Alberto Schanaider

Uso de animais em cirurgia experimental

Diversos aspectos da cirurgia experimental sao ignorados pelos pesquisadores. Este artigo enfatiza padroes eticos, detalhes anatomicos e procedimentos anestesicos com o objetivo de auxiliar na escolha adequada de animais utilizados em laboratorio para pesquisas em cirurgia e na educacao medica.

Intraperitoneal but Not Intravenous Cryopreserved Mesenchymal Stromal Cells Home to the Inflamed Colon and Ameliorate Experimental Colitis

Background and Aims Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)–induced colitis. Methods After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. Results Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. Conclusions Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Unfractionated Heparin and New Heparin Analogues from Ascidians (Chordate-Tunicate) Ameliorate Colitis in Rats

The anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-α, TGF-β, and VEGF was observed, as well as a reduction of NF-κB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin.

Prophylactic systemic P2X7 receptor blockade prevents experimental colitis

BACKGROUND The P2X7 receptor (P2X7-R) is a non-selective adenosine triphosphate-gated cation channel present in epithelial and immune cells, and involved in inflammatory response. Extracellular nucleotides released in conditions of cell stress or inflammation may function as a danger signal alerting the immune system from inflammation. We investigated the therapeutic action of P2X7-R blockade in a model of inflammatory bowel disease. METHODS Rats with trinitrobenzene sulfonic (TNBS) acid-induced colitis were treated with the P2X7-R antagonists A740003 or brilliant blue G (BBG) through intra-peritoneal (IP) or intra-colonic (IC) injection prior to colitis induction. Clinical and endoscopic follow-up, histological scores, myeloperoxidase activity, densities of collagen fibers and goblet cells were evaluated. P2X7-R expression, NF-kappa B and Erk activities, and densities of T-cells and macrophages were analyzed by immunoperoxidase. The inflammatory response was determined by measuring inflammatory cytokines in cultures of colon explants, by enzyme-linked immunosorbent assay. Colonic apoptosis was determined by the TUNEL assay. RESULTS IP-BBG significantly attenuated the severity of colitis, myeloperoxidase activity, collagen deposition, densities of lamina propria T-cells and macrophages, while maintaining goblet cell densities. IP-BBG inhibited the increase in P2X7-R expression in parallel with apoptotic rates. TNF-α and interleukin-1β stabilized in low levels, while TGF-β and interleukin-10 did not change following IP-BBG-therapy. Colonic NF-kappa-B and Erk activation were significantly lower in IP-BBG-treated animals. Prophylactic IP-A740003 also protected rats against the development of TNBS-colitis. CONCLUSIONS Prophylactic systemic P2X7-R blockade is effective in the prevention of experimental colitis, probably due to a systemic anti-inflammatory action, interfering with a stress-inflammation amplification loop mediated by P2X7-R.

Use of butyrate or glutamine in enema solution reduces inflammation and fibrosis in experimental diversion colitis

AIM To investigate whether butyrate or glutamine enemas could diminish inflammation in experimental diversion colitis. METHODS Wistar specific pathogen-free rats were submitted to a Hartmanns end colostomy and treated with enemas containing glutamine, butyrate, or saline. Enemas were administered twice a week in the excluded segment of the colon from 4 to 12 wk after the surgical procedure. Follow-up colonoscopy was performed every 4 wk for 12 wk. The effect of treatment was evaluated using video-endoscopic and histologic scores and measuring interleukin-1β, tumor necrosis factor-alpha, and transforming growth factor beta production in organ cultures by enzyme linked immunosorbent assay. RESULTS Colonoscopies of the diverted segment showed mucosa with hyperemia, increased number of vessels, bleeding and mucus discharge. Treatment with either glutamine or butyrate induced significant reductions in both colonoscopic (P < 0.02) and histological scores (P < 0.01) and restored the densities of collagen fibers in tissue (P = 0.015; P = 0.001), the number of goblet cells (P = 0.021; P = 0.029), and the rate of apoptosis within the epithelium (P = 0.043; P = 0.011) to normal values. The high levels of cytokines in colon explants from rats with diversion colitis significantly decreased to normal values after treatment with butyrate or glutamine. CONCLUSION The improvement of experimental diversion colitis following glutamine or butyrate enemas highlights the importance of specific luminal nutrients in the homeostasis of the colonic mucosa and supports their utilization for the treatment of human diversion colitis.

