The current study demonstrates that NO inhalation at 40 ppm for 23h starting at 1h after successful CPR markedly improves myocardial and neurological function and survival rate at 10 days after CA/CPR in mice. The neuroprotective effects of inhaled NO were associated with attenuation of the CA/CPR-induced abnormality in water diffusion detected using brain MRI at 1 day after CA and with prevention of caspase 3 activation in the hippocampal neurons at 4 days after CA/CPR. The salutary impact of inhaled NO on the outcome of CA/CPR was also associated with the inhibition of inflammatory cytokine induction in the brain and increased serum levels of nitrite and nitrate. Finally, deficiency of sGCα1, but not NOS3, abrogated the protective effects of inhaled NO on the 10-day survival rate, neurological function, and inflammatory cytokine induction after CA/CPR. Taken together, these observations suggest that breathing NO after successful CPR confers organ protection and improves survival, at least in part, via sGC-dependent mechanisms.
It is increasingly recognized that post–CA care after ROSC can improve the likelihood of patient survival with good neurological function. Clinical trials showed that TH conferred neuroprotective effects when it was applied for 12–24h starting minutes to hours after successful CPR from CA due to ventricular fibrillation.2,3
The apparent presence of a temporal therapeutic window after successful CPR is consistent with the observations that many of the mechanisms responsible for the post-CA brain injury are executed over hours to days following ROSC.21–24
These post-CA pathogenetic pathways include excitotoxicity, neuroinflammation, disrupted ion channel homeostasis, and membrane failure, as well as pathological activation of proteases and cell death signalling.21,22
The protective effects of breathing NO for 23h beginning 1h after successful CPR, observed in the current study, further support the notion that outcomes of sudden CA can be improved by implementing innovative therapies in the “post-CA golden hours” after successful CPR.
Conventional histopathological assessment of brain injury requires brain sections from individual animals sacrificed at separate time points after injury. These methods not only diminish the statistical power but may also introduce artifacts due to the post-mortem tissue preparation. In the current study, mice that were successfully resuscitated from 7.5 min of CA and breathed air exhibited a marked abnormality in water diffusion in the hippocampus, caudoputamen, and cortex 24h after CPR. The presence of abnormal DWI signals in the vulnerable regions of the brain 1 day after CA/CPR correlated with worse neurological function and increased apoptosis of hippocampal neurons 4 days after CPR, as well as decreased rate of survival at 10 days. In contrast, NO breathing markedly attenuated the development of abnormality in water diffusion in the brain and improved neurological outcomes and survival rate. These observations are consistent with a recent clinical study that showed that diffuse cortical abnormalities in DWI are associated with poor outcomes in patients resuscitated from CA.25
Hyperintense DWI signals indicate the presence of brain edema presumably due to disruption of ion pump function and membrane failure. The current observations, therefore, suggest that NO inhalation after successful CPR can preserve ion pump homeostasis and membrane integrity early after CA/CPR.
Although the greatest proportion of the post-CA mortality and morbidity is caused by global ischemic brain damage, the severity of myocardial dysfunction correlates with poor neurological outcome.26
We found that the degree of LV dysfunction 4 days after CPR was markedly attenuated in mice that breathed NO. These observations support the correlation between myocardial dysfunction and poor neurological outcomes and survival after CA/CPR.
RV dysfunction may also contribute to the circulatory failure after CA/CPR.27
Given the ability of inhaled NO to selectively reduce pulmonary artery pressure, it is conceivable that breathing NO improved outcomes of CA/CPR by reducing RV afterload. However, we did not find the evidence of pulmonary hypertension in WT mice 1h after CA/CPR (before initiation of NO inhalation). Because inhaled NO reduces pulmonary artery pressure only in the presence of pulmonary hypertension, it is unlikely that inhaled NO improved outcomes after CA/CPR by reducing RV afterload in our model.
Neuroinflammation triggered by the whole-body IR injury associated with CA/CPR hinder the neurological recovery from prolonged CA. We observed that CA/CPR markedly upregulated the expression of genes encoding inflammatory cytokines and NADPH oxidase in the brain of WT mice that breathed air, but not in WT mice that breathed air supplemented with NO. These observations suggest that NO inhalation prevents neuroinflammation after CA/CPR. Furthermore, these results demonstrate a correlation between neuroinflammation, neurological dysfunction, and mortality after CA/CPR.
NO elicits biological effects via sGC-dependent and/or -independent mechanisms. To determine the role of sGC in the protective effects of inhaled NO on the outcome of CA/CPR, we studied sGCα1−/−
mice. We observed that sGCα1-deficiency increased the early mortality rate (in the first 2h after CPR) when compared to WT mice after CA/CPR, consistent with our previous report.6
While the cause of these early deaths is unknown, we previously reported that sGCα1 deficiency markedly exacerbated LV dysfunction early after CA/CPR.6
After excluding the mice that died early after CPR, sGCα1−/−
mice that breathed air had 10-day survival rate comparable to that in WT mice that breathed air after CA/CPR. These observations suggest that sGC activity is critically important for initial recovery after CA/CPR but may not be necessary for long-term survival after CA/CPR. In contrast, sGCα1-deficiency abolished the ability of NO inhalation to inhibit the induction of inflammatory cytokines in the brain and to improve neurological function and 10-day survival rate after CA. These observations suggest that protective effects of inhaled NO on the outcome of CA/CPR are largely mediated via sGC-dependent mechanisms.
