Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

OBJECTIVES: Necrotizing enterocolitis is a severe multifactorial intestinal disorder that primarily affects preterm newborns, causing 20-40% mortality and morbidity. Intestinal fatty acid-binding protein has been reported to be a biomarker for the detection of intestinal injuries. Our aim was to assess intestinal tissue injury and the molecular expression of intestinal fatty acid-binding protein over time in a necrotizing enterocolitis model. METHODS: A total of 144 Newborn rats were divided into two groups: 1) Control, which received breastfeeding (n=72) and 2) Necrotizing Enterocolitis, which received formula feeding and underwent hypoxia and hypothermia (n=72). A total of six time points of ischemia (2 times a day for 3 days; 12 pups for each time point) were examined. Samples were collected for analysis of body weight, morphological and histological characteristics, intestinal weight, intestinal weight/body weight ratio, injury grade, and intestinal fatty acid-binding protein levels. RESULTS: Body and intestinal weights were lower in the Necrotizing Enterocolitis group than in the Control group (p<0.005 and p<0.0005, respectively). The intestinal weight/body weight ratio was higher in the Necrotizing Enterocolitis group than in the Control group (p<0.005) only at the sixth ischemia time point. The Necrotizing Enterocolitis group displayed higher expression of intestinal fatty acid-binding protein (p<0.0005) and showed greater tissue damage than the Control group. CONCLUSION: Intestinal fatty acid-binding protein was an efficient marker of ischemic injury to the intestine and a good correlation was demonstrated between the time of ischemic injury and the grade of intestinal injury.

Standardization of pulmonary ventilation technique using volume-controlled ventilators in rats with congenital diaphragmatic hernia / Padronização da técnica de ventilação pulmonar utilizando ventiladores com volume controlado em ratos com hérnia diafragmática congênita

OBJECTIVE: To standardize a technique for ventilating rat fetuses with Congenital Diaphragmatic Hernia (CDH) using a volume-controlled ventilator. METHODS: Pregnant rats were divided into the following groups: a) control (C); b) exposed to nitrofen with CDH (CDH); and c) exposed to nitrofen without CDH (N-). Fetuses of the three groups were randomly divided into the subgroups ventilated (V) and non-ventilated (N-V). Fetuses were collected on day 21.5 of gestation, weighed and ventilated for 30 minutes using a volume-controlled ventilator. Then the lungs were collected for histological study. We evaluated: body weight (BW), total lung weight (TLW), left lung weight (LLW), ratios TLW / BW and LLW / BW, morphological histology of the airways and causes of failures of ventilation. RESULTS: BW, TLW, LLW, TLW / BW and LLW / BW were higher in C compared with N- (p <0.05) and CDH (p <0.05), but no differences were found between the subgroups V and N-V (p> 0.05). The morphology of the pulmonary airways showed hypoplasia in groups N- and CDH, with no difference between V and N-V (p <0.05). The C and N- groups could be successfully ventilated using a tidal volume of 75 ìl, but the failure of ventilation in the CDH group decreased only when ventilated with 50 ìl. CONCLUSION: Volume ventilation is possible in rats with CDH for a short period and does not alter fetal or lung morphology. .

OBJETIVO: padronizar uma técnica para ventilar fetos de rato com HDC usando um ventilador volume-controlado. MÉTODOS: ratas grávidas foram distribuídas em: a) Controle (C); e b) Expostos a Nitrofen com HDC e sem HDC (N-). Fetos dos três grupos foram divididos aleatoriamente em subgrupos ventilados (V) ou não ventilados (NV). Os fetos foram coletados no dia 21,5 da gestação, pesados e ventilados por 30 minutos usando um ventilador volume-controlado. A seguir os pulmões foram coletados para estudo histológico. Nós avaliamos: peso corporal (PC), peso pulmonar total (PPT), peso do pulmão esquerdo (PPE), razão PPT/PC e PPE/PC, histologia morfológica das vias aéreas e as causas das falhas da ventilação. RESULTADOS: PC, PPT, PPE, LLW, PPT/PC e PPE/PC foram maiores em C em relação a N- (p<0,05) e a HDC (p<0,05), mas não houve diferenças entre os subgrupos V e NV (p>0,05). A morfologia das vias aéreas pulmonares mostrou hipoplasia nos grupos N- e HDC, não havendo diferença entre V e NV (p<0,05). Os grupos C e N- puderam ser ventilados com sucesso usando o volume corrente de 75ìl, mas a falha de ventilação no grupo HDC só diminuiu quando ventilados com 50ìl. . CONCLUSÃO: a ventilação a volume de ratos com HDC por um curto período é possível e não altera a morfologia fetal ou pulmonar. .

