Double-hit Model Research Articles

Hypothermia Does Not Reverse Cellular Responses Caused by Lipopolysaccharide in Neonatal Hypoxic-Ischaemic Brain Injury

Introduction: Bacterial lipopolysaccharide (LPS) injection prior to hypoxia-ischaemia significantly increases hypoxia-ischaemic brain injury in 7-day-old (P7) rats. In addition, therapeutic hypothermia (HT) is not neuroprotective in this setting. However, the mechanistic aspects of this therapeutic failure have yet to be elucidated. This study was designed to investigate the underlying cellular mechanisms in this double-hit model of infection-sensitised hypoxia-ischaemic brain injury. Material and Methods: P7 rat pups were injected with either vehicle or LPS, and after a 4-hour delay were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like hypoxia-ischaemic injury. Pups were randomised to the following treatments: (1) vehicle-treated pups receiving normothermia treatment (NT) (Veh-NT; n = 40), (2) LPS-treated pups receiving NT treatment (LPS-NT; n = 40), (3) vehicle-treated pups receiving HT treatment (Veh-HT; n = 38) and (4) LPS-treated pups receiving HT treatment (LPS-HT; n = 35). On postnatal day 8 or 14, Western blot analysis or immunohistochemistry was performed to examine neuronal death, apoptosis, astrogliosis and microglial activation. Results: LPS sensitisation prior to hypoxia-ischaemia significantly exacerbated apoptotic neuronal loss. NeuN, a neuronal biomarker, was significantly reduced in the LPS-NT and LPS-HT groups (p = 0.008). Caspase-3 activation was significantly increased in the LPS-sensitised groups (p < 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p < 0.001) was seen, as well as a trend towards increased microglial activation (Iba 1 expression, p = 0.051) in LPS-sensitised animals. Treatment with HT did not counteract these changes. Conclusion: LPS-sensitised hypoxia-ischaemic brain injury in newborn rats is mediated through neuronal death, apoptosis, astrogliosis and microglial activation. In this double-hit model, treatment with HT does not ameliorate these changes.

GABA/Glutamate synaptic pathways targeted by integrative genomic and electrophysiological explorations distinguish autism from intellectual disability.

Phenotypic and genetic heterogeneity is predominant in autism spectrum disorders (ASD), for which the molecular and pathophysiological bases are still unclear. Significant comorbidity and genetic overlap between ASD and other neurodevelopmental disorders are also well established. However, little is understood regarding the frequent observation of a wide phenotypic spectrum associated with deleterious mutations affecting a single gene even within multiplex families. We performed a clinical, neurophysiological (in vivo electroencephalography-auditory-evoked related potentials) and genetic (whole-exome sequencing) follow-up analysis of two families with known deleterious NLGN4X gene mutations (either truncating or overexpressing) present in individuals with ASD and/or with intellectual disability (ID). Complete phenotypic evaluation of the pedigrees in the ASD individuals showed common specific autistic behavioural features and neurophysiological patterns (abnormal MisMatch Negativity in response to auditory change) that were absent in healthy parents as well as in family members with isolated ID. Whole-exome sequencing in ASD patients from each family identified a second rare inherited genetic variant, affecting either the GLRB or the ANK3 genes encoding NLGN4X interacting proteins expressed in inhibitory or in excitatory synapses, respectively. The GRLB and ANK3 mutations were absent in relatives with ID as well as in control databases. In summary, our findings provide evidence of a double-hit genetic model focused on excitatory/inhibitory synapses in ASD, that is not found in isolated ID, associated with an atypical in vivo neurophysiological pattern linked to predictive coding.

Dynamics of hepatic gene expression and serum cytokine profiles in single and double-hit burn and sepsis animal models.

