Diogo Benchimol de Souza

Effects of Chronic Stress on Penile Corpus Cavernosum of Rats

The objective of this study was to investigate structural changes in the penile corpus cavernosum of prepubertal chronically stressed rats. Eight Wistar rats were assigned into the stress group (SG) and were submitted to 2 hours of tube restraint daily, from the fourth to the ninth week of life. Another 7 rats were used as the control group (CG). All animals were weighed weekly. At day 64, animals were sacrificed by anesthetic overdose, blood was collected for testosterone concentration by radioimmunoassay, and penis and adrenal were collected. Adrenal mass index and testosterone serum levels were used to assess the efficacy of the stress stimulus. The surface density of connective tissue and smooth muscle fibers of corpus cavernosum were measured on Masson trichromic-stained slices. Picrosirius red-stained slices were assessed under polarized light for different types of collagen. The Students t test was applied for mean comparisons, with P < .05 considered significant. Testosterone serum concentrations decreased and adrenal mass index increased, confirming the effectiveness of the stress protocol. Smooth muscle fibers of corpus cavernosum decreased from 14.07% (CG) to 8.98% (SG) (P = .02), and connective tissue increased from 53.66% (CG) to 64.47% (SG) (P = .01). Also, there was a higher level of type I collagen in the SG animals compared with the CG. Stress stimuli induced structural changes in the corpus cavernosum of rats suggestive of penile fibrosis, which may play a role in erection dysfunction.

Effects of Immobilization Stress on Kidneys of Wistar Male Rats: A Morphometrical and Stereological Analysis

This paper verifies the morphological changes induced by immobilization stress on the kidney of rats by using stereological methods. Fifteen 4-week-old Wistar male rats were randomly assigned to control (n = 7) and stressed (n = 8) groups. Stress stimuli were performed over 5 weeks by immobilization of the rats for 2 h daily in a rigid opaque plastic cylinder that restrained their movements. Increases in the adrenal mass index (p < 0.05) and decreases in serum testosterone levels (p < 0.05) demonstrated the efficacy of the stressor stimuli. Stressed rats presented diminished body weight gain when compared to controls (p < 0.05). The mean values of kidney weight, kidney volume, kidney volume index and glomerular volume density were significantly lower in the stressed group (p < 0.05); nevertheless, no significant difference was found in the cortical/medullar ratio or in the volume-weighted mean glomerular volume. The number of glomeruli per kidney was 45% lower in the stressed group (p < 0.0001), but no change in serum creatinine levels was found. However, the morphological alterations may have serious implications predisposing individuals to renal disease and hypertension in adult life.

Effect of Antioxidants on Outcome of Testicular Torsion in Rats of Different Ages

PURPOSE We assessed reproductive and testicular function in adult rats after testicular torsion created before, during and after puberty, and with vs without resveratrol or arginine treatment. MATERIALS AND METHODS Age matched rats were divided into groups, including simulated surgery without testicular torsion, 720-degree testicular torsion for 4 hours, testicular torsion with resveratrol treatment and testicular torsion with arginine treatment. To study reproductive function at age 12 weeks each rat mated with 3 females. The males were sacrificed at age 14 weeks. Spermatozoids were collected from the epididymal tail and evaluated for concentration, motility and viability. Testicular samples were collected for morphological analysis. RESULTS Reproductive function was not altered by testicular torsion but antioxidants improved potency. Compared to sham operated and contralateral samples all spermatozoid parameters from testicular torsion samples were inferior. Resveratrol and arginine did not improve spermatozoid quality or quantity in torsed testes but contralateral samples were improved by each drug. The seminiferous epithelium of rats submitted to testicular torsion during puberty was least affected. Each antioxidant partially to totally prevented the morphological alterations found in rats with untreated testicular torsion. Rats submitted to testicular torsion before puberty that were treated with antioxidants showed the fewest changes. CONCLUSIONS Testicular morphology was altered less in rats when torsion occurred earlier in life, that is during puberty. Treatment with antioxidants improved contralateral spermatozoid production and some fertility parameters. Each antioxidant also prevented testicular morphology alterations after testicular torsion. Prepubertal rats benefited most from antioxidant treatment.

