Early Redox Biomarker Changes After Stellate Ganglion Block in Traumatic Brain Injury

Background and Rationale\_\_\_\_\_ Traumatic brain injury triggers a cascade of secondary injury mechanisms, including neuroinflammation, excitotoxicity, mitochondrial dysfunction, and oxidative stress. Reactive oxygen and nitrogen species overwhelm endogenous antioxidant defenses in the hours to days following injury, contributing to lipid peroxidation, protein oxidation, and DNA damage that exacerbate neuronal loss beyond the initial mechanical insult. The stellate ganglion, located at the C7-T1 vertebral level, provides sympathetic innervation to the head, neck, and upper extremities. Blockade of this ganglion with local anesthetic has been used clinically for decades in the management of complex regional pain syndrome and other sympathetically mediated conditions. In cerebrovascular pathology, SGB has been associated with increased cerebral blood flow velocity, improved brain tissue oxygenation, and attenuation of neuroinflammatory markers. The parent study (NCT04208477) demonstrated favorable hemodynamic and oxygenation changes after SGB in TBI patients. Whether these hemodynamic effects are accompanied by measurable changes in the systemic and cerebral redox environment has not been investigated in humans. The present post hoc analysis was designed to characterize the early temporal profile of circulating oxidative-stress biomarkers in paired arterial and internal jugular venous blood after SGB in moderate-to-severe TBI. The paired sampling strategy enables estimation of trans-cerebral gradients (jugular minus arterial concentration), providing indirect information about the balance of cerebral production and clearance of redox metabolites.\_\_\_\_\_ Study Design\_\_\_\_\_ This is a secondary biomarker analysis embedded within a prospective, single-center, uncontrolled, before-and-after pilot study conducted at the surgical intensive care unit of the University Medical Centre Ljubljana, Slovenia (November 2021 to December 2023). The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (approval KME RS 0120-537/2019/15) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from the legally authorized representative of each patient, as participants were unable to consent due to their clinical condition. Twenty adult patients (aged 18-80 years) with moderate or severe TBI, defined as a Glasgow Coma Scale (GCS) score of 12 or lower before tracheal intubation, who received a single ultrasound-guided left SGB as part of the parent study, were included. Paired arterial (radial artery catheter) and right internal jugular venous blood samples were collected at three time points: immediately before SGB (baseline), 12 hours post-SGB, and 24 hours post-SGB. Samples were centrifuged, aliquoted, and stored at -80 degrees Celsius until batch analysis.\_\_\_\_\_ Biomarker Assays\_\_\_\_\_ The following redox biomarkers were measured in each paired sample: d-ROMs: Measured using the Free Radical Analytical System (FRAS 5; H\&D srl, Parma, Italy), reflecting circulating hydroperoxide-derived oxidant burden. Results expressed in Carratelli Units (U.CARR). PAT: Measured on the same FRAS 5 platform, estimating total plasma antioxidant capacity to reduce ferric ions to ferrous ions. Results expressed in U.Cor. OSI: Calculated as d-ROMs/PAT, a composite measure of the pro-oxidant/antioxidant balance. Higher values indicate greater oxidative stress. HEL: An early-phase lipid peroxidation product, measured by enzyme-linked immunosorbent assay (ELISA). Reflects oxidative modification of polyunsaturated fatty acids at an early, reversible stage. 8-OHdG: A marker of oxidative DNA damage, measured by ELISA. Reflects hydroxyl radical attack on guanine residues in DNA.\_\_\_\_\_ Statistical Analysis\_\_\_\_\_ The statistical analysis plan for the oxidative-stress endpoints was documented before laboratory assays were performed. The primary analysis tests the within-participant change from baseline in jugular venous OSI at 12 hours using a Wilcoxon signed-rank test, with the Hodges-Lehmann estimator and its 95% confidence interval as the effect measure. Secondary analyses apply repeated-measures approaches (Friedman test for time effects; Wilcoxon signed-rank tests for pairwise time-point and compartment comparisons) to d-ROMs, HEL, and 8-OHdG in both arterial and venous samples. Multiple comparisons are adjusted using the Holm-Bonferroni method. Exploratory Spearman correlations between redox biomarkers and inflammatory/brain-injury markers (IL-6, NSE, S100B, GFAP) are corrected using the Benjamini-Hochberg false discovery rate. The sample size of 20 reflects the complete parent-study cohort. No separate power calculation was performed for this exploratory secondary analysis. Variability estimates from this pilot are reported to inform future sample-size planning for a controlled trial.