Modulatory effects of branched-chain amino acid supplementation on neuroendocrine disturbances following traumatic brain injury in rats

Abstract

Traumatic brain injury (TBI) is frequently accompanied by neuroendocrine disturbances involving the hypothalamic–pituitary–thyroid (HPT) axis and vitamin D–calcium–phosphorus homeostasis. Despite increasing interest in nutritional strategies after TBI, the effects of branched-chain amino acid (BCAA) supplementation on post-injury neuroendocrine homeostasis, including thyroid hormone levels, vitamin D status, and calcium–phosphorus balance, remain insufficiently characterized. Twenty-eight male Wistar rats were randomly assigned to Control, Sham, TBI, and TBI + BCAA groups. Moderate TBI was induced using a closed-head weight-drop model. BCAA (0.26 g/kg) was administered orally after injury. Motor function was assessed using balance beam and gait analysis, and serum levels of thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus were measured. TBI induced a negative body weight change, impaired balance and gait, reduced serum thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus levels, and decreased brain and hippocampal morphometric parameters. Post-injury BCAA supplementation significantly attenuated TBI-associated body weight loss and improved balance beam scores and gait parameters relative to the untreated TBI group. In addition, BCAA treatment partially restored serum thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus levels, although values did not fully return to control levels. BCAA supplementation also partially attenuated TBI-induced changes in gross brain and hippocampal morphometric parameters. Post-injury BCAA supplementation exerted modulatory effects, as reflected by partial improvement in neuroendocrine parameters and motor performance. However, incomplete normalization indicates that BCAA supplementation supports metabolic and functional adaptation rather than full endocrine or structural recovery. These findings suggest that BCAAs may serve as a complementary nutritional strategy to mitigate post-TBI neuroendocrine dysfunction.

Introduction

Traumatic brain injury (TBI) is a leading cause of long-term disability and death across all ages globally (Manley et al. 2025). Recent estimates indicate that 50–60 million individuals sustain TBI annually. Among all common neurological disorders, TBI has the highest incidence and constitutes a major public health burden (Gu et al. 2025). TBI results from external mechanical forces impacting the head, causing injury in brain tissue (Gu et al. 2025). In survivors of TBI, a complex cascade of secondary injury mechanisms, including metabolic dysregulation, neuroendocrine and immune disturbances, inflammation, and oxidative stress, evolves across the acute, subacute, and chronic phases and contributes to long-term neurological and functional deficits (Lu et al. 2026).

One of the most important secondary effects of TBI is a major disruption of both systemic and brain metabolism. Soon after the injury, the brain shifts into a hypermetabolic state, in which energy demands increase, mitochondrial function becomes less efficient, and protein breakdown accelerates. At the same time, the brain’s ability to use energy effectively is reduced. This mismatch between energy supply and demand not only threatens neuronal survival, but also reduces the availability of the key substrates needed for tissue repair (Tabassum et al. 2025).

Branched-chain amino acids (BCAAs) are essential amino acids involved in protein turnover and energy metabolism, particularly under metabolically stressed conditions such as brain injury (Cassidy et al. 2026, Bo and Fujii 2024). In addition to this metabolic role, BCAAs can cross the blood–brain barrier via L-type amino acid transporter 1 (LAT1), directly participating in cerebral energy metabolism and the synthesis of key neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA) (Huang et al. 2025). Alterations in BCAA availability or metabolism after TBI disrupt the balance between glutamate and GABA, impair synaptic function, and ultimately impede recovery processes in both peripheral tissues and the damaged brain (Cassidy et al. 2026).

Beyond their role in cerebral energy metabolism and neurotransmitter synthesis, BCAAs may also influence neuroendocrine function through hypothalamic nutrient-sensing pathways. Recent evidence indicates that BCAA catabolism within thyrotropin-releasing hormone (TRH)-expressing neurons of the hypothalamic paraventricular nucleus regulates energy expenditure and modulates downstream activity of the hypothalamic–pituitary–thyroid (HPT) axis (Ming et al. 2025). Given that TBI disrupts cerebral energy metabolism (Tabassum et al. 2025) and can impair hypothalamic–pituitary endocrine axes, including HPT-axis function (Feldt-Rasmussen and Klose 2025, Khandelwal et al. 2025), BCAA supplementation may support neuroendocrine adaptation through mechanisms related to hypothalamic energy sensing, including: (i) providing metabolic substrates that may support energy homeostasis in hypothalamic TRH neurons, thereby potentially influencing HPT-axis activity and neuroendocrine regulation; and (ii) modulating hypothalamic nutrient-sensing pathways that regulate TRH expression and downstream neuroendocrine output (Ming et al. 2025). Nevertheless, direct evidence linking BCAA supplementation to HPT-axis modulation and broader neuroendocrine adaptation after TBI remains limited. The present study therefore aimed to explore this relationship at the level of circulating endocrine and mineral outcomes.

Clinical studies have demonstrated marked reductions in circulating triiodothyronine (T3), thyroxine (T4), and thyroid‑stimulating hormone (TSH) levels after acute brain injury, and these changes are closely correlated with injury severity and unfavorable neurological outcomes (Zhang et al. 2025). In parallel with metabolic dysregulation, TBI is associated with alterations in central neuroendocrine function, including impaired thyroid hormone signaling. Experimental and clinical evidence indicates that TBI can disrupt the HPT axis, leading to reduced thyroid hormone availability and altered thyroid hormone transport and metabolism in the brain (Khandelwal et al. 2025). Thyroid hormones are key regulators of cerebral energy metabolism (Vujovic and Farahani 2025), neurogenesis (Salas-Lucia et al. 2025), synaptic plasticity (Khandelwal et al. 2025), and myelination (Zhang et al. 2026), and their deficiency may therefore amplify metabolic stress and impair neural repair mechanisms in the injured brain (Zare et al. 2025).

In addition to thyroid hormones, Vitamin D has increasingly been recognized as a neuroactive steroid hormone with pleiotropic effects on neuronal survival, neuroinflammation, and calcium homeostasis in the central nervous system, largely mediated through activation of vitamin D receptors and modulation of neuroprotective signaling pathways. Altered vitamin D status following TBI disrupts calcium and phosphorus homeostasis, potentially exacerbating neural dysfunction through impaired synaptic transmission and neuronal excitability (Liu et al. 2025). Given the close interdependence between metabolic state and HPT axis adaptations following TBI, it is plausible that other metabolically sensitive neuroendocrine components are also affected. However, the potential contribution of pathways such as vitamin D signaling and calcium–phosphate homeostasis to post-TBI neurobiology remains comparatively under-characterized. Collectively, disturbances across this broader neuroendocrine–metabolic network may represent an underexplored driver of structural and functional deficits after TBI (Feldt-Rasmussen and Klose 2025, Khatoon 2025).

