Several types of arthritis share the common feature that the generation of inflammatory mediators leads to joint cartilage degradation. However, the shared mechanism is largely unknown. H2BK120ub1 was reportedly involved in various inflammatory diseases but its role in the shared mechanism in inflammatory joint conditions remains elusive. The present study demonstrated that levels of cartilage degradation, H2BK120ub1, and its regulator WW domain-containing adapter protein with coiled-coil (WAC) were increased in cartilage in human rheumatoid arthritis (RA) and osteoarthritis (OA) patients as well as in experimental RA and OA mice. By regulating H2BK120ub1 and H3K27me3, WAC regulated the secretion of inflammatory and cartilage-degrading factors. WAC influenced the level of H3K27me3 by regulating nuclear entry of the H3K27 demethylase KDM6B, and acted as a key factor of the crosstalk between H2BK120ub1 and H3K27me3. The cartilage-specific knockout of WAC demonstrated the ability to alleviate cartilage degradation in collagen-induced arthritis (CIA) and collagenase-induced osteoarthritis (CIOA) mice. Through molecular docking and dynamic simulation, doxercalciferol was found to inhibit WAC and the development of cartilage degradation in the CIA and CIOA models. Our study demonstrated that WAC is a key factor of cartilage degradation in arthritis, and targeting WAC by doxercalciferol could be a viable therapeutic strategy for treating cartilage destruction in several types of arthritis.

Objective To investigate the clinical manifestations,molecular genetics and gonadal pathol-ogy characteristics of patients with disorders of sex development(DSD),and to summarize the clinical experi-ence of identifying rare diseases from common symptoms.Methods The clinical data of 416 patients with DSD diagnosed and treated in the multidisciplinary center of DSD of Guangzhou Women and Children's Medical Cen-ter from May 2018 to August 2023 were retrospectively analyzed,summarized and discussed.Results Accord-ing to chromosome karyotype,416 cases of DSD were classified into three types:92 cases(22.1％)of abnormal sex chromosome karyotype,285 cases(68.5％)of 46,XY karyotype and 39 cases(9.4％)of 46,XX karyotype.Among the 92 patients with abnormal sex chromosome karyotype,59 cases were raised as males,18 cases(30.5％)complained of short penis with hypospadias and cryptorchidism.The most common karyotype was 45,X/46,XY(58 cases,63.0％).Among the 285 patients with 46,XY karyotype,238 cases were raised as males,and 63 cases(26.5％)complained of short penis and hypospadias;47 cases were raised as females,and 13 ca-ses(27.7％)complained of inguinal mass.A total of 216 patients with 46,XY karyotype were subjected to whole exome gene detection,and 155 cases(71.8％)were found to have molecular pathogenesis with the clinical phe-notype.Among the 39 patients with 46,XX karyotype,19 cases were raised as males,and 8 cases(42.1％)com-plained of short penis and hypospadias.In the 18 cases of gonad biopsy,17 cases showed testicular tissue in go-nads.Whole exome sequencing was performed in 14 cases.NR5A1 gene heterozygous mutation,SRY gene muta-tion and SOX3 gene mutation were found in 2 cases,respectively(14.3％).Twenty cases were raised as females,and 14 cases(70.0％)complained of clitoral hypertrophy.Gonad biopsy was performed in 8 cases,with 7 cases of ovotestis(87.5％)and 1 case of NR5A1 gene heterozygous mutation(14.3％).Conclusions The etiologies of DSD are complex and diverse,and the clinical manifestations are various,which can be manifested as hypospa-dias,micropenis,cryptorchidism and other common symptoms of the urinary system.Different etiologies have dif-ferent treatment options.Therefore,chromosome karyotype,molecular genetic testing and gonadal pathology can be used to clarify the cause of disease,especially for rare diseases,improve the detection rate,reduce the rate of missed diagnosis,and ensure reasonable treatment,especially sex selection.

