Effects of melatonin on cytokine release and healing of colonic anastomoses in an experimental sepsis model.

BACKGROUND: The present objective was to identify effects of early melatonin application on healing of anastomotic wound and inflammation in an experimental sepsis model. METHODS: A total of 60 Wistar albino rats were divided into 2 groups. Cecal ligation puncture (CLP) and colonic resection anastomosis were performed on both the control group and the melatonin treatment group. Both groups were divided into 3 subgroups consisting of 10 rats each. One subgroup from each group underwent re-laparotomy at the 16th hour, the next on the 3rd day, and the final subgroup on the 7th day. Presently evaluated were effects of melatonin treatment of early sepsis on interleukin-6 (IL-6), interleukin-10 (IL-10), interferon gamma (INF-γ), and C-reactive protein (CRP) levels, as well as burst pressures (BPs), collagen and hydroxyproline (OHP) content of the anastomotic segments, histopathologic healing, immunohistochemical expressions, CD34, and transforming growth factor beta (TGF-ß). RESULTS: IL-6 and INF-γ levels of the treatment group showed a significant decrease at the 16th hour and an increase on the 3rd and 7th postoperative days. IL-10 levels were significantly higher at the 16th hour and significantly lower on the 3rd and 7th postoperative days in the control group (p<0.001 for each). The treatment group also showed significantly higher capillary permeability, fibroblast proliferation, and collagen deposits (p<0.001 for each). CD34 expression was significantly increased in the treatment group on the 7th postoperative day (p=0.005). CONCLUSION: Application of melatonin in early sepsis significantly improved colonic anastomotic healing in a rat model.

Melatonin causes regression of endometriotic implants in rats by modulating angiogenesis, tissue levels of antioxidants and matrix metalloproteinases.

PURPOSE: The aim of this study was to test if melatonin causes regression of endometriotic implants and whether it influences implant levels of superoxide dismutase (SOD), malondialdehyde (MDA), vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-2 and matrix metalloproteinase (MMP)-9 in rats. METHODS: Endometriotic implants were introduced surgically to 20 female Wistar albino rats, which were either treated with melatonin via intraperitoneal injection for four weeks (melatonin group, n = 10) or with saline (control group, n = 10) after a second-look laparotomies. The main outcome measures included volume (mm(3)) and weight (mg) of explants and tissue levels of SOD, MDA, VEGF, TIMP-2 and MMP-9. RESULTS: Before and after treatment implant volumes of the melatonin group were decreased significantly (P < 0.01) while there was no significant difference between the pretreatment and posttreatment implant volumes of the control group. Moreover, weight (P < 0.05) and histologic score (P < 0.05) of implants of the melatonin-treated rats were significantly lower than controls. Activity of SOD and TIMP-2 staining in melatonin group was significantly higher (both P < 0.01) while there were significant reductions in implant levels of VEGF and MMP-9 in melatonin group (both P < 0.01) than controls. CONCLUSIONS: Melatonin induces the regression of endometriotic implants in rats by modulating implant levels of SOD, MDA, VEGF, MMP-9 and TIMP-2.

Pilot Experimental Study on the Effect of Arginine, Glutamine, and ß-Hydroxy ß-Methylbutyrate on Secondary Wound Healing.

BACKGROUND: Wound healing is a complex process, dependent on available nutrition substrates. When used together with ß-hydroxy ß-methylbutyrate, arginine and glutamine have been shown to increase collagen deposition in human subjects. However, there are no experimental investigations on the influence of this amino acid mixture with regard to secondary wound healing. The aim of this study is to investigate the effects of the supplementation of these 3 amino acids on the healing of open wounds in otherwise healthy animals. MATERIALS AND METHODS: Twelve rats were divided into control and treatment groups. Two 2-cm × 1-cm full-thickness skin defects were prepared on each subject. The rats in both groups received a diet containing 1.2 g of protein per 100 g of body weight per day. The treatment group, in addition, received 200 mg/kg L-arginine, 200 mg/kg L-glutamine, and 40 mg/kg ß-hydroxy ß-methylbutyrate every day. Wound sizes were measured every 2 days. On the 10th day, tissue samples were taken for histopathologic evaluation and also for the measurement of hydroxyproline concentrations. RESULTS: There was no statistically significant difference between mean wound sizes for the 2 groups (P > .05). There was also no statistically significant difference between the groups with regard to histological healing parameters (reepithelialization [P = 1.00], granulation tissue [P = 1.00], collagen accumulation [P = .455], inflammatory cell accumulation [P = .455], angiogenesis [P = .242]) or tissue hydroxyproline concentrations (P = .240). CONCLUSION: Diet supplemented with arginine, glutamine, and ß-hydroxy ß-methylbutyrate is not beneficial in enhancing secondary healing of open wounds in rats. Further research regarding this topic is warranted.

