PRPF19 modulates morphology and growth behavior in a cell culture model of human skin.

The skin provides one of the most visual aging transformations in humans, and premature aging as a consequence of oxidative stress and DNA damage is a frequently seen effect. Cells of the human skin are continuously exposed to endogenous and exogenous DNA damaging factors, which can cause DNA damage in all phases of the cell cycle. Increased levels of DNA damage and/or defective DNA repair can, therefore, accelerate the aging process and/or lead to age-related diseases like cancer. It is not yet clear if enhanced activity of DNA repair factors could increase the life or health span of human skin cells. In previous studies, we identified and characterized the human senescence evasion factor (SNEV)/pre-mRNA-processing factor (PRPF) 19 as a multitalented protein involved in mRNA splicing, DNA repair pathways and lifespan regulation. Here, we show that overexpression of PRPF19 in human dermal fibroblasts leads to a morphological change, reminiscent of juvenile, papillary fibroblasts, despite simultaneous expression of senescence markers. Moreover, conditioned media of this subpopulation showed a positive effect on keratinocyte repopulation of wounded areas. Taken together, these findings indicate that PRPF19 promotes cell viability and slows down the aging process in human skin.

An Early Myelosuppression in the Acute Mouse Sepsis Is Partly Outcome-Dependent.

Adult hematopoietic stem and progenitor cells (HSPCs) respond to bacterial infections by expansion to myeloid cells. Sepsis impairs this process by suppressing differentiation of stem cells subsequently contributing to an ineffective immune response. Whether the magnitude of HSPCs impairment in sepsis is severity-dependent remains unknown. This study investigated dynamics of the HSPC immune-inflammatory response in the bone marrow, splenic, and blood compartments in moribund and surviving septic mice. The 12-week-old outbred CD-1 female mice (n=65) were subjected to a cecal ligation and puncture (CLP) sepsis, treated with antibiotics and fluid resuscitation, and stratified into predicted-to-die (P-DIE) and predicted-to-survive (P-SUR) cohorts for analysis. CLP strongly reduced the common myeloid and multipotent progenitors, short- and long-term hematopoietic stem cell (HSC) counts in the bone marrow; lineage-ckit+Sca-1+ and short-term HSC suppression was greater in P-DIE versus P-SUR mice. A profound depletion of the common myeloid progenitors occurred in the blood (by 75%) and spleen (by 77%) of P-DIE. In P-SUR, most common circulating HSPCs subpopulations recovered to baseline by 72 h post-CLP. Analysis of activated caspase-1/-3/-7 revealed an increased apoptotic (by 30%) but not pyroptotic signaling in the bone marrow HSCs of P-DIE mice. The bone marrow from P-DIE mice revealed spikes of IL-6 (by 5-fold), CXCL1/KC (15-fold), CCL3/MIP-1α (1.7-fold), and CCL2/MCP-1 (2.8-fold) versus P-SUR and control (TNF, IFN-γ, IL-1ß, -5, -10 remained unaltered). Summarizing, our findings demonstrate that an early sepsis-induced impairment of myelopoiesis is strongly outcome-dependent but varies among compartments. It is suggestive that the HSCPC loss is at least partly due to an increased apoptosis but not pyroptosis.

Circulating miRNAs Associated With ER Stress and Organ Damage in a Preclinical Model of Trauma Hemorrhagic Shock.