BACKGROUND:The sclerotic zone in the femoral head is an important imaging feature in the progression of steroid-induced femoral head necrosis,which is associated with disease prognosis.Peroxisome proliferator-activated receptor γ coactivator 1α(PGC-1α)has been shown to possess biological activities such as osteogenesis,angiogenesis and anti-mitochondrial apoptosis,which may be closely related to bone repair of steroid-induced femoral head necrosis. OBJECTIVE:To screen for the differential proteins in the sclerotic zone of steroid-induced osteonecrosis of the femoral head versus the normal zone,to screen for hub proteins in the sclerotic zone,and to verify the differential expression of hub proteins in the femoral head specimens following steroid-induced femoral head necrosis,and to to explore the repair pattern of the sclerotic zone following steroid-induced femoral head necrosis. METHODS:Femoral head samples were collected from patients with steroid-induced osteonecrosis of the femoral head receiving total hip arthroplasty.The differentially expressed genes in the sclerotic zone and the normal zone were screened by Tandem Mass Tags and analyzed by GO and KEGG signaling pathways to construct a protein-protein interaction network and screen hub genes.In addition,the expression of hub genes in the sclerotic zone was verified by immunohistochemistry and western blot. RESULTS AND CONCLUSION:Quantitative protein profiling by Tandem Mass Tags revealed that 609 proteins were significantly differentially expressed(Log2FC＞1.20,Log2FC＜0.84 and P＜0.05)in the sclerotic zone of the femoral head compared with the normal zone,of which 290 proteins were upregulated and 319 proteins were downregulated.The GO and KEGG pathway enrichment analyses revealed that among the top 10 enriched pathways,Wnt signaling pathway and life-cycle regulatory pathway were closely related to bone repair;in the life-cycle regulatory pathway,PGC-1α was one of the important proteins.In addition,western blot results verified the low expression of PGC-1α and NRF1 in the sclerotic zone and high expression of Cleaved Caspase-3 in the sclerotic zone compared with the normal zone of steroid-induced femoral head necrosis specimens.Light microscopic immunohistochemical results showed the distribution of PGC-1α,NRF1 and Cleaved Caspase-3 positive expression in the sclerotic and normal zones in the femoral head tissue specimens,indicating the presence of their expression in bone trabeculae,osteoblasts and bone marrow.In contrast,the brown area of the sclerotic zone of femoral head necrosis stained darker and showed more obvious expression of Cleaved Caspase-3.To conclude,in the sclerotic zone of steroid-induced femoral head necrosis,biological behaviors including activation of osteogenesis-related pathways such as Wnt and oxidative apoptosis characterized by low expression of PGC-1 are observed.Low expression of PGC-1α in the sclerotic zone of steroid-induced femoral head necrosis may be associated with the activation of oxidative apoptosis.

Objective:To explore the risk factors of early infection patients after heart transplantation(HT)and provide references for preventing and treating early infection.Methods:From April 2018 to May 2021, clinical data were retrospectively reviewed for 95 HT recipients treated at Zhengzhou Seventh People's Hospital.They were divided into two groups of infected(n=34)and uninfected(n=61). Gender, age, disease type, preoperative IABP implantation, postoperative intra-aortic balloon pump(IABP)implantation, postoperative extracorporeal membrane oxygenation(ECMO)implantation, preoperative mechanical ventilation, preoperative leukocyte, preoperative lymphocyte, preoperative serum C-reactive protein(CRP), operative approach, APACHEⅡscore, NYHA grade, hemoglobin, cardiopulmonary bypass time, donor heart cold ischemia time, postoperative thoracic drainage tube indwelling time, postoperative gastric tube indwelling time, postoperative urinary tube indwelling time, postoperative acute rejection, postoperative ventilator assisted treatment time and postoperative ICU time.The risk factors of early infection were analyzed by univariate and multivariate Logistic regression analysis.Results:There were 34 cases of early infection after HT and 8 cases died.In infection group, preoperative hemoglobin(female <110 g/L or male <120 g/L), ECMO post-operation, 24-48 h post-operation, APACHE post-operation(>6), postoperative intrathoracic drainage tube indwelling time(≥7 d), postoperative gastric tube indwelling time(≥4 d), postoperative urinary tube indwelling time(≥5 d), postoperative acute rejection(positive), postoperative ventilator assisted treatment time(≥2 d)and postoperative ICU time(≥10 d)were 18 cases(52.94%), 8(23.53%), 30(88.24%), 22(64.71%), 18(52.94%), 20(58.82%), 4(11.76%), 21(61.76%)and 19(55.88%); uninfected group: 16 cases(26.23%), 3(4.92%), 32(52.46%), 24(39.34%), 15(24.59%), 31(34.43%), 1(1.64%), 21(34.43%)and 4(6.56%). Significant inter-group differences existed( χ2=6.778, 5.68, 12.326, 5.623, 7.740, 5.297, 4.489, 6.615, 28.947, P<0.05). Multivariate Logistic regression analysis indicated that 24-48h post-operation, APACHEⅡ score >6(β=1.024, Wald χ2=7.653, OR=2.141, OR95% CI=1.323~4.215), ECMO post-operation(β=1.783, Wald χ2=6.186, OR=5.949, OR95% CI =1.459~24.25), postoperative intrathoracic drainage tube indwelling time ≥7 d(β=0.712, Wald χ2=5.745, OR=1.054, OR95% CI=1.183~6.753), postoperative gastric tube indwelling time(β=0.832, Wald χ2=6.756, OR=1.132, OR95% CI=1.416~8.406), postoperative ventilator assisted treatment time(β=0.745, Wald χ2=6.563, OR=1.212, OR95% CI=1.289~7.346)and postoperative ICU time=1.28(β=1.325, Wald χ2=9.752, OR=2.435, OR95% CI=1.426~6.354)were independent risk factor for early infection after HT( P<0.05). Conclusions:Early infection after HT remains higher.It is significantly correlated with 24-48 h post-operation APACHE II score, ECMO post-operation, postoperative intrathoracic drainage tube indwelling time, postoperative gastric tube indwelling time, postoperative ventilator assisted treatment time and postoperative ICU time.Targeted interventions should be adopted for lowering the incidence of early infection after HT.