We simulate the pathophysiology of severe burn trauma and burn-induced sepsis, using rat models of experimental burn injury and cecal ligation and puncture (CLP) either individually (singe-hit model) or in combination (double-hit model). The experimental burn injury simulates a systemic but sterile pro-inflammatory response, while the CLP simulates the effect of polymicrobial sepsis. Given the liver׳s central role in mediating the host immune response and onset of hypermetabolism after burn injury, elucidating the alterations in hepatic gene expression in response to injury can lead to a better understanding of the regulation of the inflammatory response, whereas circulating cytokine protein expression, reflects key systemic inflammatory mediators. In this article, we present both the hepatic gene expression and circulating cytokine/chemokine protein expression data for the above-mentioned experimental model to gain insights into the temporal dynamics of the inflammatory and hypermetabolic response following burn and septic injury. This data article supports results discussed in research articles (Yang et al., 2012 [1,4]; Mattick et al. 2012, 2013 [2,3]; Nguyen et al., 2014 [5]; Orman et al., 2011, 2012 [6–8]).

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoetic Stem and Progenitor Cells

In recent clinical studies, it has been demonstrated that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). Mutation of ASXL1 leads to truncation of the protein and thereby to a loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in hematopoietic system in vitro mimics the changes observed in MDS-patients.We down regulated ASXL1 in CD34+ cord blood (CB) cells using a lentiviral approach and obtained a 40-50% reduction of ASXL1 expression. Colony forming (CFC) assays revealed that erythroid colony formation was significantly impaired (p=0.01) and, to some extent, granulocytic and macrophage colony formation (p=0.09, p=0.05 respectively). As MDS can affect all hematopoietic lineages, we first stimulated cell differentiation along the myeloid or erythroid lineage in liquid culture. Upon culturing shASXL1 CB CD34+ cells in suspension, we observed a modest reduction in expansion (two-fold at week1) under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p=0.02). The increase of cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. This phenotype is similar to what has been observed in patients, where increased cell death of progenitors occurs, and suggests that ASXL1 loss may reflect an MDS-like phenotype in this culture setting. Furthermore, as MDS is considered a hematopoietic stem cell (HSC)-driven disorder, we tested whether HSCs were affected by ASXL1 loss. Long-term culture initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, we performed cultures on stromal layers with or without cytokines. shASXL1 CB CD34+ cells cultured on MS5 stromal layer showed a modest, two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. In patients, mutations in ASXL1 are frequently accompanied by a loss of p53. Possibly, loss of p53 is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our first data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies compared to shASXL1/shSCR cells, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, when using erythroid liquid conditions, a decrease in apoptosis compared to the ASXL1 single mutation could be observed. Nevertheless, no transformation occurred and ASXL1 mutated cells were eventually lost in the double hit model despite reduced apoptosis, suggesting that the p53 axis might not be sufficient as the second hit for full transformation.In conclusion, our data indicate that mutations in ASXL1 may lead to an increase in cell death and reduced progenitor output in vitro, which may reflect disease development and progression as seen in patients. Unexpectedly, MS5 stromal did not alter the negative phenotype caused by ASXL1 knock down. Therefore, studies are ongoing to investigate whether an already established MDS microenvironment will influence ASXL1 mutation positively. To this end, we are using healthy human mesenchymal stem cells (MSC) and patient derived MDS MSCs. DisclosuresNo relevant conflicts of interest to declare.

Characterization of a double-hit murine model of acute respiratory distress syndrome.

