Localization And Expression Analysis Of BDNF And NOS In The Eyeball Of Chinese Alligator

Objective:In this study,the eyeball of Chinese alligator from Chinese Alligator Breeding Research Center of Anhui Province was used.The expressions and distributions of nitric oxide synthase?NOS?and brain-derived neurotrophic factor?BDNF?in the retina of Chinese alligator were detected by immunohistochemical and immunofluorescence staining,respectively.The present study will demonstrate the role of BDNF and NOS in the visual formation of Chinese alligator.Methods:The four amino acid sequences of NOS from four species were searched in public database NCBI GenBank?https://www.ncbi.nlm.nih.gov/protein/?,including Homo sapiens?accession no:NP₀00616.3?,Oryctolagus cuniculus?accession no:AAB68663.1?,Xenopus laevis?accession no:AAD55136.2?,and Danio rerio?accession no:NP₅71735.1?,as well as the sequences of BDNFs,including Homo sapiens?accession no:CAA62632.1?,Oryctolagus cuniculus?accession no:XP₀17201122.1?,Xenopus laevis?accession no:AAH82887.1?,Danio rerio?accession no:AAH66468.1?.The sequences were aligned using free software MEGA.The fresh eysballs of Chinese alligator were fixed with 4%paraformaldehyde and embedded using OCT.The serial slides?6?m thick?were cut under the freezing-microtome.Slides were wased three times with PBST for 10 min and then added 3%H₂O₂ and cultured at room temperature for 30 min.After washed with PBST for another three times,the primary antibody anti-BDNF or anti-nNOS were added onto the slides,and incubated overnight at 4°C.Furtherly,slides were washed three times with PBST for 10 min each time.The sections were then incubated for 1 h with second antibody?HRP-labeled goat anti-rabbit IgG antibody or HRP-labeled rabbit anti-mouse IgG antibody?at room teperature.After washing three times with PBST for 10 min,the expressions of BDNF and nNOS in retinal tissue were observed under light microscope after developting using DAB color kit.HE staining was also used for detecting the eyeball structure of the Chinese alligator.For determining the locations of BDNF and nNOS in retina of Chinese alligator,slides were blocked using 5%horse serum for 30 min at room temperature.Then,the slides were incubated with ani-BDNF and anti-nNOS antibodies?dilution at 1:200?overnight at 4°C.Subsequently,the slides were incubated with seconed antibodies?donkey anti-mouse 594 and anti-rabbit 488 antibodies?for 2 h at room temperature.After washed with PBST three times,DAPI?dilution at 1:1000?was added for nuclear staining.Fluorescent photograpy were detected under laser confocal microscopy.Results:The results showed that there was different homology among the amino acid sequences of nNOS from four species.The highest homology of NOS was from Oryctolagus cuniculus and Xenopus laevis,which was 76.70%,followed by the homology of nNOS from Xenopus laevis and Danio rerio,which was 74.02%.Moreover,the highest homology of BDNF was from Homo sapiens and Xenopus laevis,which is 85.48%,followed by the homology of BDNF from Homo sapiens and Oryctolagus cuniculus,which was 72.95%.HE staining showed clear structure of the sclera,choroid,and retina of alligator eyeball.Immunohistochemical staining of the alligator's eyeball revealed that BDNF was expressed in the choroid and retina,while NOS was mainly expressed in the retina.Immunofluorescence staining revealed that BDNF and NOS were co-expressed in the retina.Conclusion:BDNF was mainly expressed in the choroid and retinal layer of eyeball while NOS was expressed in retina layer of Chinese alligator.They were co-expressed in the retina layer,which may be involved in the regulation of the visual formation of Chinese alligator.