H₂S-Dependent Mechanisms of Caspase-3 Expression and Localization in Brain Cells of Mice with Traumatic Brain Injury

Introduction . Traumatic brain injury (TBI) is a neurotrauma widespread in animals. TBI causes a complex cascade of pathological processes: primary brain injury turns into secondary brain injury associated with inflammation, oxidative stress, excitotoxicity and apoptosis. Secondary injury aggravates the condition after injury. In this regard, the role of hydrogen sulfide (H₂S) as a gasotransmitter involved in neuromodulation, anti-inflammatory, antioxidant and anti-apoptotic processes in the central nervous system is of particular interest. Caspase-3 is an important element in TBI-induced apoptosis. H₂S has a potential to modulate the expression and activity of caspase-3 affecting the survival of nerve cells and brain recovery after TBI. However, H₂S-dependent mechanisms of caspase-3 regulation in traumatic injury are not fully investigated. The aim of the research is to study the role of H₂S in the expression and localization of caspase-3 in neurons and astrocytes of mice with TBI. Materials and Methods. The research was conducted at the Bioengineering Department of DSTU (Rostov-on-Don) from April 20 to June 1, 2024 in conditions compliant with the international and national standards. The objects of the study were 36 adult male mice divided into three groups: control group and two experimental ones. TBI was simulated by dropping a weight (200 g) on the intact skulls of mice anesthetized with chloral hydrate. During 7 days after the TBI, the animals were daily administered the sodium sulfide (Na₂S), a donor of H₂S, which can efficiently release H₂S, or the aminooxyacetic acid (AOAA), an inhibitor of cystathionine β-synthase (CBS), an enzyme responsible for the endogenous synthesis of H₂S, until the animals were withdrawn from the experiment. The use of Na₂S and AOAA enabled efficient modulation of the level of endogenous H₂S in the brain. The control group was administered physiological saline solution. Brain sections fixed in 4% paraformaldehyde (PFA) solution were incubated with antibodies to caspase-3 and to the neuronal nuclear antigen (NeuN) or to the astrocytic marker (GFAP). Colocalization was assessed using the ImageJ software. Caspase-3 expression in the brain penumbra was analysed by Western blotting using primary antibodies against caspase-3 and β-actin and secondary antibodies IgG conjugated to horseradish peroxidase. For detection, the chemiluminescence method was used. Results. The initial level of caspase-3 in the brain cells of mice under study was low. Seven days after injury, TBI had induced caspase-3 expression in neurons and glial cells of the ipsilateral injured hemisphere in animals of all groups. Administering the donor Na₂S led to decrease of caspase-3 level in neurons by 32%, whereas administering the inhibitor AOAA led to its increase by 31% compared to the injured nerve cells of animals from the control group, which were administered the physiological saline solution. This was confirmed by the values of the M1 colocalization coefficient demonstrating colocalization of caspase-3-positive cells with the neuronal nuclear antigen (NeuN). Similar effects were demonstrated in astrocytes, which were visualized using the astrocyte-specific marker GFAP. Western blot analysis confirmed these data and showed a significant decrease of caspase-3 level with administering Na₂S and its increase with that of AOAA 7 days after TBI. Discussion and Conclusion. The results of the study demonstrate that TBI leads to significant activation of caspase-3 in neurons and astrocytes of the injured hemisphere of mice brain, which means development of apoptosis in response to traumatic injury. Administering Na₂S has efficiently decreased caspase-3 level, which indicates its neuroprotective and anti-apoptotic effect. Whereas, administering the AOAA has induced an increase in caspase-3 expression, which confirms the important role of CBS and, therefore, of H₂S in regulation of cell death after TBI. The reliability of these findings was ascertained by both immunohistochemical and Western blot analysis. The obtained data contribute to better understanding the fundamental H₂S-dependent signaling mechanisms of survival and death of neurons and glial cells during traumatic injury of the nervous system. The CBS inhibitor and H₂S donor used in our study may serve a basis for development of the clinically efficient neuroprotective agents.