ObjectiveTranscranial magneto-acoustic stimulation (TMAS) is an emerging non-invasive neuromodulation technique that may provide a novel non-pharmacological intervention strategy for Parkinson's disease (PD). PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to motor impairments such as bradykinesia, tremor, and rigidity. Increasing evidence indicates that mitochondrial dysfunction and impaired mitochondrial quality control are central mechanisms underlying dopaminergic neuronal loss. In particular, abnormalities in mitophagy and mitochondrial fission-fusion balance contribute substantially to oxidative stress, energy metabolic failure, and neuronal injury. At present, most clinical treatments for PD mainly alleviate symptoms but do not effectively halt disease progression. Therefore, exploring new interventions targeting the core pathological mechanisms is of considerable significance. This study aims to investigate whether TMAS can improve neural damage and motor dysfunction in PD mice by regulating mitophagy and the fission/fusion dynamic balance, thereby providing theoretical and experimental support for its application in PD treatment. MethodsMale C57BL/6 mice were used in this study. A PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days. After model induction, mice in the intervention group received TMAS once daily for 14 consecutive days, whereas the corresponding control group received sham stimulation. The stimulation target was positioned over the primary motor cortex (M1). Motor performance was evaluated using the pole test and the open-field test. To verify the activation effect of TMAS on the target cortical region, c-Fos immunohistochemistry was performed in the M1. To assess nigral dopaminergic neuronal injury, tyrosine hydroxylase (TH) immunohistochemistry was used to quantify TH-positive neurons in the SNc. Mitochondrial function was evaluated by measuring reactive oxygen species (ROS) levels and adenosine triphosphate (ATP) content in the SNc. Western blot was further performed to determine the expression of mitophagy-related proteins, including PINK1, Parkin, LC3-II, and p62, as well as mitochondrial dynamics-related proteins, including Drp1 and Opa1. ResultsTMAS significantly increased the number of c-Fos-positive cells in M1 (P<0.000 1), indicating effective activation of neurons in the targeted cortical region. Compared with the control group, MPTP-treated mice exhibited marked motor dysfunction, including a significant reduction in total distance traveled in the open-field test (P<0.000 1) and mean speed (P=0.000 1), as well as significant prolongation of turn time and total climbing time in the pole test (P<0.000 1). These behavioral impairments were accompanied by a substantial loss of TH-positive dopaminergic neurons in the SNc, whereas TMAS significantly increased TH-positive neuron survival (P<0.000 1). In parallel, MPTP induced a pronounced increase in ROS levels and a significant reduction in ATP content, indicating severe mitochondrial dysfunction and energy metabolism impairment (P<0.01). TMAS treatment significantly improved motor performance, as reflected by the reversal of MPTP-induced impairment in the open-field and pole tests, and significantly reduced ROS accumulation (P<0.01) while restoring ATP production (P<0.001). At the molecular level, MPTP markedly downregulated PINK1 and Parkin, decreased p62 expression, increased LC3-II accumulation, elevated Drp1 expression, and reduced Opa1 expression, whereas TMAS significantly reversed these abnormalities, suggesting restoration of mitophagy-related mitochondrial quality control and re-establishment of mitochondrial fission-fusion balance. Collectively, these findings indicate that TMAS ameliorates MPTP-induced neurotoxicity and restores mitochondrial homeostasis and energy metabolism. ConclusionTMAS effectively attenuates neural damage and improves motor dysfunction in MPTP-induced PD mice. Its neuroprotective effects are closely associated with multidimensional regulation of the mitochondrial quality control system, including restoration of PINK1/Parkin-mediated mitophagy and rebalancing of Drp1/Opa1-related mitochondrial dynamics. Rather than acting only as a symptomatic neuromodulatory intervention, TMAS may influence a key pathological axis of PD by improving mitochondrial homeostasis in SNc and protecting nigral dopaminergic neurons. These findings provide experimental evidence supporting TMAS as a promising non-invasive physical intervention for PD.

