Bicuculline Methiodide Preserves Neuromuscular Integrity and Skeletal Muscle Bioenergetics Through Neuroimmune Modulation in Experimental Sepsis

Abstract

Background: Sepsis-induced systemic inflammation triggers profound metabolic and inflammatory alterations in skeletal muscle, leading to oxidative damage, mitochondrial dysfunction, and muscle wasting. However, the mechanisms linking endotoxemia to skeletal muscle metabolic failure and neuromuscular-associated alterations remain incompletely understood. The present study investigated the effects of bicuculline methiodide (BMI), a GABAA receptor antagonist, on inflammatory responses, oxidative stress, energy metabolism, and selected neural-associated markers in a lipopolysaccharide (LPS)-induced rat model of sepsis. Methods: Adult male Sprague–Dawley rats were allocated into Control, LPS, Bicuculline, and LPS + Bicuculline groups. Twenty-four hours after treatment, gastrocnemius muscle tissues were analyzed for TNF-alpha, IL-10, GABA, and malondialdehyde (MDA) levels, while ATP-related metabolites were quantified by HPLC. NeuN and synaptophysin immunoreactivity were also assessed in muscle sections. Results: LPS administration increased TNF-alpha and MDA levels, altered IL-10 concentrations, and disrupted high-energy phosphate metabolism, accompanied by changes in NeuN and synaptophysin immunoreactivity. Bicuculline co-administration reduced MDA levels, was associated with higher IL-10 concentrations than the LPS group, and markedly increased phosphocreatine levels. Conclusions: These findings indicate that BMI was associated with attenuation of selected inflammatory and oxidative alterations and with changes in skeletal muscle energy metabolites in this experimental model. Because cholinergic markers, alpha7 nicotinic acetylcholine receptor activity, vagal activity, and receptor-specific pharmacological controls were not assessed, the present findings do not establish direct activation of the cholinergic anti-inflammatory pathway or a GABAergic-cholinergic mechanism. The observed effects should therefore be considered exploratory and hypothesis-generating.

1. Introduction

Sepsis is a life-threatening syndrome characterized by organ dysfunction caused by a dysregulated host response to infection, leading to widespread inflammation, multiple organ dysfunction, and, in severe cases, septic shock and death [1,2]. Despite advances in intensive care management, sepsis remains a major global health concern because of its high morbidity, mortality, and long-term functional sequelae [2,3]. The clinical presentation of sepsis is highly heterogeneous and is influenced by the source and severity of infection, the extent of organ dysfunction, the patient’s underlying health status, and the timing of therapeutic intervention [1,3].

Skeletal muscle represents approximately 40–50% of total body mass and is among the tissues most profoundly affected by systemic inflammatory conditions [4,5]. Sepsis-induced skeletal muscle dysfunction is a common and clinically significant complication associated with muscle wasting, weakness, prolonged hospitalization, impaired recovery, and intensive care unit-acquired weakness [4,5,6,7]. Although the pathophysiological mechanisms underlying septic myopathy have not been fully elucidated, accumulating evidence suggests that inflammatory mediators, mitochondrial dysfunction, proteolytic systems, and oxidative stress play central roles in this process [4,5,6,7,8,9]. Pro-inflammatory cytokines, reactive oxygen species (ROS), and cyclooxygenase-derived mediators activate catabolic pathways, including the ubiquitin-proteasome and autophagy-lysosome systems, resulting in accelerated protein degradation and skeletal muscle proteolysis [8,9,10,11]. Furthermore, sepsis-induced muscle injury is characterized by enhanced cytokine signaling, increased oxidative stress, activation of apoptotic pathways, and suppression of protein synthesis, collectively contributing to the loss of muscle mass and function [4,7,8,9,10,11].

