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. Methanol fixation appears to be the method of choice for droplet-based scRNA-seq on neural cell populations, based on a broad analysis of how various fixation or preservation methods impact the single-cell transcriptomes of hiPSC-derived neural cells

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 (po0.05); intestine: IO=0.42; RL=0.48; RL+PTX=0.29; HS +PTX=0.26 (po0.05). The number of labeled cells for TUNEL and Bax was lower in the HS+PTX group than in the other groups (po0.05). The Bax/Bcl-2 ratio was lower in the HS+PTX group than in the other groups (po0.05). 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 mortalit

N-Acetylcysteine Reduced Ischemia and Reperfusion Damage Associated with Steatohepatitis in Mice

N-acetylcysteine (NAC) is a pharmacological alternative with great potential for reducing the deleterious effects of surgical procedures on patients with steatohepatitis. We evaluated the effect of NAC on hepatic ischemia/reperfusion (I/R) injury in C57BL/6J mice, 8 weeks-old, weighing 25–30 g, with steatohepatitis induced by a methionine- and choline-deficient (MCD) diet. Groups: MCD group (steatohepatitis), MCD-I/R group (steatohepatitis plus 30 min of 70% liver ischemia and 24 h of reperfusion), MCD-I/R+NAC group (same as MCD-I/R group plus 150 mg/kg NAC 15 min before ischemia), and control group (normal AIN-93M diet). Liver enzymes and histopathology; nitrite and TBARS (thiobarbituric acid reactive substances) levels; pro-inflammatory cytokines; antioxidants enzymes; Nrf2 (nuclear factor erythroid-2-related factor 2) expression; and apoptosis were evaluated. In the group treated with NAC, reductions in inflammatory infiltration; AST (aspartate aminotransferase), nitrite, and TBARS levels; GPx (gutathione peroxidase) activity; cytokines synthesis; and number of apoptotic cells were observed while the GR (glutathione reductase) activity was increased. No differences were observed in Nfr2 expression or in SOD (superoxide dismutase), CAT (catalase), and GST (glutathione S-transferase) activities. Thus, it may be concluded that NAC exerts beneficial effects on mice livers with steatohepatitis submitted to I/R by reducing oxidative stress, inflammatory response, and cell death

N-Acetylcysteine reduced ischemia and reperfusion damage associated with steatohepatitis in mice

This research was funded by CAPES—PROAP, 2017, Post graduate Program in Surgical Clinics; PQ10/2012—National Council for Scientific and Technological Development (CNPq) grant number: 308995/2012-0.Universidade de São Paulo. Faculdade de Medicina. Department of Surgery. Laboratory of Surgial Physiopathology. São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Department of Emergency Medicine. São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Department of Surgery. Laboratory of Surgial Physiopathology. São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Department of Surgery. Laboratory of Surgial Physiopathology. São Paulo, SP, Brazil / Universidade de São Paulo. Faculdade de Medicina. Hospital de Clinicas. Instituto Central. Department of Surgery. General and Trauma Surgery Division. São Paulo, SP, Brazil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Farmacologia. São Paulo, SP, Brazil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Farmacologia. São Paulo, SP, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microscopia Eletrônica. Ananindeua, PA, Brasil.Universidade de São Paulo. Faculdade de Medicina. Department of Surgery. Laboratory of Surgial Physiopathology. São Paulo, SP, Brazil / Universidade de São Paulo. Faculdade de Medicina. Hospital de Clinicas. Instituto Central. Department of Surgery. General and Trauma Surgery Division. São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Department of Surgery. Laboratory of Surgial Physiopathology. São Paulo, SP, Brazil / Universidade de São Paulo. Faculdade de Medicina. Hospital de Clinicas. Instituto Central. Department of Surgery. General and Trauma Surgery Division. São Paulo, SP, Brazil.N-acetylcysteine (NAC) is a pharmacological alternative with great potential for reducing the deleterious effects of surgical procedures on patients with steatohepatitis. We evaluated the effect of NAC on hepatic ischemia/reperfusion (I/R) injury in C57BL/6J mice, 8 weeks-old, weighing 25–30 g, with steatohepatitis induced by a methionine- and choline-deficient (MCD) diet. Groups: MCD group (steatohepatitis), MCD-I/R group (steatohepatitis plus 30 min of 70% liver ischemia and 24 h of reperfusion), MCD-I/R+NAC group (same as MCD-I/R group plus 150 mg/kg NAC 15 min before ischemia), and control group (normal AIN-93M diet). Liver enzymes and histopathology; nitrite and TBARS (thiobarbituric acid reactive substances) levels; pro-inflammatory cytokines; antioxidants enzymes; Nrf2 (nuclear factor erythroid-2-related factor 2) expression; and apoptosis were evaluated. In the group treated with NAC, reductions in inflammatory infiltration; AST (aspartate aminotransferase), nitrite, and TBARS levels; GPx (gutathione peroxidase) activity; cytokines synthesis; and number of apoptotic cells were observed while the GR (glutathione reductase) activity was increased. No differences were observed in Nfr2 expression or in SOD (superoxide dismutase), CAT (catalase), and GST (glutathione S-transferase) activities. Thus, it may be concluded that NAC exerts beneficial effects on mice livers with steatohepatitis submitted to I/R by reducing oxidative stress, inflammatory response, and cell death