Trigeminal Nerve Stimulation: A Novel Method of Resuscitation for Hemorrhagic Shock.

OBJECTIVES: To determine if trigeminal nerve stimulation can ameliorate the consequences of acute blood loss and improve survival after severe hemorrhagic shock. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Severe hemorrhagic shock was induced in rats by withdrawing blood until the mean arterial blood pressure reached 27 ± 1 mm Hg for the first 5 minutes and then maintained at 27 ± 2 mm Hg for 30 minutes. The rats were randomly assigned to either control, vehicle, or trigeminal nerve stimulation treatment groups. The effects of trigeminal nerve stimulation on survival rate, autonomic nervous system activity, hemodynamics, brain perfusion, catecholamine release, and systemic inflammation after severe hemorrhagic shock in the absence of fluid resuscitation were analyzed. MEASUREMENTS AND MAIN RESULTS: Trigeminal nerve stimulation significantly increased the short-term survival of rats following severe hemorrhagic shock in the absence of fluid resuscitation. The survival rate at 60 minutes was 90% in trigeminal nerve stimulation treatment group whereas 0% in control group (p < 0.001). Trigeminal nerve stimulation elicited strong synergistic coactivation of the sympathetic and parasympathetic nervous system as measured by heart rate variability. Without volume expansion with fluid resuscitation, trigeminal nerve stimulation significantly attenuated sympathetic hyperactivity paralleled by increase in parasympathetic tone, delayed hemodynamic decompensation, and improved brain perfusion following severe hemorrhagic shock. Furthermore, trigeminal nerve stimulation generated sympathetically mediated low-frequency oscillatory patterns of systemic blood pressure associated with an increased tolerance to central hypovolemia and increased levels of circulating norepinephrine levels. Trigeminal nerve stimulation also decreased systemic inflammation compared with the vehicle. CONCLUSIONS: Trigeminal nerve stimulation was explored as a novel resuscitation strategy in an animal model of hemorrhagic shock. The results of this study showed that the stimulation of trigeminal nerve modulates both sympathetic and parasympathetic nervous system activity to activate an endogenous pressor response, improve cerebral perfusion, and decrease inflammation, thereby improving survival.

Cold-inducible RNA-binding protein-derived peptide C23 attenuates inflammation and tissue injury in a murine model of intestinal ischemia-reperfusion.

BACKGROUND: Cold-inducible RNA-binding protein is a novel damage-associated molecular pattern that causes inflammation. C23, a short peptide derived from cold-inducible RNA-binding protein, has been found to have efficacy in blocking cold-inducible RNA-binding protein's activity. We hypothesized that C23 reduces inflammation and tissue injury induced by intestinal ischemia-reperfusion. METHODS: Male C57BL/6 mice were subjected to 60 minutes of intestinal ischemia by clamping the superior mesenteric artery. Immediately after reperfusion, either normal saline (vehicle) or C23 peptide (8 mg/kg body weight) was injected intraperitoneally. Four hours after reperfusion, blood, intestinal, and lung tissues were collected for analysis of inflammatory and tissue injury parameters. RESULTS: Cold-inducible RNA-binding protein levels in the intestinal tissues were significantly increased following intestinal ischemia-reperfusion. Histologic examination of the intestine revealed a significant reduction in injury score in the C23 group by 48% as compared with the vehicles after intestinal ischemia-reperfusion. The serum levels of lactate dehydrogenase and aspartate aminotransferase were increased in animals that underwent vehicle-treated intestinal ischemia-reperfusion, whereas C23-treated animals exhibited significant reductions by 48% and 53%, respectively. The serum and intestinal tissue levels of tumor necrosis factor α were elevated in vehicle-treated intestinal ischemia-reperfusion mice but decreased by 72% and 69%, respectively, in C23-treated mice. Interleukin-6 mRNA levels in the lungs were reduced by 86% in the C23-treated group in comparison to the vehicle-treated group after intestinal ischemia-reperfusion. Expression of macrophage inflammatory protein 2 and level of myeloperoxidase activity in the lungs were dramatically increased after intestinal ischemia-reperfusion and significantly reduced by 91% and 25%, respectively, in the C23-treated group. CONCLUSION: C23 has potential to be developed into a possible therapy for reperfusion injury after mesenteric ischemia and reperfusion.

SRT1720, a sirtuin 1 activator, attenuates organ injury and inflammation in sepsis.