Environmental Tobacco Smoke Exposure during Intrauterine Period, Promotes Caspase Dependent and Independent DNA Fragmentation in Sertoli-Germ Cells.

Objectives. To investigate the effect of cigarette smoke exposure during intrauterine period on neonatal rat testis. Methods. Twenty-five rats were randomized to be exposed to cigarette smoke with the Walton Smoking Machine or to room air during their pregnancies. The newborn male rats (n = 21) were grouped as group 1 (n = 15) which were exposed to cigarette smoke during intrauterine life and group 2 (n = 6) which were exposed to room air during intrauterine life. The orchiectomy materials were analyzed with TUNEL immunofluorescent staining for detection of DNA damage. To detect apoptosis, immunohistochemical analyses with caspase-3 were performed. Primary outcomes were apoptotic index and immunohistochemical scores (HSCORES); secondary outcomes were Sertoli-cell count and birth-weight of rats. Results. Sertoli cell apoptosis was increased in group 1 (HSCORE = 210.6 ± 41.9) when compared to group 2 (HSCORE = 100.0 ± 17.8) (P = 0.001). Sertoli cell count was decreased in group 1 (P = 0.043). The HSCORE for the germ cells was calculated as 214.0 ± 46.2 in group 1 and 93.3 ± 10.3 in group 2 (P = 0.001) referring to an increased germ cell apoptosis in group 1. The apoptotic indexes for group 1 were 49.6 ± 9.57 and 29.98 ± 2.34 for group 2 (P = 0.001). The immunofluorescent technique demonstrated increased DNA damage in seminiferous epithelium in group 1. Conclusions. Intrauterine exposure to cigarette smoke adversely affects neonatal testicular structuring and diminishes testicular reserve.

Effect of intraoperative platelet-rich plasma and fibrin glue application on skin flap survival.

The experiment was designed to compare the effect of intraoperative platelet-rich plasma (PRP) and fibrin glue application on skin flap survival. In this study, bilateral epigastric flaps were elevated in 24 rats. The right-side flaps were used as the control of the left-side flaps. Platelet-rich plasma, fibrin glue, and thrombin had been applied under the flap sites in groups 1, 2, and 3, respectively. Five days later, all flap pedicles were ligated. Necrotic area measurements, microangiography, and histologic and immunohistochemical evaluations were performed to compare the groups. Platelet-rich plasma reduced necrotic area percentages as compared with other groups. Histologically and microangiographically increased number of arterioles were observed in PRP groups. Thrombin when used alone increased flap necrosis. Vascular endothelial growth factor, platelet-derived growth factor, and transforming growth factor ß3 primary antibody staining showed increased neovascularization and reepithelialization in all PRP-applied flaps. This study demonstrated that PRP, when applied intraoperatively under the skin flap, may enhance flap survival. Thrombin used alone was found to be unsuitable in flap surgery.

Effect of preoperative subcutaneous platelet-rich plasma and fibrin glue application on skin flap survival.