The aim of the present study was to characterize a murine model of acute respiratory distress syndrome (ARDS) abiding by the Berlin definition of human ARDS and guidelines for animal models of ARDS. To this end, C57BL/6NCrl mice were challenged with lipopolysaccharide (LPS; 15 mg/kg, i.p.) followed 18 h later by injection of oleic acid (OA; 0.12 mL/kg, i.v.). Controls received saline injection at both time points. Haemodynamics were monitored continuously. Arterial blood gas analyses were performed just before and every 30 min after OA challenge. Ninety minutes after OA challenge, the chest of mice was scanned using micro-computed tomography (CT). Cytokine concentrations were measured in plasma samples. Lungs were harvested 90 min after OA challenge for histology, immunohistochemistry, lung weight measurements and tissue cytokine detection. A histological lung injury score was determined. Eighteen hours after LPS challenge, mice exhibited a severe systemic inflammatory response syndrome. Oxygenation declined significantly after OA injections (Pa o2 /Fi o2 283 ± 73 and 256 ± 71 mmHg at 60 and 90 min, respectively; P < 0.001). Bilateral patchy infiltrates were present on the micro-CT scans. Histology revealed parenchymal damage with accumulation of polymorphonuclear neutrophils, intra-alveolar proteinacous debris and few hyaline membranes. The lung wet : dry ratio indicated damage to the alveolar capillary membrane. Cytokine patterns evidenced a severe local and systemic inflammatory state in plasma and lung tissue. In conclusion, the described two-hit model of ARDS shows a pathological picture of ARDS closely mimicking human ARDS according to the Berlin definition and may facilitate interpretation of prospective experimental results.

A double-hit model of stress dysregulation in rats: implications for limbic corticosteroid receptors and anxious behavior under amitriptyline treatment

Adversity during early life can lead to diverging endocrine and behavioral responses to stress in adulthood. In our laboratory, we evaluated the long-term effects of early life adversity and its interaction with chronic stress during adulthood. We propose this as a model of vulnerability to dysregulation of the stress response. We hypothesized that rats subjected to both protocols would show differential expression of corticosteroid receptors measured as number of neurons immunoreactive for glucocorticoid receptors (GR) or mineralocorticoid receptors (MR), in limbic areas related to the control of anxiety-like behavior. We also evaluated the effect of amitriptyline expecting to prevent the outcomes of the model. Male Wistar rats were separated from the mother (MS) for 4.5 h every day for the first 3 weeks of life. From postnatal day 50, rats were subjected to chronic variable stress (CVS) during 24 d (five types of stressor at different times of day). During the stress protocol, the rats were administered amitriptyline (10 mg/kg i.p.) daily. MS evoked lower MR expression in the central amygdaloid nucleus and this was reversed by amitriptyline. Furthermore, CVS increased MR immunoreactivity in the hippocampal area CA2 and increased anxious behavior; both effects were prevented by the antidepressant. When MS was combined with CVS during adulthood, there was a reduction of locomotor activity, with no corrective effect of amitriptyline. The differential effects among groups could mean that MS would promote an alternative phenotype that is expressed when facing CVS (a double hit) later in life.

Human Umbilical Cord Blood Mononuclear Cells in a Double-Hit Model of Bronchopulmonary Dysplasia in Neonatal Mice

BackgroundBronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia.ObjectivesWe investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD.MethodsFor the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups.ResultsCompared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased.ConclusionThe results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.

Bronchopulmonary dysplasia in a double-hit mouse model induced by intrauterine hypoxia and postnatal hyperoxia: Closer to clinical features?

Despite increased survival of very preterm newborns, bronchopulmonary dysplasia (BPD) remains a major threat, as it affects long-term pulmonary function and neurodevelopmental outcome. Recent research focused on mechanisms of lung repair. Animal models of BPD in term rodents use postnatal hyperoxia in order to mimic features observed in very preterm human neonates: reduced alveolarization and impaired septal architecture without profound inflammatory changes. In contrast, BPD in very preterm human neonates involves prenatal hits e.g. infections and growth restriction plus postnatal ventilation. BPD induced in rodents by postnatal hyperoxia also exhibits reduced alveolarization however without septal pathology but with marked inflammation. We therefore aimed to establish an animal model combining prenatal growth restriction (FiO₂ 0.1 for 4 days) with postnatal hyperoxia (FiO₂ 0.7 for 2 weeks). In double-hit mice the development was retarded: body weight and length, lung and brain weight were significantly reduced by day P14 compared with normoxic controls. Histomorphometric analysis revealed reduced alveolarization and increased septal thickness without pronounced inflammatory lesions. A down-regulation of SftpB and SftpC genes was observed in double-hit animals compared with controls. Thus, we established a new model of BPD using pre- and postnatal hits.

