Cerebral small vessel disease （CSVD） is an important etiology of vascular cognitive impairment and stroke， and there are still many challenges in its early diagnosis and pathological mechanisms. As a type of extracellular vesicles that exist stably in body fluids and can cross the blood-brain barrier， exosomes have attracted wide attention in recent years. Studies have shown that various exosomal proteins have a potential value in the diagnosis and treatment of CSVD and related cognitive impairment， providing new research directions for the noninvasive management of these diseases. This article systematically reviews the latest advances in exosomal proteins for the diagnosis and treatment of CSVD and related cognitive impairment， in order to provide new perspectives for the application of exosomal proteins in the precise treatment of CSVD.
Biomarkers

Objective:To develop a convolutional neural network model of whole slide imaging of cerebrospinal fluid cells for rapid and accurate identification and classification of tumor cells in cerebrospinal fluid.Methods:A total of 8 692 cerebrospinal fluid cytology smears from Huashan Hospital Affiliated to Fudan University from January 2nd, 2019, to December 27th, 2024. As randomly assigned, the training set included 4 941 benign and 1 745 malignant samples, while the validation set comprised of 1 368 benign and 638 malignant samples. Whole-slide digital images were acquired using a cytopathology scanner, cells (clusters) were annotated for classification, and a deep learning model was constructed via tiled image patches for cell detection and classification. Model performance was evaluated using accuracy, sensitivity, specificity, and other indicators. The classification efficiency of manual microscopy was compared.Results:The model achieved a mean precision of 96.75% for cerebrospinal fluid cell classification. For malignant tumor cells, the classification accuracy was 96.61% (mAP=98.36%, AUC=0.97). Subtype classification accuracies for epithelial/epithelioid tumors and small round cell tumors were 97.13% (AUC=0.98) and 95.58% (AUC=0.93), respectively. Compared with manual microscopy, which took (9.70±0.82) minutes for classifying 200 cells, (18.27±1.21) minutes for 500 cells, and often exceeded 60 minutes or infeasible for full slides, the AI model took (3.46±0.49) seconds for 200 cells, (6.76±0.82) seconds for 500 cells, and a median of 48.57 seconds for full slides ( P<0.001), representing an efficiency improvement of approximately 161-170 times, significantly enhancing diagnostic efficiency. Conclusion:This fully automated hierarchical deep learning model enables efficient and accurate tumor cell identification and classification in CSF, providing an effective auxiliary tool for the rapid diagnosis of meningeal carcinomatosis.

Objective This study aimed to observe the effects of Yin-Yang balancing acupotomy intervention on knee-joint function and lower limb biomechanics in a rat model of knee osteoarthritis(KOA),and to explore the mechanisms of acupotomy when treating KOA.Methods Forty SD rats were randomly divided into a blank group,a model group,an acupotomy group,and a medication group.Except for the blank group,KOA models were established by injecting a mixed solution of 4%papain and 0.03 mol/L L-cysteine into the left knee-joint cavity.The acupotomy group received Yin-Yang balancing acupotomy interventions targeting the medial/lateral collateral ligaments and patellar ligament.The medication group received daily oral celecoxib(10 mg/(kg·d)).Interventions began on day 7 post-modeling,and occurred once weekly for 4 weeks.All rats were assessed pre-and post-intervention using the modified Lequesne MG knee-joint grading system and rotarod fatigue test.Post-intervention,in vivo DR imaging was used to measure joint space width.Cartilage morphology was evaluated via HE and safranin O-fast green staining.Ligament biomechanical tensile testing was performed.Serum and cartilage tissues were analyzed by ELISA and Western Blot for matrix metalloproteinase-13(MMP-13)expression.Results(1)Compared with the blank group,the model group showed increased modified Lequesne MG scores,reduced rotarod endurance time,and narrowed joint space(P＜0.01).(2)Compared with the model and medication groups,the acupotomy group exhibited lower Lequesne MG scores,prolonged rotarod endurance time(P＜0.05),and expanded joint space(P＜0.05).(3)The elastic modulus of ligaments in the acupotomy group showed no significant difference from those in the model group but was higher than those in the medication group.Yield strength,maximum strain,and yield-to-tensile strength ratio in the acupotomy group were higher than those in the model and medication groups(P＜0.05).(4)HE and Safranin O-Fast green staining revealed minimal inflammatory infiltration in the acupotomy group compared with the model group.Cartilage surfaces in the acupotomy group were smoother than those in the medication group.(5)ELISA showed reduced serum MMP-13 levels in the acupotomy group versus the model group(P＜0.01),and no significant differences between levels in the drug and acupotomy groups.(6)Cartilage MMP-13 expression in the acupotomy group was significantly lower than that in the model group(P＜0.01)and lower than that in the medication group(P＜0.05).Conclusions Acupotomy intervention enhances knee joint stability,improves lower limb mechanical alignment,and suppresses MMP-13 expression in KOA rats.