Circulating microRNAs (miRNA) alterations have been reported in severe trauma patients but the pathophysiological relevance of these changes is still unclear. miRNAs are critical biologic regulators of pathological events such as hypoxia and inflammation, which are known to induce endoplasmic reticulum (ER) stress. ER stress is emerging as an important process contributing to the development of single and/or multiple organ dysfunction after trauma hemorrhagic shock (THS) accompanied by impaired tissue microcirculation and inflammation. Here, we aim to bring new insights into the involvement of miRNAs associated with ER stress in THS. THS was induced in rats by a median laparotomy and blood withdrawal until mean arterial pressure (MAP) dropped to 30-35 mmHg followed by a restrictive (40 min) and full reperfusion (60 min) with Ringer's solution. Tunicamycin was used to induce ER stress. Blood samples were collected 24 h after THS for the determination of pathological changes in the blood (PCB) and circulating miRNAs. Plasma levels of circulating miRNAs were compared between THS, tunicamycin, and sham groups and correlated to biomarkers of PCB. MiRNA profile of THS animals showed that 40 out of 91 (44%) miRNAs were significantly upregulated compared to sham (p < 0.01). The data showed a very strong correlation between liver injury and miR-122-5p (r = 0.91, p < 0.00001). MiR-638, miR-135a-5p, miR-135b-5p, miR-668-3p, miR-204-5p, miR-146a-5p, miR-200a-3p, miR-17-5p, miR-30a-5p, and miR-214-3p were found positively correlated with lactate (r > 0.7, p < 0.05), and negatively with base excess (r ≤ 0.8, p < 0.05) and bicarbonate (r ≤ 0.8, p < 0.05), which are clinical parameters that reflected the shock severity. Tunicamycin significantly modified the microRNA profile of the animals, 33 out of 91 miRNAs were found differentially expressed. In addition, principal component analysis revealed that THS and tunicamycin induced similar changes in plasma miRNA patterns. Strikingly, the data showed that 15 (25.9%) miRNAs were regulated by both THS and tunicamycin (p < 0.01). This included miR-122-5p, a liver-specific microRNA, but also miR-17-5p and miR-125b-5p which are miRNAs remarkably involved in unfolded protein response (UPR)-mediating pro-survival signaling (IRE1α). Since miRNAs associated with ER stress are clearly correlated with THS, our data strongly suggest that interaction between miRNAs and ER stress is an important pathologic event occurring during THS. Overall, we consider that the miRNA profile developed in this study can provide a rationale for the development of bench-to-bedside strategies that target miRNAs in critical care diseases or be used as biomarkers in the prognosis of trauma patients.

Comparison of post-traumatic changes in circulating and bone marrow leukocytes between BALB/c and CD-1 mouse strains.

This manuscript emerged from a larger third-party funded project investigating a new poly-trauma model and its influence upon secondary sepsis. The present sub-study compared selected leukocyte subpopulations in the circulation and bone marrow after polytrauma in BALB/c versus CD-1 mice. Animals underwent unilateral femur fracture, splenectomy and hemorrhagic shock. We collected blood and bone marrow for flow cytometry analysis at 24h and 48h post-trauma. Circulating granulocytes (Ly6G+CD11+) increased in both strains after trauma. Only in BALB/c mice circulating CD8+ T-lymphocytes decreased within 48h by 30%. Regulatory T-cells (Tregs, CD4+CD25+CD127low) increased in both strains by approx. 32%. Circulating Tregs and lymphocytes (CD11b-Ly6G-MHC-2+) were always at least 1.5-fold higher in BALB/c, while the bone marrow MHC-2 expression decreased in CD-1 mice (p<0.05). Overall, immune responses to polytrauma were similar in both strains. Additionally, BALB/c expressed higher level of circulating regulatory T-cells and MHC-2-positive lymphocytes compared to CD-1 mice.

The Fluctuations of Leukocytes and Circulating Cytokines in Septic Humanized Mice Vary With Outcome.

