A NOVEL ANGIOGENESIS INHIBITOR BEVACIZUMAB INDUCES APOPTOSIS IN THE RAT ENDOMETRIOSIS MODEL
Soysal D1,*, Kızıldağ S2,*, Saatlı B1, Posacı C1, Soysal S3, Koyuncuoğlu M4, Doğan ÖE1
*Corresponding Author: Sefa Kızıldağ, Ph.D., Dokuz Eylül University, Faculty of Medicine, 35340 İnciralti, İzmir, Turkey. Tel.: +90-2324124613. Fax: +90-2322590541. E-mail:sefa.kizildag@deu.edu.tr
page: 73

MATERIALS AND METHODS

Thirty-five female Wistar albino mature rats at 8 weeks, weighing 200-250 g, were used for the study. Animals were housed in steel cages with free access to food and water. The temperature of the environment was controlled within 24 C and 12-hour light/dark cycles were maintained. The experimental procedures were approved by the institutional review board of Dokuz Eylül University, İzmir, Turkey. The induction of endometriosis was conducted by the method described by Vernon and Wilson [12]. All rats were anesthetized, using ketamine hydrochloride at a dose of 40-80 mg/kg, and 5-10 mg/kg of xylazine hydrochloric was administered intraperitoneally. The abdomen was opened through a 5 cm midline incision. The left uterine horn was ligated at both the uterotubal junction and the cervical end, and removed. The excised horn was immersed in sterile saline solution, the endometrium was exposed by bisecting along its antimesenteric axis and 5 × 5 mm sections were cut. These explants were then anchored onto the peritoneum on the left side of the ventral abdominal wall close to an artery by 5-0 polypropylene sutures. Abdominal layers were closed anatomically, using 3-0 polyglactin 910 sutures and the animals were allowed to recover from anesthesia. Three rats died in the first week of the initial surgery. Three weeks after the initial surgery, each rat was anesthetized and a midventral laparotomy was performed to determine the attachment and viability of endometrial explants. The surface areas of the explants were measured (length × width). Twenty-nine of the 32 rats (90.6%) had viable and well vascularized endometrial explants in the second laparotomy. Twenty-nine rats were randomly divided into three groups. The GnRH agonist group was treated with a single subcutaneous injection of GnRH analog, leuprolide acetate depot formulation (1 mg/kg body weight) (Lucrin Depot; Abbott Laboratories, Abbott Park, IL, USA). The Bevacizumab group was given a single intraperitoneal injection of bevacizumab (2.5 mg/kg body weight) (Avastin; Genentech/Roche, San Francisco, CA, USA). The Control group received saline solution as placebo by gastric lavage. Three weeks after treatment, a third laparotomy was performed. After a midline incision, the endometrial explants were measured and the level of the adhesions were scored macroscopically by the method defined by Linsky et al. in 1987 [13]. The severity of the adhesions was scored as follows: 0 point no resistance to separation, 0.5 point partial resistance to separation, 1 point sharp dissection was needed. The extension of the adhesions was scored as: 0 point no adhesions; 1 point 25.0% of traumatized area; 2 points 50.0% of traumatized area; 3 points total involvement. The total grade was additive, giving a range of adhesion scores from 0 to 4, which represented both extent and severity of adhesions. These measurements and evaluations were made by one operator blinded to the study. For histologic examination and apoptosis assays, endometriotic explants were excised. A small part of endometrial explant was kept for polymerase chain reaction (PCR) study and the remainder was fixed in 10.0% formalin. The formalin-fixed endometriotic foci were embedded in paraffin blocks, sectioned at ~5 mm thickness (four sections per sample), stained with hema-toxylin-eosin and CD10 (a 94 kDa zinc-dependent cell membrane metalloprotein that participates in the postsecretory processing of neuropeptides) immunohistochemistry and examined under a light microscope (Figure 1). The pathologist assessing the samples was blinded to the treatment groups. The persistence of epithelial cells in endometrial explants was semiquantitatively evaluated as follows: 3, well-preserved epithelial layer; 2, moderately preserved epithelium with leukocyte infiltrate; 1, poorly preserved epithelium (occasional epithelial cells only) and 0, no epithelium. This evaluation was based on a previous rat endometriosis study [14]. All rats were sacrificed at the end of study. Total RNA Extraction and cDNA Synthesis. Total RNA was extracted from cells using High Pure RNA isolation Kit according to the protocol provided by Roche Diagnostics (Basel, Switzerland). Complementary DNAs (cDNAs) were synthesized from 2 mg of the total RNA with SuperScript First- Strand Synthesis System for reverse transcription- PCR (RT-PCR) according to the protocol provided by Invitrogen (Carlsbad, CA, USA). The mixture was incubated at 42 C for 50 min., 72 C for 15 min. After the addition of 2 U RNase H, the PCR was per-formed in a volume of 20 μL containing 2 μL cDNA. Real-Time Reverse Transcription-Polymerase Chain Reaction. Real-time PCR was carried out using a Light Cycler 2.0 instrument and FastStart TaqMan Probe Master kit (both from Roche Diagnostics). Reactions were performed in a 20 μL volume with 5 pmol of each primer and 2 μL of cDNA template derived from reverse-transcribed RNA of the GnRH agonist group, Bevacizumab group and Control group tissue cells. A hypoxanthine phosphoribosyl- transferase (HPRT) housekeeping gene was used as endogenous control and reference gene for relative quantifications. Sequences of oligonucleotide pri-mers were as follows: HPRT (F) 5-GTG GAG ATG ATC TCT CAA CT-3, HPRT (R) 5-ACA TGA TTC AAA TCC CTG AAG-3, BAX (F) 5-AAG AAG CTG AGC GAG T-3, BAX (R) 5-GCC CAT GAT GGT TCT G-3, CYC (F) 5-TGG GTG ATG TTG AGA AAG G-3, CYC (R) 5-TTT GTT CCA GGG ATG TAC T-3, BCL XL (F) 5-GCT GGT GGT TGA CTT TC-3, BCL XL (R) 5-GGA TGG GTT GCC ATT GA-3, BCL-2 (F) 5-ACC TGA CGC CCT TCA C-3, BCL-2 (R) 5-AGG TAC TCA GTC ATC CAC-3. The same thermal profile was optimized for all primers: a pre incubation for 10 min. at 95 C, followed by 40 amplification cycles of denaturation at 95 C for 10 seconds, primer annealing at 59 C for 5 seconds, and pri-mer extension at 72 C for 10 seconds. Distilled water was included as a no template control. Melting curves were derived after 40 cycles by a denaturation step at 95 C for 10 seconds, followed by annealing at 65 C for 15 seconds, and a temperature rise to 95 C with a heating rate of 0.1 C/second and continuous fluorescence measurement. Final cooling was performed at 40 C for 30 seconds. Melting curve analyses of each sample were done using LightCycler Software version 4.0.0.23 (Roche Diagnostics). The step of relative quantification was a fully automated process done by the software, with the efficiency set at 2 and the cDNA of untreated cells defined as the calibrator. All experiments were done in triplicate. Statistical Analyses. The statistical analyses were performed using the Statistical Package for the Social Sciences version 16.0 (SPSS Inc., Chicago, IL, USA). Non normally distributed metric variables, adhesion scores, histopathologic scores and gene expression levels were analyzed by the Kruskal-Wallis test and Mann-Whitney U-test. The mean surface areas of the endometriotic explants between the same group (before and after medical treatment) were analyzed by Wilcoxons signed-rank test. p Values of <0.05 were considered statistically significant. Values were expressed as mean standard deviation (SD), unless stated otherwise.analysis



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