Objective:To evaluate the long-term effect of structured patient education and exercise therapy for knee osteoarthritis (KOA).Methods:Prospective cohort study, 162 patients with KOA were consecutively recruited from May 2016 to December 2019 to receive structured patient education and exercise therapy and were followed up 3 years after the recruitment. All the patients received two 1-hour educational courses and exercise therapy twice per week for six weeks under the supervision of physicians or physical therapists. Knee injury and osteoarthritis score (KOOS), visual analog score of pain (VAS), intermittent and constant OA pain (ICOAP), self-efficacy for arthritis were queried at 3-month and 36-month follow-up visit. We fitted linear mixed-effects models to examine the difference in scores between baseline and 3-month and 36-month visits.Results:109(67.3%) patients finished both 3-month and 36-month follow-up visits. The KOOS pain score increased from 70.8±1.7 at baseline to 79.7±1.8 at 36 months ( P<0.05). The KOOS symptom score increased from 66.8±2.0 at baseline to 74.9±2.1 at 36 months ( P<0.05). The KOOS daily function score increased from 81.7±1.4 at baseline to 87.0±1.5 at 36 months ( P<0.05). KOOS motor function score increased from 47.4±2.8 at baseline to 55.0±2.9 at 36 months ( P<0.05). The quality of life score of KOOS increased from 46.6±2.1 at baseline to 63.5±2.2 at 36 months ( P<0.05). Compared with the baseline data, there were statistically significant improvements in all subscales of KOOS in 36 months after exercise therapy intervention ( F=14.548, 8.102, 11.394, 5.687 and25.942, P<0.05). VAS pain score of left knee, VAS pain score of right knee, ICOAP score, self-efficacy pain score and other symptoms were also significantly improved ( F=17.643, 26.791, 8.290, 4.052 and 3.654, P<0.05). Conclusion:Structured patient education and exercise therapy are effective in improving knee pain and function as well as self-efficacy until as long as 36 months.

Purpose: Progressive familial intrahepatic cholestasis (PFIC) is a rare genetic autosomal recessive disease caused by mutations in ATP8B1, ABCB11 or ABCB4. Mutational analysis of these genes is a reliable approach to identify the disorder. Methods: We collected and analyzed relevant data related to clinical diagnosis, biological investigation, and molecular determination in nine children carrying these gene mutations, who were from unrelated families in South China. Results: Of the nine patients (five males, four females) with PFIC, one case of PFIC1, four cases of PFIC2, and four cases of PFIC3 were diagnosed. Except in patient no. 8, jaundice and severe pruritus were the major clinical signs in all forms. γ-glutamyl transpeptidase was low in patients with PFIC1/PFIC2, and remained mildly elevated in patients with PFIC3. We identified 15 different mutations, including nine novel mutations (p.R470HfsX8, p.Q794X and p.I1170T of ABCB11 gene mutations, p.G319R, p.A1047P, p.G1074R, p.T830NfsX11, p.A1047PfsX8 and p.N1048TfsX of ABCB4 gene mutations) and six known mutations (p.G446R and p.F529del of ATP8B1 gene mutations, p.A588V, p.G1004D and p.R1057X of ABCB11 gene mutations, p.P479L of ABCB4 gene mutations). The results showed that compared with other regions, these three types of PFIC genes had different mutational spectrum in China. Conclusion The study expands the genotypic spectrum of PFIC. We identified nine novel mutations of PFIC and our findings could help in the diagnosis and treatment of this disease.

