Luiz Felipe Forgiarini

Simulating Sleep Apnea by Exposure to Intermittent Hypoxia Induces Inflammation in the Lung and Liver

Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH). IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n = 6) or a simulated IH (SIH) (n = 6) for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF-α), inducible NO synthase (iNOS), vascular endothelial growth factor (VEGF), and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

Altered aquaporins in the brains of mice submitted to intermittent hypoxia model of sleep apnea

Rostral fluid displacement has been proposed as a pathophysiologic mechanism of both central and obstructive sleep apnea. Aquaporins are membrane proteins that regulate water transport across the cell membrane and are involved in brain edema formation and resolution. The present study investigated the effect of intermittent hypoxia (IH), a model of sleep apnea, on brain aquaporins. Mice were exposed to intermittent hypoxia to a nadir of 7% oxygen fraction. Brain water content, Aquaporin-1 and Aquaporin-3 were measured in the cerebellum and hippocampus. Hematoxylin-eosin and immunohistochemistry stainings were performed to evaluate cell damage. Compared to the sham group, the hypoxia group presented higher brain water content, lower levels of Aquaporin-1 and similar levels of Aquaporin-3. Immunoreactivity to GFAP and S100B was stronger in the hypoxia group in areas of extensive gliosis, compatible with cytotoxic edema. These findings, although preliminary, indicate an effect of IH on aquaporins levels. Further investigation about the relevance of these data on the pathophysiology of OSA is warranted.

METHIONINE- AND CHOLINE-DEFICIENT DIET INDUCES HEPATIC CHANGES CHARACTERISTIC OF NON-ALCOHOLIC STEATOHEPATITIS

CONTEXT Non-alcoholic steatohepatitis is a disease with a high incidence, difficult diagnosis, and as yet no effective treatment. So, the use of experimental models for non-alcoholic steatohepatitis induction and the study of its routes of development have been studied. OBJECTIVES This study was designed to develop an experimental model of non-alcoholic steatohepatitis based on a methionine- and choline-deficient diet that is manufactured in Brazil so as to evaluate the liver alterations resulting from the disorder. METHODS Thirty male C57BL6 mice divided in two groups (n = 15) were used: the experimental group fed a methionine- and choline-deficient diet manufactured by Brazilian company PragSoluções®, and the control group fed a normal diet, for a period of 2 weeks. The animals were then killed by exsanguination to sample blood for systemic biochemical analyses, and subsequently submitted to laparotomy with total hepatectomy and preparation of the material for histological analysis. The statistical analysis was done using the Students t-test for independent samples, with significance level of 5%. RESULTS The mice that received the methionine- and choline-deficient diet showed weight loss and significant increase in hepatic damage enzymes, as well as decreased systemic levels of glycemia, triglycerides, total cholesterol, HDL and VLDL. The diagnosis of non-alcoholic steatohepatitis was performed in 100% of the mice that were fed the methionine- and choline-deficient diet. All non-alcoholic steatohepatitis animals showed some degree of macrovesicular steatosis, ballooning, and inflammatory process. None of the animals which were fed the control diet presented histological alterations. All non-alcoholic steatohepatitis animals showed significantly increased lipoperoxidation and antioxidant enzyme GSH activity. CONCLUSION The low cost and easily accessible methionine- and choline-deficient diet explored in this study is highly effective in inducing steatosis and steatohepatitis in animal model, alterations that are similar to those observed in human livers.

N-Acetylcysteine administration confers lung protection in different phases of lung ischaemia–reperfusion injury

OBJECTIVES To verify the effects of N-acetylcysteine (NAC) administered before and after ischaemia in an animal model of lung ischaemia-reperfusion (IR) injury. METHODS Twenty-four Wistar rats were subjected to an experimental model of selective left pulmonary hilar clamping for 45 min followed by 2 h of reperfusion. The animals were divided into four groups: control group (SHAM), ischaemia-reperfusion, N-acetylcysteine-preischaemia (NAC-Pre) and NAC-postischaemia (NAC-Post). We recorded the haemodynamic parameters, blood gas analysis and histology. We measured the thiobarbituric acid reactive substances concentration; the expression of superoxide dismutase (SOD), inducible nitric oxide synthase (iNOS), nitrotyrosine, cleaved caspase 3, nuclear factor κB (NF-κB), NF-kappa-B inhibitor alpha (IκB-α), tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β); myeloperoxidase activity (MPO). RESULTS No significant differences were observed in the haemodynamic parameters, blood gas analysis and SOD activity among the groups. Lipid peroxidation was significantly higher in the IR and NAC-Pre groups (P < 0.01). The expression of nitrotyrosine, cleaved caspase 3, NF-κB, IκB-α, TNF-α and IL-1β were significantly higher in the IR group when compared with the SHAM and NAC groups (P < 0.01). The NAC-Pre group showed a significantly higher expression of these proteins when compared with the SHAM and NAC-Post groups (P < 0.05). After reperfusion, the expression of iNOS increased almost uniformly in all groups when compared with the SHAM group (P < 0.01). The histological analysis showed fewer inflammatory cells in the NAC groups. CONCLUSIONS The intravenous administration of NAC demonstrated protective properties against lung IR injury. The use of NAC immediately after reperfusion potentiates its protective effects.

