Rodent immobilization apparatus and method of use thereof

A rodent immobilizing apparatus for use during experimental procedures, dissection, and sample procurement. The apparatus has two (2) points of adherence to the dissection board, one at the head/jaw region and one at the tail region. Generally, the rodent immobilizing apparatus includes a base, a bed positioned atop the base, a bank disposed along one side of the bed, another bank disposed along the opposite side of the bed, a string/wire hook affixed to each bank with string/wire therebetween, and a tail clip/clamp positioned at an inferior portion of the bed. The bed is slidable along the banks to accommodate different sized rodents. In operation, an anesthetized/euthanized rodent is positioned supine on the bed. The bed slides along the banks, so that the string/wire is hooked within the rodent\u27s jaw. The tail is clamped to the bed, and the bed is pulled back to stretch out the rodent, thus immobilizing the rodent completely and rapidly

Rodent immobilization apparatus and method of use thereof

A rodent immobilizing apparatus for use during experimental procedures, dissection, and sample procurement. The apparatus has two (2) points of adherence to the dissection board, one at the head/jaw region and one at the tail region. Generally, the rodent immobilizing apparatus includes a base, a bed positioned atop the base, a bank disposed along one side of the bed, another bank disposed along the opposite side of the bed, a string/wire hook affixed to each bank with string/wire therebetween, and a tail clip/clamp positioned at an inferior portion of the bed. The bed is slidable along the banks to accommodate different sized rodents. In operation, an anesthetized/euthanized rodent is positioned supine on the bed. The bed slides along the banks, so that the string/wire is hooked within the rodent\u27s jaw. The tail is clamped to the bed, and the bed is pulled back to stretch out the rodent, thus immobilizing the rodent completely and rapidly

Dysregulation of CLOCK Gene Expression in Hyperoxia-induced Lung Injury

Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3−/− mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3−/− was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3−/− mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression. acute lung injury (ali) affects a large number of patients worldwide, with mortality rates reported up to 40% (16). Many patients with ALI require oxygen supplementation to maintain adequate tissue oxygenation; unfortunately, it can exacerbate the condition as it may lead to hyperoxia-induced acute lung injury (HALI) (4). Exposure to hyperoxia can have pathological effects such as lung inflammation and edema accompanied by epithelial and endothelial cell death, suggesting that oxygen supplementation, although necessary, may potentially perpetuate or exacerbate ALI (2, 3). Inflammatory cells flood the lung tissue and proinflammatory cytokines like IL-1β are produced as a result of hyperoxia-induced ALI (11). Recent reports from our laboratory suggest the IL-1β processing machinery known as the “inflammasome” plays an important role in hyperoxic lung injury (14). Inflammasomes are master switches that are involved in caspase-mediated processing of proinflammatory cytokines (14). Circadian rhythms play important roles in physiology and behavior; disruption of these rhythms can become a major cause in disease development (23). Recent evidence has implicated an important role for CLOCK genes in inflammation in relation to chronic diseases like diabetes and hypertension (17, 21). However, their role in ALI and how CLOCK gene expression is altered due to inflammation in ALI and other pulmonary associated diseases has been unexplored. The mammalian circadian CLOCK is composed of at least 10 core circadian CLOCK proteins. Circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) transcription factors form a heterodimeric complex. This complex binds to E-boxes in the promoters of various target genes, including those encoding for negative [e.g., period homolog 1 (Per1), Per2, cryptochrome 1 (Cry1), and Cry2] or positive (e.g., Bmal1) loop components, as well as target genes, including D site of albumin promoter-binding protein (DBP), nuclear receptor subfamily 1, group D, member 1 (Rev-erb-α), and peroxisome proliferator-activated receptor-γ (PPARγ). It is well known that CLOCK genes are expressed rhythmically in the suprachiasmatic nucleus of the hypothalamus, the master circadian pacemaker in mammals. Recently, it has become clear that CLOCK genes also express and function in various peripheral tissues (24). In particular, CLOCK genes exhibit circadian expression patterns in organs that play critical roles in blood pressure homeostasis, including the vasculature, more specifically, the mouse aorta (18), heart (19, 25, 27), and kidney (19, 25). However, it is unknown whether CLOCK gene expression levels are altered in mice with HALI. In the present study, we hypothesize that chronic hyperoxia can induce inflammation in mouse lungs which may alter CLOCK gene expression and lead to the development of HALI

