Inhaled nitric oxide reduces secondary brain damage after traumatic brain injury in mice

Abstract

Ischemia, especially pericontusional ischemia, is one of the leading causes of secondary brain damage after traumatic brain injury (TBI). So far efforts to improve cerebral blood flow (CBF) after TBI were not successful because of various reasons. We previously showed that nitric oxide (NO) applied by inhalation after experimental ischemic stroke is transported to the brain and induces vasodilatation in hypoxic brain regions, thus improving regional ischemia, thereby improving brain damage and neurological outcome. As regional ischemia in the traumatic penumbra is a key mechanism determining secondary posttraumatic brain damage, the aim of the current study was to evaluate the effect of NO inhalation after experimental TBI. NO inhalation significantly improved CBF and reduced intracranial pressure after TBI in male C57 Bl/6 mice. Long-term application (24 hours NO inhalation) resulted in reduced lesion volume, reduced brain edema formation and less blood-brain barrier disruption, as well as improved neurological function. No adverse effects, e.g., on cerebral auto-regulation, systemic blood pressure, or oxidative damage were observed. NO inhalation might therefore be a safe and effective treatment option for TBI patients.

Figures

Figure 1

(A) Nitric oxide (NO) inhalation does not increase intracranial pressure in healthy C57 Bl/6 mice during 30 minutes of NO inhalation (50 p.p.m.). (B) Auto-regulation is not changed by NO inhalation (iNO). (C) Expression of NO synthases in brain tissue is not altered by 24 hours of iNO. (D) iNO does not increase protein nitrosylation as assessed by immunohistochemistry. Images: × 40 magnification, inlets: × 100 magnification.

Figure 2

Nitric oxide (NO) inhalation improves cerebral blood flow and reduces intracranial hypertension after trauma without influencing systemic blood pressure. (A) Mean arterial blood pressure was equal in both groups. (B) Regional cerebral blood after trauma was markedly reduced in both groups. While in controls cerebral blood flow (CBF) remained low, it recovered in NO inhalation (iNO) mice, reaching almost baseline value toward the end of the observation period. (C) After trauma, there was an almost linear intracranial pressure (ICP) increase in untreated controls. Nitric oxide inhalation animals showed the same initial increase in ICP, but it was blunted immediately after the start of the therapy. Maximum ICP was significantly lower than in controls. (D) Posttraumatic ICP values were plotted against corresponding CBF values: Most control animals fall into the low CBF/high ICP group, while most values of iNO animals have high CBF and low ICP. *P<0.05, n=8 each.

Figure 3

Nitric oxide (NO) inhalation is neuroprotective after trauma in a dose-dependent way. (A) If initiated within 1 hour after trauma, NO inhalation (iNO) significantly reduces contusion volume at 24 hours. (B) For this neuroprotective effect it is necessary to conduct iNO over a period of 24 hours: If performed for 12 or 18 hours after trauma, lesion volume tends to be smaller, but the changes did not reach statistical significance. (C) Intermittent application of iNO (3 hours iNO, 1 hour break) could not reduce contusion volume in a significant way as continuous administration did. *P<0.05, n=8 each.

Figure 4

(A) Posttraumatic edema formation was significantly reduced after 24 hours of nitric oxide inhalation (iNO). *P<0.02, n=8 each. (B) Evans Blue extravasation in the traumatized hemisphere was significantly lower in the iNO group than in controls (n=6 each, *P<0.05).

Figure 5

Neurological outcome over 7 days after trauma is better in animals receiving nitric oxide inhalation (iNO) for the first 24 hours after trauma. (A) Performance in Neurological Severity Score (NSS) testing significantly deteriorated in both groups compared with pre-trauma values. Controls recovered slowly and still presented a marked neurological deficit at day 7, iNO animals regained neurological functions quicker than untreated mice; from day 5 on they achieved significantly better NSS results than controls. n=8 each, *P<0.05 versus control, #P<0.05 compared with corresponding pre-trauma score. (B) iNO significantly reduced contusion volume after 7 days.*P<0.05 versus control, #P<0.001 versus corresponding pre-trauma value, n=8 each.