Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27.On the third day,10 ?mol/L cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.Half of the culture medium was changed every week.The morphological changes of neuron cells were observed by light microscope.Double immuno-staining of microtubule-associated protein 2(MAP2) and karyon were applied to assess the culture purity.Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre-and postsynaptic protein markers.Results:The improved method could remarkably increase the cell number and reduce neuronal damnification.The primary culture was characte-rized by high uniformity,purity,normal synapse formation and longtime livability.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.

BACKGROUND: Anterior cruciate ligament injury accompanied by unstable rotation is a hotspot in sports medicine. Further understanding of the anatomy and biomechanics of the anterolateral ligament can play a guiding significance for the recovery of knee joint rotational stability.
OBJECTIVE:To systematicaly review current literature on the anterolateral ligament of the knee and to understand the incidence, anatomy, morphology and histology of the anterolateral ligament as wel as mechanism of anterolateral ligament injury.
METHODS: The first author searched the PubMed, Medline, CNKI, Wanfang, VIP databases for articles specificaly addressing the anterolateral ligament. Data extraction related to the incidence, anatomy, morphometry, biomechanics, and histology of the anterolateral ligament and its relation to the “Segond” fracture was performed. The retrieve time ranged from 1878 to 2015. Totaly 362 literatures were retrieved, including 342 articles in English and 20 in Chinese. According to inclusive and exclusive criteria, 42 articles were included in result analysis.
RESULTS AND CONCLUSION:The occurrence rate of the anterolateral ligament ranges from 83% to 100%, and this range occurs because of smal discrepancies in the definition of the bony insertions of the anterolateral ligament. The anterolateral ligament originates anterior and distal to the femoral attachment of the lateral colateral ligament. It spans the joint in an oblique fashion and inserts between the fibular head and Gerdy tubercle of the tibia. Exact anatomic and morphometric descriptions vary in the literature, and there are discrepancies regarding the anterolateral ligament attachment to the capsule and lateral meniscus. The anterolaterial ligament is a contributor to the stability of tibial internal rotation, and histologicaly, it exhibits paralel, crimped fibers consistent with a ligamentous microstructure. The footprint of the anterolateral ligament has been shown to be at the exact location of the Segond fracture. The anterolateral ligament is a distinct ligamentous structure at the anterolateral plane of the knee, and it is likely involved in the control of excessive tibial internal rotation that can cause the Segond fracture.

Objective The purpose of this study was to examine the clinical features, radiological and arthroscopic characteristics of mucoid degeneration of the anterior cruciate ligament (ACL) and to assess the results of arthroscopic treat?ment. Methods From January 2012 to August 2013, 32 knees (24 females and 8 males with left knee of 14 and 18 of right) were diagnosed with mucoid degeneration of the ACL and underwent arthroscopic treatment, all of whom with a mean age of 58.81±7.97 years (42-74 years). All 32 patients have pain on terminal extension, in which 12 patients have pain on both terminal flexion and extension. Arthroscopic debridement of hypertrophied ACL was performed in conjunction with notchplasty. Biopsy specimens were taken from yellowish degenerative lesions of ACL posterolateral bundles. Manual Lachman and Anterior Drawer tests were taken or measured preoperatively, as well as, visual analogue scale (VAS), range of motion (ROM) of knee, Lysh?lm scores, Western On?tario and McMaster Universities (WOMAC) scores. Results The narrow intercondylar notch show in all 32 radiographs and sagit?tal magnetic resonance image showing with its attachment thickened and ill?defined ACL. All patients received follow?up. The mean follow?up time was 21.16 ± 5.53 months (15-32 months). The mean VAS score decreased significantly from 5.75 ± 1.32 to 1.13±1.36 (t=13.44, P=0.00). Extension deficits decrease gradually from a mean angle of 11.62°±3.52° preoperatively. Almost sta?ble 6 months postoperatively and the mean score was 0.41° ± 1.01° 1 year after operation. The extension deficit improved signifi?cantly. Manual Lachman tests and Anterior Drawer tests were all negative. At the same time, Lysh?lm scores increased from 50.13 ± 11.57 to 91.97 ± 3.04 and WOMAC scores decreased from 35.13 ± 7.88 to 6.25 ± 2.78, which is statistically significant (t=-20.20, P=0.00;t=24.72, P=0.00). Conclusion Mucoid degeneration of the ACL has a typical clinical feature and the MRI find?ings are fairly specific for clinical diagnosis before arthroscopy. Arthroscopic debridement of mucoid hypertrophy of the ACL in conjunction with notchplasty can effectively provide symptomatic improvement without instability.