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and exerts its biological effects primarily through GABAA and GABAB receptors [12,13]. Bicuculline methiodide (BMI), a GABAA receptor antagonist, has been widely employed as a pharmacological tool for investigating GABAergic signaling pathways [12,14]. Beyond its classical effects on neurotransmission, emerging evidence indicates that GABAergic signaling can influence peripheral immune-cell activity, cytokine production, and inflammatory responses [13,15,16,17]. These observations provide a biological rationale for examining whether pharmacological blockade of GABAA receptors is associated with inflammatory and metabolic changes during experimental sepsis; however, the downstream pathways responsible for such effects remain uncertain.

The cholinergic anti-inflammatory pathway (CAP) is a neural mechanism through which cholinergic signaling can modulate systemic inflammation [18,19,20]. The cholinergic system also has broader roles in neuronal regulation and neurodegenerative processes [21]. Activation of CAP can suppress production of pro-inflammatory cytokines and promote anti-inflammatory signaling, with the alpha-7 nicotinic acetylcholine receptor (α7nAChR) identified as an important mediator in several experimental models [19,20,22,23,24]. These findings provide a relevant neuroimmune framework for interpreting inflammatory responses in sepsis. Nevertheless, the present study did not directly measure acetylcholine, choline acetyltransferase, α7nAChR expression or activity, or vagal activity. Accordingly, CAP or α7nAChR involvement cannot be established from the present data.

Previous experimental studies have demonstrated that pharmacological or electrical manipulation of cholinergic pathways can attenuate systemic inflammation [18,20,22,23,24,25]. These observations support the broader concept that neural and immune pathways may interact during systemic inflammation, but they do not establish that bicuculline acts through the cholinergic anti-inflammatory pathway. In the present study, the term “neuroimmune modulation” is therefore used to describe a possible conceptual framework rather than a directly demonstrated mechanism. The 1.5 mg/kg bicuculline dose and 24-h endpoint were selected to maintain comparability with our previously published study using the same experimental model [26] and to examine the acute response. This exploratory design was not intended to determine dose–response relationships or temporal dynamics.

Therefore, the present study was designed to investigate the effects of bicuculline administration on inflammatory and oxidative stress responses, high-energy phosphate metabolism, and selected neural-associated immunoreactivity in gastrocnemius muscle tissue of rats subjected to LPS-induced sepsis. We hypothesized that bicuculline administration would be associated with attenuation of inflammatory and oxidative alterations and preservation of muscle energy metabolism. Any involvement of GABAergic-cholinergic or cholinergic anti-inflammatory signaling was considered a hypothesis to be evaluated indirectly rather than a mechanism directly tested in this study.

2. Materials and Methods

Ethical approval number 2017/63 was obtained from the Istanbul Bagcilar Training and Research Hospital Animal Experiments Local Ethics Committee. Adult male Sprague–Dawley rats with an average weight of 230 g were used in the study. The rats were obtained from the Experimental Research and Skill Development Center of Istanbul Bagcilar Training and Research Hospital. Standard rat food and water were provided ad libitum, and no dietary restrictions were imposed.

2.1. Experimental Groups

The rats were divided into 4 groups:

Control Group (C) (n = 8): No substance was administered to this group, and the rats were fed ad libitum.

LPS Group (LPS) (n = 8): Rats in this group were administered 10 mg/kg intraperitoneal LPS (E. coli serotype 055:B5, Sigma-Aldrich, St. Louis, MO, USA), dissolved in 1 mL of 0.9% NaCl solution. The animals were euthanized 24 h after the first injection.

Bicuculline Group (B/Bic) (n = 8): Rats in this group were administered 1.5 mg/kg subcutaneous bicuculline methiodide (Sigma-Aldrich), dissolved in 1 mL of 0.9% NaCl solution. The animals were euthanized 24 h after administration.

LPS + Bicuculline Group (LPS + B) (n = 8): Rats in this group received 10 mg/kg intraperitoneal LPS immediately followed by 1.5 mg/kg subcutaneous bicuculline methiodide. Figure 1.

Figure 1. Diagram of experimental procedure.