L-FABP and I-FABP expression in newborn rats changes inversely in the model of necrotizing enterocolitis

PURPOSE: To determine the expression of hepatic L-FABP and intestinal I-FABP in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. METHODS: Newborn Sprague-Dawley rats were divided into four groups: Control (C1) - exclusive breastfeeding at the first and sixth procedures (C6), NEC1 - fed formula milk and submitted to hypoxia and hypothermia at the first and sixth procedures (NEC6). The newborn pups were fed twice a day for three days, for a total of six procedures. Samples were collected for morphometric evaluation (body weight, liver weight, liver weight/body weight ratio, intestinal weight and intestinal/body weight ratio) and for immunohistochemical and Western blotting analysis. The values obtained were analyzed statistically, with the level of significance set at p<0.05. RESULTS: Morphometric measurements showed reduction of body and liver weights in the NEC group (p<0.05). Both immunohistochemistry and western blotting revealed that L-FABP expression in the liver was decreased and I-FABP expression in the ileum was increased in the NEC group (p<0.05). CONCLUSION: L-FABP and I-FABP expression changed inversely in the rat NEC model. These findings can contribute to a better diagnosis of NEC in human newborns. .

Evaluation of histological changes after tracheal occlusion at different gestational ages in a fetal rat model

OBJECTIVES: To evaluate the histological changes of tracheal cartilage and epithelium caused by tracheal occlusion at different gestational ages in a fetal rat model. METHODS: Rat fetuses were divided into two groups: a) External control, composed of non-operated rats, and b) Interventional group, composed of rats operated upon on gestational day 18.5 (term = 22 days), divided into triads: 1) Tracheal occlusion, 2) Internal control and 3) Sham (manipulated but not operated). Morphological data for body weight, total lung weight and total lung weight/body weight ratio were collected and measured on gestational days 19.5, 20.5 and 21.5. Tracheal samples were histologically processed, and epithelial, chondral and total tracheal thicknesses were measured on each gestational day. RESULTS: The tracheal occlusion group exhibited an increase in total lung weight/body weight ratio (p<0.001). Histologically, this group had a thicker epithelial thickness (p<0.05) and thinner chondral (p<0.05) and total tracheal thicknesses (p<0.001). These differences were more prominent on gestational days 20.5 and 21.5. CONCLUSION: Tracheal occlusion changed tracheal morphology, increased epithelial thickness and considerably decreased total tracheal thickness. These changes in the tracheal wall could explain the development of tracheomegaly, recently reported in some human fetuses subjected to tracheal occlusion.

The role of gut-liver axis in the restriction of intrauterine growth in a model of experimental gastroschisis / O papel do eixo intestino-fígado na restrição de crescimento intra-uterino em um modelo de gastrosquise experimental

PURPOSE: To evaluate the intrauterine growth restriction (IUGR) by the expression of IR-β, IRS-1, IRS-2, IGF-IRβ and Ikappaβ in experimental model of gastroschisis. METHODS: Pregnant rats at 18.5 days of gestation were submitted to surgery to create experimental fetal gastroschisis (term = 22 days) were divided in three groups: gastroschisis (G), control (C) and sham (S). Fetuses were evaluated for body weight (BW), intestinal (IW), liver (LW) and their relations IW/BW and LW/BW. IR-β and IGF-IRβ receptors, IRS-1 and IRS-2 substrates and Ikappaβ protein were analyzed by western blotting. RESULTS: BW was lower in G, the IW and IW / BW were greater than C and S (p<0.05) groups. The liver showed no differences between groups. In fetuses with gastroschisis, compared with control fetuses, the expression of IGF-IRβ (p<0.001) and Ikappaβ (p<0.001) increased in the liver and intestine, as well as IR-β (p<0.001) which decreased in both. In contrast to the intestine, IRS-1 (p<0.001) increased in the liver and IRS-2 decreased (p<0.01). CONCLUSION: The axis of the intestine liver has an important role in inflammation, with consequent changes in the metabolic pathway of glucose can contribute to the IUGR in fetuses with gastroschisis.

