Age-Dependent Skeletal Muscle Response to Mechanical Injury Under Halothane Anesthesia in Rats: A Histological and Immunohistochemical Study

Abstract

Background

Skeletal muscle regeneration following traumatic injury is influenced by age, anesthetic exposure, and systemic inflammatory responses. We aimed to evaluate the morphological changes in skeletal muscle tissue after mechanical injury under halothane inhalation anesthesia in rats of different ages.

Materials and Methods

A total of 120 male Wistar rats aged 3, 6, and 12 months were subjected to closed soft tissue injury in the right hindlimb. Following trauma, all animals received halothane (10 ppm) as general inhalation anesthesia. Muscle biopsies were collected at 24, 72, and 168 hours postinjury. Histological, histochemical, and immunohistochemical assessments were conducted to evaluate muscle fiber edema, inflammatory infiltration, degenerative changes, and tissue regeneration.

Results

Age significantly influenced the severity and progression of morphological changes. Younger rats (3 months) exhibited faster recovery, with reduced inflammation and earlier onset of regeneration. Older rats (12 months) showed delayed and prolonged inflammation, more severe degeneration, and slower regenerative activity. The 6-month-old group demonstrated intermediate responses.

Conclusion

Halothane anesthesia, combined with mechanical trauma, reveals age-dependent differences in skeletal muscle recovery. Younger animals exhibit more efficient regenerative responses, highlighting the impact of age on postinjury healing and inflammatory modulation in skeletal muscle tissue.

 

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Keywords

age-related response - experimental trauma - halothane anesthesia - immunohistochemistry - morphological changes - skeletal muscle injury - Wistar rats

Introduction

Skeletal muscle possesses a remarkable capacity for regeneration following injury, primarily orchestrated by resident muscle stem cells, known as satellite cells. Upon activation, these cells proliferate, differentiate, and fuse to repair damaged myofibers.[1] However, this regenerative potential is not uniform across the lifespan. Aging is associated with a decline in satellite cell function, leading to delayed and often incomplete muscle repair.[2] This age-related attenuation in regenerative capacity contributes to conditions such as sarcopenia, characterized by the progressive loss of muscle mass and strength, thereby increasing the risk of disability and morbidity in the elderly population.[3]

The regenerative process of skeletal muscle is a complex interplay between various cellular and molecular mechanisms, including inflammation, extracellular matrix remodeling, and revascularization. In younger individuals, the inflammatory response postinjury is typically transient and resolves efficiently, facilitating effective tissue repair. Conversely, in aged muscle, there is a propensity for a prolonged inflammatory milieu, which can impede regeneration and promote fibrotic tissue deposition.

Anesthetic agents, particularly volatile anesthetics like halothane (2-bromo-2-chloro-1,1,1-trifluoroethane), are commonly employed in both clinical and experimental settings to induce general anesthesia. Halothane has been shown to influence various physiological processes, including modulation of calcium homeostasis within muscle cells. Specifically, halothane can activate ryanodine receptors (RyR1) in the sarcoplasmic reticulum, leading to altered calcium release and potentially affecting muscle contractility and metabolism.[4] Furthermore, prolonged exposure to halothane has been associated with changes in muscle carbohydrate metabolism, such as increased glycogenolysis and lactate accumulation, which may impact muscle recovery postinjury.[5]

Despite the widespread use of halothane in experimental models, there is a paucity of data regarding its impact on skeletal muscle regeneration, particularly in the context of aging. Understanding how halothane anesthesia influences muscle repair processes across different age groups is crucial, given the potential implications for both experimental outcomes and clinical practices involving elderly patients.

• (1) Age-related decline in skeletal muscle regeneration: Skeletal muscle regeneration is a complex process involving the activation, proliferation, and differentiation of satellite cells. With advancing age, there is a notable decline in the regenerative capacity of skeletal muscle, primarily attributed to the diminished function and number of satellite cells. This decline is further exacerbated by alterations in the muscle microenvironment, including increased fibrosis and reduced vascularization, which collectively impair muscle repair mechanisms.

