Evaluation of adhesive bone conduction hearing aid in pediatric patients with unilateral congenital aural atresia / 中华耳鼻咽喉头颈外科杂志

Objective: To evaluate the auditory efficacy and subjective satisfaction of adhesive bone conduction hearing aid in children with unilateral congenital aural atresia (UCAA). Methods: Ten subjects (5 males and 5 females) diagnosed with UCAA with an average age of 8.3 years old (ranged from 5 to 15) were included in Beijing Tongren Hospital, Capital Medical University from January to August 2019. The free sound field hearing threshold, word recognition score in quiet, speech reception threshold in noise and sound localization ability (results were measured by RMS error) tests were performed in unaided and aided situation, respectively. Subjective satisfaction questionnaires were also distributed to subjects. Paired t test and Wilcoxon signed rank test were used as statistical analysis methods. Results: The average hearing threshold in aided condition was improved by (21.9±4.4) dB (t=15.8，P<0.05). Speech recognition abilities were generally improved both under quiet and noise (P<0.05);however, when the binaural summation, squelch and head shadow effects were analyzed respectively, the binaural squelch effect was not statistically improved (P>0.05), while the other effects were improved in aided condition (P<0.05). In sound localization test, there was no significant difference of the RMS error value between the unaided and aided situation (P>0.05). The subjects got high satisfaction rates in three subjective questionnaires. Conclusion: The adhesive bone conduction hearing aid can provide significant audiological benefit for children with UCAA as well as raising the quality of their life.

Effect of γ-secretase inhibitor on middle ear ultrastructures in ovalbumin-mediated otitis media with effusion in rats / 中华耳鼻咽喉头颈外科杂志

Objective: To study the effect of the inhibitor of Notch signaling pathway-γ-secretase inhibitor DAPT on the ultrastructures of middle ear in the ovalbumin (OVA)-mediated allergic OME in vivo. Methods: Male Sprague-Dawley (SD) rats, weighing 250-300 g, were completely and randomly divided into three groups (5 rats, 10 ears in each group):(1)Control group(2)OME group(3)OME+DAPT group. Rats in the OME group underwent systemic and local sensitization by intraperitoneal and intratympanic injection of ovalbumin to make the model of OVA-induced OME. Rats in the control group were sensitized with PBS. On the basis of establishing the OME model, OME+DAPT group were intraperitoneal injected with DAPT (10 mg/kg) for seven consecutive days and were administered before intratympanic injection of ovalbumin. After the model was successfully established, endoscopy,H&E staining and scanning electron microscopy were used to study the histology and mucous-ciliary ultrastructures of the non-ciliated and ciliated mucosa in the middle ear of each group. One-way ANOVA and Tukey methods were used for statistical analysis. Results: H&E staining showed that the three groups had statistically significant differences in submucosal thickness both in non-ciliated and ciliated regions (non-ciliated area:(6.83±1.47)μm, (38.58±9.57)μm, (32.17±11.89)μm, respectively. F=107.9;cilia area:(26.69±3.22)μm, (30.41±6.75)μm, (26.76±4.06)μm, respectively. F=5.62,both P<0.01). The thickness of the submucosa in the non-ciliated area and the cilia area of the OME group were significantly thicker than that of control group (F=42.08 and 4.40,both P<0.05); the thickness of the non-ciliated area and the ciliated area in OME+DAPT group were reduced compared to OME group(F=1.55 and 2.77,both P<0.05). Scanning electron microscopy showed that the array of cilia on the middle ear mucosa was disorderly arranged and inversed, this phenomenon was relieved in the OME+DAPT group. The number of goblet cells in the control group, OME group, and OME+DAPT group were 9.87±1.92; 15.67±5.77; 10.33±1.99 respectively and the difference between them was statistically significant (F=11.43, P<0.01). The number of goblet cells in the OME group were significantly higher than those in the control group (F=9.00,P<0.01) and the number of goblet cells in the OME+DAPT group were decreased compared to those of OME group (F=8.41, P<0.01). Conclusions: The study demonstrates the pathological changes of the ultrastructure in middle ear in OVA-induced OME and the effect of the γ-secretase inhibitor on it. In OME group, the cilia are disorderly arranged and inversed, the number of goblet cell is increased and they are swelled which suggest the hypersecretion of the mucus. DAPT can regulate OVA-induced allergic inflammation and relieve pathological changes of ultrastructure in middle ear mucociliary transport system through alleviating submucosal inflammation, reducing the hypersecretion of goblet cell and the morphological damage of cilia through the Notch signaling pathway.

