We used a healthy house geckos, as indicated by their normal animal activity, body weight, anatomy, and skin color. Healthy subjects were identified by their ability to successfully catch their prey. The physical characteristics of the animals were recommended by a herpetologist from the Zoology Laboratory at the Indonesian Institute of Sciences (LIPI): Dr. rer. nat. Evy Ayu Arida.
The research method used descriptive-analytical experimental and cohort design to analyze the tissue regeneration of the house gecko tails. The source of the research animals was the Zoology Laboratory of the Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia. The research procedures included animal adaptation, an autotomization procedure, and use of a DNA primer design, RNA isolation, qPCR, hematoxylin and eosin staining, western blot, and IHC for the data collection and statistical analyses of the data. Ethical permission for the research was obtained from the Faculty of Medicine of the University of Indonesia’s (FKUI’s) Research Ethics Committee, with no. 672/UN2.F1/ETIK/VII /2.
The research was performed in the Zoology Laboratory of the Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia; the laboratory of the Histology Department, Faculty of Medicine, Universitas Indonesia; and the laboratory of the Center of Hypoxia and Oxidative Stress Studies in the Department of Biochemistry & Molecular Biology, Faculty of Medicine, Universitas Indonesia, from January 2015 to February 2018. All the animal research procedures were covered by Animal Scientific Procedures licenses.
Materials and Reagents
The materials and reagents used in our research included Cygb primary antibody obtained from Rabbit polyclonal anti-Cygb (Life-Span BioSciences LS-C312809) and anti-mouse IgG secondary antibody obtained from Trekkie Universal Link in Starr Trek Universal HR Detection System (BioCare Medical STUHRP700 H). The isolation of RNA was carried out using a MasterPure™ RNA Purification KITpicenter Illumina Company, Water-Biotechnology). The gene expression was analyzed using a KAPA SYBR FAST one-step RT qPCR kit niversal Kapa Biosystems, KK4650) and the machine for analyzing the qPCR was an Eco48 from Illumina.
The characteristics of the house gecko as the animal model included body weight (5 ± 0.5 grams), body length (10–13 cm), and tail length (3.8–4.0 cm; i.e., a tail length less than half of the body length). The International Classification of Hemidactylus platyurus includes the Reptilia class, the Squamata order, the Gekkonidae family, and the Hemidactylus genus.[22,35] Thirty three house geckos ere kept in a glass cage with a size of 40 x 20 x 30 cm3 and adapted for one week at the Zoological Herpetology Laboratory, LIPI. The house gecko cage was exposed to sunlight in a room, with a variation of 12 hours of light and 12 hours of darkness. The geckos were fed with small live insects, such as mosquitoes, cockroaches, and grasshoppers, and were given water ad libitum in a small bowl placed in the middle of the cage.
The number of animal models, based on Federer’s formula, was thirty-three, divided into 10 experimental groups and 1 control groups. Each group consisted of three geckos.
Federer’s formula: (t - 1)(r – 1) ≥ 15
t = experimental groups and control (11 groups)
r = the number of animals in each experimental group
(11- 1)(r – 1) ≥ 15, r = 3 animals/group
All the house geckos were chosen randomly for autotomization, leaving non-autotomized house geckos as the control group. The house geckos release their tails naturally. The procedures for the autotomization accorded with the protocol of the Herpetology Laboratory, LIPI. The ten autotomized house gecko groups were returned to the cage and allowed to regenerate their tails until days 1, 3, 5, 8, 10, 13, 17, 21, 25, and 30, respectively.
Tissue Collection and Analyses
The house geckos that had regenerated their tails, and the control group, were euthanized on days 1, 3, 5, 8, 10, 13, 17, 21, 25, 30, respectively, using an intravenous injection of ketamine (100 mg/mL) with a dosage of 10 mg/kg BW and xylazine (20 mg/mL) with dosage of 2 mg/kg BW. For house geckos weighing 5 grams, a mixture of ketamine and xylazine was administered in 5 uL volume .
