Journal of Neurological Sciences [Turkish] 27:(1)# 22; 035-042, 2010 http://www.jns.dergisi.org/text.php3?id=338. Research Article. Effect of Erythropoietin on ...
J.Neurol.Sci.[Turk]
Journal of Neurological Sciences [Turkish] 27:(1)# 22; 035-042, 2010 http://www.jns.dergisi.org/text.php3?id=338 Research Article Effect of Erythropoietin on Peripheral Nerve Regeneration Mustafa OZKAN1, Necati GOKMEN2, Osman YILMAZ3, Serhat ERBAYRAKTAR4, Alper BAGRIYANIK5 1
Dokuz Eylül Üniversity, Orthopaedics, izmir, Türkiye 2Dokuz Eylül Üniversity, Anesthesia and Reanimation, İzmir, Türkiye 3Dokuz Eylül Üniversity, Experimental Animal Laboratory, İzmir, Türkiye 4Dokuz Eylül Üniversity, Neurosurgery, İzmir, Türkiye 5Dokuz Eylül Üniversity, Histology and Embryology, İzmir, Türkiye Summary The aim of this study was to identify the effect of erythropoietin (EPO) on a sciatic nerve injury model. The effect of single or repeated doses was also determined. Twenty-one Wistar rats were anesthetised and the sciatic nerve was transected 1 cm above the trifurcation and the nerve was repaired with four epineural 10/0 nylon sutures placed at 90 degrees intervals under microscope magnification.The rats were divided into 4 groups as follows: the sham,the saline, the single dose EPO and the multiple dose EPO. The skin was incised and closed and no treatment was given in sham group. In the saline group, 1 mL saline was given intraperitoneally; in the single EPO group, 5000 U/kg EPO was given intraperitoneally immediately after the procedure. In the multiple EPO group, 5000 U/kg EPO was given after the procedure and the same dose was repeated after the 1st, 2nd, 3rd and 4th weeks. Functional recovery was evaluated by static sciatic functional index(SSI).Single EPO group had greater myofibril size, axon number, diameter, and ratio M than the saline group. The multiple EPO treatment was not found to be more effective than single EPO treatment. However, no significant difference was found between the single EPO, multiple EPO, and saline groups based on the 3rd and 4th postoperative month SSI scores. Thus, EPO treatment increased axonal regeneration in our study. However, repeated dose therapy was not found to be more effective than single dose therapy. The optimum dose and duration should be researched in further studies. Key words: Erythropoietin, nerve regeneration, peripheral nerve injury Periferik Sinir İyileşmesinde Eritropoietinin Etkisi Özet Bu çalışmanın amacı; siyatik sinir hasar modelinde eritropoietinin(EPO) etkisini araştırmaktır. Ayrıca tek ya da tekrarlayan eritropoietin dozlarının etkileri de araştırılmıştır. 21 wistar sıçanın siyatik siniri, anestezi sonrası trifürkasyonun 1 cm proksimalinden kesildi ve mikroskop altında, 10/0 naylon dikiş kullanılarak, 90 derece aralıklarla atılan dört adet epinöral dikiş ile tamir edildi. Sıçanlar dört gruba ayrıldı: sham, serum fizyolojik, tek doz EPO ve çoklu doz EPO. Sham grubunda cilt kesisi yapıldı ve kapatıldı. Herhangi bir tedavi verilmedi. Serum fizyolojik grubunda 1 ml serum fizyolojik intraperitoneal olarak verildi. Tek doz EPO grubunda işlem sonrası hemen 5000 U/kg EPO intraperitoneal olarak verildi. Çoklu doz EPO grubunda ise işlem sonrası 5000 U/kg EPO intraperitoneal olarak verildi ve aynı doz 1., 2., 3. ve 4. haftalarda tekrarlandı. Fonksiyonel değerlendirme Statik Siyatik Sinir fonksiyonel indeksi(SSI) ile yapıldı.
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Tek doz EPO grubunda myofibril boyutu, akson sayısı, çapı ve M oranı SF grubuna göre daha yüksek bulundu. Çoklu doz EPO tedavisinin tek doz EPO tedavisinden daha etkili olmadığı saptandı.Tek doz EPO, çoklu doz EPO ve serum fizyolojik grupları arasında, postoperatif 3. ve 4. aylardaki SSI skorlarında anlamlı fark saptanmadı. Çalışmamızda EPO tedavisinin aksonal iyileşmeyi arttırdığı saptanmıştır. Fakat çoklu doz tedavinin, tek doz tedaviden daha üstün olmadığı bulunmuştur. Optimal dozun ne olacağı ve ne kadar sürede kullanılacağı konusunda daha ileri çalışmalara ihtiyaç vardır. Anahtar Kelimeler: Eritropoietin, sinir iyileşmesi, periferik sinir hasarı Temperature was measured with the aid of a rectal probe, which was maintained 37.0 ± 0.5ºC during the entire study and was controlled under guidance of feedback to the heating lamp.
INTRODUCTION Erythropoietin (EPO) has been shown to have an effect on the peripheral nervous system. Recently the EPO receptor (EPOr) has been identified within the peripheral nervous system.(6). It has been demonstrated that endogenous mechanisms are insufficient to protect nerves after injury and exogenously administered recombinant human EPO (rhEPO) has beneficial effects to protect primary sensory neurons from apoptosis in several brain, spinal, and peripheral nerve injury models and inhibits pain in the spinal nerve crush injury model.(4,7,8,17,21) EPO expresses potent neuroprotective activity in the peripheral nervous system and Schwann cells also express EPOr, and its expression is increased after peripheral nerve injury.(7,17,21) However, knowledge about the administration of EPO or the proper doses of EPO is incomplete.
