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An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug
Published on: June 4, 2021
Inhaled thrombolytics reduce lung microclot and leukocyte infiltration after acute blood loss
Robert L Conhaim1, Kal E Watson, William F Dovi
1*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.
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 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.
Insights
Hemorrhage causes lung microthrombi and leukocyte buildup. Inhaled tissue plasminogen activator or lactated Ringer's solution clears these clots, preventing inflammation after blood loss.
Area of Science:
- Pulmonary circulation physiology
- Hemorrhage and resuscitation
- Thrombolytic therapy
Background:
- Acute blood loss leads to microthrombi and leukocyte accumulation in the pulmonary circulation.
- Hemorrhage-induced stasis in the low-pressure pulmonary circuit is hypothesized to cause spontaneous microthrombi formation.
- Leukocyte sequestration is thought to be attracted to these microthrombi.
Purpose of the Study:
- To test the hypothesis that inhaled thrombolytic agents can prevent neutrophil sequestration after blood loss.
- To investigate the spontaneous formation of microthrombi and leukocyte attraction in the pulmonary circulation following hemorrhage.
- To evaluate the efficacy of tissue plasminogen activator and lactated Ringer's solution in clearing lung microthrombi.
Main Methods:
- Rats underwent a 30% blood volume removal, followed by recovery.
- Six hours post-hemorrhage, rats were nebulized with tissue plasminogen activator, lactated Ringer's solution (LRS), or ipratropium bromide.
- Lung sections were analyzed via immunohistochemistry for microthrombi (fibrinogen) and leukocytes (CD16) using confocal microscopy.
Main Results:
- Nebulized tissue plasminogen activator and LRS significantly reduced microthrombi and leukocyte accumulation compared to controls.
- Lactated Ringer's solution demonstrated thrombolytic properties, likely through fibrin depolymerization, confirmed in vitro.
- Ipratropium bromide did not show significant thrombolytic or anti-inflammatory effects.
Conclusions:
- Lysis of pulmonary microthrombi formed after acute blood loss effectively prevents subsequent leukocyte sequestration.
- Inhaled lactated Ringer's solution presents a potential therapeutic strategy for managing post-hemorrhage pulmonary complications.
- Targeting microthrombi formation is a viable approach to mitigate inflammatory responses following significant blood loss.
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