Objective This study aimed to observe the effects of Yin-Yang balancing acupotomy intervention on knee-joint function and lower limb biomechanics in a rat model of knee osteoarthritis(KOA),and to explore the mechanisms of acupotomy when treating KOA.Methods Forty SD rats were randomly divided into a blank group,a model group,an acupotomy group,and a medication group.Except for the blank group,KOA models were established by injecting a mixed solution of 4%papain and 0.03 mol/L L-cysteine into the left knee-joint cavity.The acupotomy group received Yin-Yang balancing acupotomy interventions targeting the medial/lateral collateral ligaments and patellar ligament.The medication group received daily oral celecoxib(10 mg/(kg·d)).Interventions began on day 7 post-modeling,and occurred once weekly for 4 weeks.All rats were assessed pre-and post-intervention using the modified Lequesne MG knee-joint grading system and rotarod fatigue test.Post-intervention,in vivo DR imaging was used to measure joint space width.Cartilage morphology was evaluated via HE and safranin O-fast green staining.Ligament biomechanical tensile testing was performed.Serum and cartilage tissues were analyzed by ELISA and Western Blot for matrix metalloproteinase-13(MMP-13)expression.Results(1)Compared with the blank group,the model group showed increased modified Lequesne MG scores,reduced rotarod endurance time,and narrowed joint space(P＜0.01).(2)Compared with the model and medication groups,the acupotomy group exhibited lower Lequesne MG scores,prolonged rotarod endurance time(P＜0.05),and expanded joint space(P＜0.05).(3)The elastic modulus of ligaments in the acupotomy group showed no significant difference from those in the model group but was higher than those in the medication group.Yield strength,maximum strain,and yield-to-tensile strength ratio in the acupotomy group were higher than those in the model and medication groups(P＜0.05).(4)HE and Safranin O-Fast green staining revealed minimal inflammatory infiltration in the acupotomy group compared with the model group.Cartilage surfaces in the acupotomy group were smoother than those in the medication group.(5)ELISA showed reduced serum MMP-13 levels in the acupotomy group versus the model group(P＜0.01),and no significant differences between levels in the drug and acupotomy groups.(6)Cartilage MMP-13 expression in the acupotomy group was significantly lower than that in the model group(P＜0.01)and lower than that in the medication group(P＜0.05).Conclusions Acupotomy intervention enhances knee joint stability,improves lower limb mechanical alignment,and suppresses MMP-13 expression in KOA rats.

