Clinical outcomes prediction in kidney transplantation by use of biomarkers from hypothermic machine perfusion.

PURPOSE: The clinical outcomes of kidney transplantation from deceased donors have seen significant improvements with the use of machine perfusion (MP), now a standard practice in transplant centers. However, the use of perfusate biomarkers for assessing organ quality remains a subject of debate. Despite this, some centers incorporate them into their decision-making process for donor kidney acceptance. Recent studies have indicated that lactate dehydrogenase (LDH), glutathione S-transferase, interleukin-18, and neutrophil gelatinase-associated lipocalin (NGAL) could predict post-transplant outcomes. MATERIALS AND METHODS: Between August 2016 and June 2017, 31 deceased-donor after brain death were included and stroke was the main cause of death. Pediatric patients, hypersensitized recipients were excluded. 43 kidneys were subjected to machine perfusion. Perfusate samples were collected just before the transplantation and stored at -80ºC. Kidney transplant recipients have an average age of 52 years, 34,9% female, with a BMI 24,6±3,7. We employed receiver operating characteristic analysis to investigate associations between these perfusate biomarkers and two key clinical outcomes: delayed graft function and primary non-function. RESULTS: The incidence of delayed graft function was 23.3% and primary non-function was 14%. A strong association was found between NGAL concentration and DGF (AUC=0.766, 95% CI, P=0.012), and between LDH concentration and PNF (AUC=0.84, 95% CI, P=0.027). Other perfusate biomarkers did not show significant correlations with these clinical outcomes. CONCLUSION: The concentrations of NGAL and LDH during machine perfusion could assist transplant physicians in improving the allocation of donated organs and making challenging decisions regarding organ discarding. Further, larger-scale studies are required.

Sulforaphane and Albumin Attenuate Experimental Intestinal Ischemia-Reperfusion Injury.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) injury constitutes a severe disorder, in great part resulting from oxidative stress. Because sulforaphane and albumin were shown to increase antioxidant defenses, we evaluated the therapeutic potential of these agents in an experimental model of I/R injury. METHODS: Wistar rats were used to establish a model of intestinal I/R (35 min of ischemia, followed by 45 min of reperfusion) and were treated with albumin (5 mL/kg), sulforaphane (500 µg/kg), or saline intravenously before reperfusion. Animals that were not subjected to I/R served as the sham (laparotomy only) and control groups. Blood samples were analyzed for arterial gas, reactive oxygen species, and reactive nitrogen species using different molecular fluorescent probes. After euthanasia, ileal samples were collected for analysis, including histopathology, immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assays, and lactic dehydrogenase measurement. RESULTS: Oxygenation status and hemodynamic parameters were uniform during the experiment. The sulforaphane- or albumin-treated groups showed reduced concentrations of reactive oxygen species (P < 0.04), nitric oxide (P < 0.001), and peroxynitrite (P = 0.001), compared with I/R injury untreated animals. Treatment with sulforaphane or albumin resulted in the preservation of goblet cells (P < 0.03), reductions in histopathologic scores (P < 0.01), macrophage density (P < 0.01), iNOS expression (P < 0.004), NF-kappa B activation (P < 0.05), and apoptotic rates (P < 0.04) in the mucosa and a reduction in the concentration of lactic dehydrogenase (P < 0.04), more pronounced with sulforaphane. CONCLUSIONS: Attenuation of intestinal I/R injury in this model probably reflects the antioxidative effects of systemic administration of both sulforaphane and albumin and reinforces their use in future translational research.

Unilateral frontalis muscle paralysis reanimated by contralateral frontalis muscle through sural nerve graft connecting the two muscles (muscle-nerve-muscle neurotization technique)-A case report.

