Evaluation of Sirtuin 1 (SIRT1) and Sirtuin 3 (SIRT3) in serum and cerebrospinal fluid following fatal traumatic brain injury

Abstract

Traumatic brain injury (TBI) remains a major contributor to mortality and is frequently the subject of forensic and neuropathological inquiries. In light of ongoing advances in neuroscience and clinical diagnostics, the development of novel bioanalytical methods and data interpretation tools for diagnosing TBI in both living and deceased individuals remains a pressing need. Among the proteins of growing interest are the multifunctional and evolutionarily conserved sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), due to their adaptive roles in mitochondrial energy metabolism, aging, genomic stability, inflammation, and oncogenesis. This study aimed to determine whether SIRT1 and SIRT3 levels are elevated in post-mortem biofluids, specifically serum and cerebrospinal fluid (CSF) in fatal TBI cases in a population-based autopsy survey. A total of 40 individuals were included, equally divided into two groups, individuals who died from traumatic brain injury (n = 20) and those who died from sudden cardiac or respiratory causes (n = 20), serving as controls. Serum and CSF samples were collected approximately 24 h post-mortem and analyzed using ELISA. The findings demonstrated significantly elevated SIRT1 concentrations in both serum and CSF in the TBI group. Therefore, further analyses demonstrated significantly higher SIRT3 concentrations in serum among study group after TBI. In cases with macroscopically unclear post-mortem abnormalities, a additional assessment of SIRT1 and SIRT3 in body fluids such as serum and CSF might theoretically be utilized to supplementary determination of the cause of death and the magnitude of brain damage for forensic medicine and neuropathological purposes. These findings support the hypothesis that sirtuin expression is altered in the acute phase of TBI and may serve as a useful biomarker in elucidating post-traumatic pathophysiological mechanisms.

Introduction

Traumatic brain injury (TBI) encompasses a broad spectrum of pathological processes associated with high morbidity and, in its severe form, a markedly increased risk of mortality and poor neurological outcomes [1]. Despite considerable progress in translational neuroscience, the pathophysiological mechanisms underlying TBI remain incompletely understood, posing ongoing challenges to modern medicine in the implementation of multimodal neurointensive care, neurosurgical interventions, and personalized rehabilitation strategies [2, 3]. TBI is also a frequent subject of medico-legal investigations, including forensic autopsies and neuropathological examinations, which aim to assess the extent of brain injury and its contribution to the cause of death [4]. In both preclinical and clinical settings, current biofluid-based diagnostics for TBI utilize panels of biomarkers indicative of neuronal, astroglial, and axonal injury, typically detectable in serum and cerebrospinal fluid (CSF) [5, 6]. Currently, the family of highly conserved nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases sirtuins (silent mating-type information regulation 2), including its most prominent members such as sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) has attracted extensive attention in the fields of neuroscience and oncology, due of its adaptable role in mitochondrial energy metabolism, aging regulation, genomic stability, inflammation and oncogenesis [7, 8]. Therefore, sirtuins are viewed as cellular energy sensors that require NAD + for their enzymatic activity, where low energy state or cellular stressors in the cell increases the NAD+/reduced NAD+ (NADH) ratio, decreases nicotinamide levels as well as activates sirtuins [9–11]. The expression and immunolabeling of SIRT1 in brain parenchyma of humans and rodent covers ubiquitously prefrontal cortex, basal ganglia, hypothalamic arcuate, ventromedial, dorsomedial, and paraventricular nuclei, area postrema, brainstem and cerebellum [12]. Moreover, certain SIRT1-containing neurons are present within hippocampus (cell layers of Ammon’s horn from CA1 to CA4) as well as substantia nigra, where neurons of this two structures appear to subserve cognitive and movement functions [13]. In this case, the observed pattern indicates, that abovementioned wide neuronal expression is associated with co-presence of NAD⁺-dependent histone deacetylases that protect neural cells from several metabolic stressors [14]. The subcellular distribution of SIRT1 within brain parenchyma covers predominantly nuclear (euchromatin) localisation whereas cytosolic presence is also detected [12]. The neuroanatomical expression profile of SIRT3 resembles mainly SIRT1, excepting cerebellum, where its expression seems to be modestly lower what could be potentially linked to relatively lesser number of mitochondria present in cerebellum in comparison to other brain regions [15, 16]. The clear SIRT3 expression was observed within neurons, astrocytes and microglia [15, 17]. Therefore, the relative expression of SIRT3 is higher in cortical neurons than in astrocytes or microglia at both the protein and mRNA levels [15]. The subcellular distribution of SIRT3 in brain parenchyma is predominantly associated with mitochondria [18]. Therefore, the exclusive localisation of SIRT3 in mitochondria could be associated with its migration from nucleus during cellular stress [19]. The presence of SIRT1 and SIRT3 in neuronal tissues appears to be protective being responsible for shielding from neuronal, and specifically axonal, degeneration in the context of oxidative stress and ensuring genetic stability [20–22]. In case of TBI, along with blood-brain barrier (BBB) comes to release various neuronal, astroglial or axonal biomarkers including e.g. glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), tau protein or myelin basic protein (MBP) [23]. Therefore, several of this biomarkers seems to be associated with sirtuins [24]. It was observed, that SIRT1 could decreases tau protein acetylation in neurodegenerative models, serves as NSE binding molecule as well as direct interact with GFAP [25–27]. Similar properties was observed in case of SIRT3, what was associated with tau protein deacetylation and GFAP interaction [28]. Until now, the role of SIRT1 and SIRT3 as well as its potential application as a biomarker has not been widely discussed with reference to TBI. Given the total combined neuromolecular data tying SIRT1 and SIRT3 to cellular energy regulation and genetic stability, it appears reasonable to investigate whether its assay could serve as a viable complementary diagnostic technique for detecting TBI in forensic and medico-legal cases. To provide an accurate and current overview of the problem, we conducted a study to elucidate and determine whether elevated or decreased SIRT1 and SIRT3 concentration levels in biofluids such as blood and CSF are detected in cases of TBI in population-based autopsy screening. While previous work by our group has addressed more traditional neural and astroglial biomarkers, this study aims to provide foundational and basics data on sirtuins in forensic medicine and neuropathology setting, recognizing that future research should include also comparative analyses to better define their diagnostic and prognostic relevance. In this case, in order to describe SIRT1 and SIRT3 potential predictive function in survival outcome and the development of post-traumatic neurodegenerative illnesses, we reviewed the potential importance of this protein in TBI diagnoses in both ante- and post-mortem cases with a focus on relevant meaningful implications.

