Mouse Model Of Hemorrhagic Shock Research Articles

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

Dual phenotypic characteristics of P-selectin in a mouse model of hemorrhagic shock and hepatectomy

BackgroundMembrane-bound P-selectin induces endothelial adhesion of leucocytes and amplifies organ inflammations during major trauma, while soluble P-selectin (sP-sel) mediates survival rescue properties. This study characterized the differential effects of P-selectin in a “2-hit” model of hemorrhagic shock (HS) and partial hepatectomy (PH). Materials and methodsHS was induced by withdrawing blood (0.3 mL) directly from the mouse femoral arteries. 70% or 50% of liver volumes were resected after inducing HS. Time of survival in P-selectin deficient (Selp −/−) mice treated with and without intraperitoneal injections of recombinant P-sel IgG-Fc fusion proteins (rP-sel-Fc, 1.5 mg/kg) were recorded for up to 72h after injury. In addition, liver regeneration at 72h after HS and 50% PH was assessed in wild-type and Selp −/− mice. ResultsCompared to wild-types, Selp −/− mice had significantly higher mortality rates post HS and 70% PH, as none of these animals survived up to 48h postoperatively. The survival curve was restored in Selp −/− mice pre-treated with rP-sel-Fc. In the HS followed by 50% PH experimental arm, liver remnant growth ratios were significantly higher in Selp −/− mice (15.7 ± 3.1 vs 11.7 ± 4.9, P = 0.040). The elevated serum concentrations of alanine aminotransferase post-PH were significantly reduced in Selp −/− mice. Hepatocyte proliferation indices (CYP7a1 and PCNA) expression were enhanced and myeloperoxidase activity in the regenerated remnant liver was reduced in the Selp −/− mice. ConclusionIn critical conditions induced by HS and PH, P-selectin mediates two distinct phenotypic characteristics. Soluble-form circulating P-selectin improves survival in the acute stage of HS and extensive loss of liver parenchyma; membrane-bound P-selectin induces regional pro-inflammatory reactions in the remnant liver after the acute stage of two insults, thereby inhibiting hepatic regeneration. The results of this pre-clinical study may provide molecular mechanistic insight and clinical therapeutic applications of P-selectin in the acute and regenerative phases of traumatic hepatic injury.

IL-18BP Alleviates Anxiety-Like Behavior Induced by Traumatic Stress via Inhibition of the IL-18R-NLRP3 Signaling Pathway in a Mouse Model of Hemorrhagic Shock and Resuscitation.

Psychological distress and posttraumatic stress, including anxiety, severely influence life quality. Previously, we reported that interleukin-18 (IL-18) was involved in pyroptosis-induced emotional changes in a rodent model of hemorrhagic shock and resuscitation (HSR). Here, we aimed to continue our investigation on the role of IL-18 binding protein (IL-18BP), which exhibits excellent anti-inflammatory effects as an IL-18 negative regulator. Mice were administered with an intraperitoneal injection of IL-18BP after HSR exposure and anxiety-like behavior was examined using the open-field test and elevated plus maze test. Moreover, the following variables post-HSR were measured: (1) the activation of astrocytes; (2) pyroptosis-associated factors including cleaved caspase-1, GSDMD, IL-18; (3) the roles of IL-18 receptor (IL-18R)-NOD-like receptor pyrin domain-containing-3 (NLRP3) signal with the application of the NLRP3 specific agonist or astrocyte-specific NLRP3 knockout mice. IL-18BP administration remarkably alleviated HSR-induced anxiety-like behavior, astrocytic activation, and increases in pyroptosis-associated factors, while NLRP3 agonist nigericin partially reversed IL-18BP-induced neuroprotective effects. Astrocyte-specific NLRP3 knockout mice exhibited relatively less anxiety-like behavior. Similarly, IL-18BP exhibited an anti-pyroptosis effect in astrocytes in an in vitro model of low oxygen-glucose deprivation. These findings offer unique perspectives on HSR-induced posttraumatic stress and indicate that inhibition of IL-18R-NLRP3 signal via IL-18BP can attenuate astrocytic activation and pyroptosis, broadening the therapeutic landscape for patients with psychological distress and posttraumatic stress.