2.2. Experimental Procedure

Bicuculline methiodide was administered concurrently with LPS at 1.5 mg/kg subcutaneously. Twenty-four hours after the LPS injection, the animals were anesthetized with ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Body temperatures were measured rectally before injection and before euthanasia. Blood samples were collected from the heart for biochemical analyses, and gastrocnemius muscle tissue samples were collected for biochemical and histological analyses.

To further characterize the experimental model, rectal body temperature was measured before injections and again before the experimental procedure/euthanasia. Cardiac blood samples were evaluated for leukocyte count and selected biochemical parameters. The blood biochemical panel included AST, ALT, sodium, potassium, chloride, magnesium, calcium, phosphate, lactate dehydrogenase (LDH), creatine kinase (CK), and fibrinogen; the available values are presented in Table 1. Leukocyte-count findings from the same experimental cohort were reported in our previous publication [26].

Table 1. Blood biochemical parameters in the experimental groups.

Parameter	Control	LPS	Bicuculline	LPS + Bic
AST U/L	128.6667 ± 58.287	99.3333 ± 9.292 *	105 ± 9.899	110 ± 36.497
ALT U/L	34.667 ± 14.154	49.5 ± 4.95	34.667 ± 17.954	42.333 ± 8.737
Na+ mmol/L	144 ± 1.732	141.667 ± 1.528 *	139.333 ± 0.577 **	142.667 ± 0.577
K+ mmol/L	4.767 ± 0.25	4.525 ± 0.77	5.3 ± 1.929	4.03 ± 0.451
Cl− mmol/L	101 ± 0	99.75 ± 2.218	98.333 ± 0.578	98.333 ± 0.578
Mg2+ mmol/L	0.983 ± 0.135	1.03 ± 0.124	1.27 ± 0.470	1.2667 ± 0.266
Ca2+ mmol/L	10.533 ± 0.473	11 ± 0.337	11.2 ± 0.854	11.533 ± 0.153
Phosphate mmol/L	6.267 ± 0.808	8.425 ± 0.443	11.233 ± 4.366	10.6 ± 2.252
LDH U/L	568 ± 28.284	994.5 ± 40.37	441 ± 53.006	638 ± 82.434
CK U/L	1094.5 ± 57.276	1145.25 ± 60.176	1033.333 ± 91.167	1059.333 ± 108.921
Fibrinogen U/L	101.57 ± 55.799	402.358 ± 67.312 *	82.53 ± 45.156	118.9 ± 104.793

Values are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Gastrocnemius muscle tissue samples were homogenized to investigate TNF-α, IL-10, and GABA levels using enzyme-linked immunosorbent assay (ELISA), and thiobarbituric acid reactive substances (TBARS) levels were determined using the Buege method [27]. Whole-muscle tissue samples were placed in 10% formaldehyde for histopathological and immunofluorescence evaluations.

2.3. ELISA (Enzyme-Linked Immuno Sorbent Assay) Procedure

ELISA is a quantitative measurement method of an antigen–antibody interaction based on investigating the activity of an enzyme bound to an antibody.

The pro-inflammatory cytokine TNF-α levels were measured in muscle tissue using a rat-specific ELISA kit (Yh-biosearch, Shanghai, China).

The anti-inflammatory cytokine IL-10 levels were measured in muscle tissue using a rat-specific ELISA kit (Yh-biosearch, Shanghai, China).

GABA levels were measured in muscle tissue using a rat-specific ELISA kit (Yh-biosearch, Shanghai, China).

2.4. Malondialdehyde (MDA) Procedure

Lipid peroxidation was assessed by measuring TBARS levels. Tissue homogenates were reacted with thiobarbituric acid reagent (TBA, Sigma-Aldrich) containing trichloroacetic acid (TCA, Sigma, Kawasaki, Japan), concentrated hydrochloric acid (HCl, Merck, Darmstadt, Germany), and butylated hydroxytoluene (Sigma), boiled for 15 min, and then cooled and centrifuged. TBARS levels were measured in the supernatant fraction using spectrophotometry (Shimadzu, Kyoto, Japan) at 532 nm. TBARS concentrations were expressed as nmol/g tissue [27].