Sepsis remains a major challenge in translational research given its heterogeneous pathophysiology and the lack of specific therapeutics. The use of humanized mouse chimeras with transplanted human hematopoietic cells may improve the clinical relevance of pre-clinical studies. However, knowledge of the human immuno-inflammatory response during sepsis in humanized mice is scarce; it is unclear how similar or divergent mouse and human-origin immuno-inflammatory responses in sepsis are. In this study, we evaluated the early outcome-dependent immuno-inflammatory response in humanized mice generated in the NSG strain after cecal ligation and puncture (CLP) sepsis. Mice were observed for 32 h post-CLP and were assigned to either predicted-to-die (P-DIE) or predicted-to-survive (P-SUR) groups for retrospective comparisons. Blood samples were collected at baseline, 6 and 24 h, whereas the bone marrow and spleen were collected between 24 and 32 h post-CLP. In comparison to P-SUR, P-DIE humanized mice had a 3-fold higher frequency of human splenic monocytes and their CD80 expression was reduced by 1.3-fold; there was no difference in the HLA-DR expression. Similarly, the expression of CD80 on the bone marrow monocytes from P-DIE mice was decreased by 32% (p < 0.05). Sepsis induced a generalized up-regulation of both human and murine plasma cytokines (TNFα, IL-6, IL-10, IL-8/KC, MCP-1); it was additionally aggravated in P-DIE vs. P-SUR. Human cytokines were strongly overridden by the murine ones (approx. ratio 1:9) but human TNFα was 7-fold higher than mouse TNFα. Interestingly, transplantation of human cells did not influence murine cytokine response in NSG mice, but humanized NSG mice were more susceptible to sepsis in comparison with NSG mice (79 vs. 33% mortality; p < 0.05). In conclusion, our results show that humanized mice reflect selected aspects of human immune responses in sepsis and therefore may be a feasible alternative in preclinical immunotherapy modeling.

Effect of Coagulation Factor Concentrates on Markers of Endothelial Cell Damage in Experimental Hemorrhagic Shock.

BACKGROUND: Plasma-based resuscitation showed protective effects on the endothelial glycocalyx compared with crystalloid resuscitation. There is paucity of data regarding the effect of coagulation factor concentrates (CFC) on the glycocalyx in hemorrhagic shock (HS). We hypothesized that colloid-based resuscitation supplemented with CFCs offers a therapeutic value to treat endothelial damage following HS. METHODS: Eighty-four rats were subjected to pressure-controlled (mean arterial pressure (MAP) 30-35 mm Hg) and lab-guided (targeted cutoff: lactate >2.2. mmol/L and base deficit > 5.5 mmol/L) HS. Animals were resuscitated with fresh frozen plasma (FFP), human albumin (HA) or Ringer's lactate (RL) and RL or HA supplemented with fibrinogen concentrate (FC) or prothrombin complex concentrate (PCC). Serum epinephrine and the following markers of endothelial damage were assessed at baseline and at the end-of-observation (120 min after shock was terminated): syndecan-1, heparan sulfate, and soluble vascular endothelial growth factor receptor 1 (sVEGFR 1). RESULTS: Resuscitation with FFP had no effect on sVEGFR1 compared with crystalloid-based resuscitation (FFP: 19.3 ng/mL vs. RL: 15.9 ng/mL; RL+FC: 19.7 ng/mL; RL+PCC: 18.9 ng/mL; n.s.). At the end-of-observation, syndecan-1 was similar among all groups. Interestingly, HA+FC treated animals displayed the highest syndecan-1 concentration (12.07 ng/mL). Resuscitation with FFP restored heparan sulfate back to baseline (baseline: 36 ng/mL vs. end-of-observation: 36 ng/mL). CONCLUSION: The current study revealed that plasma-based resuscitation normalized circulating heparan sulfate but not syndecan-1. Co-administration of CFC had no further effect on glycocalyx shedding suggesting a lack of its therapeutic potential. LEVEL OF EVIDENCE: VExperimental in vivo study.

Organ-Specific Oxidative Events under Restrictive Versus Full Reperfusion Following Hemorrhagic Traumatic Shock in Rats.