RECOVERY OF ENDOTHELIOPATHY AT 24 HOURS IN AN ESTABLISHED MOUSE MODEL OF HEMORRHAGIC SHOCK AND TRAUMA.

Introduction: The endotheliopathy of trauma develops early after injury and consists of increased vascular permeability, inflammation, and dysfunctional coagulation. Persistence of these abnormalities ultimately leads to multiorgan failure. We hypothesized that extending an established 3-hour acute mouse model of hemorrhagic shock and trauma (HS/T) to a 24-hour survival model would allow for evaluation of persistent endotheliopathy and organ injury after HS/T. Methods: Adult male C57BL/6J mice underwent laparotomy, femoral artery cannulation, and blood withdrawal to induce HS to a MAP of 35 mm Hg for 90 minutes. Mice were resuscitated with either lactated Ringer's (LR) or fresh frozen plasma (FFP). Vascular permeability in the lung and gut was assessed by measuring extravasation of a fluorescent dextran dye. Lungs were evaluated for histopathologic injury, and immunofluorescent staining was used to evaluate intercellular junction integrity. Pulmonary inflammatory gene expression was evaluated using NanoString (Seattle, WA). All endpoints were evaluated at both 3 and 24 hours after initiation of shock. Results: Lactated Ringer's- and FFP-treated mice had an equal mortality rate of 17% in the 24-hour model. Lactated Ringer's-treated mice demonstrated increased vascular permeability in the lung and gut at 3 hours compared with sham mice (lung, P < 0.01; gut, P < 0.001), which was mitigated by FFP treatment (lung, P < 0.05; gut, P < 0.001). Twenty-four hours after shock, however, there were no differences in vascular permeability between groups. Similarly, although at 3 hours, the lungs of LR-treated mice demonstrated significant histopathologic injury, loss of tight and adherens junctions, and a pro-inflammatory gene expression profile at 3 hours, these endpoints in LR mice were similar to sham mice by 24 hours. Conclusions: In an established mouse model of HS/T, endotheliopathy and lung injury are evident at 3 hours but recover by 24 hours. Polytrauma models or larger animal models allowing for more severe injury coupled with supportive care are likely necessary to evaluate endotheliopathy and organ injury outside of the acute period.

Mesenchymal stem cell extracellular vesicles mitigate vascular permeability and injury in the small intestine and lung in a mouse model of hemorrhagic shock and trauma.

Hemorrhagic shock and trauma (HS/T)-induced gut injury may play a critical role in the development of multi-organ failure. Novel therapies that target gut injury and vascular permeability early after HS/T could have substantial impacts on trauma patients. In this study, we investigate the therapeutic potential of human mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC EVs) in vivo in HS/T in mice and in vitro in Caco-2 human intestinal epithelial cells. In vivo, using a mouse model of HS/T, vascular permeability to a 10-kDa dextran dye and histopathologic injury in the small intestine and lungs were measured among mice. Groups were (1) sham, (2) HS/T + lactated Ringer's (LR), (3) HS/T + MSCs, and (4) HS/T + MSC EVs. In vitro, Caco-2 cell monolayer integrity was evaluated by an epithelial cell impedance assay. Caco-2 cells were pretreated with control media, MSC conditioned media (CM), or MSC EVs, then challenged with hydrogen peroxide (H2O2). In vivo, both MSCs and MSC EVs significantly reduced vascular permeability in the small intestine (fluorescence units: sham, 456 ± 88; LR, 1067 ± 295; MSC, 765 ± 258; MSC EV, 715 ± 200) and lung (sham, 297 ± 155; LR, 791 ± 331; MSC, 331 ± 172; MSC EV, 303 ± 88). Histopathologic injury in the small intestine and lung was also attenuated by MSCs and MSC EVs. In vitro, MSC CM but not MSC EVs attenuated the increased permeability among Caco-2 cell monolayers challenged with H2O2. Mesenchymal stem cell EVs recapitulate the effects of MSCs in reducing vascular permeability and injury in the small intestine and lungs in vivo, suggesting MSC EVs may be a potential cell-free therapy targeting multi-organ dysfunction in HS/T. This is the first study to demonstrate that MSC EVs improve both gut and lung injury in an animal model of HS/T.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Mitigate Vascular Permeability and Injury in the Small Intestine and Lungs in a Mouse Model of Hemorrhagic Shock and Trauma

