Sphingolipidomics Investigation of the Temporal Dynamics after Ischemic Brain Injury.

Sphingolipids (SPLs) have been proposed as potential therapeutic targets for strokes, but no reports have ever profiled the changes of the entire range of SPLs after a stroke. This study applied sphingolipidomic methods to investigate the temporal and individual changes in the sphingolipidome including the effect of atorvastatin after ischemic brain injury. We conducted sphingolipidomic profiling of mouse brain tissue by liquid chromatography-electrospray ionization tandem mass spectrometry at 3 h and 24 h after 1 h of middle cerebral artery occlusion (MCAO), and SPL levels were compared with those of the Sham control group. At 3 h post-MCAO, ceramides (Cers) exhibited an increase in levels of long-chain Cers but a decrease in very-long-chain Cers. Moreover, sphingosine, the precursor of sphingosine-1-phosphate (S1P), decreased and S1P increased at 3 h after MCAO. In contrast to 3 h, both long-chain and very-long-chain Cers showed an increased trend at 24 h post-MCAO. Most important, the administration of atorvastatin improved the neurological function of the mice and significantly reversed the SPL changes resulting from the ischemic injury. Furthermore, we used plasma samples from nonstroke control and stroke patients at time points of 72 h after a stroke, and found a similar trend of Cers as in the MCAO model. This study successfully elucidated the overall effect of ischemic injury on SPL metabolism with and without atorvastatin treatment. The network of SPL components that change upon ischemic damage may provide novel therapeutic targets for ischemic stroke.

Low-adhesive ethylene vinyl alcohol-based packaging to xenogeneic islet encapsulation for type 1 diabetes treatment.

Transplantation of encapsulated porcine islets is proposed to treat type 1 diabetes. However, the envelopment of fibrous tissue and the infiltration of immune cells impair islet function and eventually cause implant failure. It is known that hemodialysis using an ethylene vinyl alcohol (EVOH) membrane results in minor tissue responses. Therefore, we hypothesized that using a low-adhesive EVOH membrane for encapsulation may prevent host cell accumulation and fibrous capsule formation. In this study, rat islets suspended in chitosan gel were encapsulated in bags made from highly porous EVOH membranes, and their in vitro insulin secretion function as well as in vivo performance was evaluated. The results showed that the EVOH bag did not affect islet survival or glucose-stimulated insulin secretion. Whereas naked islets were dysfunctional after 7 days of culture in vitro, islets within the EVOH bag produced insulin continuously for 30 days. Streptozotocin-induced diabetic mice were given islets-chitosan gel-EVOH implants intraperitoneally (650-800 islets equivalent) and exhibited lower blood glucose levels and regained body weight during a 4-week observation period. The transplanted mice had higher levels of serum insulin and C-peptide, with an improved blood glucose disappearance rate. Retrieved implants had minor tissue adhesion, and histology showed a limited number of mononuclear cells and fibroblasts surrounding the implants. No invasion of host cells into the EVOH bags was noticed, and the encapsulated islets were intact and positive for insulin-glucagon immunostaining. In conclusion, an EVOH bag can protect encapsulated islets, limit fibrous capsule formation, and extend graft function.

Protection against acetaminophen-induced acute liver failure by omentum adipose tissue derived stem cells through the mediation of Nrf2 and cytochrome P450 expression.

BACKGROUND: Acetaminophen (APAP) overdose causes acute liver failure (ALF) in animals and humans via the rapid depletion of intracellular glutathione (GSH) and the generation of excess reactive oxygen species (ROS) that damage hepatocytes. Stem cell therapy is a potential treatment strategy for ALF. METHODS: We isolated mesenchymal stem cells (MSCs) from mice omentum adipose tissue-derived stem cells (ASCs) and transplanted them into a mouse model of APAP-induced ALF to explore their therapeutic potential. In addition, we performed in vitro co-culture studies with omentum-derived ASCs and primary isolated hepatocytes to demonstrate the hepatoprotective effect of omentum-derived ASCs on hepatocytes that were subjected to APAP-induced damage. RESULT: ASC transplantation significantly improved the survival rate of mice with ALF and attenuated the severity of APAP-induced liver damage by suppressing cytochrome P450 activity to reduce the accumulation of toxic nitrotyrosine and the upregulation of NF-E2-related factor 2 (Nrf2) expression, resulting in an increase in the subsequent antioxidant activity. These effects protected the hepatocytes from APAP-induced damage through the suppression of downstream MAPK signal activation and inflammatory cytokine production. CONCLUSIONS: our results demonstrate that omentum-derived ASCs are an alternative source of ASCs that regulate the antioxidant response and may represent a beneficial therapeutic strategy for ALF.

Prevascularization-free Primary Subcutaneous Transplantation of Xenogeneic Islets Coencapsulated With Hepatocyte Growth Factor.