Materials and methods

Autopsy cases

The study and subsequent analyses were conducted on biofluids collected during routine autopsies performed by forensic pathologists at the Department of Forensic Medicine of the Medical University of Warsaw. A total of 40 autopsy cases (n = 40) were included in the study and divided into two ensuing groups. The study group comprised 20 cases (n = 20) involving severe and fatal head injuries (TBI). Death in this group occurred at the incident site, without any attempts at cardiopulmonary resuscitation (CPR), hospitalization, or further critical/intensive care. The control group consisted of 20 cases (n = 20) of instantaneous death due to cardiopulmonary failure (sudden cardiac arrest) without head injuries or morphological signs of brain injury at the macroscopic level. Similar to the study group, there were no emergency or intensive therapy procedures performed in these cases. Based on the collected data, it was assumed that death occurred within minutes of either a cardiological incident or head injury. Additional information regarding the circumstances of death and pattern of fatal injury/instantaneous death was obtained from available case files during the medico-legal investigation. After death was confirmed, the bodies were transported to a controlled cold storage facility at 4 °C and were stored until forensic autopsies were performed and materials were collected. Additional family testimony and medical records underwent a comprehensive qualification process based on specified literature review-based exclusion criteria. Exclusion criteria included neurological diseases: meningitis, encephalitis, Alzheimer’s disease, frontotemporal lobar degeneration, Parkinson’s disease, neuronal ceroid lipofuscinosis, amyotrophic lateral sclerosis, epilepsy, multiple sclerosis, cerebrovascular disease, bipolar disease, schizophrenia and brain tumours as well as non-neurological diseases: impaired kidney function and disease, impaired liver function and disease, rheumatoid diseases, other inflammatory and autoimmune diseases, history of infectious disease within 6 months, and history of acute trauma or surgery within 6 months, diabetes mellitus, adiposity, underweight, cachexia, previous history of malignancy (tumour) and therapy with drugs that potentially affect epigenetic pathways including corticosteroids, resveratrol, metformin and histone deacetylase (HDAC) inhibitors. Conducted strict selection was implemented with the assumption that changes in SIRT1 and SIRT3 presence and concentration level in biofluids are known to be related to a variety of neurological and non-neurological human disorders. The study and control groups were matched by age, where according to the demographic data, the average age of the deceased was 42.8 ± 1.9 years in the study group and 46.2 ± 1.4 years in the control group. There was no statistically significant difference (p >0.05) in age between the groups. The performed analyses has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and uniform requirements for manuscripts submitted to biomedical journals. The groups used in the study were the same as those used in our previous publication, where more detailed characteristics of the cases used were described [29].