Mesenchymal Stem Cell-Derived Extracellular Vesicles Mitigate Vascular Permeability and Injury in the Small Intestine and Lungs in a Mouse Model of Hemorrhagic Shock and Trauma

Murine Myeloid Progenitors Attenuate Immune Dysfunction Induced by Hemorrhagic Shock.

SummaryHemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia. We observed that transplant of NPs decreases the proportion of host neutrophils that express programmed death ligand 1 and intercellular adhesion molecule 1 in the context of prior hemorrhage. Following hemorrhage, NP transplant decreased proinflammatory cytokines in the lungs, increased neutrophil migration into the airspaces, and enhanced bacterial clearance. Further, hemorrhagic shock improved NP engraftment in the bone marrow. These results suggest that NPs hold the potential for use as a cellular therapy in the treatment and prevention of secondary infection following hemorrhagic shock.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro

BackgroundImmune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. MethodsThe normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. ResultsHemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. ConclusionsThe results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

Blockade of programmed death-ligand 1 attenuates indirect acute lung injury in mice through targeting endothelial cells but not epithelial cells

To examine the expression profile of programmed death-ligand 1 (PD-L1) on lung endothelial or epithelial cells, and to determine the specific role of PD-L1 in mouse model of indirect acute lung injury (i-ALI). Eighty male C57BL/6 mice were randomly divided into two parts (both n = 40). The effects of different administration routes on the expression of PD-L1 were observed. The mice in each part were randomly divided into sham, i-ALI, i-ALI+small interfering RNA (siRNA) random sequence control, and i-ALI+PD-L1 siRNA which could specifically inhibit PD-L1 expression groups, with 10 mice in each group. i-ALI was reproduced in a mouse model of hemorrhagic shock in combination with a subsequent cecal ligation and puncture (CLP). In sham group, only bilateral femoral arteries were ligated without catheterization or bleeding, and only cecum was separated but perforation was not ligated. Intravenous or intratracheal delivery of PD-L1 siRNA was performed 2 hours following the resuscitation to suppress the expression of PD-L1 on lung endothelial or epithelial cells. The mice in i-ALI+siRNA random sequence control group were given siRNA random sequence without inhibition effect on PD-L1 expression, and those in sham group and i-ALI group were given 100 μL phosphate buffered saline (PBS). The mice were sacrificed at 24 hours after CLP, and samples of blood, lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Expressions of PD-L1 were determined with flow cytometry. Cytokines and chemokines in plasma, lung tissue and BALF were determined by enzyme linked immunosorbent assay (ELISA). The protein concentration in plasma and BALF and the activity of myeloperoxidase (MPO) in lung tissue were quantitatively measured. The pathological changes in lung tissue were observed under light microscope. (1) Compared with sham group, PD-L1 expression on lung endothelial or epithelial cells were significantly elevated in i-ALI group [endothelial cells: (27.88±1.53)% vs. (19.64±1.03)%, epithelial cells: (58.70±8.21)% vs. (29.23±3.94)%, both P < 0.05]. (2) Mice received intravenous delivery of liposomal-encapsulated siRNA had significantly lower expression of PD-L1 on lung endothelial cells as compared with that of i-ALI group [(21.37±0.76)% vs. (27.88±1.53)%, P < 0.05]. Intratracheal delivery of naked PD-L1 siRNA mainly inhibited the PD-L1 expression on epithelial cell as compared with that of i-ALI group [(31.23±4.71) % vs. (58.70±8.21) %, P < 0.05]. The expression of PD-L1 in pulmonary microvascular endothelial cells or pulmonary epithelial cells of i-ALI mice was not affected by siRNA random sequence. (3) PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to a lower protein ratio of BALF/plasma [(4.48±0.35)×10-3 vs. (6.11±0.56)×10-3, P < 0.05] and a decreased MPO activity in lung tissue (U×μg-1×min-1: 2.48±0.47 vs. 4.56±0.52, P < 0.05) as compared with that of i-ALI group. Moreover, inflammatory mediator levels such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) in lung tissue or plasma were significantly reduced following PD-L1 suppression on endothelial cells as compared with those of i-ALI group [IL-6 (ng/g): 177.4±23.2 vs. 287.9±57.3, MCP-1 (ng/g): 839.6±91.7 vs. 1 395.7±211.9, MIP-2 (ng/g): 923.7±107.3 vs. 1 700.9±240.2 in lung tissue; IL-6 (ng/L): 950.2±192.7 vs. 1 828.2±243.6, TNF-α (ng/L): 258.7±29.1 vs. 443.0±58.1, MCP-1 (ng/L): 2 583.8±302.3 vs. 4 328.1±416.4, MIP-2 (ng/L): 1 512.9±165.6 vs. 2 005.9±85.7 in plasma, all P < 0.05], however, there was no significant change in the levels of inflammatory factors in BALF. It was shown in lung tissue histology that PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to lessened pulmonary edema and reduced immune cells emigration. Intratracheal delivery of PD-L1 siRNA for PD-L1 suppression on epithelial cells had minimal effects on protein ratio of BALF/plasma, MPO activity, inflammatory mediator expressions in lung tissue, plasma, and BALF as well as lung tissue histology. PD-L1 silencing on endothelial cells but not epithelial cells protected mice against hemorrhagic shock-sepsis induced i-ALI.

