A Novel Role for the Endocannabinoid System in Ameliorating Motivation for Alcohol Drinking and Negative Behavioral Affect after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is associated with psychiatric dysfunction-including pain, cognitive impairment, anxiety, and increased alcohol use. We previously demonstrated that inhibiting endocannabinoid degradation post-TBI with JZL184 attenuates neuroinflammation and neuronal hyperexcitability at the site of injury and improves neurobehavioral recovery. This study aimed to determine the effect of JZL184 on post-TBI behavioral changes related to psychiatric dysfunction and post-TBI neuroadaptations in brain regions associated with these behaviors. We hypothesized that JZL184 would attenuate post-TBI behavioral and neural changes in alcohol-drinking rats. Adult male Wistar rats were trained to operantly self-administer alcohol before receiving lateral fluid percussion injury. Thirty minutes post-TBI, rats received JZL184 (16 mg/kg, i.p.) or vehicle. Spatial memory (Y-maze), anxiety-like behavior (open field), alcohol motivation (progressive ratio responding), and mechanosensitivity (Von Frey) were measured 3-10 days post-injury, and ventral striatum (VS) and central amygdala (CeA) tissue were collected for western blot analysis of phosphorylated glutamate receptor subunit 1 (GluR1) and glucocorticoid receptor (GR). TBI impaired spatial memory, increased anxiety-like behavior, and increased motivated alcohol drinking. JZL184 prevented these changes. TBI also increased phosphorylated GluR1 and GR in the CeA (but not the VS) compared with sham controls. JZL184 attenuated post-TBI GR phosphorylation in the CeA. These findings suggest that TBI produces comorbid cognitive dysfunction, increased alcohol motivation, and anxiety-like behavior, possibly related to amygdala dysfunction, and these changes are prevented by systemic post-TBI endocannabinoid degradation inhibition. Thus, boosting endocannabinoid tone post-TBI may represent a viable therapeutic strategy for TBI-related psychiatric comorbidities such as alcohol use disorder and anxiety.

Alcohol's Burden on Immunity Following Burn, Hemorrhagic Shock, or Traumatic Brain Injury.

Alcohol consumption contributes to increased incidence and severity of traumatic injury. Compared with patients who do not consume alcohol, alcohol-consuming patients have higher rates of long-term morbidity and mortality during recovery from injury. This can be attributed in part to an impaired immune response in individuals who consume alcohol. Acute and chronic alcohol use can affect both the innate and adaptive immune defense responses within multiple organ systems; the combination of alcohol use and injury results in increased susceptibility to bacterial and viral pathogens. This review examines the major deleterious effects of alcohol on immunity following tissue damage or traumatic injury, with a focus on alcohol's influence on the ability of the immune and major organ systems to fight disease and to repair damaged tissues following injury.

Traumatic brain injury induces neuroinflammation and neuronal degeneration that is associated with escalated alcohol self-administration in rats.

BACKGROUND: Traumatic brain injury (TBI) affects millions of people each year and is characterized by direct tissue injury followed by a neuroinflammatory response. The post-TBI recovery period can be associated with a negative emotional state characterized by alterations in affective behaviors implicated in the development of Alcohol Use Disorder in humans. The aim of this study was to test the hypothesis that post-TBI neuroinflammation is associated with behavioral dysfunction, including escalated alcohol intake. METHODS: Adult male Wistar rats were trained to self-administer alcohol prior to counterbalanced assignment into naïve, craniotomy, and TBI groups by baseline drinking. TBI was produced by lateral fluid percussion (LFP; >2 ATM; 25ms). Alcohol drinking and neurobehavioral function were measured at baseline and following TBI in all experimental groups. Markers of neuroinflammation (GFAP and ED1) and neurodegeneration (FJC) were determined by fluorescence histochemistry in brains excised at sacrifice 19 days post-TBI. RESULTS: The cumulative increase in alcohol intake over the 15 days post-TBI was greater in TBI animals compared to naïve controls. A higher rate of pre-injury alcohol intake was associated with a greater increase in post-injury alcohol intake in both TBI and craniotomy animals. Immediately following TBI, both TBI and craniotomy animals exhibited greater neurobehavioral dysfunction compared to naïve animals. GFAP, IBA-1, ED1, and FJC immunoreactivity at 19 days post-TBI was significantly higher in brains from TBI animals compared to both craniotomy and naïve animals. CONCLUSIONS: These results show an association between post-TBI escalation of alcohol drinking and marked localized neuroinflammation at the site of injury. Moreover, these results highlight the relevance of baseline alcohol preference in determining post-TBI alcohol drinking. Further investigation to determine the contribution of neuroinflammation to increased alcohol drinking post-TBI is warranted.