Biofluid obtainment and Preparation

The medico-legal autopsy was conducted within 24 h after death, during which cerebrospinal fluid CSF and blood samples were collected for further examination. The CSF samples were obtained through a suboccipital (C₀-C₁) puncture from cistern magna, while the blood samples were collected from the vena femoralis (external puncture) after the dermal surface was decontaminated with a 90% ethanol (C₂H₅OH) solution prior to both proceedings. The blood samples were instantly centrifuged at 6000 rpm for 10 min to separate the serum, and the CSF samples were processed at 1600 rpm for 10 min to obtain the supernatant. The collected samples were stored in sterile tubes and preserved at −80˚C using specialized freezer until further analysis. Any samples that showed visible signs of hemolysis were excluded from testing.

ELISA

The presence and concentration level of selected biomarkers in sampled biofluids were evaluated utilizing double-sandwich ELISA kits. A series of assays were carried out using Human SIRT-1 (Sirtuin 1) ELISA Kit (EH3785; Wuhan Fine Biotech, Wuhan, Hubei, China) and Human SIRT-3 (Sirtuin 3) ELISA Kit (EH4451; Wuhan Fine Biotech, Wuhan, Hubei, China) according to the standardized manuals provided by the manufacturers and supplier. In brief, standards, CSF and serum samples were incubated for the required period in microplate wells pre-coated with anti-above-mentioned antibodies, followed by incubation with a biotin-labeled monoclonal anti-above-mentioned biomarkers antibody solution and incubation with a streptavidin-horseradish peroxidase (SAV-HRP) conjugate. Between each step, the plate was thoroughly washed several times with a washing buffer. After the last washing step the substrate solution, 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated for the required period. The reaction was stopped by adding an acidic solution, and the absorbance of the resulting colour product was measured by reading the ELISA plate at the wavelength (λ) of 450 nm. Concentrations of samples were determined using the standard curve. The limit of detection (LOD) and the limit of quantification (LOQ) for measured markers by ELISA assays was 0.188 ng/ml and 0.313 ng/ml for EH3785, 9.375 pg/ml, and 15.625 pg/ml for EH4451 respectively. Samples above the detection range (out of the standard curve) were diluted and reanalyzed, and the results were multiplied by the appropriate dilution factor. Dilution rates were up to 1:6 for both tests. All samples were assayed in duplicate, and average results were analyzed.

Statistical analysis

The data was analyzed and comparisons of both groups were performed using the Statistica 13.1 PL (StatSoft, Tulsa, OK, USA) and Microsoft Office 365 software package for Windows.

(Microsoft Corporation, Redmond, WA, USA). The results were considered statistically significant when p-values were less than 0.05 (p < 0.05). The Shapiro-Wilk test was used to assess whether the SIRT-1 and SIRT-3 concentration levels were normally distributed. As they were not, the groups were tested for statistically significant differences using non-parametric Mann-Whitney U test.

Results

Concentration of SIRT1 in biofluids

The results presented in Fig. 1B show an increased CSF concentration of SIRT1 in the study (head injury) group (6.03 ± 1.55 pg/ml) compared to the control group (0.18 ± 0.06 pg/ml) which was statistically significant (p = 0.0032; Mann-Whitney U test). Consecutively, the results presented in Fig. 1A show an increased serum concentration of SIRT1 in the study (head injury) group (6.45 ± 2.36 pg/ml) compared to the control group (0.50 ± 0.11 pg/ml) which was statistically significant (p = 0.0008; Mann-Whitney U test).

Fig. 1.

