank with a diameter of 100 cm and a depth

of 20 cm, maintained at a water temperature

of 24±2 °C. The tank was artificially divided

into four quadrants: northeast (NE), south-

east (SE), northwest (NW), and southwest

(SW). The escape platform was positioned at

280 Chang et al.

Investigación Clínica 67(2): 2026

the center of the SE quadrant, submerged 1

cm below the water’s surface.

During the spatial acquisition phase,

mice were placed in the water, facing the

wall, at one of four designated starting points

(N, E, S, W) and allowed to locate the hidden

platform in the southeast quadrant. After the

final trial, the swimming routes of mice from

the different experimental groups, as well as

the time spent in the target quadrant, were

recorded. The experimental data were then

collected and analyzed using EthoVision XT

Version 13.0 software.

Detection of mRNA transcription levels

Total RNA was isolated from hippocam-

pal cells of mice according to the Trizol kit

instructions, and RNA concentration was

measured. The extracted RNA was reverse-

transcribed into complementary DNA (cDNA)

using Oligo(dT) primers. Primers targeting

mouse BDNF, TrkB, and β-actin were designed

based on their complete GenBank sequenc-

es. The primer sequences are as follows:

BDNF: forward, 5′-GTGTGACAGTATTAGC-

GAGTGGG-3′; reverse, 5′-ACGATTGGG-

TAGTTCGGCATT-3′. TrkB: forward, 5′-AGA-

CAAACCCAAATTACCCTGA-3′; reverse,

5′-ACTTTTGTTCGTAGTATCCCCA-3′. β-actin:

forward, 5′-CTCATGCCATCCTGCGTCT-3′;

reverse, 5′-ACGCACGATTTCCCTCTCA-3′.

These primers were synthesized by Beijing

Tsingke Biotech Co., Ltd. Using the reverse-

transcribed cDNA as a template, PCR am-

plification was performed according to the

qPCR kit protocol. The reaction mixture (15

μL) consisted of 1.5 μL of cDNA, 7.5 μL of

2×SYBR Green Realtime PCR Master Mix,

0.5 μL of each primer (10 μmol·L⁻¹), and 5 μL

of nuclease-free water. The PCR amplification

protocol consisted of an initial denaturation

at 95°C for 30 s, followed by 39 cycles of dena-

turation at 95°C for 5 s and annealing/exten-

sion at 60°C for 30 s.

Detection of protein expression levels

To obtain total protein from hippocam-

pal tissue, we measured protein concentra-

tion using the BCA method. Next, protein

samples were mixed with 5× SDS-PAGE

loading buffer and heated for 8 minutes to

denature them. Following this step, proteins

were separated by electrophoresis on a 12%

SDS-polyacrylamide separating gel with a 5%

stacking gel. Proteins were transferred to a

PVDF membrane by wet transfer for 90 min-

utes. The membrane was blocked with 5%

skim milk for 90 minutes at room tempera-

ture with shaking. Subsequently, the mem-

brane was incubated overnight at 4°C with

primary antibodies against BDNF (1:1000)

or TrkB (1:5000). After washing five times

for 8 minutes each with TBST, the membrane

was incubated with an HRP-conjugated sec-

ondary antibody (1:10000) for 60 minutes at

room temperature. After a subsequent wash,

the signal was detected using the improved

chemiluminescence technique, with β-actin

serving as a reference control.

Statistical analysis

Data were collected from six biologi-

cal replicates, and results were reported as

mean ± standard deviation (Mean ± SD).

Statistical analyses were conducted in SPSS

version 28.0, and GraphPad Prism 10.0 was

used for data visualization. For comparisons

between two groups, an independent-samples

Student’s t-test was used. For comparisons in-

volving three or more groups, one-way analy-

sis of variance (One-way ANOVA) was applied,

followed by Tukey’s post hoc test for multiple

comparisons. A probability value (p) below

0.05 was considered statistically significant.

RESULTS

General condition of mice in each group

Before the experiment, all groups of

mice had similar overall conditions. After

the final exposure, mice in the Ctrl group ex-

hibited normal food intake, shiny fur, and a

significant increase in body weight (Fig. 2).

In sharp contrast, mice in the NPs, Mod, and

NPs+Mod groups showed reduced food in-

take and rough, lackluster fur. Although the

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 281

Vol. 67(2): 275 - 288, 2026

Fig. 2. Changes in body mass in each group. Note: vs. before poisoning, ** p<0.01, *** p<0.001; vs. Ctrl

group, ## p<0.01, ### p<0.001; vs. Mod group, & p<0.05, && p<0.01.

NPs and Mod groups showed a slight increase

in body weight, the gain was significantly less

than that in the Ctrl group. The NPs+Mod

group showed little to no increase in body

weight (Fig. 2).

