Our results showed that we have successfully produced a lung injury model using CLP and HV ventilation that could be compared with the rats exposed to LV ventilation. However, in opposition to our hypothesis, rTM did not alleviate the lung injury, as shown by the PaO2 and BALF protein levels. Moreover, although most clinical findings are consistent with rTM worsening lung injury of septic rats, the results were not statistically significant.
Model of lung injury
CLP is a traditional model for inducing bacterial sepsis, although an earlier study showed that the CLP itself did not cause lung injury (28). Two invasions are thought to be needed to induce a VILI—hence the “two-hit theory.” There are data suggesting CLP plus HV ventilation causes VILI (29). We, however, found no oxygenation deterioration in their VILI model.
We successfully produced a lung injury model by adding HV ventilation to CLP rats, which was confirmed by the decreased PaO2 level and increased protein concentration in BALF. Following the initial increase in PaO2 in all groups, the PaO2 did not decrease in the LV-ventilation groups but decreased significantly in the HV-ventilation groups, suggesting that HV ventilation causes lung injury. The reason that PaO2 was initially increased was probably due to overstretching of the lung. HV overstretches more than LV and this caused initial higher oxygenation but resulted in higher lung injury. Lung IL-6 and MCP-1 mRNA levels increased more in the CLP groups than in the sham-operation rats, indicating that our model exhibited a sepsis-associated inflammatory response. Platelet counts and the plasma TAT and D-dimer levels were similar among the rTM non-treated groups, showing that the rats had not developed DIC. Thus, rTM was administered to septic rats that did not have overt DIC.
The concentration of BALF is normalized according to its fluid (saline) volume. The BALF protein increase and bacterial infiltration of alveoli in the HV ventilation groups indicate that mechanical stretching during MV exacerbated lung inflammation in the CLP rats. The BALF TAT concentration was significantly more increased in HV ventilation groups than in the LV ventilation groups, suggesting that HV ventilation activated coagulation in the alveoli in this group, which is also one of the characteristics of the VILI lung.
TRPV4 is a stretch-activated cation channel, which was originally identified as the sodium channel in Drosophila. Recently, it was found in lung endothelial cells, epithelial cells, and alveolar macrophages. TRPV4 is activated by membrane stretching, and it causes cell spreading of alveolar macrophages. Activation of these macrophages is known to cause VILI by increasing pro-inflammatory cytokines (4). Because TRPV4 knockdown mice or administration of TRPV4 receptor antagonist prevents the development of VILI (32, 33), we measured lung TRPV4 mRNA levels and found no differences among the groups. As TRPV4 mRNA expression was not increased more in the HV ventilation groups than in the LV ventilation groups, TRPV4’s function, not expression alone, might cause VILI.
Controversy Concerning rTM
Reports of the effect of rTM on the septic model have been inconsistent among studies. For example, rTM administration blocked LPS-induced acute lung injury in one study (34), whereas it improved survival in mice lacking the lectin-like domain of rTM (35).
In our experiment, rTM did not alleviate the lung injury. This is confirmed by the PaO2 level and BALF protein leakage. There was a significant increase in BALF IL-6 in rTM-treated, HV-ventilated rats, compared with the LV-ventilated groups, while plasma IL-6 was not significantly different among groups suggesting that induction of lung local inflammation was more sensitively occurred than plasma inflammation.
BALF TAT levels were unexpectedly significantly higher in the HV/Sep/TM(+) group than in the HV/Sep/TM(−) group. We speculated that this result might also show that rTM could not alleviate lung injury in the present study. The effect of rTM on TAT generation can be projected as follows. Plasma level thrombin generation is suppressed by rTM administration in LV ventilation groups. Administration of rTM in the HV ventilation group accelerated the destruction of the lung wall and induced high-level local inflammation in addition to vascular permeability due to the systemic inflammation. This situation resulted in a greatly increased level of TAT in lung but not enough to be inactivated by the infiltrated rTM.
We offer three possible explanations for why rTM was not effective in the current VILI model. First, the timing of the administration may have been too early. With our experimental protocol, rTM was administered before inducing sepsis and DIC. Our results may indicate that too-early administration may have suppressed immunologically necessary trap formation. Recently rTM was shown to inhibit neutrophil extracellular trap (NET) formation (36). NET formation is known to have an important role in the host’s defense against bacterial infection, but it may also induce hypercoagulability. However, we have chosen this timing because our previous experiment showed that plasma rTM level were elevated after 9 h and remained elevated for 48 h after rTM injection (37). Thus, plasma level rTM was appropriate when septic VILI has been made.
Second, our protocol was based on a bacterial infection-caused VILI model that was not treated with antibiotics. rTM has an anti-inflammatory effect by increasing anti-inflammatory cytokines (e.g., IL-1β, TGFβ) in an LPS model. In the present study, TGFβ mRNA was decreased in the HV/Sep groups and was not increased by rTM administration. These data suggest that, in the presence of live bacteria, anti-inflammatory cytokines are suppressed, and rTM administration does not induce an anti-inflammatory response.
Third, the intensity of the disease was not identical among groups. CLP is a traditional method for inducing bacterial sepsis. However, the quantity of feces that spreads into the abdomen might not be equal among the subjects, and the bacteria species responsible for the infection in each rat are ultimately unknown. This situation, however, is equivalent to intestinal perforation in the clinical setting. Even if there were some diversity in the intensity of the bacterial infections, we successfully showed statistical differences in the impact of HV ventilation.
There are several limitations in this study. One is the mechanism of rTM improved oxygenation in hyperoxic VILI but not in this experiment was not clear. Infection, DIC, severity and timing are possible explanations but not sure. Further research is needed. Second, the number of the animals were small. We tried to minimize the number of animals but might be too small to show the statistical analysis. Increased number of the animals should be considered for further research.