Paralysis of the frontalis muscle is extremely difficult to reverse. The best treatment for facial paralysis reanimation which preserves spontaneity and muscle specificity is end-to-end neurorrhaphy through cross-face nerve grafting. However, it is rarely possible. Muscle-nerve-muscle (MNM) neurotization consists of an interposition of a nerve graft connecting the normal muscle to the denervated muscle. The axons of the muscle with intact innervation grow inside a neural graft towards the paralyzed muscle resulting in neurotization. The purpose of this report is to present a case of frontalis muscle paralysis reanimated by MNM neurotization. A 65-year-old female patient presented complete facial paralysis after temporomandibular joint surgery. Five months afterwards, the patient spontaneously recovered facial muscle movements except the frontalis muscle. Definitive paralysis of the frontalis muscle was diagnosed after 11 months, and MNM neurotization was chosen and performed. Three strings of sural nerve were placed in separated tunnels in the subcutaneous plane, through small skin incisions to connect the two bellies of frontalis muscle bilaterally, and then sutured into the muscle pocket of each side. The patient presented voluntary and synchronic contraction of the bilateral frontalis muscle, 4 months after neurotization. Electroneuromyography confirmed muscle contraction by contralateral stimulation. Despite its efficacy still being researched, it is a very promising technique for the reanimation of small muscles in facial paralysis.

Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats.

BACKGROUND: Previous study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor. METHODS: We investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-ß1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1ß, procollagens type I, III, and IV) for mRNA quantification. RESULTS: The group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- ß1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1ß mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1ß mRNAs, as well as less immunoreactivity of HSP-47, TGF-ß, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation. CONCLUSION: BBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

Effects of pressure support ventilation on ventilator-induced lung injury in mild acute respiratory distress syndrome depend on level of positive end-expiratory pressure: A randomised animal study.

BACKGROUND: Harmful effects of spontaneous breathing have been shown in experimental severe acute respiratory distress syndrome (ARDS). However, in the clinical setting, spontaneous respiration has been indicated only in mild ARDS. To date, no study has compared the effects of spontaneous assisted breathing with those of fully controlled mechanical ventilation at different levels of positive end-expiratory pressure (PEEP) on lung injury in ARDS. OBJECTIVE: To compare the effects of assisted pressure support ventilation (PSV) with pressure-controlled ventilation (PCV) on lung function, histology and biological markers at two different PEEP levels in mild ARDS in rats. DESIGN: Randomised controlled experimental study. SETTING: Basic science laboratory. PARTICIPANTS: Thirty-five Wistar rats (weight ±â€ŠSD, 310 ±â€Š19) g received Escherichia coli lipopolysaccharide (LPS) intratracheally. After 24 h, the animals were anaesthetised and randomly allocated to either PCV (n=14) or PSV (n=14) groups. Each group was further assigned to PEEP = 2 cmH2O or PEEP = 5 cmH2O. Tidal volume was kept constant (≈6 ml kg). Additional nonventilated animals (n=7) were used as a control for postmortem analysis. MAIN OUTCOME MEASURES: Ventilatory and mechanical parameters, arterial blood gases, diffuse alveolar damage score, epithelial integrity measured by E-cadherin tissue expression, and biological markers associated with inflammation (IL-6 and cytokine-induced neutrophil chemoattractant, CINC-1) and type II epithelial cell damage (surfactant protein-B) were evaluated. RESULTS: In both PCV and PSV, peak transpulmonary pressure was lower, whereas E-cadherin tissue expression, which is related to epithelial integrity, was higher at PEEP = 5 cmH2O than at PEEP = 2 cmH2O. In PSV, PEEP = 5 cmH2O compared with PEEP = 2 cmH2O was associated with significantly reduced diffuse alveolar damage score [median (interquartile range), 11 (8.5 to 13.5) vs. 23 (19 to 26), P = 0.005] and expressions of IL-6 and CINC-1 (P = 0.02 for both), whereas surfactant protein-B mRNA expression increased (P = 0.03). These changes suggested less type II epithelial cell damage at a PEEP of 5 cmH2O. Peak transpulmonary pressure correlated positively with IL-6 [Spearman's rho (ρ) = 0.62, P = 0.0007] and CINC-1 expressions (ρ = 0.50, P = 0.01) and negatively with E-cadherin expression (ρ = -0.67, P = 0.0002). CONCLUSION: During PSV, PEEP of 5 cmH2O, but not a PEEP of 2 cmH2O, reduced lung damage and inflammatory markers while maintaining epithelial cell integrity.

Exogenous pulmonary surfactant prevents the development of intra-abdominal adhesions in rats.