Apoptotic and inflammatory signaling via Fas and tumor necrosis factor receptor I contribute to the development of chest trauma-induced septic acute lung injury

Direct acute lung injury (ALI) is still associated with a high mortality, whereas the underlying pathomechanisms are not yet fully understood. In this regard, epithelial cell death in the lungs has been attributed an important role in the pathogenesis of this clinical entity. Based on this background here, we hypothesized that signaling through Fas and tumor necrosis factor receptor 1 (TNFR-1) is involved in mediating apoptosis and inflammation in chest trauma induced septic ALI. Male C57BL/6 mice (wild-type [WT]), male mutant mice expressing nonfunctional Fas receptor (B6.MRL-Faslpr/J [lpr]) (lpr) and male TNFR-1-deficient mice (TNFR-1(-/-)) were subjected to a model of direct ALI consisting of blunt chest trauma followed by cecal ligation and puncture.Cytokine/chemokine concentrations of plasma, bronchoalveolar lavage (BAL) fluids, and lung tissue were investigated as well as BAL protein and lung myeloperoxidase. Lung histology was assessed; lung caspase 3, TUNEL-positive cells, and apoptotic polymorphonuclear neutrophil were measured, followed by a survival study. Cytokine/chemokine levels in plasma, BAL, and lung tissue were markedly increased in WT animals following ALI, whereas lpr and TNFR-1((-/-) showed significantly decreased levels. BAL protein levels were substantially elevated following ALI, but lpr animals presented markedly diminished protein levels compared with WT and TNFR-1(-/-) animals. Lung myeloperoxidase level was only increased 12 hours after ALI in WT animals, whereas lung myeloperoxidase levels in lpr and TNFR-1(-/-) animals were not increased compared with sham. Lung histology revealed beneficial effects in lpr and TNFR-1(-/-). Lung active caspase 3 after ALI was substantially decreased in lpr and TNFR-1(-/-) mice compared with WT. Interestingly, an early but not persisting survival benefit was observed in lpr and TNFR-1 animals(-/-). Pathomechanistically, Fas and TNFR-1 signaling contributed to the apoptotic and inflammatory response in a clinically relevant double-hit model of trauma-induced septic ALI. Moreover, this was associated with a temporary survival benefit.

SILENCING OF FAS, FAS-ASSOCIATED VIA DEATH DOMAIN, OR CASPASE 3 DIFFERENTIALLY AFFECTS LUNG INFLAMMATION, APOPTOSIS, AND DEVELOPMENT OF TRAUMA-INDUCED SEPTIC ACUTE LUNG INJURY

Activation of Fas signaling is a potentially important pathophysiological mechanism in the development of septic acute lung injury (ALI). However, so far the optimal targets within this signaling cascade remain elusive. Thus, we tested the hypothesis that in vivo gene silencing of Fas, Fas-associated via death domain (FADD), or caspase 3 by intratracheal administration of small interfering RNA would ameliorate ALI in a clinically relevant double-hit mouse model of trauma induced septic lung injury. Male C57Bl/6 mice received small interfering (Fas, FADD, caspase 3) or control RNA 24 h before and 12 h after blunt chest trauma or sham procedures. Polymicrobial sepsis was induced by cecal ligation and puncture 24 h after chest trauma. Twelve or 24 h later, lung tissue, plasma, and bronchoalveolar lavage fluid were harvested. During ALI, lung apoptosis (active caspase 3 Western blotting, TUNEL staining) was substantially increased when compared with sham. Silencing of caspase 3 or FADD both markedly reduced pulmonary apoptosis. Fas- and FADD-small interfering RNA administration substantially decreased lung cytokine concentration, whereas caspase 3 silencing did not reduce lung inflammation. In addition, Fas silencing markedly decreased lung neutrophil infiltration. Interestingly, only in response to caspase 3 silencing, ALI-induced lung epithelial barrier dysfunction was substantially improved, and histological appearance was beneficially affected. Taken together, downstream inhibition of lung apoptosis via caspase 3 silencing proved to be superior in mitigating ALI when compared with upstream inhibition of apoptosis via Fas or FADD silencing, even in the presence of additional anti-inflammatory effects. This indicates a major pathophysiological role of lung apoptosis and suggests the importance of other than Fas-driven apoptotic pathways in trauma-induced septic ALI.