The regenerated tail tissue was cut in cross section using a size 11 scalpel proximal to the injury site (Fig. 5A). The analysis for each time point used a different set of animals. Each sample of tail regenerated from each individual was used for the histological analysis, IHC analysis, qPCR analysis, and western blot analysis (Fig. 5B-C) The sample was cut into longitudinal sections; the first part was used for the histological analysis (HE and IHC staining) and stored in formalin 70%, while the other sections were used for the qPCR analysis and western blot analysis, stored in RNAse solutionr analyzing the gene expression and at -800C for nalyzing the protein expression.
The gene expressions of Cygb and PGC-1α were analyzed using qPCR. The Cygb protein expression was analyzed using IHC and western blot, and the histological analysis used hematoxylin-eosin staining.
Design of Primer DNA
Tracking of the Cygb, PGC-1α, and 18S ribosome (housekeeping) genes of the house gecko (Hemidactylus platyurus) began with phylogenetic study of the species with the closest taxonomic kinship to Hemidactylus platyurus. Gesscko is taxonomically the closest species to H. platyurus. The Gecko genome was described nd deposited with the National Center for Biotechnology Information (NCBI) at https://www.ncbi.nlm.nih.gov/. These genes were analyzed with the BLAST method to find the sequences of the genes. A determination of DNA-conserved sequences was made using multiple alignments with Clustal X in the Mega7 software, and the primer DNA was designed using the Primer3 software.
Isolation of total RNA
The frozen regenerated tail tissue was crushed using a micro-homogenizer, and the isolation of total RNA was carried out using the Illumina Company’s Epicentre MasterPure™ RNA Purification Kit (www.epibio.com/applications/nucleic-acid.kits/rna/masterpure-rna-purification-kit). The isolated RNA was pipetted into a 35 μL solution of TE buffer and stored at -80°C.
Quantification PCR (qPCR)
The RNA was converted to cDNA using the KAPA SYBR FAST RT-qPCR reverse transcription system for the cDNA that was used as a template for the qPCR reactions. The expression of Cygb and PGC-1α mRNA were determined using the Livak formula, with 18S RNA as a housekeeping gene.
Hematoxylinnd Eosin (HE) Staining
Regenerated tail tissues from days 1, 3, 5, 8, 10, 13, 17, 21, 25, 30 after autotomy, and tissues from the control group, were stored in formalin overnight. The tissues were dehydrated with alcohol for 24 hours and subsequently purified with xylol for 24 hours. The tail tissues were then embedded in liquid paraffin and left to solidify into block paraffin to be cut by machine with a thickness of 4–5 µm. The sample slices were mounted on glass objectsnd incubated for 24 hours. The slices were then ready to be stained with hematoxylin-eosin.
IHC analysis was performed on the paraformaldehyde-fixed and paraffin-embedded samples. We used rabbit antibody anti-Cygb (MyBioSource and LifeSpan BioSciences) 1:1000 as the primary antibody and Trekkie Universal Link (BioCare Medical) as the secondary antibody. HRP streptavidin was used as a probe that bound to the secondary antibody. HRP was detected by DAB-Chromogen dye and visualized by ImageQuant™.
Quantitative histological analysis by ImageJ I-46 program
The Image J I-46 program was used to calculate the number of cells and measure the length width of the tissue samples. (Fig. 6A). For the length or the width of the tissue area, we set the scale for the image using the function in the Image J I-46 program, and the results of the measurements appeared as lines, automatically, in the feature (Fig. 6B).
The data for the Cygb and PGC-1α (mRNA) gene expressions was analyzed. The data for the Cygb protein expression, the tail growth length, and the histology analyses was all collected from the same individual and subjected to statistical analysis.
The data distribution was analyzed with a Kolmogorov-Smirnov test. If the data distribution was normal, a comparative analysis was carried out for every growth day group using the one-way ANOVA; if the data distribution was not normal, the comparative analysis for every group was carried out using the Kruskal-Wallis test. The differences for every group were considered to be significant at the p value < 0.05.