2.5. High-Performance Liquid Chromatography (HPLC) Analysis of Creatine, Creatine Phosphate, AMP, ADP, and ATP

Muscle tissue samples (200 mg) were homogenized in 2 mL of 0.42 M perchloric acid (HClO₄) using an Ultra-Turrax (IKA, Staufen, Germany) T25 homogenizer for 30 s. The homogenates were centrifuged at 3000 rpm for 5 min, and the resulting supernatants were neutralized with 1.0 M K₂HPO₄ prior to chromatographic analysis.

The concentrations of creatine, creatine phosphate, AMP, ADP, and ATP were determined by ion-pair reverse-phase HPLC using an Agilent 1100 system equipped with a C18 column (250 × 4.6 mm, 5 μm; Nucleodur (Macherey-Nagel, Düren, Germany). Separation was achieved under isocratic conditions with a mobile phase containing 215 mM KOH/KH₂PO₄ buffer (pH 6.25), 3 mM tetrabutylammonium phosphate, and 5% acetonitrile. Eluted compounds were monitored at 214 nm, and their concentrations were quantified by comparison with external calibration curves generated from authentic standards, as previously described by Sellevold et al. [28].

2.6. Immunofluorescence Analysis

Muscle tissues were fixed in 10% neutral-buffered formaldehyde overnight, dehydrated through graded ethanol, cleared in xylene, infiltrated with paraffin at 58 °C, embedded, and sectioned at 10 μm using a microtome. Sections were mounted on poly-L-lysine-coated slides. Immunofluorescence staining was performed to assess NeuN and synaptophysin (SYP) immunoreactivity. S100B was included in the original staining panel as an exploratory marker; however, quantitative S100B results were not available for analysis and are therefore not presented or interpreted in this manuscript. NeuN was treated as an exploratory neural-associated marker in muscle sections and was not interpreted as evidence of neuronal cell bodies within skeletal muscle. SYP was assessed as a presynaptic-associated marker. Primary antibodies included NeuN (orb48522, Biorbyt; Durham, NC, USA, Entrez: 146,713; rabbit) and SYP (orb69251, Biorbyt; Entrez: 6855; mouse). DyLight 649-conjugated goat anti-mouse IgG (H&L) (Abbkine, Wuhan, China, #A23610) and FITC-conjugated anti-rabbit secondary antibodies were used for fluorescence detection. Following deparaffinization and rehydration, antigen retrieval was performed in 1× citrate buffer (pH 6.0) by microwave heating for 20 min. Sections were permeabilized with 0.2% Tween-20, washed with PBS (pH 7.6), and incubated overnight at 4 °C with the primary antibodies. NeuN was diluted in PBS containing 2% serum, whereas SYP was diluted in PBS containing 0.3% Triton X-100/0.3% serum and 10% goat serum, respectively. After PBS washes, the appropriate secondary antibodies were applied for 2 h at room temperature. Nuclei were counterstained with DAPI, and the sections were mounted with an antifade mounting medium before fluorescence microscopy.

2.7. Statistical Analysis

Statistical analysis was performed using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA); ANOVA outputs and degrees of freedom were checked in GraphPad Prism v8. Parametric data were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test. Data are presented as mean ± standard deviation (SD). The planned sample size was n = 8 animals per group; however, the number of observations included in individual tissue analyses varied by outcome because potentially unreliable observations identified during median-based review were excluded from the relevant parameter-specific analyses. Consequently, the residual degrees of freedom and total analyzed sample size differed across outcomes and are reported with each analysis. Statistical significance was accepted at p < 0.05.

3. Results

3.1. Findings of Septic Conditions

In the LPS group, animals were either hypothermic (34.7 °C ± 0.4) or hyperthermic (38.8 °C ± 0.7), consistent with an acute systemic response in this model. Body temperatures in the control, bicuculline, and LPS + Bic groups were within standard values. Selected blood biochemical findings are summarized in Table 1. Table S1 shows the biochemical parameter findings for all groups. Compared with the other groups, the LPS group showed higher mean LDH and fibrinogen values, while AST and ALT values are presented descriptively. The leukocyte-count analysis from the same experimental cohort was reported previously; leukocyte counts were significantly lower in the LPS group than in the control, bicuculline, and LPS + bicuculline groups [26].