BACKGROUND: The aim of this study was to compare the effects of preoperative subcutaneous platelet-rich plasma and fibrin glue administration on skin flap survival. METHODS: One week before surgery; saline, platelet-rich plasma, fibrin glue, and thrombin solutions were applied under rat skin flap areas in Groups I, II, III, and IV, respectively. Unipedicled epigastric flaps were elevated in the first three groups but could not be elevated in Group IV because of preoperative abdominal skin necrosis. Necrotic area measurements, microangiography, and histological and immunohistochemical evaluations were performed. RESULTS: Platelet-rich plasma reduced the percentage of necrotic area when compared to other groups. Histologically and microangiographically an increased number of arterioles were observed in the platelet-rich plasma group. Thrombin (when used alone) caused abdominal skin necrosis. Increased expression of VEGF and PDGF was found in all platelet-rich plasma-treated flaps. There was no significant difference between groups with respect to TGF-ß3 staining intensity. CONCLUSION: In this study preoperative administration of platelet-rich plasma mimicked the pharmacological delay effect and enhanced flap survival. Individual use of thrombin was found to be unsuitable in flap surgery. LEVEL OF EVIDENCE I: This journal requires that authors assign a level of evidence to each article.

Time dependent influence of etonogestrel on the caspase-3 immunoreactivity and apoptotic indexes of rat uterus and ovaries.

Apoptosis is necessary for the balance between cell proliferation and loss. Thirty-six Wistar-Albino rats were subjected to investigate apoptotic effect of widely used implantable progestins on ovarian and uterine tissues. Rats were divided into 6 groups. In the first five groups, we applied etonogestrel (IMP) subcutaneous implants (n = 30). The rats in groups were sacrificed sequentially every 10 days after application. The rats in the last group (n = 6) were accepted as controls. Apoptotic index (AI) values and Caspase-3 immunoreactivities of ovaries and uterus were recorded. In IMP groups, AI and Hscore values in stroma and glandular epithelium of uterus, granulosa and teca-lutein cells of the ovary increased with the longer progesterone exposure. Increase in AI and Hscore values were more prominent after 30 days of exposure for teca-lutein cells of ovary. Progestins increased apoptosis in ovaries and uterus by the longer exposure. Apoptosis increased in ovaries by chronic progesterone exposure. The apoptotic effect of progestin on endometrium is clear but long-term systemic application may lead to alterations in ovarian physiology. We evaluated time dependent apoptotic effect of etonogestrel on reproductive physiology and discussed progestins effect from another point of view in this study.

Environmental tobacco smoke exposure during intrauterine period promotes granulosa cell apoptosis: a prospective, randomized study.

OBJECTIVE: To evaluate the intrauterine effect of cigarette smoke on cell death and DNA damage in follicular cells of fetal ovarian tissue. METHODS: A prospective, randomized study was conducted with 25 female wistar-albino rats. The rats were randomized to be exposed either to cigarette smoke or to room air, initiating from proestrous period and during pregnancy. Newborn female rats were categorized as Group 1 (n = 24) that had been exposed to cigarette smoke during intrauterine life and Group 2 (n = 7) that had been exposed to room air during intrauterine life. Bilateral ooferectomies were performed on the 2nd week of their life. TUNEL (in-situ Terminal Deoxynucleotidyl-Transferase Mediated dUTP-Nick-End Labeling) immunofluorescent staining and immunohistochemical analyses with caspase-3 were used for detection of DNA damage and apoptosis. Primary outcomes were apoptotic index and immunohistochemical scores (HSCORE). Secondary outcomes were ovarian follicle counts and birth weights of newborn rats. RESULTS: There was a significant increase of HSCORE and apoptotic index in Group 1. Increased immunofluorescent staining; evaluating DNA damage, with TUNEL method was observed in granulosa cells in Group 1. CONCLUSIONS: Intrauterine exposure to cigarette smoke diminishes ovarian reserve of female offspring, raising the concern about the generational impact of maternal smoking on ovarian function in the human.

The protective effect of adrenomedullin on renal injury, in a model of abdominal aorta cross-clamping.