Experimental BPD – cells from human umbilical cord blood improve lung development in a double-hit mouse model

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in neonates with low gestational age and low birth weight. Besides the consequences for the neonatal period, the disease also affects the further development of the patients with lifelong consequences like persistent lung abnormalities. BPD has a multifactorial etiology with main risk factors being prematurity, low birth weight, oxygen therapy or infection. Among other symptoms, “new” BPD often presents with alveolar arrest and impaired lung development. Several studies showed improvement of lung development in rodent models using stem cells of different origin. Objectives: To investigate the effects of human umbilical cord blood cells (buffy coat), including progenitor and stem cells on the lung development in a BPD double-hit model. Methods: We established a new double-hit animal model in the mouse, combining prenatal hypoxia (4 days) and postnatal hyperoxia (13 days), thus combining lung damage with low birth weight to get closer to the multifactorial situation in humans. The mice were intraperitoneally injected with cells from human umbilical cord blood at postnatal day seven. The mRNA expression in lung tissue was analysed for SPB, SPC, VEGF, Elastin, HIF1a, mTOR, IGF1, RARα, RARβ, RARγ, CRABP1, RBP1, TGFb1, TGFb2, TGFb3. Results: The double-hit mice were smaller at birth than normal mice and developed more slowly. The lung structure in the double-hit mice was less developed compared to normal mice. Septal surface was decreased and septa were thickened. Treatment with buffy coat cells resulted in a tendency towards better growth of the pups. Lung structure was improved compared to untreated mice. Furthermore there was a tendency to a changed expression of several genes, being the most clear for mTOR. Conclusions: Treatment with human umbilical cord blood cells has an overall positive effect on growth and development in general and lung development in particular in our double-hit mouse model for BPD. Further studies are necessary in order to further improve stem cell therapy in experimental BPD.

Distribution of Zonula Occludens‐1 and Occludin and alterations of testicular morphology after in utero radiation and postnatal hyperthermia in rats

In utero irradiation (IR) and postnatal hyperthermia (HT) exposure cause infertility by decreasing spermatogenic colony growth and the number of sperm in rats. Four groups were used: (i) Control group, (ii) HT group (rats exposed to hyperthermia on the 10th postnatal day), (iii) IR group (rats exposed to IR on the 17th gestational day) and (iv) IR+HT group. Three and six months after the procedures testes were examined by light and electron microscopy. Some degenerated tubules in the HT group, many vacuoles in spermatogenic cells and degenerated tight junctions in the IR group, atrophic tubules and severe degeneration of tight junctions in the IR+HT group were observed. ZO-1 and occludin immunoreactivity were decreased and disorganized in the HT and IR groups and absent in the IR+HT group. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in haploid, diploid and tetraploid cells in all groups. Degenerative findings were severe after 6months in all groups. The double-hit model may represent a Sertoli cell only model of infertility due to a decrease in spermatogenic cell and alterated blood-testis barrier proteins in rat.

