Publications by authors named "Luis Fernando Tirapelli"

28 Publications

  • Page 1 of 1

Expression of MicroRNAs (miR-15b, miR-16, miR-138, miR-221, and miR-222) as Biomarkers of Endothelial Corpus Cavernosum Dysfunction in a Diabetic Alcoholic Murine Model.

Sex Med 2021 Apr 3;9(2):100326. Epub 2021 Mar 3.

Division of Urology, University of São Paulo, Ribeirão Preto Medical School, Surgery and Anatomy, Ribeirao Preto, São Paulo, Brazil.

Introduction: MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression and are related to endothelial dysfunction (EnD). Recently, miRNAs have also been explored as potential biomarkers and target molecular therapy of erectile dysfunction (ED). Could the miRNAs be the tip of the iceberg of chronic arterial disease foreshadowed by the ED?

Aim: To investigate the expression of miR-15b, miR-16, miR-138, miR-221, and miR-222 in corpus cavernosum (CC) and peripheral blood in a rat model of endothelium dysfunction secondary to diabetes (DM) and alcohol consumption to assess potential endothelial lesion biomarkers.

Methods: Twenty males Wistar rats were divided into 4 groups: control group (C), alcohol consumption group (A), diabetic group (D), diabetic-alcohol consumption group (D + A). DM was alloxan-induced and alcohol consumption was through progressive increase of ethanol concentration in drinkable water. After 7 weeks, miRNAs expressions from CC and blood sample were evaluated by real-time PCR. Functional assessment of CC was performed in an acetylcholine endothelium-dependent relaxation pharmacological study.

Main Outcome Measure: miRNA expression in CC and blood were evaluated; pharmacological study in CC strips was conducted to validate EnD.

Results: We found that 3 miRNAs (miR-16, miR-221, and miR-222) were downregulated in the CC in the D+A group, while all 5 miRNAs were downregulated in the blood of D and D + A groups. The endothelium-dependent relaxation induced by acetylcholine was significantly decreased in groups A, D, and D + A. Diagnostic accuracy estimated by AUC, to discriminating groups A, D, and D + A from controls, was superior to >0.9 in all plasmatic miRNAs.

Conclusion: miRNAs downregulation was identified in both CC and blood notably in DM associated with alcohol consumption animals (D + A), the greatest endothelial injury potential group. Serum miRNAs have also demonstrated high diagnostic accuracy properties in predicting CC relaxation dysfunction labeling EnD. RB Tiraboschi, FSL Neto, DP da Cunha Tirapelli, et al. Expression of MicroRNAs (miR-15b, miR-16, miR-138, miR-221, and miR-222) as Biomarkers of Endothelial Corpus Cavernosum Dysfunction in a Diabetic Alcoholic Murine Model. Sex Med 2021;9:100326.
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http://dx.doi.org/10.1016/j.esxm.2021.100326DOI Listing
April 2021

Chronic alcoholism associated with diabetes induced apoptosis in the corpus cavernosum of rats.

Acta Cir Bras 2020 Sep 7;35(8):e202000805. Epub 2020 Sep 7.

PhD, Associate Professor, Department of Surgery and Anatomy, FMRP-USP, Ribeirao Preto-SP, Brazil. Conception and design of the study, critical revision, supervised all phases.

Purpose: To evaluate the effects of alcohol exposure and diabetes on apoptotic process in the corpus cavernosum.

Methods: Forty eight male Wistar rats were divided into four groups: control, diabetic, alcoholic and diabetic-alcoholic. Samples of the corpus cavernosum were prepared to study protein expression of apoptotic genes (Caspases-3 and 9) by immunohistochemistry and Real-Time PCR.

Results: The immunoreactivity of Caspases-3 and -9 was diffuse and higher in the treated groups though there was no significant difference between the experimental groups, only when compared with the control group. An increase was observed in the gene expression of Caspases-9 in the diabetic and ethanol-diabetic groups when compared with control and ethanol groups.

Conclusions: The association of these factors (ethanol and diabetes) probably can affect the apoptosis mechanism in lesions of the cavernous tissue in the rat penis. Both gene and protein expression of Caspase-9 in diabetic and ethanol-diabetic groups suggest the involvement of the apoptosis cascade from this study model.
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http://dx.doi.org/10.1590/s0102-865020200080000005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478468PMC
September 2020

Morphological and molecular analysis of apoptosis in the corpus cavernosum of rats submitted to a chronic alcoholism model.

Acta Cir Bras 2020 3;35(3):e202000305. Epub 2020 Jun 3.

PhD, Associate Professor, Department of Surgery and Anatomy, FMRP-USP, Ribeirao Preto-SP, Brazil. Final approval.

Purpose: To evaluate the effect of chronic alcoholism on morphometry and apoptosis mechanism and correlate with miRNA-21 expression in the corpus cavernosum of rats.

Methods: Twenty-four rats were divided into two experimental groups: Control (C) and Alcoholic group (A). After two weeks of an adaptive phase, rats from group A received only ethanol solution (20%) during 7 weeks. The morphometric and caspase-3 immunohistochemistry analysis were performed in the corpus cavernosum. The miRNA-21 expression was analyzed in blood and cavernous tissue.

Results: Chronic ethanol consumption decreased cavernosal smooth muscle area of alcoholic rats. The protein expression of caspase 3 in the corpus cavernosum was higher in A compared to the C group. There was no difference in the expression of miRNA-21 in serum and cavernous tissue between the groups.