Intra-abdominal adhesions are major post-operative complications for which no effective means of prevention is available. We aimed to evaluate the efficacy of exogenous pulmonary surfactant administration in the prevention of post-operative abdominal adhesions. Rats were randomly assigned to undergo laparotomy (L) or gastroenterostomy (GE) and then treated with surfactant (groups L-S and GE-S, respectively). Intra-abdominal adhesions, collagen fibre content, metalloproteinase (MMP)-9, expression of growth factors (TGF-ß, KGF and VEGF), type III procollagen (PCIII) and pro-caspase 3, as well as isolectin B4 and ED1-positive cells expressing MMP-9, were evaluated. Groups treated with surfactant (GE-S and L-S) exhibited fewer adhesions. A significant reduction in collagen fibre content was observed in GE-S compared to GE animals (P < 0.001). In situ and gelatin zymography analysis showed higher MMP-9 expression and activity in the GE-S group compared to the GE group (P < 0.05). ED1-positive cell counts were significantly higher in the GE-S group (P < 0.001) than in the GE group. Virtually all cells positive for ED1 were MMP-9+. Double-labelling of MMP-9 with IB4 showed no significant differences between GE-S and GE groups. TGF-ß, KGF, PCIII and pro-caspase-3 mRNA expression decreased significantly in GE-S compared to GE animals (P < 0.05). Surfactant administration also reduced apoptosis in the GE-S group. These findings suggest that surfactant reduces the intra-abdominal adhesions triggered by laparotomy and gastrointestinal anastomosis, thus preventing fibrosis formation at the peritoneal surfaces. This preclinical study suggests an innovative treatment strategy for intra-abdominal adhesions with surfactant and to endorse its putative mechanism of action.

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.

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-1ß stabilized in low levels, while TGF-ß 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.

Reduced flow after tubularized incised plate urethroplasty--increased fibrogenesis, elastin fiber loss or neither?

PURPOSE: Low urinary flow rates are common after tubularized incised plate urethroplasty but the etiology remains unclear and may be related to low urethral compliance due to abnormal collagen concentrations and/or fewer elastic fibers in the healed urethral plate. We hypothesized that inserting a preputial mucosal graft over the dorsal raw area after the midline incision may avoid scarring and improve urethral compliance. MATERIALS AND METHODS: Adult rabbits were submitted to tubularized incised plate urethroplasty with or without inlay preputial graft according to a previously described protocol. Tissular concentrations of collagens I, III, IV, VI, VIII and XIII were measured. Histomorphometric analysis was used to quantify elastic fibers in the urethra. Tubularized incised plate urethroplasty with and without inlay preputial graft was compared to normal rabbit urethras (controls). RESULTS: mRNA concentrations for collagens I, II and XIII were similar between controls and operated rabbits. The proportions between collagens I and III were 1.05, 0.87 and 1.21, respectively, in controls and animals undergoing tubularized incised plate urethroplasty with and without inlay preputial graft. mRNA concentrations for collagen IV and collagens VI/VIII tended to be higher and lower, respectively, in the operated urethras, despite showing statistical significance only for collagen VIII in animals undergoing tubularized incised plate urethroplasty with inlay preputial graft vs controls (p=0.02). The operated animals did not demonstrate a reduced number of elastic fibers in the urethral tissues compared to controls. CONCLUSIONS: Elastic fiber number and distribution were similar between tubularized incised plate urethroplasty cases and controls, suggesting that decreased concentrations of elastic fibers do not explain the reduced urethral compliance after tubularized incised plate urethroplasty. The raw area determined by the dorsal urethral incision regenerated after standard tubularized incised plate urethroplasty, while cicatrization with fibrosis occurred in correspondence to the grafted areas after tubularized incised plate urethroplasty with inlay preputial graft.

The biological effects of higher and lower positive end-expiratory pressure in pulmonary and extrapulmonary acute lung injury with intra-abdominal hypertension.