Objective:To develop a convolutional neural network model of whole slide imaging of cerebrospinal fluid cells for rapid and accurate identification and classification of tumor cells in cerebrospinal fluid.Methods:A total of 8 692 cerebrospinal fluid cytology smears from Huashan Hospital Affiliated to Fudan University from January 2nd, 2019, to December 27th, 2024. As randomly assigned, the training set included 4 941 benign and 1 745 malignant samples, while the validation set comprised of 1 368 benign and 638 malignant samples. Whole-slide digital images were acquired using a cytopathology scanner, cells (clusters) were annotated for classification, and a deep learning model was constructed via tiled image patches for cell detection and classification. Model performance was evaluated using accuracy, sensitivity, specificity, and other indicators. The classification efficiency of manual microscopy was compared.Results:The model achieved a mean precision of 96.75% for cerebrospinal fluid cell classification. For malignant tumor cells, the classification accuracy was 96.61% (mAP=98.36%, AUC=0.97). Subtype classification accuracies for epithelial/epithelioid tumors and small round cell tumors were 97.13% (AUC=0.98) and 95.58% (AUC=0.93), respectively. Compared with manual microscopy, which took (9.70±0.82) minutes for classifying 200 cells, (18.27±1.21) minutes for 500 cells, and often exceeded 60 minutes or infeasible for full slides, the AI model took (3.46±0.49) seconds for 200 cells, (6.76±0.82) seconds for 500 cells, and a median of 48.57 seconds for full slides ( P<0.001), representing an efficiency improvement of approximately 161-170 times, significantly enhancing diagnostic efficiency. Conclusion:This fully automated hierarchical deep learning model enables efficient and accurate tumor cell identification and classification in CSF, providing an effective auxiliary tool for the rapid diagnosis of meningeal carcinomatosis.

Objective:To examine the effects of Zishenwan Prescription on bile acid metabolism in mice with diabetic encephalopathy; To explore its mechanism of improvement of diabetic encephalopathy.Methods:Male C57BL/6J mice were used to replicate the mouse model of type 2 diabetes mellitus by using high-fat chow and a single intraperitoneal injection of streptozotocin (120 mg/kg). The mice were screened for diabetic encephalopathy by using the Morris water maze test after 8 weeks of continuous stimulation with hyperglycemia, and were divided into model group and Zishenwan Prescription group according to random number table method, with 12 mice in each group. The mice in the Zishenwan Prescription group were treated with the crude extract of Zishenwan Prescription (9.36 g/kg) by gavage, and the normal group and the model group were treated with the same volume of distilled water once a day for 8 weeks. At the end of the treatment, Morris water maze test was used to investigate the cognitive function of diabetic encephalopathy mice; cresyl violet staining was used to detect the number of granule neurons in the hippocampus; serum and feces were collected to detect the content of bile acids by liquid-liquid coupling; hepatic bile acid synthase CYP7a1 and CYP27a1, farnesol X receptor (FXR), fibroblast growth factor 15 (FGF15), fibroblast growth factor receptor 4 (FGFR4), and ileocecal apical sodium-dependent bile acid transporter protein (ABST) mRNA levels were detected by using fluorescence quantitative PCR assay.Results:Compared with the model group, mice in the Zishenwan Prescription group had shorter evasion latency time ( P<0.05 or P<0.01), decreased time to first reach the platform ( P<0.01), increased number of times to traverse the platform ( P<0.01), and reduced neuronal cell damage in hippocampal area; mice in the Zishenwan Prescription group showed decreased serum and fecal total bile acid content ( P<0.05 or P<0.01); the liver CYP7a1 and CYP27a1 mRNA expressions increased ( P<0.01), and FXR and FGF15 mRNA expressions decreased ( P<0.01); ileal ABST mRNA expression decreased ( P<0.01). Conclusion:Zishenwan Prescription may regulate bile acid metabolism, inhibit FRX-FGF15/FGFR4 signaling and ABST expression to promote new bile acid synthesis and conjugated bile acid reabsorption, and thus improve cognitive function in diabetic encephalopathy mice.