Background aim: Reperfusion after hemorrhagic traumatic shock (HTS) is often associated with complications that are partly ascribed to the formation of reactive oxygen species (ROS). The aim of our study was to compare the effects of restrictive reperfusion (RR) to rapid full reperfusion (FR) on ROS formation and/or oxidative events. MATERIALS AND METHODS: Anesthetized male rats were randomly subjected to HTS followed by FR (75 mL/kg/h) or RR (30 mL/kg/h for 40 min, followed by 75 mL/kg/h) with Ringer's solution (n = 8/group). Compartment-specific ROS formation was determined by infusion of ROS scavenger 1-hydroxy-3-carboxy-2,2,5,5-tetramethyl-pyrrolidine hydrochloride (CP-H) during resuscitation, followed by electron paramagnetic resonance spectroscopy. Sham-operated animals (n = 8) served as controls. The experiment was terminated 100 min post-shock. RESULTS: Mean arterial pressure was significantly higher in the FR compared to the RR group during early reperfusion. Only RR animals, not FR animals, showed significantly higher ROS concentrations in erythrocytes (1951 ± 420 vs. 724 ± 75 AU) and in liver (474 ± 57 vs. 261 ± 21 AU) compared to sham controls. This was accompanied by elevated alanine aminotransferase and creatinine levels in RR animals compared to both shams and FR animals, while lipid peroxidation products (thiobarbituric acid reactive substances) were significantly increased only in the kidney in the FR group (p < 0.05). RR animals showed significantly higher plasma peroxiredoxin-4 values when compared to the FR group (20 ± 2 vs. 14 ± 0.5 RLU). CONCLUSION: Restrictive reperfusion after HTS is associated with increased ROS formation in erythrocytes and liver compared to sham controls. Moreover, the restrictive reperfusion is associated with a more pronounced injury to the liver and kidney, which is likely mediated by other than lipid peroxidation process and/or oxidative stress reactions.

Secretome Conveys the Protective Effects of ASCs: Therapeutic Potential Following Hemorrhagic Shock?

OBJECTIVES: We tested whether resuscitation supplemented with rat adipose-derived stem cells (ASCs) or secretome (conditioned media) of ASCs can ameliorate inflammation, cell/organ injury, and/or improve outcome after hemorrhagic traumatic shock (HTS). INTERVENTIONS: Rats were subjected to HTS and a resuscitation protocol that mimics prehospital restrictive reperfusion followed by an adequate reperfusion phase. Twenty minutes into the restrictive reperfusion, animals received an intravenous bolus of 2 × 10 cells (ASC group) or the secretome produced by 2 × 10 ASCs/24 h (ASC-Secretome group). Controls received the vehicle (Vehicle group). All rats were observed for 28-day survival. MEASUREMENTS AND MAIN RESULTS: HTS-induced inflammation represented by IL-6 was inhibited in the ASC (80%, P < 0.001) and in ASC-Secretome (59%, P < 0.01) group at 48 h compared with Vehicle group. At 24 h, HTS-induced liver injury reflected in plasma alanine aminotransferase was ameliorated by 36% (P < 0.001) in both the ASC and ASC-Secretome groups when compared with the Vehicle. There was no effect on kidney function and/or general cell injury markers. HTS induced a moderate 28-day mortality (18%) that was prevented (P = 0.08) in the ASC but not in the ASC-Secretome group (12%). CONCLUSIONS: Our data suggest that the ASC-secretome supplemented resuscitation following HTS, in the absence of the stem cells, exerts anti-inflammatory and liver protective effects. Given its ease of preparation, storage, availability, and application (in contrast to the stem cells) we believe that the cell-free secretome has a better therapeutic potential in the early phase of an acute hemorrhagic shock scenario.

Experimental Models of Endotheliopathy: Impact of Shock Severity.