OBJETIVO: Avaliar a restrição de crescimento intra-uterino (RCIU) pela expressão de IR-β, IRS-1, IRS-2, IGF-IRβ e a via inflamatória do Ikappaβ no modelo de gastrosquise experimental. MÉTODOS: Ratas grávidas com 18,5 dias de gestação foram submetidas a cirurgia experimental para criar gastrosquise fetal (termo = 22 dias) e os fetos foram divididos em três grupos: gastrosquise (G), controle (C) e sham (S). Os fetos foram avaliados quanto ao peso corporal (BW), intestinal (IW), fígado (LW) e suas relações IW/BW e LW/BW. Os receptores IR-β e IGF-IRβ, os substratos IRS-1 e IRS-2 e a proteína Ikappaβ foram analisados por western blotting. RESULTADOS: O BW de G foi menor, o IW e IW/BW foram superiores a C e S (p < 0.05). O fígado não apresentou diferenças entre os grupos. Nos fetos com gastrosquise, quando comparados com fetos controles, a expressão de IGF-IRβ (p<0.001) e Ikappaβ (p<0.001) aumentou no fígado e intestino, assim como IR-β (p<0.001) que diminuiu em ambos. Inversamente ao intestino, IRS-1 (p<0.001) aumentou no fígado e IRS-2 diminuiu (p<0.01). CONCLUSÃO: O eixo do intestino fígado tem um papel importante na inflamação, com consequentes alterações na via metabólica de glicose que pode contribuir para a RCIU em fetos com gastrosquise.

Corticosteroid effect upon intestinal and hepatic interleukin profile in a gastroschisis rat model / Efeito do corticoesteróide sobre o perfil das interleucinas intestinais e hepáticas no modelo de gastrosquise em ratos

PURPOSE: To evaluate the effect of corticosteroids on intestinal and liver interleukin profile in an experimental model of gastroschisis in fetal rats. METHODS: Sprague-Dawley rats at 19.5 days of gestation had its fetuses operated for the creation of gastroschisis. Two groups of fetuses were studied with and without maternal administration of dexamethasone. Each group was composed of fetuses who underwent gastroschisis (G), control fetuses without manipulation (C) and sham fetuses (S). A dosage of the following interleukins was carried out in fetal intestinal and liver tissues: IL-1, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). The differences between the groups and subgroups were tested by ANOVA with Tukey post-test, with significant values of p<0.05. RESULTS: Dexamethasone led to an increase in intestinal and liver IL-6 (p<0.05) and a decrease in intestinal TNF-α (p<0.001) in fetuses with gastroschisis. CONCLUSION: Corticosteroids had an effect on the intestinal interleukin profile and a small effect on the liver interleukin profile due to immunological immaturity of the fetus, and also of fetuses with gastroschisis. The steroid action may not be exclusively anti-inflammatory, but also pro-inflammatory, varying with time of pregnancy.

OBJETIVO: Avaliar a ação do corticosteroide no perfil de interleucinas intestinais e hepáticas no modelo experimental de gastrosquise em fetos de ratos. MÉTODOS: Ratas Sprague-Dawley com 19,5 dias de gestação tiveram fetos operados para criação de gastrosquise. Dois grupos de fetos foram estudados: com e sem administração materna de dexametasona. Cada grupo foi composto por fetos submetidos a gastrosquise (G), fetos controles sem manipulação (C) e fetos sham (S). Realizou-se a dosagem das seguintes interleucinas no tecido intestinal e hepático fetal: IL-1, IL-6, IL-10, fator de necrose tumoral-alfa (TNF-α) e interferon-gama (IFN-γ). As diferenças entre os grupos e subgrupos foram testadas pelo teste de ANOVA com pós-teste de Tukey, com valores significativos de p<0,05. RESULTADOS: A dexametasona levou a um aumento da IL-6 intestinal e hepática (p<0,05) e a uma diminuição do TNF-α intestinal (p<0,001) em fetos com gastrosquise. CONCLUSÃO: O corticosteróide apresentou efeito sobre o perfil de IL intestinal e pouco na hepática, devido a imaturidade imunológica dos fetos e também dos fetos com gastrosquise a ação do esteróide pode não ser exclusivamente anti-inflamatória, mas também pró inflamatória.