Age-Dependent Changes in AMPK Metabolic Pathways in the Lung in a Mouse Model of Hemorrhagic Shock.

The development of multiple organ failure in patients with hemorrhagic shock is significantly influenced by patient age. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which coordinates metabolic repair during cellular stress. We investigated whether AMPK-regulated signaling pathways are age-dependent in hemorrhage-induced lung injury and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) affords lung protective effects. Male C57/BL6 young mice (3-5 mo), mature adult mice (9-12 mo), and young AMPKα1 knockout mice (3-5 mo) were subjected to hemorrhagic shock by blood withdrawing, followed by resuscitation with shed blood and lactated Ringer's solution. Plasma proinflammatory cytokines were similarly elevated in C57/BL6 young and mature adult mice after hemorrhagic shock. However, mature adult mice exhibited more severe lung edema and neutrophil infiltration, and higher mitochondrial damage in alveolar epithelial type II cells, than did young mice. No change in autophagy was observed. At molecular analysis, the phosphorylation of the catalytic subunit AMPKα1 was associated with nuclear translocation of peroxisome proliferator-activated receptor γ co-activator-α in young, but not mature, adult mice. Treatment with AICAR ameliorated the disruption of lung architecture in mice of both ages; however, effects in mature adult mice were different than young mice and also involved inhibition of nuclear factor-κB. In young AMPKα1 knockout mice, AICAR failed to improve hypotension and lung neutrophil infiltration. Our data demonstrate that during hemorrhagic shock, AMPK-dependent metabolic repair mechanisms are important for mitigating lung injury. However, these mechanisms are less competent with age.

Lack of species-specific difference in pulmonary function when using mouse versus human plasma in a mouse model of hemorrhagic shock.