Metabolic and neuroendocrine dysregulation after TBI ultimately manifests as measurable impairments in motor coordination, gait, and balance, as well as structural alterations within vulnerable brain regions (Khandelwal et al. 2025, Enslow et al. 2025). The hippocampus is highly vulnerable to a wide range of metabolic and endocrine insults, which is partly attributed to its high energy demand and sensitivity to hormonal dysregulation (Davidson and Stevenson 2024). Disruption of thyroid hormone signaling has been associated with structural alterations in the brain, including changes in hippocampal morphology (Alevyzaki et al. 2025). Integrative approaches that combine behavioral and structural assessments help clarify how underlying pathophysiological mechanisms translate into functional neurological outcomes after TBI (Wiegand et al. 2022).

Previous studies demonstrated that dietary BCAA supplementation restores hippocampal function and ameliorates cognitive impairment in rodent TBI models (Cole et al. 2010), and subsequent work established the minimum effective dose and treatment duration (Elkind et al. 2015). Although BCAA supplementation has shown promising effects on functional recovery and neuroprotection in experimental and limited clinical studies of TBI (Sharma et al. 2018), most studies have focused primarily on behavioral outcomes or isolated metabolic parameters. The potential role of BCAAs in modulating neuroendocrine function—particularly thyroid hormones, vitamin D status, and mineral homeostasis—remains poorly understood. We hypothesized that post-injury BCAA supplementation would attenuate TBI-induced neuroendocrine disturbances and that these biochemical improvements would be associated with enhanced functional recovery and preservation of gross brain and hippocampal morphometric parameters. Moreover, the concurrent assessment of biochemical, functional, and gross morphometric alterations after BCAA supplementation remains limited. Therefore, the present study aimed to evaluate the effects of post-injury BCAA supplementation on neurological function, gait performance, serum thyroid hormones, 25-hydroxyvitamin D, calcium and phosphorus levels, as well as brain and hippocampal morphometry in a rat model of TBI.

Materials and methods

Animal

Experiments were performed on 28 adult male Wistar rats (10–12 weeks old, 300–350 g) obtained from the Animal Center of Ilam University. Rats were maintained under a 12-h light/dark cycle, with ad libitum access to standard pellet diet and water, and housed in polypropylene cages under controlled environmental conditions (24 ± 1 °C, 55 ± 5% humidity). All experimental procedures and protocols for animal studies were approved by the Animal Care and Use Committee of Ilam University in accordance with international guidelines on the ethical use of animals (Ethics approval code: IR.ILAM.REC.1404.030). Experiments were designed to minimize the number of rats required.

Experimental design

The experimental design, group allocation, and treatment protocol are summarized in Table 1. Upon arrival, all 28 rats were assigned unique identification numbers (1–28). Rats were allocated to the four experimental groups (Control, Sham, TBI, and TBI + BCAA; n = 7 per group) by simple randomization using a standard random number table. The sample size was selected with reference to previous experimental TBI studies (Heffernan et al. 2013), while also adhering to ethical principles aimed at minimizing animal use. In addition, a sensitivity power analysis was performed using G*Power 3.1.9.7 for a one-way ANOVA with four groups, α = 0.05, statistical power = 0.80, and a total sample size of 28. This analysis indicated that the present study was powered to detect large group effects of f ≥ 0.677. To minimize observer bias, behavioral and gross morphometric assessments were performed by investigators blinded to group allocation throughout the study. Specifically, rats were identified only by individual code numbers, and group assignments were known only to one investigator (J.C.) who was not involved in behavioral testing or gross morphometric measurements. Balance beam performance was scored independently by two blinded observers, and the average score was used for analysis. Footprint parameters were measured from inked paper tracks coded by animal number, with the investigator performing the measurements unaware of treatment assignment. Similarly, gross morphometric measurements of brain and hippocampal dimensions were performed by a blinded investigator. Group codes were revealed only after completion of all data collection and primary measurements. The experimental timeline is illustrated in Fig. 1.

Table 1.

Experimental design and treatment protocol

Group	Description	TBI Model	BCAA Dose (g/kg)	Route of Administration	Treatment Duration
Control	Intact rats without surgery or treatment	–	–	–	–
Sham	Rats underwent anesthesia, scalp incision, skull exposure, and metallic disc placement without weight-drop impact	–	–	–	–
TBI	Rats subjected to TBI induced by the Closed-head weight-drop method	Closed-head weight-drop	–	–	–
TBI + BCAA	Closed-head weight-drop injured rats treated with BCAAs	Closed-head weight-drop	0.26	Oral gavage	Once daily on Days 1–5 plus a booster dose on Day 10

Fig. 1.

Experimental timeline. Following habituation (Day − 1) and baseline assessment (Day 0), TBI was induced and BCAA (0.26 g/kg) administered daily on Days 1–5 plus a booster on Day 10. Behavioral testing occurred Days 1–6; terminal procedures on Day 11

Anesthesia and induction of TBI

The rats were anesthetized via intraperitoneal injection (i.p.) of a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg) (both from Alfasan, Woerden, The Netherlands) and were allowed to breathe spontaneously. A moderate TBI was induced using a weight-drop closed-head injury model, as described in recent experimental studies (Sağlam-Çifci et al. 2025). Briefly, the rats were placed in the prone position on the table and supported on a 10-cm foam bed to allow deceleration after impact. A midline incision was made on the scalp, and the coronal and lambdoid sutures were identified. A metallic disc (10 mm in diameter and 3 mm in thickness) was fixed to the skull using bone wax at the midline between the cranial sutures. A lead weight (300 g) was allowed to fall freely from a height of 70 cm through a vertical tube onto the metal disc to induce brain injury. Following impact, the metallic disc was removed, the surgical area was cleaned, and the skin was sutured. Sham-operated rats underwent the same anesthesia, scalp incision, skull exposure, and metallic disc placement procedures, but the weight-drop impact was not applied. No craniotomy was performed before impact.

BCAA treatment

A mixture of BCAAs (L-leucine, Cat No. 105360; L-isoleucine, Cat No. 105362; and L-valine, Cat No. 108495) was prepared in equimolar amounts. BCAAs (Sigma-Aldrich Inc., St. Louis, MO, USA) were administered by oral gavage at a total dose of 0.26 g/kg body weight. The dose, equimolar ratio, and route of administration were adapted from Elkind et al. (2015) (Elkind et al. 2015), who demonstrated that once-daily oral gavage of 0.26 g/kg BCAAs in a mouse TBI model produced significant cognitive recovery, equivalent to that achieved with 100 mM BCAA-supplemented drinking water. This dosing approach is also consistent with the BCAA treatment paradigms summarized in a systematic review of BCAA supplementation after TBI (Sharma et al. 2018). Although the original study was conducted in mice, the same dose was used as an exploratory starting dose in the present rat model because it had previously shown efficacy in an experimental TBI setting. The treatment schedule of once-daily administration on post-injury Days 1–5 was guided by the finding of Elkind et al. that at least five consecutive days of BCAA administration are required to improve cognitive recovery after TBI, whereas shorter durations are insufficient (Elkind et al. 2015). The single booster dose on Day 10 was included as an exploratory extension of the treatment protocol, based on evidence that the beneficial effects of BCAA treatment may not persist after treatment discontinuation (Elkind et al. 2015). This booster dose was intended to provide additional BCAA availability before terminal outcome assessment on Day 11.