• (2) Inflammaging and its impact on muscle repair: The term “inflammaging” describes the chronic, low-grade inflammation that develops with age, characterized by elevated levels of proinflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and C-reactive protein. This persistent inflammatory state adversely affects muscle regeneration by disrupting the balance between proinflammatory and anti-inflammatory signals, leading to impaired satellite cell function and increased muscle catabolism.[6]

• (3) Effects of halothane anesthesia on skeletal muscle: Halothane, a volatile anesthetic, has been widely used in both clinical and experimental settings. Studies have indicated that prolonged exposure to halothane can lead to alterations in muscle metabolism, including increased glycogen breakdown and lactate accumulation, which may adversely affect muscle recovery postinjury. Furthermore, halothane has been shown to influence calcium homeostasis within muscle cells, potentially impacting muscle contractility and regeneration.[5]

• (4) Interplay between aging, inflammation, and anesthesia: The combined effects of aging, chronic inflammation, and exposure to anesthetic agents like halothane create a challenging environment for muscle regeneration. In aged individuals, the prolonged inflammatory response and altered muscle metabolism due to halothane exposure may synergistically impair muscle repair processes, leading to delayed recovery and increased susceptibility to muscle degeneration.[7]

 

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Objective

This study aims to investigate the morphological changes in skeletal muscle tissue following mechanical injury under halothane inhalation anesthesia in rats of varying ages. By employing histological, histochemical, and immunohistochemical analyses, we seek to elucidate the age-dependent effects of halothane on muscle regeneration, inflammation, and degeneration.

 

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Materials and Methods

The study involved a total of 120 clinically healthy Wistar rats divided into three age groups: 3-month-old (n = 40), 6-month-old (n = 40), and 12-month-old (n = 40) males, with body weights ranging from 150 to 240 g. All animals were housed under standard laboratory conditions according to the international sanitary and ethical animal care guidelines. Identification was performed using age-specific body markings.

A closed soft tissue injury was induced in the right hind thigh region of each rat to simulate mechanical trauma. Subsequently, all animals were subjected to general inhalation anesthesia using halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) at a concentration of 10 ppm. Biopsy samples of the lower limb skeletal muscles were collected at 24 hours (day 1), 72 hours (day 3), and 168 hours (day 7) after trauma for morphological evaluation.

Muscle tissue samples were processed for histological, histochemical, and immunohistochemical analyses. The following morphological parameters were assessed: muscle fiber edema, inflammatory infiltration, degenerative changes, and tissue regeneration capacity. Microscopy was performed using hematoxylin and eosin staining, with additional histochemical markers as needed. Immunohistochemistry involved detection of key markers involved in inflammation and regeneration (e.g., CD68 for macrophages, MyoD for regeneration).

Immunohistochemistry

For immunohistochemical analysis, muscle tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed using citrate buffer (pH 6.0) at 95°C for 20 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Sections were then incubated overnight at 4°C with primary antibodies: anti-CD68 (Abcam, ab955, United Kingdom) at a concentration of 1:100, and anti-MyoD (Santa Cruz Biotechnology, sc-760, United States) at a concentration of 1:200. Following primary antibody incubation, sections were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 30 minutes at room temperature. Staining was visualized using diaminobenzidine chromogen, and sections were counterstained with hematoxylin.

Immunohistochemical staining was evaluated semiquantitatively using a scoring system based on staining intensity and the percentage of positive cells. The scoring system was defined as follows: 0: no staining; 1 + : weak staining; 2 + : moderate staining; and 3 + : strong staining. All slides were independently evaluated by two researchers, and the final scores were averaged for consistency.

All procedures adhered to the principles of humane treatment and ethical research practices for laboratory animals (National Research Council, 2011).

 

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Results

Morphological Alterations in Skeletal Muscle Postinjury and Anesthesia

Quantitative and qualitative histological analyses revealed significant age- and time-dependent variations in muscle tissue response following mechanical injury under halothane anesthesia. These morphological parameters included muscle fiber edema, inflammatory cell infiltration, degenerative alterations, and regenerative activity, assessed at 24 hours (day 1), 72 hours (day 3), and 168 hours (day 7) posttrauma.

• (1) Muscle fiber edema

At 24 hours postinjury, 3-month-old rats exhibited moderate muscle fiber swelling, with an average edema score of 2.5 ± 0.3 (on a semiquantitative scale of 0–4). This edema intensified to a distinct peak by day 3 (3.8 ± 0.2), before regressing by day 7 (1.4 ± 0.4), indicating a transient but pronounced acute phase response ([Figs. 1] and [2]).