Bonebridge implantation combined with simultaneous bilateral auricle reconstruction for bilateral congenital aural atresia / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the feasibility and safety of auricle reconstruction combined with Bonebridge implantation for bilateral aural atresia patients. Methods: A retrospective analysis was conducted for 36 cases(72 ears) who underwent Bonebridge implantation combined with bilateral auricle reconstruction from February 1, 2017 to January 15, 2020. All cases were bilateral congenital aural atresia and underwent Nagata auricle reconstruction for both sides simultaneously. Bonebridge implantations were performed during the second stage of auricle reconstruction. Results: All 36 patients healed well and had no surgical complications when discharged. The preoperative average bone conduction threshold of the patients was(8.5±5.8) dB HL and postoperative bone conduction threshold was (8.4±5.2) dB HL. There was no significant change after the implantation (P=0.724). The preoperative average air conduction threshold of was(64.9±7.4)dB HL and postoperative air conduction threshold was (24.0±5.3) dB HL, which had a significant change after the implantation (P<0.001). The hearing threshold with Bonebridge significantly decreased by 40.9 dB HL compared with the preoperative air conduction threshold(P<0.001). The speech recognition rate of monosyllable words, disyllabic words and short sentences in quiet environment increased by 62.5%, 63.5% and 72.2% respectively. The differences were statistically significant (P<0.001). The speech recognition rate of monosyllabic words, disyllabic words and short sentences in noise environment were significantly increased by 55.9%, 58.9% and 69.9% respectively (P<0.001). After a follow-up of 18.3 months in average, the hearing results were stable and the aesthetic outcomes were satisfied. One patient had implant rupture and healed after revision surgery. Conclusions: With an integrated surgical procedure, patients with bilateral congenital aural atresia can complete bilateral auricle reconstruction and hearing implantation within six months. This integrated surgical procedure is safe and efficient, with a stable hearing improvement and good appearance.

F4.8 visual miniature nephroscope for the diagnosis and treatment of hematospermia / 中华男科学杂志

<p><b>Objective</b>To explore the practicability and safety of the F4.8 visual miniature nephroscope in the diagnosis and treatment of hematospermia.</p><p><b>METHODS</b>This study included 12 cases of refractory hematospermia accompanied by perineal or lower abdominal pain and discomfort. All the patients failed to respond to two months of systemic anti-inflammatory medication and local physiotherapy. Seminal vesicle tumor and tuberculosis were excluded preoperatively by rectal seminal vesicle ultrasonography, MRI or CT. Under epidural anesthesia, microscopic examination was performed with the F4.8 miniature nephroscope through the urethra and ejaculatory duct orifice into the seminal vesicle cavity, the blood clots washed out with normal saline, the seminal vesicle stones extracted by holmium laser lithotripsy and with the reticular basket, the seminal vesicle polyps removed by holmium laser ablation and vaporization, and the seminal vesicle cavity rinsed with diluted iodophor after operation.</p><p><b>RESULTS</b>Of the 10 patients subjected to bilateral seminal vesiculoscopy, 3 with unilateral and 2 with bilateral seminal vesicle stones were treated by holmium laser lithotripsy, saline flushing and reticular-basket removal, 2 with seminal vesicle polyps by holmium laser ablation and vaporization, and the other 3 with blood clots in the seminal vesicle cavity by saline flushing for complete clearance. The 2 patients subjected to unilateral seminal vesiculoscopy both received flushing of the seminal vesicle cavity for clearance of the blood clots. The operations lasted 10－55 (25 ± 6) minutes. There were no such intra- or post-operative complications as rectal injury, peripheral organ injury, and external urethral sphincter injury. The urethral catheter was removed at 24 hours, anti-infection medication withdrawn at 72 hours, and regular sex achieved at 2 weeks postoperatively. The patients were followed up for 6－20 (7 ± 2.3) months, during which hematospermia and related symptoms disappeared in 10 cases at 3 months and recurrence was observed in the other 2 at 4 months after surgery but improved after antibiotic medication.</p><p><b>CONCLUSIONS</b>The F4.8 visual miniature nephroscope can be applied to the examination of the seminal vesicle cavity and treatment of seminal vesicle stones and polyps, with the advantages of minimal invasiveness, safety and reliability.</p>

