In our experimental model of LTx, the infusion of the free radical scavenger, hydrogen sulfide, is associated with a significant decrease in the presence of ROS and a trend toward an increased level of cGMP. eNOS and iNOS expression were similar between the two groups. Additionally, physiological measures of lung performance including oxygenation, ventilation, and mPAP were similar.
LTx is often complicated by severe respiratory failure secondary to IRI. This injury is thought to be partially mediated by ROS. Thus, a therapy that can decrease the level of these damaging molecules has potential clinical utility in LTx. Although the levels of ROS declined in both groups over time, our experiment demonstrated a greater decline in ROS levels in the group treated with H2
S. Although ROS have been previously associated with IRI, in our experiment, reduction in ROS levels was not associated with improved clinical lung function.1–4
Several explanations are possible.
First, it is possible that ROS are not a critical mediator of gas exchange or pulmonary vascular reactivity in the early reperfusion period. Other studies have also found free radical scavengers to be ineffective at improving oxygenation and reducing pulmonary pressures in models of LTx.18
Although ROS have been previously associated with IRI in LTx, it’s possible that disruption of other biochemical pathways and modulation of inflammatory mediators are more clinically relevant in the early reperfusion period. Free radical damage is primarily mediated by direct tissue injury and the up-regulation of pro-apoptotic pathways through the modulation of intracellular calcium levels.3
While this damage may be important to long-term graft function, it may not affect early clinical performance. Further investigation with longer periods of reperfusion is warranted.
Second, it is possible that our model produces more severe lung injury than can be ameliorated by post-injury treatment. In our past experience with this model, we have found the rabbit lungs to be highly resistant to ischemic injury, leading us to use prolonged ischemia (18 hours) to ensure lung damage. However, while the lungs tend to be resistant to injury to a point, at approximately 18 hours, they appear to collapse, sustaining often irrecoverable injury. It is possible that our current model causes such severe injury that the additional oxidative stress caused by ROS is not clinically relevant. Thus mitigating ROS damage by decreasing ROS levels may not produce a clinically significant difference. Given these limitations, future investigation will make use of less severe injury as well as pre-ischemia H2S treatment, aimed at preventing injury caused by prolonged ischemia.
Third, it is possible that our dose of H2
S was insufficient. In the literature, two different forms of intravenous H2
S donors have been utilized over a wide range of concentrations and in various different animal models. Initial bolus doses range from 100–3,000 ug/kg followed by maintenance infusion doses of 0.5–3 mg/kg/hour.9, 19–21
However, high doses of H2
S can cause acute lung injury.17, 22
Wary of causing iatrogenic lung injury, we chose a modest dose used by other investigators interested in cardiopulmonary function.8, 11
However, review of the temporal trends in ROS levels in our experiments suggests that the initial bolus dose was possibly too low as ROS levels do not differ between the groups until after 2 hours of continuous H2
S infusion. We suspect that a greater bolus dose would lead to an earlier differential in the presence of ROS, possibly leading to less free radical damage in the treated group and thus a greater clinical difference. This idea is supported by previous work that suggests there is a very narrow therapeutic window for effective administration of H2
S after IRI.20
Previous research suggests that elevated levels of cGMP are associated with improved outcomes in LTx, mediated by decreased ROS levels, improved immunologic cell regulation, increased perfusion through vascular dilation, and the activation of cytoprotective mediators.1, 23, 24
In our experiment, infusion of H2
S was associated with increased levels of cGMP although this did not reach statistical significance. Nitric oxide synthases are an important pathway involved in the production of cGMP. However, in our experiment, levels of eNOS and iNOS were similar between the two groups. Previous research from our laboratory suggests that in LTx, eNOS may be protective and iNOS may be injurious.1
S has been previously associated with the upregulation of NOS 3 and the downregulation of iNOS through modulation of endogenous carbon monoxide and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).5, 20
In these experiments, eNOS and iNOS levels were not significantly affected by H2
S but cGMP levels were increased, suggesting either a mechanism of cGMP production outside the canonical nitric oxide (NO) pathway or a type II error. The relationship between NO and H2
S is not well understood, meriting further exploration.
