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Inhaled Thrombolytics Reduce Lung Microclot and Leukocyte Infiltration After Acute Blood Loss

Conhaim, Robert L.*†; Watson, Kal E.*; Dovi, William F.; Bates, Melissa L.

doi: 10.1097/SHK.0000000000000149
Basic Science Aspects

ABSTRACT We 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 reanesthetized 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 nonnebulized 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.

*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

Received 25 Nov 2013; first review completed 12 Dec 2013; accepted in final form 27 Jan 2014

Address reprint requests to Robert L. Conhaim, PhD, H5/301-BX3236, 600 Highland Ave, Madison, WI 53792. E-mail:

This study was supported by the Veterans Health Administration, Office of Research and Development, Department of Veterans Affairs, Biomedical Laboratory Research and Development Service (R.L.C., principal investigator).

The authors have no conflicts of interest to declare.

The contents do not represent the views of the Department of Veterans Affairs or the US Government.

© 2014 by the Shock Society