Objective Risk factor of radial and horizontal tear for posterior horn of medial meniscus is analyzed in present study.Methods A total of 390 patients diagnosed with medial meniscus tear received during January 2011 to December 2012 were of retrospective analysis.94 cases with radial tear of medial meniscus posterior horn and 95 horizontal cases were chosen as research objects.Age,gender,duration of symptoms,body weight index,trauma history,posterior slop of tibia plateau,knee valgus angle and Outerbridge cartilage classification of patients with radial or horizontal tear were recorded and analyzed.Multifactor unconditioned Logistic regression analysis was employed to analyze risk factor of meniscus radial tear.Results Significant difference could be spotted in the analysis of gender (x2=9.059,P=0.003),body weight index (t=2.549,P=0.012),knee valgus angle (t=-5.609,P=0.000) and Outerbridge cartilage classification (x2=42.300,P=0.000) between patients with radial or horizontal tear.Meanwhile,no significant difference could be spotted in the analysis of trauma history (x2=0.368,P=0.544),posterior slop of tibia plateau (x2=1.021,P=0.312),age (t=-1.228,P=0.221) and symptom duration (t=0.272,P=0.786).According to the results of multi-factor analysis,valgus angle (OR=12.581,P=0.001),age (OR=0.875,P=0.026) and Outerbridge cartilage classification (OR=33.790,P=0.000; OR=15.558,P=0.000; OR=39.891,P=0.000; OR=91.041,P=0.000) were risk factors of meniscus radial tear.Conclusion High incidence of posterior horn of medial meniscus tear was found in patients with senile osteoarthritis.The incidence of radial tear of medial meniscus posterior horn in elderly patients with knee varus or serious articular cartilage was higher than that of horizontal tear.

Objective To evaluate the risk of hyperuricemia in patients younger than 45 with coronary artery disease.Methods Six hundred and seventy-seven patients with coronary artery disease under 45 years old at disease onset were recruited retrospectively.The subjects were divided into hyperuricemia group (n=164) and normal uric acid group (n=513).Patients with coronary artery disease with hyperuricemia were analyzed for clinical characteristics.The risk of hyperuricemia was analyzed on the severity of coronary lesions.T-test,x2 test and Logistic regression analysis were used for statistical analysis.Results Comparing with patients with normal uric acid,patients in the hyperuricemia group were male predominant and more prevalent with hypertension [197(38.5％) and 93(56.7％) ; x2=16.85,P＜0.01],hypedipidemia [274(53.4％) and 130 (80.7％); x2=37.58,P＜0.01],and cardiac dysfunction [24 (4.7％) and 17 (10.4％); x2=7.18,P=0.01].Hyperuricemia increased the risk of coronary artery disease complicated with cardiac dysfunction [ORlcorrection=2.66,95％CI (1.28,5.53)],and it also increased the risk of coronary artery disease concurrent with cardiac arrhythmia [OR2correction=1.28,95％CI (1.00,1.65)] revealed by multivariate regression analysis.Conclusion In young patients under 45 years old with coronary artery disease,hyperuricemia is the independent risk factor for coronary artery disease complicated with cardiac dysfunction and arrhythmia.

