Background
Ischemia is the subject of investigation in many experimental studies on neuropathies
representing neural changes in diabetes mellitus, vasculitis or vasculopathies, vascular
occlusion by emboli or thrombosis, and trauma. Histological changes including endoneurial
edema, demyelination, axonal degeneration, and diffuse loss of nerve fibers as well
as behavioral and electro-physiologic studies are the commonly used measures by researchers
to evaluate the outcome of ischemia/reperfusion (I/R) injuries. Setting up a model
that is easily performable and induces expected para-clinical and clinical changes
in the target organ, is a prerequisite to simulate clinical conditions under laboratory
circumstances.
Various models of unilateral I/R injury of rat sciatic nerve have been developed.
In one method described by Mitsui et al. [[1 ]], the abdominal aorta, the right iliac and femoral arteries, and all identifiable
collateral vessels supplying the right sciatic-tibial nerve are ligated for 3 hrs
and then reperfused for different time intervals. In another model introduced by Saray
and his colleagues [[2 ]], only femoral artery and vein – just distal to the inguinal ligament – are clamped
for 3 hrs followed by reperfusion. The former is time consuming and requires advanced
surgical techniques and instruments and the latter, albeit easily performable, does
not produce severe injury [[3 ]]. The aim of this study was to develop a practical model producing serious neurologic
deficits and still technically feasible. For this purpose, clamping of both the femoral
artery and ipsilateral common iliac artery was performed to induce sciatic nerve I/R.
Materials and methods
Thirty Spraque-Dawley male rats were housed in a temperature-controlled room (25 ±
1°C) and maintained on a 12 h light/dark cycle with free access to food and water.
All experiments were performed in accordance with institutional guidelines for animal
care and use and also “Principles of laboratory animal care” were followed. The animals
weighing 150–200 g were randomly divided into six groups: one control group and five
I/R groups at different time intervals of reperfusions (0 h, 3 h, 1 d, 4 d, 7 d).
All animals were anesthetized with ketamine (50 mg/kg) and xylazine (4 mg/kg) and
subjected to laparotomy. In the I/R groups, the right common iliac artery and the
right femoral artery – just distal to the inguinal ligament – were clamped for 3 hrs
using two Yasargil aneurysm clips providing 125 g (1.24 N) force. Reperfusion was
checked under a microscope at the distal site of clamping after removing the clips.
Using a rectal probe inserted 5 cm into the rectum, the deep rectal temperature was
monitored and maintained at 36.5 ± 1°C by a thermal pad. All procedures were carried
out for the sham-operated group except for arterial clamping. After certain time intervals
of reperfusion, the function of the hind limb was assessed for each animal in terms
of behavioral score based on gait, grasp, paw position, and pinch sensitivity, while
the score for each index varied from 0 (no function) to 3.0 (normal function) except
for pinch sensitivity ranging from 0 to 2 [[4 ]]. Behavioral scoring was performed by two separate observers blinded to the status
of the rats and the average scores were recorded. Then, the sciatic nerve was fixed
in situ for 30 min using 4% formaldehyde in phosphate buffer (pH 7.4) and then, trifurcation
of the sciatic nerve was removed, embedded in paraffin, and finally, sections were
stained with hematoxylin, eosin and tri-chrome gomori for light-microscopy studies,
and were graded for ischemic fiber degeneration (IFD) and edema [[5 ]]. The sections were graded 0 to 4 for IFD based on the percentage of IFD as follows:
≤ 2%, 3–25%, 26–50%, 51–75%, and >75%, respectively ([Table 1 ]). Three sections in row from each specimen and three random fields at high power
field (HPF) level from each section were chosen and the results were averaged. Grading
for edema was based on severity and distribution, where 0 = normal, 1 = mild edema,
2 = moderated edema, 3 = severe edema, and 4 = severe and widespread edema ([Table 1 ]).
Table 1
Histopathological scoring system for quantification of edema and Ischemia Fiber Degeneration
(IFD)
Histopathologic grade
Edema
IFD (ischemic fiber degeneration)
0
normal
≤ 2%
1
Mild edema
3–25%
2
Moderate edema
26–50%
3
Severe edema
51–75%
4
Severe and widespread edema
>75%
Non-parametric Kruskal-Wallis (KW) and Mann-Whitney U (MWU) tests were used to compare
behavioral and pathologic scores (i.e. edema and IFD grades) among the groups. Data
throughout the manuscript are presented as median. All statistical analyses were performed
utilizing the SPSS software, version 13.0.
