- Short report
- Open Access
Ipsilateral common iliac artery plus femoral artery clamping for inducing sciatic nerve ischemia/reperfusion injury in rats: a reliable and simple method
© Nouri et al. 2008
Received: 29 September 2008
Accepted: 22 December 2008
Published: 22 December 2008
The aim of this study was to develop a practical model of sciatic ischemia reperfusion (I/R) injury producing serious neurologic deficits and being technically feasible compared with the current time consuming or ineffective models. Thirty rats were divided into 6 groups (n = 5). Animal were anesthetized by using ketamine (50 mg/kg) and xylazine (4 mg/kg). Experimental groups included a sham-operated group and five I/R groups with different reperfusion time intervals (0 h, 3 h, 1 d, 4 d, 7 d). In I/R groups, the right common iliac artery and the right femoral artery were clamped for 3 hrs. Sham-operated animals underwent only laparotomy without induction of ischemia. Just before euthanasia, behavioral scores (based on gait, grasp, paw position, and pinch sensitivity) were obtained and then sciatic nerves were removed for light-microscopy studies (for ischemic fiber degeneration (IFD) and edema). Behavioral score deteriorated among the ischemic groups compared with the control group (p < 0.01), with maximal behavioral deficit occurring at 4 days of reperfusion. Axonal swelling and IFD were found to happen only after 4 and 7 days, respectively. Our observations led to an easy-to-use but strong enough method for inducing and studying I/R injury in peripheral nerves.
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. , 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 , 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 . 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).
Histopathological scoring system for quantification of edema and Ischemia Fiber Degeneration (IFD)
IFD (ischemic fiber degeneration)
Severe and widespread edema
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.
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  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.
- Mitsui Y, Schmelzer JD, Zollman PJ, et al.: Hypothermic neuroprotection of peripheral nerve of rats from ischaemia-reperfusion injury. Brain 1999,122(Pt 1):161–169.View ArticlePubMedGoogle Scholar
- Saray A, Can B, Akbiyik F, et al.: Ischemia-Reperfusion injury of the peripheral nerve: an experimental study. Microsurg 1999, 19:374–380.View ArticleGoogle Scholar
- Nouri M, Rasouli M, Rahimian R, et al.: Femoral vessels cross-clamping: the reliability of a method for sciatic nerve ischemia-reperfusion injury. Microsurg 2007,27(3):206–207.View ArticleGoogle Scholar
- Iida H, Schmelzer JD, Schmeichel AN, et al.: Peripheral nerve ischemia: reperfusion injury and fiber degeneration. Exp Neurol 2003, 184:997–1002.View ArticlePubMedGoogle Scholar
- Kihara M, Zollman PJ, Schmelzer JD, et al.: The influence of dose of microspheres on nerve blood flow, electrophysiology, and fiber degeneration of rat peripheral nerve. Muscle Nerve 1993, 16:1383–1389.View ArticlePubMedGoogle Scholar
- Zollman PJ, Awad O, Schmelzer JD, et al.: Effect of ischemia and reperfusion in vivo on energy metabolism of rat sciatic-tibial and caudal nerves. Exp Neurol 1991, 114:315–320.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.