ObjectiveTranscranial magneto-acoustic stimulation (TMAS) is an emerging non-invasive neuromodulation technique that may provide a novel non-pharmacological intervention strategy for Parkinson's disease (PD). PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to motor impairments such as bradykinesia, tremor, and rigidity. Increasing evidence indicates that mitochondrial dysfunction and impaired mitochondrial quality control are central mechanisms underlying dopaminergic neuronal loss. In particular, abnormalities in mitophagy and mitochondrial fission-fusion balance contribute substantially to oxidative stress, energy metabolic failure, and neuronal injury. At present, most clinical treatments for PD mainly alleviate symptoms but do not effectively halt disease progression. Therefore, exploring new interventions targeting the core pathological mechanisms is of considerable significance. This study aims to investigate whether TMAS can improve neural damage and motor dysfunction in PD mice by regulating mitophagy and the fission/fusion dynamic balance, thereby providing theoretical and experimental support for its application in PD treatment. MethodsMale C57BL/6 mice were used in this study. A PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days. After model induction, mice in the intervention group received TMAS once daily for 14 consecutive days, whereas the corresponding control group received sham stimulation. The stimulation target was positioned over the primary motor cortex (M1). Motor performance was evaluated using the pole test and the open-field test. To verify the activation effect of TMAS on the target cortical region, c-Fos immunohistochemistry was performed in the M1. To assess nigral dopaminergic neuronal injury, tyrosine hydroxylase (TH) immunohistochemistry was used to quantify TH-positive neurons in the SNc. Mitochondrial function was evaluated by measuring reactive oxygen species (ROS) levels and adenosine triphosphate (ATP) content in the SNc. Western blot was further performed to determine the expression of mitophagy-related proteins, including PINK1, Parkin, LC3-II, and p62, as well as mitochondrial dynamics-related proteins, including Drp1 and Opa1. ResultsTMAS significantly increased the number of c-Fos-positive cells in M1 (P<0.000 1), indicating effective activation of neurons in the targeted cortical region. Compared with the control group, MPTP-treated mice exhibited marked motor dysfunction, including a significant reduction in total distance traveled in the open-field test (P<0.000 1) and mean speed (P=0.000 1), as well as significant prolongation of turn time and total climbing time in the pole test (P<0.000 1). These behavioral impairments were accompanied by a substantial loss of TH-positive dopaminergic neurons in the SNc, whereas TMAS significantly increased TH-positive neuron survival (P<0.000 1). In parallel, MPTP induced a pronounced increase in ROS levels and a significant reduction in ATP content, indicating severe mitochondrial dysfunction and energy metabolism impairment (P<0.01). TMAS treatment significantly improved motor performance, as reflected by the reversal of MPTP-induced impairment in the open-field and pole tests, and significantly reduced ROS accumulation (P<0.01) while restoring ATP production (P<0.001). At the molecular level, MPTP markedly downregulated PINK1 and Parkin, decreased p62 expression, increased LC3-II accumulation, elevated Drp1 expression, and reduced Opa1 expression, whereas TMAS significantly reversed these abnormalities, suggesting restoration of mitophagy-related mitochondrial quality control and re-establishment of mitochondrial fission-fusion balance. Collectively, these findings indicate that TMAS ameliorates MPTP-induced neurotoxicity and restores mitochondrial homeostasis and energy metabolism. ConclusionTMAS effectively attenuates neural damage and improves motor dysfunction in MPTP-induced PD mice. Its neuroprotective effects are closely associated with multidimensional regulation of the mitochondrial quality control system, including restoration of PINK1/Parkin-mediated mitophagy and rebalancing of Drp1/Opa1-related mitochondrial dynamics. Rather than acting only as a symptomatic neuromodulatory intervention, TMAS may influence a key pathological axis of PD by improving mitochondrial homeostasis in SNc and protecting nigral dopaminergic neurons. These findings provide experimental evidence supporting TMAS as a promising non-invasive physical intervention for PD.