INTRODUCTION: Mechanical ventilation with high positive end-expiratory pressure (PEEP) has been used in patients with acute respiratory distress syndrome (ARDS) and intra-abdominal hypertension (IAH), but the role of PEEP in minimizing lung injury remains controversial. We hypothesized that in the presence of acute lung injury (ALI) with IAH: 1) higher PEEP levels improve pulmonary morphofunction and minimize lung injury; and 2) the biological effects of higher PEEP are more effective in extrapulmonary (exp) than pulmonary (p) ALI. METHODS: In 48 adult male Wistar rats, ALIp and ALIexp were induced by Escherichia coli lipopolysaccharide intratracheally and intraperitoneally, respectively. After 24 hours, animals were anesthetized and mechanically ventilated (tidal volume of 6 mL/kg). IAH (15 mmHg) was induced and rats randomly assigned to PEEP of 5 (PEEP5), 7 (PEEP7) or 10 (PEEP10) cmH2O for 1 hour. RESULTS: In both ALIp and ALIexp, higher PEEP levels improved oxygenation. PEEP10 increased alveolar hyperinflation and epithelial cell damage compared to PEEP5, independent of ALI etiology. In ALIp, PEEP7 and PEEP10 increased lung elastance compared to PEEP5 (4.3 ± 0.7 and 4.3 ± 0.9 versus 3.1 ± 0.3 cmH2O/mL, respectively, P <0.01), without changes in alveolar collapse, interleukin-6, caspase-3, type III procollagen, receptor for advanced glycation end-products, and vascular cell adhesion molecule-1 expressions. Moreover, PEEP10 increased diaphragmatic injury compared to PEEP5. In ALIexp, PEEP7 decreased lung elastance and alveolar collapse compared to PEEP5 (2.3 ± 0.5 versus 3.6 ± 0.7 cmH2O/mL, P <0.02, and 27.2 (24.7 to 36.8) versus 44.2 (39.7 to 56.9)%, P <0.05, respectively), while PEEP7 and PEEP10 increased interleukin-6 and type III procollagen expressions, as well as type II epithelial cell damage compared to PEEP5. CONCLUSIONS: In the current models of ALI with IAH, in contrast to our primary hypothesis, higher PEEP is more effective in ALIp than ALIexp as demonstrated by the activation of biological markers. Therefore, higher PEEP should be used cautiously in the presence of IAH and ALI, mainly in ALIexp.

Endoluminal ultrasound biomicroscopy as a reliable tool for in vivo assessment of colonic inflammation in rats.

PURPOSE: Endoscopic ultrasound (EUS) imaging of the colon is an important diagnostic tool for early neoplasia, although usually restricted to the rectum in inflammatory bowel disease (IBD). This study aimed to evaluate the ability of an endoluminal ultrasound biomicroscopic (eUBM) system to detect and characterize lesions simulating Crohn's disease in the colon of rats in vivo. METHODS: Colitis was induced with trinitrobenzene sulfonic acid instillated in the distal colon. Eighteen Wistar rats were submitted to eUBM in three time points: week 1 group (18 animals examined on day 3 after colitis induction), week 2 group (12 animals on days 3 and 10), and week 3 group (7 animals on days 3, 10, and 17). This design yielded distinct inflammation intensities. Three untreated rats were used for acquisition of control images. Scores were used for comparison with histology. RESULTS: Scores for eUBM and histology in the different moments of examination achieved a Spearman's rank correlation coefficient of 0.87 (p < 0.001). Findings of wall thickening presented positive predictive value (PPV) and sensitivity of 94 and of 100 %, respectively. Superficial and deep ulcers presented a PPV of 89 and 80 %, respectively, and negative predictive values of 100 and 85 %, respectively. CONCLUSION: Accurate detection and analysis of the lesions was achieved. The model is essential for the clinical development of the technique and a reproducible method for the evaluation of experimental colitis. eUBM might be applicable in different segments of the gut, developing into a novel adjunct method for IBD evaluation.

Heterologous mesenchymal stem cells successfully treat femoral pseudarthrosis in rats.