Clinical studies have demonstrated that the early and empiric use of plasma improves survival after hemorrhagic shock. We have demonstrated in rodent models of hemorrhagic shock that resuscitation with plasma is protective to the lungs compared with lactated Ringer's solution. As our long-term objective is to determine the molecular mechanisms that modulate plasma's protective effects in injured bleeding patients, we have used human plasma in a mouse model of hemorrhagic shock. The goal of the current experiments is to determine if there are significant adverse effects on lung injury when using human versus mouse plasma in an established murine model of hemorrhagic shock and laparotomy. Mice underwent laparotomy and 90 minutes of hemorrhagic shock to a mean arterial pressure (MAP) of 35 ± 5 mm Hg followed by resuscitation at 1× shed blood using either mouse fresh frozen plasma (FFP), human FFP, or human lyophilized plasma. Mean arterial pressure was recorded during shock and for the first 30 minutes of resuscitation. After 3 hours, animals were killed, and lungs collected for analysis. There was a significant increase in early MAP when mouse FFP was used to resuscitate animals compared with human FFP or human lyophilized plasma. However, despite these differences, analysis of the mouse lungs revealed no significant differences in pulmonary histopathology, lung permeability, or lung edema between all three plasma groups. Analysis of neutrophil infiltration in the lungs revealed that mouse FFP decreased neutrophil influx as measured by neutrophil staining; however, myeloperoxidase immunostaining revealed no significant differences in between groups. The study of human plasma in a mouse model of hemorrhagic shock is feasible but does reveal some differences compared with mouse plasma-based resuscitation in physiologic measures such as MAP postresuscitation. Measures of end organ function such as lung injury appear to be comparable in this acute model of hemorrhagic shock and resuscitation.

Enhanced Innate Inflammation Induced by Anti-BTLA Antibody in Dual Insult Model of Hemorrhagic Shock/Sepsis.

Sepsis following hemorrhagic shock is a common clinical condition, in which innate immune system suffers from severe suppression. B and T lymphocyte attenuator (BTLA) is an immune-regulatory coinhibitory receptor expressed not only on adaptive, but also on innate immune cells. Our previous data showed that BTLA gene deficient mice were protected from septic mortality when compared with wild-type control C57BL/6 mice. Here, we extended our study by treating C57BL/6 mice with an anti-BTLA monoclonal antibody (clone 6A6; reported to have the ability to neutralize or agonize/potentiate BTLA signaling) in a mouse model of hemorrhagic shock (Hem) followed by sepsis induced by cecal ligation and puncture (CLP); positing initially that if BTLA engagement was neutralized, like gene deficiency, an anti-BTLA mAb would have the similar effects on the inflammatory response/morbidity in these mice after such insults. Here, we report that BTLA expression is elevated on innate immune cells after Hem/CLP. However, anti-BTLA antibody treatment increased cytokine (TNF-α, IL-12, IL-10)/chemokine (KC, MIP-2, MCP-1) levels and inflammatory cells (neutrophils, macrophages, dendritic cells) recruitment in the peritoneal cavity, which in turn aggravated organ injury and elevated these animals' mortality in Hem/CLP. When compared with the protective effects of our previous study using BTLA gene deficient mice in a model of lethal septic challenge, we further confirmed BTLA's contribution to enhanced innate cell recruitment, elevated IL-10 levels, and reduced survival, and that engagement of antibody with BTLA potentiates/exacerbates the pathophysiology in Hem/sepsis.

Histone Deacetylase Inhibition and IκB Kinase/Nuclear Factor-κB Blockade Ameliorate Microvascular Proinflammatory Responses Associated With Hemorrhagic Shock/Resuscitation in Mice.

To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and IκB kinase/nuclear factor-κB signaling blockade by IκB kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo. Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock. Research laboratory at university teaching hospital. Endothelial cells and C57BL/6 male mice. Endothelial cells were incubated with tumor necrosis factor-α in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 μg/g body weight) or BAY11-7082 (400 μg per mouse). Mice were killed 1 hour and 4 hours after resuscitation. Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-α-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-κB, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation. Suppression of histone deacetylase activity and blockade of IκB kinase/nuclear factor-κB signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.