The study cited as reference [26] used the same animals and experimental model, with the previous publication focusing on brain-related outcomes. The current manuscript presents skeletal-muscle findings together with the available blood biochemical values to provide additional, but not comprehensive, characterization of the model. These measures do not replace a full systemic assessment of sepsis severity, including circulating inflammatory cytokines and lactate.

3.2. Cytokine Findings

A significant overall difference was detected in tissue TNF-α levels among the experimental groups (F(3,23) = 15.64, p < 0.0001). Tukey’s post hoc analysis demonstrated that the LPS group exhibited significantly higher TNF-α concentrations than the control and bicuculline groups (both p < 0.001). TNF-α levels in the LPS + B group remained significantly elevated compared with the control group (p < 0.05). Importantly, no significant difference was observed between the LPS and LPS + B groups, indicating that bicuculline co-administration did not statistically normalize TNF-α concentrations relative to LPS alone. No significant differences were observed between the bicuculline and LPS + B groups or between the control and bicuculline groups (Figure 2A).

Figure 2. (A,B): TNF-α and IL-10 levels in gastrocnemius muscle tissue. Data are presented as mean ± SD. The number of observations included varied by outcome (TNF-α: total n = 27; IL-10: total n = 28) after parameter-specific exclusion of potentially unreliable observations. One-way ANOVA followed by Tukey’s multiple-comparison test was used. * p < 0.05; for the indicated comparisons.

Analysis of IL-10 levels also revealed a significant effect of treatment (F(3,24) = 4.701, p = 0.0101). According to Tukey’s multiple-comparison test, IL-10 concentrations in the LPS group were significantly lower than those in both the bicuculline and LPS + B groups (both p < 0.05). No statistically significant differences were identified between the control and LPS groups, control and bicuculline groups, control and LPS + B groups, or bicuculline and LPS + B groups (Figure 2B). Thus, the principal cytokine finding associated with bicuculline was the increase in IL-10 relative to the LPS group rather than a statistically demonstrated normalization of TNF-α.

3.3. GABA ELISA Findings

One-way ANOVA demonstrated significant differences in tissue GABA levels among the experimental groups (F(3,16) = 6.103, p = 0.0057). Post hoc comparisons revealed that both the LPS and bicuculline groups displayed significantly higher GABA concentrations than the control group (p < 0.01 and p < 0.05, respectively). In contrast, GABA levels in the LPS + B group did not differ significantly from those in the control, LPS, or bicuculline groups. Likewise, no significant difference was observed between the LPS and bicuculline groups (Figure 3A). The increase in GABA in the bicuculline group was not accompanied by a corresponding reduction in GABA in the LPS + B group. Therefore, the present data do not establish a simple inverse relationship between GABAA receptor blockade and tissue GABA concentration. The observed changes may reflect altered local GABA production, release, uptake, metabolism, or compensatory responses; these possibilities were not directly tested.

Figure 3. (A,B): GABA and MDA levels in gastrocnemius muscle tissue. Data are presented as mean ± SD; the total analyzed sample size was n = 24 for each outcome after parameter-specific exclusion of potentially unreliable observations. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ** p < 0.01; *** p < 0.001 for the indicated comparisons.

3.4. Lipid Peroxidation Findings

Tissue MDA levels differed significantly among the experimental groups (F(3,16) = 25.18, p < 0.0001). Tukey’s multiple-comparison analysis showed that the LPS group had significantly higher MDA concentrations than both the control and bicuculline groups (both p < 0.001). Moreover, MDA levels were significantly reduced in the LPS + B group compared with the LPS group (p < 0.001). No significant differences were detected between the control and bicuculline groups, the control and LPS + B groups, or the bicuculline and LPS + B groups (Figure 3B).