Fig. 1 The image clearly shows elongated, striated muscle fibers, characteristic of skeletal muscle tissue. The wavy appearance of the fibers is associated with traumatic injury. Stained with hematoxylin and eosin. Magnification: 10 × 40.

Fig. 2 Muscle fiber edema over time by age group. This line graph shows the severity of muscle fiber edema in skeletal muscle tissue across different age groups (3, 6, and 12 months) at days 1, 3, and 7 postinjury under halothane anesthesia. Scores are presented as mean ± standard deviation on a semiquantitative scale.

In contrast, 6-month-old rats showed localized edema initially (1.7 ± 0.2), which progressively increased by day 3 (2.9 ± 0.3), followed by a partial resolution on day 7 (1.8 ± 0.3).

Twelve-month-old rats demonstrated a markedly attenuated edema response on day 1 (1.1 ± 0.1), yet swelling persisted longer, with moderate edema still present on day 7 (2.0 ± 0.3), suggesting delayed clearance of interstitial fluid.

• (2) Inflammatory cell infiltration

Inflammatory infiltration was quantified by the density of immune cells (neutrophils and macrophages) per high-power field (HPF). The youngest group showed a moderate infiltration of 45 ± 7 cells/HPF at day 1, peaking at 75 ± 5 cells/HPF by day 3, and declining sharply to 20 ± 4 cells/HPF by day 7.

Middle-aged rats exhibited a higher basal infiltration (55 ± 6 cells/HPF) on day 1, which remained elevated at 70 ± 8 cells/HPF on day 3, and showed a slower decline to 35 ± 6 cells/HPF by day 7.

Older rats (12 months) displayed significantly higher and prolonged inflammatory infiltration: 60 ± 8 cells/HPF at day 1, 85 ± 9 cells/HPF at day 3, and sustained infiltration of 50 ± 7 cells/HPF at day 7 (p < 0.05 compared with younger groups). This suggests an age-dependent exacerbation and persistence of inflammatory processes ([Figs. 3] and [4]).

Fig. 3 Peak inflammation at day 3 across age groups. Bar graph comparing inflammatory cell infiltration at day 3 postinjury among the three age groups.

Fig. 4 Inflammatory cell infiltration over time by age group. This line graph depicts the density of inflammatory cells (neutrophils and macrophages) per high-power field (HPF) in skeletal muscle tissue across different age groups (3, 6, and 12 months) at days 1, 3, and 7 postinjury under halothane anesthesia. Data are presented as mean ± standard deviation.

 

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CD68 Expression

To further characterize the inflammatory response, we assessed the expression of CD68, a marker for macrophages. In 3-month-old rats, CD68 expression was moderate at day 1 (score 2.0 ± 0.3), peaked at day 3 (score 3.0 ± 0.2), and significantly decreased by day 7 (score 1.0 ± 0.2), indicating a robust but transient macrophage presence. For 6-month-old rats, CD68 expression was slightly higher at day 1 (score 2.5 ± 0.3), reaching a peak at day 3 (score 3.0 ± 0.3), and showed a slower decline by day 7 (score 1.5 ± 0.3). In contrast, 12-month-old rats exhibited persistently high CD68 expression, with scores of 2.8 ± 0.4 at day 1, peaking at 3.0 ± 0.4 at day 3, and remaining elevated at 2.5 ± 0.3 by day 7 (p < 0.05 compared with younger groups). These findings suggest a prolonged and unresolved macrophage presence in older animals, contributing to chronic inflammation.

• (3) Degenerative changes

As shown [Figs. 5] and [6], degenerative features such as muscle fiber necrosis, vacuolization, and sarcoplasmic disorganization were markedly age-dependent. At day 1, 3-month-old rats showed moderate degenerative changes (score 2.7 ± 0.4), which peaked at day 3 (3.9 ± 0.3), then subsided substantially by day 7 (1.2 ± 0.2).

Fig. 5 In the upper part of the image, fibers with reduced staining intensity are observed, indicating signs of injury. In contrast, the fibers in the lower part of the micrograph are stained bright reddish, reflecting the dense structure of muscle tissue (under local anesthesia). Stained with hematoxylin and eosin. Magnification: 10 × 40.