ObjectiveTo investigate the correlation of MRI features and phenotypes and genetic mutations in Pelizaeus-Merzbacher disease.Methods Sixteen boys with clinical diagnosis of PelizaeusMerzbacher disease (PMD) were included in this study.Their ages ranged from 22 months to 9 years.They were examined by pediatric neurologists,and clinical classification was made according to the symptoms and physical signs.An experienced radiologist reviewed the cranial MRI images and analyzed the brain involvement,including pallidus globus,pyramidal tract,corpus callosum,cerebellar white matter,semiovale centrum,brain atrophy and ‘ tigroid sign’.ResultsThere were 8 patients with classic form,7 patients with transitional form and one patient with connatal form.They all showed diffuse delayed myelination in the white matter,with involvement of pallidus globus in 13 cases,pyramidal tract in 7 cases,corpus callosum in 11 cases,cerebellar white matter in 7 cases,semiovale centrum in 12 cases.Cerebral atrophy was found in 5 patients and eerebellar atrophy was found in one patient.Five cases depicted ' tigroid sign'.In patients with PLP1 gene point mutation,pyramidal tract and cerebellar white matter involvement showed a high incidence.Cerebellar white matter lesions were relatively frequent in children with transitional form and connatal form.In contrast,‘ tigroid sign' was often related to classic form,which indicated a better myelination and outcome.ConclusionPMD patients show distinct imaging features in their brains,which may be correlated with the phenotype and genetic mutation.

Objective: To study the protection of IL 1ra in cultured developing neuron injury following Mg 2+ free induced epileptiform discharges. Methods: Rat embryo cortical neurons cultured for 6 d and 17 d were directly exposed to Mg 2+ free media, or pretreated with IL 1 receptor antagonist or NMDA receptor antagonists before being exposed to Mg 2+ free media, and then returned to regular media.MTT assay was used to study mitochondrial function injury, laser scanning confocal microscope to measure [Ca 2+ ]i, and real time RT PCR to detect gene mRNA expression. Results:(1) MTT conversion rates were higher in neurons pre and co treated with 10 mg/L IL 1ra than those of neurons with only Mg 2+ free treatment in neurons cultured for 17 d, but not in neurons cultured for 6 d.(2) [Ca 2+ ]i was lower in neurons pre and co treated with 10 mg/L IL 1ra than those of neurons with only Mg 2+ free treatment, either in neurons cultured for 6 d or in neurons cultured for 17 d, and the effects of IL 1ra on [Ca 2+ ]i change were different between neurons cultured for 6 d and neurons cultured for 17 d.(3) Pre and co treated with 10 mg/L IL 1ra NR1 mRNA expression increase induced by Mg 2+ free treatment was decreased, either in neurons cultured for 6 d or neurons cultured for 17 d, and this effect showed no difference between neurons cultured for 6 d and 17 d; Pre and co treated with 10 mg/L IL 1ra NR2A mRNA expression increase induced by Mg 2+ free treatment in neurons cultured for 17 d was decreased, and NR2A mRNA expression showed no difference between IL 1ra group and age matched control group, but have no effect on neurons cultured for 6 d; Pre and co treated with 10 mg/L IL 1ra have NR2B mRNA expression increase induced by Mg 2+ free treatment was not affected, either in neurons cultured for 6 d or neurons cultured for 17 d. Conclusion:Neuroprotection of IL 1Ra in seizure induced injury is age dependent. The mech anism of the neuroprotection of IL 1Ra includes down regulation of [Ca 2+ ]i and others.