3.5. Findings of High Energy Compounds, Creatine and Creatine Phosphate

HPLC analysis of muscle tissues revealed significant alterations in energy metabolites among the experimental groups. LPS administration led to a significant decrease in ATP levels compared with the control group (p < 0.05). Conversely, ADP levels were significantly elevated in the LPS group compared with the control (p < 0.05), bicuculline, and LPS + B groups. Creatine levels in the LPS group also differed significantly from the control (p < 0.01) and bicuculline groups. Regarding AMP levels, the control group exhibited the highest concentrations (p < 0.001), followed by a significant increase in the bicuculline-alone group compared with the LPS and LPS + B groups (p < 0.05). Notably, phosphocreatine (CreatineP) levels remained relatively stable across the control, LPS, and bicuculline groups but demonstrated a marked increase in the LPS + B group (p < 0.001) compared with all other groups (Figure 4). The marked increase in phosphocreatine occurred without a statistically demonstrated parallel restoration of ATP. Thus, the phosphocreatine finding should not be interpreted as direct evidence of improved mitochondrial ATP production. An alternative interpretation is that phosphocreatine accumulated because its utilization or transfer of high-energy phosphate to ATP was altered under the combined treatment condition. The present measurements cannot distinguish between increased phosphocreatine synthesis, reduced utilization, or altered creatine kinase flux.

Figure 4. AMP, ADP, ATP, creatine, phosphocreatine (CreatineP), and ADP/ATP ratio levels in gastrocnemius muscle tissue were measured by HPLC in all experimental groups. Data are presented as mean ± SD (n = 8/group). One-way ANOVA followed by Tukey’s multiple-comparison test was used. * p < 0.05; ** p < 0.01; *** p < 0.001 for the indicated comparisons.

Assessment of Neurosynaptic Integrity via NeuN and Synaptophysin Expression:

Immunofluorescence imaging and subsequent densitometric analysis demonstrated changes in neural-associated immunoreactivity in the muscle sections. LPS exposure was associated with a significant reduction in NeuN signal and synaptophysin immunoreactivity compared with the control and bicuculline groups (p < 0.05). In the LPS + B group, NeuN immunoreactivity was preserved relative to the LPS group, and synaptophysin immunoreactivity showed an increase relative to the LPS group. These findings indicate alterations in neural-associated markers within the muscle sections. However, because NeuN is primarily a neuronal nuclear marker and skeletal muscle does not contain neuronal cell bodies, NeuN staining in muscle should not be interpreted as direct evidence of neuronal survival or neuromuscular-junction integrity. Similarly, synaptophysin immunoreactivity alone cannot establish functional neuromuscular transmission. These results are therefore considered exploratory structural observations and require confirmation using markers such as neurofilament or neuromuscular-junction-specific markers in future studies. (Figure 5, Figure 6 and Figure 7).

Figure 5. Muscle tissue sections were stained using DAPI, NeuN, and synaptophysin antibodies at ×10 magnification. (C) Control, LPS, (B) Bicuculline, and (L + B) LPS + Bicuculline groups.

Figure 6. Muscle tissue sections were stained with synaptophysin antibody at ×40 magnification. (C) Control, (L) LPS, (B) Bicuculline, and (L + B) LPS + Bicuculline groups.

Figure 7. H-scores of synaptophysin immunoreactivity in gastrocnemius muscle tissue sections from the experimental groups. (C) Control, LPS, (B) Bicuculline, and (LPS + B) LPS + Bicuculline groups. Data are presented as mean ± SD (n = 8/group). One-way ANOVA followed by Tukey’s multiple-comparison test was used. Statistical annotations indicate the pairwise comparisons shown in the figure. * p < 0.05.