Fig. 6 Degenerative changes over time by age group. This line graph illustrates the progression of degenerative changes (necrosis, vacuolization, and sarcoplasmic disorganization) in skeletal muscle tissue across different age groups (3, 6, and 12 months) at days 1, 3, and 7 postinjury under halothane anesthesia. Scores are presented as mean ± standard deviation on a semiquantitative scale.

Six-month-old rats had significantly greater degeneration at day 3 (4.2 ± 0.4), with a slower regression by day 7 (2.5 ± 0.3).

The 12-month group showed the most severe and persistent degeneration, with scores of 3.5 ± 0.3 on day 1, escalating to 4.5 ± 0.5 by day 3, and remaining elevated at 3.0 ± 0.4 by day 7.

• (4) Muscle tissue regeneration

Regeneration was assessed by immunohistochemical detection of MyoD-positive satellite cells and histological evidence of new myofiber formation ([Fig. 7]).

Fig. 7 Muscle regeneration (MyoD+ cells/HPF) over time by age group. This line graph demonstrates the regenerative activity, quantified by the number of MyoD-positive satellite cells per high-power field (HPF), in skeletal muscle tissue across different age groups (3, 6, and 12 months) at days 1, 3, and 7 postinjury under halothane anesthesia. Data are presented as mean ± standard deviation.

In 3-month-old rats, regenerative activity initiated early, with 15 ± 3 MyoD+ cells/HPF on day 1, increasing to 40 ± 4 cells/HPF by day 3, and maintained at 35 ± 3 cells/HPF on day 7.

Six-month-old rats exhibited delayed regenerative onset: 10 ± 2 cells/HPF at day 1, rising to 30 ± 4 at day 3, and 25 ± 3 at day 7.

Twelve-month-old rats demonstrated significantly impaired regeneration, with low MyoD+ cell counts (7 ± 1 at day 1), moderate increase by day 3 (20 ± 3), and minimal maintenance at day 7 (15 ± 2), indicating slower and less efficient regenerative processes (p < 0.01 compared with younger groups).

 

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Statistical Analysis

Statistical comparisons using two-way analysis of variance followed by Tukey's post hoc test demonstrated significant main effects of both age and time on all measured parameters (p < 0.001). Age-related differences were most pronounced at days 3 and 7, highlighting the delayed resolution of inflammation and impaired regeneration in older rats.

 

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Predictive Insights

These findings predict that skeletal muscle repair following injury under halothane anesthesia is considerably less efficient in aged subjects due to prolonged inflammation and slower regenerative initiation. Clinically, this suggests elderly patients may require tailored perioperative management to mitigate muscle damage and optimize recovery.

 

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Discussion

The present study elucidates the complex interplay between age, inhalation anesthesia with halothane, and the regenerative capacity of mechanically injured skeletal muscle tissue. Our results demonstrate a clear age-dependent modulation of muscle morphology characterized by differential dynamics of edema, inflammation, degeneration, and regeneration over the first week postinjury. These findings corroborate and extend current understanding of skeletal muscle repair under anesthesia, revealing critical implications for both experimental protocols and clinical management.

Our data indicate that younger rats (3 months) exhibit a robust but transient edema and inflammatory response, with efficient resolution and early initiation of regeneration as evidenced by peak MyoD-positive satellite cell counts at day 3. This aligns with previous literature showing that youthful skeletal muscle maintains a tightly regulated inflammatory response that promotes efficient satellite cell activation and myogenesis.[8] The observed edema peak (3.8 ± 0.2) and inflammatory infiltration (75 ± 5 cells/HPF) at day 3 are consistent with a well-orchestrated acute phase necessary for debris clearance and signaling.[9]

In contrast, middle-aged (6 months) and older (12 months) rats exhibited prolonged edema and higher sustained inflammatory infiltration, reaching 85 ± 9 cells/HPF at day 3 in the oldest group. This protracted inflammatory milieu likely impairs the transition from inflammation to regeneration, as evidenced by delayed and diminished satellite cell activation (MyoD+ cells dropping to 15 ± 2 by day 7 in 12-month-olds). Chronic low-grade inflammation, or inflammaging, has been extensively implicated in attenuated muscle repair and increased fibrosis in aged muscles.[10] [11] Our results quantitatively support these findings, highlighting a near doubling of inflammatory cell persistence in older rats compared with the youngest cohort.