Anesthetic experimental device for small animal

PURPOSE The difficulty to anesthetize small laboratory animals with vaporizer prompted us to go in search of new materials, and create new techniques. The improved equipment of anesthesia we looked for should be low cost, practical, versatile, and its management serve ethical, teaching, and research purposes. METHODS The new components of the equipment were: the vaporizer, the unidirectional valve, the glass cylinder filled with water, the flow guidance y-shape tube, the flow regulators, the mask modifications, and another free airway for emergency occurrence. A test was done with 30 Wistar rats, Rattus norvegicus albinus, divided into three groups with 10 rats for each one. Groups 1, 2 and 3 were anesthetized with Ether, Halothane and Sevoflurane respectively, using the new gadget. The anesthetic induction time, the breathing rhythm alteration during an anesthesia pre-established time (10 minutes), and the recovery time were observed. RESULTS The equipment enabled an easy handling, and fulfilled a larger safeness and stability during the induction and anesthetic management. The test showed it was possible to make use of several anesthetic agents. CONCLUSION The device is effective, and turns the anesthesia procedure into a very easy practice with low-cost. It should be recommended for experimental surgery, teaching and research.

Propofol and N-Acetylcysteine attenuate oxidative stress induced by intestinal ischemia/reperfusion in rats: Protein carbonyl detection by immunoblotting

PURPOSE To evaluate the antioxidant effect of Propofol and N-Acetylcysteine (NAC) on intestinal ischemia/reperfusion (I/R) in rats by determining carbonyl protein level. METHODS Forty Wistar rats were randomly assigned into the following groups: Control; Sham; I/R with Propofol; I/R with Propofol and NAC; I/R with Ketamine and Xylazine. The I/R groups underwent 60 minutes of ischemia and an equal period of reperfusion. Blood samples, collected by cardiac punction, were centrifuged for plasma obtainment. Protein carbonyl level in plasma samples was determined by immunoblotting. RESULTS No significant difference in protein carbonyl level was found between Control and Sham groups (P>0.05). The highest reduction in protein carbonyl level (P<0.05) was obtained with the administration of Propofol and NAC (Group 4) in intestinal I/R procedure. CONCLUSION The administration of Propofol and NAC showed the best antioxidant effect on oxidative stress in rats that underwent intestinal I/R procedure, suggesting a synergistic interaction.

Oxidized tissue proteins after intestinal reperfusion injury in rats

PURPOSE To analyse if the carbonyl proteins measurement could be validated as a method that allows the identification of an intestinal oxidative stress after ischemia and reperfusion injury. METHODS Twenty-five male Wistar rats (n = 21) weighting 200 to 250 g were divided into three groups. Group I--control (n = 10). Group II--sham (n = 5) and Group III (n = 10) subjected to 60 minutes of intestinal ischemia and equal period of reperfusion. For this purpose it was clamped the superior mesenteric artery in its distal third. Histological changes and carbonyl protein levels were determined in the samples of all groups. In group III, samples of both normal and reperfused ileal segment were studied. RESULTS All the reperfused segments showed mucosal and submucosal swelling and inflammatory infiltrate of the lamina propria. Levels of carbonyl protein rose in group III, including in the non-ischemic segments. The sensitivity and specificity of the carbonyl protein tissue levels were respectively 94% and 88%. CONCLUSION The carbonyl protein method is a useful biologic marker of oxidative stress after the phenomenon of intestinal ischemia and reperfusion in rats. It was also noteworthy that the effects of oxidative stress could be seen far from the locus of the primary injury.