Target organ cross talk in cardiorenal syndrome: animal models

The combination of chronic kidney disease (CKD) and heart failure (HF) is associated with an adverse prognosis. Although clinical studies hint at a specific bidirectional interaction between HF and CKD, insight into the pathogenesis of cardiorenal syndrome (CRS) remains limited. We review available evidence on cardiorenal interactions from animal models of CKD and HF and discuss several studies that employed a "double-hit" model to research organ cross talk between the heart and kidneys. Regarding cardiac changes in CKD models, parameters of cardiac remodeling are equivocal and cardiac systolic function generally remains preserved. Structural changes include hypertrophy, fibrosis, and microvasculopathy. In models of HF, data on renal pathology are mostly limited to functional hemodynamic changes. Most double-hit models were unable to show that combined renal and cardiac injury induces additive damage to both organs, perhaps because of the short study duration or absence of organ failure. Because of this lack of "dual-failure" models, we have developed two rat models of combined CKD and HF in which renal dysfunction induced by a subtotal nephrectomy preceded cardiac dysfunction. Cardiac dysfunction was induced either functionally by nitric oxide depletion or structurally by myocardial infarction. In both models, we found that cardiac remodeling and failure were worse in CKD rats compared with controls undergoing the same cardiac insult. Variables of renal damage, like glomerulosclerosis and proteinuria, were also further worsened by combined cardiorenal injury. These studies show that target organ cross talk does occur in CRS. These models may be useful for interventional studies in rats.

Role of Activated Neutrophils in Chest Trauma–Induced Septic Acute Lung Injury

More than 50% of severely injured patients have chest trauma. Second insults frequently result in acute lung injury (ALI), with sepsis being the main underlying condition. We aimed to develop a standardized, reproducible, and clinically relevant double-hit mouse model of ALI induced by chest trauma and polymicrobial sepsis and to investigate the pathophysiologic role of activated neutrophils. Lung contusion was applied to C57Bl/6 mice via a focused blast wave. Twenty-four hours later, sepsis was induced by cecal ligation and puncture. For polymorphonuclear leukocyte (PMN) depletion, animals received intravenous injections of PMN-depleting antibody. In response to blunt chest trauma followed by sepsis as well as after sepsis alone, a significant local and systemic inflammatory response with increased cytokine/chemokine levels in lung and plasma was observed. In contrast, lung apoptosis was markedly elevated only after a double hit. Intra-alveolar neutrophils and total bronchoalveolar lavage protein concentrations were markedly increased following isolated chest trauma or the combined insult, but not after sepsis alone. Lung myeloperoxidase activity was enhanced only in response to the double hit accompanied by histological disruption of the alveolar architecture, lung congestion, and marked cellular infiltrates. Neutrophil depletion significantly diminished lung interleukin 1β and interleukin 6 concentrations and reduced the degree of septic ALI. Here we have established a novel and highly reproducible mouse model of chest trauma-induced septic ALI characterizing a clinical relevant double-hit scenario. In particular, the depletion of neutrophils substantially mitigated the extent of lung injury, indicating a pathomechanistic role for neutrophils in chest trauma-induced septic ALI.

The dynamics of the early inflammatory response in double-hit burn and sepsis animal models

Severe burn trauma is generally associated with bacterial infections, which causes a more persistent inflammatory response with an ongoing hypermetabolic and catabolic state. This complex biological response, mediated by chemokines and cytokines, can be more severe when excessive interactions between the mediators take place. In this study, the early inflammatory response following the cecum ligation and puncture (CLP) or its corresponding control treatment (sham-CLP or SCLP) in burn (B) male rats was analyzed by measuring 23 different cytokines and chemokines. Cytokines and chemokines, including MCP-1, IP-10, leptin, TNF-α, MIP-1α, IL-18, GMCSF, RANTES and GCSF were significantly altered in both B+CLP and B+SCLP groups. IL-10 and IL-6 were significantly up-regulated in the B+CLP group when compared to the B+SCLP group. Down regulation of leptin and IP-10 concentrations were found to be related to surgery and/or infection. IL-18 and MCP-1 were elevated in all groups including previously published single injury models receiving similar treatments. In this study, insult-specific mediators with their characteristic temporal patterns were elucidated in double hit models.

