Protective effects of verapamil on skeletal muscle ischemia–reperfusion injury and the influence of administration timing in a rat model

Authors

Abstract

Aim: Ischemia–reperfusion (I/R) injury of skeletal muscle is a major cause of morbidity in reconstructive and orthopedic procedures. This study aimed to investigate the protective effects of verapamil on skeletal muscle I/R injury and to evaluate how the timing of administration (before ischemia vs. before reperfusion) influences its efficacy.
Materials and Methods: Forty male rats were divided equally into four groups: Sham, I/R, Verapamil Pre-Ischemia, and Verapamil Pre-Reperfusion. Ischemia was induced by clamping the femoral artery for 150 minutes, followed by 20 minutes of reperfusion. Verapamil (0.4 mg/kg,i.v) was administered either immediately before ischemia or just prior to reperfusion. Malondialdehyde (MDA) levels were measured as a marker of oxidative stress, and muscle samples were examined histopathologically for necrosis, eosinophilia, striation loss, and neutrophil infiltration.
Results: MDA levels and histopathological damage scores were significantly elevated in the I/R group compared to the sham group (p < 0.001). Verapamil treatment reduced both biochemical and histologic indices of injury, with the pre-ischemic administration producing the greatest reduction in MDA (0.38 ± 0.05 nmol/mg) and histologic scores (p < 0.001 vs. I/R). A strong positive correlation was found between MDA levels and total histological damage (r = 0.84, p < 0.001).
Discussion: Verapamil significantly attenuated skeletal muscle I/R injury by reducing oxidative stress and structural damage. The timing of administration was crucial, as pre-ischemic treatment offered superior protection compared to pre-reperfusion dosing. These results support calcium-channel blockade as a potential pharmacologic strategy to mitigate I/R-related muscle injury in surgical settings where ischemia is anticipated.

Keywords

Introduction

Ischemia–reperfusion injury (IRI) of skeletal muscle is a major challenge in orthopedic and reconstructive surgery, particularly in situations of vascular occlusion, tourniquet use, trauma, or flap transplantation. During the ischemic phase, cellular ATP depletion, ionic imbalance (including intracellular Ca²⁺ overload), and hypoxia set the stage for damage; on reperfusion, a paradoxical burst of reactive oxygen species (ROS) and inflammatory responses amplifies tissue injury [1]. In the context of skeletal muscle, reperfusion leads to lipid peroxidation, neutrophil infiltration, microvascular dysfunction, and ultimately cell necrosis or apoptosis [2, 3].
Calcium-channel blockers such as verapamil have been explored as potential protective agents in IRI by virtue of their ability to modulate intracellular calcium influx, stabilize cell membranes, and reduce oxidative stress. In experimental models of cardiac I/R injury, verapamil has been shown to attenuate ROS generation and myocardial infarct size through activation of SIRT1 and downstream antioxidant signaling [4]. Although less well studied in skeletal muscle, similar pathophysiologic mechanisms—calcium overload, oxidative lipid damage, and neutrophil-mediated injury—suggest the possibility of a protective role.
Another important variable in IRI research is the timing of pharmacologic intervention. Some data indicate that administration of protective agents before ischemia (preconditioning) may confer superior benefit compared to administration at reperfusion [5]. In skeletal muscle models, the window for intervention appears narrow: once reperfusion begins, ROS and calcium overload progress rapidly [6]. Thus, the precise timing of verapamil administration may determine the extent of muscle salvage.
In this study, we aimed to investigate the protective effect of verapamil on skeletal muscle IRI in a rat model, and to evaluate how the timing of administration (pre-ischemia versus pre-reperfusion) influences outcomes. We hypothesize that verapamil given before ischemia will yield greater attenuation of oxidative damage and histopathologic injury than the same dose given just prior to reperfusion.

Materials and Methods

Experimental Design and Groups
Forty male Sprague–Dawley rats, weighing 208–212 gr at the start of the experiment, were obtained from the experimental animal facility of Selçuk University, Meram Faculty of Medicine. The animals were housed in standard polypropylene cages under controlled conditions (12 h light–12 h dark cycle, ambient temperature 22±2 °C, relative humidity ~50 %) and allowed free access to standard pellet diet and water. The ischemia– reperfusion model used in this study has been widely validated in skeletal muscle injury research [7, 8]. Rats were randomly assigned (n = 10 per group) into four experimental groups:
- Sham group (Group 1): Animals underwent anesthesia and surgical exposure, but no femoral artery occlusion (ischemia) or reperfusion.
- Ischemia–Reperfusion (I/R) group (Group 2): Animals underwent 150 minutes of femoral artery clamping followed by 20 minutes of reperfusion.