BACKGROUND: Hemorrhagic shock (HS) followed by resuscitation is often associated with sympathoadrenal activation (SAA) and endothelial damage (ED). OBJECTIVE: We aimed to evaluate the impact of HS alone on the magnitude of SAA and consecutive ED, and to characterize potential targets for a standardized and reproducible model of HS-induced endotheliopathy in rats. METHODS: Rats were subjected either to a volume-controlled HS (40% of total blood volume: v-HS group) or to a laboratory-guided HS (l-HS) targeting base deficit (BD) more than 5.5 mmol/L and/or lactate more than 2.2 mmol/L using a pressure-controlled volume loss. RESULTS: At the end of shock, mean arterial pressure was significantly higher in the v-HS than the l-HS group (36 ±â€Š5.6 vs. 30 ±â€Š3.0 mmHg; P < 0.01). Base deficit and lactate were higher in l-HS than the v-HS group (BD: 9.5 ±â€Š2.5 vs. 3.0 ±â€Š1.0 mmol/L; P < 0.001; lactate: 4.1 ±â€Š1.3 vs. 1.6 ±â€Š0.6 mmol/L; P < 0.001). sVEGFR-1 and syndecan-1 were approximately 50% higher in the l-HS than the v-HS group (% changes vs. baseline: 160 ±â€Š10 vs. 116 ±â€Š36; P < 0.01; 170 ±â€Š37 vs. 113 ±â€Š27; P < 0.001). Adrenaline was 2-fold higher in l-HS than the v-HS group (1964 ±â€Š961% vs. 855 ±â€Š451%; P < 0.02, respectively). Moreover, linear regression analysis revealed an independent association of shock severity BD with syndecan-1 (rho = 0.55, P = 0.0005), sVEGFR1 (rho = 0.25, P < 0.05), and adrenaline (rho = 0.31, P = 0.021). CONCLUSIONS: Our findings indicate that ED has already occurred during HS without reperfusion; intensity is strongly related to the severity of HS and consecutive SAA; and severity may appropriately be targeted and standardized in a HS model controlled by biological endpoints such as BD and/or lactate.

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis.

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n = 90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments.

TNF-α release capacity is suppressed immediately after hemorrhage and resuscitation.

PURPOSE: It has been suggested that patients with traumatic insults are resuscitated into a state of an early systemic inflammatory response. We aimed to evaluate the influence of hemorrhagic shock and resuscitation (HSR) upon the inflammatory response capacity assessed by overall TNF-α secretion capacity of the host compared to its release from circulating leukocytes in peripheral circulation. METHODS: Rats (8/group) subjected to HS (MAP of 30-35 mmHg for 90 min followed by resuscitation over 50 min) were challenged with Lipopolysaccharide (LPS), 1 µg/kg intravenously at the end of resuscitation (HSR-LPS group) or 24 h later (HSR-LPS24 group). Control animals were injected with LPS without bleeding (LPS group). Plasma TNF-α was measured at 90 min after the LPS challenge. In addition, whole blood (WB) was obtained either from healthy controls (CON) immediately after resuscitation (HSR), or at 24 h post-shock (HSR 24). WB was incubated with LPS (100 ng/mL) for 2 h at 37 °C. TNF-α concentration and LPS binding capacity (LBC) was determined. RESULTS: Compared to LPS group, HSR followed by LPS challenge resulted in suppression of plasma TNF-α in HSR-LPS and HSR-LPS24 groups (1835 ± 478, 273 ± 77, 498 ± 200 pg/mL, respectively). Compared to CON the LPS-induced TNF-α release capacity of circulating leukocytes ex vivo was strongly declined both at the end of resuscitation (HSR) and 24 h later (HSR24) (1012 ± 259, 313 ± 154, 177 ± 63 ng TNF/mL, respectively). The LBC in WB was similar between CON and HSR and only moderately enhanced in HSR24 (57 ± 6, 56 ± 6, 71 ± 5 %, respectively). CONCLUSION: Our data suggest that the overall inflammatory response capacity is decreased immediately after HSR, persisting up to 24 h, and is independent of LBC.

A non-lethal traumatic/hemorrhagic insult strongly modulates the compartment-specific PAI-1 response in the subsequent polymicrobial sepsis.

INTRODUCTION: Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes. METHODS: Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE). RESULTS: In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2-4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction. CONCLUSIONS: Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.

Relationship between age/gender-induced survival changes and the magnitude of inflammatory activation and organ dysfunction in post-traumatic sepsis.