Renal injury induced by aortic ischemia-reperfusion (IR) is an important factor in the development of postoperative acute renal failure following abdominal aortic surgery. The aim of this study was to examine the effect of adrenomedullin (AM) on kidney injury induced by infrarenal abdominal aortic IR in rats. Thirty-two Wistar Albino rats were randomized into four groups (eight per group) as follows: Control group, IR group (120-minute ischemia and 120-minute reperfusion), IR + AM group (a bolus intravenously of 0.05 µg/kg/min AM), and control + AM group. At the end of the experiment, blood and kidney tissue specimens were obtained for biochemical analysis. Immunohistological evaluation of the rat kidney tissues was also done. IR significantly increased (p < 0.05 vs control group) and AM significantly decreased (p < 0.05 vs. IR group) all of the biochemical parameters. Immunohistological evaluation showed that AM attenuated morphological changes as apoptosis associated with kidney injury. The results of this study indicate that AM attenuates both biochemically and immunohistopathologically kidney injury induced by aortic IR in rats.

Reconstruction of cranial bone defects using Struthio camelus eggshell.

BACKGROUND: There are many synthetic materials for the treatment of bone defects, which have their own advantages and disadvantages. We aimed to compare the efficacy of ostrich eggshell, which is cheap and easily available, and demineralized bone matrix in healing of cranial bone defects. METHODS: A full-thickness circular bone defect was created in the frontal bone of 40 Wistar rats. Group 1 was the operative control group. In group 2, demineralized bone matrix applied into the defects; in group 3, Struthio camelus (ostrich) eggshell implants (OSIs) were applied into the defects; and in group 4, ostrich eggshell powders were applied into the defects. Computed tomographic analysis was performed to evaluate the healing of bone defects, the bone density, the OSI area measurements, and the OSI volume and density. At the end of the 24th week, all rats were killed. New bone formation, infection, resorption, and tissue reactions were evaluated. RESULTS: Ostrich eggshell implants were slightly resorbed, integrated with bone, stable, and supplied good cranial completeness. Ostrich eggshell powders were totally resorbed at the sixth month. There were no significant differences between control and ostrich eggshell groups in new bone formation. CONCLUSIONS: Ostrich eggshell did not seem to be an osteoproductive material, but it has some important advantages as an implant. Ostrich eggshell has a strong structure, is cheap, is shaped easily, and does not cause tissue reaction or infection. Ostrich eggshell could be a good alternative graft material for craniomaxillofacial procedures. Further studies are required to find out the potential use of the ostrich eggshell in craniomaxillofacial reconstructions.

Feasibility of demineralized bone matrix for craniomaxillofacial contour restoration.

Demineralized bone matrix (DBM) could be a good alternative for craniomaxillofacial contour restoration, especially in perialar, malar, temporal, and frontal regions. In this study, the histologic behavior of DBM was investigated in different tissue planes to determine its proper application plane for restoration of craniomaxillofacial contour deformities and defects.Forty Wistar rats were divided into 6 groups: (1) 0.3 mL of 0.9% saline was injected into the subperiosteal plane of the cranium, (2) 0.3 mL of DBM was implanted into the subperiosteal plane of the cranium, (3) 0.3 mL of 0.9% saline was injected into the subdermal plane on the left inguinal region, (4) 0.3 mL of DBM was implanted into the subdermal plane on the right inguinal region, (5) 0.3 mL of 0.9% saline was injected between the left external and internal oblique muscles, and (6) 0.3 mL of DBM was implanted between the right external and internal oblique muscles. At the 8th week half of the rats and at 16th week the remaining rats were killed in each group, and tissue samples were harvested. Histological and immunohistochemical evaluation revealed new bone tissue and bone marrow formation in all planes that DBM was given.Demineralized bone matrix can provide satisfactory results in craniomaxillofacial contour deformities including forehead, temporal, and malar augmentations, as well as mental and perialar augmentations and saddle nose corrections, with supraperiosteal or deep subcutaneous applications. However, superficial applications must be avoided because of the possibility of palpation, because it induces hard bone tissue formation in all tissue planes.

Neuroprotective effects of gabapentin on spinal cord ischemia-reperfusion injury in rabbits.

