FK506和骨髓间充质干细胞治疗后肢异体移植大鼠

doi:10.3969/j.issn.1673-5374.2012.34.005

中国神经再生研究(英文版) ›› 2012, Vol. 7 ›› Issue (34): 2681-2688.doi: 10.3969/j.issn.1673-5374.2012.34.005

• 原著:周围神经损伤修复保护与再生 • 上一篇下一篇

FK506和骨髓间充质干细胞治疗后肢异体移植大鼠

河北省，承德市，承德医学院附属医院骨科，067000

收稿日期:2012-10-11 修回日期:2012-11-29 出版日期:2012-12-05 发布日期:2012-11-29
基金资助:
国家自然科学基金项目 30801171;河北省自然科学基金项目C2009001013， H2012406015.

Use of FK506 and bone marrow mesenchymal stem cells for rat hind limb allografts

Youxin Song¹, Zhujun Wang², Zhixue Wang³, Hong Zhang⁴, Xiaohui Li², Bin Chen¹

Department of Orthopedics, Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, China

Received:2012-10-11 Revised:2012-11-29 Online:2012-12-05 Published:2012-11-29
Contact: Bin Chen, M.D., Department of Orthopedics, Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, Chinadrchenbin@yahoo.cn
About author:Youxin Song☆, M.D., Department of Orthopedics, Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, China
Supported by:
This work was supported by the National Natural Science Foundation of China, No. 30801171; the Natural Science Foundation of Hebei Province, No. C2009001013 and No. H2012406015.

摘要/Abstract

摘要：

为探讨骨髓间充质干细胞在免疫宽容环境中对神经再生和肢体功能恢复的影响，实验将Dark Agouti大鼠后肢异体移植至Lewis大鼠上，每天肌肉注射FK506，同时移植骨髓间充质干细胞至损伤神经。结果显示异体移植后未进行治疗大鼠在移植后10d移植肢体坏死和脱落。而FK506和骨髓间充质干细胞联合治疗后12个月则使坐骨神经中髓鞘轴突和许旺细胞的数量增多，腓肠肌肌肉变性等病理变化减轻，后肢异体移植大鼠后肢感觉明显恢复，坐骨神经功能指数明显增加，后肢肌肉质量增加，且治疗效果优于自体移植和FK506单独治疗。说明FK506改善了细胞抑制的免疫微环境，FK506联合骨髓间充质干细胞治疗能明显促进损伤坐骨神经再生，改善异体移植后肢感觉和运动功能的恢复。

关键词: FK506, 骨髓间充质干细胞, 异体移植, 后肢移植, 功能恢复, 感觉功能, 运动功能, 周围神经损伤, 再生, 神经再生

Abstract:

Dark Agouti rat donor hind limbs were orthotopically transplanted into Lewis rat recipients to verify the effects of bone marrow mesenchymal stem cells on neural regeneration and functional recovery of allotransplanted limbs in the microenvironment of immunotolerance. bone marrow mesenchymal stem cells were intramuscularly (gluteus maximus) injected with FK506 (tacrolimus) daily, and were transplanted to the injured nerves. Results indicated that the allograft group not receiving therapy showed severe rejection, with transplanted limbs detaching at 10 days after transplantation with complete necrosis. The number of myelinated axons and Schwann cells in the FK506 and FK506 + bone marrow mesenchymal stem cells groups were significantly increased. We observed a lesser degree of gastrocnemius muscle degeneration, and increased polymorphic fibers along with other pathological changes in the FK506 + bone marrow mesenchymal stem cells group. The FK506 + bone marrow mesenchymal stem cells group showed significantly better recovery than the autograft and FK506 groups. The results demonstrated that FK506 improved the immune microenvironment. FK506 combined with bone marrow mesenchymal stem cells significantly promoted sciatic nerve regeneration, and improved sensory recovery and motor function in hind limb allotransplant.

Key words: FK506 (tacrolimus), bone marrow mesenchymal stem cells, allotransplant, hind limb transplant, function recovery, sensory function, motor function, peripheral nerve injury, regeneration, neural regeneration

. FK506和骨髓间充质干细胞治疗后肢异体移植大鼠[J]. 中国神经再生研究(英文版), 2012, 7(34): 2681-2688.