Abstract 13557: Regulation of PBEF Release by NAD Glycohydrolase CD38

Introduction: Our previous work suggests that PBEF is an early blood marker inversely related to hypothermia protection and resuscitation survival in a mouse model of hemorrhagic shock. Given that neutrophils are a potential high-concentration source of blood PBEF, we studied mechanisms of PBEF release from isolated human neutrophils that could help design novel resuscitation strategies. Methods: Human blood neutrophils were isolated by Percoll density gradient. PBEF secretion into neutrophil supernatant was measured by ELISA. Results: Non-stimulated neutrophils (i.e. exposed to serum-starved conditions) released PBEF. PBEF in supernatant was observed at 5 min and plateaued after 30 min at 37oC. This release was not due to cell injury since LDH was unchanged, nor was it blocked by the classical secretion inhibitors, brefeldin A (blocks ER-Golgi transport) or nocodazole (blocks microtubule polymerization). As PBEF product NAD+ is consumed by multiple intra- and ecto-NADases, we determined whether inhibition of NADase activity impairs PBEF release. Nicotinamide, a global inhibitor of all NADases, blocked PBEF secretion by 73%, suggesting NADases promote PBEF secretion. To identify the specific NADase in PBEF secretion, neutrophils were treated with inhibitors for PARP, sirtuin, CD38 or mono-ADP-ribosyltransferase. We found that inhibition of CD38 by tannic acid or apigenin was able to block PBEF secretion in neutrophils (10.2 ± 2.8 ng/ml for control vs 1.8 ± 0.2 ng/ml for tannic acid at 100 μM, P &lt; 0.01), but not by inhibitors of the other NADases. This observation indicated that CD38 glycohydroase activity is required for PBEF secretion. To further support the role of tannic acid in CD38 inhibition, tannic acid was shown to inhibit ε-ADP ribose production when neutrophils were treated with ε-NAD+. Finally, PBEF secretion was significantly enhanced by oxidative stress using H2O2 exposure. Conclusions: PBEF is constitutively released from neutrophils and this release is regulated by CD38 NAD glycohydrolase activity. Changes in human neutrophil microenvironment, especially oxidative stress, result in significant PBEF release within a timeframe that could account for the blood changes seen during mouse cardiovascular resuscitation.

Fresh frozen plasma and spray-dried plasma mitigate pulmonary vascular permeability and inflammation in hemorrhagic shock.

In retrospective and prospective observational studies, fresh frozen plasma (FFP) has been associated with a survival benefit in massively transfused trauma patients. A dry plasma product, such as spray-dried plasma (SDP), offers logistical advantages over FFP. Recent studies on FFP have demonstrated that FFP modulates systemic vascular stability and inflammation. The effect of SDP on these measures has not been previously examined. This study compares SDP with FFP using in vitro assays of endothelial function and in vivo assays of lung injury using a mouse model of hemorrhagic shock (HS) and trauma. FFP, SDP, and lactated Ringer's (LR) solution were compared in vitro using assays of endothelial cell (EC) permeability, cytokine production and content, gene expression, as well as tight and adherens junction stability. All resuscitation products were also compared in a murine model of HS. Mean arterial pressures and physiologic measures were assessed. Pulmonary vascular permeability was measured using tagged dextran. Lung tissues were stained for CD68, VE-cadherin, and occludin. Treatment of ECs with FFP and SDP, but not LR, preserved the integrity of EC monolayers in vitro and resulted in similar EC gene expression patterns and cytokine/growth factor production. FFP and SDP also reduced HS-induced pulmonary vascular permeability in vivo to the same extent. In mice with HS, mean arterial pressures and base excess were corrected by both FFP and SDP to levels observed in sham-treated mice. Treatment after HS with FFP and SDP but not LR solution reduce alveolar wall thickening, leukocyte infiltration, and the breakdown of EC junctions, as measured by staining for VE-cadherin, and occludin. Both FFP and SDP similarly modulate pulmonary vascular integrity, permeability, and inflammation in vitro and in vivo in a murine model of HS and trauma.