The left limbs of all animals were prepared and the sciatic nerve was transected 1 cm above the trifurcation.(Fig. 1a,b) The nerve was repaired with four epineural 10/0 nylon interrupted sutures placed at 90 degrees intervals under microscope magnification. The rats were divided into 4 groups as follows: 1) the sham group (SH; n=3), 2) the saline group (S; n=6), 3) the single dose EPO group (single EPO; n=6), and 4) the multiple dose EPO group (multiple EPO; n=6). Sham group was used as the control group to determine the borders of normal in study groups. The skin was incised and closed and no treatment was given in this group. In the S group, 1 ml saline was given intraperitoneally and in the singleEPO group, 5000 U/kg EPO was given intraperitoneally immediately after the procedure. In the multipleEPO group, 5000U/kg EPO was given after the procedure and the same dose was repeated after the 1st, 2nd, 3rd, and 4th weeks postoperatively. Functional and histologic assessments were also carried out.
The aim of this study was to identify the effect of EPO on a sciatic nerve injury model. In addition, the effects of single or repeated doses of EPO in the sciatic nerve injury model were also studied. MATERIAL AND METHODS The study protocol was approved by the Animal Research Committee. Twenty-one Wistar rats, weighing between 200 and 250 g, were used in this study. Animals were maintained on a 12-h light/dark cycle at a room temperature of 20ºC- 22ºC, with water and food freely available.
At the end of the 4th month, the sciatic nerve was harvested under deep barbiturate anesthesia using sodium pentobarbital injected intraperitoneally at a dose of 50 mg/kg. The sciatic nerves were removed 5 mm distal and 5 mm proximal to the lesion. The contralateral sciatic nerve was also removed and fixed with 10% formaldehyde. After routine histologic
Anesthesia was induced with 2-3% halothane in oxygen and was maintained with 1.5% halothane in 100% O2 delivered through a face mask during the experiment. 36
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processing and embedding in paraffin blocks, 4 mm cross-sections were made with a Leica/Reichert-Jung Rotary microtome (Leica, Köln, Germany), and then stained with hematoxylin eosin and toluidine blue. For morphometric analysis, the entire endoneurial area of each stained cross-section of a sciatic nerve was digitized at a magnification of 100 (immersion objective) using an Olympus BH-2 (Tokyo, Japan) fully motorized microscope and the images were transferred onto a computer using a color
video camera (JVC head model No:TK890E, Tokyo, Japan), video capture and appropriate preparations were also digitally photographed. The desired measurements were obtained using an UTHSCSA image tool for Windows, version 3.00. During morphometry, the following parameters were measured:(12) 1) mean number of axons per standard fascicle area (500 µm2), 2) mean axon diameter per standard fascicle area (500 µm2), and 3) ratio M (total axon area/total fascicle area).
Figure 1: a: The left limbs of all animals were prepared and the sciatic nerve was transected 1 cm above the trifurcation. Fiure 1b: The nerve was repaired with four epineural 10/0 nylon interrupted sutures placed at 90 degrees intervals under microscope magnification.
analysis software as developed by Wayne Rasband National Institutes of Health, Bethesda, MD, USA. At least 10 myofibrils from each animal were measured.
The tissues were fixed in 10% buffered formalin overnight and embedded in paraffin after routine tissue processing for quantification of myofibril size. Serial sections (5 µM) were cut for histologic staining. The area of the muscle tissue containing the most atrophic fibers on a representative slide from each animal was circled to ensure adequate and representative sampling for quantification. The mean muscle fiber area was measured using a computer-assisted image analyzer system consisting of a microscope with a 10x objective (Labophot –2; Nikon, Tokyo, Japan) equipped with a high resolution video camera (VKC220E, Hitachi, Tokyo, Japan). The images on the computer were analyzed with image
Evaluation of functional recovery: Functional recovery was assessed by walking track analysis.(2) Video analysis of the standing static sciatic functional index (SSI), as described by Bervar,(3) as calculated preoperatively, postoperatively, and repeated after the 3rd and 4th months postoperatively.(Fig 2) The following calculation was used for measuring SSI:(3) SSI = 108.44 toe spread factor (TSF) + 31.85 intermediary toe spread factor (ITF) - 5.49.
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Figure 2: Evaluation of functional recovery by measuring SSI (static sciatic functional index) scores. Animal is standing or periodic rest on a flat transparent surface. View from the bottom, the hind feet are contact with the transparent surface, A:normal feet B: injured left hind limb.
Statistical analysis was carried out using SPSS for Windows, version 11.0. Results are presented as the mean ± standard deviation. All variables were analyzed using the Kruskal Wallis test followed by the Mann–Whitney U test, when indicated. Within comparisons were done using the Friedman test, followed by the Wilcoxon test, when indicated. P values < 0.05 were considered significant.
diameter than S group but it was not statistically significant (p>0.05; Table 1). Ratio M is a ratio of total axon area to total fasicle area in each nerve stump. Axon number and diameter were calculated as mean number of axons and axon diameter per standard fascicle area. So Ratio M is considered as a controlling parameter. The singleEPO and multipleEPO groups had a greater ratio M than the S group (p0.05; Table 1).
RESULTS Morphometry: At the end of the 4th month, the number of axons per standard fascicle area was calculated and the singleEPO group had a statistically significant higher axon number than the S and multipleEPO groups (p0.05; Table 1).
Myofibril size: The multipleEPO group had greater myofibril size when compared with the S and singleEPO groups (p