Laparoscopic Partial Nephrectomy Under Warm Ischemia Reduces the Glomerular Density in a Pig Model

PURPOSE To investigate the number of nephrons, using unbiased stereological method, after warm ischemia for laparoscopic partial nephrectomy in a pig model. METHODS Fourteen pigs underwent left laparoscopic partial nephrectomy, and four animals were used as controls (not operated). Renal vessels were clamped, and 25% of kidney length was resected. The animals were euthanized after 2 weeks, and the kidneys were collected. Thus, we analyzed 14 left kidneys submitted to warm ischemia, 14 right kidneys from the same animals but not submitted to ischemia and eight kidneys from control animals. Renal fragments were processed using routine histological methods. The total operative time and the time of warm ischemia were recorded. Glomerular mean volume and glomerular density were quantified by stereological methods. Creatinine serum levels were assessed preoperatively and before euthanasia. RESULTS Surgical time was 71 ± 17 minutes, and ischemia time was 16 ± 5 minutes. The mean glomerular volume in the left kidneys was higher when compared with controls and to right kidneys (p<0.05). In addition, the glomerular density was reduced in the left kidneys (p<0.05) when compared with controls and right kidneys. Nevertheless, creatinine serum levels after 2 weeks of surgery were not different from the preoperative levels. No difference was found for stereological measurements between controls and right kidneys. CONCLUSION Stereological determination of glomerular density can be used as an accurate and objective method for studies regarding renal damage from ischemia. Warm ischemia during laparoscopic partial nephrectomy in pigs determined a significant reduction of glomerular density in the ipsilateral remaining parenchyma.

Kidney Healing After Laparoscopic Partial Nephrectomy Without Collecting System Closure in Pigs

OBJECTIVES To access the kidney healing after laparoscopic partial nephrectomy without closing of the collecting system in pigs. METHODS Fourteen pigs underwent left partial laparoscopic nephrectomy, with removal of 25% of the kidney length at caudal pole (n = 7) or at cranial pole (n = 7). Briefly, the surgical technique involved a transperitoneal laparoscopic access, en bloc vascular clamping of renal vessels, tissue excision with cold scissor and monopolar energy hemostasis of only the parenchyma, leaving the collecting system opened, with no insertion of a double-J catheter. The animals were clinically evaluated during 14 days, and afterward were killed. Serum levels of urea and creatinine were assessed prior and at different moments after surgery. Macroscopic necropsy analysis, a retrograde ex vivo pyelogram and a histologic study of the operated renal poles were performed. RESULTS The animals did not show any postoperative clinical alterations. Serum levels of urea and creatinine showed a slight raising at the second postoperative day with gradual decreasing to preoperative levels. At necropsy, the abdominal cavity was normal, with normal quantity and aspect of peritoneal liquid. No signs of urine leakage were found. The operated renal pole was always involved by a perirenal fibrosis with adherences to adjacent organs. CONCLUSIONS The pig kidney collecting system healed well without any kind of suture or internal drainage. Therefore, we concluded that the pig kidney is not an adequate model for research on which the collecting system healing is an important aspect to be considered.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

PURPOSE Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. MATERIALS AND METHODS Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. RESULTS For all analyzed parameters Sham and Pp groups presented no statistical difference. CONCLUSION When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats.

Penile Histomorphometrical Evaluation in Hypertensive Rats Treated with Sildenafil or Enalapril Alone or in Combination: A Comparison with Normotensive and Untreated Hypertensive Rats

INTRODUCTION Erectile dysfunction (ED) is frequently associated to hypertension and antihypertensive drugs; however, the penile morphological aspects on these situations are poorly known. AIM Evaluate the penile morphology of untreated hypertensive rats and rats treated with enalapril or sildenafil alone or in combination to verify the hypothesis that morphological alterations promoted by hypertension on corpus cavernosum could be ameliorated by the use of angiotensin-converting enzyme inhibitors and/or phosphodiesterase type 5 inhibitors. METHODS Fifty male rats were assigned into five groups: normotensive rats, untreated spontaneously hypertensive rats (SHRs), and SHR treated with enalapril or sildenafil alone or in combination. Blood pressure was measured weekly. At the conclusion of the study, the rats were euthanized, and their penises were collected for histomorphometrical analysis. MAIN OUTCOME MEASURES The cross-sectional areas of the penis, tunica albuginea, and corpus cavernosum were measured. The density of the corpus cavernosum structures was quantified. RESULTS Both groups of SHR rats treated with enalapril became normotensive. Untreated SHR showed no difference in penile and cavernosal cross-sectional area compared with normotensive rats; however, those rats treated with enalapril or sildenafil alone demonstrated an increase in these parameters. Rats receiving combination therapy showed no cross-sectional area differences compared with normotensive rats. Cavernosal connective tissue density was increased, while the sinusoidal spaces were diminished in untreated SHR. All treatments were effective in maintaining connective tissue density in comparison with normotensive animals. Cavernosal smooth muscle density was similar in all groups, with the exception of the combination therapy group, which demonstrated a reduction in smooth muscle. CONCLUSIONS Hypertension promoted structural alterations in the corpus cavernosum that may be related to ED. Enalapril- and sildenafil-treated animals had preservation of normal corpus cavernosum structure and an increase in penile and cavernosal cross-sectional area. The combination of these drugs showed less benefit than individual use.