Neurological and behavioral assessments

Balance beam walking test

The animals’ ability to maintain balance was evaluated using the balance beam walking test. Performance was scored using a 6-point scale ranging from 0 (normal balance) to 6 (severe impairment), as detailed in Table 2 (Zheng et al. 2024). Assessments were performed before surgery (Day 0) and daily from Day 1 to Day 6 post-injury.

Table 2.

Balance beam walking test scoring system

Test	Score
Posture Balance	0
Catching the Edges of the BEAM	1
One Limb Falls Down from the BEAM	2
Two Limbs Fall Down from the BEAM and Hang for More than 40 s	3
Attempts to Balance but Falls Down After More than 40 s	4
Attempts to Balance but Falls Down After More than 20 s	5
No Attempt to Balance and Falls Off the BEAM	6

Footprint test

Gait analysis was performed using the footprint test, a well-established method for assessing locomotor function in rodents. Rats were acclimated to the testing environment one day prior to the experiments. Footprint testing was performed at the pre-operation baseline, immediately after surgery, and daily from Day 1 to Day 6 post-injury. During testing, the forelimbs and hindlimbs were coated with non-toxic, water-based acrylic paint, and the animals were allowed to walk along a paper-lined runway (100 cm long × 8.5 cm wide) leading to a dark enclosure. Each animal performed three consecutive runs, and stride length (ST), hind-base width (HBW), front-base width (FBW), and overlap of fore- and hind-footprints (OV) were measured from at least four consecutive steps per run (Fig. 2). The mean value of all runs was calculated for each parameter for each animal (Kamal et al. 2025). A detailed description and definition of gait parameters are provided in Table 3.

Fig. 2.

Footprint parameters stride length (ST), hind-base width (HBW), front-base width (FBW), and overlap of fore- and hind-footprints (OV)

Table 3.

Definitions of footprint test parameters

Index	Test
Stride length (ST)	The average distance between consecutive strides
Hind-base width (HBW)	The average distance between left And right hind footprints
Front-base width (FBW)	The average distance between left And right front footprints
Overlap of front/hind footprint (OV)	Distance from front footprint/hind footprint overlap

Brain and hippocampal tissue collection and gross morphological assessment

On Day 11 post-injury, 24 h after the booster BCAA dose, rats were deeply anesthetized with ketamine–xylazine (50/10 mg/kg, i.p.). Following terminal blood collection by cardiac puncture, the animals were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Brains were carefully removed after opening the skull and post-fixed in 4% paraformaldehyde. After removal of surrounding connective tissue, the whole brain and hippocampus were dissected and weighed using a digital analytical balance with 0.001 g precision. Linear dimensions of the whole brain, including length, width, and height, were measured using a vernier caliper.

Due to the curved anatomical structure of the hippocampus, its linear dimensions were measured by gently tracing the longest axis using a flexible thread, which was subsequently straightened and measured using a millimeter-scale ruler. Brain and hippocampal volumes were estimated using the fluid displacement method based on the principle of liquid displacement.

Body weight measurement and blood collection

Body weight was recorded at baseline before TBI/Sham surgery and again on Day 11 post-injury, 24 h after the booster BCAA dose and immediately before terminal anesthesia. Body weight change was calculated for each animal as Day 11 body weight minus baseline body weight. Positive values indicate weight gain, whereas negative values indicate weight loss. Rats were deeply anesthetized with ketamine–xylazine (50/10 mg/kg, i.p.), and terminal blood samples were collected by cardiac puncture before transcardial perfusion to avoid contamination from surrounding tissues. The collected blood was allowed to clot at room temperature for 15–30 min. To separate the serum, samples were centrifuged at 1500 g for 10 min at 4 °C in a refrigerated centrifuge (Hettich, Germany). The serum was then carefully collected from gel clot tubes, aliquoted, and stored at − 70 °C. These samples were subsequently used to perform assays for thyroid hormones, calcium, phosphorus, and 25-hydroxyvitamin D.

Determination of serum thyroid hormone and 25-hydroxyvitamin D concentrations

Serum concentrations of thyroid hormones and 25-hydroxyvitamin D were measured using commercial ELISA kits (Pishtaz Teb, Iran; CAT numbers: TSH: [CAT No: PT-TSH-96], T3: [CAT No: PT-T3-96], T4: [CAT No: PT-T4-96], 25-hydroxyvitamin D: CAT No: PT-25-OH VIT D (TR)-96]) according to the manufacturer’s instructions, and absorbance was read with a microplate reader (BioTek, USA). The assays are based on a competitive antibody-binding principle, in which samples and standards are incubated in microplate wells pre-coated with antibodies for T3, T4, TSH, or 25-OH vitamin D. Signal detection was achieved using horseradish peroxidase (HRP) and 3,3’,5,5’-tetramethylbenzidine (TMB) as the chromogenic substrate. Optical density was measured at 450 nm. All measurements were performed in duplicate to ensure accuracy and reproducibility. Standard curves were generated using a four-parameter logistic (4PL) nonlinear regression model in GraphPad Prism, in accordance with the manufacturer’s instructions. Optical density (OD) values of standards were fitted to the 4PL model, and sample concentrations were interpolated from the standard curve. For each sample, duplicate OD values were averaged prior to concentration calculation. Only duplicate measurements with a coefficient of variation (CV) < 10% were accepted for analysis.

Determination of serum calcium and phosphorus levels

Serum calcium and phosphorus levels were measured using an automated biochemical analyzer (Biotecnica BT1500, Italy). The calcium concentration was determined using a colorimetric method based on the Arsenazo III complex (Adin Tashkhis Teb, Iran; CAT No: 292B001), which produces a colored product that can be quantified by measuring the absorbance at 650 nm. Phosphorus levels were assessed using the phosphomolybdate method (Adin Tashkhis Teb, Iran; CAT No: 440011), a colorimetric reaction that produces a blue color, which is measured at 340 nm. The samples were processed according to the manufacturer’s instructions, and all measurements were performed in duplicate to ensure accuracy and reproducibility.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.6.1 (GraphPad Software, CA, USA). The normal distribution of all data was assessed using the Shapiro–Wilk test. Body weight change, serum 25-hydroxyvitamin D, thyroid hormone levels, calcium, phosphorus, and brain and hippocampal gross morphometric parameters were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Balance beam walking test and footprint test data were analyzed using two-way repeated-measures ANOVA followed by Tukey’s post hoc test. When appropriate, Geisser–Greenhouse correction was applied for repeated-measures analyses. Pearson correlation analyses were performed to explore associations between endocrine markers, gross morphometric outcomes, and behavioral performance within the injured cohorts only (TBI and TBI + BCAA groups; n = 14). These correlation analyses were considered exploratory and interpreted cautiously. Data were expressed as mean ± SD, and statistical significance was set at P < 0.05. Each data point represents one independent biological replicate (one individual animal). For biochemical assays, duplicate technical measurements were averaged before statistical analysis. No outlier exclusion was performed.