Effect of nitrofen in the final stages of development of the diaphragm muscle in rats / Efeito do nitrofen na fase final do desenvolvimento da musculatura do diafragma em ratos

PURPOSE: To evaluate the expression of myosin in muscle fibers of the diaphragm in experimental congenital diaphragmatic hernia (CDH). METHODS: Fetuses of pregnant rats were divided into four groups: External Control (EC), composed of non-manipulated rats; Nitrofen, composed of pregnant rats that received 100 mg of nitrofen (2,4-dichloro-4'nitrodiphenyl ether) diluted in olive oil on gestational day (GD) 9.5, whose fetuses developed CDH (N+) or not (N-), and Olive Oil Placebo (OO), composed of pregnant rats that received the oil on the same GD. The fetuses were collected on GD 18.5, 19.5, 20.5 and 21.5 (term = 22 days). We obtained body weight (BW) and photographed the diaphragm area (DA), hernia area (HA) and subsequent calculated the HA/DA ratio in N+ group. Samples of Diaphragm muscle were processed for histological staining with H/E and immunohistochemistry (IHQ) for myosin.} RESULTS: The fetuses of N- and N+ groups had decreased BW and DA compared to EC and OO groups (p <0.001). HA was decreased on GD 18.5 compared to 21.5 (p <0.001) and the HA/DA ratio showed no difference. IHQ showed decreased expression of myosin in nitrofen groups. CONCLUSION: CDH induced by nitrofen model contributes to the understanding of muscularization in the formation of the diaphragm where the myosin expression is decreased.

OBJETIVO: Avaliar a expressão da miosina na muscularização do diafragma na hérnia diafragmática congênita (CDH) experimental. MÉTODOS: Fetos de ratas foram divididos em quatro grupos: Controle Externo (EC), composto de ratas não manipuladas; Nitrofen, composto de ratas que receberam 100 mg de nitrofen (2,4-dicloro-4'nitrodifenil éter) diluído no azeite no dia de gestação (GD) 9.5, cujos fetos desenvolveram CDH (N+) ou não (N-) e Placebo óleo de oliva (OO), composto de ratas que ingeriram apenas óleo no mesmo GD. Os fetos foram coletados com 18,5, 19,5, 20,5 e 21,5 GD (termo = 22 dias). Foi obtido o peso corporal (BW) e tiradas fotografias da área do diafragma (DA), da hérnia (HA) e calculada a relação HA/DA no grupo N+. Amostras de diafragmas foram processadas histologicamente para coloração com H/E e imunohistoquímica. RESULTADOS: Os fetos dos grupos N- e N+ tiveram BW e DA diminuídos em relação aos grupos EC e OO (p<0.001). Só houve diferença na HA entre os GD 18.5 e 21.5 (p<0.001) e a relação HA/DA não mostrou diferença entre os grupos. A imunohistoquímica mostrou menor expressão de miosina nos grupos que receberam nitrofen. CONCLUSÃO: O modelo de CDH induzida por nitrofen contribui para entender a muscularização na formação do diafragma onde a expressão da miosina está diminuída.

Validation of protocol of experimental necrotizing enterocolitis in rats and the pitfalls during the procedure / Validação do modelo de enterocolite necrotizante experimental em ratos e as armadilhas durante sua execução

PURPOSE: To describe the difficulties of implementing the protocol of experimental necrotizing enterocolitis (NEC) in order to obtain a larger number of newborns affected with the disease and a lower mortality. METHODS: Term Sprague-Dawley newborns rats (22 days) were divided into four groups of 12 fetuses each (n = 48): EC - breastfed newborns; IH - breastfed newborns and subjected to a stress protocol by ischemia and hypothermia; ESB - formula-fed newborns (Esbilac®, PetAg, Hampshire, IL, USA) and NEC - formula-fed newborns and subjected to stress protocol. The parameters set for the study protocol were: milk concentration (0.19 g ml or 0.34 g/ml), diet instilled volume (according to body weight - 200 kcal/day/Kg - or progressive, according to acceptance), weight (gain, loss or maintenance) and duration of the experiment (72 hours or 96 hours). Data of body weight (BW), intestinal weight (IW) and the IW/BW ratio were obtained. Samples of terminal ileum were collected and analyzed by the degree of injury to the intestinal wall. Statistically significance was set to p<0.05. RESULTS: The established protocol with less mortality and increased number of NEC was with Esbilac® at a concentration of 0.19 g/ml of diet instilled volume of 0.1 ml, every 3 hours, for 72 hours. All infants fed with artificial milk lost weight. In the degree score of intestinal injury, the ESB, IH and NEC groups were considered positive for NEC with greater histological injury in the latter. CONCLUSION: The described NEC protocol in rats allowed a greater survival of puppies with a greater number of animals affected by the disease.