Objective:To construct a prognosis associated micro RNA(miRNA) prediction model based on bioinformatics analysis and evaluate its application value in pancreatic cancer patients.Methods:The retrospective cohort study was conducted. The clinicopathological data of 171 pancreatic cancer patients from the Cancer Genome Atlas (TCGA) (https: //cancergenome.nih.gov/) between establishment of database and September 2017 were collected. There were 93 males and 78 females, aged from 35 to 88 years, with a median age of 65 years. Of the 171 patients, 64 had complete clinicopathological data. Patients were allocated into training dataset consisting of 123 patients and validation dataset consisting of 48 patients using the random sampling method, with a ratio of 7∶3. The training dataset was used to construct a prediction model, and the validation dataset was used to evaluate performance of the prediction model. Nine pairs of miRNA sequencing data (GSE41372) of pancreatic cancer and adjacent tissues were downloaded from Gene Expression Omnibus database. The candidate miRNAs were selected from differentially expressed miRNAs in pancreatic cancer and adjacent tissues for LASSO-COX regression analysis based on the patients of training dataset. A prognosis associated miRNA prediction model was constructed upon survival associated miRNAs which were selected from candidate differentially expressed miRNAs. The performance of prognosis associated miRNA prediction model was validated in training dataset and validation dataset, the accuracy of model was evaluated using the area under curve (AUC) of the receiver operating characteristic curves and the efficiency was evaluated using the consistency index (C-index). Observation indicarors: (1) survival of patients; (2) screening results of differentially expressed miRNAs; (3) construction of prognosis associated miRNA model; (4) validation of prognosis associated miRNA model; (5) comparison of clinicopathological factors in pancreatic cancer patients; (6) analysis of factors for prognosis of pancreatic cancer patients; (7) comparison of prediction performance between prognosis associated miRNA model and the eighth edition TNM staging. Measurement data with normal distribution were represented as Mean± SD, comparison between groups was analyzed by the student- t test, and comparison between multiple groups was analyzed by the AVONA. Measurement data with skewed data were represented as M (range), and comparison between groups was analyzed using the Mann-Whitney U test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test. Ordinal data were analyzed using the rank sum test. Correlation analysis was conducted based on count data to mine the correlation between prognosis associated miRNA model and clinicopathological factors. COX univariate analysis and multivariate analysis were applied to evaluate correlation with the results described as hazard ratio ( HR) and 95% confidence interval ( CI). HR<1 indicated the factor as a protective factor, HR>1 indicated the factor as a risk factor, and HR equal to 1 indicated no influence on survival. The Kaplan-Meier method was used to draw survival curve and calculate survival rates, and the Log-rank test was used for survival analysis. Results:(1) Survival of patients: 123 patients in the training dataset were followed up for 31-2 141 days, with a median follow-up time of 449 days. The 3- and 5-year survival rates were 16.67% and 8.06%. Forty-eight patients in the validation dataset were followed up for 41-2 182 days, with a median follow-up time of 457 days. The 3- and 5-year survival rates were 15.63% and 9.68%. There was no significant difference in the 3- or 5-year survival rates between the two groups ( χ2=0.017, 0.068, P>0.05). (2) Screening results of differentially expressed miRNAs. Results of bioinformatics analysis showed that 102 candidate differentially expressed miRNAs were selected, of which 63 were up-regulated in tumor tissues while 39 were down-regulated. (3) Construction of prognosis associated miRNA model: of the 102 candidate differentially expressed miRNAs, 5 survival associated miRNAs were selected, including miR-21, miR-125a-5p, miR-744, miR-374b, miR-664. The differential expression patterns of pancreatic cancer to adjacent tissues were up-regulation, up-regulation, down-regulation, up-regulation, and down-regulation, respectively, with the fold change of 4.00, 3.43, 3.85, 2.62, and 2.35. A prognostic expression equation constructed based on 5 survival associated miRNAs = 0.454×miR-21 expression level-0.492×miR-125a-5p expression level-0.49×miR-744 expression level-0.419×miR-374b expression level-0.036×miR-664 expression level. (4) Validation of prognosis associated miRNA model: The C-index of prognosis associated miRNA model was 0.643 and 0.642 for the training dataset and validation dataset, respectively. (5) Comparison of clinicopathological factors in pancreatic cancer patients: results of COX analysis showed that the prognosis associated miRNA model was highly related with pathological T stage and location of pancreatic cancer ( Z=45.481, χ2=10.176, P<0.05). (6) Analysis of factors for prognosis of pancreatic cancer patients: results of univariate analysis showed that pathological N stage, radiotherapy, molecular targeted therapy, score of prognosis associated miRNA model were related factors for prognosis pf pancreatic cancer patients ( HR=2.471, 0.290, 0.172, 2.001, 95% CI: 1.012-6.032, 0.101-0.833, 0.082-0.364, 1.371-2.922, P<0.05). Results of multivariate analysis showed that molecular targeted therapy was an independent protective factor for prognosis of pancreatic cancer patients ( HR=0.261, 95% CI: 0.116-0.588, P<0.05) and score of prognosis associated miRNA model≥1.16 was an independent risk factor for prognosis of pancreatic cancer patients ( HR=1.608, 95% CI: 1.091-2.369, P<0.05). (7) Comparison of prediction performance between prognosis associated miRNA model and the eighth edition TNM staging: in the training dataset, there was a significant difference in the prediction probability for 3- and 5-year survival of pancreatic cancer patients between prognosis associated miRNA model and the eighth edition TNM staging ( Z=-1.671, -1.867, P<0.05). The AUC of the prognosis associated miRNA model and the eight edition TNM staging for 3- and 5-year survival prediction was 0.797, 0.935 and 0.737 , 0.703, with the 95% CI of 0.622-0.972, 0.828-1.042 and 0.571-0.904 , 0.456-0.951. The C-index was 0.643 and 0.534. In the validation dataset, there was a significant difference in the prediction probability for 3- and 5-year survival of pancreatic cancer patients between prognosis associated miRNA model and the eighth edition TNM staging ( Z=-1.729, -1.923, P<0.05). The AUC of the prognosis associated miRNA model and the eight edition TNM staging was 0.750, 0.873 and 0.721 , 0.703, with the 95% CI of 0.553-0.948, 0.720-1.025 and 0.553-0.889, 0.456-0.950, respectively. The C-index was 0.642 and 0.544. Conclusions:A prognosis associated miRNA prediction model can be constructed based on 5 survival associated miRNAs in pancreatic cancer patients, as a complementation to current TNM staging and other clinicopathological parameters, which provides individual and accurate prediction of survival for reference in the clinical treatment.