Behavioral experiments

The effect on the sucrose preference rate

in mice

A significant reduction in sucrose pref-

erence was observed in mice from the NPs,

Mod, and NPs+Mod groups after the final

exposure, compared with the Ctrl group (p<

0.05). Furthermore, mice in the NPs+Mod

group showed a marked decline in sucrose

preference compared with the Mod group (p

<0.05) (Fig. 3A).

The effect on the spontaneous activity

of mice in the open field test

After the final exposure, mice in the

NPs, Mod, and NPs+Mod groups showed re-

duced spontaneous activity in the open field

test compared with the Ctrl group (Fig. 3B).

This reduction was evidenced by a signifi-

cant decrease in time spent in the central

area, a concomitant increase in time spent

near the walls, and reductions in total dis-

tance traveled and average speed (p<0.05).

Moreover, compared with the Mod group,

the NPs+Mod group exhibited a more pro-

nounced decrease in spontaneous activity,

characterized by an even greater preference

for the periphery and further reductions in

total distance traveled and average speed (p

<0.05) (Fig. 3C).

The effect on immobility time during

the forced swim test in mice

After the final exposure, the NPs-treat-

ed group, the Mod group, and the combined

NPs+Mod treatment group exhibited behav-

iors indicative of despair. This was evidenced

by a significantly longer immobility duration

in these groups than in the control group.

The statistical analysis confirmed that these

differences were significant (p<0.05). Fur-

thermore, when comparing the NPs+Mod

group to the Mod group, the mice in the

NPs+Mod group exhibited a longer duration

of immobility, and this difference was also

statistically significant (p<0.05) (Fig. 3D).

Morphological changes in hippocampal

neurons

After the last exposure, hippocampal

neurons in the control group of mice were

organized in an orderly manner, with normal

intercellular spaces, consistent morphology,

intact structures, numerous Nissl bodies, and

282 Chang et al.

Investigación Clínica 67(2): 2026

Fig. 3. Behavioral changes in each group. A: Sugar water preference test. B: Open field test (blue line: mouse

path; absence of line: mouse did not enter that area). C: Autonomous activity. D: Immobility time.

Note: vs. Ctrl group, ## p<0.01, ### p<0.001; vs. Mod group, && p<0.01.

uniform, pronounced staining. In contrast,

the NPs group, Mod group, and NPs+Mod

group showed disordered cell arrangements,

enlarged intercellular spaces, substantial

neuronal loss, and lighter staining of Nissl

bodies. Importantly, the NPs+Mod group ex-

hibited more pronounced alterations than

the Mod group (Fig. 4).

The Morris water maze experiment

After the final exposure to the toxin,

mice in the NPs, Mod, and NPs+Mod groups

showed a significant decline in spatial mem-

ory capacity compared with the Ctrl group.

This decline was evidenced by a significantly

shorter time spent in the target quadrant

and fewer platform location crossings during

the probe trial (p<0.05). Furthermore, com-

pared with the Mod group, the NPs+Mod

group exhibited even more severe spatial

memory impairment, with a further signifi-

cant reduction in both time spent in the

target quadrant and the number of platform

crossings (p<0.05) (Figs. 5 and 6).

Changes in the expression levels of BDNF

and TrkB in the hippocampal tissue

After the final exposure, reductions in

BDNF and TrkB mRNA transcription and

protein expression were observed in the hip-

pocampal tissue of mice in the NPs, Mod,

and NPs+Mod groups compared with the

Ctrl group, demonstrating statistically sig-

nificant differences (p<0.05). Furthermore,

the NPs+Mod group showed significantly

greater reductions in BDNF and TrkB mRNA

and protein levels than the Mod group (p<

0.05) (Fig. 7).

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 283

Vol. 67(2): 275 - 288, 2026

Fig. 4. Pathological changes of hippocampal neurons in each group. Nissl staining, ×100.

Fig. 5. Morris water maze test in each group. Blue line: mouse path; absence of line: mouse did not enter

that area.

Fig. 6. Target quadrant dwell time in each group.

Note: vs. Ctrl group, ## p<0.01, ### p<

0.001; vs. Mod group, && p<0.01.

DISCUSSION

Global ecological and health implications

of NPs exposure

In recent years, the widespread pres-

ence and ongoing release of NPs have be-

come a major global environmental issue.

Their high mobility enables migration across

aquatic environments, terrestrial ecosys-

tems, and atmospheric layers, ultimately

disrupting ecological balance and posing

significant threats to human health through

bioaccumulation and trophic transfer. A no-

table concern is the confirmed neuroinvasive

capability of NPs, as evidenced by their abil-

ity to cross the blood-brain barrier and ac-

cumulate in the central nervous system 11.

