Nerve injuries in extremity surgery occur usually by crush or tension type rather than incision or rupture. Orthopedic surgeons strive these type problems while treating long bone fracture and some times after surgical operations. Demyelinization and remyelinization, axonal degeneration and regeneration, focal, multifocal or diffuse nerve fiber loss and endoneural edema may be encountered due to crush injury 123. It is also known that free oxygen radicals increase and cause tissue damage due to the tissue destruction after the injury 34.

There is an extensive degeneration of the distal segment, known as Wallerian Degeneration after an axonal lesion 1. The proximal stump that is connected to the cell body can regenerate to reinnervate the target organs especially in the peripheral nervous system. Although this process is often facilitated by a permissive environment in the periphery, some factors can impede normal return to function, such as the distance from injury site, metabolic disturbances, age and type of lesion 5678. Experimentally, a lot of medications were used in rat crush injury models such as steroids, nonsteroidal anti-inflammatory drugs and vitamins 91011. Some antioxidants such as Acetyl-L carnitine (ALCAR), FK506, polyethylene glycol (PEG) are used experimentally in treatment of nerve crush injuries 121314.

Angiotensin-converting enzyme (ACE) inhibitors are drugs with different structures and activities used to treat heart failure and hypertension 15. Zofenopril and captopril are the only ACE inhibitors with sulphydryl groups (SH) and consequent potential antioxidant activity 16. This activity may contribute to the notable cardio- and endothelium protective effects of Zofenopril 17.

In this study, we have evaluated the effect of Zofenopril on functional recovery following sciatic nerve crush injury in rats.

The experimental protocols have been reviewed and approved by our University Animal Care and Ethic Committee. All efforts were made to minimize the number of animals used and their distress. 21 adult Sprague-Dawley rats weighing 250–275 g underwent unilateral (right) sciatic nerve crush. Test animals in group Z received Zofenopril (15 mg/kg/day for 7 days) (n = 7), group S received normal saline for 7 days following surgery (n = 7), and group C control animals (n = 7). The animals were kept in standard room conditions and fed with standard rat diet and water ad libitum.

All of the operations were performed under the microscope by same surgeon. The right lateral thigh was operated, after shaving and preparing the skin with 10% povidone iodine. The sciatic nerve was exposed by opening the fascial plane between the gluteal and femoral musculature via a longitudinal incision. Under kethamine anesthesia, the sciatic nerve of 21 rats was exposed at mid-thigh level and either crushed for 30 seconds with a pair of jewelers forceps (n = 16). The wound was sutured in layers and the animals were allowed to recover.

At 2^nd and 6^th weeks, all animals were evaluated for sciatic functional index (SFI) by walking tract analysis (WTA) and electromyelography (EMG).

At 6 weeks after the evaluation, in order to confirm the nerve recovery, all animals were euthanatized by cervical dislocation. A 10-mm-long sample of the right sciatic nerve segment centered to the lesion was removed, fixed, and prepared for light and electron microscopic examination. From seven random of these rats, a 10-mm-long sample of the left sciatic nerve segment without any injury was removed, fixed, and prepared for histopathological examination and histomorphometry of myelinated nerve fibers.

Severe anatomical and functional disorders can be seen after peripheral nerve injury. This type of injury frequency is increasing with technology in industrialized societies. Nerve injuries in extremity represent usually by crush or tension type rather than incision or rupture in surgery or trauma. Spontaneous regeneration through the distal nerve stump with good functional return can be expected after this type of injury 2021. This type of nerve injuries are treated pharmacological agents instead of surgery.