Objective To summarize the phenotypic features of an unrecognized leukoencephalopathy in infants sharing same clinical features,and to better understand the disease and provide new evidence for identification of new leukoencephalopathy. Methods Clinical and follow-up data of 13 patients with unrecognized infantile leukoen-cephalopathy were collected from Peking University First Hospital from January, 2006 to December, 2014. Results (1) There were 7 male and 6 female. The average age of onset was 11 months (4-25 months). Thirty-eight percent (5/13 cases) of patients had incentives before the onset;all of the cases had acute onset and rapid motor function regression. Fifteen percent (2/13 cases) of the patients suffered from seizures in the course of the disease. Patients′condition became stable,and cognition and motor function improved gradually 1 month after onset. No patient died till the last follow-up. (2) Imaging features:magnetic resonance imaging (MRI) of the patients was characterized by im-plicating deep white matter,presenting T1 hypointense,T2 and fluid attenuated inversion recovery ( FLAIR) hyperin-tense in the periventricular area. All of MRI showed massive and symmetric lesions with heterogeneous signal and cystic degeneration. DWI showed patch or massive hyperintense in some of the lesions. The follow-up MRI showed the original lesions decreased in 88% ( 8/9 cases ) of patients, and white matters atrophied in 55% ( 5/9 cases ) of patients;the cystic degeneration still existed and even expanded;DWI showed regional linear or spot hyperintense in 88% (8/9 cases) of patients,which was smaller than before,and distributed around the original lesions. Conclusions The patients with leukoencephalopathy caused by unknown pathogenic gene were much likely to be mitochondrial leukoencephalopathy. This study provided evidence for further exploration of new pathogenic genes causing leu-koence-phalopathy.

Objective To analyze and determine the clinical, molecular pathology and genetic features of a Chinese family with dystrophinopathy. Methods Clinical data of the proband and his family members were collected. Immunohistochemistry staining was performed on muscular biopsy tissues with antimerosin, emerin and the N, C and central rod domains of dystrophin. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes. Multiplex ligation-dependent probe amplification (MLPA) was used to test Duchenne muscular dystrophy (DMD) gene to determine the ways and sites of genetic mutation, and analyze the relationships between genotype and phenotype. Results Patients from this family were clinically diagnosed as muscular dystrophy, and they presented serious manifestations although the immunohistochemistry analysis for the proband exhibited partial loss of dystrophin staining, and positive expression with merosin and emerin. Further test with MLPA detected the loss of exons 45--54 in DMD gene in the proband, while his mother had heterozygositic loss in exons 45--54. Conclusions The losses of exons 45--54 in the proband are all derived from his mother, who carries genetic mutation with normal phenotype. He has been diagnosed as dystrophinopathy. At the same time, his partial loss of dystrophin is not parallel to the out-of-frame mutation of the gene and his severe clinical manifestations. Abnormal expression of dystrophin is the pathological basis for dystrophinopathy phenotype. Its clinical outcome depends not only on the degree of the protein expression, but also on the function of the sites where the DMD gene less occurs.

Objective To investigate the characteristic of MRS in patients suffered diffuse axonal injury (DAI)in acute and suba-cute stage,and the correlation between MRS changes and the severity of disease.Methods We reported MRI and proton magnetic resonance spectroscopy studies of 3 6 head-inj ured patients in acute and subacute stage.Proton magnetic resonance spectra were ac-quired from the white matter and gray matter of bibateral frontal lobe that on conventional MRI appeared normal by using 2D MRSI at 3.0T MRI .30 volunteers as contronl were studied at the same time.Results In patient group,N-acetylaspartate/creatine ratios were (2.14±0.15)and (1.71±0.08)choline/creatine ratios were(1.35±0.13)and (1.03±0.08)for the white matter and the gray matter,respectivily.The brain N-acetylaspartate/creatine ratio was reduced and the choline/creatine ratio was increased in pa-tient group compared with the controls.The increase in the choline/creatine ratio was significant even in the moderate and severe in-j ured groups.Furthermore,there was a significantly correlation between the severity of head inj ury and the N-acetylaspartate/cho-line ratio,and changes in patients with metabolite ratios of the GCS score had a strong correlation.Conclusion We conclude that DAI patients with normal conventional imaging manifestations of the frontal lobe appear metabolite ratio change,suggesting the existence of local cerebral inj ury,and it has a strong correlation between the prognosis and MRS changes.MRS may provide an useful method that can tell us the severity of the brain inj ury in patients with DAI .