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Direct Peritoneal Resuscitation Alters Leukocyte Infiltration in the Lung After Acute Brain Death

Weaver, Jessica L.*,†; Matheson, Paul J.; Matheson, Amy; Downard, Cynthia D.*; Garrison, Richard Neal*; Smith, Jason W.*

doi: 10.1097/SHK.0000000000001069
Basic Science Aspects
Editor's Choice

Background: Brain death is associated with significant lung injury and inflammation. This has been associated with worse long-term outcomes for transplanted lungs. Direct peritoneal resuscitation (DPR) reduces systemic inflammation in brain death and improves lung procurement rate. The effect of DPR on macrophage and neutrophil infiltration in the lungs is not known.

Methods: Male Sprague–Dawley rats had a 4F Fogarty catheter inserted into the skull and the balloon inflated until brain death was achieved. Rats were resuscitated with normal saline to maintain a mean arterial pressure of 80 mmHg (targeted intravenous fluid, TIVF) and DPR animals received an intraperitoneal injection of commercial peritoneal dialysis solution. Rats were sacrificed at 0, 2, 4, and 6 h after brain death. Protein levels were assessed using quantitative ELISA. Leukocytes were quantified using flow cytometry and immunohistochemistry.

Results: At all time points, DPR downregulated multiple inflammatory cytokines including IFN-γ, TNF-α, IL-1α, and IL-6. Adhesion molecules ICAM, E-selectin, and P-selectin were increased above sham at 4 and 6 h after brain death and reduced with DPR, whereas VCAM was reduced at 2 and 6 h. Infiltration of macrophages and neutrophils were trended downward at 6 h with DPR, though this difference was not statistically significant.

Conclusions: Animals that received TIVF alone had significant increases in inflammatory cytokines within the lung tissue, leading to adhesion molecule expression and ultimately leukocyte infiltration. Each stage of inflammation was affected by DPR. Using DPR in brain dead organ donors shows promise as a way to reduce lung injury and inflammation.

*University of Louisville Department of Surgery, Louisville, Kentucky

Robley Rex Veterans Affairs Medical Center, Louisville, Kentucky

Address reprint requests to Jessica L. Weaver, MD, PhD, 550 S Jackson St, ACB 2nd floor, Rm A2J19, Louisville, KY 40292. E-mail: jlweav08@louisville.edu

Received 2 July, 2017

Revised 25 July, 2017

Accepted 20 November, 2017

Presented as a poster at the 2017 Shock Society annual meeting in Fort Lauderdale, FL.

The authors report no conflicts of interest.

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INTRODUCTION

Brain death occurs when cerebral swelling or bleeding within the skull causes increased intracranial pressure, forces the brain to herniate through the foramen magnum, leading to brain stem infarction (1). This process is associated with a number of physiologic changes within the body. Initially, there is a significant increase in the release of catecholamines, causing increased heart rate and blood pressure. After this there is a loss of sympathetic outflow, causing peripheral blood vessels to lose their resting vascular tone, leading to profound hypotension (1, 2).

Brain death is associated with significant lung injury. In addition to the contusion and aspiration that frequently accompany any initial trauma, brain dead patients frequently develop neurogenic pulmonary edema (1). This edema is theorized from two possible sources: hypertension and increased capillary pressure causing direct pulmonary capillary damage (3), or increased capillary permeability due to α-adrenergic stimulation (4). Brain death also causes significant systemic inflammation as well as inflammation and apoptosis within the lung tissue (4). When these lungs are procured for transplantation, this inflammation leads to worse ischemia–reperfusion injury (5) and higher rates of rejection (6). In part because of this inflammation and edema, only 20% of lungs from brain dead donors are considered suitable for transplantation (7).

This lung damage can be worsened by resuscitation after brain death. Large amounts of intravenous (IV) crystalloid fluid is often given to support blood pressure, but this can worsen lung edema (2). Direct peritoneal resuscitation (DPR) is an innovative method of resuscitation that allows for resuscitation with less IV fluid. This process involves introducing hyperosmotic fluid directly into the abdomen in addition to conventional resuscitation. The hyperosmolarity of the solution causes visceral vasodilation (8) that reverses the intestinal ischemia normally associated with shock (9). We have previously demonstrated that use of DPR in a rat model of brain death leads to improved visceral organ blood flow, and reduced serum levels of multiple inflammatory cytokines (10). This study also showed that DPR caused a significant reduction in organ edema, particularly in the lungs, likely due to the reduction in the amount of IVF required (10). This may be part of the mechanism for how use of DPR in human organ donors led to an improvement in the rate of lung procurement (11). We hypothesized that these anti-inflammatory effects of DPR would lead to a reduction in the infiltration of macrophages and neutrophils into the lung after brain death.

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MATERIALS AND METHODS

Animal model

Animals were maintained at the Veterans Affair Hospital Research Facility in Louisville, KY, which is approved by the American Association for the Accreditation of Laboratory Animal Care. This protocol was approved by the Institutional Animal Care and Use Committee. All experiments were performed using male Sprague–Dawley rats 250 to 300 g. They were given food and water ad lib and were acclimated using 12-h light/dark cycles for at least 7 days before use.

Body temperature was maintained at 37.0 ± 1.0°C with a rectal probe and a servocontrolled heating pad. Anesthesia was initiated with intraperitoneal (IP) pentobarbital and a tracheostomy was inserted. Then the right carotid artery and jugular vein were isolated and cannulated with PE-50 tubing. The rat was then placed prone and a drill was used to create a small opening in the skull. A 4F Fogerty catheter was slowly inserted into the epidural space. The rat was then returned to the supine position and allowed to equilibrate for 20 min.

