Practical reasons (project coordinator has resigned and started new employment).

Aneurysmal subarachnoid haemorrhage (SAH) carries a high morbidity and mortality, which is in part due to the development of secondary brain injury. The mechanisms behind this remain incompletely understood, but oxidative/nitrosative stress and disturbances in vasoregulatory mechanisms are believed to be involved. The present study aims to characterise the transcerebral exchange of oxidative/nitrosative stress markers and nitric oxide metabolites during the early phase after SAH compared to healthy volunteers, including the influence of induced changes in arteriel oxygen tension.

BACKGROUND:

Aneurysmal subarachnoid haemorrhage (SAH) carries a high morbidity and mortality. This is in large part due to the development of secondary brain injury, including the complication delayed cerebral ischaemia (DCI), which affects 30% of initial survivors. The mechanisms behind secondary brain injury after SAH are incompletely understood.

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, 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 after SAH, 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 (and a key mechanism behind DCI), others argue that an overproduction of NO mediated by iNOS is maladaptive response leading to aggravated tissue injury due to nitrosative stress.

The transcerebral (i.e., arterial to jugular venous) exchange of NO metabolites and its interrelationship with oxidative stress has never been studied in patients with SAH. The investigators hypothesise that SAH is associated with an initial reduction in the cerebrovascular bioavailability of NO due to scavenging by free radicals. This 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 and NO depletion, ultimately leading to ischaemic brain injury and poor outcome.

HYPOTHESES:

The present explorative study will characterise the transcerebral exchange of oxidative/nitrosative stress markers and NO metabolites during the early phase after SAH as compared to healthy volunteers. Additionally, it will examine the influence of these disturbances on indices of brain ischemia and metabolic dysfunction as assessed by multimodal neuromonitoring, and the influence of induced changes in arterial oxygen tension (PaO2). The investigators hypothesise that:

1. Patients will have a greater cerebral efflux of oxidative/nitrosative stress markers and a lower cerebrovascular bioavailability of NO compared to healthy controls.
2. SAH patients will exhibit a progressive decrease in the transcerebral release of oxidative/nitrosative stress markers and a corresponding increase in the cerebrovascular bioavailability of NO in the days following ictus.
3. A greater clinical severity of SAH will be associated with a greater transcerebral release of oxidative/nitrosative stress markers and a lower cerebrovascular bioavailability of NO.
4. A lower brain tissue oxygen tension (PbtO2) and a larger burden of brain tissue hypoxia (defined as the percentage of monitoring time with a PbtO2 <20mmHg) will be associated with a greater cerebral efflux of oxidative/nitrosative stress markers and a lower cerebral bioavailability of NO.
5. A greater microdialysate lactate/pyruvate ratio and a larger burden of brain metabolic crisis (defined as the percentage of monitoring time with a lactate/pyruvate ratio >40 and glucose concentration <0,7 mmol/l) will be associated with a greater cerebral efflux of oxidative/nitrosative stress markers and a lower cerebral bioavailability of NO.
6. Induced mild hypoxia will be associated with an increased transcerebral release of markers of oxidative/nitrosative stress and a reduction in the cerebrovascular bioavailability of NO compared to baseline, while induced mild hyperoxia will have the opposite effect.

METHODS:

The study is a prospective physiological study which will include patients with SAH admitted to the Neurointensive Care Unit (NICU) at Rigshospitalet. Patient inclusion will be continued until we have obtained complete data on 20 patients in the interventional substudy (see below), or until the 1st of April 2024, at which point inclusion will be halted and data will be analysed irrespective of the number of included patients. In addition, 12 healthy subjects will be included to serve as a control group.

Practical conduct of the study, patients:

Patients will have an arterial catheter inserted shortly after admission as part of routine care. In addition, as early as possible after closure of the aneurysm, a retrograde internal jugular venous catheter (RJV catheter) will be inserted for sampling of cerebral venous blood. Vital parameters will be monitored according to standard practice in the NICU, and patients will undergo continuous multimodal neuromonitoring of intracranial pressure (ICP), PbtO2, and brain metabolism (cerebral microdialysis). Additionally, jugular bulb blood pressure will be monitored on the RJV-catheter for at least 24 hours in all patients.