Youxin Song, Zhujun Wang, Zhixue Wang, Hong Zhang, Xiaohui Li, Bin Chen. Use of FK506 and bone marrow mesenchymal stem cells for rat hind limb allografts[J]. Neural Regeneration Research, 2012, 7(34): 2681-2688.

参考文献

[1] Jones JW, Gruber SA, Barker JH, et al. Successful hand transplantation. One-year follow-up. Louisville Hand Transplant Team. N Engl J Med. 2000;343(7):468-473.

[2] Jones NF. Concerns about human hand transplantation in the 21st century. J Hand Surg Am. 2002;27(5):771-787.

[3] Dubernard JM, Owen E, Herzberg G, et al. Human hand allograft: report on first 6 months. Lancet. 1999; 353(9161):1315-1320.

[4] Vögelin E. Hand transplantation-fiction or reality? Ther Umsch. 2011;68(12):730-734.

[5] Kaufman CL, Ouseph R, Blair B, et al. Graft vasculopathy in clinical hand transplantation. Am J Transplant. 2012; 12(4):1004-1016.

[6] Arai K, Hotokebuchi T, Miyahara H, et al. Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival. Transplantation. 1989; 48(5):782-786.

[7] Ishikane S, Ohnishi S, Yamahara K, et al. Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia. Stem Cells. 2008;26(10):2625-2633.

[8] Huang WC, Lin JY, Wallace CG, et al. Vascularized bone grafts within composite tissue allotransplants can autocreate tolerance through mixed chimerism with partial myeloablative conditioning: an experimental study in rats. Plast Reconstr Surg. 2010;125(4):1095-1103.

[9] Muramatsu K, Kuriyama R, Kato H, et al. Prolonged survival of experimental extremity allografts: a new protocol with total body irradiation, granulocyte-colony stimulation factor, and FK506. J Orthop Res. 2010;28(4): 457-461.

[10] Muramatsu K, Moriya A, Hashimoto T, et al. Immunomodulatory effects of pre-irradiated extremity allograft in the rodent model. J Plast Reconstr Aesthet Surg. 2012;65(7):950-955.

[11] Goto T, Kino T, Hatanaka H, et al. Discovery of FK-506, a novel immunosuppressant isolated from Streptomyces tsukubaensis. Transplant Proc. 1987;19(5 Suppl 6):4-8.

[12] Tocci MJ, Matkovich DA, Collier KA, et al. The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. J Immunol. 1989;143(2): 718-726.

[13] Song YX, Muramatsu K, Kurokawa Y, et al. Functional recovery of rat hind-limb allografts. J Reconstr Microsurg. 2005;21(7):471-476.

[14] Ide C, Nakai Y, Nakano N, et al. Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat. Brain Res. 2010;1332:32-47.

[15] Mata M, Alessi D, Fink DJ. S100 is preferentially distributed in myelin-forming Schwann cells. J Neurocytol. 1990;19(3):432-442.

[16] Paik SK, Lee DS, Kim JY, et al. Quantitative ultrastructural analysis of the neurofilament 200-positive axons in the rat dental pulp. J Endod. 2010;36(10):1638-1642.

[17] Doolabh VB, Mackinnon SE. FK506 accelerates functional recovery following nerve grafting in a rat model. Plast Reconstr Surg. 1999;103(7):1928-1936.

[18] Jensen JN, Brenner MJ, Tung TH, et al. Effect of FK506 on peripheral nerve regeneration through long grafts in inbred swine. Ann Plast Surg. 2005;54(4):420-427.

[19] Rustemeyer J, van de Wal R, Keipert C, et al. Administration of low-dose FK 506 accelerates histomorphometric regeneration and functional outcomes after allograft nerve repair in a rat model. J Craniomaxillofac Surg. 2010;38(2):134-140.

[20] Toll EC, Seifalian AM, Birchall MA. The role of immunophilin ligands in nerve regeneration. Regen Med. 2011;6(5):635-652.

[21] Yan Y, Sun HH, Mackinnon SE, et al. Evaluation of peripheral nerve regeneration via in vivo serial transcutaneous imaging using transgenic Thy1-YFP mice. Exp Neurol. 2011;232(1):7-14.