- Verapamil Pre-Ischemia group (Group 3): Animals received intravenous verapamil (0.4 mg/kg) immediately prior to the onset of ischemia; then underwent 150 minutes of femoral artery clamping and 20 minutes of reperfusion [4].
- Verapamil Pre-Reperfusion group (Group 4): Animals underwent
150 minutes of femoral artery clamping, then received the same dose of verapamil (0.4 mg/kg) immediately prior to the onset of reperfusion, followed by 20 minutes of reperfusion (Supplementary Figure S1).
Anesthesia and Surgical Procedure
All animals were anaesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). After confirmation of adequate anesthesia (lack of pedal reflex, stable respiration), the right femoral region was shaved, aseptically prepared, and a longitudinal incision was made to expose the femoral artery. In groups 2–4, a non-traumatic microvascular clamp was applied to the right femoral artery to induce ischemia (150 min). Visual confirmation, such as loss of distal femoral pulse or blanching of the distal limb, was used to verify successful occlusion. Reperfusion was initiated by removal of the clamp at the end of the ischemia period; restoration of flow was confirmed by return of pulse and color to the distal limb. The incision was temporarily covered with moist gauze and maintained at physiologic temperature (37 °C) using a heated pad during both ischemia and reperfusion phases. The sham group underwent identical exposure without clamping.
Drug Administration
Verapamil hydrochloride (prepared in sterile saline) was administered via tail vein (0.4 mg/kg in 0.2 mL sterile saline). In the pre-ischemia group, the drug was given immediately before clamp placement. In the pre-reperfusion group, the drug was given immediately before clamp removal [4, 9, 10].
Tissue Collection
At the end of the reperfusion period (20 minutes), animals were euthanized under deep anesthesia by cardiac puncture (blood collection) followed by decapitation. The gastrocnemius muscle from the right lower limb was excised, washed in ice-cold saline, and divided into two parts: one portion fixed in 10 % buffered formalin for histopathologic examination, and the other snap- frozen in liquid nitrogen and stored at -80 °C for biochemical assays.
Biochemical Analysis
Frozen muscle tissue samples were homogenized in phosphate- buffered saline (PBS, pH 7.4) and centrifuged at 10,000×g for 15 minutes at 4 °C. The supernatants were used for measurement of malondialdehyde (MDA) levels, via the thiobarbituric acid reactive-substances (TBARS) assay (nmol/mg tissue). Protein concentration was determined by the Bradford method to normalize MDA values. All assays were performed in duplicate. Histopathologic Analysis Formalin-fixed muscle samples were processed through standard paraffin embedding, sectioned at 5 µm thickness, and stained with hematoxylin & eosin (H&E). An experienced blinded pathologist evaluated the sections for the following parameters: myofibre necrosis, eosinophilia, loss of striation, nuclear pyknosis, neutrophil infiltration, and interstitial oedema. Each parameter was scored semi-quantitatively on a scale from 0 (absent) to 3 (severe). The total histologic damage score was derived by summing the individual parameter scores (maximum possible score: 15) [11, 12].
Statistical Analysis
Data are presented as mean ± standard deviation (SD) or median (interquartile range [IQR]) depending on distribution. The Shapiro–Wilk test was used to assess normality. For comparisons between groups, the Kruskal–Wallis test was used for non-parametric data; if significant, pair-wise comparisons were performed with the Mann–Whitney U test with Bonferroni correction. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA).
Ethical Approval
This experimental study was approved by the Ethics Committee of Meram University, Faculty of Medicine, Konya, Türkiye (Date: 2002-06-08, No: İ01-143-23). All animal procedures were conducted in compliance with institutional guidelines for the care and use of laboratory animals and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocol adhered to the ethical principles outlined in the Declaration of Helsinki.

Results

Malondialdehyde (MDA) levels, a marker of lipid peroxidation, differed significantly among groups (p < 0.001). The highest MDA concentrations were observed in the I/R group, while both verapamil-treated groups showed markedly lower levels. The reduction was most pronounced when verapamil was administered before ischemia, indicating stronger protection against oxidative stress (Table 1).
Histological evaluation of the gastrocnemius muscle demonstrated extensive structural disruption, fiber necrosis, and neutrophil infiltration in the I/R group. In contrast, verapamil pretreatment markedly preserved myofibrillar integrity and reduced inflammatory cell infiltration. Semi-quantitative scores for nuclear pyknosis, eosinophilia, striation loss, and neutrophil infiltration are summarized below (Table 2, Figure 1).
Correlation analysis revealed significant positive associations between oxidative stress and histopathological injury severity. Malondialdehyde (MDA) levels showed a strong positive correlation with the total histologic damage score (Spearman’s r = 0.84, p < 0.001), indicating that increased lipid peroxidation was closely linked to overall tissue injury. Additionally, MDA levels correlated significantly with neutrophil infiltration (r = 0.78, p < 0.001) and eosinophilia (r = 0.72, p < 0.001).