Results
Using KW test, behavioral, IFD, and edema scores of I/R groups differed significantly
from those of control (p < 0.01, p < 0.001, and p < 0.001 respectively). Behavioral
assessment was not reliable in 0 h reperfused group, because the animals were still
under the effect of anesthetic drugs which would interfere with the behavioral outcome.
Behavioral function was normal in control group (total score = 11) at any given time.
Loss of function was observed in all I/R groups compared with control (group 3 h:
score 3.00; group 1 d: score 6.00; group 4 d: score 4.00; group 7 d: score 6.00; MWU,
p < 0.01 vs. control) ([Fig. 1A ]). Animals at 4 days showed the worst behavioral outcome, though this was not statistically
significant (MWU, p > 0.05). Pathologic changes were significant in the reperfused
groups compared with control group ([Fig. 2 ]). Fiber degeneration was observed only in group 7 d (group 7 d; 3.00; MWU, p < 0.01)
([Fig. 1B ]). Epineural edema was observed from day 1 on, but marked endoneural edema occurred
at 4 (group 4 d; 2.00; MWU, p < 0.01) and 7 days (group 7 d; 4.00; MWU, p < 0.01)
([Fig. 1C ]). Specimens from group 7 d had more severe edema compared with group 4 d (MWU, p
< 0.05).
Figure 1 . (A) Behavioral score worsens as time passes with the worst outcome at 4 days. (B)
IFD occurred only after 7 days of reperfusion while (C) edema happened earlier at
4 days, but was still more severe at 7 days.
Figure 2 . There were axonal swelling, and fiber degeneration at 7 days compared with control
group.
Discussion
The results of our experiment promised of a newly introduced method for induction
of I/R injury in peripheral nerve. Our method produced profound ischemic changes at
light microscopic level with its maximal effect elicited at 7 days and a considerable
behavioral deficit with its peak at 4 days.
In previous studies, three phases were reported at the light microscopic level [[4 ],[6 ]]. The first phase was reportedly happens in 0 h and 3 h groups and did in our study
with only minimal axonal changes observed. Group 1 d in our study also showed slight
edema, being mostly epineurial, but still no noticeable endoneurial changes. The second
phase in our study was also in concordance with the aforementioned studies showing
IFD and profound axonal edema at 7 days. Iida [[4 ]] also reported a third phase of fiber regeneration with only minimal edema and fiber
debris at 28 and 42 days of reperfusion; but, as our study included groups up to 7
days, changes of third phase were not observed.
Regarding the behavioral score, our results were slightly different from the previous
models. Iida et al. reported maximal behavioral deficit to happen at day 7 of reperfusion,
while in our model this occurred at day 4 with a slight recovery at day 7. As Iida
and his colleagues’ study did not include a 4 day group, we can not make a judgment
whether our model induces an earlier maximal deficiency. They used a 20 score scale
to investigate behavioral performance and observed score 4 at 7 days (20% of the max)
while in our study score 6 of an 11 score scale (54% of the max) was seen at 7 days.
Although, inter-observer bias should be taken into consideration, it seems that our
model failed to induce a neurologic deficiency so severe as the above-mentioned method.
This could be explained by the extent of arteries cross clamped in their method. In
spite of this difference, the simplicity of our modified method and its still-considerable
induced functional defect in the limb make it rational to apply in experimental studies.
Briefly, our results show that common iliac artery and femoral artery clamping induces
I/R injury in rat sciatic nerve. This method provides the obvious advantage of producing
an easily induced moderate to severe behavioral deficit over the current methods and
can be used in experimental studies designed to evaluate the effect of therapeutic
candidates on I/R injury in peripheral nerves.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MN participating in drafting the manuscript. RR participating in drafting the manuscript
and statistical analysis. GF participating in statistical analysis and study design.
MRR: Preparing the revised paper and drafting the manuscript. SMR participating in
setting up the method.
ABF participating in setting up the method. FAA carried out pathologic assessment.
ARD participated in the design and cordination of the study. All authors read and
approved the manuscript.