Results

Effects of TBI and BCAA supplementation on body weight change

Body weight change from baseline to Day 11 was calculated for each animal as Day 11 body weight minus baseline body weight to account for baseline inter-animal variability (Fig. 3). One-way ANOVA revealed a significant effect of group on body weight change [F‌ (3, 24) = 2900, P < 0.0001]. Control and Sham animals showed positive body weight change over the study period (13.71 ± 0.95 g and 12.00 ± 1.15 g, respectively), whereas the TBI group exhibited marked body weight loss (− 36.71 ± 1.49 g), which was significantly lower than both Control and Sham groups (P < 0.0001). BCAA supplementation significantly attenuated this loss in the TBI + BCAA group (− 11.57 ± 0.97 g) compared with the untreated TBI group (P < 0.0001), although body weight change remained significantly lower than in the Control group.

Fig. 3.

Effects of TBI and BCAA supplementation on body weight change (Day 11 minus baseline). Data are mean ± SD (n = 7/group). One-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Effects of BCAA supplementation on balance beam performance

To assess balance and motor coordination following TBI, animals were evaluated using the balance beam walking test (Fig. 4). At baseline (Day 0), no significant differences in balance beam scores were observed among the experimental groups, indicating comparable pre-injury motor performance. Using Geisser–Greenhouse correction where appropriate, two-way repeated-measures ANOVA revealed significant main effects of group [F (3, 24) = 840.1, P < 0.0001] and time [F (4.703, 112.9) = 99.18, P < 0.0001], as well as a significant group × time interaction [F (14.11, 112.9) = 34.14, P < 0.0001]. The Control group maintained stable balance beam scores close to zero throughout the post-injury period. In contrast, the TBI group exhibited markedly elevated balance beam scores from Day 1 onward, with mean values remaining high across all post-injury time points compared with the Control group. Rats in the TBI + BCAA group showed balance beam scores comparable to those of the untreated TBI group on Day 1; however, a progressive reduction in scores was observed from Day 4 through Day 6. By the end of the observation period (Day 6), balance beam scores in the TBI + BCAA group were significantly lower than those in the TBI group (P < 0.01).

Fig. 4.

Effects of BCAA supplementation on balance beam performance after TBI. Daily assessment Days 1–6 post-injury. Data are mean ± SD (n = 7/group). Two-way repeated measures ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Effects of BCAA supplementation on gait parameters

Gait performance was evaluated using the footprint test to assess locomotor coordination following TBI (Fig. 5). No significant differences were observed among the experimental groups during the pre-operation assessment or immediately after surgery, indicating comparable baseline and immediate post-operative gait performance. Significant gait impairments were observed from Day 1 post-injury in the injured groups. Using Geisser–Greenhouse correction where appropriate, two-way repeated-measures ANOVA revealed significant main effects of group and time, as well as a significant group × time interaction effect for all gait parameters.

Fig. 5.

Gait parameters assessed by footprint test after TBI. (A) Stride length (ST), (B) front base width (FBW), (C) hind base width (HBW), (D) forepaw/hindpaw overlap (OV). Measurements at baseline, post-surgery, and Days 1–6. Data are mean ± SD (n = 7/group). Two-way repeated measures ANOVA with Tukey’s post hoc. TBI significantly impaired all parameters vs. Control/Sham throughout Days 1–6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

For stride length, significant main effects of group [F (3, 24) = 63.36, P < 0.0001] and time [F (4.006, 96.13) = 70.92, P < 0.0001] were observed, along with a significant group × time interaction [F (12.02, 96.13) = 32.52, P < 0.0001]. A marked reduction in stride length was observed in both injured groups on Day 1 compared with the Sham group (P < 0.0001; Fig. 5A). While stride length remained significantly reduced in the TBI group throughout the observation period, rats in the TBI + BCAA group exhibited a progressive increase in stride length beginning on Day 3. By Day 6, stride length in the TBI + BCAA group did not differ significantly from that of the Control group (P > 0.05).

For front base width, two-way repeated-measures ANOVA revealed significant main effects of group [F (3, 24) = 14.96, P < 0.0001] and time [F (2.670, 64.08) = 39.51, P < 0.0001], as well as a significant group × time interaction [F (8.010, 64.08) = 15.85, P < 0.0001]. For hind base width, significant main effects of group [F (3, 24) = 227.1, P < 0.0001] and time [F (3.961, 95.06) = 57.94, P < 0.0001] were observed, along with a significant group × time interaction [F (11.88, 95.06) = 35.01, P < 0.0001]. TBI also resulted in significant narrowing of front base width and hind base width from Day 1 onward (P < 0.001; Fig. 5B and C). In the TBI + BCAA group, front base width showed a significant increase from Day 5; however, for hind base width, this significant recovery was observed earlier, starting from Day 4. By Day 6, both parameters in the treated group reached values not significantly different from Control levels, whereas both parameters remained consistently reduced in the untreated TBI group.

For forepaw-hindpaw overlap, significant main effects of group [F (3, 24) = 18.32, P < 0.0001] and time [F (2.875, 68.99) = 35.14, P < 0.0001] were observed, along with a significant group × time interaction [F (8.624, 68.99) = 14.24, P < 0.0001]. Inter-limb coordination, assessed by forepaw–hindpaw overlap, was significantly impaired following TBI (P < 0.0001; Fig. 5D). Although overlap values remained low in the TBI group throughout the study period, a significant increase was observed in the TBI + BCAA group from Day 5 onward. At Day 6, overlap values in BCAA-treated rats were significantly higher than those in the TBI group, although still lower than Control levels.

Effects of BCAA supplementation on thyroid hormone levels

The standard curves for TSH, T3, and T4 showed appropriate assay performance and were used to calculate their respective serum concentrations (Fig. 6A, B and C). One-way ANOVA revealed significant differences among the experimental groups for all thyroid hormone parameters (P < 0.0001). For TSH, a significant main effect of group was observed [F (3, 24) = 2029, P < 0.0001]. Following TBI, a marked reduction in serum TSH levels was observed compared to the Control group (0.14 ± 0.02 mIU/L vs. 4.72 ± 0.15 mIU/L, P < 0.0001; Fig. 6A). Administration of BCAAs after injury significantly increased TSH levels relative to the untreated TBI group (3.69 ± 0.13 mIU/L vs. 0.14 ± 0.02 mIU/L, P < 0.0001), although values remained lower than those of the Control group.

