Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells.

The main critical step in single-cell transcriptomics is sample preparation. Several methods have been developed to preserve cells after dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the cell types to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single-cell RNA-seq on neural and glial cells derived from induced pluripotent stem cells. Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell, it strongly affects the cellular composition and induces the expression of stress and apoptosis genes. In contrast, methanol fixed samples display a cellular composition similar to fresh samples and provide a good cell quality and little expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single-cell transcriptomics experiments on neural cell populations.

Hypertonic saline and pentoxifylline enhance survival, reducing apoptosis and oxidative stress in a rat model of strangulated closed loop small bowel obstruction.

OBJECTIVES: Intestinal obstruction has a high mortality rate when therapeutic treatment is delayed. Resuscitation in intestinal obstruction requires a large volume of fluid, and fluid combinations have been studied. Therefore, we evaluated the effects of hypertonic saline solution (HS) with pentoxifylline (PTX) on apoptosis, oxidative stress and survival rate. METHODS: Wistar rats were subjected to intestinal obstruction and ischemia through a closed loop ligation of the terminal ileum and its vessels. After 24 hours, the necrotic bowel segment was resected, and the animals were randomized into four groups according to the following resuscitation strategies: Ringer's lactate solution (RL) (RL-32 ml/kg); RL+PTX (25 mg/kg); HS+PTX (HS, 7.5%, 4 ml/kg), and no resuscitation (IO-intestinal obstruction and ischemia). Euthanasia was performed 3 hours after resuscitation to obtain kidney and intestine samples. A malondialdehyde (MDA) assay was performed to evaluate oxidative stress, and histochemical analyses (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL], Bcl-2 and Bax) were conducted to evaluate kidney apoptosis. Survival was analyzed with another series of animals that were observed for 15 days. RESULTS: PTX in combination with RL or HS reduced the MDA levels (nmol/mg of protein), as follows: kidney IO=0.42; RL=0.49; RL+PTX=0.31; HS+PTX=0.34 (p<0.05); intestine: IO=0.42; RL=0.48; RL+PTX=0.29; HS+PTX=0.26 (p<0.05). The number of labeled cells for TUNEL and Bax was lower in the HS+PTX group than in the other groups (p<0.05). The Bax/Bcl-2 ratio was lower in the HS+PTX group than in the other groups (p<0.05). The survival rate on the 15th day was higher in the HS+PTX group (77%) than in the RL+PTX group (11%). CONCLUSION: PTX in combination with HS enhanced survival and attenuated oxidative stress and apoptosis. However, when combined with RL, PTX did not reduce apoptosis or mortality.

Oxidative stress on ischemia/reperfusion injury in mice with non-alcoholic hepatic steatosis or steatohepatitis.

PURPOSE: To evaluate the oxidative stress, resulting from ischemia and hepatic reperfusion, in mice with non-alcoholic hepatic steatosis and steatohepatitis. METHODS: C57BL/6 male mice were used. Part of them were ob/ob mice, and the other part was fed with standard or MCD diets - this last used to develop steatohepatitis. The animals - MCD-I/R, ob/ob-I/R and I/R groups - were submitted to 30 minutes of partial hepatic ischemia, followed by reperfusion for 24 hours. The blood was collected, for biochemical analysis of AST, and the liver removed for assessment of TBARS and nitrite, and of histology. RESULTS: After the I/R, the animal fed with MCD diet presented higher AST levels (MCD-I/R: 967±349U/L / ob/ob-I/R: 606±18 U/L / I/R: 311±172 U/L), TBARS (MCD-I/R: 7±1 nM/mg protein / ob/ob-I/R: 3±1 nM/mg protein / I/R: 3±1 nM/mg protein) and nitrite (MCD-I/R: 614±87 µg/mL / ob/ob-I/R: 512±81 µg/mL / I/R: 459±29 µg/mL) than the ob/ob mice, when both groups were compared to animals fed with standard diet. Regarding histology, the steatosis level (azonal macrovesicular steatosis of level 3 - >66%) and hepatic fibrosis (periportal and perisinusoidal of level 2) was also more intense, but both animal models presented lobular inflammation of level 3 (>66%). CONCLUSIONS: The murine model fed with MCD diet is suitable for the assessment of oxidative stress in hepatic I/R injury associated with the nonalcoholic fatty liver disease. Although both murine models showed inflammatory infiltrate and macro and micro vesicular steatosis.