Discussion

In this rat model of skeletal muscle ischemia–reperfusion (I/R), verapamil reduced biochemical and histopathological indices of tissue injury, with the most pronounced benefit when administered before ischemia. These findings align with the current understanding that I/R injury in striated muscle is driven by a surge of reactive oxygen species (ROS), calcium overload, endothelial dysfunction, and an amplified inflammatory response at reperfusion that culminate in myocyte necrosis and microvascular failure. By limiting calcium entry and secondarily dampening oxidative stress, calcium-channel blockade provides a biologically plausible strategy for attenuation of I/R damage. Our data support this concept and additionally underscore the importance of timing, with pre-ischemic administration outperforming pre-reperfusion dosing [13].
Contemporary reviews of skeletal muscle I/R emphasize that reperfusion is not simply restorative but pathologic— characterized by ROS bursts, mitochondrial dysfunction, neutrophil recruitment, and microvascular plugging that together dictate injury severity and clinical outcomes. The microvasculature is a key determinant of parenchymal survival, and interventions that stabilize endothelial function and temper leukocyte–endothelial interactions are consistently protective in preclinical models. Our observation of lower neutrophil infiltration and improved structural preservation with verapamil is consistent with this microvascular paradigm [14].
Direct data on verapamil in skeletal muscle I/R are limited, but there is mounting evidence from the heart and other organs that verapamil mitigates I/R injury by curbing Ca²⁺ influx and attenuating oxidative/apoptotic signaling. In murine myocardial I/R, verapamil activates SIRT1-linked antioxidant pathways and reduces infarct size; more recent work suggests modulation of apoptosis (e.g., JAK2/STAT signaling) during hypoxia/reoxygenation. Although organ contexts differ, the shared mechanisms—calcium overload and ROS-coupled injury—support a translatable protective role for verapamil in skeletal muscle. Our MDA and histology results are directionally concordant with these mechanistic reports [4].
Beyond verapamil, 2023 pharmacology overview highlights calcium antagonists (verapamil, diltiazem, nifedipine) as one class among several with reproducible anti-I/R effects across organs by reducing calcium-dependent injury—again consistent with our findings [15].
The superiority of pre-ischemic dosing observed here resonates with the broader conditioning literature: preconditioning strategies (ischemic or pharmacologic) prime antioxidant defenses, preserve mitochondrial integrity, and blunt the initial wave of injury at the moment of reperfusion. While post- conditioning can still confer benefit, its window is narrow and often less robust. Our data mirror this asymmetry: verapamil before ischemia produced greater reductions in oxidative (MDA) and histological injury than the same dose given just prior to reperfusion [16].
We observed strong positive correlations between MDA—a canonical lipid peroxidation marker—and composite histologic scores, reinforcing that oxidative damage tracks with tissue- level injury. Methodologically, recent assessments emphasize that MDA remains widely used, especially when measured with validated spectrophotometric or chromatographic approaches, but that preanalytical and analytical rigor is essential to avoid over- or underestimation. These considerations support our choice of MDA while also motivating future inclusion of complementary redox readouts (e.g., 4-HNE adducts, isoprostanes) [17].
Recent work in lower-extremity I/R underscores multifactorial injury (oxidative stress, mitochondrial dysfunction, inflammation) and surveys a broadening armamentarium— antioxidants, metabolic modulators, and mitochondria-targeted approaches—with several agents lowering MDA and neutrophil infiltration in rodent muscle. Our results are consistent with that direction of travel and suggest that calcium-channel blockade deserves renewed attention as a pragmatic adjunct, particularly where tourniquet or vascular clamping is anticipated [18].
For settings such as limb revascularization, free-flap surgery, or prolonged tourniquet use, pre-operative pharmacologic prophylaxis is attractive if it is safe, inexpensive, and simple to administer. Verapamil meets these criteria but carries hemodynamic constraints (hypotension, bradycardia) that would need careful peri-operative management. The “timing effect” observed here implies that protocols aiming to pre-treat before ischemia (analogous to pharmacologic preconditioning) may maximize benefit; if pre-treatment is not feasible, rapid delivery before reperfusion may still provide partial protection. Controlled dose-finding, safety monitoring, and comparative trials against other agents (e.g., antioxidants or mitochondria- centric therapies) are warranted [15].

Limitations

Strengths include a standardized I/R protocol with blinded histopathology and concordant biochemical–morphologic signals. Limitations are the single-dose design, short reperfusion window, and reliance on one oxidative biomarker. Mechanistically, we did not interrogate mitochondrial dynamics, apoptotic signaling, or microvascular function directly—targets highlighted by recent reviews—nor did we evaluate systemic repercussions (e.g., remote organ injury). Future studies should incorporate longer reperfusion intervals, hemodynamic monitoring, and mechanistic readouts (SIRT1 activity, JAK2/ STAT signaling, mitochondrial fission/fusion indices) to clarify how verapamil confers protection in skeletal muscle [14].

Conclusion

In conclusion, verapamil reduced oxidative (MDA) and histopathological injury in skeletal muscle I/R, with pre- ischemic dosing outperforming pre-reperfusion dosing. These data, integrated with contemporary mechanistic literature on ROS, calcium overload, and microvascular dysfunction, support calcium-channel blockade as a rational, time-sensitive adjunct to mitigate skeletal muscle I/R injury and justify translational evaluation in limb surgery contexts.

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