Systemic Nitrosative/Oxidative Stress in Patients with Acute Brain Injury

Acute brain injury due to traumatic brain injury (TBI), intracerebral haemorrhage (ICH), and aneurysmal subarachnoid haemorrhage (SAH) carries a high morbidity and mortality, in part due to the development of secondary brain injury. The mechanisms behind secondary brain injury are incompletely understood, but oxidative/nitrosative stress and disturbances in the metabolism of the vasodilator nitric oxide (NO) are believed to be involved. The aim of the present study is to characterise systemic changes in markers of oxidative/nitrosative stress and NO metabolism in the early phase after acute brain injury, and to examine their relationship to clinical course, neurological outcome, and mortality.

BACKGROUND:

Acute brain injury due to traumatic brain injury (TBI), intracerebral haemorrhage (ICH), and aneurysmal subarachnoid haemorrhage (SAH) is a major cause of mortality and permanent disability worldwide. Irrespective of its aetiology, acute brain injury is associated with a widespread activation of cellular and biochemical processes which can aggravate the damage after the primary injury - this is termed secondary brain injury.

Nitric oxide (NO) is a potent endogenous vasodilator produced from arginine by the enzyme nitric oxide synthase (NOS), which exists in three isoforms: endothelial, neuronal, and inducible NOS (eNOS, nNOS and iNOS). In conditions of inflammation and oxidative stress (e.g. in acute brain injury), free radicals may react with NO to form peroxynitrite (ONOO-), which is highly reactive and can directly damage biological macromolecules such as lipids and proteins. This phenomenon, i.e. an increased production of reactive nitrogen species potentially leading to cellular damage, is termed nitrosative stress.

It is widely believed that oxidative/nitrosative stress and associated disturbances in the metabolism of NO are involved in the development of secondary brain injury, but the exact role of these mechanisms remains incompletely understood. While some authors believe that NOS dysfunction and a resultant low NO bioavailability is an important cause of secondary brain injury, others argue that an overproduction of NO mediated by iNOS is maladaptive response leading to aggravated tissue injury due to nitrosative stress.

The investigators hypothesise that acute brain injury is associated with an immediate elevation in circulating biomarkers of oxidative stress and a reduction in the bioavailability of NO due to formation of peroxynitrite (nitrosative stress), and that this represents an important mechanism behind the development of secondary brain injury. This decrease in NO availability could contribute to a vicious cycle in which a resulting increase in microvascular resistance, cerebral hypoperfusion, and brain tissue hypoxia further increases free radical production. However, it is further hypothesised that the initial decrease in NO availability is followed by an iNOS-mediated increase in NO metabolites in the subsequent days after injury. The present explorative study will attempt to characterise these changes and their role in patients with acute brain injury.

HYPOTHESES:

1. Patients will have the highest levels of oxidative/nitrosative stress markers and lowest levels of NO metabolites immediately after ictus, with a progressive reduction in oxidative/nitrosative stress markers and increase in NO metabolites over the subsequent days.
2. The degree of oxidative/nitrosative stress will be associated with an unfavourable clinical course (e.g., episodes of neuroworsening), poor neurological outcome, and death.
3. Patients with a higher disease severity (e.g., a higher World Federation of Neurological Surgeons Score for patients with SAH) will have a greater degree of oxidative/nitrosative stress compared to patients with a lower disease severity.
4. The degree of oxidative/nitrosative stress will be associated with the degree of biomarker-determined neurovascular unit injury.
5. The degree of oxidative/nitrosative stress will be associated with evidence of systemic organ dysfunction.
6. The degree of oxidative/nitrosative stress and relative NO-depletion is associated with brain tissue hypoxia, brain metabolic crisis, and cortical spreading depolarisations (in a subset of patients undergoing multimodal neuromonitoring).

METHODS:

The study is a single-center, prospective, explorative, observational study, which will include 50 patients with SAH, 50 patients with ICH, and 50 patients with TBI admitted to the Neurointensive Care Unit (NICU) at Rigshospitalet, Copenhagen. Patient inclusion will continue until the planned number of patients have been enrolled, or until the 1st of May 2023, at which point inclusion will be halted and data will be analysed irrespective of the number of included patients.

Arterial blood samples will be collected at 3 time points: day 0-2 (early), day 3-5 (intermediate) and day 6-8 (late) after admission. If no arterial catheter is available, central venous or peripheral venous samples may be drawn as an alternative. Blood samples will only be collected during admission to the NICU and/or intermediate care unit, and sample collection will be halted in case of discharge to another department.

Demographical, clinical and paraclinical data will be obtained from each patients' electronic medical records. Data from multimodal neuromonitoring (i.e., intracranial pressure, brain tissue oxygenation, cerebral microdialysis, and/or electrocorticography) will be collected continuously along with physiological parameters when available. Neurological outcome (as determined by the modified Rankin Scale) will be determined at 6 months in connection with an outpatient follow-up visit at the hospital or through telephone interviews.

BIOCHEMICAL ANALYSES:

Blood samples will be analysed for the following markers of oxidative stress: the ascorbate radical, lipid hydroperoxides, myeloperoxidase, and the antioxidants glutathione, α/γ-tocopherol, α/β-carotene, retinol and lycopene.

The following NO metabolites will be determined: total plasma NO concentration (nitrate (NO3-) + nitrite (NO2-) + S-nitrosothiols (RSNO)) and total red blood cell bound NO (nitrite (NO2-) + nitrosyl haemoglobin (HbNO) + S-nitrosohaemoglobin (HbSNO)). In addition, 3-nitrotyrosine will be determined as a surrogate marker for peroxynitrite.

The following biomarkers of neurovascular unit injury will be determined: S100ß, glial fibrillary acidic protein, neuron-specific enolase, ubiquitin carboxy-terminal hydrolase L1, neurofilament light-chain and total tau.

Eligibility Criteria:
Inclusion Criteria:

* Admission to the Neurointensive Care Unit (NICU) at Rigshospitalet
* Diagnosis of TBI, spontaneous ICH or aneurysmal SAH
* Initiation of blood sampling possible within 3 days after ictus
* Expected length of stay in the NICU and/or intermediate care unit of ≥48 hours
* Closest relatives understand written and spoken Danish

Exclusion Criteria:

* Brain death before inclusion
* Expected death within 24 hours
* ICH secondary to other causes (e.g., a tumour or arteriovenous malformation)
* SAH secondary to other causes (e.g., a mycotic aneurysm or arteriovenous malformation)