RESUMO
BACKGROUND: Acute pancreatitis is an inflammation of the pancreatic glandular parenchyma that causes injury with or without the destruction of pancreatic acini. Clinical and experimental evidence suggest that certain systemic proinflammatory mediators may be responsible for initiating the fundamental mechanisms involved in microglial reactivity. Here, we investigated the possible repercussions of acute pancreatitis (AP) on the production of inflammatory mediators in the brain parenchyma focusing on microglial activation in the hippocampus. METHODS: The acute pancreatic injury in rats was induced by a pancreas ligation surgical procedure (PLSP) on the splenic lobe, which corresponds to approximately 10% of total mass of the pancreas. Blood samples were collected via intracardiac puncture for the measurement of serum amylase. After euthanasia, frozen or paraffin-embedded brains and pancreas were analyzed using qRT-PCR or immunohistochemistry, respectively. RESULTS: Immunohistochemistry assays showed a large number of Iba1 and PU.1-positive cells in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus of the PLSP group. TNF-α mRNA expression was significantly higher in the brain from PLSP group. NLRP3 inflammasome expression was found to be significantly increased in the pancreas and brain of rats of the PLSP group. High levels of BNDF mRNA were found in the rat brain of PLSP group. In contrast, NGF mRNA levels were significantly higher in the control group versus PLSP group. CONCLUSION: Our findings suggest that AP has the potential to induce morphological changes in microglia consistent with an activated phenotype.
Assuntos
Pancreatite , Ratos , Animais , Pancreatite/metabolismo , Microglia/metabolismo , Doença Aguda , Hipocampo/metabolismo , Pâncreas/metabolismo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Ischemic stroke represents a significant global health concern, necessitating thorough investigations and the utilization of stroke animal models to explore novel treatment modalities and diagnostic imaging techniques. NEW METHOD: Ultrasound biomicroscopy (BMU), operating at a center frequency of 21 MHz, along with ultrasound contrast agents (UCAs), was used to quantify microcirculation cerebral blood flow in a rat model of ischemic stroke. The microcirculation parameters were derived from time intensity curve (TIC) plots obtained based on UCA-bolus kinetics. RESULTS: Semiquantitative perfusion-related parameters were assessed. The TIC curves showed differences in amplitude when compared intra-animal between the left and right sides, and three situations were observed: normal perfusion, hypoperfusion, and nonperfusion. ROC analysis of delays between the left and right time intensity peak (TIP) for regions of interest (ROIs) in the control and stroke-hypoperfusion groups revealed an optimal cutpoint of 0.39 s to indicate when hypoperfusion is occurring in rats, with a sensitivity of 93.33 % and a specificity of 80 %. COMPARISON WITH EXISTING METHOD(S): Ultrasound perfusion imaging through the temporal bone window has been clinically applied to stroke patients using a UCA bolus for TIC analysis. TIC parameters were correlated with MRI- and CT-based measurements. CONCLUSIONS: This investigation quantified cerebral blood flow in a rat model of ischemic stroke by measuring microcirculation parameters. The study demonstrated the efficacy of this approach as a valuable tool for conducting preclinical studies.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Ratos , Animais , Meios de Contraste , Microscopia Acústica , Acidente Vascular Cerebral/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Encéfalo/irrigação sanguínea , Ultrassonografia/métodos , PerfusãoRESUMO
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.