[22] Gold BG. FK506 and the role of immunophilins in nerve regeneration. Mol Neurobiol. 1997;15(3):285-306.

[23] Yang X, Ewald ER, Huo Y, et al. Glucocorticoid-induced loss of DNA methylation in non-neuronal cells and potential involvement of DNMT1 in epigenetic regulation of Fkbp5. Biochem Biophys Res Commun. 2012;420(3): 570-575.

[24] Lyons WE, Steiner JP, Snyder SH, et al. Neuronal regeneration enhances the expression of the immunophilin FKBP-12. J Neurosci. 1995;15(4): 2985-2994.

[25] Pereira U, Boulais N, Lebonvallet N, et al. Mechanisms of the sensory effects of tacrolimus on the skin. Br J Dermatol. 2010;163(1):70-77.

[26] Szydlowska K, Gozdz A, Dabrowski M, et al. Prolonged activation of ERK triggers glutamate-induced apoptosis of astrocytes: neuroprotective effect of FK506. J Neurochem. 2010;113(4):904-918.

[27] Shin HJ, Jeon BT, Kim J, et al. Effect of the calcineurin inhibitor FK506 on K+-Cl- cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus. J Neural Transm. 2012;119(6):669-677.

[28] Grand AG, Myckatyn TM, Mackinnon SE, et al. Axonal regeneration after cold preservation of nerve allografts and immunosuppression with tacrolimus in mice. J Neurosurg. 2002;96(5):924-932.

[29] Konofaos P, Burns J, Terzis JK. Effect of low-dose FK506 after contralateral C7 transfer to the musculocutaneous nerve: a study in rats. J Reconstr Microsurg. 2010;26(4): 225-233.

[30] Navarro X, Udina E, Ceballos D, et al. Effects of FK506 on nerve regeneration and reinnervation after graft or tube repair of long nerve gaps. Muscle Nerve. 2001;24(7): 905-915.

[31] Yeh C, Bowers D, Hadlock TA. Effect of FK506 on functional recovery after facial nerve injury in the rat. Arch Facial Plast Surg. 2007;9(5):333-339.

[32] Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 2001;282(1): 148-152.

[33] Akiyama Y, Radtke C, Kocsis JD. Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci. 2002;22(15):6623-6630.

[34] Koopmans G, Hasse B, Sinis N. Chapter 19: The role of collagen in peripheral nerve repair. Int Rev Neurobiol. 2009;87:363-379.

[35] Yang Y, Yuan X, Ding F, et al. Repair of rat sciatic nerve gap by a silk fibroin-based scaffold added with bone marrow mesenchymal stem cells. Tissue Eng Part A. 2011;17(17-18):2231-2244.

[36] Seth R, Revenaugh PC, Kaltenbach JA, et al. Facial nerve neurorrhaphy and the effects of glucocorticoids in a rat model. Otolaryngol Head Neck Surg. 2012;147(5): 832-840.

[37] Shichinohe H, Kuroda S, Maruichi K, et al. Bone marrow stromal cells and bone marrow-derived mononuclear cells: which are suitable as cell source of transplantation for mice infarct brain? Neuropathology. 2010;30(2):113-122.

[38] Thompson DM, Meloche M, Ao Z, et al. Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy. Transplantation. 2011;91(3):373-378.

[39] Rustemeyer J, Dicke U. Allografting combined with systemic FK506 produces greater functional recovery than conduit implantation in a rat model of sciatic nerve injury. J Reconstr Microsurg. 2010;26(2):123-129.

[40] Azizi S, Mohammadi R, Amini K, et al. Effects of topically administered FK506 on sciatic nerve regeneration and reinnervation after vein graft repair of short nerve gaps. Neurosurg Focus. 2012;32(5):E5.

[41] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[42] Chen B, Song Y, Liu Z. Promotion of nerve regeneration in peripheral nerve by short-course FK506 after end-to-side neurorrhaphy. J Surg Res. 2009;152(2):303-310.

[43] Bain JR, Mackinnon SE, Hudson AR, et al. The peripheral nerve allograft: a dose-response curve in the rat immunosuppressed with cyclosporin A. Plast Reconstr Surg. 1988;82(3):447-457.