Endobronchial perfluorocarbon administration decreases lung injury in an experimental model of ischemia and reperfusion

OBJECTIVE To verify the effects of liquid endobronchial perfluorocarbon (PFC) administered before reperfusion in an animal model of lung ischemia-reperfusion injury. METHODS Eighteen Wistar rats were subjected to an experimental model of selective left pulmonary artery clamping for 45 min followed by reperfusion for 2 h. The animals were divided into three groups: the ischemia-reperfusion (IR) group, the sham group, and the PFC group. We recorded the hemodynamic parameters, blood gas analysis, and histology. A Western blot assay was used to measure the inducible nitric oxide synthase, caspase 3, and nuclear factor қB (subunit p65) activities. Lipid peroxidation was assessed by the thiobarbituric acid reactive substances assay and the activity of the antioxidant enzyme superoxide dismutase. RESULTS No significant differences were observed in lipid peroxidation among the groups. The superoxide dismutase activity was increased (P < 0.05) in the PFC-treated group. The expressions of nuclear factor қB, inducible nitric oxide synthase, and caspase 3 were significantly lower in the PFC group than in the IR group (P < 0.05). The histologic analysis showed a reduction in lung injuries in the PFC group compared with the sham and IR groups. CONCLUSION The use of endobronchial PFC reduces the inflammatory response, preserves the alveolar structure, and protects the lungs against the hazardous effects of ischemia-reperfusion injuries.

Aminoguanidine reduces oxidative stress and structural lung changes in experimental diabetes mellitus

We evaluated the effect of aminoguanidine on pulmonary oxidative stress and lung structure in an experimental model of diabetes mellitus. Thiobarbituric acid reactive substances (TBARS), histology and arterial blood gases were evaluated in animals with diabetes mellitus (DM group), animals with diabetes mellitus treated with aminoguanidine (DM+AG group), and controls. The TBARS levels were significantly higher in the DM group than in the control and DM+AG groups (2.90 ± 1.12 vs. 1.62 ± 0.28 and 1.68 ± 0.04 nmol/mg protein, respectively), as was PaCO2 when compared with that of the control group (49.2 ± 1.65 vs. 38.12 ± 4.85 mmHg), and PaO2 was significantly higher in the control group (104.5 ± 6.3 vs. 16.30 ± 69.48 and 97.05 ± 14.02 mmHg, respectively). In this experimental model of diabetes mellitus, aminoguanidine reduced oxidative stress, structural tissue alterations, and gas exchange.

Oxidative Stress and Pulmonary Changes in Experimental Liver Cirrhosis

The use of carbon tetrachloride (CCl4) in rats is an experimental model of hepatic tissue damage; which leads to fibrosis, and at the long term, cirrhosis. Cirrhosis is the consequence of progressive continued liver damage, it may be reversible when the damaging noxae have been withdrawn. The aim of this study is to evaluate the changes caused by cirrhosis in lung and liver, through the experimental model of intraperitoneal CCI4 administration. We used 18 male Wistar rats divided into three groups: control (CO) and two groups divided by the time of cirrhosis induction by CCI4: G1 (11 weeks), G2 (16 weeks). We found significant increase of transaminase levels and lipid peroxidation (TBARS) in liver and lung tissue and also increased antioxidant enzymes SOD and CAT, as well as the expression of TNF-α and IL-1β in the lung of cirrhotic animals. We observed changes in gas exchange in both cirrhotic groups. We can conclude that our model reproduces a model of liver cirrhosis, which causes alterations in the pulmonary system that leads to changes in gas exchange and size of pulmonary vessels.