Alcohol exposure after mild focal traumatic brain injury impairs neurological recovery and exacerbates localized neuroinflammation.

Traumatic brain injury (TBI) represents a leading cause of morbidity and mortality among young individuals. Alcohol abuse is a risk factor associated with increased TBI incidence. In addition, up to 26% of TBI patients engage in alcohol consumption after TBI. Limited preclinical studies have examined the impact of post-injury alcohol exposure on TBI recovery. The aim of this study was to determine the isolated and combined effects of TBI and alcohol on cognitive, behavioral, and physical recovery, as well as on associated neuroinflammatory changes. Male Sprague-Dawley rats (∼300g) were subjected to a mild focal TBI by lateral fluid percussion (∼30PSI, ∼25ms) under isoflurane anesthesia. On day 4 after TBI, animals were exposed to either sub-chronic intermittent alcohol vapor (95% ethanol 14h on/10h off; BAL∼200mg/dL) or room air for 10days. TBI induced neurological dysfunction reflected by an increased neurological severity score (NSS) showed progressive improvement in injured animals exposed to room air (TBI/air). In contrast, TBI animals exposed to alcohol vapor (TBI/alcohol) showed impaired NSS recovery throughout the 10-day period of alcohol exposure. Open-field exploration test revealed an increased anxiety-like behavior in TBI/alcohol group compared to TBI/air group. Additionally, alcohol-exposed animals showed decreased locomotion and impaired novel object recognition. Immunofluorescence showed enhanced reactive astrocytes, microglial activation, and HMGB1 expression localized to the injured cortex of TBI/alcohol as compared to TBI/air animals. The expression of neuroinflammatory markers showed significant positive correlation with NSS. These findings indicated a close relationship between accentuated neuroinflammation and impaired neurological recovery from post-TBI alcohol exposure. The clinical implications of long-term consequences in TBI patients exposed to alcohol during recovery warrant further investigation.

Predator odor stress alters corticotropin-releasing factor-1 receptor (CRF1R)-dependent behaviors in rats.

Humans with stress-related anxiety disorders exhibit increases in arousal and alcohol drinking, as well as altered pain processing. Our lab has developed a predator odor stress model that produces reliable and lasting increases in alcohol drinking. Here, we utilize this predator odor stress model to examine stress-induced increases in arousal, nociceptive processing, and alcohol self-administration by rats, and also to determine the effects of corticotropin-releasing factor-1 receptors (CRF1Rs) in mediating these behavioral changes. In a series of separate experiments, rats were exposed to predator odor stress, then tested over subsequent days for thermal nociception in the Hargreaves test, acoustic startle reactivity, or operant alcohol self-administration. In each experiment, rats were systemically injected with R121919, a CRF1R antagonist, and/or vehicle. Predator odor stress increased thermal nociception (i.e., hyperalgesia) and acoustic startle reactivity. Systemic administration of R121919 reduced thermal nociception and hyperarousal in stressed rats but not unstressed controls, and reduced operant alcohol responding over days. Stressed rats exhibited increased sensitivity to the behavioral effects of R121919 in all three tests, suggesting up-regulation of brain CRF1Rs number and/or function in stressed rats. These results suggest that post-stress alcohol drinking may be driven by a high-nociception high-arousal state, and that brain CRF1R signaling mediates these stress effects.

Nonhuman primate lung decellularization and recellularization using a specialized large-organ bioreactor.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.