BACKGROUND: This study evaluated the effectiveness of treating pseudarthrosis in rats by using bone marrow cell suspensions or cultures of bone marrow mesenchymal stromal cells METHODS: Thirty-eight specific pathogen-free (SPF) animals were randomly assigned to four groups: Group 1, Control, without surgical intervention; Group 2 (Placebo), experimental model of femoral pseudarthrosis treated only with saline solution; Group 3, experimental model of femoral pseudarthrosis treated with heterologous bone marrow cells suspension; Group 4, experimental model of femoral pseudarthrosis treated with cultures of heterologous mesenchymal stromal cells from bone marrow. When pseudarthrosis was confirmed by simple radiological studies, digital radiography and histopathology after a 120-day postoperative period, Groups 2, 3 and 4 were treated as above. At 30, 60 and 90 days after the treatment, all animals were evaluated by simple radiological studies, and at the end of the experiment, the animals were assessed by computed axial tomography and anatomopathological and histomorphometric examinations. RESULTS: Injected cells were detected in the areas affected by pseudarthrosis using scintigraphy within the first 24 hours after their administration. After 60 days, the animals of Group 3 showed callus formation while the animals of Group 4 presented periosteal reaction and had some consolidated areas. In contrast, Group 2 showed a predominance of fibro-osteoid tissue. After 90 days, bone consolidation and remodeling was observed in all animals from Group 3 whereas animals from Group 4 exhibited partial consolidation and those ones from Group 2 persisted with pseudarthrosis. CONCLUSION: The treatment with heterologous bone marrow cells suspension proved to be effective in the treatment of pseudarthrosis whereas cultures of heterologous bone marrow mesenchymal stromal cells did not show the same potential to aid bone healing.

Rat thyroid graft transplantation after cryopreservation with scintigraphic standardization for an experimental study.

PURPOSE: Budget cuts among other factors undermine the use of state-of-the-art equipment by many research groups. This doesn't mean that their scientific data are not reliable or top-notch. Resort to adaptations is a recurrent need in their reality. The aim of this study was to assess whether scintigraphy with 99mTcO4 is effective in evaluating the functionality of thyroid grafts after cryopreservation in rats. MATERIAL AND METHODS: 24 rats were randomly distributed into 3 groups: Control Group (CG), without surgical procedure, Hypothyroidism Group (HTG), submitted to total thyroidectomy, and Transplanted Group (TG), with total thyroidectomy and cryopreservation of the thyroid gland for 7 days followed by grafting of a thyroid lobe. A protocol using a gamma camera imaging was conducted fourteen weeks after transplantation, and the whole body 99mTc, focusing on the topic of heterotopic thyroid uptake was evaluated. RESULTS: The images acquired had good quality with no noise and artifacts that could jeopardize its analysis. On the 14th day, HTG displayed no thyroid uptake, and the TG had a clear uptake of the thyroid graft in the topography of the biceps femoris muscle. Presented data also showed that both equipment spatial resolution and alignment (4.375 mm) did not interfere with the physiological uptake of 99mTc by the thyroid graft. CONCLUSION: The viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy were successfully accessed by the scintigraphy protocol developed.

Laparoscopic Pancreatectomy in Rats: The Development of an Experimental Model.

INTRODUCTION: several pancreatectomy techniques in rats have been described and utilized for research concerning the pancreas endocrine and exocrine functions. However, we did not find a description of any kind of laparoscopic pancreatectomy in rats in the consulted databases. The objective of this study is to describe a laparoscopic splenic lobe pancreatectomy in rats. METHODS: ten Wistars rats weighting more then 300 g were operated for standardizing the laparoscopic pancreatosplenectomy technique, aided by previous descriptions of laparoscopic splenectomy and open pancreatectomy in rats. Adjustments have been progressively adopted for technical refinement. RESULTS: In five animals a low-cost rat laparoscopic set was used. In other five animals we used a standard laparoscopic set. Three rats died early due to different causes: transection of the gastroesophageal junction, hemorrhagic shock and inadvertent colonic injury. The postoperative period of the other seven rats was uneventful until the seventh postoperative day. DISCUSSION: laparoscopic distal pancreatectomy and splenectomy in rats is feasible and safe, even with a low-cost set, in which the results were alike the standard laparoscopic set.

Bone marrow cells obtained from cirrhotic rats do not improve function or reduce fibrosis in a chronic liver disease model.