Conclusion: Chronic ethanol consumption reduced smooth muscle area and increased caspase 3 in the corpus cavernosum of rats, without altered serum and cavernosal miR-21 gene expression.
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http://dx.doi.org/10.1590/s0102-865020200030000005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282493PMC
June 2020

Analysis of Caspase-9 protein and microRNAs miR-21, miR-126 and miR-155 related to the apoptosis mechanism in the cerebellum of rats submitted to focal cerebral ischemia associated with an alcoholism model.

Arq Neuropsiquiatr 2019 24;77(10):689-695. Epub 2019 Oct 24.

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Ribeirão Preto SP, Brasil.

Objective: This study aimed to analyze the cerebellum of rats submitted to an experimental focal cerebral ischemia, by middle cerebral artery occlusion for 90 minutes, followed by reperfusion for 48 hours, associated with an alcoholism model.

Methods: Fifty adult Wistar rats were used, subdivided into five experimental groups: control group (C): animals submitted to anesthesia only; sham group (S): animals submitted to complete simulation of the surgical procedure; ischemic group (I): animals submitted to focal cerebral ischemia for 90 minutes followed by reperfusion for 48 hours; alcoholic group (A): animals that received daily absolute ethanol diluted 20% in water for four weeks; and, ischemic and alcoholic group (I + A): animals receiving the same treatment as group A and, after four weeks, submitted to focal cerebral ischemia for 90 minutes, followed by reperfusion for 48 hours. The cerebellum samples were collected and immunohistochemical analysis of Caspase-9 protein and serum analysis by RT-PCR of microRNAs miR-21, miR-126 and miR155 were performed.

Results: The expression of Caspase-9 was higher in groups I, A and I + A. In the microRNAs analyses, miR-126 was higher in groups A and I + A, miR-155 was higher in groups I and I + A.

Conclusions: We conclude that apoptosis occurs in the cerebellar cortex, even if it is distant from the ischemic focus, and that microRNAs 126 and 155 show a correlation with cellular apoptosis in ischemic rats and those submitted to the chronic alcohol model.
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http://dx.doi.org/10.1590/0004-282X20190126DOI Listing
July 2020

Expression of MicroRNAs miR-145, miR-181c, miR-199a and miR-1183 in the Blood and Hippocampus of Patients with Mesial Temporal Lobe Epilepsy.

J Mol Neurosci 2019 Dec 31;69(4):580-587. Epub 2019 Jul 31.

Department of Surgery and Anatomy, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.

The aim of this study was to analyze the expression profiles of the microRNAs (miRNAs) miR-145, miR-181c, miR-199a and miR-1183 in the hippocampus and blood of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) and to investigate whether these can be used as diagnosis and prognosis biomarkers for epilepsy. Hippocampus and blood samples were collected from 20 patients with MTLE-HS, ten of whom had a favorable surgical outcome (Engel I) and ten with an unfavorable surgical outcome (Engel III-IV). Hippocampus samples from autopsied individuals with no neurological or psychiatric medical history (necropsy samples) and blood samples from healthy individuals were used as controls. Real-time quantitative PCR (RQ-PCR) was used to analyze miRNA expression. The results showed that the expressions of these miRNAs differed quantitatively in the hippocampus and blood of patients with MTLE-HS in comparison to the respective control. This difference was most pronounced for miR-145, which was hypo-expressed in the hippocampus and hyper-expressed in the blood of MTLE-HS patients. MiRNAs miR-145, miR-181c, miR-199a and miR-1183 were hyper-expressed in the blood of patients with MTLE-HS. No statistical differences in the levels of these miRNAs in the blood or hippocampus were found between Engel I patients and Engel III-IV patients. These results suggest that the analyzed microRNAs are potential circulating biomarkers for epilepsy diagnosis.
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http://dx.doi.org/10.1007/s12031-019-01386-wDOI Listing
December 2019

Morphological and immunohistochemical analysis of proteins CASPASE 3 and XIAP in rats subjected to cerebral ischemia and chronic alcoholism.

Acta Cir Bras 2018 Aug;33(8):652-663

Assistant Professor, Department of Surgery and Anatomy, Medical School, USP, Ribeirao Preto-SP, Brazil. Design of the study, manuscript writing.

Purpose: To evaluate histopathological and ultrastructural changes and expression of proteins related to apoptosis CASPASE 3 and XIAP after experimental induction of temporary focal cerebral ischemia (90 minutes) due to obstruction of the middle cerebral artery in alcoholism model.

Methods: Forty adult Wistar rats were used, subdivided into 5 experimental groups: control group (C); Sham group (S); Ischemic group (I); Alcoholic group (A); and Ischemic and Alcoholized group (I+A): animals submitted to the same treatment of group A and after four weeks were submitted to focal cerebral ischemia during 90 minutes, followed by reperfusion of 48 hours. Were processed for histopathological analysis and immunohistochemistry (for the protein expression of CASPASE -3 and XIAP).

Results: Greater histopathological changes were observed in the animals of groups I and I+A in the three areas analyzed. The neuronal loss was higher in the medial striatum region of the animals of groups I and I + A. The protein expression of CASPASE -3 was higher than that of XIAP in the groups I and I + A for both proteins.

