Lauryn Kohut

Alterations in Nitric Oxide and Cytokine Production with Airway Inflammation in the Absence of IL-10

IL-10 is an anti-inflammatory cytokine that suppresses NO synthase (NOS) and production of NO; its lack may promote NO production and alterations in cytokines modulated by NO with allergic airway inflammation (AI), such as IL-18 and IL-4. Therefore, we induced AI in IL-10 knockout (−/−) and IL-10-sufficient C57BL/6 (C57) mice with inhaled OVA and measured airway NO production, as exhaled NO (ENO) and bronchoalveolar lavage fluid nitrite levels. ENO and nitrite levels were elevated significantly in naive IL-10−/− mice as compared with C57 mice. With AI, ENO and nitrite levels increased in C57 mice and decreased in IL-10−/− mice. IL-18 production fell with both AI and addition of S-nitroso-N-acetyl-d,l-penicillamine (a NO donor) but was not significantly increased by chemical NOS inhibition by l-N5-(1-iminoethyl)-ornithine. IL-4 AI was increased significantly (up to 10-fold greater) in the absence of IL-10 but was reduced significantly with chemical inhibition of NOS. Airway responsiveness was lower in IL-10−/− mice and was associated with alteration in production of NO and IL-4. Thus, IL-4 production was increased, and likely decreased NO production, in a way not predicted by the absence of IL-10. Inhibition of IL-4 production, with inhibition of NOS in the absence of IL-10, demonstrated the importance of a NO and IL-4 feedback mechanism regulating this interaction.

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality. Many studies have begun to assess these changes in the reactivity of the immune system following trauma. Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses. The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible. This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone. The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution. Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity.

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival. Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals. Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

TLR9 deficiency in parenchymal or immune cells is associated with reduced tissue damage in a trauma model

Temp (C) 0.2 0.0 0.2 0.1 0.7 0.4 0.8 0.3 1.6 0.1 1.6 0.2 HR (bpm) 4 39 11 4 92 3 7 42 1 35 5 WBCs 1 0.9 2 13 0.4 4 0.7 5 1.3 6 0.8 LF/HF 0.9 0.36 0.54 0.41 2.25 1.70 1.9 0.95 2.03 1.53 5.18 0.92 TNF (pg/mL) 6 09 49 32 5 17 244 22 191 64 IL-6 (pg/mL) 15 12 4 02 2 19 77 35 323 46 320 28 Significant vs placebo. CONCLUSIONS: Both vital sign changes and HRV markers of autonomic imbalance were seen at LPS doses 2 to 4 times lower than those required for significant elevations in TNF, IL-6, and WBC. This finding suggests that endotoxinemia is associated with changes in autonomic balance well before overt acute inflammation arises. Furthermore, it suggests the possibility of unidentified signaling mechanisms that partially precede proinflammatory cytokines as the causal agents of early vital sign and autonomic changes.