The proportion of SIRT1 and SIRT3 concentration levels in obtained biofluids such as serum and CSF. Data are presented as a mean value ± SEM. *differs from the control group, **p < 0.001, ***p < 0.0001

Concentration of SIRT3 in biofluids

The results presented in Fig. 1D show an increased CSF concentration of SIRT3 in the study head injury) group (2.78 ± 0.76 pg/ml) compared to the control group (0.14 ± 0.06 pg/ml) which was not statistically significant (p = 0.1176; Mann-Whitney U test). Consecutively, the results presented in Fig. 1C show an increased serum concentration of SIRT3 in the study (head injury) group (111.80 ± 70.81 pg/ml) compared to the control group (0.74 ± 0.28 pg/ml) which was statistically significant (p = 0.008; Mann-Whitney U test).

Discussion

To the best of our knowledge, no data have been available regarding changes in SIRT1 and SIRT3 levels in central and peripheral biofluids following fatal TBI. In the present study, we demonstrated that the study group (individuals with head injury) exhibited significantly higher SIRT1 concentrations in both serum and CSF compared to the control group. Furthermore, we observed significantly elevated SIRT3 concentrations in the serum of the study group, while CSF levels of SIRT3 did not differ significantly from those in the control group. As outlined in the methodology, the stringent case selection and qualification process along with the exclusion of conditions previously shown to influence SIRT1 and SIRT3 levels may have impacted the results obtained [30]. This underscores a key finding, serum and CSF concentrations of SIRT1 and SIRT3 vary in the context of TBI, as shown in our post-mortem analysis. Despite the well-documented pleiotropic roles of SIRT1 and SIRT3 in neurobiological processes, data on their specific involvement in the pathophysiology of TBI in clinical settings remain scarce [24]. To date, most insights have been derived from preclinical rodent models employing proteomic, transcriptomic, or immunofluorescence techniques. These studies have explored the neuroprotective effects of various pharmacological agents through the modulation of sirtuin expression; however, translational outcomes in human clinical contexts are still lacking. According to the potential TBI-related expression profile of SIRT1, one of the very few available clinical studies was conducted by Teertam et al., focusing on the related topic of brain ischemia/stroke. In this study, increased SIRT1 expression was observed in the brains of stroke patients [31]. Similarly, in a study by Yang et al., conducted on rats using a lateral fluid percussion (LFP) brain injury model, it was found that TBI markedly increased SIRT1 expression, reaching a peak at 12 h post-trauma [32]. Our findings, along with the data summarized above, suggest that SIRT1 may be actively upregulated or released in response to TBI, potentially playing a role in early post-traumatic molecular processes [33]. SIRT1 is known to play a neuroprotective role via orchestration of neuroinflammatory response, mitochondrial functions and apoptosis [34]. Its elevated levels in CSF indicate either upregulated expression in direct damaged brain tissue or passive release from injured neurons and astrogial cells. The simultaneous increase in serum levels supports the hypothesis that SIRT1 may be systemically released or cross the disrupted BBB or glymphatic pathway following TBI, thus indicating responses from both central and peripheral compartments [35]. Regarding SIRT3, a study using a controlled cortical impact (CCI) model in normal and aged mice demonstrated a significant increase in SIRT3 protein expression at 3, 6, and 12 h after trauma [36]. Conversely, a separate study by Chen et al. reported decreased SIRT3 expression in rats subjected to CCI compared to sham-operated animals [37]. In the context of acute intracerebral hemorrhage (ICH), Yan et al. conducted a prospective cohort study involving 105 patients (n = 105), examining serum SIRT3 levels in relation to initial hematoma volume, Glasgow Coma Scale (GCS) score, and other clinical parameters [38]. They found that SIRT3 serum levels were independently and negatively correlated with GCS score, hematoma volume, and the presence of intraventricular hemorrhage (IVH). Moreover, plasma SIRT3 levels were reduced in patients with a poor prognosis (hematoma volume < 30 ml) [38]. Similar findings of reduced SIRT3 expression were also reported in rodent models of subarachnoid hemorrhage (SAH) [39, 40]. The concentration levels of SIRT3 demonstrated a markedly distinct expression pattern. While CSF levels were elevated in the TBI group, the increase did not reach statistical significance. This may suggest a more modest or delayed release from CNS tissues, or inherently lower baseline expression of SIRT3 within the CNS. In contrast, serum levels of SIRT3 were significantly elevated in TBI patients, indicating a considerable systemic response. Given that SIRT3 is a