These data show that isoflurane anesthesia prevents the development of ALI, in part, through inhibition of the platelet ADP pathway. Our standard T/HS model was shown to induce ALI as evidenced by BALF protein levels, and histologically, by imunofluorescence. However, animals pre-treated with isoflurane prior to T/HS developed no lung injury. Platelet Mapping was employed on whole blood obtained from animals pre-treated with isoflurane to determine isoflurane’s in-vivo effect on platelets. Platelet Mapping confirmed a specific ADP-pathway inhibition, and no change was observed in the AA-pathway. To determine if ADP-pathway inhibition was involved in the mechanism of protection, animals were pre-treated with a platelet ADP-receptor antagonist (clopidogrel) and subjected to T/HS. This resulted in protection from developing ALI, suggesting that isoflurane’s antiplatelet effects are significant in its post-injury protective properties.
Since these studies are performed at intermediate altitude, with a lower partial pressure of oxygen, our model incorporates the return of heparinized shed blood during resuscitation to prevent critical anemia and early mortality. Our prior work has shown that heparinized shed blood contains activated platelet microaggregates, and thus, exacerbates ALI compared to other models which do not use the return of heparinized shed blood.6
In our prior study, thrombelastography-based platelet mapping demonstrated thrombin-independent platelet activation without the addition of AA or ADP. This current study demonstrated the capability of isoflurane to overcome the platelet activation observed in heparinized shed blood, and specifically inhibit platelets through the ADP pathway.
Although most of the protective effects of isoflurane in sepsis17,18
injuries have been attributed to the neutrophil, the effect on the platelet has been largely overlooked. The emerging role of platelets as a key mediator of ALI has been shown in TRALI, sepsis, and T/HS models, and continues to gain clinical support.4–6
Moreover, there is substantial evidence that a coupling of both the inflammatory and innate immune systems are necessary for the development of ALI.19–21
Thus, platelets may be the principle link, since uncoupling this system through platelet inhibition prevents the neutrophil-mediated injury. Recent evidence has shown that the platelet-endothelium interaction mediated through platelet P-selectin is vital in the pathogenesis of ALI.22
Platelet activation results in platelet P-selectin expression and platelet-leukocyte aggregations through P-selectin glycoprotein ligand-I (PSGL-1) expression on the leukocyte, and this activation may induce a pro-thrombotic state in the microcirculation through the release of leukocyte tissue factor.23
Since pulmonary capillary microthrombi have been implicated in the development of ALI/ARDS,14,15
this phenomenon could explain the microthrombi observed in patients who develop ALI/ARDS. In this animal model, isoflurane eliminated pulmonary microthrombi observed following T/HS.
Halogenated ethers, therefore, with known anti-inflammatory properties, and now with known anti-platelet effects could be a potential therapeutic in the prevention of post-traumatic ALI through this uncoupling effect. Animals pre-treated with an ADP-receptor antagonist had a greater BALF protein leak compared to animals pre-treated with isoflurane. This may be attributed to the dual inhibitory effects of isoflurane on both coagulation and inflammation. Most studies exploring the protective effects of halogenated ethers have involved cardiothoracic surgery patients, since volatile anesthetics have been shown to protect the myocardium against ischemic-reperfusion injury.8
These studies have demonstrated that isoflurane reduces neutrophil adherence to the endothelium,9,10
and attenuates the neutrophil-mediated injury.24,25
In addition, isoflurane reduces the release of tumor necrosis factor-α, and inhibits cytokine-induced death of endothelial and smooth muscle cells.26
These factors, along with anti-platelet properties, make halogenated ethers a potential therapeutic against the development of ALI.
Clinically, since patients cannot be pre-treated with isoflurane prior to their injury, we explored if isoflurane given post-injury could offer protection. Animals were, therefore, given isoflurane at the initiation of resuscitation. Although this did not decrease pulmonary capillary leak to T/SS levels, there was a significant reduction observed, further implicating isoflurane as a potential post-injury therapeutic. Full anesthetic doses of halogenated ethers may not be required to see these effects, since sub-anesthetic doses can decrease platelet-neutrophil interactions in human subjects.11
With the use of any drug in in-vivo models, unintentional effects must be assumed, and controlling for these effects is difficult. Animals not receiving isoflurane received sodium pentobarbital as an anesthetic. We attempted to minimize possible confounding effects by studying both isoflurane and pentobarbital control and T/SS groups. Other limitations of this study include the lack of both dose-response and timing relationships. Our primary purpose was to determine if isoflurane would decrease ALI in a clinically relevant animal model, and to determine if isoflurane had anti-platelet properties supporting other models of lung injury. In fact, increasing the isoflurane may have additional protective effects in the lung, as well as in other organs. On the other hand, further derangement in platelet function may have adverse effects, particularly in the context of uncontrolled cavitary bleeding or intracranial hemorrhage.
In spite of these acknowledged limitations, we have shown that pre-treatment with isoflurane abolishes T/HS-induced ALI, inhibits platelets specifically through the ADP pathway, and that treatment initiated at the start of resuscitation has a significant reduction of pulmonary capillary leak. Due to what is known about the pathogenesis of ALI and the mechanistic effects of halogenated ethers, further investigation is warranted to determine if volatile anesthetics have any clinical therapeutic potential. A large body of clinical evidence supports their use in cardiothoracic surgery, and therefore, these volatile anesthetics may be a viable option in post-injury surgeries or may be given in sub-anesthetic doses by paramedics prior to arrival to the emergency department.