Lung Functional and Biologic Responses to Variable Ventilation in Experimental Pulmonary and Extrapulmonary Acute Respiratory Distress Syndrome

Objectives: The biologic effects of variable ventilation may depend on the etiology of acute respiratory distress syndrome. We compared variable and conventional ventilation in experimental pulmonary and extrapulmonary acute respiratory distress syndrome. Design: Prospective, randomized, controlled experimental study. Settings: University research laboratory. Subjects: Twenty-four Wistar rats. Interventions: Acute respiratory distress syndrome was induced by Escherichia coli lipopolysaccharide administered intratracheally (pulmonary acute respiratory distress syndrome, n = 12) or intraperitoneally (extrapulmonary acute respiratory distress syndrome, n = 12). After 24 hours, animals were randomly assigned to receive conventional (volume-controlled ventilation, n = 6) or variable ventilation (n = 6). Nonventilated animals (n = 4 per etiology) were used for comparison of diffuse alveolar damage, E-cadherin, and molecular biology variables. Variable ventilation was applied on a breath-to-breath basis as a sequence of randomly generated tidal volume values (n = 600; mean tidal volume = 6 mL/kg), with a 30% coefficient of variation (normal distribution). After randomization, animals were ventilated for 1 hour and lungs were removed for histology and molecular biology analysis. Measurements and Main Results: Variable ventilation improved oxygenation and reduced lung elastance compared with volume-controlled ventilation in both acute respiratory distress syndrome etiologies. In pulmonary acute respiratory distress syndrome, but not in extrapulmonary acute respiratory distress syndrome, variable ventilation 1) decreased total diffuse alveolar damage (median [interquartile range]: volume-controlled ventilation, 12 [11–17] vs variable ventilation, 9 [8–10]; p < 0.01), interleukin-6 expression (volume-controlled ventilation, 21.5 [18.3–23.3] vs variable ventilation, 5.6 [4.6–12.1]; p < 0.001), and angiopoietin-2/angiopoietin-1 ratio (volume-controlled ventilation, 2.0 [1.3–2.1] vs variable ventilation, 0.7 [0.6–1.4]; p < 0.05) and increased relative angiopoietin-1 expression (volume-controlled ventilation, 0.3 [0.2–0.5] vs variable ventilation, 0.8 [0.5–1.3]; p < 0.01). In extrapulmonary acute respiratory distress syndrome, only volume-controlled ventilation increased vascular cell adhesion molecule-1 messenger RNA expression (volume-controlled ventilation, 7.7 [5.7–18.6] vs nonventilated, 0.9 [0.7–1.3]; p < 0.05). E-cadherin expression in lung tissue was reduced in volume-controlled ventilation compared with nonventilated regardless of acute respiratory distress syndrome etiology. In pulmonary acute respiratory distress syndrome, E-cadherin expression was similar in volume-controlled ventilation and variable ventilation; in extrapulmonary acute respiratory distress syndrome, however, it was higher in variable ventilation than in volume-controlled ventilation. Conclusions: Variable ventilation improved lung function in both pulmonary acute respiratory distress syndrome and extrapulmonary acute respiratory distress syndrome. Variable ventilation led to more pronounced beneficial effects in biologic marker expressions in pulmonary acute respiratory distress syndrome compared with extrapulmonary acute respiratory distress syndrome but preserved E-cadherin in lung tissue only in extrapulmonary acute respiratory distress syndrome, thus suggesting lower damage to epithelial cells.

Desenvolvimento de um novo modelo experimental de síndrome do compartimento abdominal

OBJECTIVE To describe an experimental, unprecedented model that mimics the abdominal compartment syndrome (ACS). METHODS twenty rats were randomly divided into four groups. To simulate ACS intra-abdominal hypertension (IAH) was induced by inserting cotton surgical dressing (Zobec ®), 15x15cm (intra-abdominal pressure constant and equal to 12 mmHg) associated with hypovolemia induced by withdrawing blood, keeping mean arterial pressure (MAP) around 60 mmHg (HYPO). To dissociate the effects of those IAH-induced hypovolemia per se, two other groups were analyzed: one with only with IAH and another with only hypovolemia. The simulation group (sham) underwent the same surgical procedure performed earlier, however, the levels of intra-abdominal pressure and MAP were kept in 3 mmHg and 90 mmHg, respectively. RESULTS By analyzing the impact of IAH on the small intestine, we observed necrosis of the villi, congestion, and neutrophilic infiltration. Hypovolemia induced only inflammation and edema of the villi. However, the association of IAH and HYPO led to hemorrhagic infarction, besides worsening of the aforementioned parameters. CONCLUSION This model was effective in inducing ACS expressed by the effects found in the small intestine.