Immunohistochemistry staining for macrophages using anti-CD68 and neutrophils using anti-MPO is shown in Figure 6. Evaluation by a blinded pathologist showed that the 6-h TIVF samples had moderate-to-severe edema and hemorrhage within the lung tissue, whereas the 0- and 6-h DPR had mild-to-moderate levels of each. Both macrophage and neutrophils increased in the tissue at 6 h compared with time 0, and were more likely to be found in the intra-alveolar spaces than in the 0- or 6-h DPR samples. The DPR samples also showed a small decrease in the overall quantity of macrophages and neutrophils present.
These data help delineate the series of events that lead to lung inflammation and injury after brain death. The first step in the inflammatory cascade after brain death is an increase in inflammatory cytokines within the lung. TNF-α is an important part of the inflammatory response. When found in the lungs, it can stimulate production of other cytokines (17), activate neutrophils, and increase endothelial permeability (18). Levels of TNF-α increased rapidly after brain death, with significantly higher levels after only 2 h. IL-1α and IL-1β are also common markers of inflammation, and when elevated in the lung have been associated with respiratory failure (17). Both were increased as soon as 2 h after brain death. One of the most common markers of cell stress, damage, and inflammation is IL-6, which is produced by innate immune cells and endothelial cells among other cell types (19). Our data show that levels of IL-6 also increased soon after brain death in the lung. This is a similar to the findings of Wauters et al., who found an increase in inflammatory lung cytokine between 1 and 3 h after brain death in mice (20). This rapid rise in each of these potent mediators after brain death indicates how quickly the inflammatory response is activated after brain death.
Several of the inflammatory cytokines studied, including IL-1 and TNF-α, increase the expression of leukocyte adhesion molecules (21). These are a group of molecules that help leukocytes move from the blood vessels and into the tissues. Adhesion molecules ICAM and VCAM are expressed on vascular endothelium and facilitate rolling and spreading of leukocytes in preparation for moving through the vessel wall (22). Expression of ICAM was increased at 4 h and VCAM at 6 h after brain death and both decreased with DPR. This timing is consistent with findings from Skrabal et al. who found that ICAM increased in the lung between 1 and 6 h after brain death (23). E-selectin and P-selectin are expressed on the surface of endothelial cells and serve to help capture leukocytes from the bloodstream (22). Levels of both were increased 4 h after brain death and decreased with DPR. Although there are many stimuli for the expression of this group of molecules, inflammatory cytokines such as IL-1 and TNF-α are some of the most potent. As DPR downregulates this inflammatory signal within the lungs, it is not surprising that the expression of these molecules is also downregulated.
The primary role of these leukocyte adhesion molecules is to facilitate leukocytes moving into the tissues. Our data show a clear progression of the inflammatory cytokines rising shortly after brain death in the lung, triggering adhesion molecule expression at 4 h after brain death, and ultimately leading to macrophage and neutrophil infiltrate at 6 h after brain death. This is consistent with previous studies that have found significant leukocyte infiltration in the lungs at 6 h (20); however, by examining multiple time points, we have demonstrated that instead of a gradual increase over time, this increase is a rapid one that occurs between 4 and 6 h after brain death. These findings are exciting because they suggest that the leukocyte response to brain death in the lung is not a gradual response to the initial inflammatory signal, but is being triggered by a specific event that occurs prior. Although we have identified the rise in leukocyte adhesion molecules as one of these signals, it is likely not the only one. Further study may reveal other signals around this time that are part of this complex pathway, and may serve as therapeutic targets.
Just as DPR downregulated the cytokines and leukocyte adhesion molecules, it also exerts an effect on the leukocytes. Although the results of the flow cytometry were not statistically significant, they did suggest a downward trend in both macrophage and neutrophil infiltration with DPR. The lack of statistical significance is likely due to the relatively small group size. An in vivo model is intrinsically variable, and counting individual cells in a sample of tissue will also produce widely variable results, thus our study was likely underpowered to detect this difference. Although this downward trend by itself would have little meaning, the findings on IHC staining certainly show a clear reduction with DPR at 6 h, as well as a reduction in lung tissue edema. When these data are taken together with the cytokine and leukocyte adhesion molecule data, they make a compelling argument for the effectiveness of DPR in altering leukocyte infiltration.
Our findings vary from those found in other types of shock. After hemorrhagic shock, several authors have found increased neutrophil infiltration and ICAM expression increased 3 h after hemorrhage in rats and mice (24, 25). Other authors have examined lungs after ischemia and reperfusion injury, and found increased macrophages, neutrophils, and ICAM after 2 to 4 h in rats (26, 27). Sheridan et al. found increased lung neutrophil accumulation after 4 h in a rat model of sepsis (28). Although these studies are variable, they all suggest that, compared with the data in this study, macrophages and neutrophils infiltrate into the lungs earlier in other types of injury when compared with brain death. This could be due to a number of factors that make brain death different from other types of injury, such as the neural and hormonal changes. In addition, previous studies have shown that inflammation increased both with hemorrhage and resuscitation (24, 29), so it is also possible that the resuscitation after brain death, which relies more heavily on large volumes of crystalloid solution, may be an additional source of these differences.
Systemic inflammation after brain death is hypothesized to arise from multiple sources. It has been suggested that the process of brain death is associated with damage to the blood–brain barrier, and that inflammatory mediators are released from the brain itself (30, 31). It has also been theorized that the endothelium can be damaged by hypertension during the sympathetic surge (4) or by ischemia/reperfusion injury that leads to endothelial activation (32). We and other authors have suggested that some of this inflammation arises from the intestine (33, 34) because ischemia has been demonstrated to cause inflammation in other types of shock (35). This may explain how DPR, a therapy applied in the abdomen and that maintains intestinal blood flow, is able to reduce systemic inflammation and produce changes within the lungs.
There are multiple limitations of our study. Most importantly, the sample size is relatively small for an intrinsically variable in vivo model. This may be why the differences in leukocyte infiltration at 6 h failed to reach statistical significance despite the results visualized on IHC suggesting a large change. In addition, the experiment only examines the first 6 h of brain death in rats. As time from brain death increases, the rats become progressively more unstable and thus these results are more difficult to obtain. However, it is certainly possible that DPR's effects would be altered as time from application increased. Perhaps most importantly, this is a rat model and thus may not translate directly to a human model. Repeated experiments in humans would be needed to determine the correlating timeline. Also, our rat resuscitation was limited to IVF resuscitation that maintained blood pressure, whereas human brain dead organ donors receive more complex treatments including pressor agents and hormone drips, which may further alter leukocyte activity and were not used in our brain death model. Finally, we have demonstrated strong evidence of association between the use of DPR, and a reduction in lung injury and leukocyte infiltration after brain death. However, further studies are needed to definitively demonstrate a causation between these findings. Future studies will focus on further alterations to resuscitation and the subsequent effects of lung inflammation. Such alterations may include changes in IV resuscitation fluid, or adding anti-inflammatory mediators such as prostacyclins to the DPR solution, and could also use markers of ventilation such as arterial blood gas to assess outcomes.
In conclusion, our data suggest that by using DPR in the resuscitation of brain dead donors and maintaining the blood flow to the intestine during brain death, this decreases the permeability of the intestine and subsequent release of inflammatory mediators. This, in turn, leads to a reduction in inflammatory messaging within the lung, which decreases the infiltration of macrophages and neutrophils into the tissue. Although further study is needed to confirm the causative nature of these findings, use of DPR shows great potential as a way to reduce lung injury and inflammation after brain death.
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