Subchronic MK-801 treatment and post-weaning social isolation in rats: Differential effects on locomotor activity and hippocampal long-term potentiation

Subchronic NMDA receptor antagonist treatment and post-weaning social isolation are two animal models of schizophrenia symptoms. However, behavioral and physiological changes following a combination of these two procedures have not been investigated. Thus, we examined effects of a novel, “double hit” model combining these two treatments, comparing them to standard models involving only NMDA antagonist treatment or social isolation. Male, Sprague–Dawley rats were either group-housed or maintained in social isolation (starting at postnatal day [PD] 21 and continuing throughout the study). Each housing condition was further subdivided into two groups, receiving either subchronic treatment with either saline or MK-801 (0.5 mg/kg, i.p., 2 × day for seven days starting at PD 56). Post-weaning social isolation increased locomotor activity (assessed at PD 70) in response to a novel environment and an acute amphetamine injection, while subchronic MK-801 increased only amphetamine induced locomotor activity. Subsequent electrophysiological experiments (under urethane anesthesia) assessing changes in plasticity of hippocampal synapses showed that subchronic MK-801 treatment resulted in an increase in long-term potentiation in area CA1 in response to high frequency stimulation of the contralateral CA3 area, while housing condition had no effect. No other changes in hippocampal electrophysiology (input–output curves, paired-pulse facilitation) were observed. These data are the first to demonstrate an enhancement in hippocampal long-term plasticity in vivo following subchronic MK-801 administration, an effect that may be related to the well-characterized changes in glutamatergic and GABAergic systems seen after subchronic NMDA receptor blockade. That lack of additive or synergistic effects in the “double hit model” suggests that combining isolation and subchronic MK-801 treatment does not necessarily produce greater behavioral or physiological dysfunction than that seen with either treatment alone.

Acute Lung Injury in Patients with Severe Brain Injury: A Double Hit Model

The presence of pulmonary dysfunction after brain injury is well recognized. Acute lung injury (ALI) occurs in 20% of patients with isolated brain injury and is associated with a poor outcome. The "blast injury" theory, which proposes combined "hydrostatic" and "high permeability" mechanisms for the formation of neurogenic pulmonary edema, has been challenged recently by the observation that a systemic inflammatory response may play an integral role in the development of pulmonary dysfunction associated with brain injury. As a result of the primary cerebral injury, a systemic inflammatory reaction occurs, which induces an alteration in blood-brain barrier permeability and infiltration of activated neutrophils into the lung. This preclinical injury makes the lungs more susceptible to the mechanical stress of an injurious ventilatory strategy. Tight CO2 control is a therapeutic priority in patients with acute brain injury, but the use of high tidal volume ventilation may contribute to the development of ALI. Establishment of a therapeutic regimen that allows the combination of protective ventilation with the prevention of hypercapnia is, therefore, required. Moreover, in patients with brain injury, hypoxemia represents a secondary insult associated with a poor outcome. Optimal oxygenation may be achieved by using an adequate FiO2 and by application of positive end-expiratory pressure (PEEP). PEEP may, however, affect the cerebral circulation by hemodynamic and CO2-mediated mechanisms and the effects of PEEP on cerebral hemodynamics should be monitored in these patients and used to titrate its application.

204: Acid is the priming insult for the double hit model of aspiration induced lung injury

Introduction: Aspiration induced lung injury is the third commonest cause of ARDS. However, only 30% of witnessed gastric aspiration lead to ARDS. Previous data from our laboratory has indicated a synergistic lung injury from CASP (combination of acid and particulate matter) compared to either gastric acid alone(ACID) or small non-acidified gastric particles (SNAP). The following experiments were conducted to study the pathogenesis of the synergistic lung injury in CASP by varying the sequential timing and order of ACID and SNAP components.