Age/gender may likely influence the course of septic complications after trauma. We aimed to characterize the influence of age/gender on the response of circulating cytokines, cells and organ function in post-traumatic sepsis. We additionally tested whether post-traumatic responses alone can accurately predict outcomes in subsequent post-traumatic sepsis. A mouse 2-hit model of trauma/hemorrhage (TH, 1(st) hit) and cecal ligation and puncture (CLP, 2(nd) hit) was employed. 3, 15 and 20 month (m) old female (♀) and male (♂) CD-1 mice underwent sublethal TH followed by CLP 2 days later. Blood was sampled daily until day 6 post-TH and survival was followed for 16 days. To compare general response patterns among groups, we calculated two scores: the inflammatory response (including KC, MIP-1α, TNFα, MCP-1, IFNγ, IL-1ß,-5,-6,-10) and the organ dysfunction score (Urea, ALT, AST and LDH). Moreover, mice were retrospectively divided into survivors (SUR) and dying (DIE) based on post-CLP outcome. In general, females survived better than males and their survival did not correspond to any specific estrus cycle phase. Pre-CLP phase: the post-TH inflammatory score was weakest in 3 m♂ but there were no changes among remaining groups (similar lack of differences in the organ dysfunction score). TH induced a 40% increase of IFNγ, MIP-1α and IL-5 in 15 m♂ SUR (vs. DIE) but predictive accuracy for post-CLP outcomes was moderate. Post-CLP phase: while stable in males, inflammatory response score in 15 m and 20 m females decreased with age at day 1 and 2 post-CLP. SUR vs. DIE differences in inflammatory and organ dysfunction score were evident but their magnitude was comparable across age/gender. Nearly identical activation of the humoral inflammatory and organ function compartments, both across groups and according to sepsis severity, suggests that they are not directly responsible for the age/gender-dependent disparity in TH-CLP survival in the studied young-to-mature population.

Xylazine-/diazepam-ketamine and isoflurane differentially affect hemodynamics and organ injury under hemorrhagic/traumatic shock and resuscitation in rats.

Most experimental studies on hemorrhage and trauma are performed under anesthesia. We determined the effects of three commonly used anesthetic regimens on hemodynamics and organ damage under normal and hemorrhagic/traumatic shock (HTS) conditions in rats. Animals were anesthetized with ketamine/diazepam (K/D), ketamine/xylazine (K/X), or isoflurane (ISO). Hemorrhagic/traumatic shock was induced by a midline laparotomy, bleeding to a mean arterial pressure of 30 to 35 mmHg until decompensation, followed by restrictive and adequate phases of resuscitation. The experiment was terminated 120 min after the completion of resuscitation. Under normal conditions, K/D anesthesia resulted in higher mean arterial pressure and heart rate than K/X and higher systemic vascular resistance index (SVRI) than ISO. Stroke volume was significantly lower in K/D group than in K/X and ISO groups. Under normal conditions, ISO anesthesia was accompanied by the highest cardiac index. During shock and resuscitation, heart rate remained higher in the K/D than K/X. During shock, SVRI decreased in the K/D group but increased in K/X and ISO groups. After resuscitation, SVRI was lower, and cardiac index was higher in the ISO group than in the K/D group. Despite higher shed blood volume, the rats anesthetized with ISO did not decompensate within the time frame compared with other groups. Cellular damage (plasma creatine kinase, lactate dehydrogenase, uric acid) was more pronounced with K/D compared with ISO. Histological examinations revealed frequent HTS-induced damage to adrenals, kidney, and liver of animals anesthetized with K/D and K/X but not with ISO. Anesthetics differentially affect HTS-induced hemodynamic alterations and organ injury. Thus, when interpreting data from HTS models, the individual effect of anesthetics should be considered.

Nitric oxide-supplemented resuscitation improves early gastrointestinal blood flow in rats subjected to hemorrhagic shock without late consequences.

BACKGROUND: we have shown that hemorrhage/resuscitation altered gastrointestinal blood flow (GI-BF) and that gastric perfusion did not recover after resuscitation. This study aimed to determine the effect of nitric oxide (NO) supplemented resuscitation on the mean arterial blood pressure (MAP), GI-BF, and outcome after hemorrhagic shock. METHODS: rats were subjected to hemorrhage and resuscitation with/without the NO-donor S-nitroso human serum albumin (S-NO-HSA). GI-BF was determined using colored microspheres. RESULTS: NO supplementation significantly decreased MAP at the end of resuscitation. At the same time point, the GI-BF has significantly increased in the stomach, duodenum, and colon. Two hours after treatment discontinuation, there was no difference in either MAP or GI-BF between NO-supplemented and control groups. The survival times indicated that S-NO-HSA treatment was noninferior compared with control. CONCLUSIONS: NO-supplemented resuscitation improves the GI-BF during the early stage of resuscitation without a negative impact on short-/long-term survival despite a transient MAP decrease.