4. Discussion

The present study demonstrates that bicuculline methiodide was associated with attenuation of selected inflammatory and oxidative alterations, changes in high-energy phosphate metabolism, and changes in neural-associated immunoreactivity in skeletal muscle during LPS-induced experimental sepsis. LPS exposure increased TNF-α and lipid peroxidation and altered IL-10 and energy-metabolite profiles, while bicuculline co-administration was associated particularly with lower MDA, higher IL-10 relative to the LPS group, and a marked increase in phosphocreatine. Because direct measurements of cholinergic signaling and α7nAChR activity were not performed, these findings should not be interpreted as proof that bicuculline acts through the cholinergic anti-inflammatory pathway.

Sepsis is increasingly recognized as a disorder involving coordinated inflammatory, neuroendocrine, and immunometabolic responses rather than a simple cytokine-mediated syndrome [2,3,18,19,20]. Excessive activation of innate immune pathways can contribute to reactive oxygen species generation, mitochondrial dysfunction, metabolic disturbances, and skeletal muscle wasting [4,5,6,7,8,9,10,11]. In the present model, LPS increased TNF-α and MDA and altered high-energy phosphate metabolites, consistent with a metabolically and oxidatively stressed muscle environment. The observed changes support the presence of muscle-associated metabolic consequences of endotoxemia but do not by themselves establish a causal sequence between inflammation, mitochondrial dysfunction, and muscle weakness.

The inflammatory findings require careful interpretation. Bicuculline co-administration was associated with higher IL-10 concentrations than the LPS group, whereas TNF-α remained significantly higher in the LPS + B group than in the control group and did not differ significantly from the LPS group. Thus, the present data support a selective change in the inflammatory profile rather than complete normalization of inflammation. Although GABAergic signaling can influence immune-cell activity [13,15,16], the present study did not measure acetylcholine, choline acetyltransferase, α7nAChR, vagal activity, or downstream CAP signaling. The possibility that bicuculline-related effects involve neuroimmune pathways therefore remains a hypothesis rather than a demonstrated mechanism.

Oxidative stress represents another important component of sepsis-associated muscle injury [4,7,8,9,10,11]. Increased lipid peroxidation can damage membrane components and impair cellular metabolism. Consistent with this concept, LPS administration significantly elevated MDA levels in skeletal muscle, whereas bicuculline co-administration significantly reduced MDA compared with LPS alone. This finding indicates an association between bicuculline treatment and reduced lipid peroxidation. However, MDA/TBARS measurement alone cannot determine whether bicuculline acts as a direct antioxidant, indirectly reduces oxidative stress through altered inflammatory signaling, or affects another upstream process. A direct antioxidant action is therefore an alternative explanation that should be considered alongside neuroimmune hypotheses.

An important aspect of this study was the evaluation of high-energy phosphate metabolites together with inflammatory biomarkers. Sepsis significantly reduced ATP levels while increasing ADP concentrations, consistent with altered cellular energy balance [7,29,30,31]. Interestingly, phosphocreatine increased markedly in the LPS + Bicuculline group without a statistically demonstrated parallel restoration of ATP. Phosphocreatine functions as an intracellular high-energy phosphate buffer, but its accumulation does not necessarily indicate improved ATP production. One possible explanation is altered utilization or transfer of phosphocreatine through the creatine kinase system, resulting in accumulation despite persistent limitations in ATP availability. Because creatine kinase activity, mitochondrial respiration, and phosphocreatine flux were not measured, the mechanism underlying this accumulation cannot be determined from the present data [32].

Changes in NeuN and synaptophysin immunoreactivity were also observed in the muscle sections. Synaptophysin is a presynaptic vesicle-associated protein, whereas NeuN is primarily a neuronal nuclear marker [33,34]. The use of NeuN in skeletal muscle therefore requires caution because skeletal muscle does not contain neuronal cell bodies. In the present study, NeuN immunoreactivity is reported as an exploratory neural-associated staining pattern rather than evidence of neuronal survival. Likewise, synaptophysin staining alone cannot establish neuromuscular-junction integrity or functional transmission. The apparent preservation of these signals after bicuculline treatment is hypothesis-generating and should be tested in future studies using neurofilament and neuromuscular-junction-specific markers.