Conclusion: The expression of XIAP was slightly higher where the histopathological changes and expression of CASPASE -3 was less evident.
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http://dx.doi.org/10.1590/s0102-865020180080000001DOI Listing
August 2018

High expression of XIAP and Bcl-2 may inhibit programmed cell death in glioblastomas.

Arq Neuropsiquiatr 2017 Dec;75(12):875-880

Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Ribeirão Preto SP, Brasil.

Glioblastoma (GBM) is the most malignant glioma and represents 29% of all brain tumors. Tumorigenesis is intimately connected with characteristics acquired in the physiologic pathway of cellular death.

Objective: In the present study, the expression of anti-apoptotic (XIAP and Bcl-2) and apoptotic (cytochrome C, caspase 9, APAF-1), caspase 3 and the Smac/DIABLO genes related to the apoptosis pathway were evaluated in 30 samples of glioblastoma.

Methods: The gene expression was evaluated in 30 glioblastomas (WHO grade IV) and compared to 10 white matter control samples with real-time PCR.

Results And Conclusion: There were higher expressions of XIAP (p = 0.0032) and Bcl-2 (p = 0.0351) in the glioblastoma samples compared to the control samples of normal brain. These results raise the question of whether Bcl-2 and XIAP genes can be responsible for the inhibition of programmed cell death in glioblastomas. Moreover, they provide additional information capable of allowing the development of new target therapy strategies.
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http://dx.doi.org/10.1590/0004-282X20170156DOI Listing
December 2017

High expression of anti-apoptotic genes in grade I and II meningiomas.

Arq Neuropsiquiatr 2017 04;75(4):209-215

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Ribeirão Preto SP, Brasil.

Objective: To evaluate the expression of c-FLIP, XIAP, Bcl-2, caspase 3, 8 and 9, cytochrome c, APAF 1 and Smac/DIABLO genes related to apoptosis pathways.

Methods: The gene expression was evaluated in 30 meningiomas (WHO grades I and II) and in 10 normal samples (from arachnoid tissue) through PCR-RT.

Results: The results showed higher expression of anti-apoptotic genes in meningiomas when compared to the control group, which had a low expression of pro-apoptotic genes.

Conclusion: There is a possible block in the activation of caspases through the intrinsic apoptosis pathway in meningiomas. c-FLIP modulates caspase 8 and, by inhibiting its activation due to the lack of connection with the receiver, there is a block to the FAS activation of apoptosis by its extrinsic pathway.
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http://dx.doi.org/10.1590/0004-282X20170027DOI Listing
April 2017

Expression profiles of eNOS, iNOS and microRNA-27b in the corpus cavernosum of rats submitted to chronic alcoholism and Diabetes mellitus.

Acta Cir Bras 2017 Jan;32(1):38-45

Associate Professor, Division of Urology, Department of Surgery and Anatomy, School of Medicine of Ribeirao Preto, USP, Ribeirao Preto-SP, Brazil. Concept, design, intellectual and scientific content of the study; critical revision; supervised all phases of the study.

Purpose: : To evaluate the expression of endothelial and inducible NOS in addition to the miRNA-27b in the corpus cavernosum and peripheral blood of healthy rats, diabetic rats, alcoholic rats and rats with both pathologies.

Methods: : Forty eight Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D) and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study protein expressions of eNOS and iNOS by immunohistochemistry and expression of miRNA-27b in the corpus cavernosum and peripheral blood.

Results: : Immunohistochemistry for eNOS and iNOS showed an increase in cavernosal smooth muscle cells in the alcoholic, diabetic and alcoholic-diabetic groups when compared with the control group. Similarly, the mRNA levels for eNOS were increased in cavernosal smooth muscle (CSM) in the alcoholic, diabetic and alcoholic-diabetic groups and miRNA-27b were decreased in CSM in the alcoholic, diabetic and alcoholic-diabetic groups.

Conclusion: : The major new finding of our study was an impairment of relaxation of cavernosal smooth muscle in alcoholic, diabetic, and alcoholic-diabetic rats that involved a decrease in the nitric oxide pathway by endothelium-dependent mechanisms accompanied by a change in the corpus cavernosum contractile sensitivity.
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http://dx.doi.org/10.1590/s0102-865020170105DOI Listing
January 2017

Expression of NMDA receptor and microRNA-219 in rats submitted to cerebral ischemia associated with alcoholism.

Arq Neuropsiquiatr 2017 Jan;75(1):30-35

Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Ribeirão Preto SP, Brasil.

Objective:: To evaluate the microRNA-219 and NMDA expression in brain tissue and blood of animals subjected to cerebral ischemia associated with alcoholism.

Methods:: Fifty Wistar rats were divided into groups: control, sham, ischemic, alcoholic, and ischemic plus alcoholic. The expression of microRNA-219 and NMDA were analyzed by real-time PCR.

Results:: When compared to the control group, the microRNA-219 in brain tissue was less expressed in the ischemic, alcoholic, and ischemic plus alcoholic groups. In the blood, this microRNA had lower expression in alcoholic and ischemic plus alcoholic groups. In the brain tissue the NMDA gene expression was greater in the ischemic, alcoholic, and ischemic plus alcoholic groups.

Conclusion:: A possible modulation of NMDA by microRNA-219 was observed with an inverse correlation between them.
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http://dx.doi.org/10.1590/0004-282X20160188DOI Listing
January 2017

Morphological and immunohistochemical analysis of apoptosis in the cerebellum of rats subjected to focal cerebral ischemia with or without alcoholism model.