Objective To investigate the temporal changes in pathological damage and macrophage polarization in liver and spleen tissues of mice infected with Angiostrongylus cantonensis, and to preliminarily unravel the peripheral immune responses during the early stage of A. cantonensis infection. Methods Forty female BALB/c mice at ages of 6 to 8 weeks were randomly divided into four groups, including the control group and 7-, 14-, and 21-day infection groups, with 10 mice in each group. Each mouse in the infection groups was inoculated with 30 third-stage (L3) larvae of A. cantonensis by oral gavage, and five mice were randomly selected from each infection group on days 7, 14, and 21 post-infection, while mice in the control group were given the same volume of physiological saline and five mice were randomly selected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled. The histopathological changes of mouse liver and spleen tissues were observed using hematoxylin and eosin (HE) staining, and the percentage of positive staining area and the co-localization positive rates of the macrophage surface antigens F4/80, CD86, and CD206 were quantified in mouse liver and spleen tissues using immunohistochemical and immunofluorescence staining. In addition, five mice were collected from each infection group on days 7, 14, and 21 post-infection, and five mice were collected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled for detection of macrophage markers CD86 and CD206 and macrophage phenotyping using flow cytometry, and the expression of M1 macrophage markers, including inducible nitric oxide synthase (Nos2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and M2 markers, including arginase 1 (Arg1), mannose receptor C-type 1 (Mrc1) and chitinase-like protein 3 (Chil3) was quantified in mouse liver and spleen tissues using real-time quantitative PCR (RT-qPCR) assay. Results Proliferative lesions of the hepatocyte were observed in mouse liver tissues and the follicular structures of the mouse spleen white pulp were disrupted 21 days post-infection with A. cantonensis. Immunohistochemical staining showed that there were significant differences in the percentages of F4/80, CD86 and CD206 positive staining areas in the liver and spleen tissues among the four groups of mice (F = 242.40, 197.14, 183.19, 157.65, 242.35 and 146.24; all P values < 0.001), and the percentages of positive staining in the liver and spleen tissues of mice in the 14-day infection group [(4.45 ± 0.51)%, (3.74 ± 0.67)%, (8.32 ± 0.72)%, (16.56 ± 1.14)%, (11.62 ± 0.52)%, and (8.29 ± 0.72)%, respectively] and the 21-day infection group [(3.70 ± 0.11)%, (3.22 ± 0.43)%, (11.53 ± 1.03)%, (12.59 ± 1.05)%, (9.02 ± 0.83)%, and (11.67 ± 1.10)%, respectively] were higher than in the control group [(0.35 ± 0.16)%, (0.40 ± 0.02)%, (0.93 ± 0.05)%, (2.78 ± 0.26)%, (2.33 ± 0.20)%, and (1.85 ± 0.20)%, respectively] (all P values < 0.05). Immunofluorescence staining showed significant differences in the positive rates of F4/80 co-localization with CD86 and CD206 in mouse liver and spleen tissues among the four groups (F = 24.42, 25.28, 54.51 and 130.55; all P values < 0.001). Flow cytometry detected significant differences in the proportions of CD86⁺ and CD206⁺ macrophages in mouse liver and spleen tissues among the four groups (F = 67.98, 18.41, 29.77, 172.80; all P values < 0.001), and the proportions of CD206⁺ macrophages in the liver and spleen of the 21-day infection group were significantly higher than those in the control group [(9.25 ± 2.55)% vs (3.83 ± 0.72)%, and (4.22 ± 0.56)% vs (0.47 ± 0.18)%, respectively] (both P values < 0.05). In addition, RT-qPCR assay quantified significant differences in the relative mRNA expression of M1 macrophage markers (IL-1β, TNF-α and Nos2) and M2 macrophage markers (Arg1, Chil3 and Mrc1) in mouse liver and spleen tissues among the four groups (F = 41.30, 31.82, 199.33, 19.96, 62.01, 119.76, 23.67, 95.90, 72.27, 82.59, 123.41 and 29.75; all P values < 0.05). Conclusions A. cantonensis infection may cause progressive pathological damage in mouse liver and spleen tissues, accompanied by dynamic temporal changes in macrophage polarization. M1 macrophage polarization predominates at the early stage of A. cantonensis infection and shifts towards M2 polarization at the later stages, suggesting that M2 polarization may participate in immune regulation at late stages of A. cantonensis infection by suppressing excessive inflammatory responses and promoting tissue repair.