Subcutaneous pouch is a potential site for islet transplantation. However, insufficient oxygen supply remains challenging. Pretreatment of neovascularization using basic fibroblast growth factor can solve this, but it needs 2× operations. We developed a device that contains rat islets in chitosan gel packed in a bag made of highly biocompatible ethylene vinyl alcohol copolymer porous membrane. This study investigated whether coencapsulation of hepatocyte growth factor (HGF) with islets in the device enables novel method of prevascularization-free primary subcutaneous transplantation. METHODS: In vitro experiments examined slow release of HGF from the chitosan gel and islet-protection effect of HGF against hypoxia. In the latter, rat islets with/without HGF (200 ng/mL) was cultured in 1% oxygen. In in vivo experiment, fabricated device with/without HGF (10 µg/device) containing rat islets was primarily transplanted to streptozotocin-induced diabetic mice subcutaneously. RESULTS: In vitro experiments showed sustained release of HGF for 28 d and alleviating effect of HGF on cell death and glucose-responsive insulin release after hypoxic culture. Islet + HGF mice, but not islet-alone mice, showed decreased nonfasting blood glucose and regained body weight after transplantation. In intraperitoneal glucose tolerance test, islet + HGF mice exhibited decreased fasting blood glucose (200 ± 55 mg/dL) and good blood glucose disappearance rate (K value) (0.817 ± 0.101) comparing to normal mice (123 ± 28 mg/dL and 1.074 ± 0.374, respectively). However, in islet-alone mice, fasting blood glucose was high (365 ± 172 mg/dL) and K value was indeterminable. Serum insulin in islet + HGF mice (1.58 ± 0.94 µg/L) was close to normal mice (1.66 ± 0.55 µg/L), whereas those in islet-alone mice (0.279 ± 0.076 µg/L) and diabetic mice (0.165 ± 0.079 µg/L) were low. Immunohistochemical examination showed intact insulin- and glucagon-positive islets in retrieved devices with HGF, but no intact islet was found in the device without HGF. CONCLUSIONS: HGF could enhance islet survival in hypoxia and enhance in vivo function of encapsulated islets after primary subcutaneous transplantation.

Effect of Basic Fibroblast Growth Factor on Xenogeneic Islets in Subcutaneous Transplantation-A Murine Model.

BACKGROUND: Subcutaneous pockets provide an extrahepatic transplant site for islet grafting to treat type 1 diabetes. However, a hypoxic environment may cause central necrosis to islets and lead to graft failure. Our previous studies focused on a pre-treated subcutaneous site with basic fibroblast growth factor (bFGF) for the formation of vascular bed. In addition to neovascularization, bFGF was also shown to protect islets against oxidative stress and chemical-induced damage in vitro. Accordingly, we propose that subcutaneous islet transplantation with a bFGF-slow releasing device simultaneously can improve islet survival in vivo. METHODS: A bFGF-impregnated collagen sheet was implanted in the right back of a streptozotocin-induced diabetic mouse for neovascularization. After 10 days, the sheet was removed and the rat islet-embedding gel within the immune-isolation device was transplanted (2-time operation [OP]). In another group, the diabetic mice received bFGF-impregnated gel with rat islets within the immune-isolation device simultaneously (1-time OP). RESULTS: Diabetic mice in 2-time OP group experienced a decrease in their non-fasting blood glucose level for a period of 10 days, and the glucose levels were lower than those of untreated diabetic mice post-implantation. However, the mice in the 1-time OP group remained hyperglycemic post-operation and showed no improvements in body weight or the area under curve in intraperitoneal glucose tolerance test. Furthermore, mice in the 2-time OP had relatively higher serum insulin levels with improved renal and metabolic biomarkers. CONCLUSION: Our findings suggest that bFGF had no beneficial effect on a 1-time operation in subcutaneous islet transplantation.

Using precursor ion scan of 184 with liquid chromatography-electrospray ionization-tandem mass spectrometry for concentration normalization in cellular lipidomic studies.

Cellular lipidomic studies have been favored approaches in many biomedical research areas. To provide fair comparisons of the studied cells, it is essential to perform normalization of the determined concentration before lipidomic analysis. This study proposed a cellular lipidomic normalization method by measuring the phosphatidylcholine (PC) and sphingomyelin (SM) contents in cell extracts. To provide efficient analysis of PC and SM in cell extracts, flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) with a precursor ion scan (PIS) of m/z 184 was used, and the parameters affecting the performance of the method were optimized. Good linearity could be observed between the cell extract dilution factor and the reciprocal of the total ion chromatogram (TIC) area in the PIS of m/z 184 within the dilution range of 1- to 16-fold (R2 = 0.998). The calibration curve could be used for concentration adjustment of the unknown concentration of a cell extract. The intraday and intermediate precisions were below 10%. The accuracy ranged from 93.0% to 105.6%. The performance of the new normalization method was evaluated using different numbers of HCT-116 cells. Sphingosine, ceramide (d18:1/18:0), SM (d18:1/18:0) and PC (16:1/18:0) were selected as the representative test lipid species, and the results showed that the peak areas of each lipid species obtained from different cell numbers were within a 20% variation after normalization. Finally, the PIS of 184 normalization method was applied to study ischemia-induced neuron injury using oxygen and glucose deprivation (OGD) on primary neuronal cultured cells. Our results showed that the PIS of 184 normalization method is an efficient and effective approach for concentration normalization in cellular lipidomic studies.