Bioinformatic prediction and validation of conserved microRNAs and their target genes in Rehmannia glutinosa / 中草药

Objective: In order to establish the material basis for future studies on the biological function of miRNAs, we aimed to predict novel miRNAs from EST sequences of Rehmannia glutinosa by using bioinformatic strategies. Methods: Since most of the plant miRNAs were conserved in plant species, all plant miRNAs deposited in miRNase were aligned to the 93 172 EST sequences generated by next generation high-throughput RNA sequencing technology and the putative miRNA precursors were screened according to serious criteria. Results: Eight novel rehmannia miRNAs were identified which belonged to eight different families that were further validated by real-time PCR analysis. Then the eight rehmannia miRNAs were subjected to target prediction analysis, and the results showed that the target genes encoded the proteins related to root growth, metabolism, stress responses and other processes. Conclusion: The miRNAs and their target genes identified in this study will provide clues to their biological functions in R. glutinosa.

Bone marrow stem cell-derived astrocytes are involved in glia limitans formation in rats after brain injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the involvement of bone marrow stem cell-derived astrocytes (BMDSCs) in the formation of glia limitans after brain injury.</p><p><b>METHODS</b>In a female SD rat model of brain injury, green fluorescence protein (GFP)-labeled BMDSCs from male SD rats were transplanted via the caudal vein 24 h after the injury. The rats were sacrificed at 2, 4 and 8 weeks after the transplantation, and immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed to observe the astrocytes. The fluorescence emitted by GFP was observed to identify the presence of the bone marrow-derived stem cells, and the GFAP(+)/GFP(+) cells in the glia limitnas were detected under fluorescence microscopy. RESULTS The GFAP(+)/GFP(+) cells were found in the glia limitans between the brain lesion and normal brain tissue.</p><p><b>CONCLUSION</b>Bone marrow stem cell-derived astrocytes is involved in glia limitans formation after brain injury, which can be of significance in brain injury recovery and implantation of engineered materials.</p>

RNA interference mediated silencing of alpha-synuclein in MN9D cells and its effects on cell viability / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability.</p><p><b>METHODS</b>We identified two 19-nucleotide stretches within the coding region of the alpha-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR/H1/TO vector and transfected into MN9D dopaminergic cells. alpha-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT).</p><p><b>RESULTS</b>The mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability.</p><p><b>CONCLUSION</b>Our results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse alpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.</p>

Astrocytes protect MN9D neuronal cells against rotenone-induced oxidative stress by a glutathione-dependent mechanism / 生理学报

Astrocytes maintain homeostasis of neuronal microenvironment, provide metabolic and trophic support to neurons and modulate neuronal responses to injury. Rotenone specifically inhibits mitochondrial complex I, and long exposure to rotenone may increase the risk for Parkinson's disease (PD) and cause Parkinsonism. However, little is known about the role of astrocytes in the process of rotenone-induced dopaminergic neuron injury. In order to investigate this issue, we used MN9D cells as a cell model of dopaminergic neurons and rotenone as a toxin to initiate mitochondrial deficiency. MN9D cells treated with the normal medium or astrocyte-conditioned medium (ACM) were exposed to different concentrations of rotenone for different time followed by cell viability measurement by MTT assay. Besides, various concentrations of ACM and temporally different treatments were devised to evaluate protective efficiency of ACM. Growth curve of cells in the normal medium or ACM was continuously assessed by cell counting for 8 d. The influence of rotenone and ACM on cellular oxidative stress was determined by DCFH-DA staining followed by flow cytometric analysis. Glutathione (GSH) content after treatment of ACM or rotenone was measured by GSH assay kit. Our results showed that rotenone decreased viability of MN9D cells in a dose-dependent manner and ACM treatment significantly attenuated rotenone toxicity at each concentration. No significant difference in growth rate was observed between the normal medium and ACM treatment. Four concentrations of ACM, namely 1/3ACM, 1/2ACM, 2/3ACM and pure ACM, all displayed protection, increasing cell viability to (124.15+/-0.79)%, (126.59+/-0.82) %, (125.84+/-0.61) % and (117.15+/-1.63) % of the cells exposed directly to rotenone, respectively. Treatment with ACM through the whole experiment except the initial 24 h, 24 h before or at the same time of rotenone addition all exerted protective effects, with cell viability being (110.11+/-2.52)%, (113.30+/-2.36) %, (114.42+/-2.00)% of the cells exposed directly to rotenone, respectively. Conversely, ACM treatment 12 h after rotenone addition had no protective effect, with cell viability being (102.54+/-1.36)% of the cells exposed directly to rotenone. Moreover, ACM treatment up-regulated GSH level in MN9D cells nearly twofold. Incubation with 100 nmol/L rotenone for 24 h depleted GSH level by nearly two thirds of the control, but ACM treatment mitigated the drop of GSH level, maintaining its content at (147.83+/-0.63)% of the control. Consistent with GSH change, rotenone administration resulted in a positive rate of 96.24% of DCF staining, implying a great extent of oxidative stress, whereas treatment with ACM reduced the extent of oxidative stress to a positive rate of 78.31%. Taken together, these findings suggest that astrocytes protect MN9D cells from oxidative stress caused by rotenone, and GSH partially accounts for the protection. Therefore, astrocytes may play a protective role in the process of PD.