Objective: To examine the characteristics of physical activity among secondary vocational school students and its associations with depressive symptoms, anxiety symptoms, and their co-existence, so as to provide evidence for promoting their mental health. Methods: From September to December 2024, a questionnaire survey was conducted on 5 141 secondary vocational school students using convenience sampling method across 16 secondary vocational schools in 8 provinces/municipalities, including Fujian, Chongqing, Guangdong, Guangxi, Hubei, Jiangxi, Shanghai and Zhejiang. A self designed questionnaire, International Physical Activity Questionnaire Short Form, Patient Health Questionnaire-9 and Generalized Anxiety Disorder Scale-7 were used to assess basic information, physical activity characteristics, and depressive and anxiety symptoms. Chi-square test and Logistic regression model were used to analyze the associations of physical activity characteristics with depressive symptoms, anxiety symptoms, and their co-existence. Results: Among secondary vocational school students, 22.2% had depressive symptoms, 12.2% had anxiety symptoms, and 10.5% had both conditions. After adjustment for confounding factors such as sex, grade, and family structure, Logistic regression analyses showed that a high physical activity level, meeting the recommendation for daily moderate to vigorous physical activity (MVPA), and a concentrated physical activity pattern were associated with lower odds of depressive symptoms, anxiety symptoms, and their co-existence ( OR =0.70-0.81, all P <0.05). Regular weekly MVPA and a regular physical activity pattern were associated only with lower odds of depressive symptoms ( OR =0.79, 0.70, both P <0.05). Sex stratified analyses showed that meeting the recommendation for daily MVPA and a concentrated physical activity pattern were associated with lower odds of depressive symptoms, anxiety symptoms, and their co-existence among boys ( OR =0.60-0.71); a high physical activity level, regular weekly MVPA, and a regular physical activity pattern were associated with lower odds of depressive symptoms among boys ( OR =0.65-0.78)(all P <0.05). Meanwhile, a high physical activity level, meeting the recommendation for daily MVPA, regular weekly MVPA, and regular and concentrated physical activity patterns were associated with lower odds of depressive symptoms in female students ( OR =0.72-0.79) (all P <0.05). Conclusions Physical activity characteristics are associated with depressive symptoms, anxiety symptoms, and their co-existence among secondary vocational school students. Schools should optimize physical activity promotion strategies according to sex specific characteristics to reduce the occurrence of depressive and anxiety symptoms among secondary vocational school students.

Bruch's membrane opening(BMO)is the true anatomical border of the optic nerve head and provides higher anatomical accuracy, structural stability and measurement repeatability than traditional assessments based on the clinically visible optic disc margin. Optical coherence tomography(OCT)serves as a key technical tool for quantitative assessment of BMO-associated structural parameters. With the development of spectral-domain OCT(SD-OCT)and swept-source OCT(SS-OCT), parameters such as BMO-minimum rim width(BMO-MRW), BMO-minimum rim area(BMO-MRA), BMO area, offset, tilt, and torsion have emerged as important imaging biomarkers for evaluating optic nerve head morphology and for assisting in the diagnosis, staging, and longitudinal monitoring of glaucoma, myopia, and pathological myopia. Traditionally, the acquisition of these parameters has relied heavily on manual segmentation, which is limited by low efficiency, strong subjectivity, and considerable inter-observer variability. In recent years, artificial intelligence has been increasingly applied to OCT image segmentation, quality control, cross-device calibration, and automated quantitative analysis, thereby improving efficiency, objectivity, and reproducibility, particularly in eyes with complex optic disc morphology. This review summarizes recent advances in the BMO-related terminology standardization, imaging and quantitative methodologies, quality control,artificial intelligence-assisted analysis, as well as clinical evidence of its application in glaucoma, myopia and pathological myopia. Particular emphasis is placed on their potential value in the differential diagnosis and follow-up evaluation of highly myopic eyes with glaucoma, aiming to provide a reference for the standardized clinical application and future translational use of BMO-related parameters.