Objective:To evaluate the effect of Kuntai capsules combined with progestin-primed ovarian stimulation (PPOS) protocol on the ovarian response, laboratory parameters and embryo transfer outcomes of expected poor ovarian response (POR) patients undergoing invitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. Methods:A retrospective cohort study was performed at Center for Reproductive Medicine, Jiangxi Maternal and Child Health Hospital from June 2020 to July 2023. A total of 1 733 expected POR patients were enrolled and divided into the PPOS only group (control group) and the Kuntai capsules combined with PPOS regimen group (trial group). After a 1∶1 propensity score matching, 423 cases were included in each group. Further analyses and comparisons were made between the two groups, concerning the ovarian stimulation outcomes, embryo laboratory parameters and pregnancy rates after the first frozen-thawed embryo transfer cycles. The primary outcome measure was the number of oocytes retrieved.Results:No statistically significant difference was found in baseline characteristics after matching between the two groups (all P>0.05). Compared with control group, the number of oocytes retrieved did not differ significantly in the trial group ( P=0.295). The level of luteinizing hormone (LH) on the trigger day [3.3 (2.2, 5.0) U/L], the number of available blastocysts formed [1 (0, 1)] and the available blastocyst formation rate [46.2% (85/184)] in the trial group were significantly higher than those in control group [2.9 (1.9, 4.5) U/L, P=0.004; 0 (0, 1), P=0.034; 30.0% (48/160), P=0.002]. However, there were no significant differences in the duration and dosage of gonadotropin used, progesterone and estradiol levels on the trigger day, incidence of premature LH surge, number/rate of fertilized oocytes, number/rate of cleaved embryos, number/rate of high-quality embryos, as well as rate of unavailable embryos (all P>0.05). Compared with control group, the trial group demonstrated comparable implantation rate, clinical pregnancy rate, early miscarriage rate, ongoing pregnancy rate and live birth rate after the first frozen-thawed embryo transfer cycles (all P>0.05). Conclusion:Compared with the PPOS only regimen, the combination of Kuntai capsules and PPOS did not increase the number of oocytes retrieved but could increase the number and rate of available blastocysts formed in expected POR patients.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.

Objective:To evaluate the effect of Kuntai capsules combined with progestin-primed ovarian stimulation (PPOS) protocol on the ovarian response, laboratory parameters and embryo transfer outcomes of expected poor ovarian response (POR) patients undergoing invitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. Methods:A retrospective cohort study was performed at Center for Reproductive Medicine, Jiangxi Maternal and Child Health Hospital from June 2020 to July 2023. A total of 1 733 expected POR patients were enrolled and divided into the PPOS only group (control group) and the Kuntai capsules combined with PPOS regimen group (trial group). After a 1∶1 propensity score matching, 423 cases were included in each group. Further analyses and comparisons were made between the two groups, concerning the ovarian stimulation outcomes, embryo laboratory parameters and pregnancy rates after the first frozen-thawed embryo transfer cycles. The primary outcome measure was the number of oocytes retrieved.Results:No statistically significant difference was found in baseline characteristics after matching between the two groups (all P>0.05). Compared with control group, the number of oocytes retrieved did not differ significantly in the trial group ( P=0.295). The level of luteinizing hormone (LH) on the trigger day [3.3 (2.2, 5.0) U/L], the number of available blastocysts formed [1 (0, 1)] and the available blastocyst formation rate [46.2% (85/184)] in the trial group were significantly higher than those in control group [2.9 (1.9, 4.5) U/L, P=0.004; 0 (0, 1), P=0.034; 30.0% (48/160), P=0.002]. However, there were no significant differences in the duration and dosage of gonadotropin used, progesterone and estradiol levels on the trigger day, incidence of premature LH surge, number/rate of fertilized oocytes, number/rate of cleaved embryos, number/rate of high-quality embryos, as well as rate of unavailable embryos (all P>0.05). Compared with control group, the trial group demonstrated comparable implantation rate, clinical pregnancy rate, early miscarriage rate, ongoing pregnancy rate and live birth rate after the first frozen-thawed embryo transfer cycles (all P>0.05). Conclusion:Compared with the PPOS only regimen, the combination of Kuntai capsules and PPOS did not increase the number of oocytes retrieved but could increase the number and rate of available blastocysts formed in expected POR patients.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.