Implementation of a free cataract surgery program in rural China: a community-based randomized interventional study.

PURPOSE: To identify effective methods to increase the number of cataract surgeries in a rural setting in Pucheng County of Shaanxi Province, northwestern China. DESIGN: Community-based randomized interventional study. PARTICIPANTS: Four hundred thirty-two patients 50 years of age or older with operable cataract who had not undergone surgery 3 months after participation in a cataract outreach screening program. METHODS: Three hundred fifty-five (82.2%) patients eligible for surgery, but not scheduling it on their own, were contacted and were assigned randomly into 4 groups. Participants in group 1 (n = 86) were given informative reminders by telephone or in person by a trained facilitator about undergoing low-cost cataract surgery. Group 2 (n = 86) was offered free cataract surgery. Group 3 (n = 90) was offered free surgery and reimbursement of transportation expenses. Group 4 (n = 93) was provided with free rides from home to hospital in addition to the reminder and free surgery. MAIN OUTCOME MEASURES: Number of participants undergoing cataract surgery after interventions. RESULTS: In total, 94 patients (26.5%) underwent cataract surgery after interventions. In group 1, 13 patients (14.4%) underwent surgery, which was significantly lower than the number in group 2 (n = 25 [27.8%]; P = 0.027), group 3 (n = 28 [31.1%]; P = 0.012), and group 4 (n = 26 [28%]; P = 0.038). There were no significant differences between groups 2 and 3 (P = 0.768) or between groups 2 and 4 (P = 0.869). CONCLUSIONS: Provision of free cataract surgery was twice as effective as giving patients an informative reminder when it came to increasing the uptake of cataract surgery. However, offering reimbursement of transportation expenses or provision of free rides had minimal added impact on the response rate of participants to undergo cataract surgery.

[Adenovirus-mediated delivery of bcl-2 gene attenuates cisplatin-induced degeneration of cultured spiral ganglion cells.].

OBJECTIVE: To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC). METHODS: SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software. RESULTS: SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration. CONCLUSIONS: Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

Identification and characterization of pannexin expression in the mammalian cochlea.

The gap junction in vertebrates is encoded by the connexin gene family. Recently, a new gene family termed pannexin (Panx) has been identified in vertebrates and found to encode gap junctional proteins as well. To date, three pannexin isoforms (Panx1, 2, and 3) have been cloned from mouse and human genomes. In this study, expression of pannexins in the mouse and rat cochlea was investigated. Polymerase chain reaction and Western blot analysis showed that all three pannexin isoforms were expressed in the cochlea. Immunofluorescent staining showed that Panx1 expression was extensive. In the organ of Corti, Panx1 labeling was found in supporting cells, including pillar cells, Hensen cells, Claudius cells, and Boettcher cells. Both surface plaque-like punctate labeling and diffuse-cytoplasmic labeling were visible. However, the labeling was weak and rare in Deiters cells. No labeling was found in the hair cells. Intense labeling for Panx1 was also observed in the interdental cells in the spiral limbus, the inner and outer sulcus cells, and the type II fibrocytes in the spiral prominence and central region in the cochlear lateral wall. In addition, Panx1 labeling was detectable in Reissner's membrane and strial blood vessel cells. Panx2 labeling was restricted to the basal cells in the stria vascularis and was also detectable in the spiral ganglion neurons. However, no overlapping labeling for Panx1 and Panx2 was observed. Finally, Panx3 labeling was exclusively observed in the cochlear bone. Thus, Panx1, 2, and 3 are abundantly expressed in the mammalian cochlea and demonstrate distinct cellular distributions. Like connexins, they may play an important role in hearing.

Cellular characterization of Connexin26 and Connnexin30 expression in the cochlear lateral wall.

Gap junctions in the cochlear lateral wall, which consists of the stria vascularis (SV) and spiral ligament (SPL), are important for generating a positive endocochlear potential and high potassium concentration in the endolymph. In this study, the cellular expression of connexin 26 (Cx26) and Cx30 in the cochlear lateral wall of rats and guinea pigs was examined by immunofluorescent staining and confocal microscopy. Co-labeling for Kir4.1 revealed that the stria intermediate cells had extensive labeling for Cx26 and Cx30 with a leaf-like distribution. Cx26 and Cx30 also co-distributed hexagonally around the basal cells. However, no labeling was observed in the marginal cells. In the SPL, punctate Cx26 and Cx30 labeling was distributed along vertical lines orthogonal to the cochlear longitudinal direction. Intense labeling for Cx26 and Cx30 was found in type II fibrocytes in the spiral prominence and central region, but Cx26 labeling was absent in the middle region just beneath the SV, where only Cx30 labeling was observed. Outer sulcus (OS) cells and their root processes also exhibited intense labeling for Cx26 and Cx30. Neither Cx26 nor Cx30 was immunopositive in the hyaline region beneath the OS, in the subcentral region (type IV fibrocytes), or in the tension (type III) fibrocytes beneath the bone. Cx26 and Cx30 labeling was also absent in the lateral wall blood vessels. Thus, Cx26 and Cx30 have distinct cell-specific distributions in the SV and SPL, suggesting that they can form different pathways for transporting ions/nutrients in the cochlear lateral wall.

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein.

OBJECTIVE: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. METHODS: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. RESULTS: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. CONCLUSION: We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

[Inhibitory effects of small interfering RNA specific to protein kinase CK2a on the growth of laryngeal carcinoma cells].

OBJECTIVE: To assess the effect of small interfering RNA (siRNA) specific to protein kinase CK2a on proliferation and apoptosis of Hep-2 cell line. METHODS: siRNA expression plasmid psiRNA-hH1neo-CK2 specific to protein kinase CK2a and non-specific siRNA expression plasmid psiRNA-hH1neo-cont were constructed respectively, and then were transfected into Hep-2 cells by lipofectamine methods. Protein kinase CK2a mRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. RESULTS: Protein kinase CK2a mRNA and protein expressions were significantly decreased in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). The Hep-2 cells grew slowly after transfected with psiRNA-hH1neo-CK2(P < 0.05). Obvious subdiploid peaks were found in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). CONCLUSIONS: siRNA expression plasmid specific to protein kinase CK2a suppressed the protein kinase CK2a expression and the proliferation of Hep-2, and induced apoptosis of Hep-2 cells.

[Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells].

OBJECTIVE: To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro. METHODS: Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR. RESULTS: The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC. CONCLUSIONS: The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.