ObjectiveTo generate cynomolgus and rhesus monkey models carrying the Booroola fecundity (FecB) variant of bone morphogenetic protein receptor type 1B (BMPR1B) gene (designated BMPR1B^FecB) using adenine base edit technology and to explore genetic improvement strategies for enhancing the reproductive performance of laboratory macaques. MethodsFour single-guide RNAs (sgRNAs) were designed to target the BMPR1B c.746A>G locus in cynomolgus and rhesus monkeys. The sgRNAs were combined with the ABE8e-SpRY base editor, and the resulting editing complexes were microinjected into zygotes of both macaque species. Following in vitro culture of the injected embryos, nested PCR amplification was performed to detect the base-editing efficiency at the target site to screen for highly efficient sgRNAs. Subsequently, normally developing gene-edited embryos were transferred into the oviducts of surrogates. Seven days after birth, ear skin or peripheral blood samples were collected from the newborn monkeys for genotyping and mosaicism analysis, and the growth, development, and health status of the gene-edited monkeys were monitored. ResultsAmong the four candidate sgRNAs, sgRNA4 was identified as achieving 100% editing efficiency at the c.746A>G site. A total of 17 live monkey offspring were obtained via embryo transfer, including 8 cynomolgus monkeys and 9 rhesus monkeys. Genotyping results showed that all 8 newborn cynomolgus monkeys carried the c.746A>G point mutation, with an editing efficiency of 100% (8/8), of which 37.50% (3/8) were mosaics. Among the 9 newborn rhesus monkeys, 8 individuals carried the c.746A>G mutation, with an editing efficiency of 88.89% (8/9), of which 75.00% (6/8) were mosaics. ConclusionThe ABE8e-SpRY base-editing system enables precise editing at the c.746A>G (p.Q249R) locus of the BMPR1B gene in both cynomolgus and rhesus monkey embryos, resulting in the successful establishment of laboratory monkey models carrying the BMPR1B^FecB variant. These models provide a valuable foundation for future studies of the regulatory role of the BMPR1B^FecB variant in reproductive performance at the non‑human primate level, as well as for exploring genetic improvement strategies to enhance reproductive performance in laboratory monkeys.

Metabolic dysfunction-associated fatty liver disease （MAFLD） has become the most common chronic liver disease worldwide. Its pathological process is centered on the “two-hit” theory， with gut-liver axis dysregulation running through the entire disease course from steatosis to liver cirrhosis and even hepatocellular carcinoma， and it forms an extensive cross-system regulatory mechanism with the metabolic， cardiovascular， renal， and psychological and nervous systems through multi-dimensional pathways such as the “gut-liver-brain axis”， the “liver-kidney axis”， and the “liver-heart axis”. Disease progression is not limited to the liver itself， and it also involves functional imbalance of multiple organ systems throughout the body. This article systematically elaborates on the association mechanism of “liver pathological progression-multi-axis regulation-comorbidity occurrence” in MAFLD， proposes synergistic management strategies integrating early screening based on risk stratification， targeted intervention， and multidisciplinary diagnosis and treatment， and analyzes the limitations of current research and future development directions， in order to provide a theoretical basis and practical guidance for precise diagnosis and treatment and individualized management of MAFLD.

Objective Using cockroach surveillance data in Nanjing from 2017 to 2024, we have descriptively analyzed population characteristics and changes in insecticide resistance among Blattella germanica, and have established a seasonal Autoregressive Integrated Moving Average (ARIMA) model to predict monthly population densities in Nanjing spanning the year 2025. Methods B. germanica density and infestation rate were monitored using the sticky trap method, and insecticide resistance was assessed using the residual contact method. Our ARIMA model was applied to fit and estimate cockroach density fluctuations over time. Results From 2017 to 2024, B. germanica accounted for 94.90% of all cockroach specimens captured in Nanjing, with an average density of 0.37 cockroaches per trap and an average infestation rate of 5.56%. Significant differences in density and infestation rate were observed among different habitats (P＜0.001). Significant downward annual trends in both density and infestation rate were found (P＜0.05). ARIMA was found to be the optimal model for fitting the density data from 2017 to 2024 (0, 1, 1) × (0, 1, 1)1 2. Conclusions From 2017 to 2024, B. germanica was overwhelmingly the dominant cockroach species in Nanjing, with both its density and infestation rate declining annually. A positive correlation was observed between the amount and proportion of insecticide use and the development of insecticide resistance (P＜0.05). The established model demonstrated good performance in predicting monthly B. germanica densities in Nanjing throughout 2025, providing a valuable reference for future surveillance and control efforts.