Inhaled NO may exert systemic effects via interaction with circulating bone marrow (BM)-derived cells (e.g. leukocytes) as they transit lungs. Alternatively, some NO, once inhaled, may escape scavenging by hemoglobin and be converted to relatively stable NO-metabolites (e.g., nitrite, S-nitrosothiols) that can regenerate NO in the periphery and directly protect neurons.28,29
In fact, in the present study, we found that breathing NO increased levels of nitrite and nitrate 24h after CA/CPR. We previously reported that neutrophils are required for inhaled NO to reduce MI size in WT mice subjected to transient left coronary artery occlusion.8
Along these lines, we recently observed that NO breathing markedly decreased MI size in WT but not in sGCα1−/−
Furthermore, breathing NO decreased MI size in chimeric sGCα1−/−
mice carrying WT BM generated by BM transplantation. These results raise the possibility that the neuroprotective effects of inhaled NO after CA/CPR maybe mediated by BM-derived cells in a sGC-dependent manner.
Our data does not exclude the possibility that sGC-independent mechanisms could contribute to the protective effects of inhaled NO on peripheral organs after CA/CPR. It is conceivable that NO modifies functions of enzymes and ion channels in a sGC-independent manner.7,31
For example, ischemic preconditioning has been shown to protect cardiomyocytes from subsequent IR injury by preventing Ca2+
overload via S-nitrosylation-mediated inhibition of L-type Ca2+
channel α1 subunit.32
Further studies are warranted to elucidate the mechanisms responsible for the protective effects of inhaled NO on the outcome after CA/CPR.
From the viewpoint of translating the current results into clinical benefit, it is of particular importance that NO inhalation started 1h after successful CPR and continued for 23h markedly improves neurological and myocardial function and survival rate 10 days after CA/CPR. For example, NO inhalation can be started after patients are transferred to hospital and informed consent obtained. To date, TH is the only therapeutic approach that is proven to improve outcomes after CA/CPR when applied hours after successful CPR.2,3
Since body temperature of mice were allowed to decrease to ~30°C during NO inhalation in the first 24h after CA/CPR in the current study, our data suggests that NO breathing may confer protection in the setting of mild hypothermia. Nonetheless, effects of combination of inhaled NO with TH, compared to either alone, on outcomes after CA/CPR remains to be formally determined in future studies.
There are several limitations in the current study. The induction of CA by bolus administration of potassium chloride may have limited clinical relevance. However, we believe this model provides a valuable platform for elucidating the molecular mechanisms of organ dysfunction associate with CA/CPR and the impact of inhaled NO on the post-CA syndrome. All mice were anesthetized when subjected to CA/CPR. It is possible that drugs used to induce anesthesia may impact outcomes of CA/CPR.
In summary, the current study revealed robust protective effects of NO inhalation on the outcome of CA/CPR in mice. Breathing NO at 40 ppm for 23h starting 1h after successful CPR markedly improved myocardial and neurological function and survival rate 10 days after CA/CPR, at least in part, via sGC-dependent mechanisms. The ability of “delayed” NO breathing to prevent the post-CA brain injury and promote survival in mice, if extrapolated to human beings, is highly clinically relevant and may serve as the experimental basis for future clinical trials in which effects of inhaled NO on the outcome after CA/CPR are examined. We anticipate that the established safety profile of NO inhalation33
will enable the rapid translation of findings in animal models to patients suffering from the post-CA syndrome.
Sudden cardiac arrest is one of the leading causes of death worldwide. Despite advances in resuscitation techniques, fewer than 8% of the 300,000 adults who experience cardiac arrest in the US each year survive to hospital discharge, and up to 60% of survivors have long lasting neurological deficits. While therapeutic hypothermia has proven effective in clinical studies, no pharmacological agent is available to improve outcome from cardiac arrest. Although originally developed as a selective pulmonary vasodilator, inhaled NO has been shown to have systemic effects in a variety of pre-clinical and clinical studies without causing systemic vasodilation. In the current study, we found that breathing a low concentration of NO starting 1h after successful CPR for 23h markedly improves long-term neurological and cardiac outcomes and survival in mice subjected to cardiac arrest and CPR. The ability of NO breathing to improve outcomes after cardiac arrest when begun after CPR, if extrapolated to human beings, makes inhaled NO a practical therapeutic approach, which can be initiated after patients are transferred to hospital. Furthermore, because inhaled NO does not cause systemic hypotension, in contrast to systemic NO-donors, it is uniquely suited for the treatment of post-cardiac arrest patients in whom blood pressure is often unstable. We anticipate that the established safety profile of NO inhalation will enable the rapid translation of findings in animal models to patients suffering from the post-cardiac arrest syndrome.