Protective effect of maternal immunoglobulin G against NMDA-induced neurotoxicity on hippocampus neurons / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate possible protective effect of maternal immunoglobulin G (IgG) against N-methyl-D-aspartate-mediated neurotoxicity on primary-cultured rat hippocampal neurons and the mechanism of the effect.</p><p><b>METHODS</b>An in vitro system had been developed for the study of hippocampal neurons. Intracellular lactic dehydrogenase (LDH) release was used as a marker to measure the rates of neuronal damage. The cells were stained with Trypan blue to measure the rate of neuronal death.</p><p><b>RESULTS</b>N-methyl-D-aspartate (NMDA) at a concentration of 50 micromol/L resulted in increased release of LDH and the cell mortality (P < 0.01, respectively). Maternal IgG of different concentration (10 mg/L, 100 mg/L) inhibited NMDA-induced intracellular LDH release (P < 0.01, respectively) and cell mortality (P < 0.05, 0.01, respectively), and larger dose had stronger effect (P < 0.05).</p><p><b>CONCLUSIONS</b>Maternal IgG had protective effect on primary-cultured rat hippocampal neurons injured by NMDA and the effect was dose-dependent.</p>

N-terminal of ?-Synuclein Involved in Regulation of Mitochondrial Function / 中国生物工程杂志

Objective: To identify the functional domain of ?-Synuclein in affecting mitochondrial function and how the function to be impaired,especially,the mitochondrial membrane potential and the release of Cytochrome c.Methods: Harvest of ?-Syn-N and ?-Syn-△N by PCR,then subcloned into the pCMV-Myc mammalian expression vector.The recombinant plasmids were transfected into HEK293T cells by Lipofectamine 2000.After detecting the protein expression by Western blot,the functional domain was detected by co-immunoprecipitation.The mitochondrial membrane potential through flow cytometry and immunofluorescence,at the same time,the release of Cytochrome c through flow cytometry to detect.Results: The recombinant plasmids were constructed successfully.CO-IP has proved that N-terminal may be the functional domain of ?-Synuclein in affecting mitochondria.Over-expression of N-terminal could depolarize the mitochondrial membrane potential and induce the Cytochrome c releasing in MN9D cells.Conclusion: N-terminal may be the functional domain of ?-synuclein and over-expression of N-terminal could decrease mitochondrial activity.

The Influence of ?-Synuclein Overexpression on Mitochondrial Membrane Structure with Atomic Force Microscopy / 中国生物工程杂志

Objective:To identify the effect of ?-synuclein overexpression on mitochondrial membrane structure with atomic force microscopy. Methods:?-syn expression was mediated by AAV (adeno-associated viral vector) and Recombinant AAV/?-syn and AAV/LacZ viral particles were stereotaxically injected in the left side of rat substantia nigra (SN) for rat model of ?-synuclein overexpression. Mitochondria were isolated from rats SN of Brain. Mitochondria were analysis with JC-1 staining,atomic force microscopy and Western blot. Results:By 16 weeks post-infection of AAV-?-syn,the level of ?-syn increased about 2 times in mitochondrial fraction with Western blot and mitochondrial membrane potential (??) decreased with JC-1 staining. Furthermore,mitochondria swelling and porous like structure formed on the mitochondrial membrane with atomic force microscopy. Conclusion:The data suggested that ?-syn could accumulate in mitochondria,might form mitochondrial membrane pores and lead to ?? decreases. ?-syn might lead to mitochondrial dysfunction in Parkinson's disease.