OBJECTIVETo compare the differences in the clinical therapeutic effects on reflux esophagitis among the combined therapy ofdecoction (the decoction for resolving the turbid, detoxification and reducing the pathologic upwardin short) and acupuncture, omeprazole and Chinese herbal medicine.

METHODSNinety patients were randomized into 3 groups, 4 cases of them were dropped off. Finally, there were 29 cases in the combined therapy group with acupuncture and the decoction, 29 cases in the western medication group and 28 cases in the Chinese herbal medicine group in the statistical analysis. In the combined therapy group with acupuncture and the decoction, the decoction was prescribed recurrence rate. The therapeutic effects are better than the simple application of either Chinese herbal medicine or omeprazole. for oral administration. Additionally, acupuncture was applied to Neiguan (PC 6), Zusanli (ST 36), Zhongwan (CV 12), Ganshu (BL 18), Danshu (BL 19) and Taichong (LR 3). The decoction was applied one dose a day and acupuncture was once a day. In the western medication group, omeprazole capsules, 20 mg were prescribed for oral administration, twice a day. In the Chinese herbal medicine group, the decoction was simply applied. The treatment was 8 weeks in the 3 groups and the follow-up visit was 6 months. The score of reflux disorder questionnaire (RDQ) and the changes in esophageal mucosa under gastroscope were observed before and after treatment; the clinical therapeutic effects and recurrence rate were evaluated in the 3 groups.