Acta Cir Bras 2016 Sep;31(9):629-637

Associate Professor, Department of Surgery and Anatomy, School of Medicine of Ribeirao Preto, USP, Brazil. Conception, design, intellectual and scientific content of the study; manuscript writing, critical revision; supervision of all phases of the study.

Purpose: : To evaluated histopathological changes, morphometric and expression of proteins CASPASE-3, BCL-2 and XIAP related to apoptosis in the cerebellum after induction of temporary focal cerebral ischemia followed by reperfusion, with or without a model of chronic alcoholism.

Methods: : Fifty Wistar rats were used and divided into: control group (C), sham group (S), ischemic group (I), alcoholic group (A), and ischemic and alcoholic group (IA). The cerebellum samples collected were stained for histopathological and morphometric analysis and immunohistochemistry study.

Results: : Histopathological changes were observed a greater degree in animals in groups A and IA. The morphometric study showed no difference in the amount of cells in the granular layer of the cerebellum between the groups. The expression of CASPASE-3 was higher than BCL-2 and XIAP in the groups A and IA.

Conclusion: : We observed correlation between histopathological changes and the occurrence of apoptosis in cerebellar cortex.
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http://dx.doi.org/10.1590/S0102-865020160090000009DOI Listing
September 2016

Straight sinus: ultrastructural analysis aimed at surgical tumor resection.

J Neurosurg 2016 08 8;125(2):494-507. Epub 2016 Jan 8.

Division of Neurosurgery and.

OBJECTIVE Accurate knowledge of the anatomy of the straight sinus (SS) is relevant for surgical purposes. During one surgical procedure involving the removal of part of the SS wall, the authors observed that the venous blood flow was maintained in the SS, possibly through a vein-like structure within the dural sinus or dural multiple layers. This observation and its divergence from descriptions of the histological features of the SS walls motivated the present study. The authors aimed to investigate whether it is possible to dissect the SS walls while keeping the lumen intact, and to describe the histological and ultrastructural composition of the SS wall. METHODS A total of 22 cadaveric specimens were used. The SS was divided into three portions: anterior, middle, and posterior. The characteristics of the SS walls were analyzed, and the feasibility of dissecting them while keeping the SS lumen intact was assessed. The thickness and the number of collagen fibers and other tissues in the SS walls were compared with the same variables in other venous sinuses. Masson's trichrome and Verhoeff's stains were used to assess collagen and elastic fibers, respectively. The data were analyzed using Zeiss image analysis software (KS400). RESULTS A vein-like structure independent of the SS walls was found in at least one of the portions of the SS in 8 of 22 samples (36.36%). The inferior wall could be delaminated in at least one portion in 21 of 22 samples (95.45%), whereas the lateral walls could seldom be delaminated. The inferior wall of the SS was thicker (p < 0.05) and exhibited less collagen and greater amounts of other tissues-including elastic fibers, connective tissue, blood vessels, and nerve fibers (p < 0.05)-compared with the lateral walls. Transmission electron microscopy revealed the presence of muscle fibers at a level deeper than that of the subendothelial connective tissue in the inferior wall of the SS, extending from its junction with the great cerebral vein to the confluence of sinuses. CONCLUSIONS The presence of a structure within the SS that can maintain the venous blood flow despite the dural wall might be considered an anatomical variation. The greater thickness of the inferior wall of the SS compared with the lateral walls is mainly due to the presence of larger amounts of tissues other than collagen. Delamination of the inferior wall of the SS was mostly possible in its inferior wall, but an attempt to delaminate the lateral walls is not recommended. Ultrastructural assessment corroborated a recent report of the presence of muscle fibers in the inferior wall of the SS.
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http://dx.doi.org/10.3171/2015.6.JNS15584DOI Listing
August 2016

Guanylate cyclase inhibition by methylene blue in circulatory shock caused by acute necrotizing pancreatitis: a word of caution based on a porcine model.

Rev Col Bras Cir 2013 Nov-Dec;40(6):480-9

Objective: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs.

Methods: acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05).

Results: amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group.

Conclusion: preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.
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http://dx.doi.org/10.1590/s0100-69912013000600011DOI Listing
April 2015

Effect of photodynamic therapy on the skin using the ultrashort laser ablation.

J Biophotonics 2014 Aug 11;7(8):631-7. Epub 2013 Apr 11.

Physics Institute of Sao Carlos IFSC, University of Sao Paulo, Biophotonics Laboratory, Sao Carlos-SP, Brazil.

Photodynamic Therapy (PDT) with 5-aminolevulinic acid (ALA) is known to be limited for applications in tumours of large volume mainly due to the limited penetration of topical photosensitization. The results show that micro-holes created using a femtosecond laser before PDT significantly increased the depth of PDT effect in the healthy tissue. The combination of ultrashort laser ablation technique with PDT showed an important scientific breakthrough related to transportation and delivery of drugs into the deeper regions of the tissue.
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http://dx.doi.org/10.1002/jbio.201300024DOI Listing
August 2014

Pre, intra and post-ischemic hypothermic neuroprotection in temporary focal cerebral ischemia in rats: morphometric analysis.

