Pseudofracture: An Acute Peripheral Tissue Trauma Model

Abstract

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. [1][2][3][4][5][6][7][8][9] Many studies have begun to assess these changes in the reactivity of the immune system following trauma. [10][11][12][13][14][15] 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. [16][17][18][19][20][21] The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible. [22][23][24][25][26][27][28][29][30] 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. 31 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. [32][33][34][35][36] The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37 Protocol

Tissue damage negatively regulates LPS-induced macrophage necroptosis

“…23,24 We demonstrate that LPS acting through TLR4 promotes Mϕ necroptosis, as expected. However, release of HMGB1 from damaged tissue signals via RAGE signaling to upregulate caveolin-1 expression and so induces Mϕ surface TLR4 internalization, which results in amelioration of LPS-TLR4-induced Mϕ necroptosis.…”

“…The PF model consists of two parts: a musclecrush injury to the hind limbs, followed by injection of a bone solution into these injured muscles. 24 In the current study, we used the PF model to elucidate the influence of tissue damage on LPS-induced Mϕ necroptosis, and revealed that HMGB1 within the BCM acts through RAGE signaling and is a major component mediating tissue damage-suppressed Mϕ necroptosis in response to LPS. Necroptosis is a regulated form of inflammatory cell death.…”

“…To determine Mϕ death response to posttrauma infection, wild-type (WT) mice were first subjected to PF, a validated model of long-bone fracture. 23,24 To further confirm that necroptosis was occurring in the Mϕ/ monocytes, cells were double stained with the RIPK1 and RIPK3. 26 Colocalization of RIPK1 and RIPK3 was visualized by confocal microscopy and represents the RIPK1 and RIPK3 association to form the necrosome.…”

“…PF is a reproducible murine model of sterile musculoskeletal trauma that allows for evaluation of post-traumatic immune responses. 24 The challenges in experimental murine trauma modeling include technical difficulties in fracture fixation in mice and reproducibility. The PF model overcomes these difficulties by immunologically mimicking an extremity fracture environment, whereas allowing freedom of movement in the animals and long-term survival without continual, prolonged use of anesthesia.…”

“…The recipient mouse was anesthetized with ketamine (50 mg/kg BW) and xylazine (5 mg/kg BW) and the thighs were squeezed with a hemostat for 30 s to induce a soft-tissue lesion, followed by injection of 0.15 ml of the BCM in the tissue lesion area. 24 Sham animals underwent the same anesthesia procedure and injection of 0.15 ml of normal saline at the thighs. In some animals, at 12 h after the injection of BCM or saline, were intraperitoneally administered LPS (2 mg/kg BW in 0.2 ml of PBS).…”