67.09 UCH-L1 IS A SERUM BIOMARKER FOR PERIPHERAL NERVE INJURY IN A MODEL OF EXTREMITY ISCHEMIA

Posted on January 31, 2019

A. P. Bercz1, M. C. Morris1, F. Kassam1, R. Veile1, L. Friend1, R. Schuster1, T. A. Pritts1, A. T. Makley1, M. D. Goodman1  1University Of Cincinnati,Department Of Surgery, College Of Medicine,Cincinnati, OH, USA

Introduction:  Ubiquitin carboxy-terminal hydroxylase L1 (UCH-L1) is specifically expressed by neurons and its release reflects the neuronal response to injury. In humans and murine models, UCH-L1 is elevated in serum and cerebrospinal fluid following traumatic brain injury (TBI), hypoxic encephalopathy, and intracerebral hemorrhage, but it is unknown whether UCH-L1 is specific to central nervous system injury. Our lab has recently demonstrated that UCH-L1 is significantly elevated in a murine polytrauma model, regardless of TBI. In this study, we hypothesized that UCH-L1 would function as a marker of peripheral nerve injury induced by extremity ischemia.

Methods:  Mice were anesthetized mice with pentobarbital and the left femoral vessels isolated. Mice were then subjected to either femoral artery isolation alone (sham), femoral artery ligation, femoral vein ligation, femoral artery ligation after 60 minutes of controlled hemorrhagic shock followed by resuscitation with shed blood, or four hours of ischemia with a femoral artery clamp followed by reperfusion (I/R). Mice were sacrificed at 4, 24, or 72 hours after treatment. Serum was analyzed using enzyme-linked immunosorbent assays for UCH-L1, neuron specific enolase (NSE), and creatine kinase (CK), the latter two being known biomarkers of neuronal and muscle injury, respectively. The bilateral quadriceps muscles were harvested and analyzed using hematoxylin and eosin staining.

Results: UCH-L1 was significantly increased in the hemorrhagic shock/resuscitation group at 4 hours, compared to the sham, artery ligation, vein ligation, and I/R groups (Figure 1). No other groups had significant UCH-L1 elevations compared to Sham at 4 hours. In the setting of hemorrhagic shock, UCH-L1 elevation persisted through 72 hours. NSE was not as sensitive to these ischemic changes. CK was altered in a pattern similar to that of UCH-L1 in its temporal response profile and was significantly increased in the hemorrhagic shock/resuscitation group with respect to the other groups at 4 hours. Hematoxylin and eosin staining of the quadriceps revealed muscle necrosis in the hemorrhagic shock/resuscitation group only, demonstrated by increased inflammatory infiltrate surrounding enucleated fascicles.

Conclusion: The acute elevation of UCH-L1 in response to isolated, controlled hemorrhagic shock and resuscitation suggests that UCH-L1 is not specific for TBI or central nervous system injury. The lack of NSE elevation in response to femoral vessel ligation suggests that NSE may be a more specific biomarker for TBI than peripheral nerve injury. UCH-L1 may be utilized as an early marker of neuronal injury in the context of hemorrhagic shock, polytrauma, and isolated extremity ischemia.

 

67.08 The Role of Chemoprophylactic Agents in Modulating Hypercoagulability after Traumatic Brain Injury

Posted on January 31, 2019

F. Kassam1, M. C. Morris1, A. Bercz1, R. Veile1, L. Friend1, N. Beckmann1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Department Of Surgery, College Of Medicine,Cincinnati, OH, USA

Introduction:  The pathophysiology behind the subacute but persistent hypercoagulable state following traumatic brain injury is poorly understood but contributes to morbidity induced by venous thromboembolism (VTE). Because platelets and their microvesicles have been hypothesized to play a role in posttraumatic hypercoagulability, administration of commonly utilized agents for inflammation, VTE chemoprophylaxis, and sphingolipid modulation may ameliorate this coagulability. We hypothesized that utilization of aspirin, ketorolac, amitriptyline, unfractionated heparin, and enoxaparin would modulate the platelet and whole blood coagulation response following traumatic brain injury.

Methods:  A standard weight-drop system was utilized to induce concussive traumatic brain injury in mice. Following injury, mice were randomized into drug treatment groups to receive aspirin (100 mg/kg), ketorolac (5 mg/kg), amitriptyline (10 mg/kg), heparin (75 IU/kg), enoxaparin (3 mg/kg), or saline control (100µL) at 2 and 8 hours post-TBI. Mice were then sacrificed at 6 or 24 hours after injury and blood was drawn via cardiac puncture to determine coagulability by thromboelastometry (EXTEM and FIBTEM), platelet function testing with impedance aggregometry, and microvesiscle enumeration utilizing nanoparticle tracking analysis.

Results: Thromboelastometry results demonstrated that the platelet contribution to maximum clot firmness (%MCF-Platelet) at 6 hours (Figure 1) was significantly higher in mice that received aspirin (69%, p<0.002) or amitriptyline (68%, p<0.007) compared to mice that received saline (57%). At 24 hours, the %MCF-Platelet remained significantly higher in mice that received amitriptyline (66%, p=0.04) compared to those that received saline (63%). The overall ADP- and arachidonic acid-induced platelet aggregation was significantly lower in mice receiving ketorolac,  aspirin, and amitriptyline compared to mice receiving saline at 6 hours post-injury. By  24 hours after injury, mice that received aspirin (40 a.u., p<0.005), ketorolac (38 a.u., p=0.02), and enoxaparin (35 a.u., p=0.04) had significantly lower ADP-induced platelet aggregation than saline control mice (54 a.u.). However, there was no difference in the total or percentage of platelet-derived (CD41+) microvesicles between any treatment group at both 6 and 24 hours.

Conclusion: Following traumatic brain injury, amitriptyline decreased platelet aggregability and increased contribution to clot in a manner similar to aspirin. The effect of amitriptyline on platelet function reflects a possible role of acid sphingomyelinase in the hypercoagulability observed following injury and suggests sphingolipid metabolism as a novel target for multimodal VTE chemoprophylaxis. Additionally, inhibition of platelet reactivity may be an underappreciated benefit of low molecular weight heparins, such as enoxaparin, compared to unfractionated heparin use.