BACKGROUND: The objective of this study was to evaluate the therapeutic potential of bone marrow cells (BMCs) obtained from cirrhotic donors in a model of chronic liver disease. METHODS: Chronic liver injury was induced in female Wistar rats by the association of an alcoholic diet with intraperitoneal injections of carbon tetrachloride. BMCs obtained from cirrhotic donors or placebo were injected through the portal vein. Blood analysis of alanine aminotransferase (ALT) and albumin levels, ultrasound assessment including the measurement of the portal vein diameter (PVD) and liver echogenicity, histologic evaluation with hematoxylin and eosin and Sirius red staining, and quantification of collagen deposition were performed. RESULTS: ALT and albumin blood levels showed no significant differences between the experimental groups two months after injection. Additionally, no significant variation in PVD and liver echogenicity was found. Histological analysis also showed no significant variation in collagen deposition two months after placebo or BMC injection. CONCLUSION: This study suggests that, even though BMC therapy using cells from healthy donors has previously shown to be effective, this is not the case when BMCs are obtained from cirrhotic animals. This result has major clinical implications when considering the use of autologous BMCs from patients with chronic liver diseases.

Cryopreserved Rat Thyroid Autotransplantation in the Treatment of Postoperative Hypothyroidism.

To verify the viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy in the treatment of postoperative hypothyroidism. Thirty-two Wistar rats were randomly assigned into groups (G) with eight animals each: control (CG); simulation (SG); hypothyroidism (HTG) and transplanted (TG). At the beginning and in the 13th week of the experiment, serum levels of total T3, free T4, TSH and calcium were determined. In both the first and 14th weeks, scintigraphic examinations, 99m-Tc pertechnetate radioisotope biodistribution and histopathology were performed. In the 14th week, the expression of proliferating cell nuclear antigen (PCNA) and cellular apoptosis (caspase-3) were also evaluated. In the 13th week, the transplanted animals had normal serum levels of total T3 and free T4. TSH levels showed a tendency towards normality. In the 14th week, scintigraphic exams displayed graft isotopic uptake in all animals in the TG group. Histological examinations 13 weeks after transplantation showed the viability and functionality of thyroid follicles. PCNA revealed significant immunoreactivity of the graft (p < 0.001) when the TG was compared to the CG. There was no difference between CG and TG considering the expression of activated caspase-3. The experimental study confirmed the viability and functionality of thyroid autotransplantation implanted in skeletal muscle with evidence of cell proliferation without cellular apoptosis. This surgical strategy was effective in the treatment of postoperative hypothyroidism.

Translational Application of Fluorescent Molecular Probes for the Detection of Reactive Oxygen and Nitrogen Species Associated with Intestinal Reperfusion Injury.

Acute mesenteric ischemia, caused by an abrupt interruption of blood flow in the mesenteric vessels, is associated with high mortality. When treated with surgical interventions or drugs to re-open the vascular lumen, the reperfusion process itself can inflict damage to the intestinal wall. Ischemia and reperfusion injury comprise complex mechanisms involving disarrangement of the splanchnic microcirculatory flow and impairment of the mitochondrial respiratory chain due to initial hypoxemia and subsequent oxidative stress during the reperfusion phase. This pathophysiologic process results in the production of large amounts of reactive oxygen (ROS) and nitrogen (RNS) species, which damage deoxyribonucleic acid, protein, lipids, and carbohydrates by autophagy, mitoptosis, necrosis, necroptosis, and apoptosis. Fluorescence-based systems using molecular probes have emerged as highly effective tools to monitor the concentrations and locations of these often short-lived ROS and RNS. The timely and accurate detection of both ROS and RNS by such an approach would help to identify early injury events associated with ischemia and reperfusion and increase overall clinical diagnostic sensitivity. This abstract describes the pathophysiology of intestinal ischemia and reperfusion and the early biological laboratory diagnosis using fluorescent molecular probes anticipating clinical decisions in the face of an extremely morbid disease.

[Effects of rocuronium, sugammadex and rocuronium-sugammadex complex on coagulation in rats]. / Efeitos do rocurônio, sugamadex e complexo rocurônio­sugamadex sobre a coagulação em ratos.