ObjectiveTo investigate the effects of Xijiao Dihuangtang （XJDHT） on mice with sepsis and cellular models of sepsis and explore its molecular mechanism in alleviating sepsis-induced inflammatory responses via regulating pyruvate kinase M2 （PKM2）-mediated one-carbon metabolism pathway. MethodsForty C57BL/6N mice were randomly divided into four groups： normal group， model group， low-dose XJDHT group （7.7 g·kg^-1）， and high-dose XJDHT group （15.4 g·kg^-1）. After one week of continuous gavage， sepsis was induced using cecal ligation and puncture （CLP） in groups except the normal group. 24 h after the surgery， mortality rates in all groups were recorded， and serum cytokines were measured by enzyme linked immunosorbent assay （ELISA）. Lung histopathology was examined by hematoxylin-eosin （HE） staining. During the in vitro experiment， the human monocytic leukemia cell line （THP-1） was exposed to various concentrations of XJDHT and treated with lipopolysaccharide （LPS） at a final concentration of 2 mg·L^-1 for 24 h. Cell apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay. Protein levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， B-cell lymphoma 2 （Bcl-2）， and Bcl-2-associated X protein （Bax） were measured by Western blot. Transcriptome sequencing was performed to analyze differentially expressed genes in all groups and conduct gene ontology （GO） enrichment. Key genes in the one-carbon metabolism pathway， including pyruvate kinase M2 （PKM2）， 5-methyltetrahydrofolate-homocysteine methyltransferase （MTR）， and phosphoglycerate dehydrogenase （PHGDH）， were verified by Western blot. A PKM2 inhibition model was established using shikonin for further protein expression analysis. ResultsAnimal experiments showed that compared with the normal group， the model group exhibited significantly elevated body temperature and lung pathology （P<0.01） and increased serum TNF-α and IL-1β levels （P<0.01）. High-dose XJDHT reduced body temperature and lung tissue damage （P<0.01） and significantly decreased serum TNF-α and IL-1β levels （P<0.01）. Low-dose XJDHT treatment showed no significant temperature change （P<0.01） but reduced serum TNF-α and IL-1β levels （P<0.01）. Transcriptome sequencing and Western blot revealed significant differences in the expression of TNF-α， IL-1β， and one-carbon metabolism genes （PKM2， MTR， and PHGDH） （P<0.01）. Cell experiments demonstrated that compared to the normal group， the model group showed elevated protein expressions of TNF-α and IL-1β in THP-1 cells （P<0.01）， decreased Bcl-2/Bax ratio， and increased apoptosis （P<0.01）. Transcriptome sequencing and Western blot revealed significant differences in the expression of TNF-α， IL-1β， and one-carbon metabolism genes （PKM2， MTR， and PHGDH） （P<0.01）. Compared to the model group， high-dose XJDHT significantly increased Bax/Bcl-2 ratio and PHGDH protein expression （P<0.01） and effectively reduced cell apoptosis （P<0.01） while down-regulating protein expressions of TNF-α， IL-1β， PKM2， and MTR （P<0.01）. Low-dose XJDHT moderately increased Bax/Bcl-2 ratio and PHGDH protein expression （P<0.05）， reduced apoptosis （P<0.05）， and decreased IL-1β and MTR protein levels （P<0.05， P<0.01）， but there were no significant changes in TNF-α and PKM2 expression. After PKM2 inhibition by shikonin in THP-1 cells， the expression of protein related to one-carbon metabolism was detected. Compared with the blank group， the LPS-induced model group showed significantly upregulated PKM2 and MTR protein expression （P<0.01） and downregulated PHGDH expression （P<0.01）. Compared with the model group， shikonin treatment significantly reduced PKM2 expression （P<0.05）， increased PHGDH expression （P<0.01）， and decreased MTR expression （P<0.05）. ConclusionXJDHT can inhibit the release of inflammatory factors in sepsis， and its mechanism is related to the intervention of the PKM2-regulated one-carbon metabolism pathway in macrophages.

