BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective:To observe the maximum tongue pressure and study the oropharyngeal activity during swallowing of patients with nasopharyngeal carcinoma (NPC) after radiotherapy so as to correlate the maximum tongue pressure with swallowing function.Methods:The mean maximum tongue pressure of nineteen NPC patients with dysphagia was measured at the anterior (TA), middle (TM) and posterior (TP) positions, followed by video fluoroscopy. Oral transit time (OTT), upper esophageal sphincter(UES)opening time (UOT) and UES opening range (UOR) were correlated with the mean maximum tongue pressures.Results:The maximum pressure at the TM and TP positions was significantly negatively correlated with OTT, but there was no significant correlation with the anterior readings. The maximum pressures at all three tongue positions were, however, positively correlated with UOT and UOR.Conclusion:The maximum pressure at the TA, TM and TP positions is strongly correlated with the swallowing function of NPC patients.

Objective:To explore the sensitivity and specificity of the simplified cough provoking test in predicting aspiration among patients with dysphagia.Methods:A total of 115 aspirating patients were evaluated using the simplified cough provoking test. Based on the overt and silent aspiration observed through video fluorography, the sensitivity and specificity of the test for predicting each type were quantified.Results:The subjects presented 44 cases of overt aspiration and 71 of silent aspiration. The simplified cough provoking test found that 36 had a normal cough reflex, 22 had a weakened reflex and 57 had none. When the cough′s strength indicated a normal cough reflex, the sensitivity of the simplified cough provoking test predicting silent aspiration was 70.4% and its specificity was 84.1%. The Youden coefficient was 0.545. When the cough reflex was adjudged as abnormal, the test′s sensitivity was 85.9% in predicting silent aspiration and its specificity was 59.1% with a Youden coefficient of 0.45.Conclusion:The simplified cough provoking test has good sensitivity and specificity in identifying silent aspiration. It can effectively screen populations at risk of dysphagia.

Objective:To investigate the incidence of anxiety and depression after radiotherapy for nasopharyngeal carcinoma among persons suffering from dysphagia, and to analyze the related factors.Methods:A total of 143 persons with dysphagia after radiotherapy for nasopharyngeal carcinoma were studied. They completed a general information questionnaire and were evaluated using the hospital anxiety and depression scale.Results:Anxiety was detected in 52 of the subjects (36.3%) and depression in 61 (42.7%). Multivariate regression analysis showed that the average total anxiety score was significantly related to whether the respondent used a gastric tube and whether they lived in a religious household. The average total depression score was related to gastric tube use and to the respondent′s district of residence.Conclusion:Persons with dysphagia after radiotherapy for nasopharyngeal carcinoma often suffer anxiety and depression. The use of a gastric tube increases the risk. The depression of rural residents and those with religious beliefs tends to be milder.

Objective: To identify the pathophysiological characteristics of cortical stroke survivors′ swallowing. Methods: Sixty cortical stroke survivors with dysphagia and cognitive impairment were enrolled into the observation group, while another 16 with dysphagia but without cognitive impairment formed the unimpaired control group and 16 healthy counterparts were selected for a normal control group. Each subject was recorded videofluoroscopically while swallowing 5ml of a liquid of medium consistency. The occurrence of refusing to eat, mouth opening difficulty, incomplete oral closure, residue in the oral cavity, residue in the pharyngeal cavity, leakage and aspiration were observed. Each subject′s swallowing time and kinematic parameters were analyzed from the fluoroscopic videos. Results: The incidence of refusing to eat (37.5%) and/or incomplete mouth closure (68.75%) were significantly higher in the observation group than in the other two groups. The incidence of difficulty in opening the mouth (37.5%), residue in the oral cavity (81.25%), residue in the pharyngeal cavity (56.25%), leakage (56.25%) and aspiration (50%) of the observation group were significantly higher in the observation group than among the normal controls, but were not significantly different from those incidences among the group without cognitive impairment. The average oral transit time and soft palate elevation time of the observation group were significantly longer than those of the other two groups. The observation group′s average hyoid movement time was significantly longer than that of the normal control group, but not significantly different from that of the group without cognitive impairment. There were no significant differences among the groups in average upper esophageal sphincter opening time, larynx closure time or the kinematic parameters. Conclusions Stroke survivors with dysphagia and cognitive impairment present dysphagia characteristic of oral phase swallowing difficulties.