During interventions, transcranial Doppler ultrasound (TCD) will be used to determine middle cerebral artery mean flow velocity as a surrogate measure of cerebral blood flow. The study will consist of an observational and an interventional substudy, which are described individually below.

Observational substudy: Paired blood samples (i.e., simultaneous blood samples from the RJV and arterial catheters) will be drawn 1) immediately after placement of the RJV catheter (expected: day 0-2 after admission), 2) during interventions (see below), and 3) shortly before removal of the RJV catheter (expected: day 3-6 after admission). In addition to blood sampling, patients with an existing external ventricular drain will undergo sampling of CSF, and patients with a cerebral microdialysis catheter will undergo sampling of excess microdialysate for explorative purposes.

Interventional substudy: Mild hypo- and hyperoxia will be induced by in a randomised order by changing ventilator settings (the fraction of inspired oxygen), aiming at a PaO2 of 9-10 kPa for mild hypoxia and 13-14 kPa for mild hyperoxia (standard treatment target: 10-12 kPa). Blood samples will be drawn at baseline (normoxia) and after 60 minutes of each intervention (3 paired blood samples in total).

Practical conduct of the study, healthy controls:

Experiments in healthy subjects will be conducted in the NICU. Instrumentation and monitoring is similar to what is described above: an arterial- and RJV catheter will be inserted, and subjects will undergo TCD monitoring as an index of cerebral blood flow in addition to standard cardiorespiratory monitoring.

After instrumentation and a short rest period, a baseline evaluation will be conducted, after which subjects will undergo interventions in a randomised order: Hypo- and hyperoxia will be induced using a tight-fitting mask connected to a non-rebreathing valve, an end-tidal CO2 monitor, and a reservoir supplied through compressed gas cylinders. The fraction of inspired oxygen will be titrated, aiming at a PaO2 of 9-10 kPa for mild hypoxia and 13-14 kPa for mild hyperoxia.. Additionally, healthy subjects will undergo a third intervention where the fraction of inspired oxygen will be increased to 100%. Isocapnia will be ensured by adding CO2 to the inspired air ad hoc. Blood samples will be drawn at baseline (normoxia) and after 60 minutes of mild hypoxia, 60 minutes of mild hyperoxia, and 60 minutes of "severe hyperoxia".

BIOCHEMICAL ANALYSES:

At each sampling time point, a small amount of blood will immediately be analysed for levels of blood gases and acid-base status. The remaining biological samples will be centrifuged, aliquoted, and stored at -80°C until analysis.

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.

Additionally, as part of explorative substudies, blood samples from certain patients may be analysed for leukocyte subsets using mass cytometry, as well as markers of endothelial injury (plasma syndecan-1, soluble thrombomodulin, and PECAM-1).

Inclusion Criteria (patients):

• Age ≥ 18 years
• Admission to the NICU at Rigshospitalet
• Diagnosis of aneurysmal SAH
• Need for sedation and mechanical ventilation after the aneurysm has been secured
• Initiation of study possible ≤3 days after the ictus
• Closest relatives understand written and spoken Danish or English

Exclusion Criteria (patients):

• Brain death before inclusion
• Expected death within 24 hours
• Failed or conservative treatment of the aneurysm
• Severe acute lung failure with a PaO2/FiO2-ratio ≤16 kPa
• Severe chronic lung failure with habitual long-term oxygen therapy
• Habitual treatment with medication directly affecting NO metabolism (e.g., sildenafil)

Inclusion Criteria (patients):

• Age 40-60 years
• 50/50 sex distribution (6 men and 6 women)
• Healthy (including no prior cerebrovascular disease)
• No regular medication or recreational drug use
• Understands written and spoken Danish or English

Twenty patients with severe SAH and twelve pseudo-matched healthy controls.

Data from each individual participant will be available after publication of planned manuscripts, with a valid reason, and after signing a data processing agreement.

The approved study protocol will be available upon request until publication of the study results.

Valid reason and contact with author.