The cochlear auditory epithelium contains two types of sound receptors, inner hair cells (IHCs) and outer hair cells (OHCs). Mouse models for labelling juvenile and adult IHCs or OHCs exist; however, labelling for embryonic and perinatal IHCs or OHCs are lacking. Here, we generated a new knock-in Fgf8^P2A-3×GFP/+ (Fgf8^GFP/+) strain, in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements. After confirming that GFP expression accurately reflects the expression of Fgf8, we successfully obtained both embryonic and neonatal IHCs with high purity, highlighting the power of Fgf8^GFP/+. Furthermore, our fate-mapping analysis revealed, unexpectedly, that IHCs are also derived from inner ear progenitors expressing Insm1, which is currently regarded as an OHC marker. Thus, besides serving as a highly favorable tool for sorting early IHCs, Fgf8^GFP/+ will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.
Animals ; Mice ; Hair Cells, Auditory, Inner ; Cochlea/metabolism* ; Hair Cells, Auditory, Outer/metabolism* ; Disease Models, Animal ; Fibroblast Growth Factor 8/metabolism*

OBJECTIVETo compare the effects of flipping moxibustion ofmedicine combined with western medication and simple western medication for rheumatoid arthritis with cold dampnesssyndrome.

METHODSEighty patients were randomly assigned into an observation group and a control group, 40 cases in each group. Oral methotrexate (1 time a week) and sulfasalazine (twice a day except the day for methotrexate) were used in the two groups. Patients in the observation group were treated with flipping moxibustion ofmedicine, twice a week. The main acupoints were Shenshu (BL 23), Guanyuan (CV 4), Zusanli (ST 36), Yinlingquan (SP 9), and the matching acupoints were in the meridians related to the disease location. All the treatment was given for continuous 4 weeks. The TCM symptom score, visual analogue scale (VAS) score, blood sedimentation (ESR), rheumatoid factor (RF) and C-reactive protein (CRP) were observed before and after treatment. The effect was evaluated.

RESULTSThe total effect rate in the observation group was 95.0% (38/40), which was better than 77.5% (31/40) in the control group (<0.05). After treatment, the VAS score, TCM symptom score, RF, ESR, CRP levels decreased in the two groups (all<0.05), with better effects in the observation group (all<0.05). The adverse reactions in the observation group were lower than those in the control group.

CONCLUSIONFlipping moxibustion ofmedicine combined with western medication for rheumatoid arthritis with cold dampnesssyndrome are better than simple western medication, which are safer and more effective.

BACKGROUND: Neural stem cell transplantation is an emerging therapeutic option in the recovery of neural lesions and neurodegenerative diseases. Neural stem cell culture and differentiation lay a foundation for the further study. OBJECTIVE: To improve the techniques for the isolation, cultivation, differentiation and identification of neural stem cells, and to explore the biological characteristics of cells. METHODS: The neural stem cells from C57BL/6 fetal rats were isolated and cultured in vitro using neurophere culture method followed by morphological and ultrastucture examination. The growth curve and cell cycle of passage 3 cells were drawn and analyzed. Nestin expression was tested by immunofluorescence. Neural stem cells induced in 1% and 10% fetal bovine serum were identified using anti-GFAP, anti-βⅢ-tubulin and anti-MBP by immunofluorescence. RESULTS AND CONCLUSION: The neurospheres exhibited strong cell proliferation ability. Under transmission electron microscope, there was a high nuclear/cytoplasmic ratio in the neural stem cells, indicating a low differentiation degree. Immunofluorescence analysis revealed that neural stem cells were positive for Nestin. The induced cells were positive for GFAP, βⅢ-tubulin, and MBP, indicating these cells were induced to differentiate into astrocytes, neurons and oligodendrocytes, and there were more neurons in 1% fetal bovine serum than those in 10% fetal bovine serum. In conclusion, we could successfully isolate neural stem cells in C57BL/6 mice, and low concentration of fetal bovine serum contributes to more neurons differentiated from neural stem cells.

Purpose After the angiotripsy treatment of tumor,tumor blood vessels have different degrees of regeneration,leading to incomplete tumor necrosis,which may be related to the high expression of HIF-lα.To explore the feasibility of microbubble with HIF-1α antisense oligonucleotide combined with ultrasound inhabit tumor growth.Materials and Methods Twenty-four rabbits with VX2 tumor were randomly assigned to three groups:microbubbles with HIF-1α antisense oligonucleotide plus ultrasound (HMB+US) (n=8),microbubbles plus ultrasound (MB+US) (n=8),therapeutic ultrasound alone without microbubbles (US) (n=8).Pulsed focused ultrasound was delivered directly to the tumor surface for 10 minutes during intravenous infusion of microbubbles with HIF-1α antisense oligonucleotide in the experimental group.The control groups were applied only microbubbles or saline injection.The procedure was repeated every 72 hours until the 30th day.Contrast enhanced US and grey scale ultrasonography were acquired after every treatment to get the tumor perfusion images,size and volume measurements.Results The average maximum diameter of the HMB+US,MB+US,and the US groups grows from (1.13±0.19) cm,(1.17±0.21) cm,(1.22±0.17) cm to (1.60±0.45) cm,(2.11 ±0.57) cm,(3.43 ± 0.71) cm within a 30d-experimental period,respectively.No statistical difference in average diameter was observed among three groups before treatment (P＞0.05).The average maximum diameter of the HMB+US group was significantly smaller than that of the two control groups at the end of experiment (P＜0.05),and the average maximum diameter of the MB+US group was significantly smaller than that of the US group (P＜0.05).Conclusion Microbubbles with HIF-1α antisense oligonucleotide can enhance vascular damage effect ofangiotripsy,which can be a novel method for tumor treatment.