Fig. 6.

Effects of TBI and BCAA on serum thyroid hormones at Day 11. (A) TSH, (B) T3, (C) T4. Data are mean ± SD (n = 7/group). One-way ANOVA with Tukey’s post hoc. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Consistent with the TSH findings, for T3, one-way ANOVA revealed a significant main effect of group [F (3, 24) = 7695, P < 0.0001]. TBI induced a substantial decrease in circulating T3 levels compared to the Control and Sham groups (P < 0.0001; Fig. 6B). Specifically, T3 levels dropped significantly in the TBI group (0.79 ± 0.01 pg/mL), compared to the Control group (1.80 ± 0.01 pg/mL). The BCAA intervention significantly mitigated this decline, resulting in higher T3 levels compared to the untreated TBI group (1.70 ± 0.01 pg/mL vs. 0.79 ± 0.01 pg/mL, P < 0.0001).

A similar pattern was observed for serum T4 levels. For T4, one-way ANOVA also revealed a significant main effect of group [F (3, 24) = 2547, P < 0.0001]. TBI induced a significant reduction in T4 concentrations in the TBI group (1.10 ± 0.01 µg/dL), compared to the Control group (1.70 ± 0.01 µg/dL, P < 0.0001; Fig. 6C). Post-injury administration of BCAAs partially attenuated this reduction, with the TBI+BCAA group exhibiting significantly higher T4 levels than the untreated TBI group (1.50 ± 0.01 µg/dL vs. 1.10 ± 0.01 µg/dL, P < 0.0001).

Effects of BCAA supplementation on serum 25-hydroxyvitamin D levels

A standard curve was generated and used to calculate serum 25-hydroxyvitamin D concentrations (Fig. 7). One-way ANOVA revealed significant differences among the experimental groups [F (3, 24) = 906.4, P < 0.0001]. The TBI group exhibited a marked reduction in serum 25-hydroxyvitamin D levels compared with the Control group (12.56 ± 1.54 ng/mL vs. 75.77 ± 3.11 ng/mL, P < 0.0001). Post-injury BCAA supplementation significantly increased serum 25-hydroxyvitamin D levels in the TBI + BCAA group compared with the untreated TBI group (35.11 ± 3.18 ng/mL vs. 12.56 ± 1.54 ng/mL, P < 0.0001); however, 25-hydroxyvitamin D concentrations in the TBI + BCAA group remained significantly lower than those observed in the Control group.

Fig. 7.

Effects of TBI and BCAA on serum 25-hydroxyvitamin D at Day 11. Data are mean ± SD (n = 7/group). One-way ANOVA with Tukey’s post hoc. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Effects of BCAA supplementation on serum calcium and phosphorus levels

Serum calcium and phosphorus concentrations were measured on Day 11 post-injury to evaluate mineral homeostasis following TBI (Fig. 8). One-way ANOVA revealed significant differences among the experimental groups for both calcium and phosphorus levels (P < 0.0001).

Fig. 8.

Effects of TBI and BCAA on serum minerals at Day 11. (A) Calcium, (B) Phosphorus. Data are mean ± SD (n = 7/group). One-way ANOVA with Tukey’s post hoc. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

For calcium, a significant main effect of group was observed [F (3, 24) = 119.5, P < 0.0001]. Serum calcium levels were significantly reduced in the TBI group compared with the Control and Sham groups (3.89 ± 0.63 mg/dL vs. 10.09 ± 0.62 mg/dL and 9.67 ± 0.84 mg/dL, respectively; P < 0.0001; Fig. 8A). Post-injury administration of BCAAs resulted in significantly higher serum calcium concentrations in the TBI + BCAA group compared with the untreated TBI group (8.41 ± 0.62 mg/dL vs. 3.89 ± 0.63 mg/dL, P < 0.0001); however, calcium levels remained significantly lower than those observed in the Control and Sham groups.

Similarly, for phosphorus, one-way ANOVA revealed a significant main effect of group [F (3, 24) = 3376, P < 0.0001]. Serum phosphorus concentrations were markedly decreased in the TBI group compared with the Control and Sham groups (0.61 ± 0.06 mg/dL vs. 8.69 ± 0.14 mg/dL and 8.68 ± 0.14 mg/dL, respectively; P < 0.0001; Fig. 8B). BCAA supplementation significantly increased serum phosphorus levels in injured rats compared with the untreated TBI group (3.43 ± 0.30 mg/dL vs. 0.61 ± 0.06 mg/dL, P < 0.0001), although phosphorus concentrations in the TBI + BCAA group remained significantly lower than Control and Sham values.

Effects of BCAA supplementation on brain and hippocampal morphometry

Gross morphometric parameters of the brain and hippocampus were evaluated on Day 11 post-injury to assess macroscopic morphometric alterations following TBI (Fig. 9). One-way ANOVA revealed significant differences among the experimental groups for all measured morphometric parameters (P < 0.001).

Fig. 9.

Representative gross morphology of rat hippocampus (left) and brain (right) used for morphometric measurements (see Tables 4 and 5)

In the hippocampus, TBI resulted in significant reductions in hippocampal weight, volume, width, and length compared with the Control group (Table 4). BCAA supplementation significantly increased hippocampal weight, volume, width, and length in the TBI + BCAA group compared with the untreated TBI group. Hippocampal weight, width, and length in the TBI + BCAA group were not significantly different from Control values, whereas hippocampal volume remained significantly lower than Control and Sham values, indicating incomplete restoration of this parameter.

Table 4.

Effects of traumatic brain injury and BCAA supplementation on hippocampal morphometric parameters

	Hippocampal
	Weight (HW, mg)	Volume (HV, mm3)	Width (HWid, mm)	Length (HL, mm)
Control	126.5 ± 0.59	180.0 ± 0.83	6.10 ± 0.20	11.50 ± 0.34
Sham	125.6 ± 0.41	179.0 ± 0.87	6.33 ± 0.40	11.20 ± 0.35
TBI	117.2 ± 0.68*	165.4 ± 2.23*	5.03 ± 0.17*	10.53 ± 0.3*
TBI + BCAA	120.1 ± 0.98#	170.0 ± 2.77*#	5.80 ± 0.23#	11.43 ± 0.3#

Similar trends were observed in whole-brain morphometric analyses (Table 5). TBI induced significant reductions in brain volume, width, length, and height compared with the Control group. BCAA treatment significantly increased brain volume, width, length, and height in injured rats compared with the untreated TBI group. However, brain volume in the TBI + BCAA group remained significantly lower than that observed in Control animals, whereas brain width, length, and height were comparable to Control and Sham values.