Arq Neuropsiquiatr 2012 Aug;70(8):609-16

Division of Neurosurgery, Department of Surgery and Anatomy, Ribeirão Preto Medical School, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Objective: To evaluate the neuroprotection of mild hypothermia, applied in different moments, in temporary focal cerebral ischemia in rats.

Methods: Rats was divided into Control (C), Sham (S), Ischemic-control(IC), Pre-ischemic Hypothermia (IH1), Intra-ischemic Hypothermia (IH2), and Post-ischemic Hypothermia (IH3) groups. Morphometry was performed using the KS400 software (Carl Zeiss®) in coronal sections stained by Luxol Fast Blue. Ischemic areas and volumes were obtained.

Results: Statistically, blue areas showed difference for C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.01; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH vs. IH2 (p=0.39; p=0.85; p=0.63). Red areas showed difference between C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.009; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH1 vs. IH2 (p=0.48; p=0.27; p=0.68). Average ischemic areas and ischemic volumes showed difference between IC vs. IH1 and IC vs. IH2 (p=0.0001 and p=0.0011), and no difference between IC vs. IH3 and IH1 vs. IH2 (p=0.57; p=0.79).

Conclusion: Pre-ischemic and intra-ischemic hypothermia were shown to be similarly neuroprotective, but this was not true for post-ischemic hypothermia.
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http://dx.doi.org/10.1590/s0004-282x2012000800010DOI Listing
August 2012

Nitric oxide synthase in heart and thoracic aorta after liver ischemia and reperfusion injury: an experimental study in rats.

Exp Clin Transplant 2012 Feb;10(1):43-8

Department of Surgery and Liver Transplantation, University of Pernambuco, Brazil.

Objectives: We tested the effects of liver reperfusion in the immunohistochemical expression of nitric oxide synthase on the thoracic aorta and the heart.

Materials And Methods: We randomized 24 male Wistar rats into 3 groups: (1) control; (2) R2 group, with 60 minutes of partial (70%) liver ischemia and 2 hours of global liver reperfusion; (3) and R6 group, with 60 minutes of partial liver ischemia and 6 hours of global liver reperfusion.

Results: In the heart, there was little, diffuse immunohistochemical endothelial staining; immunohistochemical inducible nitric oxide synthase staining was expressed in the adventitia layer of intramyocardial vessels in both cases, with a time-dependent but not statistically significant increase. In the thoracic aorta, a time-dependent decrease in endothelial nitric oxide synthase expression in the muscular layer after reperfusion, which was statistically significant in R6 versus the control. Positive immunostaining for inducible nitric oxide synthase was seen in the muscular and endothelial layers, and this varied from moderate in the control group, to light in the endothelium in groups R2 and R6.

Conclusions: We observed changes that may be implicated in heart injury and impairment of aortal tone after liver ischemia and reperfusion injury.
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http://dx.doi.org/10.6002/ect.2011.0055DOI Listing
February 2012

Apoptosis in glioma cells treated with PDT.

Photomed Laser Surg 2011 May 23;29(5):305-9. Epub 2011 Jan 23.

Department of Surgery and Anatomy, University of São Paulo (USP), Brazil.

Background: Despite significant advances in neurosurgical techniques, the median survival time of patients with glioblastoma has improved little over the past 50 years and remains less than one year. Photodynamic therapy (PDT) is presently established as a widely accepted modality for the treatment of a variety of solid tumors.

Objectives: This study evaluated the effect of PDT-Photogem(®) on five glioma cell lines (U87, U138, U251, U343, and T98G).

Methods: The experiments were carried out in 25-cm(3) flasks with different groups of cells seeded at a density of 1 × 10(5) cells per flask. After 3 h, the medium was removed, and the cells were incubated for 4 h with Photogem (5 μg/mL). After the incubation time, the photosensitizer-containing medium was removed and the cells were irradiated with LED (630 nm, 25 mW/cm(2), 25 J/cm(2)) devices for 17 min. For the final steps of the PDT, the cells were returned to the incubator and kept at 37°C with 5% CO(2) for 24 h, the cell viability assay was assessed using the trypan blue method, and the expression of Caspase 3 mRNA levels was assessed by real-time quantitative PCR.

Results: Upon PDT-Photogem(®) treatment, viable cells, as evaluated by the trypan blue dye-exclusion method, decreased in two cell lines (U87 and U138) but not in the other three. Apoptosis, as assessed by the expression of caspase-3 mRNA levels, was at least partly involved in the death mechanism of the cell lines.

Conclusions: Collectively, our results indicated that PDT-Photogem(®) can act in glioma cells, thus encouraging new experiments in this field.
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http://dx.doi.org/10.1089/pho.2009.2649DOI Listing
May 2011

Caspase-3 and Bcl-2 expression in glioblastoma: an immunohistochemical study.

Arq Neuropsiquiatr 2010 08;68(4):603-7

Department of Surgery and Anatomy, Medicine School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

The unfavorable prognosis of malignant gliomas can also be explained by the incomplete knowledge of their molecular pathways. Studies regarding the regulatory process of apoptosis in glioblastoma (GBM), the most common malignant glioma, are few, and better knowledge of the expression of pro and anti-apoptotic proteins could collaborate with the development of new treatments founded on molecular basis. The objective of this study was to evaluate by immunohistochemistry the expression of caspase-3 and Bcl-2 in 30 samples of GBMs. The expression of caspase-3 (mean 17.67%) was lower than Bcl-2 (mean 30.92%), a statistically significant result (p<0.0001), suggesting low apoptotic activity in these tumors. Other studies of proteins related to the intrinsic and extrinsic pathway of apoptosis are required to provide additional information of this mechanism in GBMs.
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http://dx.doi.org/10.1590/s0004-282x2010000400023DOI Listing
August 2010

Expression of HSP70 in cerebral ischemia and neuroprotetive action of hypothermia and ketoprofen.