BACKGROUND: Sepsis affects 800,000 patients in the United States annually with a mortality rate of up to 30%. Recent studies suggest that sepsis-associated metabolic derangements due to hypoxic tissue injury, impaired oxygen utilization, and mitochondrial dysfunction contribute to mortality. Sirtuin 1 (Sirt1) is a crucial modulator of energy metabolism during starvation states and has anti-inflammatory effects. Here, we hypothesized that SRT1720, a Sirt1 activator, could attenuate the severity of sepsis. MATERIALS AND METHODS: Male C57BL/6 mice (20-25 g) were subjected to cecal ligation and puncture (CLP) to induce sepsis. SRT1720 (5 or 20 mg/kg BW) or 10% dimethyl sulfoxide (vehicle) in 0.2-mL saline was injected intravenously at 5 h after CLP. Control animals were not subjected to any surgery. Blood and liver samples were harvested at 20 h after CLP for analysis. RESULTS: Administration of SRT1720 markedly reduced the serum levels of tissue injury markers (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase) and renal injury markers (blood urea nitrogen and creatinine) in a dose-dependent manner after CLP. Furthermore, the levels of proinflammatory cytokines interleukin (IL)-1ß and IL-6 in the serum and liver were significantly inhibited by SRT1720 treatment after CLP. SRT1720 treatment resulted in a significantly decreased mRNA expression of inflammasome components (nucleotide oligomerization domain-like receptor protein 3, adapter apoptosis-associated speck-like protein containing caspase-recruitment domain, IL-1ß, and IL-18) in the liver, compared with the vehicle group. CONCLUSIONS: SRT1720 treatment attenuates multiorgan injury in septic mice. SRT1720 treatment also decreases the production of proinflammatory cytokines and reduces inflammasome activation. Thus, pharmacologic stimulation of Sirt1 may present a promising therapeutic strategy for sepsis.

Milk fat globule-epidermal growth factor-factor VIII-derived peptide MSP68 is a cytoskeletal immunomodulator of neutrophils that inhibits Rac1.

BACKGROUND: Prolonged neutrophil infiltration leads to exaggerated inflammation and tissue damage during sepsis. Neutrophil migration requires rearrangement of their cytoskeleton. Milk fat globule-epidermal growth factor-factor VIII-derived short peptide 68 (MSP68) has recently been shown to be beneficial in sepsis-induced tissue injury and mortality. We hypothesize that MSP68 inhibits neutrophil migration by modulating small GTPase Rac1-dependent cytoskeletal rearrangements. METHODS: Bone marrow-derived neutrophils (BMDNs) or whole lung digest isolated neutrophils were isolated from 8 to 10 wk old C57BL/6 mice by Percoll density gradient centrifugation. The purity of BMDN was verified by flow cytometry with CD11b/Gr-1 staining. Neutrophils were stimulated with N-formylmethionine-leucine-phenylalanine (f-MLP) (10 nM) in the presence or absence of MSP68 at 10 nM or cecal ligation and puncture (CLP) was used to induce sepsis, and MSP68 was administered at 1 mg/kg intravenously. Cytoskeletal organization was assessed by phalloidin staining, followed by analysis using fluorescence microscopy. Activity of the Rac1 GTPase in f-MLP or CLP-activated BMDN in the presence or absence of MSP68 was assessed by GTPase enzyme-linked immunosorbent assay. Mitogen-activated protein (MAP) kinase activity was determined by western blot densitometry. RESULTS: BMDN treatment with f-MLP increased cytoskeletal remodeling as revealed by the localization of filamentous actin to the periphery of the neutrophil. By contrast, cells pretreated with MSP68 had considerably reduced filamentous actin polymerization. Cytoskeletal spreading is associated with the activation of the small GTPase Rac1. We found BMDN-treated with f-MLP or that were exposed to sepsis by CLP had increased Rac1 signaling, whereas the cells pretreated with MSP68 had significantly reduced Rac1 activation (P < 0.05). MAP kinases related to cell migration including pp38 and pERK were upregulated by treatment with f-MLP. Upregulation of these MAP kinases was also significantly reduced after pretreatment with MSP68 (P < 0.05). CONCLUSIONS: MSP68 downregulates actin cytoskeleton-dependent, Rac1-MAP kinase-mediated neutrophil motility. Thus, MSP68 is a novel therapeutic candidate for regulating inflammation and tissue damage caused by excessive neutrophil migration in sepsis.

Growth arrest-specific protein 6 protects against renal ischemia-reperfusion injury.