RESULTSIn 4 and 8 weeks of treatment, RDQ scores in the 3 groups were all reduced as compared with those before treatment (all<0.05). In 4 weeks of treatment, RDQ score in the combined therapy group with acupuncture and Chinese herbal medicine was lower than that in the western medication group (<0.05). In 8 weeks of treatment, RDQ score in the combined therapy group with acupuncture and Chinese herbal medicine was lower than those in the western medication group and the Chinese herbal medicine group (both<0.05). In follow-up visit for 6 months, the recurrence rate in the combined therapy group with acupuncture and the decoction was lower than those in the other two groups (both<0.05). In 8 weeks of treatment, the total effective rate for clinical symptoms and that observed under gastroscope in the combined therapy group with acupuncture and the decoction were all better than those in the western medication group and the Chinese herbal medicine group (all<0.05).

CONCLUSIONSThe combined therapy ofdecoction and acupuncture achieve the definite therapeutic effects on reflux esophagitis, relieve the symptoms, protect gastric mucosa and reduce the.

BACKGROUND:Col agen type II and proteoglycan loss are two most obvious manifestations of cartilage damage in the onset of osteoarthritis. Changes in col agen type II and proteoglycan as the main components of cartilage matrix directly lead to cartilage degeneration and subsequently result in osteoarthritis. How to reverse or prevent the development of this process becomes the focus of medical research. OBJECTIVE:To observe the effect of warming yang and benefiting marrow recipe on the expression of col agen type II and proteoglycan in the articular cartilage of knee osteoarthritis rabbits as wel as to further explore the mechanism underlying chondrocyte protection. METHODS:Ninety-six New Zealand rabbits, aged 9 months old, male and female, were selected to prepare osteoarthritis models in extension position using cast immobilization method, and were randomly divided into four groups:blank group (untreated), model group (simple modeling), Chinese medicine group (intragastric administration of extracts of warming yang and benefiting marrow recipe, 24 mL/kg/d) and western medicine group (intragastric administration of glucosamine hydrochloride, 24 mL/kg/d). Intragastric administration was done once a day for 6 weeks. RT-PCR technology was used to observe the effect of warming yang and benefiting marrow recipe on the expression of col agen type II and proteoglycans in the articular cartilage, and pathological examination was also done. RESULTS AND CONCLUSION:The cartilage surface was smooth in the blank group and Chinese medicine group, with uniform toluidine blue staining, but in the model group and western medicine group, the cartilage surface was rough and the toluidine blue staining was extremely uneven with obvious loss of surface and middle layer dying. The expressions of cartilage proteoglycan and col agen type II in the model group were significantly lower than those in the blank group (P<0.01) as wel as in the Chinese medicine group and western medicine group (P<0.05). In addition, the expressions of cartilage proteoglycan and col agen type II in the Chinese medicine group were higher than those in the western medicine group (P<0.05). These findings indicate that the recipe of warming yang and benefiting marrow can enhance the expressions of col agen type II and proteoglycan, which can maintain the normal col agen phenotype and protect the articular cartilage.

Objective To develop a method of studying fiber projecting in the spinal cord duiring chicken embryo development.Methods At embryonic incubation 3 day (E3), pCAGGS-green fluorescent protein (GFP) plasmid was injected into the spinal cord using in vivo electroporation.Three days after transfection (E6), GFP-positive embryos were collected under a stereo fluorescence microscope .Subsequently , the spinal cord was separated from the embryos and cut from the roof plate as an open book .After fixed with 4%paraformaldehyde ( PFA) for one hour , the opened spinal cords were used for immunohistochemistry with N-cadherin antibody and with DAPI for nuclei .Finally, the nerve fiber projecting was photographed and analyzed under a fluorescence microscope . Results Based on the opened spinal cord and immunostaining in the cryosection , we observed that the nerve fibers projected across the midline of the floor plate and reached to the sulcus terminalis along the white matter of the contra side .The immunoreaction against N-cadherin indicated that overexpression of GFP has no significant effect on chicken embryonic development .Conclusion A new method to study fiber projecting in the developing chicken spinal cord is established successfully in this study .