1C,?,D).D). factor (TNF), subsequently stimulating nociceptive neurons. Extensive preclinical evidence supports the role of spinal microglia in mediating nociceptive sensitization in male rodent neuropathic and inflammatory pain models, but female mice do not require microglia activation to sustain pain hypersensitivity at levels equivalent to that of male mice after nerve injury or inflammation.[16,17,29,30] Complex regional pain syndrome (CRPS) usually develops after a regional injury and presents with distal limb nociceptive, vascular, and bone changes that exceed the expected clinical course of the inciting injury in both magnitude and duration, frequently resulting in significant motor impairment and Fmoc-Val-Cit-PAB-PNP disability. Interestingly, Fmoc-Val-Cit-PAB-PNP there is a 3:1 female to male ratio for the development of CRPS after injury.[4,26] Distal limb fracture is the most common cause of CRPS,[4,26] and we have developed a tibia fracture rodent model closely resembling CRPS. Distal tibia fractured male rats and mice casted for 3C4 weeks develop hindpaw allodynia, unweighting, warmth, edema, increased spontaneous protein extravasation, and regional periarticular bone loss. The tibia fracture model has been used to investigate the wide ranging effects of limb trauma on Fmoc-Val-Cit-PAB-PNP pronociceptive cutaneous and spinal neuropeptide signaling,[6,8,35,37] sympathetic nervous system activation,, mast cell infiltration,  keratinocyte [28,35,37] and microglia [14,27] activation, pronociceptive inflammatory mediator (IL-1, IL-6, TNF, and nerve growth factor (NGF)) production in skin and spinal cord,[5,11,15,24,25,36] and pronociceptive autoantibody dependent immune responses.[7,13] The innate immune system is the initial nonspecific response of the body to infection and utilizes effector cells such as monocytes, macrophages, dendritic cells, keratinocytes, microglia, and natural killer cells. The adaptive immune response is specific to a particular pathogen or antigen and is mediated by T cells and antibody producing B cells. Post fracture pain behaviors in male mice transition from being initially dependent on both innate and adaptive inflammatory mechanisms at 3 Fmoc-Val-Cit-PAB-PNP weeks after fracture to being entirely mediated by antibody responses at 12 weeks after fracture and spontaneously resolving by 21 weeks post fracture. Furthermore, serum or IgM antibodies from wild-type fracture male mice have pronociceptive effects in the fracture limb when injected into muMT fracture male mice lacking B cells and antibodies. IgM antibody levels peak at 12C18 weeks post-fracture and then decline. We postulate that fracture induces expression of neoantigens in fracture limb skin, sciatic nerve, and cord, which trigger B cells to secret IgM antibodies that bind those antigens, initiating nociceptive sensitization. The current study investigates sex and hormone effects on the temporal evolution of peripheral and central innate and adaptive pronociceptive immune responses to tibia fracture in mice. 2.?Materials and methods 2.1. Animals. These experiments were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA, USA) and followed the animal subjects guidelines of the International Association for the Study of Pain. Three-month-old male and female C57BL/6J mice (#000664, Jackson Laboratory, Bar Harbor, ME) were designated the wild-type (WT) mice and muMT mice lacking mature B cells and immunoglobulin, on a C57BL/6J congenic background (#002288, Jackson Laboratory, Bar Harbor, ME) were used in these experiments. The animals were housed 4 per group under pathogen-free conditions with soft bedding Rabbit Polyclonal to SDC1 and were given food and water em ad libitum /em , with a 12:12 light:dark cycle. During the experimental period the animals were fed Teklad lab rodent diet 2018 (Harlan Laboratories, Indianapolis, IN), which contains 1.0% calcium, 0.7% phosphorus, and 1.5 IU/g vitamin D3, and were kept under standard conditions with a 12-h light-dark cycle. Data collection was conducted blind to group assignment. 2.2. Surgery. The fracture model was performed in 3 month-old male and female mice as previously Fmoc-Val-Cit-PAB-PNP described.  Under isoflurane anesthesia a hemostat was used to make a closed fracture of the right tibia just distal to the middle of the tibia. The hindlimb was then wrapped in casting tape (Delta-Lite, BSN Medical, Hamburg, Germany) so the hip, knee and ankle were all fixed. After fracture and casting, the mice were subcutaneously given 2 days of buprenorphine (0.1 mg/kg) and enrofloxacin (5 mg/kg) as well as 1.0 ml of normal saline. At 3 weeks after surgery the mice were anesthetized with isoflurane and the solid eliminated. All mice experienced union in the fracture site by manual inspection. Ovariectomy or sham surgery was performed 2 weeks prior to fracture in female mice under isoflurane anesthesia. A 1.