Assuntos
Hipotireoidismo/cirurgia , Complicações Pós-Operatórias/cirurgia , Glândula Tireoide/transplante , Tireoidectomia/efeitos adversos , Animais , Hipotireoidismo/sangue , Hipotireoidismo/etiologia , Masculino , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Ratos , Ratos Wistar , Tiroxina/sangue , Transplante Autólogo , Tri-Iodotironina/sangueRESUMO
BACKGROUND: Dehiscence of intestinal anastomosis results in high morbidity and mortality. The aim of this study was to investigate the effects of locally administered adipose tissue-derived mesenchymal stromal cells in a model of high-risk colonic anastomosis in rats. METHODS: Seven days after induction of colitis with 2,4,6-trinitrobenzene sulfonic acid, Wistar rats were submitted to a transection of the descending colon followed by end-to-end anastomosis and were then treated with 2×106 adipose tissue-derived mesenchymal stromal cells (from the preperitoneal fat) or an acellular culture solution instilled onto the surface of the anastomosis. At day 14, after macroscopic survey of the abdominal cavity, the anastomotic area was submitted to histologic and immunohistochemical analysis, evaluation of myeloperoxidase activity, fibrosis, epithelial integrity, NF-κ B activation, expression of inflammatory cytokines, and extracellular matrix-related genes. RESULTS: Anastomotic leakage and mortality associated with high-risk anastomosis decreased with treatment with adipose tissue-derived mesenchymal stromal cells (P < .03). Application of adipose tissue-derived mesenchymal stromal cells resulted in lower histologic scores (P = .011), decreased deposition of collagen fibers (P = .003), preservation of goblet cells (P = .033), decreased myeloperoxidase activity (P = .012), decreased accumulation of CD4+ T-cells (P = .014) and macrophages (P = .011) in the lamina propria, a decrease in the number of apoptotic cells (P = .008), and the activation of NF-κ B (P = .036). Overexpression of IL-17, TNF-α , IFN-γ, and metalloproteinases in the acellular culture solution-treated, high-risk anastomosis group decreased (P < .05) to near normal values with adipose tissue-derived mesenchymal stromal cells treatment. CONCLUSION: Improvements in outcomes of a high-risk colonic anastomosis with adipose tissue-derived mesenchymal stromal cells therapy reflect the immunomodulatory activity and healing effect of these cells, even after just topical administration and reinforces their use in future translational research.
Assuntos
Fístula Anastomótica/prevenção & controle , Colite/cirurgia , Colo/cirurgia , Gordura Intra-Abdominal/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Fístula Anastomótica/etiologia , Animais , Colite/induzido quimicamente , Modelos Animais de Doenças , Humanos , Masculino , Ratos , Ratos Wistar , Resultado do Tratamento , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
PURPOSE: To evaluate the most frequent surgical techniques of high-risk colorectal anastomoses in rats. METHODS: Wistar rats were enrolled in three different models comprising inflammatory (TNBS enema), vascular (portal vein occlusion) or obstructive (a non-ischemic constricting ring) mechanisms associated with colonic anastomosis that had accomplished after these former lesions. Histological analyses (Hematoxylin and eosin and Picrosirius red) were performed. RESULTS: All anastomoses techniques were associated with risk factors and had complications, mainly anastomotic leakage. In Study 1, the use of a pharmacological agent, trinitrobenzene sulfonic acid (TNBS) mimicked an inflammatory bowel disease such as Crohn's disease with 50% of anastomosis leakage, the higher percentage among all models tested. In Study 2, after portal ischemia followed by reperfusion it was observed a dense neutrophil infiltrate in the midst of necrotic tissue and fibrin at the anastomotic site and 5 days after the anastomosis, no collagen was produced. In Study 3, 5 days after the mechanical obstruction some denuded areas of epithelium with marked oedema of mucosa and submucosa were seen, at the anastomotic site and anastomosis group showed some reduction of collagen density when compared with Control/Sham group. CONCLUSION: All the experimental surgical techniques tested in rats were associated with high-risk colorectal anastomoses and were useful to study colonic anastomotic healing and intestinal leakage.
