• Robert L Conhaim, Kal E Watson, William F Dovi, and Melissa L Bates.
• *Department of Surgery, University of Wisconsin School of Medicine and Public Heath; †William S. Middleton Memorial Veterans Hospital; and ‡Department of Pediatrics, University of Wisconsin School of Medicine and Public Heath, Madison, Wisconsin.
• Shock. 2014 Jun 1;41(6):528-36.

AbstractWe showed previously that a 30% blood loss in rats, without resuscitation, caused significant accumulation of microthrombi and leukocytes within the pulmonary circulation by 24 h. We hypothesized that the microthrombi formed spontaneously as a consequence of hemorrhage-induced stasis within the low-pressure pulmonary circuit and that the leukocytes were attracted to them. This suggested that elimination of the microthrombi, using an inhaled thrombolytic agent, could prevent the neutrophil sequestration after blood loss. To test this hypothesis, we removed 30% of the calculated blood volume from isoflurane-anesthetized, male Sprague-Dawley rats (350-500 g) over 5 min and allowed them to recover. Six hours later, we re-anesthetized the rats and nebulized tissue plasminogen activator (80 or 320 µg/kg), lactated Ringer's solution (LRS), or ipratropium bromide (i-bromide) into their lungs. We used i-bromide as a control after we discovered that nebulized LRS had thrombolytic properties. At 24 h, we removed and fixed the lungs and prepared sections for immunohistochemistry using antibodies against fibrinogen (microthrombi) and CD16 (leukocytes). Digital images of each section were obtained using a confocal microscope. Pixel counts of the images showed significantly less accumulation of microthrombi and leukocytes in lungs nebulized with tissue plasminogen activator or LRS than in non-nebulized lungs or in lungs nebulized with i-bromide (P ≤ 0.05). Lactated Ringer's solution becomes positively charged when nebulized (unlike i-bromide), suggesting that it eliminated microthrombi by fibrin depolymerization. We confirmed this using an in vitro assay. Our results demonstrate that lyses of microthrombi that accumulate in the lung after acute blood loss prevent subsequent leukocyte sequestration.

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