Hypertonic saline and pentoxifylline enhance survival, reducing apoptosis and oxidative stress in a rat model of strangulated closed loop small bowel obstruction

OBJECTIVES: Intestinal obstruction has a high mortality rate when therapeutic treatment is delayed. Resuscitation in intestinal obstruction requires a large volume of fluid, and fluid combinations have been studied. Therefore, we evaluated the effects of hypertonic saline solution (HS) with pentoxifylline (PTX) on apoptosis, oxidative stress and survival rate. METHODS: Wistar rats were subjected to intestinal obstruction and ischemia through a closed loop ligation of the terminal ileum and its vessels. After 24 hours, the necrotic bowel segment was resected, and the animals were randomized into four groups according to the following resuscitation strategies: Ringer's lactate solution (RL) (RL-32 ml/kg); RL+PTX (25 mg/kg); HS+PTX (HS, 7.5%, 4 ml/kg), and no resuscitation (IO-intestinal obstruction and ischemia). Euthanasia was performed 3 hours after resuscitation to obtain kidney and intestine samples. A malondialdehyde (MDA) assay was performed to evaluate oxidative stress, and histochemical analyses (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL], Bcl-2 and Bax) were conducted to evaluate kidney apoptosis. Survival was analyzed with another series of animals that were observed for 15 days. RESULTS: PTX in combination with RL or HS reduced the MDA levels (nmol/mg of protein), as follows: kidney IO=0.42; RL=0.49; RL+PTX=0.31; HS+PTX=0.34 (p<0.05); intestine: IO=0.42; RL=0.48; RL+PTX=0.29; HS+PTX=0.26 (p<0.05). The number of labeled cells for TUNEL and Bax was lower in the HS+PTX group than in the other groups (p<0.05). The Bax/Bcl-2 ratio was lower in the HS+PTX group than in the other groups (p<0.05). The survival rate on the 15th day was higher in the HS+PTX group (77%) than in the RL+PTX group (11%). CONCLUSION: PTX in combination with HS enhanced survival and attenuated oxidative stress and apoptosis. However, when combined with RL, PTX did not reduce apoptosis or mortality.

Oxidative stress on ischemia/reperfusion injury in mice with non-alcoholic hepatic steatosis or steatohepatitis

Abstract Purpose: To evaluate the oxidative stress, resulting from ischemia and hepatic reperfusion, in mice with non-alcoholic hepatic steatosis and steatohepatitis. Methods: C57BL/6 male mice were used. Part of them were ob/ob mice, and the other part was fed with standard or MCD diets - this last used to develop steatohepatitis. The animals - MCD-I/R, ob/ob-I/R and I/R groups - were submitted to 30 minutes of partial hepatic ischemia, followed by reperfusion for 24 hours. The blood was collected, for biochemical analysis of AST, and the liver removed for assessment of TBARS and nitrite, and of histology. Results: After the I/R, the animal fed with MCD diet presented higher AST levels (MCD-I/R: 967±349U/L / ob/ob-I/R: 606±18 U/L / I/R: 311±172 U/L), TBARS (MCD-I/R: 7±1 nM/mg protein / ob/ob-I/R: 3±1 nM/mg protein / I/R: 3±1 nM/mg protein) and nitrite (MCD-I/R: 614±87 µg/mL / ob/ob-I/R: 512±81 µg/mL / I/R: 459±29 µg/mL) than the ob/ob mice, when both groups were compared to animals fed with standard diet. Regarding histology, the steatosis level (azonal macrovesicular steatosis of level 3 - >66%) and hepatic fibrosis (periportal and perisinusoidal of level 2) was also more intense, but both animal models presented lobular inflammation of level 3 (>66%). Conclusions: The murine model fed with MCD diet is suitable for the assessment of oxidative stress in hepatic I/R injury associated with the nonalcoholic fatty liver disease. Although both murine models showed inflammatory infiltrate and macro and micro vesicular steatosis.