Introduction
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 [[1],[2],[3]]. It is also known that free oxygen radicals increase and cause tissue damage due
to the tissue destruction after the injury [[3],[4]].
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 [[5],[6],[7],[8]]. Experimentally, a lot of medications were used in rat crush injury models such
as steroids, nonsteroidal anti-inflammatory drugs and vitamins [[9],[10],[11]]. Some antioxidants such as Acetyl-L carnitine (ALCAR), FK506, polyethylene glycol
(PEG) are used experimentally in treatment of nerve crush injuries [[12],[13],[14]].
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.
Methods
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 2nd and 6th 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.
Walking tract analysis
Functional recovery was analyzed using a WTA, and quantified using the sciatic functional
index (SFI) [[18]]. Rats were tested at 14th and 42nd days after injury. Paw-prints were recorded by painting the hind paws with black
ink and having them walk along an 8 × 80 cm corridor, lined with white paper. The
paw-prints were collected. Paw length and toe spread were measured. SFI was calculated
according to the following Medinacelli formula [[19]]:
Where ETS is the experimental toe spread, NTS the normal toe spread, EPL the experimental
paw length, and NPL is the normal paw length.
Motor nerve conduction velocity (MNCV)
At the 14th and 42nd days after crush injury, the MNCV studies were performed under general anesthesia,
and were carried out with a Neuromatic 2000 M/C Neuro-Myograph (Dantec Elektronic
Medicinsk Og Videnskabeligt Maleudstyr A/S, Skovlunde, Denmark). The sciatic nerve
was percutaneously stimulated with supramaximal stimulus intensity through monopolar
needle electrodes, proximal to the injury site at the level of the sciatic notch,
and distal to the lesion at the level of the ankle. Square wave stimulus pulses of
500 μsec in duration were delivered at 1 Hz. Recorded signals were amplified with
an alternating current-coupled preamplifier with filters at 1 Hz and 10 KHz. The latency
of the evoked muscle action potentials were recorded from the intrinsic foot muscles
with surface electrodes. Finally, the distance between the two sets of stimulating
electrodes was measured on the skin with a ruler to the nearest 1 mm, and the conduction
velocity was calculated. Both experimental (right) and normal (left) nerves were measured.
Morphological analysis
The crushed sciatic nerves were immersed immediately just after sacrification in a
drop of fixation solution, containing freshly prepared, ice cold 4% paraformaldehyde
for an hour. Then, they were incubated at 0.5% saccharose solution in PBS buffer overnight.
and embedded on cryomatrix (Shandon). 10 μm thick transverse frozen sections were
cut using a cryomicrotome (Leica, CM1900). Sections were kept in a humidified chamber
with wet gauze. 10 μL blocks solution, including 0.1% triton-X, was added to each
section. Panaxonal marker NE 14 (anti-nfh antibody) is used for immunhistochemical
staining as primary and anti Mouse IgG 488 antibody as secondary. Macroscopical nerve
evaluation has been performed according to regenerated axon number by immunoflourescent
technique. The sections were analyzed using confocal microscope (Zeiss LSM 510 Meta).
Crushed, proximal and distal to crushed area of the sciatic nerve were sectioned two
times and the averages used for evaluation. They were compared for immunoreactivity
with image analysis. Staining intensity of the crushed, proximal, distal regions were
recorded as percentile. Each group of experimental rats analyzed statistically.
Statistical Analysis
The data were expressed as means ± SD. Distributions of the data of the groups were
assessed with one-sample Kolmogorov-Smirnov Z test and were found normal (P > 0.05).
One-way analysis of variance (ANOVA) was performed on the data to examine differences
among groups. If a significant group effect was found, a Tukey HSD test was used to
identify the location of differences between groups. A p value less than 0.05 was
statistically significant. Independent Student t test was used to compare EMG values
of intact extremity and operated extremity.
Results
Walking-track analysis
The SFI was greatly decreased for both control and experimental groups 14 days post-injury,
and began showing signs of recovery on day 42nd. The SFI values of group Z and S (p = 0.037) and C (p = 0.034) were significantly
higher degree in the second week ([Figure 1]). At sixth week SFI values were close to each other in all groups. There was not
a statistical difference between groups (p = 0.278). SFI values for 2nd and 6th weeks are given in [Table 1] and [Table 2].