Table 5.

Effects of traumatic brain injury and BCAA supplementation on brain morphometric parameters

	Brain
	Volume (BV, mm3)	Width (BWid, mm)	Length (BL, mm)	Height (BH, mm)
Control	1350.75 ± 4.1	21.57 ± 0.7	21.42 ± 0.3	14.74 ± 0.14
Sham	1311.63 ± 8.3	21.6 ± 0.6	21.45 ± 0.37	14.69 ± 0.12
TBI	1203.25 ± 11.4*	20.3 ± 0.8*	20.04 ± 0.62*	13.71 ± 0.07*
TBI + BCAA	1288.5 ± 6.5*#	20.94 ± 0.9#	21.45 ± 0.36#	14.23 ± 0.14#

Data are presented as mean ± SD (n = 7/group). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test. *P < 0.05 vs. Control; ^#P < 0.05 vs. TBI.

Data are presented as mean ± SD (n = 7/group). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test. *P < 0.05 vs. Control; ^#P < 0.05 vs. TBI.

Effects of BCAA supplementation on the correlation between neuroendocrine markers, morphometric outcomes, and motor performance

To explore the functional relationship between biochemical restoration, structural preservation, and behavioral recovery following TBI and BCAA supplementation, Pearson correlation analyses were performed exclusively on the injured cohorts (TBI and TBI + BCAA groups, n = 14). This approach prevents the generation of spurious correlations driven by healthy control clusters.

A strong negative correlation was observed between serum TSH levels and balance beam scores on Day 6 (r = -0.89, P < 0.0001; Fig. 10A), as well as between serum T3 levels and balance beam scores (r = -0.89, P < 0.0001; Fig. 10B), indicating that greater endocrine recovery was highly associated with reduced motor impairment. Similarly, vitamin D preservation robustly correlated with better balance performance (r = -0.88, P < 0.0001; Fig. 10C). Beyond balance, motor coordination in gait was also tightly linked to metabolic status; higher T3 levels (r = 0.91, P < 0.0001; Fig. 10E) and vitamin D levels (r = 0.93, P < 0.0001; Fig. 10F) were significantly correlated with longer stride lengths.

Fig. 10.

Pearson correlations between neuroendocrine markers, morphometry, and motor performance in injured groups (TBI and TBI+BCAA; n = 14). Balance beam scores (Day 6) vs. (A) TSH, (B) T3, (C) vitamin D, (D) hippocampal volume. Stride length (Day 6) vs. (E) T3, (F) vitamin D. Lines show linear regression; shaded areas are 95% CI

Furthermore, structural preservation significantly correlated with functional outcomes, as demonstrated by the strong negative relationship between hippocampal volume and balance beam scores (r = -0.72, P < 0.01; Fig. 10D). Overall, these exploratory correlations indicate that animals with higher endocrine marker levels and greater gross hippocampal volume tended to show better motor performance. However, these associations do not establish causality and should be interpreted cautiously. Furthermore, given the clear separation between untreated and BCAA-treated injured groups, these correlations may partially reflect between-group differences in endocrine and behavioral recovery rather than exclusively representing within-group linear associations.

Discussion

TBI induces marked impairments in motor coordination and balance through disruption of sensorimotor neural circuits, particularly within the motor cortex and cerebellum. Damage to these regions compromises the initiation, coordination, and fine-tuning of voluntary movements, thereby impairing postural control and gait stability (Zhang et al. 2025). At the pathway level, TBI disrupts the corticospinal tract, which transmits motor commands to peripheral muscles, and cerebellar circuits that integrate proprioceptive input and regulate balance (Li et al. 2025, Hadi et al. 2025, Yang et al. 2024). In parallel, post-traumatic neuroinflammation exacerbates synaptic dysfunction within these motor networks, limiting adaptive plasticity (Liu et al. 2025). Together, these mechanisms contribute to persistent gait and balance deficits by limiting the brain’s capacity for functional compensation and circuit reorganization (Orr et al. 2024).

Against this background of circuit-level dysfunction and impaired sensorimotor integration following TBI, the gradual improvement in motor coordination and balance observed with BCAA supplementation suggests a modulatory effect on post-injury recovery rather than an immediate neuroprotective action. Notably, BCAA-treated rats did not show early normalization of balance beam or gait parameters but demonstrated progressive improvement during the later post-injury period, consistent with metabolic adaptation and activity-dependent plasticity.

BCAAs can cross the blood–brain barrier via the LAT1 transporter and serve as critical substrates for cerebral energy metabolism and neurotransmitter synthesis. By supporting glutamatergic and dopaminergic signaling and improving mitochondrial substrate availability, BCAAs may enhance neuronal excitability and synaptic efficiency in motor cortex and cerebellar circuits. In addition, BCAAs regulate protein turnover and activate mechanistic target of rapamycin (mTOR)-dependent pathways involved in synaptic remodeling and axonal maintenance, processes essential for the functional reorganization of damaged motor networks (Li and Seugnet 2025, Wu 2025). Collectively, these mechanisms provide a plausible explanation for the time-dependent improvement in gait symmetry, stride length, and balance performance in BCAA-treated animals, suggesting enhanced functional integration rather than complete structural recovery.

TBI was associated with a marked reduction in body weight, reflecting the profound systemic metabolic stress induced by injury. Systemic metabolic dysfunction following TBI, often manifesting as significant weight loss, is driven by a state of hypermetabolism, neuroendocrine derangements including insulin resistance, and a pro-inflammatory catabolic state coupled with inadequate nutritional intake (Oft et al. 2024). TBI-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis and alterations in glucocorticoid receptor (GR) signaling trigger a systemic metabolic stress response. These pathological changes accelerate proteolysis and promote a negative nitrogen balance, leading to significant muscle wasting and loss of body mass (Gadwala and Ghosh 2025). Furthermore, glutamate-mediated dysregulation within the lateral hypothalamus disrupts energy homeostasis and appetite control, which may contribute to the significant nutritional decline and weight loss observed during the acute and subacute phases of injury (Dickerman et al. 2025).

BCAA supplementation partially attenuated TBI-induced weight loss, indicating an improvement in systemic metabolic balance rather than full restoration of energy homeostasis. Given the established role of BCAAs in supporting protein synthesis, limiting muscle proteolysis, and providing alternative energy substrates under catabolic conditions, the observed weight recovery likely reflects enhanced peripheral metabolic support (Wu 2025). However, the incomplete normalization of body weight suggests that short-term BCAA supplementation alone is insufficient to fully reverse the complex neuroendocrine and inflammatory drivers of post-TBI catabolism.