NLRP3 Deletion Protects from Hyperoxia-induced Acute Lung Injury

Inspiration of a high concentration of oxygen, a therapy for acute lung injury (ALI), could unexpectedly lead to reactive oxygen species (ROS) production and hyperoxia-induced acute lung injury (HALI). Nucleotide-binding domain and leucine-rich repeat PYD-containing protein 3 (NLRP3) senses the ROS, triggering inflammasome activation and interleukin-1β (IL-1β) production and secretion. However, the role of NLRP3 inflammasome in HALI is unclear. The main aim of this study is to determine the effect of NLRP3 gene deletion on inflammatory response and lung epithelial cell death. Wild-type (WT) and NLRP3−/− mice were exposed to 100% O2 for 48–72 h. Bronchoalveolar lavage fluid and lung tissues were examined for proinflammatory cytokine production and lung inflammation. Hyperoxia-induced lung pathological score was suppressed in NLRP3−/− mice compared with WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNFα, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 were attenuated in NLRP3−/− mice. NLRP3 deletion decreased lung epithelial cell death and caspase-3 levels and a suppressed NF-κB levels compared with WT controls. Taken together, this research demonstrates for the first time that NLRP3-deficient mice have suppressed inflammatory response and blunted lung epithelial cell apoptosis to HALI. acute lung injury (ALI) is characterized by severe alveolar damage resulting from an acute inflammatory response that leads to proinflammatory cytokine production, neutrophil, macrophage infiltration, and edema. The most severe form of ALI is acute respiratory distress syndrome (ARDS), which is a major cause for admission to critical care units. Hyperoxia therapy is a necessary part of treatment for patients with acute and chronic cardiovascular and pulmonary diseases. However, prolonged exposure to hyperoxia could deteriorate ALI (19, 47). Currently, there are several animal models available to study the mechanism of ALI. The hyperoxia-induced acute lung injury (HALI) animal model became widely used to study human ALI after Cochrane et al. (7) revealed the increase of oxidants in the lungs of patients with ARDS. It is now well established that there are clinically relevant similarities between the animal model of HALI and human lung injury (26). However, the molecular mechanisms that initiate and amplify the lung inflammation in response to inhaled oxygen are not well understood. IL-1β is one of the most potent early cytokines found in ALI patients, and it induces the production of other cytokines (12). The proinflammatory cytokine IL-1β is also known to be one of the most biologically important inflammatory mediators in the air space of patients with early ALI (35). Interestingly, IL-1β can also act as an important activator and prosurvival cytokine for neutrophils (37). However, mechanisms that initiate IL-1β processing in ALI are not clearly defined. Martinon et al. (25) first reported in 2002 that caspase-1-mediated processing of IL-1β is mediated by the nucleotide-binding domain and leucine-rich repeat PYD-containing protein 3 (NLRP3) inflammasome. The NLRP3 inflammasome is a multiprotein complex, which contains NLRP3, the caspase recruitment domain containing protein Cardinal, apoptosis-associated speck-like protein (ASC), and caspase-1 (33). NLRP3 inflammasome is implicated in sensing stress caused by reactive oxygen species (9, 32). Recently we showed that hyperoxia induces inflammasome activation (21, 22). However, whether inhibition or deletion of NLRP3 inflammasome is critical to confer protection against HALI has not been studied yet. Since the HALI model is thoroughly characterized in terms of reactive oxygen species involvement, assessing the effect of NLRP3 deletion on HALI will provide important information about how NLRP3 plays a role in HALI and might result in novel therapeutic strategies to treat ALI. In this study for the first time we used NLRP3-deficient mice to identify the role of inflammasomes in hyperoxia-induced lung injury

Inflammasome Inhibition Suppresses Alveolar Cell Permeability Through Retention of Neuregulin-1 (NRG-1)

Background: Neuregulin (NRG)-1-human epidermal receptor (HER)-2 signaling pathway is a key regulator of IL-1β-mediated pulmonary inflammation and epithelial permeability. The inflammasome is a newly discovered molecular platform required for caspase-1 activation and maturation of IL-1β. However, the role of the inflammasome in NRG-1-HER2 signaling-mediated alveolar cell permeability is unknown. Methods: The inflammasome was activated or inhibited in THP-1 cells; supernatants from these cells were added to A549 cells and human small airway epithelial cells (HSAEC). The protein expression of NRG-1 and phospho-HER2 (pHER2) were measured by Western blot analysis and epithelial permeability was measured using Lucifer yellow dye. Results: Results reveal that alveolar permeability in A549 cells and HSAEC is increased when treated with supernatants of inflammasome-activated THP-1 cells. Alveolar permeability is significantly suppressed when treated with supernatant of inflammasome-inhibited THP-1 cells. Inflammasome-mediated permeability is decreased when A549 cells and HSAEC are pretreated with IL-1β receptor antagonist (IL-1βRA). In addition, HER2 kinase inhibitor AG825 or NRG-1 inhibitor TAPI inhibits inflammasome-mediated permeability in A549 cells and HSAEC demonstrating critical roles of IL-1β, NRG-1, and HER2 in inflammasome-mediated alveolar permeability. Conclusion: These findings suggest that inflammasome-induced alveolar cell permeability is mediated by NRG-1/HER2 signaling through IL-1β regulation