Baseline measurements were taken 10 min apart after the equilibration period was complete. After the second measurement, the epidural catheter was inflated at 1 mL/h. When the sympathetic surge and subsequent hypotension indicating brain death was seen, the tracheostomy was connected to the ventilator and the rat was ventilated with room air at a tidal volume of 1.5 mL and a rate of 90 breaths/min. Simultaneously, intravenous normal saline (NS) was infused at whatever rate needed to maintain a mean arterial pressure (MAP) of 80 mmHg. All rats received this targeted IV fluid (TIVF) resuscitation. Animals scheduled for DPR received a single IP injection of 30 mL of 2.5% glucose-based clinical peritoneal dialysis solution (Delflex; Fresenius USA, Inc. Ogden, Utah). The TIVF animals did not receive any intraperitoneal control solution such as normal saline because we have previously demonstrated that this does not have any effect (12). Continued anesthesia was not necessary as the rats were brain dead at this point. Sham animals had the cannulas placed, but the epidural catheter was not inflated so the rats never experienced brain death.

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Tissue collection

Animals were sacrificed immediately after the time of resuscitation (time 0), 2, 4, or 6 h postresuscitation with n = 8 per group. Sham rats were sacrificed at 2 h. After sacrifice, lung tissue was snap-frozen in liquid nitrogen. In addition, 250 mg of fresh lung tissue was cut into small pieces, and then allowed to digest with a 0.1% collagenase solution for 30 min at 37°C. The solution was then strained through a 70 μm cell strainer to remove excess tissue. The cells were washed in Dulbecco's phosphor-buffered saline (DPBS) by centrifuging the tubes, extracting the supernatant, and resuspending the cell pellet in a 1% paraformaldehyde solution. This was allowed to fix for 20 min at room temperature. The cells were then washed with DPBS and stored in a solution of 70% DPBS, 20% calf serum, and 10% glycerol and stored at −80°C.

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Flow cytometry

To prepare samples for flow cytometry, 500 μL of the thawed tissue digestion was stained using the Foxp3 Staining Buffer Set (Invitrogen, Carlsbad, Calif) that stains for surface and intracellular proteins. In brief, antibodies used included Anti-CD163:FITC (Bio-Rad Laboratories, Hercules, Calif); Anti-CD68:Alexa Fluor 700 (Bio-Rad); Anti-CD80:APC (Life Technologies, Waltham, Mass); Anti-CD11b:PE-Cy 7 (BD Bioscience); and Anti-RP-1:PE (BD Biosciences, San Jose, Calif). Neutrophils were identified by the presence of CD11b and RP-1 (13, 14) and macrophages by CD68 (15). Macrophage activation was represented CD80 for M1 activation and CD163 for M2 activation (15, 16). Flow cytometry was formed using a Becton Dickinson LSR II detector with lasers at 405 nm, 488 nm, and 633 nm.

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Protein quantification

Analysis of protein levels was measured using quantitative ELISAs (LifeSpan Biosciences, Seattle, Wash). In brief, tissues samples were lysed in buffer in the TissueLyser II (Qiagen, Germantown, Md). Samples were then mixed in a 96-well plate with the provided buffer and substrate according to the manufacturer's instructions. After an appropriate incubation period, plates were read at the recommended wavelength with a spectrophotometer. Results were then analyzed using SigmaPlot (Systat Software Inc, San Jose, Calif) and comparisons between multiple groups were made using ANOVA with P ≤ 0.05.

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RESULTS

Levels of cytokines and chemokines in the lung tissue are displayed in Table 1. Multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-1α, IL-6, and IL-18, were increased after brain death and decreased at both 2 and 4 h with the addition of DPR. Anti-inflammatory mediators IL-4 and IL-10 were also increased at 2 and 4 h compared with sham and decreased with DPR. Levels of IL-2 were equivalent at all time points. Levels of IL-5 and IL-13 were reduced with DPR at 2 h but unchanged by DPR at 4 h.

Table 1

Table 1

Leukocyte adhesion molecules ICAM, VCAM, E-selectin, and P-selectin are shown in Figure 1. Levels of ICAM, E-selectin, and P-selectin remained equivalent to sham levels at 2 h, then significantly increased at 4 and 6 h in the TIVF group, and were decreased with the addition of DPR. Protein levels of VCAM were increased at 2 and 6 h compared with sham and decreased with DPR. Levels of VCAM were equivalent to sham at 4 h with and without DPR.

Fig. 1

Fig. 1

Results of the flow cytometry analysis for are shown in Figures 2–4. In the TIVF group there is a large increase in the number of macrophages and neutrophils that infiltrate the tissue at 6 h. Use of DPR blunted this increase, though the difference between these two points failed to reach statistical significance. Use of DPR increased M1 activation at 2 h compared with TIVF alone, and decreased M2 activation at 2 and 4 h, but both M1 and M2 activation were equivalent in both groups by 6 h. Neutrophil activation is represented by MPO activity (Fig. 5), and this was also decreased with DPR at 2, 4, and 6 h.

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Fig. 5

Fig. 5

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.

Fig. 6

Fig. 6

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DISCUSSION

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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Keywords:

Donor resuscitation; lung transplant; macrophage; neutrophil; transplant rejection

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