OBJECT: Extensive research has been focused on neuroprotection after spinal cord trauma to alleviate the effects of secondary injury. This study aims to investigate the neuroprotective effects of gabapentin in an experimental spinal cord ischemia reperfusion injury. METHODS: Thirty-two adult male New Zealand white rabbits received spinal cord ischemic injury using the aortic occlusion model. Animals were divided into 4 groups (sham, control, low-dose, and high-dose treatment groups; 8 rabbits in each group). High (200 mg/kg) and low (30 mg/kg) doses of gabapentin were administered to the animals in the treatment groups after spinal cord ischemic injury. Neurological status of the animals, ultrastructural findings in injured tissue samples, and levels of tissue injury markers in these 2 groups were compared with findings in the animals that did not receive the ischemic procedure (sham-operated group) and those that received normal saline after administration of ischemia. RESULTS: Regarding levels of tissue injury marker levels after ischemic injury, animals in the gabapentin-treated groups demonstrated better results than animals in the other groups. The ultrastructural findings and caspase-3 activity were similar. The treatment groups demonstrated better results than the other groups. CONCLUSIONS: Gabapentin demonstrated significant neuroprotection after early phases of ischemic injury. Further studies with different experimental settings including neurological outcome are required to achieve conclusive results.

Adrenomedullin attenuates aortic cross-clamping-induced myocardial injury in rats.

BACKGROUND: In this study we investigate the effects of adrenomedullin on myocardial injury after ischemia-reperfusion (I/R) after abdominal aortic surgery. METHODS: Thirty-two Wistar rats were randomized into 4 groups (n = 8) as follows: control group (sham laparotomy), the aortic I/R group, aortic I/R plus adrenomedullin group (underwent aortic I/R periods, and received a bolus intravenous injection of .05 µg/kg/min adrenomedullin), and the control plus adrenomedullin group. RESULTS: Biochemical analysis showed that aortic I/R significantly increased (P < .05) the plasma levels of troponin-I and tumor necrosis factor-α, and the myocardial tissue levels of malondialdehyde, superoxide dismutase, catalase, and angiotensin II, whereas aortic I/R plus adrenomedullin significantly decreased these same factors (P < .05). Aortic I/R significantly increased (P < .05) myocardial tissue levels of nitric oxide whereas aortic I/R plus adrenomedullin significantly increased the same factor (P < .05). CONCLUSIONS: These results indicate that adrenomedullin has protective effects against myocardial injury induced by abdominal aortic I/R in rats.

Anti-apoptotic and neuroprotective effects of α-lipoic acid on spinal cord ischemia-reperfusion injury in rabbits.

PURPOSE: Radical oxygen species produced after injury counteracts antioxidant activity and frequently causes severe oxidative stress for the tissues. Alpha-lipoic acid is a powerful metabolic antioxidant with immunomodulatory effects which provides neuroprotection. The aim of this study is to investigate the neuroprotective and anti-apoptotic effects of alpha-lipoic acid on spinal cord ischemia-reperfusion. METHODS: Twenty-four adult, male, New Zealand rabbits were divided into sham (n = 8), control (n = 8), and treatment groups (n = 8). The abdominal aorta was clamped for 30 min by an aneurysm clip, approximately 1 cm below the renal artery and 1 cm above the iliac bifurcation in control and treatment groups. Only laparotomy was performed in the sham group. Twenty-five cubic centimeters of saline in control group and 100 mg/kg lipoic acid were administered intraperitoneally in the treatment group after closure of the incision. The animals were killed 48 h later. Spinal cord segments between L2 and S1 were harvested for analysis. Levels of nitric oxide, glutathione, malondialdehyde, advanced oxidation protein products, and superoxide dismutase were analyzed as markers of oxidative stress and inflammation. Caspase-3 activity was analyzed to detect the effect of lipoic acid on apoptosis. RESULTS: In all measured parameters of oxidative stress, administration of lipoic acid significantly demonstrated favorable effects. Both plasma and tissue levels of nitric oxide, glutathione, malondialdehyde, and advanced oxidation protein products significantly changed in favor of antioxidant activity. There was no significant difference between the plasma superoxide dismutase levels of the groups. Histopathological evaluation of the tissues also demonstrated significant decrease in cellular degeneration and infiltration parameters after lipoic acid administration. However, lipoic acid has no effect on caspase-3 activity. CONCLUSIONS: Although further studies considering different dose regimens and time intervals are required, the results of the present study prove that alpha-lipoic acid has favorable effects on experimental spinal cord ischemia-reperfusion injury.

