Session V - Polytrauma

An Investigation of the Influence of Sequential Fat Embolism, Hemorrhage, and Resuscitation on Pulmonary Pathogenesis in an Animal Model of Trauma

Michael Blankstein, MD; Emil H. Schemitsch, MD; Robert J. Byrick, MD; Masaki Nakane, MD; Annie K.W. Bang, PhD; John Freedman, MD; Robin R. Richards, MD; David Bell, MD; St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada

Purpose: The objective of this study was to assess the contribution of pulmonary fat embolism caused by intramedullary femoral canal pressurization to the development of acute lung injury in the presence of resuscitated hemorrhagic shock. This clinically relevant model will allow us to evaluate the potential of fat embolism in enhancing lung injury following hypotension. We hypothesized that sequential insults of hemorrhagic shock, resuscitation, and pulmonary fat embolism lead to pulmonary dysfunction that is greater than either injury alone, as indicated by the following parameters: an amplified initiation of a systemic inflammatory response and a greater spectrum of diffuse lung inflammatory reaction.

Methods: After anesthetic administration and preparation, New Zealand White rabbits were randomly assigned into one of four groups: resuscitated hemorrhagic shock + fat embolism (HR/FE), resuscitated hemorrhagic shock (HR), fat embolism (FE), and control. In the HR/FE group (N = 6), hypovolemic shock was induced via constant carotid bleeding. Mean arterial pressure (MAP) was maintained at 30 to 40 mmHg for 1 hour. Animals were resuscitated with the entire volume of shed blood titrated with an additional volume of saline solution necessary to restore baseline MAP values. After a 1-hour stabilization period, both distal femoral condyles were exposed through a medial parapatellar approach to the right knee in preparation for fat embolism induction. After drilling into the intramedullary cavity in a retrograde fashion, the canal was successively reamed with use of 3.5-, 4-, and 4.5-mm diameter T-handle reamers. The intramedullary canal was pressurized via a standardized injection of 1 to 1.5 ml of low viscosity bone cement. The patella was then reduced and the incision was closed. In the HR group (N = 6), after the 1-hour stabilization period following resuscitated shock, a sham knee incision was made but was closed immediately without drilling, reaming, or pressurization. In the FE group (N = 8), prior to fat embolism induction, the animals were ventilated for a 3-hour period. This time corresponds to the time it took to establish shock, resuscitate, and stabilize the animals in the HR group. In the control group (N = 7), after the 3-hour ventilation period, a sham knee incision was made but was immediately closed without further manipulations. Animals were mechanically ventilated for 4 hours after surgical closure.

For flow cytometric evaluation of neutrophil activation, 0.5 ml of blood was mixed with an anticoagulant at a final concentration of 1:10. Samples were stained with conjugated monoclonal antibodies, and neutrophils were identified by characteristic CD45 fluorescence and light scatter properties. A minimum of 10,000 events were acquired for each sample with use of a FACSCalibur model flow cytometer equipped with a 488-nm argon ion laser, and data were analyzed with CellQuest software.

After the 4-hour monitoring period, the rabbits were sacrificed by an intravenous overdose of pentobarbital. A postmortem thoracotomy was performed, and the left lung was fixed in inflation with 10% buffered formalin at a pressure of 25 cm of fixative. After fixation, the sample was sectioned sagittally, and three stratified random blocks of known size were taken from the mid-sagittal slice of each lung. The specimens were embedded in paraffin, processed for histologic examination and cut at 5-mm thickness.

Results: Three animals died in the HR/FE group immediately after canal pressurization and were excluded from the study. We assessed neutrophil activation via CD11b mean channel fluorescence (MCF) increase as compared with baseline, and CD11b MCF was only significantly elevated in the HR/FE group at 2 and 4 hours after knee manipulation. Furthermore, histologic assessment displayed significantly greater infiltration of alveoli by polymorphonuclear leukocytes as compared with the control group only in the HR/FE group.

Conclusion/Significance: Our histologic findings support our previous findings that FE by itself does not cause lung injury, as there were no apparent differences in our histologic markers of lung injury between the control and FE animals. However, in the HR and HR/FE groups, a higher number of neutrophils were seen infiltrating into the alveolar spaces than into those of the control group, and in the HR/FE group this increase was statistically significant. The expression of CD11b (a cell surface adhesion molecule that is up-regulated on activated neutrophils) can be used as an early indicator of acute inflammatory reactions preceding lung injury. Our white blood cell flow cytometric studies complemented our histologic findings and demonstrated CD11b MCF measurements to be significantly higher only in the HR/FE group at 4 hours after knee manipulation, as compared with baseline. In support of our hypothesis, the combined HR/FE did elicit two significant synergistic responses: greater neutrophil activation and enhanced alveolar infiltration. These parameters were not elevated in the FE or HR groups alone. These findings indicated the initiation of an inflammatory response when resuscitated shock was combined with fat embolism and may play a role in the development of fat embolism syndrome.