Our findings demonstrate a pronounced reduction in serum TSH, T3, and T4 levels after TBI, consistent with HPT axis disruption. Damage to hypothalamic and pituitary structures, together with inflammatory and metabolic stress signals, can suppress TSH secretion and impair peripheral thyroid hormone production (Feldt-Rasmussen and Klose 2025). Reduced thyroid hormone availability limits cerebral energy metabolism, mitochondrial function, and synaptic plasticity, thereby exacerbating functional deficits after injury (Khandelwal et al. 2025).

BCAA supplementation partially restored circulating thyroid hormone levels, suggesting an indirect modulatory effect on neuroendocrine regulation. Rather than acting directly on the thyroid gland, BCAAs may support HPT axis function by sustaining hypothalamic energy sensing and TRH expression, thereby preserving thyroid hormone–dependent metabolic homeostasis (Ming et al. 2025). Improved mitochondrial substrate availability and reduced cellular energy deficiency may partially restore energy-dependent signaling pathways and facilitate neuroendocrine recovery (Zong et al. 2024). Nevertheless, the incomplete normalization of TSH, T3, and T4 levels indicates that HPT axis dysfunction after TBI is multifactorial and not fully reversible through metabolic supplementation alone.

The partial restoration of thyroid hormones, 25-hydroxyvitamin D, and mineral balance with BCAA supplementation may involve several interconnected mechanisms. However, these remain plausible hypotheses rather than directly demonstrated pathways in this study.

First, BCAAs may provide critical metabolic support to neuroendocrine tissues experiencing energy stress after TBI. Neuroendocrine cells within the hypothalamic–pituitary axis rely on energy‑intensive, ATP‑dependent mechanisms for regulated hormone secretion, including vesicle trafficking and Ca²⁺‑dependent exocytosis (Clarke 2015, Li and Sheng 2022). Following TBI, hypermetabolism and mitochondrial dysfunction compromise energy supply to these structures. BCAAs are catabolized via branched-chain aminotransferases (BCAT) and the branched-chain α-keto acid dehydrogenase (BCKDH) complex to generate acetyl‑CoA and succinyl‑CoA, which enter the TCA cycle and support cerebral energy metabolism. This transamination reaction also supplies nitrogen for glutamate synthesis in the brain (Jeter et al. 2013). The partial recovery of TSH, T3, and T4 levels in BCAA-treated animals (Fig. 6) may reflect metabolic stabilization of hypothalamic–pituitary function, although tissue energy status was not directly assessed. Second, BCAA catabolism within hypothalamic thyrotropin-releasing hormone (TRH)-expressing neurons has been shown to regulate energy expenditure and modulate HPT-axis activity in metabolic disease models. Ming et al. demonstrated that altered BCAA metabolism in TRH neurons changes TRH expression and downstream thyroid hormone output (Ming et al. 2025). Although this mechanism has not been directly tested in TBI, the significant correlation between serum TSH levels and motor recovery (Fig. 10A) suggests a functional link between neuroendocrine restoration and behavioral improvement. In the context of TBI-induced hypothalamic dysfunction, BCAA supplementation may support energy-dependent TRH synthesis (Ming et al. 2025), normalize hypothalamic nutrient-sensing pathways that regulate downstream neuroendocrine output (Timper and Brüning 2017), and maintain the functional integrity of circuits controlling thyroid axis activity (Joseph-Bravo et al. 2015). However, direct measurement of hypothalamic TRH expression would be required to confirm this mechanism.

Third, the systemic metabolic benefits of BCAA supplementation may indirectly contribute to the stabilization of systemic endocrine homeostasis through coordinated regulation of energy metabolism, insulin signaling, and substrate utilization (Bo and Fujii 2025, Lynch and Adams 2014). BCAAs regulate whole-body protein synthesis and inhibit muscle proteolysis, and the significant attenuation of body weight loss in BCAA-treated rats (Fig. 3) reflects this systemic anabolic effect. Improved metabolic status may contribute to a more favorable biochemical environment for hepatic and renal vitamin D hydroxylation—both energy-dependent processes as well as intestinal absorption of calcium and phosphorus (Christakos et al. 2016). The parallel improvements in vitamin D, calcium, and phosphorus levels (Figs. 7 and 8) are consistent with such systemic metabolic support, although upstream inflammatory and endocrine markers were not measured.

Vitamin D functions as a neuroactive steroid hormone with important roles in calcium homeostasis, neuroinflammation, and synaptic function (Liu et al. 2025). TBI resulted in a marked reduction in serum 25-hydroxyvitamin D levels, indicating disruption of vitamin D metabolism following injury. Post-traumatic vitamin D deficiency may result from impaired hepatic and renal hydroxylation, altered endocrine signaling, and inflammation-mediated changes in vitamin D binding and utilization (Gáll and Székely 2021). Reduced vitamin D availability can negatively impact neuronal calcium regulation, synaptic transmission, and neuroprotective signaling pathways, thereby contributing to both motor and cognitive dysfunction after TBI (Liu et al. 2025).

BCAA supplementation partially increased serum 25-hydroxyvitamin D levels, suggesting an improvement in systemic metabolic regulation rather than direct modulation of vitamin D pathways. Improved nutritional status and attenuation of catabolic stress may indirectly support vitamin D metabolism; however, the persistence of subnormal 25-hydroxyvitamin D levels underscores the complexity of post-traumatic endocrine disturbances and suggests that targeted vitamin D supplementation may be required for full restoration (Xu et al. 2025).

Consistent with the observed reduction in 25-hydroxyvitamin D levels, TBI also induced significant decreases in serum calcium and phosphorus concentrations. Calcium and phosphorus homeostasis is regulated by vitamin D and parathyroid hormone signaling, and disruption of this axis may impair neuronal excitability, synaptic transmission, and intracellular signaling (Liu et al. 2025). Altered mineral balance can further exacerbate neuromuscular dysfunction, which may secondarily impair motor coordination and gait performance (Hartman et al. 2025).

BCAA supplementation significantly improved serum calcium and phosphorus levels compared with untreated injured animals, although full normalization was not achieved. This partial recovery may be associated with enhanced intestinal absorption and improved metabolic utilization due to better nutritional and energy status (Zhou et al. 2018). Nevertheless, the incomplete restoration of mineral homeostasis suggests that calcium–phosphorus imbalance after TBI is not solely driven by energy deficiency but also involves sustained endocrine and inflammatory alterations.

Notably, the temporal pattern of motor recovery in BCAA-treated animals paralleled the partial restoration of neuroendocrine parameters, suggesting a potential functional linkage between metabolic normalization and behavioral improvement. Thyroid hormones are well-established regulators of cerebellar development and motor function, whereas calcium homeostasis is essential for normal cellular function, transmission of nerve impulses, acetylcholine release at the neuromuscular junction, and muscle contraction (Ishii et al. 2021, Tageldin and Martin 2020). The partial restoration of these parameters may therefore have contributed to the observed motor recovery.