Assuntos
Fístula Anastomótica , Colo/cirurgia , Reto/cirurgia , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Fístula Anastomótica/diagnóstico por imagem , Fístula Anastomótica/patologia , Animais , Modelos Animais de Doenças , Ratos , Ratos Wistar , CicatrizaçãoRESUMO
Abstract Purpose: To evaluate the most frequent surgical techniques of high-risk colorectal anastomoses in rats. Methods: Wistar rats were enrolled in three different models comprising inflammatory (TNBS enema), vascular (portal vein occlusion) or obstructive (a non-ischemic constricting ring) mechanisms associated with colonic anastomosis that had accomplished after these former lesions. Histological analyses (Hematoxylin and eosin and Picrosirius red) were performed. Results: All anastomoses techniques were associated with risk factors and had complications, mainly anastomotic leakage. In Study 1, the use of a pharmacological agent, trinitrobenzene sulfonic acid (TNBS) mimicked an inflammatory bowel disease such as Crohn's disease with 50% of anastomosis leakage, the higher percentage among all models tested. In Study 2, after portal ischemia followed by reperfusion it was observed a dense neutrophil infiltrate in the midst of necrotic tissue and fibrin at the anastomotic site and 5 days after the anastomosis, no collagen was produced. In Study 3, 5 days after the mechanical obstruction some denuded areas of epithelium with marked oedema of mucosa and submucosa were seen, at the anastomotic site and anastomosis group showed some reduction of collagen density when compared with Control/Sham group. Conclusion: All the experimental surgical techniques tested in rats were associated with high-risk colorectal anastomoses and were useful to study colonic anastomotic healing and intestinal leakage.
Assuntos
Animais , Ratos , Reto/cirurgia , Colo/cirurgia , Fístula Anastomótica/patologia , Fístula Anastomótica/diagnóstico por imagem , Cicatrização , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Ratos Wistar , Modelos Animais de DoençasRESUMO
Purpose: To evaluate the most frequent surgical techniques of high-risk colorectal anastomoses in rats. Methods: Wistar rats were enrolled in three different models comprising inflammatory (TNBS enema), vascular (portal vein occlusion) or obstructive (a non-ischemic constricting ring) mechanisms associated with colonic anastomosis that had accomplished after these former lesions. Histological analyses (Hematoxylin and eosin and Picrosirius red) were performed. Results: All anastomoses techniques were associated with risk factors and had complications, mainly anastomotic leakage. In Study 1, the use of a pharmacological agent, trinitrobenzene sulfonic acid (TNBS) mimicked an inflammatory bowel disease such as Crohns disease with 50% of anastomosis leakage, the higher percentage among all models tested. In Study 2, after portal ischemia followed by reperfusion it was observed a dense neutrophil infiltrate in the midst of necrotic tissue and fibrin at the anastomotic site and 5 days after the anastomosis, no collagen was produced. In Study 3, 5 days after the mechanical obstruction some denuded areas of epithelium with marked oedema of mucosa and submucosa were seen, at the anastomotic site and anastomosis group showed some reduction of collagen density when compared with Control/Sham group. Conclusion: All the experimental surgical techniques tested in rats were associated with high-risk colorectal anastomoses and were useful to study colonic anastomotic healing and intestinal leakage.(AU)
Assuntos
Animais , Ratos , Anastomose Cirúrgica/veterinária , Colo/cirurgia , Cirurgia Colorretal/veterinária , Ratos Wistar , Modelos AnimaisRESUMO
Abstract Purpose: To evaluate the actual incidence of both microlithiasis and acute cholecystitis during treatment with intravenous ceftriaxone in a new rabbit model. Methods: New Zealand rabbits were treated with intravenous ceftriaxone or saline for 21 days. Ultrasound monitoring of the gallbladder was performed every seven days until the 21st day when histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), pro-caspase-3 and CD68, liver enzyme biochemistry, and chromatography analysis of the bile and sediments were also performed. Results: All animals treated with ceftriaxone developed acute cholecystitis, confirmed by histopathology (P<0.05) and biliary microlithiasis, except one that exhibited sediment precipitation. In the group treated with ceftriaxone there was an increase in pro-caspase-3, gamma-glutamyl transpeptidase concentration, PCNA expression and in the number of cells positive for anti-CD68 (P<0.05). In the ceftriaxone group, the cholesterol and lecithin concentrations increased in the bile and a high concentration of ceftriaxone was found in the microlithiasis. Conclusion: Ceftriaxone administered intravenously at therapeutic doses causes a high predisposition for lithogenic bile formation and the development of acute lithiasic cholecystitis.