predominantly mitochondrial protein involved in maintaince of mitochondrial membrane potential (ΔΨ_mt), intracellular acidification and oxidative stress regulation, its peripheral upregulation might reflect hypothetically mitochondrial dysfunction in systemic tissues secondary to TBI or multiorgan stress response [41]. The observed divergence between serum and CSF SIRT3 levels suggests compartmentalized regulation and indicates that serum SIRT3 may serve as a more sensitive biomarker of systemic oxidative stress rather than direct CNS injury [42]. Although all individuals in this study were classified as having isolated TBI without extracranial polytrauma, it is important to note that SIRT3 is also expressed in non-neural tissues, including skeletal muscle and vascular endothelium. Therefore, it is plausible that mitochondrial disruption in extracerebral cranial tissues, such as the scalp, periosteum, or cervical musculature contributed to the elevated serum SIRT3 levels. In contrast, the relatively modest CSF concentrations may also reflect the limited time for intrathecal accumulation, or differences in sirtuin kinetics and BBB dynamics. This discrepancy underscores the need for caution when interpreting serum sirtuin levels as CNS-specific markers in acute post-TBI settings. Currently, data on the post-mortem kinetics and preanalytical stability of sirtuins in biofluids are limited. Available evidence suggests that sirtuins are relatively stable under controlled storage conditions, however, differences between ante-mortem and post-mortem expression remain poorly characterized. Furthermore, the post-mortem interval (PMI) may differentially affect protein degradation and release kinetics in serum and CSF. Given that SIRT3 is a mitochondrial protein, it may be particularly susceptible to changes in cellular membrane integrity and autolysis [43]. In addition, the influence of tube type, clotting time, and temperature on the measured concentrations of SIRT1 and SIRT3 has not yet been standardized. In our study, all samples were collected, processed, and stored under identical conditions to minimize variability; however, the lack of reference kinetic data limits the interpretation of absolute values [44]. Future studies should systematically address these preanalytical variables to validate sirtuins as reliable post-mortem biomarkers. Pathologically, neurodegenerative diseases and TBI share overlapping mechanisms, including oxidative stress, mitochondrial dysfunction, and neuroinflammation. Our findings support the hypothesis that sirtuin expression is altered in the acute phase of TBI and may offer value in understanding post-traumatic pathophysiology, monitoring the efficacy of neuroprotective interventions (e.g., therapeutic hypothermia, antioxidant agents), or even predicting clinical outcomes. To the best of our knowledge, this is the first study to evaluate SIRT1 and SIRT3 levels in a post-mortem context following fatal TBI. In cases with macroscopically unclear post-mortem abnormalities, assessment of SIRT1 and SIRT3 in body fluids such as serum and CSF may theoretically support supplementary determination of the cause of death and the extent of brain injury. However, this study has several limitations, especially when viewed beyond the strict forensic medicine perspective. Future studies involving larger cohorts are warranted to validate whether CSF and serum levels of SIRT1 and SIRT3 can serve as reliable biochemical markers of TBI. Furthermore, it is essential to elucidate the kinetic expression and temporal dynamics of SIRT1 and SIRT3 concentrations in CSF and serum after TBI, and to evaluate the evolution of their levels during the post-injury period. To facilitate the practical translation of our findings and to explore the potential diagnostic and therapeutic roles of SIRT1 and SIRT3 in TBI pathogenesis, robust clinical evidence is needed. In summative assessment, following the first investigation of SIRT1 and SIRT3 in the context of TBI under forensic conditions, we observed a measurable increase in their concentrations in post-mortem serum and CSF samples. These findings support the hypothesis that SIRT1 and SIRT3 are released into various biofluids following TBI. Taken together, SIRT1 and SIRT3 emerge as promising proteins warranting further investigation within the interconnected fields of neuropathology, forensic medicine, neurology, and neurosurgery.

Funding

No funding was received for conducting this study.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The data are not publicly available due to privacy or ethical restrictions.

Declarations

Ethical approval

According to Polish law, the consent of the bioethics committee is not required for this type of research.

Human ethics and consent to participate declarations

Not applicable.

Conflict of interest

The authors declare that they have no conflict of interest.

Financial disclosure statement

None.

Clinical trial number

Not applicable.