A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

The Ohio State University Wexner Medical Center

Most adult humans have been infected with Epstein-Barr virus (EBV) and carry the latent virus. The EBV genome codes for several proteins that form an early antigen complex important for viral replication; one of these proteins is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The EBV-encoded dUTPase can induce sickness responses in mice. Because stress can increase latent virus reactivation, we hypothesized that chronic restraint would exacerbate sickness behaviors elicited by EBV-encoded dUTPase. Male Swiss-Webster mice were injected daily for 15 days with either saline or EBV-encoded dUTPase. Additionally, half of the mice from each condition were either restrained for 3h daily or left undisturbed. Restraint stress impaired learning and memory in the passive avoidance chamber; impaired learning and memory was due to EBV-encoded dUTPase injected into restrained mice. EBV-encoded dUTPase induced sickness responses and restraint stress interacts with EBV-encoded dUTPase to exacerbate the sickness response. These data support a role for EBV-encoded dUTPase and restraint stress in altering the pathophysiology of EBV independent of viral replication.

Epstein-Barr virus (EBV)-encoded dUTPase and chronic restraint induce impaired learning and memory and sickness responses.

The development and exacerbation of depression and anxiety are associated with exposure to repeated psychosocial stress. Stress is known to affect the bidirectional communication between the nervous and immune systems leading to elevated levels of stress mediators including glucocorticoids (GCs) and catecholamines and increased trafficking of proinflammatory immune cells. Animal models, like the repeated social defeat (RSD) paradigm, were developed to explore this connection between stress and affective disorders. RSD induces activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis activation, increases bone marrow production and egress of primed, GC-insensitive monocytes, and stimulates the trafficking of these cells to tissues including the spleen, lung, and brain. Recently, the observation that these monocytes have the ability to traffic to the brain perivascular spaces and parenchyma have provided mechanisms by which these peripheral cells may contribute to the prolonged anxiety-like behavior associated with RSD. The data that have been amassed from the RSD paradigm and others recapitulate many of the behavioral and immunological phenotypes associated with human anxiety disorders and may serve to elucidate potential avenues of treatment for these disorders. Here, we will discuss novel and key data that will present an overview of the neuroendocrine, immunological and behavioral responses to social stressors. 

Peripheral and central effects of repeated social defeat stress: monocyte trafficking, microglial activation, and anxiety.

Psychosocial stress is associated with altered immunity, anxiety and depression. Previously we showed that repeated social defeat (RSD) promoted microglia activation and social avoidance behavior that persisted for 24days after cessation of RSD. The aim of the present study was to determine if imipramine (a tricyclic antidepressant) would reverse RSD-inducedsocial avoidance and ameliorate neuroinflammatory responses. To test this, C57BL/6 mice were divided into treatment groups. One group from RSD and controls received daily injections of imipramine for 24days, following 6 cycles of RSD. Two other groups were treated with saline. RSD mice spent significantly less time in the interaction zone when an aggressor was present in the cage. Administration of imipramine reversed social avoidance behavior, significantly increasing the interaction time, so that it was similar to that of control mice. Moreover, 24days of imipramine treatment in RSD mice significantly decreased stress-induced mRNA levels for IL-6 in brain microglia. Following ex vivo LPS stimulation, microglia from mice exposed to RSD, had higher mRNA expression of IL-6, TNF-α, and IL-1β, and this was reversed by imipramine treatment. In a second experiment, imipramine was added to drinking water confirming the reversal of social avoidant behavior and decrease in mRNA expression of IL-6 in microglia. These data suggest that the antidepressant imipramine may exert its effect, in part, by down-regulating microglial activation. 

Imipramine attenuates neuroinflammatory signaling and reverses stress-induced social avoidance.