Assuntos
Animais , Ratos , Ceftriaxona/efeitos adversos , Colecistectomia , Colelitíase/induzido quimicamente , Colecistite Aguda/induzido quimicamente , Antibacterianos/efeitos adversos , Ceftriaxona/administração & dosagem , Colelitíase/metabolismo , Colecistectomia Laparoscópica , Colecistite Aguda/metabolismo , Modelos Animais de Doenças , Pesquisa Translacional Biomédica , Administração Intravenosa , Vesícula Biliar/patologia , Antibacterianos/administração & dosagemRESUMO
Purpose: To evaluate the actual incidence of both microlithiasis and acute cholecystitis during treatment with intravenous ceftriaxone in a new rabbit model. Methods: New Zealand rabbits were treated with intravenous ceftriaxone or saline for 21 days. Ultrasound monitoring of the gallbladder was performed every seven days until the 21st day when histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), pro-caspase-3 and CD68, liver enzyme biochemistry, and chromatography analysis of the bile and sediments were also performed. Results: All animals treated with ceftriaxone developed acute cholecystitis, confirmed by histopathology (P < 0.05) and biliary microlithiasis, except one that exhibited sediment precipitation. In the group treated with ceftriaxone there was an increase in pro-caspase-3, gamma-glutamyl transpeptidase concentration, PCNA expression and in the number of cells positive for anti-CD68 (P < 0.05). In the ceftriaxone group, the cholesterol and lecithin concentrations increased in the bile and a high concentration of ceftriaxone was found in the microlithiasis. Conclusion: Ceftriaxone administered intravenously at therapeutic doses causes a high predisposition for lithogenic bile formation and the development of acute lithiasic cholecystitis.(AU)
Assuntos
Animais , Adulto , Coelhos , Ceftriaxona/administração & dosagem , Ceftriaxona/efeitos adversos , Colecistite Aguda/etiologia , Colelitíase/etiologia , Pesquisa Translacional Biomédica/métodos , Modelos AnimaisRESUMO
PURPOSE: To evaluate the actual incidence of both microlithiasis and acute cholecystitis during treatment with intravenous ceftriaxone in a new rabbit model. METHODS: New Zealand rabbits were treated with intravenous ceftriaxone or saline for 21 days. Ultrasound monitoring of the gallbladder was performed every seven days until the 21st day when histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), pro-caspase-3 and CD68, liver enzyme biochemistry, and chromatography analysis of the bile and sediments were also performed. RESULTS: All animals treated with ceftriaxone developed acute cholecystitis, confirmed by histopathology (P<0.05) and biliary microlithiasis, except one that exhibited sediment precipitation. In the group treated with ceftriaxone there was an increase in pro-caspase-3, gamma-glutamyl transpeptidase concentration, PCNA expression and in the number of cells positive for anti-CD68 (P<0.05). In the ceftriaxone group, the cholesterol and lecithin concentrations increased in the bile and a high concentration of ceftriaxone was found in the microlithiasis. CONCLUSION: Ceftriaxone administered intravenously at therapeutic doses causes a high predisposition for lithogenic bile formation and the development of acute lithiasic cholecystitis.