Changes in tissue GABA concentrations further indicate that GABAergic signaling is altered in this experimental model. Both LPS and bicuculline alone were associated with higher tissue GABA concentrations than control animals, whereas the LPS + B group did not differ significantly from the other groups. Importantly, these measurements do not demonstrate that bicuculline lowers GABA signaling. Tissue GABA concentration is determined by multiple processes, including synthesis, release, uptake, metabolism, and compartmentalization. The present study did not assess any of these processes directly. Therefore, the GABA findings should be interpreted as evidence of altered tissue GABA homeostasis rather than as proof of a specific receptor-mediated mechanism.

Collectively, the present findings suggest that bicuculline administration is associated with attenuation of selected inflammatory and oxidative changes and with alterations in skeletal muscle energy metabolism during experimental sepsis. The data are compatible with a potential role for GABAergic and broader neuroimmune interactions, but they do not establish direct GABAergic-cholinergic coupling or activation of the cholinergic anti-inflammatory pathway. Alternative mechanisms, including direct or indirect effects on oxidative stress and cellular metabolism, remain possible. The present study should therefore be viewed as an exploratory investigation generating mechanistic hypotheses for future receptor-specific and functional studies.

Several limitations should be acknowledged. First, the study used a relatively small sample size, a single bicuculline dose, and a single 24-h experimental time point. These parameters were selected to align with our previous study using the same model [26], but the present design cannot establish dose–response relationships, toxicity across doses, or temporal dynamics. Second, systemic sepsis characterization remained incomplete: leukocyte-count findings from the same experimental cohort were reported previously [26], and selected blood biochemical values are now presented in Table 1; however, circulating inflammatory cytokines and lactate were not measured, and the available biochemical findings do not provide a comprehensive assessment of sepsis severity. Third, no direct measurements of acetylcholine, choline acetyltransferase, α7nAChR expression or activity, or vagal activity were performed, and no receptor-specific agonist/antagonist experiments were conducted. Consequently, a direct role for the cholinergic anti-inflammatory pathway cannot be established. Fourth, the marked phosphocreatine increase was not accompanied by a demonstrated ATP recovery, and mitochondrial respiration or creatine kinase flux was not measured. Fifth, NeuN is not an ideal marker for intramuscular neural structures, and synaptophysin alone does not establish neuromuscular-junction function. S100B staining was included in the original staining panel but was not quantitatively analyzed in the present dataset and therefore was not interpreted. Finally, an experimental GABA agonist arm would provide an important complementary test of receptor-directionality; this was not included in the present study. Future studies incorporating dose–response and time-course designs, systemic severity markers, receptor-specific pharmacology, cholinergic measurements, mitochondrial functional assays, and validated neuromuscular markers are warranted.

5. Conclusions

The present study demonstrates that bicuculline methiodide administration is associated with attenuation of selected inflammatory and oxidative alterations and with changes in high-energy phosphate metabolism in skeletal muscle during LPS-induced experimental sepsis. Bicuculline co-administration reduced MDA levels, was associated with higher IL-10 concentrations than the LPS group, and markedly increased phosphocreatine levels. Changes in NeuN and synaptophysin immunoreactivity were also observed, although these markers require cautious interpretation in skeletal muscle.

These findings indicate that septic skeletal muscle injury involves concurrent inflammatory, oxidative, and metabolic disturbances. The present results are compatible with the possibility that modulation of GABAergic or other neuroimmune processes contributes to these changes; however, direct involvement of cholinergic signaling, α7nAChR, vagal activity, or the cholinergic anti-inflammatory pathway was not demonstrated.

Although the precise mechanisms require further investigation, the present data provide a basis for future studies examining whether GABAergic manipulation influences skeletal muscle injury during sepsis. Studies using receptor-specific agonists and antagonists, direct measurements of cholinergic signaling, validated neuromuscular-junction markers, systemic sepsis severity measures, and mitochondrial functional assays will be important for determining the mechanisms and translational relevance of the observed associations.