Arq Neuropsiquiatr 2010 08;68(4):592-6

Departament of Surgery and Anatomy, Medicine School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Heat shock proteins (HSPs) are molecular chaperones that bind to other proteins to shepherd them across membranes and direct them to specific locations within a cell. Several injurious stimuli can induce Hsp70 expression, including ischemia. This study aimed to investigate the pattern of expression of protein (immunohistochemistry) and gene (real-time PCR) Hsp70 in experimental focal cerebral ischemia in rats by occlusion of the middle cerebral artery for 1 hour and the role of neuroprotection with hypothermia (H) and ketoprofen (K). The infarct volume was measured using morphometric analysis defined by triphenyl tetrazolium chloride. It was observed increases in the protein (p=0.0001) and gene (p=0.0001) Hsp70 receptor in the ischemic areas that were reduced by H (protein and gene: p<0.05), K (protein: p<0.001), and H+K (protein: p<0.01 and gene: p<0.05). The Hsp70 increases in the ischemic area suggests that the Hsp70-mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, H and K are directly involved with the Hsp70.
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http://dx.doi.org/10.1590/s0004-282x2010000400021DOI Listing
August 2010

Expression of apoptosis in human saphenous vein grafts in restoration of blood flow through coronary bypass surgery.

Rev Bras Cir Cardiovasc 2009 Jul-Sep;24(3):312-7

FMRP - USP.

Objective: To investigate the possible role of apoptosis on brief distensions of human saphenous veins at different pressures.

Methods: Fresh isolated grafts of human saphenous vein were assigned as control or distended (D) for fifteen seconds at 100, 200 and 300 mmHg. The degree of apoptotic caspases 3, 8, 9 and anti-apoptotic protein Bcl-2 expression were assessed by immunohistochemistry.

Results: Fresh isolated segments of distended human saphenous veins presented similar apoptotic protein expression when compared with control veins. However, the Bcl-2 expression was significantly higher in the 300 mmHg distended segments compared with the control vein.

Conclusion: These findings show that intact segments of human saphenous veins submitted to distensions at different pressures have similar apoptotic proteins expression when compared with non-distended control veins. Therefore, brief distensions commonly performed during surgical harvesting do not trigger apoptosis, and probably are not involved on the physiopathological mechanisms that lead to graft failure.
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http://dx.doi.org/10.1590/s0102-76382009000400009DOI Listing
June 2010

Surgical management of glomus jugulare tumors: a proposal for approach selection based on tumor relationships with the facial nerve.

J Neurosurg 2010 Jan;112(1):88-98

Department of Neurosurgery, Evangelic Medical School, Federal University of Parana, Curitiba, Parana, Brazil.

Object: The goal of this paper is to analyze the extension and relationships of glomus jugulare tumor with the temporal bone and the results of its surgical treatment aiming at preservation of the facial nerve. Based on the tumor extension and its relationships with the facial nerve, new criteria to be used in the selection of different surgical approaches are proposed.

Methods: Between December 1997 and December 2007, 34 patients (22 female and 12 male) with glomus jugulare tumors were treated. Their mean age was 48 years. The mean follow-up was 52.5 months. Clinical findings included hearing loss in 88%, swallowing disturbance in 50%, and facial nerve palsy in 41%. Magnetic resonance imaging demonstrated a mass in the jugular foramen in all cases, a mass in the middle ear in 97%, a cervical mass in 85%, and an intradural mass in 41%. The tumor was supplied by the external carotid artery in all cases, the internal carotid artery in 44%, and the vertebral artery in 32%. Preoperative embolization was performed in 15 cases. The approach was tailored to each patient, and 4 types of approaches were designed. The infralabyrinthine retrofacial approach (Type A) was used in 32.5%; infralabyrinthine pre- and retrofacial approach without occlusion of the external acoustic meatus (Type B) in 20.5%; infralabyrinthine pre- and retrofacial approach with occlusion of the external acoustic meatus (Type C) in 41%; and the infralabyrinthine approach with transposition of the facial nerve and removal of the middle ear structures (Type D) in 6% of the patients.

Results: Radical removal was achieved in 91% of the cases and partial removal in 9%. Among 20 patients without preoperative facial nerve dysfunction, the nerve was kept in anatomical position in 19 (95%), and facial nerve function was normal during the immediate postoperative period in 17 (85%). Six patients (17.6%) had a new lower cranial nerve deficit, but recovery of swallowing function was adequate in all cases. Voice disturbance remained in all 6 cases. Cerebrospinal fluid leakage occurred in 6 patients (17.6%), with no need for reoperation in any of them. One patient died in the postoperative period due to pulmonary complications. The global recovery, based on the Karnofsky Performance Scale (KPS), was 100% in 15% of the patients, 90% in 45%, 80% in 33%, and 70% in 6%.