Table 1
EMG results for 2nd week, *: Group Z is significantly different.
|
Zofenopril (Group Z) (n = 7)
|
Saline (Group S) (n = 7)
|
Control (Group C) (n = 7)
|
P
|
|
SFI (mean ± sd, range)
|
-12.84 ± 2.86 [(-16.92) – (-8.62)]
|
-22.88 ± 5.03* [(-32.88)-(-17.55)]
|
-23.02 ± 10.53* [(-39.51)-(-12.52)]
|
0.019
|
|
Latency (msec, mean ± sd, range)
|
1.51 ± 0.28 (1.10–1.90)
|
2.08 ± 0.23* (1.80–2.50)
|
1.90 ± 0.36* (1.40–2.40)
|
0.007
|
|
Amplitude (mV, mean ± sd, range)
|
8.77 ± 2.08 (5.70–11.50)
|
5.12 ± 1.39* (3.60–7.90)
|
4.94 ± 1.34* (3.80–7.50)
|
< 0.001
|
Table 2
EMG results for 6th week,
|
Zofenopril (Group Z) (n = 7)
|
Saline (Group S) (n = 7)
|
Control (Group C) (n = 7)
|
P
|
|
SFI (mean ± sd, range)
|
-7.19 ± 2.38 [(-12.28) – (-5.67)]
|
-9.50 ± 3.35 [(-14.11)-(-5.53)]
|
-12.20 ± 8.90 [(-31.74)-(-5.67)]
|
0.278
|
|
Latency (msec, mean ± sd, range)
|
1.33 ± 0.23 (1.00–1.60)
|
1.70 ± 0.31 (1.40–2.20)
|
1.60 ± 0.46 (1.20–2.40)
|
0.147
|
|
Amplitude (mV, mean ± sd, range)
|
12.61 ± 2.69 (9.10–16.00)
|
11.03 ± 3.52 (6.30–17.00)
|
10.31 ± 2.88 (6.90–14.40)
|
0.374
|
Figure 1
Sciatic function index (SFI) results for 2nd week, CI. Confidence intervale.
The EMG studies of the Subjects on the 14th day showed that right sciatic nerve has a severe injury according to left (intact)
side that is statistically different (paired t test) (T = -3.31 P = 0.016).
The EMG measurement of rats in the second week for the latency significant degree
between the groups are different (p = 0.007). The latency in the 2nd week of the group Z was significantly lower than group S (p = 0.006) and C (p = 0,045)
([Figure 2]). But this difference disappeared in the 6th week (p = 0,147). EMG results for 2nd and 6th weeks are given in [Table 1] and [2]. The amplitude values are examined, similar to the latency, there was a significant
difference between the groups (p < 0,001) at 2nd week, but not on the 6th week (p = 0,374) ([Figure 3]).
Figure 2
EMG results for 2nd week, (latency), CI. Confidence intervale.
Figure 3
EMG results for 2nd week, (amplitude), CI. Confidence intervale.
Morphological analysis results
In all groups, lesion area, the proximal and distal parts of the lesion were estimated
microscopically. The number of the fibrils found decreased in the distal to lesion
nerve in all groups ([Figure 4]). The lowest regenerated fibril number estimated in group C, and highest in group
Z.
Figure 4
NFH immunoreactivity in the sections of proximal, middle (crush site) and distal parts
of the sciatic nerves from animals in control (C), saline (S) and zofenopril (Z) groups. The lowest regenerated fibril number estimated in group C, and highest in group
Z.
Discussion
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
[[20],[21]]. 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 [[9],
[10],
[11],
[12],
[13],
[14]]. 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 [[26],[27]].
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 [[20],[21]]. 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 [[1],
[2],
[3],
[4],
[5]]. 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 [[28],[29]]. 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 2nd week. We believe that this medication accelerates nerve crush injury healing in rats.
Our findings in SFI and EMG studies in 2nd 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.
Conclusion
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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
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.