Minocycline and doxycycline, but not tetracycline, mitigate liver and kidney injury after hemorrhagic shock/resuscitation.

Despite recovery of hemodynamics by fluid resuscitation after hemorrhage, development of the systemic inflammatory response and multiple organ dysfunction syndromes can nonetheless lead to death. Minocycline and doxycycline are tetracycline derivatives that are protective in models of hypoxic, ischemic, and oxidative stress. Our aim was to determine whether minocycline and doxycycline protect liver and kidney and improve survival in a mouse model of hemorrhagic shock and resuscitation. Mice were hemorrhaged to 30 mmHg for 3 h and then resuscitated with shed blood followed by half the shed volume of lactated Ringer's solution containing tetracycline (10 mg/kg), minocycline (10 mg/kg), doxycycline (5 mg/kg), or vehicle. For pretreatment plus posttreatment, drugs were administered intraperitoneally prior to hemorrhage followed by second equal dose in Ringer's solution after blood resuscitation. Blood and tissue were harvested after 6 h. Serum alanine aminotransferase (ALT) increased to 1,988 and 1,878 U/L after posttreatment with vehicle and tetracycline, respectively, whereas minocycline and doxycycline posttreatment decreased ALT to 857 and 863 U/L. Pretreatment plus posttreatment with minocycline and doxycycline also decreased ALT to 849 and 834 U/L. After vehicle, blood creatinine increased to 134 µM, which minocycline and doxycycline posttreatment decreased to 59 and 56 µM. Minocycline and doxycycline pretreatment plus posttreatment decreased creatinine similarly. Minocycline and doxycycline also decreased necrosis and apoptosis in liver and apoptosis in both liver and kidney, the latter assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) and caspase 3 activation. Lastly after 4.5 h of hemorrhage followed by resuscitation, minocycline and doxycycline (but not tetracycline) posttreatment improved 1-week survival from 38% (vehicle) to 69% and 67%, respectively. Minocycline and doxycycline were similarly protective when given before as after blood resuscitation and might therefore have clinical efficacy to mitigate liver and kidney injury after resuscitated hemorrhage.

Toll-like receptor 4 regulates platelet function and contributes to coagulation abnormality and organ injury in hemorrhagic shock and resuscitation.

Growing evidence indicates that the presence of toll-like receptor 4 (TLR4) on platelets is a key regulator of platelet number and function. Platelets exposed to TLR4 agonists may serve to activate other cells such as neutrophils and endothelial cells in sepsis and other inflammatory conditions. The functional significance of platelet TLR4 in hemorrhagic shock (HS), however, remains unexplored. Using thromboelastography and platelet aggregometry, we demonstrate that platelet function is impaired in a mouse model of HS with resuscitation. Further analysis using cellular-specific TLR4 deletion in mice revealed that platelet TLR4 is essential for platelet activation and function in HS with resuscitation and that platelet TLR4 regulates the development of coagulopathy after hemorrhage and resuscitation. Transfusion of TLR4-negative platelets into mice resulted in protection from coagulopathy and restored platelet function. Additionally, platelet-specific TLR4 knockout mice were protected from lung and liver injury and exhibited a marked reduction in systemic inflammation as measured by circulating interleukin-6 after HS with resuscitation. We demonstrate for the first time that platelet TLR4 is an essential mediator of the inflammatory response as well as platelet activation and function in HS and resuscitation.