Assuntos
Antibacterianos/efeitos adversos , Ceftriaxona/efeitos adversos , Colecistectomia , Colecistite Aguda/induzido quimicamente , Colelitíase/induzido quimicamente , Administração Intravenosa , Animais , Antibacterianos/administração & dosagem , Ceftriaxona/administração & dosagem , Colecistectomia Laparoscópica , Colecistite Aguda/metabolismo , Colelitíase/metabolismo , Modelos Animais de Doenças , Vesícula Biliar/patologia , Coelhos , Pesquisa Translacional BiomédicaRESUMO
Olfactory ensheathing cells (OECs) are a type of specialized glial cell currently considered as having a double function in the nervous system: one regenerative, and another immune. Streptococcus pneumoniae is a major agent of severe infections in humans, including meningitis. It is commonly found in the nasopharynx of asymptomatic carriers, and, under certain still unknown conditions, can invade the brain. We evaluated whether pneumococcal cells recovered from lysed OECs and microglia are able to survive by manipulating the host cell activation. An intracellular-survival assay of S. pneumoniae in OECs showed a significant number of bacterial CFU recovered after 3 h of infection. In contrast, microglia assays resulted in a reduced number of CFU. Electron-microscopy analysis revealed a large number of pneumococci with apparently intact morphology. However, microglia cells showed endocytic vesicles containing only bacterial cell debris. Infection of OEC cultures resulted in continuous NF-κB activation. The IFN-γ-induced increase of iNOS expression was reversed in infected OECs. OECs are susceptible to S. pneumoniae infection, which can suppress their cytotoxic mechanisms in order to survive. We suggest that, in contrast to microglia, OECs might serve as safe targets for pneumococci, providing a more stable environment for evasion of the immune system.
Assuntos
Microglia/citologia , Bulbo Olfatório/citologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Animais , Células Cultivadas , Contagem de Colônia Microbiana , Interferon gama/metabolismo , Microglia/metabolismo , Microglia/microbiologia , Microscopia Eletrônica , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Bulbo Olfatório/metabolismo , Bulbo Olfatório/microbiologia , RatosRESUMO
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.
Assuntos
Peritônio/cirurgia , Surfactantes Pulmonares/farmacologia , Aderências Teciduais/prevenção & controle , Animais , Caspase 3/genética , Caspase 3/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Gastroenterostomia , Regulação da Expressão Gênica , Laparotomia , Lectinas/genética , Lectinas/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Peritônio/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Aderências Teciduais/genética , Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Acute pancreatitis (AP) is an inflammatory disorder that can affect adjacent and/or remote organs. Some evidence indicates that the production of reactive oxygen species is able to induce AP. Protein carbonyl (PC) derivatives, which can also be generated through oxidative cleavage mechanisms, have been implicated in several diseases, but there is little or no information on this biomarker in AP. We investigated the association between some inflammatory mediators and PC, with the severity of ischemia-reperfusion AP. Wistar rats (n = 56) were randomly assigned in the following groups : control; sham, 15- or 180-min clamping of splenic artery, with 24 or 72 h of follow-up. The relationships between serum level of PC and thiobarbituric acid reactive species (TBARS) to myeloperoxidase (MPO) activity in tissue homogenates and to cytokines in culture supernatants of pancreatic samples were analyzed. MPO activity was related to the histology scores and increased in all clamping groups. Tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin-6 were higher in the 180-min groups. Significant correlations were found between MPO activity and the concentrations of TNF-α and IL-1ß. PC levels increased in the 15-min to 24-h group. TBARS levels were not altered substantially. MPO activity and TNF-α and IL-1ß concentrations in pancreatic tissue are correlated with AP severity. Serum levels of PC appear to begin to rise early in the course of the ischemia-reperfusion AP and are no longer detected at later stages in the absence of severe pancreatitis. These data suggest that PC can be an efficient tool for the diagnosis of early stages of AP.
Assuntos
Biomarcadores/análise , Pancreatite Necrosante Aguda/diagnóstico , Pancreatite Necrosante Aguda/patologia , Carbonilação Proteica , Traumatismo por Reperfusão/patologia , Animais , Citocinas/análise , Modelos Animais de Doenças , Feminino , Peroxidase/análise , Ratos WistarRESUMO
Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.