Efeitos da metilprednisolona na atividade inflamatória e estresse oxidativo nos pulmões de ratos com morte cerebral

OBJECTIVE: To evaluate the effects that early and late systemic administration of methylprednisolone have on lungs in a rat model of brain death. METHODS: Twenty-four male Wistar rats were anesthetized and randomly divided into four groups (n = 6 per group): sham-operated (sham); brain death only (BD); brain death plus methylprednisolone (30 mg/kg i.v.) after 5 min (MP5); and brain death plus methylprednisolone (30 mg/kg i.v.) after 60 min (MP60). In the BD, MP5, and MP60 group rats, we induced brain death by inflating a balloon catheter in the extradural space. All of the animals were observed and ventilated for 120 min. We determined hemodynamic and arterial blood gas variables; wet/dry weight ratio; histological score; levels of thiobarbituric acid reactive substances (TBARS); superoxide dismutase (SOD) activity; and catalase activity. In BAL fluid, we determined differential white cell counts, total protein, and lactate dehydrogenase levels. Myeloperoxidase activity, lipid peroxidation, and TNF-α levels were assessed in lung tissue. RESULTS: No significant differences were found among the groups in terms of hemodynamics, arterial blood gases, wet/dry weight ratio, BAL fluid analysis, or histological score-nor in terms of SOD, myeloperoxidase, and catalase activity. The levels of TBARS were significantly higher in the MP5 and MP60 groups than in the sham and BD groups (p < 0.001). The levels of TNF-α were significantly lower in the MP5 and MP60 groups than in the BD group (p < 0.001). CONCLUSIONS: In this model of brain death, the early and late administration of methylprednisolone had similar effects on inflammatory activity and lipid peroxidation in lung tissue.

Endobronchial Perfluorocarbon Reduces Inflammatory Activity before and after Lung Transplantation in an Animal Experimental Model

Background. The aim of this study was to evaluate the use of liquid perfluorocarbon (PFC) as an adjuvant substance for lung preservation and assess its role in pulmonary protection after transplantation. Methods. Seventy-two rat lungs were flushed with low-potassium dextran (LPD) solution and randomized into three main groups: control with LPD alone and experimental with 3 (PFC3) and 7 mL/kg (PFC7) of endobronchial PFC instilled just after harvest. Each group was divided into four subgroups according to preservation time (3, 6, 12, and 24 hours). Afterwards, we performed lung transplantation using rat lungs preserved for 12 hours with LPD alone or with 7 mL/kg of endobronchial PFC. Results. There was a significant increase in oxidative stress in the control group at 6 h of cold ischemic time compared with the PFC3 and PFC7 groups. The apoptotic activity and NF-κB expression were significantly higher in the control group compared with the PFC groups at 3, 12, and 24 h of cold preservation. After transplantation, the NF-κB, iNOS, and nitrotyrosine expression as well as caspase 3 activity were significantly lower in the PFC groups. Conclusion. The use of endobronchial PFC as an adjuvant to the current preservation strategy improved graft viability.

Peritoneal Free Autologous Fat Graft for the Control of Pulmonary Air Leaks in Emphysematous Rat Lungs

BACKGROUND Persistent pulmonary air leak is the most frequent complication after lung resection, resulting in an increase in postoperative morbidity and mortality. We evaluated the viability, integration, and efficacy of a free peritoneal fat graft as a method for controlling air leak in normal and emphysematous rat lungs. METHODS Sixty Wistar rats were divided into two groups: elastase-produced lung emphysema (n=30) and control (normal) lungs (n=30). Pulmonary air leak was produced by puncture of the right lower lobe, and aerostasis was attempted by means of intrapulmonary injection of autologous free peritoneal fat graft. Rats in each group (n=6) were randomly allocated to subgroups and were sacrificed at 7, 14, 21, 30, and 60 days. Then, lungs were removed for histology, morphometry, vessel identification and counting, and immunohistochemistry for caspase 3, vascular endothelial growth factor, and factor VIII. RESULTS Tissue integration of the free fat grafts was found in all animals in both groups. Vessels stained with India ink inside the fat grafts were present at all assessment periods in both groups. Vascular endothelial growth factor expression was significantly higher in all periods in the emphysema group compared with normal lungs (p<0.001). There was a significant increase in caspase 3 expression in the emphysema group at 7, 21, 30, and 60 days (p<0.001). Factor VIII showed a significant increase (p<0.001) at 30 and 60 days in emphysematous lungs. CONCLUSIONS The use of free peritoneal fat graft was able to control the air leaks in normal and emphysematous rat lungs, with persisting graft viability for as long as 60 days after implantation.