Study on the Role of ?-synuclein in Mitochondria Dysfunction Caused by Small Dosage Rotenone / 中国生物工程杂志

Mitochondrial dysfunction has been implicated in the aetiology of sporadic Parkinson's disease but its role in the disease mechanism remains unclear.To investigate the effect of synuclein on mitochondrial dysfunction induced by rotenone.The human dopaminergic SH-SY5Y cells were used as a cell model.The cells over-expressed the wild-type ?-synuclein were treated with complex I inhibitor rotenone.The cell viability,complex I activity,Mitochondrial swelling and O2-content were tested at different time point-1w,2w,4w after rotenone treated.CCK-8 test results showed that the cell viability of overexpressed ?-synuclein(SH-SY5Y-Syn)was much lower than the control group(SH-SY5Y-Ctr).After administrating with rotenone about 1w or 2w the cell viability of SH-SY5Y-Syn became higher than that of SH-SY5Y-Ctr.On the 4th week the results were contrary to the first 2 weeks.Similar results were got when test the mitochondrial function.In the first 2 weeks after roteoone administrating,the mitochondrial function of SH-SY5Y-Syn was better than that of SH-SY5Y-Ctr.This suggest that the ?-synuclein could protect the mitochondrial against the injury induced by rotenone in the early stage-1w,2w,while this effect disappeared in the final stage-4w.

Overexpression of alpha-synuclein in SH-SY5Y cells partially protected against oxidative stress induced by rotenone / 生理学报

Both genetic and environmental factors are involved in the pathogenesis of Parkinsonos disease (PD). Epidemiological studies showed that environmental factors shared with the common mechanisms of resulting in alpha-synuclein aggregation by inhibiting complex I of mitochondria and leading to oxidative stress. To investigate the relationship between alpha-synuclein and oxidative stress, we used human dopaminergic SH-SY5Y cells transfected with alpha-synuclein-enhanced green fluorescent protein (EGFP). alpha-synuclein gene expression was determined by immunocytochemistry and real-time quantitative PCR. Both SH-SY5Y and alpha-synuclein overexpressed SH-SY5Y (SH-SY5Y/Syn) cells were treated with various concentrations of rotenone for different time. Cell viability and oxidative stress were detected by MTT assay and DCF assay. Superoxide dismutase (SOD) activity was assessed with xanthine peroxidase method. Cell apoptosis was detected with flow cytometry. Results showed that alpha-synuclein gene was constantly overexpressed in SH-SY5Y/Syn cells. After treatment with rotenone, both cell viability and complex I activity in these cells were reduced in a concentration-dependent manner. Oxidative stress was also found in these cells. Compared with SH-SY5Y cells, SOD activity in SH-SY5Y/Syn cells was increased distinctly (P<0.05) and alpha-synuclein significantly attenuated rotenone-induced cell apoptosis. These results suggest that the alpha-synuclein overexpression in SH-SY5Y cells has a tendency to partially resist oxidative stress induced by rotenone and this response may assist cell survival.

Expression and assessment of double genes of tyrosine hydroxylase gene and aromatic L-amino acid decarboxylase gene in vitro / 生理学报

The characteristic pathological changes of Parkinson s disease (PD) include a severe loss of dopamine neurons in the substantia nigra and a severe decrease in dopamine in the striatum. Since the expression of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) in the biosynthetic pathway for dopamine are low, a promising approach to the gene therapy of PD is to augment the gene expression of the enzymes in the biosynthetic pathway for dopamine. In the present study, human TH and AADC genes were reconstructed into retrovirous vectors pLHCX and pLNCX(2) respectively. Then pLHCX/TH and pLNCX(2)/AADC were transfected into packaging cell line PA317 with liposome. PA317/TH and PA317/AADC were selected by different antibiotics. Gene expression was examined by methods of immunohistochemistry and in situ hybridization. The catalytic activity of two cloned gene enzymes was assessed in vitro by HPLC-EC. Immunocytochemical staining showed that TH and AADC were expressed efficiently in vitro. Both TH and AADC mRNA were transcripted in PA317 cell lines by using in situ hybridazation. HPLC-EC experiments revealed that the transfected cells produced a significantly higher level of dopamine and L-dopa than the untransfected cells. The two genetically modified cells could improve the production of L-dopa and dopamine in response to suitable substrate. The present results suggest that not only recombinant TH and AADC genes are successfully expressed in vitro, but also the enzymes have respective functional activities. These results have set up a way for in vivo gene therapy of PD with TH and AADC genes.