Peritoneal inflammation in pigs is associated with early mitochondrial dysfunction in liver and kidney.

The objective of this study was to investigate early effects of peritoneal inflammation on the mitochondrial function in the vital organs, liver and kidney, and their relation to inflammatory and oxidative stress mediators. The study was performed on 14 domestic pigs. Peritoneal inflammation was induced in anesthetized pigs after a midline laparotomy by autologous feces. Fluid resuscitation maintained a MAP above 60 mmHg. Animals were sacrificed 12 h later, and tissue samples were obtained to determine mitochondrial function, mRNA levels of relevant genes [inducible NO synthase (iNOS), inducible HO (HO-1), tumor necrosis factor-alpha (TNF-alpha)], generation of reactive oxygen species (ROS), and HO-1 activity. We found impaired mitochondrial function in both liver and kidney, based on decreased state 3 respiration in the liver and increased states 2 and 4 respiration in the kidney at 12 h. This was accompanied by increased TNF-alpha protein in the blood and up-regulation of TNF-alpha mRNA in the liver. Free iron was elevated in the liver but not in the kidney. In the kidney, mitochondrial ROS production was increased. Nitric oxide levels in blood remained unchanged, corresponding to unchanged levels of iNOS mRNA expression in liver and kidney. Similarly, HO-1 mRNA and heme oxygenase (HO)-activity were unchanged. The inflammatory response in the absence of characteristic septic symptoms was not associated with morphological organ damage at this early time point. Peritoneal inflammation in pigs caused mitochondrial dysfunction in liver and kidney, preceding signs of organ damage. We did not find proof that mitochondrial dysfunction was due to increased levels of either nitric oxide (NO) or products of HO, but it was accompanied by increased levels of oxidative stress markers.

Release and hemodynamic influence of nitro-glycerine-derived nitric oxide in endotoxemic rats.

BACKGROUND AND AIM: Nitric oxide released from nitro-glycerine (NG) has been considered to improve the microcirculation. Septic conditions are, however, associated with excessive formation of nitric oxide (NO), which is formed from l-arginine by the inducible NO synthase (iNOS) activity. Since the characteristics and influence of NG-derived NO in sepsis remains unclear, the major aims of the present study were to quantify the release and to determine the effects of NO formed from NG on systemic blood pressure under endotoxemic conditions. MATERIAL AND METHODS: Four hours following endotoxin challenge (10 mg/kg intraperitoneally), rats received an infusion of NG (0.5 or 5.0 micromol/kg/h) over 45 min. We determined the changes in blood pressure and the NO concentrations generated in brain, heart, intestine, kidney, liver, and lung by means of NO trapping and EPR technique. RESULTS: NG infusion in control rats and endotoxin challenge decreased systemic blood pressure to the same extent. However, in rats subjected to endotoxin challenge NG infusion did not affect the blood pressure. The endotoxin-induced increase in tissue NO concentrations were found to be 15-folds higher than tissue levels of NO following NG infusion. CONCLUSION: Our results suggest that under endotoxic shock conditions in rats NG may not additionally affect the systemic blood pressure. This may relate to the excessive tissue NO levels induced by endotoxin that are not further increased by NG.

Nonspecific increase of systemic neuron-specific enolase after trauma: clinical and experimental findings.