Assuntos
Lectinas Tipo C/biossíntese , Lectinas de Ligação a Manose/biossíntese , Neuroglia/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Receptores de Superfície Celular/biossíntese , Animais , Western Blotting , Citometria de Fluxo , Imuno-Histoquímica , Receptor de Manose , Ratos , Ratos WistarRESUMO
The use of bone marrow mononuclear cells (BMMCs) has been shown as a putative efficient therapy for stroke. However, the mechanisms of therapeutic action are not yet completely known. Mannose receptor (MR) is a subgroup of the C-type lectin receptor superfamily involved in innate immune response in several tissues. Although known primarily for its immune function, MR also has important roles in cell migration, cell debris clearance and tissue remodeling during inflammation and wound healing. Here we analyzed MR expression in brains of rats one week after induction of unilateral focal cortical ischemia by thermocoagulation in blood vessels of sensorimotor cortex. Additionally, we evaluated possible changes in such expression in cortices of rats subjected to ischemia plus treatment with BMMCs. Our results showed high expression of MR in an unknown GFAP(+) cell type and in phagocytic macrophages/microglia within the lesion boundary zone whereas in the non-injured (contralateral) cortical parenchyma, low levels of MR expression were observed. Moreover, therapy with BMMCs induced overexpression of MR in ipsilateral (injured) cortex. Previous studies from our group have shown functional recovery and decreased neurodegeneration in BMMC-treated rats in the same model of focal cortical ischemia. Thus, we suggest that ischemic injury induces large increase in MR expression as part of a mechanism for clearance of damage-associated molecular patterns (DAMPs). In addition, induction of MR overexpression by BMMCs might increase the efficiency of clearance, being one of the protective mechanisms of these cells.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Astrócitos/metabolismo , Isquemia Encefálica/terapia , Proteína Glial Fibrilar Ácida/metabolismo , Receptor de Manose , Microglia/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores Mitogênicos/metabolismoRESUMO
Olfactory ensheathing cells (OECs) are a special glia that ensheath olfactory receptor axons that enter the brain via olfactory phila, thus, providing a potential route for access of pathogens. Streptococcus pneumoniae (Sp), that has a capsule rich in mannosyl residues, is the most common cause of rhinosinusitis that may evolve to meningitis. We have tested whether OECs in vitro express the mannose receptor (MR), and could internalize Sp via MR. Cultures were infected by a suspension of Sp (ATCC 49619), recognized by an anti-Sp antibody, in a 100:1 bacteria:cells ratio. Competition assays, by means of mannan, showed around a 15-fold reduction in the number of internalized bacteria. To verify whether MR could be involved in Sp uptake, OECs were reacted with an antibody against the MR C-terminal peptide (anti-cMR) and bacteria were visualized with Sytox Green. Selective cMR-immunoreaction was seen in perinuclear compartments containing bacteria whereas mannan-treated cultures showed an extremely low percentage of internalized bacteria and only occasional adhered bacteria. Our data suggest the involvement of MR in adhesion of bacteria to OEC surface, and in their internalization. Data are also coherent with a role of OECs as a host cell prior to (and during) bacterial invasion of the brain.