The distinct physicochemical characteristics

of NPs, including a remarkably high specific

surface area and surface energy, promote

strong interactions with receptors on neural

membranes (Fig. 2). Such interactions may

impair neuronal function, leading to mood

changes and cognitive challenges 12.

284 Chang et al.

Investigación Clínica 67(2): 2026

Fig. 7. Changes in the expression levels of BDNF and TrkB. A: Protein expression levels. B: mRNA transcrip-

tion levels. Note: vs. Ctrl group, # p<0.05, ## p<0.01, ### p<0.001; vs. Mod group, & p<0.05,

&& p<0.01.

Given the neuroinvasive potential of

NPs, there is growing concern about their

role in mental health disorders, particularly

depression 13. The implications of depres-

sion for public health are significant; the

global prevalence of depression increased

by 34%, and the prevalence of major depres-

sive disorder rose to 8%. The proportion

of adolescents suffering from depression

increased from 24% during 2001-2010 to

37% between 2011-2020 14. According to

the report, in 2016, depression was among

the leading global causes of disability. We

therefore use the CUMS model, a widely

used preclinical research framework with

good construct validity and lasting behav-

ioral outcomes. This model is highly ap-

propriate for studying the pathogenesis of

depression8. The objective of this study was

to investigate the neurotoxicological ef-

fects of NPs and to assess their impact on

depression-like behaviors and cognitive per-

formance in CUMS mice. We also evaluated

the role of the BDNF/TrkB signaling path-

way in these processes.

Behavioral alterations induced by NPs

exposure in mice

The current study employed an exten-

sive behavioral battery to systematically

investigate the effect of NPs exposure on

depression-like behavior in mice 15. Our re-

sults reveal that exposure to NPs causes

substantial behavioral changes that corre-

late with states analogous to depression, as

determined by three established behavioral

assays. Reduced sucrose preference indi-

cates that exposure to NPs may disrupt the

reward-processing machinery, a core fea-

ture of anhedonia (Fig. 3). The changes in

movement and reduced exploration of the

center zone in the open field test may also

reflect increased generalized psychomotor

slowing or anxiety-like behaviors. Clinical

reports of psychomotor agitation or retarda-

tion are consistent with these results among

depressed persons he changes in movement

and reduced exploration of the center zone

in the open field test may reflect either gen-

eralized psychomotor slowing or anxiety-like

behaviors, which is consistent with clinical

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 285

Vol. 67(2): 275 - 288, 2026

reports of psychomotor disturbances in de-

pressed individuals (Fig. 3). The increased

immobility time in the forced swim test, of-

ten interpreted as behavioral despair, further

supports the emergence of a depression-like

phenotype following NPs exposure (Fig. 3).

Collectively, the consistent findings across

these diverse behavioral paradigms robustly

demonstrate that NPs exposure induces a

depression-like state in mice. Our finding

that NPs exposure intensifies the behavioral

outcomes of CUMS is particularly signifi-

cant, indicating that NPs exposure not only

triggers depression-like behaviors but also

amplifies the effects of CUMS. These results

indicate that exposure to NPs may interfere

with emotional regulation in mice and exac-

erbate characteristics associated with stress-

induced depression.

Cognitive impairment induced by NPs

exposure in mice

The hippocampus is a pivotal structure

within the limbic system, widely connected

to brain regions such as the prefrontal cor-

tex and amygdala, and is critically involved

in emotional processing and cognitive func-

tions, particularly learning and memory16.

Our histological observations show that

exposure to NPs causes severe neuropatho-

logical alterations in the hippocampus, in-

cluding disorganized neuronal arrangement,

widened intercellular spaces, extensive neu-

ronal loss, and markedly reduced Nissl stain-

ing. These morphological alterations were

more severe in the NPs+Mod group than in

the Mod group alone, strongly implying that

NPs exposure exacerbates CUMS-induced

hippocampal neuropathology, thereby con-

tributing to the observed emotional dysregu-

lation (Fig. 4).

Consistent with the hippocampus’s

critical role in cognition, hippocampal im-

pairment also substantially affects spatial

memory 17. The Morris water maze test as-

sesses spatial learning and memory in mice.

Findings showed that, compared with the

control group, the NPs group, Mod group,

and NPs+Mod group spent less time in the

target quadrant, took fewer paths within

that quadrant, and exhibited reduced goal-

directed movement. Additionally, compared

with the Mod group, the NPs+Mod group

spent even less time in the target quadrant

and took fewer paths. This strongly indi-

cates that exposure to NPs alone impairs

hippocampal-related learning and memory

functions and compounds cognitive deficits

caused by CUMS (Figs. 5 and 6).