Objective To investigate the changes in eosinophil counts and the activation of microglial cells in the brain tissues of mice at different stages of Angiostrongylus cantonensis infection, and to examine the role of microglia in regulating the progression of angiostrongyliasis and unravel the possible molecular mechanisms. Methods Fifty BALB/c mice were randomly divided into the control group and the 7-d, 14-d, 21-day and 25-d infection groups, of 10 mice in each group. All mice in infection groups were infected with 30 stage III A. cantonensis larvae by gavage, and animals in the control group was given an equal amount of physiological saline. Five mice were collected from each of infection groups on days 7, 14, 21 d and 25 d post-infection, and 5 mice were collected from the control group on the day of oral gavage. The general and focal functional impairment was scored using the Clark scoring method to assess the degree of mouse neurological impairment. Five mice from each of infection groups were sacrificed on days 7, 14, 21 d and 25 d post-infection, and 5 mice from the control group were sacrificed on the day of oral gavage. Mouse brain tissues were sampled, and the pathological changes of brain tissues were dynamically observed using hematoxylin and eosin (HE) staining. Immunofluorescence staining with eosinophilic cationic protein (ECP) and ionized calcium binding adaptor molecule 1 (Iba1) was used to assess the degree of eosinophil infiltration and the counts of microglial cells in mouse brain tissues in each group, and the morphological parameters of microglial cells (skeleton analysis and fractal analysis) were quantified by using Image J software to determine the morphological changes of microglial cells. In addition, the expression of M1 microglia markers Fcγ receptor III (Fcgr3), Fcγ receptor IIb (Fcgr2b) and CD86 antigen (Cd86), M2 microglia markers Arginase 1 (Arg1), macrophage mannose receptor C-type 1 (Mrc1), chitinase-like 3 (Chil3), and phagocytosis genes myeloid cell triggering receptor expressed on myeloid cells 2 (Trem2), CD68 antigen (Cd68), and apolipoprotein E (Apoe) was quantified using real-time quantitative reverse transcription PCR (RT-qPCR) assay in the mouse cerebral cortex of mice post-infection. Results A large number of A. cantonensis larvae were seen on the mouse meninges surface post-infection, and many neuronal nuclei were crumpled and deeply stained, with a large number of bleeding points in the meninges. The median Clark scores of mouse general functional impairment were 0 (interquartile range, 0), 0 (interquartile range, 0.5), 6 (interquartile range, 1.0), 14 (interquartile range, 8.5) points and 20 (interquartile range, 9.0) points in the control group and the 7-d, 14-d, 21-d and 25-d groups, respectively (H = 22.45, P < 0.01), and the median Clark scores of mouse focal functional impairment were 0 (interquartile range, 0), 2 (interquartile range, 2.5), 7 (interquartile range, 3.0), 18 (interquartile range, 5.0) points and 25 (interquartile range, 6.5) points in the control group and the 7-d, 14-d, 21-d and 25-d groups, respectively (H = 22.72, P < 0.01). The mean scores of mice general and focal functional impairment were all higher in the infection groups than in the control group (all P values < 0.05). Immunofluorescence staining showed a significant difference in the eosinophil counts in mouse brain tissues among the five groups (F = 40.05, P < 0.000 1), and the eosinophil counts were significantly higher in mouse brain tissues in the 14-d (3.08 ± 0.78) and 21-d infection groups (5.97 ± 1.37) than in the control group (1.00 ± 0.28) (both P values < 0.05). Semi-quantitative analysis of microglia immunofluorescence showed a significant difference in the counts of microglial cells among the five groups (F = 17.66, P < 0.000 1), and higher Iba1 levels were detected in mouse brain tissues in 14-d (5.75 ± 1.28), 21-d (6.23 ± 1.89) and 25-d infection groups (3.70 ± 1.30) than in the control group (1.00 ± 0.30) (all P values < 0.05). Skeleton and fractal analyses showed that the branch length [(162.04 ± 34.10) μm vs. (395.37 ± 64.11) μm; t = 5.566, P < 0.05] and fractal dimension of microglial cells (1.30 ± 0.01 vs. 1.41 ± 0.03; t = 5.266, P < 0.05) were reduced in mouse brain tissues in the 21-d infection group relative to the control group. In addition, there were significant differences among the 5 groups in terms of M1 and M2 microglia markers Fcgr3 (F = 48.34, P < 0.05), Fcgr2b (F = 55.46, P < 0.05), Cd86 (F = 24.44, P < 0.05), Arg1 (F = 31.18, P < 0.05), Mrc1 (F = 15.42, P < 0.05) and Chil3 (F = 24.41, P < 0.05), as well as phagocytosis markers Trem2 (F = 21.19, P < 0.05), Cd68 (F = 43.95, P < 0.05) and Apoe (F = 7.12, P < 0.05) in mice brain tissues. Conclusions A. cantonensis infections may induce severe pathological injuries in mouse brain tissues that are characterized by massive eosinophil infiltration and persistent activation of microglia cells, thereby resulting in progressive deterioration of neurological functions.