Conclusions: Radical removal of glomus jugulare tumor can be achieved without anterior transposition of the facial nerve. The extension of dissection, however, should be tailored to each case based on tumor blood supply, preoperative symptoms, and tumor extension. The operative field provided by the retrofacial infralabyrinthine approach, or the pre- and retrofacial approaches, with or without closure of the external acoustic meatus, allows a wide exposure of the jugular foramen area. Global functional recovery based on the KPS is acceptable in 94% of the patients.
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http://dx.doi.org/10.3171/2008.10.JNS08612DOI Listing
January 2010

ICAM-1 (Lys469Glu) and PECAM-1 (Leu125Val) polymorphisms in diffuse astrocytomas.

Clin Exp Med 2009 Jun 21;9(2):157-63. Epub 2009 Mar 21.

Department of Surgery and Anatomy, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP 14040-903, Brazil.

Cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) play an important role in glioma invasion and angiogenesis. The aim of this study was to investigate whether specific genetic polymorphisms of ICAM-1 and PECAM-1 could be associated with glioma development and progression. Single-nucleotide polymorphism in codon 469 of ICAM-1 and codon 125 of PECAM-1 were examined in 158 patients with astrocytomas and 162 controls using polymerase chain reaction and restriction enzyme analysis. The distribution of PECAM-1 polymorphic genotypes in astrocytomas did not show any significant difference. However, a specific ICAM-1 genotype (G/G, corresponding to Lys469Glu) exhibited higher frequency in grade II astrocytomas compared to controls, grade III, and grade IV astrocytomas; suggesting that this polymorphism could be involved in the development of grade II astrocytomas.
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http://dx.doi.org/10.1007/s10238-009-0040-6DOI Listing
June 2009

Biochemical evaluation of focal non-reperfusion cerebral ischemia by middle cerebral artery occlusion in rats.

Arq Neuropsiquiatr 2008 Sep;66(3B):725-30

Department of Surgery and Anatomy, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Cerebral ischemia is an important event in clinical and surgical neurological practice since it is one of the diseases that most compromise the human species. In the present study 40 adult rats were submitted to periods of focal ischemia of 30, 60 and 90 min without reperfusion and animals submitted to a sham procedure were used as controls. We analyzed the levels of ATP, malondialdehyde and caspase-3. No significant differences in the biochemical measurements were observed between the right and left brain hemispheres of the same animal in each experimental group. Reduced ATP levels were observed after the three periods of ischemia compared to the sham group. No significant increase in malondialdehyde or caspase-3 levels was observed. Despite significant changes in ATP levels, the results indicated cell viability in the ischemic region as shown by the low rates of lipid peroxidation and apoptosis, findings probably related to the lack of reperfusion.
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http://dx.doi.org/10.1590/s0004-282x2008000500023DOI Listing
September 2008

Renal ischemia and reperfusion injury: influence of chorpromazine on renal function and lipid peroxidation.

Acta Cir Bras 2008 ;23 Suppl 1:42-6; discussion 46

Division of Urology, Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo, SP, Brazil.

Purpose: To evaluate the influence of chlorpromazine (CPZ) on renal function and lipid peroxidation in a rat model of kidney ischemia/reperfusion injury.

Methods: Forty eight Wistar rats underwent a laparotomy for hilar clamping of left kidney with a bulldog clamp for 60 minutes followed by organ reperfusion and contralateral nephrectomy. Of these, 26 received 3mg/kg of CPZ intravenously 15 minutes before renal ischemia (G-E) while the remaining 22 were used as ischemic control group (G-C). Eleven rats of G-E and 8 of G-C were followed for blood urea nitrogen and creatinine determinations before renal ischemia and at 1st, 4th and 7th postoperative days. Samplings of left renal tissue were obtained at 5 minutes (5 rats from each group) and 24 hours (9 G-C and 10 of G-E) of reperfusion for malondialdehy (MDA) content determination. Controls of renal MDA content were determined in kidneys harvested from 6 additional normal rats.

Results: Acute renal failure occurred in all animals but levels of BUN and creatinine were significantly lower in G-E (p<0.001). MDA content rose strikingly at 5 minutes of reperfusion in both groups (p>0.05) and returned near to normal levels 24 hours later.

Conclusion: CPZ conferred partial protection of renal function to kidneys submitted to ischemia/reperfusion injury that seems to be not dependent on inhibition of lipid peroxidation.
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http://dx.doi.org/10.1590/s0102-86502008000700008DOI Listing
June 2009

Cyclosporine action on kidneys of rats submitted to normothermic ischaemia and reperfusion.

Acta Cir Bras 2008 ;23 Suppl 1:36-41; discussion 41

Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo, SP, Brazil.

Purpose: To verify if rat kidneys lesioned by ischaemia followed by reperfusion are affected by cyclosporine A (CsA).

Methods: Male Wistar rats were randomly divided into three groups, control (GS) and experimental (G1 and G2). G1 was subdivided in two: G1A composed of animals submitted to 60 minutes ischaemia and G1C with the same ischaemic procedure associated to 20 mg/kg/day CsA. Group G2 was subdivided and treated in the same way as G1 except that ischaemia was applied only for 40 minutes. Clamping the left renal artery followed by right side nephrectomy induced kidney ischaemia. Serum urea and creatinine were quantified on the day of surgery (D0) and in the following day (D1). Twenty four hours after reperfusion the left kidney was removed and histologically analyzed.

