Major lower limb injuries are associated with considerable morbidity and mortality, and trauma is the second leading cause of major lower limb amputation commonly affecting young adults. In the present study we will analyze the role of IL-1beta, IL-6, TNF-alpha, MMP-1, -2, -3, -9, TIMP-1 and -2 in predicting the fate and the healing process of a mangled extremity. Our study will be conducted at the Policlinico Umberto I and San Giovanni-Addolorata. All consecutive patients emergently admitted in the next 12 months for a mangled lower limb (Gustilo IIIC) due to a blunt trauma will be selected after the exclusion of patients who eventually die in the emergency room before any type of treatment and those with a penetrating trauma. We estimate to include at least 40 patients. Demographics, hematochemical tests, IL-1beta (IL-1b), IL-6, TNF-alpha, MMP-1, -2, -3, -9, TIMP-1 and -2 plasma levels at admittance and thereafter every day for the next 6 days, associated injuries and computed tomography (CT) scan findings at admission will be evaluated. The patients will undergo different types of treatment and vascular/orthopedics reconstructions. A segment of the perilesional tissue will be harvested for further biologic analysis. Three different levels of biological analyses will be performed to evaluate the influence and prognostic values of the cytokines and metalloproteinases: 1-plasma level concentration with ELISA technique; 2-Western blot and RT-PCR from the perilesional tissue to quantitatively calculated the RNA expression.
The treatment of a mangled limbs require the simultaneous competence of several different surgical specialties because of the concomitant vascular, orthopaedic, soft tissue and nerve injuries. The failure rate is high and no specific parameters at present indicate the limb to attempt a salvage to those to directly amputate. However, several biochemical markers are produced and released after blunt trauma involving the lower limb, which might indicate the fate of the limb and regulated the subsequent process of healing. It is well known that the activation of the inflammatory cascade is the primary response to injury. The inflammatory reaction is mediated by inflammatory cytokines such as IL-1b, IL-6, TNF-alpha, whereas the healing process is closely related to the balance between extracellular matrix (ECM) synthesis and degradation. ECM degradation is mediated by matrix MMPs, a family of extracellular endopeptidases. MMP-1, -2, -3 and -9 play a major role in this process because of their affinity for collagen type IV and laminin, the key components of the basement membrane. MMP expression can be induced by cytokines, growth factors, stress, or inflammation. Cytokines such as IL-1, IL-6 and TNF-alpha have been shown to up-regulate MMP in several cell types, including fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs). Conceptually, the levels of circulating inflammatory cytokines could provide an indirect picture of the fate of the injuries limb and the healing process. In this clinical setting, the analysis of circulating inflammatory cytokines could represent a method to predict and monitor these events, which are unpredictable with the current scales.
If our hypotheses will result correct we will have a panel of haematological tests able to predict limb salvage and a successful process of healing. Therefore, monitoring patients affected with mangled limb with a panel of plasma biomarkers might be predictive of limb salvage and wound healing because these markers may reflect a local inflammatory status reflecting the generalized inflammatory profile of the injuries patients.