BACKGROUND AND OBJECTIVES: Sugammadex is an alternative pharmacological drug capable of reversing neuromuscular blockades without the limitations that are presented by anticholinesterase drugs. Coagulation disorders that are related to treatment with sugammadex were reported. The exact mechanism of the effects on coagulation are not fully understood. The objective of this research is to evaluate the effects of rocuronium, sugammadex and the rocuronium-sugammadex complex on coagulation in an experimental model in rats. METHODS: This is an experimental randomized animal study. Wistar rats were randomly assigned into the following groups: the Control Group; the Ssal Group - 0.5 mL of intravenous saline; the Sugammadex Group - intravenous sugammadex (100 mg.kg-1); and the Rocuronium-Sugammadex Group - intravenous solution with rocuronium (3.75 mg.kg-1) and sugammadex (100 mg.kg-1). Anesthesia was performed by using isoflurane with controlled ventilation. Coagulation factors were measured 10 minutes after the end of the preoperative preparation and 30 minutes after the administration of the drugs in accordance with the chosen groups. RESULTS: Platelet counts, prothrombin times and activated partial thromboplastin times were similar between the groups and between the moments within each group. There were reductions in the plasma fibrinogen levels between sample times 1 and 2 in the Rocuronium-Sugammadex group (p=0.035). CONCLUSIONS: The rocuronium-sugammadex complex promoted reductions in plasma fibrinogen counts, although the levels were still within normal limits.

Assessment of remnant liver function and volume after selective ligation of portal vein and hepatic artery in a rat model.

PURPOSE: To evaluate liver regeneration after selective ligation of portal vein and hepatic artery by 3D Computed Tomography in an experimental model. METHODS: Sixteen Wistar rats were randomized into four equal groups: Group I- control (sham), Group II- isolated selective ligation of the hepatic artery, Group III- isolated selective ligation of the portal vein and Group IV- combined ligation of portal vein and hepatic artery. Before procedure and five days after a 3D CT Scan was performed to analyze the hypertrophy, weight and function of the remnant liver. RESULTS: The largest regeneration rate and increase of weight in the hypertrophied lobe was detected in group IV, the first with an average of 3.99 (p=0.006) and the last varying from 6.10g to 9.64g (p=0.01). However, total liver weight and the R1 ratio (Hypertrophied Lobe Weight/Total Liver Weight) was higher in group III (P<0.001) when compared with groups I, II and IV and showed no difference between them. The immunohistochemical examination with PCNA also found higher percentages with statistical significance differences in rats of groups III and IV. It was possible to confirm a strong correlation between hypertrophied lobe weight and its imaging volumetric study. Liver function tests only showed a significant difference in serum gamma-glutamyltransferase and phosphorous. CONCLUSION: There is a largest liver regeneration after combined ligation of portal vein and hepatic artery and this evidence may improve the knowledge of surgical treatment of liver injuries, with a translational impact in anima nobile.

Glucose tolerance in the proximal versus the distal small bowel in Wistar rats.

BACKGROUND: Type 2 diabetes is an epidemic and insulin resistance is the central etiology of this disease. Obesity increases insulin resistance and glucose intolerance and also exacerbates metabolic abnormalities present in type 2 diabetes. Bariatric surgery is the most effective treatment for severe obesity. Most reported series show that return to euglycemia and normal insulin levels occur days after gastric bypass and biliopancreatic diversion, long before major weight loss has taken place. The mechanisms underlying this dramatic reversal of type 2 diabetes following these bariatric procedures are not well understood. METHODS: Twelve Wistar rats were fed with a palatable hyperlipidic diet for 12 weeks. Body weight, glucose, and intraperitoneal glucose tolerance test were measured regularly. On day 91, they were randomized in two groups (hindgut and controls) and operated. Twenty-one days later, the tests were done again and the hindgut group re-operated. A duodenal exclusion was done. The results of an intraperitoneal glucose tolerance test were compared after the procedures. RESULTS: Body weight increased regularly in all the rats. Some rats developed hyperglycemia 28 days after beginning hyperlipidic diet, but these levels returned to baseline on days 56 and 84. The glucose tolerance test showed an improvement in glycemic levels in the hindgut group 21 days after the first operation (151 +/- 21mg/dl). After the second operation, despite weight loss, the glucose tolerance test of the foregut group worsened again (267 +/- 53 mg/dl) (p < 0.01). CONCLUSION: Comparing the "hindgut hypothesis" and the "foregut hypothesis", our data show an improvement in the 30 min glucose tolerance test in the hindgut group.