How to Quantify Penile Corpus Cavernosum Structures with Histomorphometry: Comparison of Two Methods

The use of morphometrical tools in biomedical research permits the accurate comparison of specimens subjected to different conditions, and the surface density of structures is commonly used for this purpose. The traditional point-counting method is reliable but time-consuming, with computer-aided methods being proposed as an alternative. The aim of this study was to compare the surface density data of penile corpus cavernosum trabecular smooth muscle in different groups of rats, measured by two observers using the point-counting or color-based segmentation method. Ten normotensive and 10 hypertensive male rats were used in this study. Rat penises were processed to obtain smooth muscle immunostained histological slices and photomicrographs captured for analysis. The smooth muscle surface density was measured in both groups by two different observers by the point-counting method and by the color-based segmentation method. Hypertensive rats showed an increase in smooth muscle surface density by the two methods, and no difference was found between the results of the two observers. However, surface density values were higher by the point-counting method. The use of either method did not influence the final interpretation of the results, and both proved to have adequate reproducibility. However, as differences were found between the two methods, results obtained by either method should not be compared.

Histomorphometric comparison of the human, swine, and ovine collecting systems

The ovine kidney has been recently determined to be a better model than the swine kidney for the study of collecting system healing after partial nephrectomy. However, there is no histological study comparing the collecting systems of these species. To compare human, swine, and ovine collecting systems using histomorphometry. The collecting systems of 10 kidneys from each species (human, swine, and ovine) were processed for histomorphometry. The thickness of the three layers (mucosal connective tissue, submucosal muscular, and adventitial connective tissue) were measured. The densities of smooth muscle fibers, elastic system fibers, and cells were also measured. Additionally, blood vessel density in the adventitial connective tissue was measured. Analysis of the collecting systems from the three species presented several differences. The adventitial connective tissue from the swine samples was thicker, with more blood vessels and smooth muscle fibers, compared with that from the human and ovine samples. Swine also had higher density of elastic fibers on the submucosal muscular layer. Ovine and human collecting systems shared several similar features, such as blood vessel and elastic fiber density in all layers and the density of cellular and muscular fibers in the submucosal muscular and adventitial connective tissue layers. The collecting system of the ovine kidney is more similar to that of the human kidney compared with that of the swine kidney. This may explain the differences between the healing mechanisms in swine and those in humans and sheep after partial nephrectomy. Anat Rec, 299:967–972, 2016.

Leptin and leptin receptor expressions in prostate tumors may predict disease aggressiveness

PURPOSE The aim of this study was to evaluate the expression of leptin and its receptor in histological sections of prostate tumors, and their association with prognostic factors. METHODS A total of 532 surgical specimens from prostate cancer were studied. After histopathological diagnosis, the samples were included in tissue microarrays containing cores from tumor and non-tumor (benign prostatic hyperplasia) areas. These were immunostained with anti-leptin and anti-leptin-receptor antibodies. Objective and subjective analyses were performed. Students-t-test and ANOVA were used to compare mean values, and linear regression was used to evaluate the correlation between histological results and prognostic indicators. RESULTS Leptin receptor expression was reduced in tumors with a positive surgical margin, urethral margin involvement, and seminal vesicles invasion. Further, there was a negative correlation between the expression of leptin receptor in tumor areas and the sum of prognostic factors, suggesting that leptin receptor may predict the aggressiveness of disease. CONCLUSION Our findings suggest that leptin receptor expression is a potential prognostic factor for PCa. Further investigation is needed to support the use of leptin receptor as a novel biomarker, although leptin itself does not seem to predict the aggressiveness of prostate cancer.