The administration of H2
S has been associated with cytoprotective effects in several models of IRI. In a model of myocardial infarction, Sodha et al.8, 11
demonstrated that H2
S was associated with decreased infarct size, preservation of cardiac function, decreased apoptosis, and decreased inflammatory mediators. Similar cytoprotective effects have been demonstrated in IRI models of liver, kidney, and skin transplantation.7, 14–16
Previous research has also demonstrated H2
S to be protective of injury in several models of acute lung injury. In a model of ventilator-induced lung injury, Faller et al.25
demonstrated that inhaled H2
S combined with mild hypothermia was associated with decreased pulmonary edema, neutrophil accumulation, and pro-inflammatory cytokine production. Similarly, in a model of smoke-induced lung injury, Esechie et al.21
found infused H2
S to be associated with decreased mortality and improved gas exchange. Several studies have verified that H2
S is associated with the decreased production of inflammatory mediators and a decreased immune response.26, 27
While we did not directly assay for immunologic or inflammatory molecules, the decreased levels of ROS exhibited in our experiment suggest a significantly blunted inflammatory response in the presence of H2
Additionally, several investigators have reported that H2
S may cause pulmonary artery relaxation, reducing pulmonary artery pressure, particularly in models of hypoxic pulmonary hypertension.28, 29
Although we were not able to replicate this finding in our experiments, it is of particular importance in LTx where many of the recipients experience pulmonary hypertension preoperatively secondary to their underlying disease and then postoperatively secondary to IRI.
Despite the potential advantages of H2
S in LTx, some caution is warranted. High levels of H2
S can be directly toxic to the lungs, causing acute lung injury and/or triggering apnea through regulation of CO2
Moreover, Perry et al.26
have shown that H2
S can inhibit airway smooth muscle cells in human lung tissue in asthmatic patients. Although this may be beneficial in the setting of asthma, airway smooth muscle may be damaged during the LTx procedure, requiring proliferation and regeneration of these cells. Such a process may be inhibited by H2
Finally, recent studies suggest that H2
S can be used to lower cellular metabolism, inducing a state of suspended-animation that allows animals to survive under hypoxic conditions.5, 30–32
In clinical LTx, hypothermic storage is used to decrease allograft metabolism and protect the lungs from ischemic injury. Interestingly, Blackstone et al.31
have shown that H2
S administration can reduce cellular metabolism and allow mice to survive severely hypoxic conditions. Though such research is in its early stages, the prospect of inducing a state of suspended-animation that protects organs from injury during ischemia and reperfusion by lowering their metabolic oxygen requirement is appealing and merits further investigation.
In this study, we utilized an ex vivo model of lung transplantation. We did not induce brain death in the donors nor did we utilize immunosuppression. Therefore, we did not completely replicate the in vivo LTx procedure. Moreover, since we only conducted our experiment over 2 hours, we cannot evaluate long-term effects.
The ideal method of delivery, dosage, or duration of H2
S treatment, particularly in lung models, is unknown and wide ranges have been utilized in experimental models.9, 19–21
Furthermore, though a significant literature exists surrounding the use of hydrogen sulfide donors rather than H2
S gas, it is possible that the inhaled, gaseous form would produce better clinical results, particularly in LTx where a local effect would be possible, avoiding the potential complications of systemic administration. In future experiments, we plan to investigate the utilization of inhaled H2
Moreover, our experiment may be limited by small sample size and thus susceptible to a type II error. Finally, we believe our experimental model is successful in producing severe lung injury; however, we speculate that this injury may be too severe, particularly for a model aimed at evaluating post-injury treatments. In the future, we plan to subject the lungs to shorter periods of ischemia and focus on therapies that prevent injury before it occurs.