Deletion of ASK1 Protects against Hyperoxia-Induced Acute Lung Injury

<div><p>Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase kinase (MAP3K) family, is activated by various stimuli, which include oxidative stress, endoplasmic reticulum (ER) stress, calcium influx, DNA damage-inducing agents and receptor-mediated signaling through tumor necrosis factor receptor (TNFR). Inspiration of a high concentration of oxygen is a palliative therapy which counteracts hypoxemia caused by acute lung injury (ALI)-induced pulmonary edema. However, animal experiments so far have shown that hyperoxia itself could exacerbate ALI through reactive oxygen species (ROS). Our previous data indicates that ASK1 plays a pivotal role in hyperoxia-induced acute lung injury (HALI). However, it is unclear whether or not deletion of ASK1 <i>in vivo</i> protects against HALI. In this study, we investigated whether ASK1 deletion would lead to attenuation of HALI. Our results show that ASK1 deletion <i>in vivo</i> significantly suppresses hyperoxia-induced elevation of inflammatory cytokines (i.e. IL-1β and TNF-α), cell apoptosis in the lung, and recruitment of immune cells. In summary, the results from the study suggest that deletion of ASK1 in mice significantly inhibits hyperoxic lung injury.</p></div

Resolvins Decrease Oxidative Stress Mediated Macrophage and Epithelial Cell Interaction through Decreased Cytokine Secretion

<div><p>Background</p><p>Inflammation is a key hallmark of ALI and is mediated through ungoverned cytokine signaling. One such cytokine, interleukin-1beta (IL-1β) has been demonstrated to be the most bioactive cytokine in ALI patients. Macrophages are the key players responsible for IL-1β secretion into the alveolar space. Following the binding of IL-1β to its receptor, “activated” alveolar epithelial cells show enhanced barrier dysfunction, adhesion molecule expression, cytokine secretion, and leukocyte attachment. More importantly, it is an important communication molecule between the macrophage and alveolar epithelium. While the molecular determinants of this inflammatory event have been well documented, endogenous resolution processes that decrease IL-1β secretion and resolve alveolar epithelial cell activation and tissue inflammation have not been well characterized. Lipid mediator Aspirin-Triggered Resolvin D1 (AT-RvD1) has demonstrated potent pro-resolutionary effects <i>in vivo</i> models of lung injury; however, the contribution of the alveoli to the protective benefits of this molecule has not been well documented. In this study, we demonstrate that AT-RvD1 treatment lead to a significant decrease in oxidant induced macrophage IL-1β secretion and production, IL-1β-mediated cytokine secretion, adhesion molecule expression, leukocyte adhesion and inflammatory signaling.</p><p>Methods</p><p>THP-1 macrophages were treated with hydrogen peroxide and extracellular ATP in the presence or absence of AT-RvD1 (1000–0.1 nM). A549 alveolar-like epithelial cells were treated with IL-1β (10 ng/mL) in the presence or absence of AT-RvD1 (0.1 μM). Following treatment, cell lysate and cell culture supernatants were collected for Western blot, qPCR and ELISA analysis of pro-inflammatory molecules. Functional consequences of IL-1β induced alveolar epithelial cell and macrophage activation were also measured following treatment with IL-1β ± AT-RvD1.</p><p>Results</p><p>Results demonstrate that macrophages exposed to H₂O₂ and ATP in the presence of resolvins show decreased IL-1β production and activity. A549 cells treated with IL-1β in the presence of AT-RvD1 show a reduced level of proinflammatory cytokines IL-6 and IL-8. Further, IL-1β-mediated adhesion molecule expression was also reduced with AT-RvD1 treatment, which was correlated with decreased leukocyte adhesion. AT-RvD1 treatment demonstrated reduced MAP-Kinase signaling. Taken together, our results demonstrate AT-RvD1 treatment reduced IL-1β-mediated alveolar epithelial cell activation. This is a key step in unraveling the protective effects of resolvins, especially AT-RvD1, during injury.</p></div