Downregulation of Hepatic Cytochrome P-450 Isoforms and PPAR-γ: Their Role in Hepatic Injury and Proinflammatory Responses in a Double-Hit Model of Hemorrhage and Sepsis

The "double-hit" model of hemorrhage and sepsis mimics the critically ill patient admitted to the surgical intensive care unit. Although the protein expression of a cytochrome (CYP) P-450 isoform CYP1A2 is reduced in the late stage of sepsis, the effect of hemorrhage on CYP isoforms and the anti-inflammatory nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has not been investigated. We hypothesized that hemorrhage down-regulates CYP isoforms and PPAR-gamma in the liver, which plays an important role in producing tissue injury and proinflammatory responses after the subsequent sepsis (i.e., double-hit). Male Sprague Dawley rats were divided into four groups. Animals in the double-hit group underwent hemorrhage (40 +/- 2 mmHg for 90 min) followed by fluid resuscitation. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) 20 h after hemorrhage, and the animals were sacrificed 4 h after CLP. Rats in the hemorrhage-alone group were sacrificed 20 h after the insult. Rats in the CLP-alone group were sacrificed 4 h after the onset of sepsis. Animals in the sham-operated group underwent neither hemorrhage nor CLP. The gene expression of P-450 isoforms (i.e., CYP1A2 and 2C11) and PPAR-gamma in the liver was determined using RT-PCR. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, and proinflammatory cytokines (i.e., IL-6, TNF-alpha) were also assessed. In the hemorrhage-alone group, hepatic mRNA expression of CYP1A2, CYP2C11, and PPAR-gamma was significantly down-regulated 20 h after the initial stress compared with sham-operated rats. Double-hit did not appear to further decrease CYP and PPAR-gamma gene expression. In contrast, serum levels of AST, ALT, lactate, IL-6, and TNF-alpha did not change significantly in either hemorrhaged or septic animals. Those organ injury indicators and cytokines, however, were significantly elevated after the double-hit of hemorrhage and sepsis. Hepatic CYP1A2, CYP2C11, and PPAR-gamma were down-regulated after the initial stress (hemorrhage). These down-regulated CYPs and PPAR-gamma seem to work as important factors contributing to the progression of organ injury and proinflammatory responses after the second stress (CLP).

Androstenediol ameliorates alterations in immune cells cytokine production capacity in a two-hit model of trauma-hemorrhage and sepsis

Although administration of androstenediol (a metabolite of dehydroepiandrosterone) following trauma-hemorrhage (T-H) produces beneficial effects on inflammatory cytokines and organ function, it remains unknown whether this metabolite has any salutary effects in preventing alterations in immune cell cytokine production following a combined insult of T-H and sepsis. To examine this, male rats underwent laparotomy, hemorrhagic shock (mean BP 40 mmHg for 90 min) and resuscitation or sham operation. Androstenediol (1 mg/kg BW i.v.) or vehicle was administered at the end of resuscitation. Twenty hrs after T-H or sham operation, sepsis was induced by cecal ligation and puncture (CLP). Five hours thereafter, plasma cytokine levels and cytokine production of various immune cells were determined. In a separate set of experiments, survival was monitored for 10 days after the induction of sepsis. Administration of androstenediol markedly decreased plasma IL-6 and TNF-α levels following T-H and CLP. Furthermore, it prevented the increased production of IL-6 and TNF-α by Kupffer cells and alveolar macrophages and attenuated the decrease in IL-6 and TNF-α production by splenic macrophages; however, it had no significant effects on the depressed IL-6 and TNF-α production by PBMC following T-H and CLP. The depressed IL-2 and IFN-γ production by splenocytes under those conditions was attenuated by the administration of androstenediol. Furthermore, survival rate following T-H and subsequent sepsis was improved by androstenediol treatment. Since androstenediol administration following T-H attenuated cytokine production and reduced mortality in a double-hit model of T-H and sepsis, this agent appears to be a novel and useful adjunct for maintaining the immune cell functions following T-H and for decreasing the mortality rate from subsequent susceptibility to sepsis.