Augmenter of Liver Regeneration (ALR) Is a Novel Biomarker of Hepatocellular Stress/Inflammation: In Vitro, In Vivo and In Silico Studies

The liver is a central organ involved in inflammatory processes, including the elaboration of acute-phase proteins. Augmenter of liver regeneration (ALR) protein, expressed and secreted by hepatocytes, promotes liver regeneration and maintains viability of hepatocytes. ALR also stimulates secretion of inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and nitric oxide from Kupffer cells. We hypothesized that ALR may be involved in modulating inflammation induced by various stimuli. We found that hepatic ALR levels are elevated at 24 h, before or about the same time as an increase in the mRNA expression of TNF-α and IL-6, after portacaval shunt surgery in rats. Serum ALR also increased, but significantly only on d 4 when pathological changes in the liver become apparent. In rats, serum ALR was elevated after intraperitoneal administration of lipopolysaccharide alone and in a model of gram-negative sepsis. Serum ALR increased before alanine aminotransferase (ALT) in endotoxemia and in the same general time frame as TNF-α and IL-6 in the bacterial sepsis model. Furthermore, mathematical prediction of tissue damage correlated strongly with alterations in serum ALR in a mouse model of hemorrhagic shock. In vitro, monomethyl sulfonate, TNF-α, actinomycin D and lipopolysaccharide all caused increased release of ALR from rat hepatocytes, which preceded the loss of cell viability and/or inhibition of DNA synthesis. ALR may thus serve as a potential diagnostic marker of hepatocellular stress and/or acute inflammatory conditions.

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Reply We are responding to concerns raised in this Letter to the Editor that the solvent used during delivery of the specific caspase 1 inhibitor, AC-YVAD-cmk, to mice may have contributed to the observation that caspase 1 inhibition significantly increased the mortality in burn mice. We understand their concerns but wish to assure them and other readers that the concentration of solvent used in our studies was significantly lower than what has been reported to cause inflammation and increases in immune cell apoptosis in mice. The referenced study used 8 mg/kg of dimethyl sulfoxide (DMSO) before significant changes in immune cell apoptosis were observed (1). The amount of DMSO given with our AC-YVAD-cmk treatment was 0.5 mg/kg (2). This is 16-fold less than what was reported to cause apoptosis in mice. Other studies using DMSO to study changes in inflammatory cytokine production were done in vitro or at higher doses than what we used to deliver AC-YVAD-cmk (1, 3). Also, the stimulus used to induce inflammation was bacterial lipopolysaccharide (LPS), and the inflammatory reactions caused by LPS and trauma are not similar. In addition, one of the cytokines reported to be induced by DMSO exposure and LPS was interleukin 1β (IL-1β) (3). It is important to restate in this letter that we clearly showed that giving AC-YVAD-cmk in DMSO potently blocked injury-induced IL-1β production in vivo (2). Moreover, we performed in vivo dosing studies to demonstrate that giving AC-YVAD-cmk in DMSO to mice at doses that specifically reduced injury-induced caspase 1 activation led to significant mortality in burn mice. Finally, in control experiments comparing saline to vehicle (0.5 mg/kg DMSO), we found no measurable effect on mortality (n = 10 mice/group over 7 days) or systemic levels of injury-induced IL-6 production. We thank them for their opinions and interest in our research report. In closing, based on our findings, we are currently expanding our efforts to determine how inflammasome activation by injury protects mice from injury-induced complications. We recently found that caspase 1−/− mice also showed an enhanced inflammatory phenotype at 1 day after injury. Furthermore, a recent study using caspase 1−/− mice confirmed our findings showing that that caspase 1 was protective in a trauma mouse model of hemorrhagic shock with combined femur fracture (4). Thus, we are keenly interested in determining if stimulating inflammasome or caspase 1 activation could be used to protect from the acute complications of traumatic injuries. Akinori Osuka Marc Hanschen Veit Stoecklein James Lederer Brigham and Women’s Hospital Biomedical Research Institute and Harvard Medical School Boston, Massachusetts

The role of TLR4 and free fatty acids on hepatocellular injury: an obese mouse model of hemorrhagic shock

The role of TLR4 and free fatty acids on hepatocellular injury: an obese mouse model of hemorrhagic shock