Allergic eosinophilic asthma is a chronic condition causing airway remodeling resulting in lung dysfunction. We observed that expression of sirtuin 2 (Sirt2), a histone deacetylase, regulates the recruitment of eosinophils after sensitization and challenge with a triple antigen: dust mite, ragweed, and Aspergillus fumigatus (DRA). Our data demonstrate that IL-4 regulates the expression of Sirt2 isoform 3/5. Pharmacological inhibition of Sirt2 by AGK2 resulted in diminished cellular recruitment, decreased CCL17/TARC, and reduced goblet cell hyperplasia. YM1 and Fizz1 expression was reduced in AGK2-treated, IL-4-stimulated lung macrophages in vitro as well as in lung macrophages from AGK2-DRA-challenged mice. Conversely, overexpression of Sirt2 resulted in increased cellular recruitment, CCL17 production, and goblet cell hyperplasia following DRA challenge. Sirt2 isoform 3/5 was upregulated in primary human alveolar macrophages following IL-4 and AGK2 treatment, which resulted in reduced CCL17 and markers of alternative activation. These gain-of-function and loss-of-function studies indicate that Sirt2 could be developed as a treatment for eosinophilic asthma.

Sirtuin 2 enhances allergic asthmatic inflammation.

Ischemia/reperfusion injury (IRI) can occur during liver surgery. Endogenous catalase is important to cellular antioxidant defenses and is critical to IRI prevention. Pegylation of catalase (PEG-CAT) improves its therapeutic potential by extending plasma half-life, but systemic administration of exogenous PEG-CAT has been only mildly therapeutic for hepatic IRI. Here, we investigated the protective effects of direct intrahepatic delivery of PEG-CAT during IRI using a rat hilar clamp model.

Intrahepatic Delivery of Pegylated Catalase Is Protective in a Rat Ischemia/Reperfusion Injury Model.

Meeting the metabolic demands of donor livers using normothermic ex vivo liver perfusion (NEVLP) preservation technology is challenging. The delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has been reported to decrease the metabolic demand in models of ischemia and cold preservation. We evaluated the therapeutic potential of DADLE by investigating its ability to protect against oxidative stress and hepatic injury during normothermic perfusion.

[D-Ala, D-Leu] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion.

Idiopathic pulmonary fibrosis is a deadly disease characterized by excessive extracellular matrix deposition in the lungs, resulting in decreased pulmonary function. Although epithelial cells and fibroblasts have long been the focus of idiopathic pulmonary fibrosis research, the role of various subpopulations of macrophages in promoting a fibrotic response is an emerging target. Healthy lungs are composed of two macrophage populations, tissue-resident alveolar macrophages and interstitial macrophages, which help to maintain homeostasis. After injury, tissue-resident alveolar macrophages are depleted, and monocytes from the bone marrow (BM) traffic to the lungs along a CCL2/CCR2 axis and differentiate into monocyte-derived alveolar macrophages (Mo-AMs), which is a cell population implicated in murine models of pulmonary fibrosis. In this study, we sought to determine how IL-1R-associated kinase-M (IRAK-M), a negative regulator of TLR signaling, modulates monocyte trafficking into the lungs in response to bleomycin. Our data indicate that after bleomycin challenge, mice lacking IRAK-M have decreased monocyte trafficking and reduced Mo-AMs in their lungs. Although IRAK-M expression did not regulate differences in chemokines, cytokines, or adhesion molecules associated with monocyte recruitment, IRAK-M was necessary for CCR2 upregulation following bleomycin challenge. This finding prompted us to develop a competitive BM chimera model, which demonstrated that expression of BM-derived IRAK-M was necessary for monocyte trafficking into the lung and for subsequent enhanced collagen deposition. These data indicate that IRAK-M regulates monocyte trafficking by increasing the expression of CCR2, resulting in enhanced monocyte translocation into the lung, Mo-AM differentiation, and development of pulmonary fibrosis.

Brenda F. Reader

Comprehensive Transplant Center

The Ohio State University Wexner Medical Center

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

Brenda F. Reader

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The Ohio State University Wexner Medical Center

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

Comprehensive Transplant Center