BACKGROUND: Renal injury caused by ischemia-reperfusion (I/R) often occurs after shock or transplantation. Growth arrest-specific protein 6 (Gas6) is a secreted protein that binds to the TAM-Tyro3, Axl, Mer-family tyrosine kinase receptors, which modulate the inflammatory response and activate cell survival pathways. We hypothesized that Gas6 could have a protective role in attenuating the severity of renal injury after I/R. MATERIALS AND METHODS: Adult mice were subjected to 45 min of bilateral renal ischemia. Recombinant mouse Gas6 (rmGas6, 5 µg per mouse) or normal saline (vehicle) was administered intraperitoneally 1 h before ischemia and all subjects were sacrificed at 23 h after I/R for blood and tissue analysis. The expression of protein and messenger RNA (mRNA) was assessed by Western blotting and quantitative polymerase chain reaction, respectively. RESULTS: Treatment with rmGas6 significantly decreased serum levels of creatinine and blood urea nitrogen by 29% and 27%, respectively, improved the renal histologic injury index, and reduced the apoptosis in the kidneys, compared with the vehicle. Renal mRNA levels of interleukin 1ß, interleukin 6, tumor necrosis factor α, keratinocyte-derived chemokine and macrophage inflammatory protein 2 were decreased significantly by 99%, 60%, 53%, 58%, and 43%, with rmGas6 treatment, respectively. After I/R, renal I-kappa-B α levels were reduced by 40%, whereas they returned to sham levels with rmGas6 treatment. The mRNA levels of inducible nitric oxide synthase and cyclooxygenase 2 were reduced by 79% and 70%, respectively, whereas the expression of cyclin D1 was increased by 2.1-fold in the rmGas6-treated group, compared with the vehicle. CONCLUSIONS: Gas6 suppresses the nuclear factor κB pathway and promotes cell proliferation, leading to the reduction of inflammation and protection of renal injury induced by I/R.

Novel resveratrol analogues attenuate renal ischemic injury in rats.

BACKGROUND: Renal ischemia-reperfusion (I/R) is a severe clinical complication with no specific treatment. Resveratrol has been shown as a promising experimental agent in renal I/R due to its effect on cellular energy metabolism, oxidative stress, and inflammation. Recently, we identified two biologically active resveratrol analogues (RSVAs), RSVA405 and RSVA314. We hypothesized that both RSAVs would attenuate I/R-induced renal injury. METHODS: Adult male rats were subjected to renal I/R through bilateral renal pedicle clamping for 60 min, followed by reperfusion. RSVA405 (3 mg/kg Body Weight), RSVA314 (3 mg/kg Body Weight), or vehicle (10% dimethyl sulfoxide and 33% Solutol in phosphate buffered saline) were administered by intraperitoneal injection 1 h before ischemia. Blood and renal tissues were collected 24 h after I/R for evaluation. RESULTS: Administration of RSVA405 and RSVA314 significantly reduced the serum levels of renal dysfunction and injury markers, including creatinine, blood urea nitrogen, aspartate aminotransferase, and lactate dehydrogenase, compared with vehicle. The protective effect of RSVA405 and RSVA314 was also reflected on histologic evaluation. Both RSVAs reduced the number of apoptotic cells by more than 60% as determined by transferase dUTP nick end labeling assay, compared with vehicle. The renal adenosine triphosphate levels of the vehicle group was decreased to 52.4% of control, whereas those of the RSVA405 and RSVA314 groups were restored to 72.3% and 79.6% of control, respectively. Both RSVAs significantly reduced the protein expression of inducible nitric oxide synthase and nitrotyrosine and the messenger RNA levels of tumor necrosis factor-α, interleukin-6, and interleukin-1ß. CONCLUSIONS: RSVA405 and RSVA314 attenuate I/R-induced renal injury through the modulation of energy metabolism, oxidative stress, and inflammation.

Growth arrest-specific protein 6 attenuates neutrophil migration and acute lung injury in sepsis.