The incomplete restoration of brain and hippocampal gross morphometric parameters highlights a dissociation between functional improvement and structural recovery after TBI. While BCAA supplementation was associated with improvements in motor coordination and gait performance, these functional gains should be interpreted cautiously. Motor recovery during the early post-TBI period may partly reflect spontaneous recovery processes, resolution of post-traumatic swelling and diaschisis, or compensatory behavioral strategies rather than true functional recovery (Hylin et al. 2017). These functional gains were not accompanied by full normalization of brain volume or hippocampal dimensions. Moreover, the absence of histological data prevents attribution of behavioral improvements to specific neural substrates such as neuronal survival, axonal integrity, or synaptic reorganization. This dissociation suggests that BCAAs may predominantly support functional compensation within surviving neural circuits rather than reversing tissue loss, although this interpretation remains speculative without cellular-level evidence (Hylin et al. 2017). Structural recovery after TBI is a prolonged process involving complex cellular and molecular events that extend beyond the acute and subacute phases of injury (Hylin et al. 2017). Therefore, the partial gross morphometric changes observed here should be considered preliminary and hypothesis-generating pending histological confirmation.

The translational relevance of these findings for clinical TBI management warrants critical consideration. Although our results support the biological plausibility of BCAA supplementation as a complementary strategy after TBI, several important caveats must be acknowledged before clinical extrapolation. First, the rodent dose used in this study (0.26 g/kg) corresponds to approximately 3 g of BCAAs for a 70-kg adult human after body surface area conversion (Reagan-Shaw et al. 2008), which is within the range of doses reported in human TBI studies, but the optimal dosage, duration, and timing of BCAA therapy after TBI remain to be established (Sharma et al. 2018). Second, oral gavage is not feasible in many acute severe TBI patients (e.g., those with impaired consciousness or requiring mechanical ventilation); therefore, enteral or parenteral formulations would be necessary (Hansen et al. 2008). Third, although BCAAs are generally considered safe, high-dose supplementation may pose risks in patients with hepatic dysfunction and may competitively reduce aromatic amino acid transport across the blood-brain barrier, potentially affecting serotonergic and catecholaminergic neurotransmission (Fernstrom 2005). Fourth, clinical evidence supporting BCAA therapy in TBI remains limited; the most relevant studies involved small cohorts receiving intravenous BCAAs with only modest improvements in disability scores, and large-scale randomized controlled trials are lacking (Aquilani et al. 2005, Aquilani et al. 2008). Fifth, rat TBI models do not fully recapitulate the heterogeneity of clinical TBI, including variations in injury mechanism, severity, patient age, sex, and comorbidities (Zhao et al. 2023). Finally, our findings demonstrate only partial and short-term effects, raising questions about whether BCAA supplementation alone would be sufficient for clinically meaningful long-term recovery. Therefore, BCAA supplementation should currently be regarded as a promising complementary strategy requiring further preclinical and clinical validation rather than an established therapeutic intervention.

Limitations of the study

The present study has several limitations that should be acknowledged. First, only male rats were included; therefore, potential sex-dependent differences in neuroendocrine, behavioral, and structural responses to TBI and BCAA supplementation were not evaluated. Sex hormones interact with the HPT axis and may modulate thyroid hormone signaling, vitamin D metabolism, and calcium–phosphorus homeostasis following injury. Second, the observation period was relatively short (11 days post-injury), which allowed assessment of acute and early subacute effects but was insufficient to determine long-term neuroendocrine recovery or chronic structural remodeling after TBI. Third, endocrine and mineral parameters were measured at a single terminal time point, limiting our ability to assess temporal dynamics of neuroendocrine recovery. Additionally, upstream regulators such as hypothalamic TRH expression, pituitary TSH immunoreactivity, inflammatory mediators (e.g., IL-1β, TNF-α), and other neuroendocrine pathways were not directly assessed, preventing mechanistic confirmation of the proposed BCAA-neuroendocrine interactions. Fourth, the absence of a BCAA-only control group and a vehicle-gavaged TBI group limits the ability to fully distinguish specific effects of BCAA supplementation from potential confounding factors, including baseline nutritional effects of BCAAs in healthy animals and non-specific stress or absorptive effects of gavage administration. Finally, structural assessment was restricted to gross morphometric measurements of the brain and hippocampus without detailed histopathological or stereological analyses. Therefore, the morphometric findings should be interpreted as exploratory observations rather than definitive evidence of structural recovery. Future studies should include both sexes, longer follow-up periods (e.g., 4–8 weeks post-injury), additional control groups (BCAA-only and vehicle-gavaged TBI), direct measurement of hypothalamic TRH and pituitary hormone expression, inflammatory marker profiling, mitochondrial function assays, and quantitative stereological analyses of neuronal survival and synaptic density to better define the mechanisms and long-term efficacy of BCAA supplementation after TBI.

Conclusions

The present study demonstrates that TBI induces a coordinated disruption of motor function, metabolic homeostasis, and neuroendocrine regulation, resulting in persistent deficits in gait, balance, and systemic energy balance. Within the early acute/subacute observation window of this study, BCAA supplementation was associated with a time-dependent attenuation of functional impairments. Importantly, the beneficial effects of BCAA supplementation were primarily reflected in functional and metabolic outcomes rather than complete structural recovery, indicating that BCAAs support adaptive neuroplastic and metabolic responses within surviving neural circuits rather than reversing tissue loss. The dissociation observed between functional improvement and morphometric recovery highlights the multifactorial nature of post-traumatic brain repair and underscores the limits of short-term metabolic intervention.

Collectively, these findings position BCAA supplementation as a modulatory strategy that enhances functional integration and metabolic resilience after TBI, while also emphasizing that full recovery of neuroendocrine balance and tissue-level structural integrity likely requires confirmation through long-term and histological studies. Future studies should explore the integration of metabolic supplementation with targeted endocrine and neuroregenerative interventions to optimize recovery trajectories following TBI.

Author contributions

J.C.: Conceptualization, Methodology, Resources, Supervision, and Writing – review & editing.R.M.: Investigation, Data curation, Formal analysis, Validation, Visualization, Writing – original draft, and Writing – review & editing.M.H.: Investigation, Data curation, Formal analysis, Validation, Visualization, and Writing – review & editing.F.A.: Investigation, Data curation, and Writing – review & editing.B.K.: Investigation, Data curation, and Writing – review & editing.All authors read and approved the final version of the manuscript.

Funding

This research was supported by the University of Ilam.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All experimental procedures were approved by the Animal Ethics Committee of Ilam University (Approval No.: IR.ILAM.REC.1404.030).