OBJETIVO: Relatar as dificuldades da execução do protocolo de enterocolite necrosante (ECN) experimental a fim de obter um maior número de neonatos comprometidos com a doença e menor mortalidade. MÉTODOS: Neonatos de ratas Sprague-Dawley nascidos a termo (22 dias) foram divididos em 4 grupos de 12 fetos cada (n=48): EC - neonatos amamentados pela mãe; IH - neonatos amamentados pela mãe e submetidos a estresse por isquemia e hipotermia, ESB - neonatos alimentados por leite artificial (Esbilac®, PetAg, Hampshire, IL, USA) e NEC - neonatos alimentados com fórmula e submetidos a protocolo de estresse. Os parâmetros estabelecidos para o protocolo de estudo foram: concentração do leite (0,19 g/ml ou 0,34 g/ml), volume de dieta instilada (de acordo com ganho de peso - 200 kcal/dia/kg - ou progressivo, de acordo com aceitação), peso (ganho, perda ou manutenção) e duração do experimento (72 h ou 96 h). Dados de peso corporal (BW), peso intestinal (IW) e a relação IW/BW foram obtidos. Amostras de íleo terminal foram coletadas e analisadas pelo grau de lesão da parede intestinal. Os dados foram analisados estatisticamente com p <0,05. RESULTADOS: O protocolo estabelecido com menor mortalidade e maior número de ECN foi com Esbilac® na concentração de 0,19 g/ml, volume de dieta instilada de 0,1ml, a cada 3 horas, durante 72 horas. Todos os neonatos alimentados com leite artificial perderam peso. Na escala do grau de lesão, os grupos ESB, IH e NEC foram considerados positivos para NEC com maior lesão histológica no último. CONCLUSÃO: O protocolo de NEC experimental em ratos estabelecido possibilitou uma maior sobrevivência dos neonatos com o maior numero de animais acometidos pela doença.

Modelo experimental para restrição do crescimento fetal em ratos: efeito sobre o glicogênio hepático e morfometria intestinal e renal / Experimental rat model for fetal growth restriction: effects on liver glycogen and intestinal and renal morphometry

OBJETIVO: avaliar a eficácia do modelo de RCIU por ligadura da artéria uterina simulando insuficiência placentária em ratos. MÉTODOS: fetos de ratas prenhes Sprague-Dawley foram divididos em três grupos: RCIU (restrição de crescimento intrauterino), com fetos submetidos à ligadura da artéria uterina com 18,5 dias de gestação (termo = 22 dias), C-RCIU (controle da restrição), com fetos do corno contralateral à ligadura, CE (Controle Externo), com fetos de ratas sem manipulação. Com 21,5 dias de gestação, foi realizada cesárea, os fetos foram pesados e dissecados para análise morfométrica e histológica do fígado, intestino e rins. RESULTADOS: os dados morfométricos avaliados mostraram o peso corpóreo (PC), hepático (PH) e intestinal (PI) dos fetos com RCIU menor que C-RCIU e CE (p<0,001). O peso placentário (PP), renal (PR) e as relações PH/PC, PI/PC e PR/PC não se alteraram. A espessura renal foi menor nos fetos com RCIU (p<0,001) e houve diminuição da camada mucosa e submucosa intestinal (p<0,05). A avaliação histológica mostrou diminuição do glicogênio hepático nos fetos com RCIU em relação aos grupos C-RCIU e CE. CONCLUSÕES: o modelo descrito foi eficiente e causou RCIU fetal simétrica com diminuição da maioria dos órgãos, especialmente do peso hepático, e alteração nos depósitos de glicogênio.

PURPOSE: to evaluate the effectiveness of the IUGR model by uterine artery ligation mimicking placental insufficiency in rats. METHODS: sprague-Dawley rat fetuses were divided into three groups: IUGR (intrauterine growth restriction), with fetuses in the right horn of pregnant rats subjected to right uterine artery ligation at 18.5 days of gestation (term = 22 days); C-IUGR (control of restriction), with control fetuses in the left horn, and EC (external control), with fetuses of intact rats. Animals were harvested by cesarean section at day 21.5 days of gestation. Fetuses were weighed and then sacrificed. The intestine, liver, kidney and placenta were weighed and dissected for morphometric and histological analysis. RESULTS: the morphometric data showed decreased body weight (BW), liver weight (LW) and intestinal weight (IW) of fetuses with IUGR compared to C-IUGR and EC (p<0.001). The placental weight (PW), renal weight (RW) and LW/BW, IW/BW, and RW/BW ratios did not change. IUGR fetuses had decreased kidney thickness (p<0.001) and decreased thickness of the intestinal mucosa and submucosa (p<0.05). Histological evaluation showed reduction of liver glycogen storage in fetuses with IUGR compared to C-IUGR and CE. CONCLUSIONS: the model described was efficient and caused symmetric fetal IUGR with decreased size of most organs, especially the liver, and changes in glycogen stores.