For this purpose, many pharmacological agents are tried experimentally and successful results were reported 91011121314. However, these studies did not go beyond the experimental studies. The healing process after nerve injury is reduced mainly free oxygen radicals rather than inflammation and edema 2. Therefore, in recent years many researchers started to stand on the antioxidant mechanism. Antioxidant materials contribute nerve regeneration via free oxygen radicals scavenging effect 22. Antioxidant enzymes such as superoxide dismutase and catalase and GSH-Px are found in mammalian organisms and protect cells from toxic effects of free radicals. While free radicals production, lipid peroxidation develops on cell membrane and this can lead to final cell death. The protective antioxidant enzyme activity increases in response to free radical formation. There are many experimental studies available showing free radicals production and importance of lipid peroxidation on cell membrane injury in nervous system injuries. Free radicals induced traumatic cell damage is basic mechanism of cell death. Nevertheless, catalase and GSH-Px traumatic damage such as the FOR cleaners provide partial improvement. 23

Studies using the photo-oxidation of riboflavin sensitized by dianisidine to generate active oxygen species have clearly defined the remarkable difference in the antioxidant action of SH-containing compared with non-SH-containing, ACE inhibitors 24. The SH-containing, ACE inhibitors zofenopril, captopril, epicaptopril (the stereoisomer of captopril, which is devoid of ACE inhibitory properties) and fentiapril were found to be effective scavengers of non-superoxide free radicals, while four non-SH-containing ACE inhibitors were inactive. The protective effects from free radical-induced cell damage of SH-containing ACE inhibitors have also been assessed in cultured endothelial cells exposed to a superoxide anion and hydroxyl radical generating system 25. Pre-incubation of the cells with captopril, epicaptopril or zofenopril produced a concentration dependent (10 – 200 μM) inhibition of malonyldialdehyde formation. Both loss of cell viability and membrane blebbing were reduced by SH-containing ACE inhibitors at concentrations as low as 10 μM. In contrast, lisinopril and enalaprilat were ineffective at concentrations up to 200 μM.

Because of known antioxidant and free oxygen radicals scavenging effect of Zofenopril; it is used in experimental studies on ischemia-reperfusion damages in brain, kidney, heart and liver tissue 2627.

It has higher lipophilic effect than other ACE inhibitors with the long-term tissue penetration features tissue. Thus the long duration of effect is provided. In this way, and vascular tissue ACE myocardium and other drugs inhibition effects last much longer and has been shown to be effective 16.

Sunderland second-degree injury or axonotmesis means a breakdown of the axon and distal Wallerian degeneration but keeping of the continuity of the endoneural sheath. Spontaneous regeneration through the distal nerve stump with good functional return can be expected after this type of injury 2021. As the restored pattern of innervations is identical to the original, the study of this nerve lesion provides a good model for establishing the ontogeny of functional nerve recovery.

Electrophysiological, morphological and histologic studies were used for evaluation of experimental peripheric nerve regeneration 12345. But none of them was enough to determine the nerve recovery. Medinacelli at al. reported walking gait analysis for rat sciatic nerve. Later this method is modified and named as sciatic functional index 3.

The SFI increased and normal values were achieved at week 7 after sciatic nerve injury. Several authors reported nearly same results whose studies have also shown normal walking patterns only after the first month of post crush 2829. In contrast to these experiments, some authors reported a full recovery at the third and fourth weeks 30. The difference in the rate of motor functional recovery may relate to the pathophysiologic response of peripheral nerves to the magnitude of different crushing loads 31.

In this study, the SFI in Zofenopril group was significantly higher than other groups in 2^nd week. We believe that this medication accelerates nerve crush injury healing in rats. Our findings in SFI and EMG studies in 2^nd week support this improvement. In the second week after injury and the EMG test results done in six weeks on the morphological analysis results support these findings.

As a result, Zofenopril has been found effective in promoting nerve regeneration in sciatic nerve crush injury rat model. These molecules can be used also for the human injured nerve but additional work is needed.

The authors declare that they have no competing interests.

AMK designed the study and performed experimental operations. AD and VB performed statistical analyses. HY and MAG had performed final operations and specimen collection of this experimental study. MK had performed linguistic and technical corrections. All authors read and approved the final manuscript.