Halothane's modulatory effects on skeletal muscle physiology, particularly through RyR1-mediated calcium dysregulation and altered metabolic pathways, may exacerbate age-related regenerative deficits. Previous studies have demonstrated halothane-induced calcium leakage that compromises excitation-contraction coupling and can trigger proteolytic pathways leading to muscle fiber degeneration.[12] This mechanism may explain the more pronounced degenerative scores in older animals (4.5 ± 0.5 at day 3), suggesting halothane anesthesia compounds the intrinsic vulnerability of aged muscle to injury ([Fig. 2]).

Moreover, metabolic disturbances caused by halothane, such as enhanced glycogenolysis and lactate accumulation, could impede satellite cell function by altering the muscle microenvironment's bioenergetic status.[13] Such effects may underlie the delayed regenerative kinetics observed in 12-month-old rats. These findings emphasize the need to carefully consider anesthetic choice and duration in experimental designs and surgical procedures involving aging populations.

The age-dependent prolongation of edema and inflammation combined with delayed regeneration under halothane anesthesia predicts an increased risk for chronic muscle dysfunction and fibrosis in elderly subjects. Clinically, this supports emerging evidence that elderly patients undergoing surgery exhibit slower muscle recovery and greater postoperative morbidity.[14] The study advocates for tailored perioperative strategies, including anti-inflammatory treatments or alternative anesthetics with less myotoxic potential, to optimize recovery.

Experimentally, these data caution that halothane anesthesia may introduce confounding factors in muscle injury models, particularly when age is a variable. Future research should investigate anesthetic protocols minimizing muscle metabolic disruption, as well as interventions that modulate inflammation and satellite cell activation in aged muscle.

While our study provides important insights, limitations include the restriction to three age groups and a single anesthetic agent. Expanding the age range and comparing volatile anesthetics could further clarify differential impacts on muscle regeneration. Molecular analyses of cytokine profiles, fibrosis markers, and satellite cell niche components would enrich understanding of underlying mechanisms.

In summary, halothane inhalation anesthesia significantly influences skeletal muscle regeneration postinjury in an age-dependent manner. Younger animals mount an effective, transient inflammatory response and rapid regeneration, whereas older animals experience prolonged inflammation, greater degeneration, and delayed recovery. These findings underscore the importance of considering both age and anesthetic effects in muscle injury research and clinical practice, predicting a need for tailored therapeutic approaches to improve outcomes in aging populations.

 

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Conclusion

This study demonstrates that inhalational anesthesia with halothane significantly modulates the morphological and regenerative responses of mechanically injured skeletal muscle in an age-dependent manner. Younger rats (3 months) exhibit rapid resolution of edema and inflammation coupled with early regenerative activation, while older rats (12 months) show prolonged inflammatory infiltration, exacerbated degenerative changes, and delayed muscle regeneration. These findings highlight the compounding effects of age and anesthetic exposure on muscle repair processes. Clinically, this suggests that advanced age may predispose patients to impaired postoperative muscle recovery under halothane anesthesia, underscoring the need for age-specific anesthetic management and therapeutic strategies to enhance muscle healing. Further studies are warranted to elucidate molecular pathways involved and to explore alternative anesthetic agents with reduced myotoxicity.

 

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Conflict of Interest

None declared.

Acknowledgments

The authors would like to extend their gratitude to laboratory members and professors of Bukhara State Medical Institute for their valuable insights and constructive feedback on this review.

Data Availability Statement

No data sets were generated or analyzed during the current study.

Authors' Contributions

U.B.F. and T.T.B. wrote the manuscript. K.D.A. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Ethical Approval

This study was reviewed and approved by the Local Ethics Committee of the Bukhara State Medical Institute on April 4, 2025 (Protocol No. 5044, Bukhara city), submitted by Bobirjon Fayzillayevich Umurov (specialty 14.00.02–Morphology), under the supervision of Prof. D.A. Khasanova (DSc in Medical Sciences). Written informed consent was obtained from all patients.

Patients' Consent

All necessary documentation was provided, and ethical standards for conducting preclinical studies were confirmed. The Committee recommended the initiation of the research.

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Publication History

Article published online:
25 August 2025