Objective: To compare a new agar thickener with xanthan gum as a thickener in treating dysphagia patients with nasopharyngeal carcinoma after radiotherapy. Methods: Twenty nasopharyngeal carcinoma patients with dysphagia after radiotherapy were asked to swallow moderately and extremely thick liquids thickened with the agar and xanthan gum, and their swallowing was recorded with a videofluoroscope. Results: The average pharyngeal constriction ratio when swallowing agar thickener was significantly lower than when swallowing the traditional thickener. The average oral transit time, the initiation of pharyngeal swallowing were both significantly quicker. There was no significant difference in the average penetration aspiration scale scores between the two thickeners. In the subjective evaluation, the agar thickener was adjudged smoother and with better residual mouthfeel than the xanthan gum, but the scent of the xanthan gum was preferred. Conclusion The new agar thickener is smooth and not sticky. It produces faster transport with less oropharyngeal residue. It can be widely used among nasopharyngeal carcinoma patients with dysphagia after radiotherapy.

Objective To evaluate the characteristics of dysphagia after brain stem infarction,and to determine the mechanism of aspiration.Methods The fluoroscopic videos of 12 dysphagia patients who had suffered brain stem infarction and 10 healthy counterparts were analyzed quantitatively using a digital analysis system.Each participant was requested to twice swallow 5ml of thick liquid.The observations included the oral transit time (OTT),the swallow response time (SRT),the hyoid movement time (HMT),the upper esophageal sphincter opening time (UOT) and the laryngeal closure time (LCT).An 8-point penetration-aspiration scale (PAS) was used to evaluate the severity of aspiration,and the results were correlated with the other 5 quantitative observations.Results The average OTT [(3.091±1.803)s],HMT [(1.498±0.550)s] and LCT [(0.651±0.186)s] of the brain stem infarction patients were all significantly longer than those of the healthy controls.However,no significant differences were found between the patients and the healthy volunteers in terms of SRT or UOT.Aspiration severity was significantly correlated with SRT but not with LCT.Conclusion Dysphagia after brain stem infarction involves both the oral and pharyngeal phases.OTT,HMT and LCT can be used to quantify dysphagia after brain stem infarction,while SRT is a predictor of aspiration.

Objective To study the surface electromyographic signal characteristics of submental and infrahyoid muscles when swallowing in patients with nasopharyngeal carcinoma (NPC) following radiotherapy.Methods Fifteen post-radiotherapy NPC patients with pharyngeal dysphagia confirmed by videofluoroscopie swallowing study (VFSS) were selected into the patient group,while another 15 healthy subjects were recruited into the healthy group.Surface electomyography (sEMG) was used to collect sEMG signals from submental muscles and infrahyoid muscles during both dry and 3 ml thick liquid swallowing,and sEMG duration,average amplitude and peak amplitude parameters were compared between the two groups.Results Compared with healthy subjects,the patient group showed significantly longer sEMG duration of submental muscles and infrahyoid muscles during dry swallowing and 3 ml thick liquid swallowing (P＜0.05).Moreover,significantly longer sEMG duration of submental muscles and infrahyoid muscles was observed when swallowing 3 ml thick liquid,compared with dry swallowing (P＜0.05).The average sEMG amplitude and peak amplitude of submental muscles and infrahyoid muscles of patients when dry swallowing and 3 ml thick liquid swallowing were both significantly higher than those of healthy subjects (P＜0.05).Conclusion Submental muscles and infrahyoid muscles manifested enhanced contractions during swallowing in NPC patients with dysphagia,which played a compensatory role after radiotherapy.

Objective To evaluate the relationship between muscle fibrosis and the displacement of the hyoid bone while swallowing among patients with nasopharyngeal carcinoma (NPC) after radiation therapy.Methods Twenty-six NPC patients with dysphagia were recruited.Shear wave elastography was performed to assess the fibrosis of the mylohyoid muscle.The horizontal and vertical displacement of the hyoid bone were measured based on the videofluoroscopic swallowing study,and the severity of dysphagia was determined using the penetration-aspiration scale (PAS).The correlation between the modulus value of the mylohyoid muscle and the horizontal and vertical displacement of the hyoid bone were analyzed,respectively.Receiver-operating characteristic (ROC) curve were constructed to assess the diagnostic ability of modulus value for the severity of dysphagia above grade 5 of PAS.Results For patients whose PAS was higher than grade 5,the modulus value of mylohyoid muscle was negatively correlated with the displacement of hyoid bone,while such correlation was absent for patients whose PAS grading were less than or equal to 5.Sensitivity and specificity by modulus value to detect dysphagia were 80％ and 81.8％,respectively,with the best cut-off value of 14.37 kPa.Conclusion The reduction of hyoid bone displacement can be partially attributed to muscle fibrosis for post-radiation NPC patients with severe dysphagia.The modulus value measured by Shear wave elastography could be used as a supplementary way to monitor the development of dysphagia.