Hemorrhagic shock has recently been shown to cause shedding of a carbohydrate surface layer of endothelial cells known as the glycocalyx. This shedding of the glycocalyx is thought to be a mediator of the coagulopathy seen in trauma patients. Clinical studies have demonstrated increases in shed glycocalyx in the blood after trauma, and animal studies have measured glycocalyx disruption in blood vessels in the lung, skeletal muscle, and mesentery. However, no study has measured glycocalyx disruption across a wide range of vascular beds to quantify the primary locations of this shedding. Methods: In the present study, we used a rat model of hemorrhagic shock and resuscitation to more comprehensively assess glycocalyx disruption across a range of organs. Glycocalyx disruption was assessed by fluorescent-labeled wheat germ agglutinin or syndecan-1 antibody staining in flash frozen tissue. Results: We found that our model did elicit glycocalyx shedding, as assessed by an increase in plasma syndecan-1 levels. In tissue sections, we found that the greatest glycocalyx disruption occurred in vessels in the lung and intestine. Shedding to a lesser extent was observed in vessels of the brain, heart, and skeletal muscle. Liver vessel glycocalyx was unaffected, and kidney vessels, including the glomerular capillaries, displayed an increase in glycocalyx. We also measured reactive oxygen species (ROS) in the endothelial cells from these organs, and found that the greatest increase in ROS occurred in the two beds with the greatest glycocalyx shedding, the lungs, and intestine. We also detected fibrin deposition in lung vessels following hemorrhage-resuscitation. Conclusions: We conclude that the endothelium in the lungs and intestine are particularly susceptible to the oxidative stress of hemorrhage-resuscitation, as well as the resulting glycocalyx disruption. Thus, these two vessel beds may be important drivers of coagulopathy in trauma patients.
The endothelial glycocalyx forms an anti-thrombotic layer on the apical surface of endothelial cells and maintains the selective permeability barrier of blood vessels. Ischemia reperfusion injury like hemorrhagic shock is shown to cause glycocalyx damage. We have previously shown that mitochondrial reactive oxygen species (mitoROS) mediate glycocalyx damage in cultured endothelial cells. Of note, angiotensin II elevates endothelial mitoROS, suggesting a possible exacerbation of glycocalyx damage in hypertensive patients. It is unknown, however, whether mitoROS mediate glycocalyx damage in vivo . We hypothesize that mitoROS mediate glycocalyx damage in a rat model of hemorrhagic shock.We investigated the effect of mitochondrial ROS on the endothelial glycocalyx in vivo , in a rat model of hemorrhagic shock. In anesthetized rats, mean arterial pressure was reduced to 40 mmHg by withdrawing blood from the femoral artery and kept at 40 mmHg for 30 minutes. The rats were then resuscitated with IV (jugular vein) Ringer’s lactate solution for 30 minutes more. Sham rats received the vascular lines only. Syndecan-1 in the plasma was increased after 30 minutes of resuscitation with LR in hemorrhage/resuscitation (H/R) rats (p=0.02) but was not elevated in Sham rats (p=0.52). Resuscitation with IV mitoROS scavenger mitoTEMPOL significantly blunted this increase (5.9 pg/ml vs. 8.7 pg/ml, p=0.03). At the organ level, the glycocalyx was decreased in the endothelium of muscle (p=0.0444) and intestine (P=5.47 * 10 -7 ) vascular beds in H/R rats vs. Sham rats. Our findings show that mitoROS mediate the glycocalyx damage after H/R. This mechanism suggests possible therapies that target mitoROS generation. Future work will investigate whether preexisting hypertension increases glycocalyx damage during H/R due to exacerbated mitoROS levels.
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