Sepsis is an acute inflammatory condition that can result in multiple organ failure and acute lung injury. Growth arrest-specific protein 6 (Gas6) is a broad regulator of the innate immune response involved with the nuclear factor κB signaling pathway. We hypothesized that Gas6 could have a protective role in attenuating the severity of acute lung injury and sepsis. Male mice were subjected to sepsis by cecal ligation and puncture (CLP) after which recombinant murine Gas6 (rmGas6; 5 µg/mouse) or normal saline (vehicle) was administered intravenously. Blood and lung tissues were collected at 20 h after CLP for various measurements. Treatment with rmGas6 significantly reduced serum levels of the injury markers aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, as well as proinflammatory cytokines interleukin 6 (IL-6) and IL-17, compared with the vehicle group (P < 0.05). The parenchyma of the lungs damaged by CLP was attenuated by rmGas6 treatment. Lung mRNA levels of tumor necrosis factor α, IL-1ß, IL-6, IL-17, and macrophage inflammatory protein 2 (MIP-2) were decreased by 60%, 86%, 82%, 93%, and 82%, respectively, with rmGas6 treatment as determined by real-time reverse transcriptase-polymerase chain reaction (P < 0.05). The degradation of IκB-α induced by CLP in the lungs was inhibited by rmGas6 treatment. The number of neutrophils and myeloperoxidase activity in the lungs were significantly reduced in the rmGas6 group. Moreover, rmGas6 reduced the in vitro migration of differentiated human promyelocytic HL60 cells by 64%. Finally, the 10-day survival rate of mice subjected to CLP was increased from 31% in the vehicle group to 67% in the rmGas6 group (P < 0.05). Thus, Gas6 has potential to be developed as a novel therapeutic agent to treat patients with sepsis and acute lung injury.

Wnt agonist attenuates liver injury and improves survival after hepatic ischemia/reperfusion.

The Wnt/ß-catenin signaling pathway is well characterized in stem cell biology and plays a critical role in liver development, regeneration, and homeostasis. We hypothesized that pharmacologic activation of Wnt signaling protects against hepatic ischemia/reperfusion (I/R) injury through its known proliferative and antiapoptotic properties. Sprague-Dawley rats underwent 70% hepatic ischemia by microvascular clamping of the hilum of the left and median lobes of the liver for 90 min, followed by reperfusion. Wnt agonist (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine, 5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) in 0.5 mL was injected i.p. 1 h before ischemia or infused i.v. over 30 min right after ischemia. Blood and tissue samples from the pretreated groups were collected 24 h after reperfusion, and a survival study was performed. Hepatic expression of ß-catenin and its downstream target gene Axin2 were decreased after I/R, whereas Wnt agonist restored their expression to sham levels. Wnt agonist blunted I/R-induced elevations of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase and significantly improved the microarchitecture of the liver. The cell proliferation determined by Ki67 immunostaining significantly increased with Wnt agonist treatment, and inflammatory cascades were dampened in Wnt agonist-treated animals, as demonstrated by attenuations in interleukin 6, myeloperoxidase, inducible nitric oxide synthase, and nitrotyrosine. Wnt agonist also significantly decreased the amount of apoptosis, as evidenced by decreases in both TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining as well as caspase 3 activity levels. Finally, the 10-day survival rate was increased from 27% in the vehicle group to 73% in the pretreated Wnt agonist group and 55% in the Wnt agonist postischemia treatment group. Thus, we propose that direct Wnt/ß-catenin stimulation may represent a novel therapeutic approach in the treatment of hepatic I/R.

Bone morphogenetic protein-4 inhibits heat-induced apoptosis by modulating MAPK pathways in human colon cancer HCT116 cells.

Cancer thermotherapy and radiofrequency ablation (RFA) have been adopted as modalities for treating various kinds of cancer. We have previously demonstrated that bone morphogenetic protein-4 (BMP-4) is up-regulated in colonic adenocarcinoma. Here, we investigated whether an increase of BMP-4 expression changes cellular response to heat treatment in human colon cancer HCT116 cells. BMP-4 overexpressing HCT116 cells generated by stable transfection showed a significantly increased survival rate and a decreased apoptotic rate in comparison to empty vector controls after heat treatment at 45 degrees C for 20min. The expression levels and pattern of HSP90, HSP70, and HSP27 after heat treatment were similar between these two cell lines. There was no difference in expression levels of Bcl-2 and Bax in these two cell lines and their expression remained unchanged after heat treatment. Both activities of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were stimulated by heat in these cells. Comparatively, BMP-4 overexpressing cells had an intense and prolonged ERK activation, while a less intense and short JNK activation. Correspondingly, treatment of BMP-4 overexpressing cells with noggin, a BMP-4 antagonist, resulted in a reduction of heat-activated ERK but an increase of heat-activated JNK and significantly increased heat-induced apoptotic rate. These results indicate that BMP-4 can protect colon cancer cells from heat-induced apoptosis through enhancing the activation of ERK as well as inhibiting the activation of JNK.