The long-term effects of progesterone-only contraceptives on endometrium and ovary in rats.

Progestin-only (p-only) contraceptives often cause breakthrough bleeding for unknown reasons. In this study, we aimed to evaluate the long-term effects of p-only contraceptives to gain a better understanding of breakthrough bleeding mechanism. Wistar rats were divided into etonorgesterel implant (Group 1, n = 25), depot medroxyprogesterone acetate injectable (Group 2, n = 25), and control groups (n = 5). Five rats each from groups 1 and 2 were examined every 10 days for up to 50 days after the medication. Uteri and ovaries were removed and prepared for immunohistochemistry and scanning electron microscopy. The tissue nitric oxide (NO) levels were determined by Griess reaction. Dynamic changes of endometrial estrogen and progesterone receptor immunoreactivity were observed in a time-dependent manner in groups 1 and 2. The number of endometrial pinopodes, which are small endometrial protrusions, increased in both groups. There was no difference between groups for the estrogen receptor in the surface epithelium of the ovary. Estrogen-alpha and progesterone receptor in follicular cells decreased in a time-dependent manner. The granulosa cells underwent atrophic and were disorganized. Decreased levels of uterine tissue NO were determined in groups 1 and 2. The effect of some p-only contraceptives make some dynamic changes in the endometrium, ovaries, steroid hormone receptors, cell morphology, and biochemical features of the tissues during their use.

Metformin regresses endometriotic implants in rats by improving implant levels of superoxide dismutase, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinase-9.

OBJECTIVE: We sought to test if metformin could regress endometriotic explants in rats. STUDY DESIGN: After inducing endometriotic implants and randomization of female Wistar albino rats, they were given 25 and 50 mg/kg/day of oral metformin in group A (n = 9) and B (n = 8), respectively, for 28 days. Group C (n = 9) was given saline as placebo. RESULTS: Mean volume, weight, and histologic score of implants in groups A (P < .01, P < .05, and P < .05, respectively) and B (P < .01, P < .05, and P < .05, respectively) were significantly lower than in group C. The activity of superoxide dismutase and tissue inhibitor of metalloproteinase-2 staining in groups A (P < .05 and P < .01, respectively) and B (P < .01 and P < .01, respectively) was significantly higher than in the control group. Moreover, there were more significant reductions in implant levels of vascular endothelial growth factor and matrix metalloproteinase-9 in groups A (both P < .001) and B (both P < .001) than in group C. CONCLUSION: Metformin causes regression of endometriotic implants in rats.

Comparison of cyanoacrylate-assisted arteriotomy closure with conventional closure technique.

BACKGROUND: We aimed to compare conventional suture closure of arteriotomy with N-butyl-cyanoacrylate-assisted suture closure. METHODS: Forty Wistar rats were randomly divided into two groups. Standard arteriotomy was performed to the abdominal aorta through a midline incision. In the first group, arteriotomy was closed by 3 stitches with 45 degrees between each and in the second by two stitches with 0.1 ml (12-12.5 mg) cyanoacrylate. Amount of blood loss, operation time and severity of myointimal hyperplasia by immunohistochemistry on aorta segments were measured on postoperative days 7 and 30. RESULTS: Mean anastomotic time was 13.5 +/- 1.64 in the first and 13.0 +/- 1.75 min in the second group (p = 0.356). Operation time was 23.45 +/- 3.63 in the control and 21.0 +/- 3.09 min in the second group (p = 0.027). Mean amount of bleeding was 473.75 +/- 260.5 in the first and 327.5 +/- 155.36 microl in the second group (p = 0.037). Intimal thickness on the 7th day was 80.62 +/- 7.92 in the first and 83.24 +/- 3.42 microm in the second group, and on the 30th day was 81.64 +/- 5.11 in the first and 88.77 +/- 11.03 microm in the second group. The early and late intimal thicknesses were similar (p = 0.35 and 0.87, respectively). CONCLUSION: Reconstruction of arteriotomies with fewer sutures in combination with cyanoacrylate is a safe method associated with less blood loss and shorter operation time. It also does not lead to increased myointimal hyperplasia.