NPs exposure causes neuronal damage

in mice

BDNF is a signaling protein essential

for neuroplasticity and is primarily found in

the central nervous system. It regulates neu-

roregeneration and neuroprotection by ac-

tivating the TrkB receptor. Evidence shows

that activating the BDNF/TrkB signaling

pathway can reduce ischemic stroke damage

while promoting the production of vascular

endothelial cells 18,19. BDNF exerts prolifera-

tive, trophic, and maturational effects on di-

verse neurons, promoting neuronal growth

and the repair of neural structures; its ex-

pression levels can serve as a direct indicator

of neural functional recovery 20,21. Research

by Liu et al. 20 indicated that enhancing the

BDNF/TrkB signaling pathway may reduce

neurotoxicity caused by tetrahydropalma-

tine, which can manifest as depression, anxi-

ety, and cognitive deficits. Decreased expres-

sion of BDNF is associated with neuronal

injury that could disrupt brain function and

cause depression 22. In this research, qPCR

and Western blotting were employed to in-

vestigate mRNA transcription and protein

expression levels of BDNF and TrkB in hip-

pocampal tissues from mice across groups

following the final exposure. The findings

indicate that exposure to NPs significantly

decreased the expression of both BDNF and

TrkB. Furthermore, compared to the Mod

group, the NPs+Mod group exhibited a more

pronounced reduction in BDNF and TrkB

levels. This downregulation of the BDNF/

TrkB signaling pathway likely underlies the

286 Chang et al.

Investigación Clínica 67(2): 2026

observed neuronal damage in the hippo-

campus and, consequently, the exacerbated

impairment of neuronal function, as well as

the associated depression-like behavior and

cognitive deficits, in CUMS mice exposed to

NPs (Fig. 7).

In conclusion, this study demonstrates

that NPs exposure exacerbates neurotoxic-

ity in CUMS mice, worsening depression-like

behavior and cognitive impairment. Mecha-

nistic analysis indicates that these effects

are mediated primarily through disruption

of the BDNF/TrkB signaling pathway, re-

sulting in significant hippocampal neuronal

damage. These findings provide crucial in-

sight into how NPs affect the central nervous

system, offer a scientific basis for under-

standing NPs-induced neuropathology, and

suggest potential therapeutic targets for

stress-related mood and cognitive disorders.

The present study has several limita-

tions that warrant consideration. We used a

specific polystyrene NP model with a defined

size, which does not capture the vast hetero-

geneity of environmental NPs; thus, our find-

ings may not be fully generalizable to other

particle types. Furthermore, although the

study focused on the BDNF/TrkB pathway,

the observed neurotoxicity is likely multifac-

torial, potentially involving interrelated pro-

cesses such as oxidative stress, neuroinflam-

mation, and cell death that were not fully

elucidated here. Future studies should use

more diverse NP models and employ multi-

omics approaches to systematically delin-

eate the complex interplay of downstream

mechanisms.

Funding

This work was funded by the Project of

the Joint Fund for Innovative Development

of the Natural Science Foundation of Hubei

Province (No. 2025AFD411); the Key Project

of the Special Research Fund of the School

of Wuguan Medical College, Hubei Institute

of Science and Technology (No. 2020WG06);

the Faculty Specialized Research Fund Proj-

ect of Medicine, Dental, and Optometric

Medicine, Hubei Institute of Science and

Technology (No. 2021WG10); and the Hubei

Province Key R&D Plan (No. 2022BCE011).

ORCID ID of the authors

• Dewei Chang (DC):

0009-0006-8282-9577

• Miao Xu (MX):

0009-0008-0645-7243

• Wenning Shi (WS):

0009-0007-4977-4014

• Yan He (YH):

0009-0001-1725-3317

• Zhe Wu (ZW):

0009-0000-0465-8748

• Zhifeng Ning (ZN):

0000-0002-6700-161X

• Yanling Sun (YS):

0009-0000-1301-3730

• Jianguo Lv (JL):

0009-0005-0674-4814

Author's contributions

DC, MX, YS and JL conceiving and de-

signing the study; DC, MX, WS, YH, ZW and

ZN collecting the data; DC, MX, WS, YH, ZW

and ZN analyzing and interpreting the data;

DC, MX, WS, ZW, ZN and YS writing the man-

uscript; DC, MX, WS, YH, YS and JL provid-

ing critical revisions that are important for

the intellectual content. All authors approv-

ing the final version of the manuscript.

Conflict of interests

All authors declare that they have no

conflicts of interest.

Ethics approval

This study was approved by the Ethics

Committee of Hubei University of Science

and Technology (Approval Certificate ID:

Neurotoxic effects of NPs on depression-like behavior and cognitive function in mice 287

Vol. 67(2): 275 - 288, 2026

HBUST-IACUC-2024-11-010). The approval

certificate is available upon request.

Availability of data and materials

The datasets used and/or analyzed dur-

ing the current study are available from the

corresponding author on reasonable request.

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