Assuntos
Endocitose/fisiologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Neuroglia/microbiologia , Infecções Pneumocócicas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Aderência Bacteriana/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Immunoblotting , Imuno-Histoquímica , Receptor de Manose , Neuroglia/imunologia , Neuroglia/metabolismo , Ratos , Ratos Wistar , Rinite/imunologia , Rinite/microbiologia , Sinusite/imunologia , Sinusite/microbiologiaRESUMO
The mannose receptor (MR) is a transmembrane glycoprotein, postulated to be a link between innate and adaptive immunity. MR is expressed in several cell types but no information is available on that for Schwann cells (SC). We show that rodent SC in primary cultures take up the MR ligand mannosyl/bovine serum albumin-fluorescein isothiocyanate (man/BSA-FITC) in a highly specific manner and bind an antibody against the C-terminus of the murine macrophage MR (anti-cMR). After incubation with man/BSA-FITC, flow cytometry demonstrates 90% positive SC, a dose-dependent increase in tagged cellular components and near total inhibition of the neoglycoprotein uptake by D-mannose or by the mannosylated protein horseradish peroxidase (HRP). Western blot for MR shows that SC share a unique protein of about 180 kDa with peritoneal resident macrophages. Treatment of cultured SC with interferon-gamma (IFN-gamma) or dexamethasone (DM) followed by the addition of man/BSA-FITC and analysis by flow cytometry shows down- or upregulation, respectively, of man/BSA-FITC uptake. Our results show that SC express the MR in a prospectively functional state and suggest an antigen-presenting function of SC, compatible with a role in infectious/inflammatory states of the peripheral nervous system.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe II/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Células de Schwann/imunologia , Células de Schwann/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Células Cultivadas , Dexametasona/farmacologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Glicoproteínas/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Peroxidase do Rábano Silvestre/metabolismo , Interferon gama/farmacologia , Macrófagos Peritoneais/metabolismo , Manose/metabolismo , Receptor de Manose , Camundongos , Ratos , Ratos Wistar , Células de Schwann/efeitos dos fármacos , Soroalbumina Bovina/metabolismoRESUMO
Complex carbohydrate structures are essential molecules of infectious microbes and host cells, and are involved in cell signaling associated with inflammatory and immune responses. The uptake of mannose-tailed glycans is usually carried out by macrophages, dendritic cells (DCs), and other professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation, and to promote T cell effector responses. Since Schwann cells (SCs) have been proposed as immunocompetent cells, we investigated whether a human cell line (ST88-14 cells) could bind mannosylated ligands in a specific manner. The saturation of uptake of mannosylated molecules by ST88-14 cells and the internalization and distribution pathway of these ligands were tested by cytometry and confocal plus electron microscopy, respectively. This uptake showed a dose-dependent increase, the saturation point being reached at high concentrations of mannosyl residues/240 mM mannose. Merging of man/BSA-FITC and S100 labeling showed their partial, but, significant colocalization. Ultrastructural analysis of ST88-14 cells after incubation with HRP-colloidal gold, without or with subsequent chasing at 37C, showed an initial location on the cell surface and temperature- and time-dependent internalization of the probe. Our findings suggest an efficient mannosylated ligand uptake system through putative lectin(s) that may be operational in inflammatory and immune responses.
Assuntos
Manose/metabolismo , Células de Schwann/metabolismo , Linhagem Celular Tumoral , Endocitose/imunologia , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Ouro/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Peroxidase do Rábano Silvestre/ultraestrutura , Humanos , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Lectinas Tipo C/ultraestrutura , Ligantes , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/ultraestrutura , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/ultraestrutura , Proteínas S100/metabolismo , Células de Schwann/ultraestruturaRESUMO
Leishmania amazonensis, an obligatory intracellular parasite, survives internalization by macrophages, but no information is available on the involvement of microglia. We have investigated microglia-protozoa interactions in mixed glial cultures infected with promastigote forms of L. amazonensis after lipopolysaccharide (LPS) or dexamethasone (DM) treatment. After 2 hr of exposure to parasites in control cultures, there was a small number of infected microglia (1%). Preincubation with LPS or DM led to 14% or 60% of microglial cells with attached parasites, respectively. DM treatment resulted in 39% of microglial cells with internalized parasites (controls or LPS-treated cells had < or =1%). Scanning electron micrographs showed numerous filopodia in DM-treated cells, whereas these projections were rarely observed in LPS-treated or control cells. DM treatment also affected the intramicroglial survival of Leishmania. In control cultures, internalized parasites, tagged with an anti-lipophosphoglycan (anti-LPG) antibody, showed fragmented DNA [terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL+)] after 4 hr of interaction, but changes seemed slightly delayed in DM-treated cultures. After 12 hr, there were no LPG+/TUNEL+ profiles in controls, whereas rare LPG+ profiles still persisted in DM-treated cells. Our results suggest that microglia are highly effective in the elimination of Leishmania and that the process can be effectively studied by LPG/TUNEL double labeling.