Caspase-3 and VEGF immunopositivity in seminiferous tubule germ cells in cases of obstructive and non-obstructive azoospermia in smokers versus non-smokers.

PURPOSE: To investigate immunostaining pattern of caspase-3, an apoptosis marker, and vascular endothelial growth factor (VEGF), an hypoxia marker in testis biopsy specimens collected either from smoking or non-smoking patients with azoospermia. METHODS: Testis biopsy specimens were obtained from thirty seven non-smoker and thirty eight smoker patients. Using immunochemistry technique, caspase-3 and VEGF were evaluated in all intratubular spermatogenic and interstitial Leydig cells. RESULT(S): Caspase-3 expression was significantly increased in germ cells in maturation arrest specimens in smoker azoospermic patients. No statistically significant difference was present between smokers and non-smokers for caspase-3 expression in Sertoli cell. However, the VEGF immunopositive Leydig cells were statistically higher in smokers. There were no differences between groups in terms of germ cell immunopositivity. CONCLUSION: Our results support the hypothesis that increased apoptosis contributes significantly to impaired spermatogenesis. We conjecture that germ cell apoptosis may be augmented by hypoxic microenvironments and environmental toxicants in smoking azoospermic men.

Heterogeneous appearance of VEGF (vascular endothelial growth factor) immunopositivity in cyst capsules of endometrioma.

The aim of the research was to reveal vascular endothelial growth factor (VEGF) immunolocalization in endometrioma cysts and endometrial tissues. The study group (group1) included 15 patients laparoscopically operated on for endometrioma and the control group (group 2) included 13 patients prepared for diagnostic laparoscopy for primary infertility. Biopsies from endometrioma cyst capsules, disease-free peritoneum and pipelle biopsies from the endometrium were taken from group 1. Biopsies from parietal peritoneum and endometrium were taken from group 2. Results showed VEGF immunoreactivity of peritoneal biopsies of group 1 was more intense than that of the control biopsies. A positive correlation was seen between the diameter of cyst capsules and VEGF labeling intensity and as the size of cyst enlarged, the appearance of non-homogeneous distribution of VEGF immunolocalization became more frequent. We conclude that the variation of VEGF immunolocalization in endometrioma cysts may be attributed to other possible angiogenic molecules in the pathogenesis and may cause unexpected responses to anti-angiogenic therapies.

Vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) immunoreactivities in rat ovaries and uterine tubes after tubal ligation: a controlled immunohistochemical study.

OBJECTIVE: To evaluate the effects of tubal ligation on ovarian and tubal tissues by means of immunohistochemical evaluation of two hypoxia related mediators: vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). DESIGN: Fourteen Sprague-Dawley female rats were divided into two groups: a tubal ligation (Pomeroy technique) was carried out on rats in group 1 (n = 7) whereas those in group 2 served as controls (n = 7). Salpingo-oophorectomy was performed in group 1 during the second oestrous period following tubal ligation. Rats in group 2 were submitted to a salpingo-oophorectomy, as well. VEGF and iNOS immunoreactivities in ovarian and tubal tissues were evaluated by means of immunohistochemistry. Immunohistochemical scores and number of antral follicles were compared. RESULTS: In the ovary, VEGF immunoreactivity was significantly more intense in the granulosa (p = 0.002) and the theca cells (p = 0.001) of rats in group 1 but, in ovarian medulla (p = 0.259) and germinal epithelium (p = 0.209), it was not significantly different from that of rats in group 2. The iNOS immunoreactivity in ovarian granulosa cells (p = 0.073) and germinal epithelial cells (p = 0.805) did not differ between the two groups. The cytoplasmic VEGF (p = 0.001) and iNOS (p = 0.017) immunoreactivities in the uterine tube, were significantly more intense in group 1. However, VEGF immunoreactivity in the lamina propria of the uterine tube (p = 0.209) was of similar intensity in both groups. CONCLUSION: Tubal ligation may lead to supraphysiological hypoxia as evidenced by increased VEGF and iNOS immunoreactivities in ovarian and tubal tissues.