The aim of this clinical and experimental study was to determine whether systemic neuron-specific enolase (NSE) is a useful early marker of traumatic brain injury (TBI) and whether NSE is affected by ischemia/reperfusion damage of abdominal organs. Our study included patients with and without TBI (verified by computerized tomography) admitted within 6 h after trauma and male Sprague-Dawley rats with ischemia and reperfusion of the abdominal organs liver, gut, or kidney. Thirty-eight study patients included 13 with isolated TBI and 18 patients with multiple trauma and TBI. Seven patients had multiple trauma but no TBI. Fifteen rats were anaesthetized and subjected to isolated ischemia of the liver, gut, or kidney (n = 5 each) for 1 h, followed by reperfusion for 3 h. In patients, NSE increased over 2-fold versus the upper normal limit (10 microg/L) within 6 h after trauma, regardless of whether TBI had occurred or not. In rats, NSE increased over 3-fold versus laboratory controls during ischemia of the liver and kidney (both P < 0.0005), but not of the gut. NSE increased over 2-fold after onset of reperfusion of the liver and kidney (both P < 0.05), but not of the gut and increased over 3-fold after 3 h of reperfusion of the liver, gut (both P < 0.005), and kidney (P < 0.0005). Our data show that systemic NSE increases to similar degrees with and without TBI. Therefore, NSE is not a useful early marker of TBI in multiple trauma.

Neuron-specific-enolase is increased in plasma after hemorrhagic shock and after bilateral femur fracture without traumatic brain injury in the rat.

Neuron-specific enolase (NSE) is an acknowledged marker of traumatic brain injury. Several markers originally considered reliable in the setting of traumatic brain injury have been challenged after having been studied more extensively. The aim of our experimental study was to determine whether NSE is a reliable marker of traumatic brain injury early after trauma. Hemorrhagic shock was achieved by bleeding anesthetized rats to a mean arterial pressure (MAP) of 30-35 mmHg through a femoral catheter until incipient decompensation. MAP was maintained at 30-35 mmHg until 40% of shed blood had been administered as Ringer's solution and was then increased and maintained at 40-45 mmHg for 40 min by further administration of Ringer's solution, mimicking the phase of inadequate preclinical resuscitation. Blood samples were drawn at the end of the 40-min period of inadequate resuscitation. Femur fracture was achieved in anesthetized rats by bilateral application of forceps. Blood samples were drawn 30 and 60 min after fracture. Hemorrhagic shock caused NSE increase versus laboratory controls at the end of inadequate resuscitation (P < 0.01). Bilateral femur fracture caused NSE increase versus laboratory controls 30 min after fracture, which was significant 60 min after fracture (P < 0.01). During femur fracture, MAP remained at a level that is not associated with shock in rats. Our findings show for the first time that NSE increases after hemorrhagic shock as well as after femur fracture without hemorrhagic shock in rats. From a clinical point of view, these findings indicate that NSE cannot be considered a reliable marker of traumatic brain injury early after trauma in cases associated with hemorrhagic shock and/or femur fracture.

Release of S100B differs during ischemia and reperfusion of the liver, the gut, and the kidney in rats.

S100B, an acknowledged marker of brain damage, is increased post-traumatically in plasma. The aim of this study was to investigate the diagnostic value of S100B release in experimental local extracranial ischemia and reperfusion. Anesthetized rats underwent laparotomy and ligation of the afferent blood vessels to the liver, gut, or kidney to achieve local ischemia in each organ separately. After 60 min of ischemia, ligatures were removed and resuscitation was performed for 3 h. S100B was determined in plasma by immunoluminometric assay 55, 65, and 240 min after the onset of ischemia (5 min before reperfusion and 5 min and 3 h after the onset of reperfusion). During ischemia of the liver, S100B increased before ligature removal and reperfusion, reaching significance early after the onset of reperfusion and remaining almost unchanged throughout reperfusion. In contrast, S100B did not increase during ischemia of the gut or kidney before ligature removal or during early reperfusion but increased significantly to similar levels as during reperfusion of the liver 240 min after the onset of ischemia (after 3 h of reperfusion). Our findings show for the first time that S100B increases during local extracranial ischemia and reperfusion. These experimental findings support the concept that brain damage is not necessarily the cause of increased S100B. Although S100B has been an acknowledged marker of brain damage for years, our experimental clinically relevant data indicate that S100B is, in fact, not specific as a marker of brain damage in the setting of local ischemia and reperfusion of the liver, gut, and kidney because local ischemia and reperfusion of these organs cause an S100B increase per se.