Objective To investigate the efficiency of intraclot microbubbles (MB) combined with urokinase (UK) mediated ultrasound (US) thrombolysis.Metho ds Fifty clots prepared by bovine whole blood were equally divided into 5 groups and were treated in a circulation system with collateral circulation tube.The clots were treated by US,MB and UK in group 1,by US and MBingroup 2,by US and UK in group 3 and by UK in group 4.The group5 was the control group without any treatment.The thrombolysis rate of each group was measured and compared.Residual clots were histologically observed with hematoxylin-eosin staining and immunofluorescence.Results The thrombolysis rate of group 1 ([73.64±14.16]％) was significantly higher than group 2 ([47.97± 11.66]％),group 3 ([57.33±8.65]％),group 4 ([50.85±9.63]％),and group 5 ([29.76±18.06] ％,all P＜0.05).Histological examination of the clots in group 1 showed multiple thrombolysis foci with clots collapse,and the degradation of fibrin network was further confirmed by immunofluorescence.Conclusion The intraclot MB mediated US thrombolysis combined with UK can enhance the rate of thrombolysis in vitro experiment.

Objective To explore the effectiveness of different peak negative pressure ultrasound irradiated microbubble destruction on the control of rat blood brain barrier permeability.Methods Thirty healthy SD rats were divided into 5 groups according to different ultrasound pressure level (62 kPa,86 kPa, 1 81 kPa,324 kPa,708 kPa).After intravenous injection of microbubble,the rats were underwent ultrasonic irradiation for 9 minutes,and the contralateral hemispheres were served as controls.The permeability of blood brain barrier was determined by the quantitative analysis of Evans blue and lanthanum nitrate extravasation.Results Quantitative analysis results revealed a leakage in Evans blue about(4.52 ± 0.1 1 )μg/g and (10.56±0.13)μg/g in group 1 81 kPa and 708 kPa,respectively.A wide endothelial cell gap with lanthanum nitrate trace leaked out was shown at the group of ultrasound pressure of 1 81 kPa,and erythrocytes extravasations were found when ultrasound pressure at 708 kPa.Conclusions The pressure amplitude in microbubble enhanced cavitation can effectively regulate and control blood-brain barrier opening in rats.

BACKGROUNDIntravascular microbubble-enhanced acoustic cavitation is capable of disrupting the vascular walls of capillaries and small vessels. This study was designed to investigate the impact of microbubble-enhanced, pulsed and focused ultrasound (MEUS) on the blood perfusion of subcutaneous VX2 tumors in rabbits.

METHODSSubcutaneous VX2 cancers in twenty New Zealand rabbits were treated by combining high-pressure amplitude, pulsed and focused therapeutic ultrasound (TUS) and intravenous microbubble injections. The TUS transducer was operated with a peak negative pressure of 4.6 MPa and a duty cycle of 0.41%. Controls were subcutaneous VX2 cancers treated with TUS or microbubbles only. Contrast-enhanced ultrasound (CEUS) and intravenous Evans Blue (EB) perfusion were performed to assess the tumor circulation. The tumor microvascular disruption was assessed by histological examination.

RESULTSCEUS showed that the tumor circulation almost vanished after MEUS treatment. The average peak grayscale value (GSV) of tumor CEUS dropped significantly from 84.1±22.4 to 15.8±10.8 in the MEUS-treated tumors but no significant GSV changes were found in tumors in the two control groups. The mean tumor EB content of the MEUS-treated tumors was significantly lower than that of the controls. Histological examination found scattered tumor microvascular disruption with intercellular edema after MEUS treatment.

CONCLUSIONThe tumor circulation of VX2 cancers can be arrested or significantly reduced by MEUS due to microvascular disruption.

Objective To analyze the normal hepatic contrast perfusion blocked by ultrasound excited microbubble cavitation using the visual scoring method. Methods Twenty-four healthy New Zealand rabbits were divided into the microbubbles group (MB + US), the simple ultrasound group (US) and the sham group. The MB + US group was insonated by US and intravenous injection of lipid microbubbles. Microbubble was replaced by saline in the US group and sham US exposure was used in the sham group. US contrast liver perfusion imaging was performed before and 0 min,15 min,30 min,45 min,60 min,24 h after treatment in each group. Results The visual perfusion scores of each group before treatment were no statistical difference ( P >0. 05). The visual score of pre-treatment significantly higher than that of post 0 min, 15 min in the MB+ US group ( P<0. 05), but no difference with post 30 min,45 min,60 min and 24 h ( P >0. 05). There were no statistical significance between all the time points of the US and the sham groups. Conclusions Ultrasound excited microbubble cavitation can temporarily and significantly interrupt liver blood perfusion in the visual score analysis.

Objective To investigate the effect of microbubble (MB) contrast agents impacted by different parameters such as acoustic pressure, frequency, duration of exposure, and MB concentration under flowing condition. Methods A capillary flow mimic model was set up for observation and analysis of MB displacement and aggregation under stereomicroscope. Results The displacement and aggregation of ultrasonic occurred significantly at the frequency of 2.0 MHz than 1.0 MHz and 0.5 MHz. Under low acoustic pressure, MBs were not visually disrupted but the flow slowed down. The aggregation and deflection applying to MB was stronger in the tube happened at the MB concentration of 7×10~7/ml than 7×10~5/ml, but did not when the concentration rose to 7×10~9/ml because of the high viscosity. The ultrasound exposure time could not affect significantly in displacement and MB aggregation. Conclusion MB contrast agents can be manipulated under some ultrasound parameters. It is expected to be physically modulated in blood vessels, in order to increase targeted adhesions for many therapeutic purposes.