Results: Group GS had normal values for urea and creatinine both on D0 and D1 and did not show structural alterations. Renal function was not significantly different when G2C was compared to GS (p>0.05). Tissue lesions were smaller in G2C than in the other groups.

Conclusions: Renal function was protected by CsA, which also reduced tissue lesions in the kidneys of rats submitted to 40 minutes ischaemia.
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http://dx.doi.org/10.1590/s0102-86502008000700007DOI Listing
June 2009

Compensatory renal growth and mitochondrial function: the influence of warm ischemia and reperfusion.

Acta Cir Bras 2008 ;23 Suppl 1:31-5; discussion 35

Division of Urology, Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo, SP, Brazil.

Purpose: To evaluate the influence of ischemia/reperfusion injury on renal compensatory growth (CGR) and mitochondrial function.

Methods: Forty five Wistar rats were divided in 3 groups: Control Group (GC) - 21 rats were submitted to a sham laparotomy and sacrificed at 1st (6 rats) and 7th (15 rats) postoperative days to evaluate the dry weight of both kidneys and their growth during 1 week (6 rats) and to quantify mitochondrial respiration (9 rats); Group 1 (G1) - 12 rats underwent right nephrectomy and were sacrificed 7 days later for analysis of renal mitochondrial function (6 rats) and dry weight (6 rats). Group 2 (G2) - renal warm ischemia for 60 minutes followed by right nephrectomy was performed in 12 rats; they were sacrificed 7 days later to evaluate renal mitochondrial function (6 rats) and dry weight (6 rats).

Results: Dry weight (mg) of left kidneys at 7th day: GC - 219+/-18, G1 - 281+/-23 and G2 - 338+/-39 (GCxG1 p<0.01; GCxG2 p<0.001; G1xG2 p<0.01). State 4 mitochondrial respiration rate and respiratory control ratio (RCR) were similar in all groups (p>0.05). State 3 respirations (mM/min/mg) in GC, G1 and G2 was respectively: 99+/-23, 132+/-22 and 82+/-44 (p<0.02; the only statistical difference noted was between groups G1xG2 - p<0.05).

Conclusions: Following unilateral nephrectomy CRG is associated with an increase in state 3 of mitochondrial respiration. Renal ischemia/reperfusion injury enhances the CRG provoked by unilateral nephrectomy but such enhancement seems independent on mitochondrial respiration.
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http://dx.doi.org/10.1590/s0102-86502008000700006DOI Listing
June 2009

Histopathology and laser autofluorescence of ischemic kidneys of rats.

Lasers Med Sci 2009 May 26;24(3):397-404. Epub 2008 Jun 26.

Department of Surgery and Anatomy, Medical School of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.

The purpose of this research was to evaluate the severity of renal ischemia/reperfusion injury as determined by histology and by laser-induced fluorescence (LIF) with excitation wavelengths of 442 nm and 532 nm. Wistar rats (four groups of six animals) were subjected to left renal warm ischemia for 20, 40, 60 and 80 min followed by 10 min of reperfusion. Autofluorescence was determined before ischemia (control) and then every 5-10 min thereafter. Tissue samples for histology were harvested from the right kidney (control) and from the left kidney after reperfusion. LIF and ischemia time showed a significant correlation (p<0.0001 and r(2)=0.47, and p=0.006 and r(2)=0.25, respectively, for the excitation wavelengths of 442 nm and 532 nm). Histological scores showed a good correlation with ischemia time (p<0.0001). The correlations between optical spectroscopy values and histological damage were: LIF at 442 nm p<0.0001, LIF at 532 nm p=0.001; IFF (peak of back scattered light/LIF) at 442 nm p>0.05, and IFF at 532 nm p>0.05. After reperfusion LIF tended to return to preischemic basal levels which occurred in the presence of histological damage. This suggests that factors other than morphological alterations may have a more relevant effect on changes observed in LIF. In conclusion, renal ischemia/reperfusion changed tissue fluorescence induced by laser. The excitation light of 442 nm showed a better correlation with the ischemia time and with the severity of tissue injury.
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http://dx.doi.org/10.1007/s10103-008-0578-7DOI Listing
May 2009

Ultrastructural study of the lateral ventricle choroid plexus in experimental hydrocephalus in Wistar rats.

Arq Neuropsiquiatr 2007 Dec;65(4A):974-7

Department of Surgery and Anatomy, Medicine School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Hydrocephalus is one of the most frequent and complex neurological diseases characterized by the abnormal buildup of cerebrospinal fluid (CSF) in the ventricles of the brain, due to an altered CSF dynamics. To detect possible ultrastructural alterations of the lateral ventricles choroid plexus (responsible for the CSF production), rats seven days after birth were submitted to an intracisternal injection of 20% kaolim (hydrated aluminum silicate) for the hydrocephalus induction. Twenty-eight or 35 days after injection, injected animals and respective controls were processed for observation under a transmission electron microscopy. Alterations found: presence of concentric cell membrane fragments, larger number of primary and secondary lysossomes, vacuoles, and cytoplasmic vesicles, and an enlargement of the intercellular space and between the basolateral interdigitation of the choroid epithelium. The alterations observed are probably associated to an increase of the ventricular pressure, inducing morpho-functional effects on the choroid plexus integrity.
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http://dx.doi.org/10.1590/s0004-282x2007000600010DOI Listing
December 2007