[44] de Medinaceli L, Freed WJ, Wyatt RJ. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol. 1982;77(3):634-643.

引言

材料方法

Design

A randomized, controlled animal study.

Time and setting

Experiments were performed at the Affiliated Hospital of Chengde Medical College in China from October 2010 to November 2011.

Materials

Animals

Genetically inbred rats with the major histocompatibility complex were used. Graft donors were adult Dark Agouti rats (genetic expression, RT1a; Seiwa Experimental Animals, Beijing, China; SCXK 2006-0009). Rats were specific pathogen free, male, aged 12 weeks old, and weighed 200–250 g. Recipients were 12 weeks old, male Lewis rats (genetic expression, RT1l; Seiwa Experimental Animals, Beijing, China, SPF level, SCXK 2006-0009), and weighed 200–250 g. Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[41].

Drugs

FK506 (purity 98.7%, powder, used for animals, Shengtianyu Company, Wuhan, China) was dissolved in saline and kept in a refrigerator at 4°C.

Methods

Culture of BMSCs

After sacrifice with ketamine, BMSCs were obtained from the thigh bone of Lewis rats (n = 5). The marrow suspension was seeded on 100-mm dishes in Dulbecco’s modified Eagle’s medium-high glucose (Gibco, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (Sigma, St Louis, MO, USA). Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium was replaced every 4 days.

Model establishment of rat hind limb transplantation

The rat hind limb transplantation model described by Muramatsu et al ^[10] was used. Rats were anesthetized with a ketamine/xylazine cocktail and operated upon aseptically. The donor’s right hindlimb, including bone, muscle, femoral vessels, sciatic nerve and skin, was amputated at the midfemoral level. A similar amputation was made in the recipient rat. After the donor’s hind limb was orthotopically transplanted into the recipient, osteosynthesis was performed, using an 18-gauge needle as an intramedullary rod. The donor femoral artery and vein were anastomosed end-to-end to the recipient’s femoral artery and vein with 10-0 nylon using standard microsurgical techniques, whereas the sciatic nerve was sutured epineurally with 8-0 nylon. Thigh muscles were approximated using 4-0 nylon sutures, and the skin envelope of the recipient rats was used to cover the denuded thigh and lower leg of the donor limb. For autografts, the right hindlimb of Lewis rats was elevated, similarly amputated, and returned to the same site, and the femoral vessels and sciatic nerve were repaired in a similar manner (Figure 8). Recipient rats were inspected daily for one year. They were housed in a plastic cage where they could walk freely.

FK506 therapy and BMSC transplantation
After surgery, the allograft groups were treated with intramuscular (gluteus maximus) injection of FK506 at a dose of 1 mg/kg per day[10, 13] to prevent rejection until sacrificed (12 months).

BMSC suspension (1 × 107/µL; 10 µL) was injected into the injured nerve. The distal end of the injured nerve was injected (5 mm distance from the injured nerve point) at four points, the needle was maintained in every point for 1 minute and removed slowly (Figure 9).

Cutaneous pain reaction testing for the recovery of sensory function in rats

At 1, 3, 6, 9, and 12 months after surgery, modified pain reaction tests^{[13, 42]} were performed in a quiet room. The recipient was held in one hand with its hind limbs hanging downward. After the rat became quiet and stopped struggling, the transplanted hind limb was pinched with a towel clip on the middle of the foot. This procedure was repeated five times. The grades of pain reaction were described according to the following: grade 0, no reaction to pinch; grade 1, little reaction to pinch, with a very slow reaction speed without violent struggling; grade 2, strong pain reaction to pinch stimulation with a reaction speed faster than grade 1, and violent flouncing when pinch was performed; and grade 3,with faster and more sensitive pain reaction than grade 2 (normal)^{[13, 42]}.

Walking track analysis of rat sciatic nerve function

The sciatic nerve functional recovery was assessed using the sciatic function index. The procedure was repeated when an unsatisfactory result was obtained. Bain et al ^[43] and De Medinaceli et al ^[44] have described a detailed method, but some steps were modified in the present experiment. Walking tracks were obtained and were 8.2 cm × 42 cm, made of cardboard with walls and darkened at one end. Paper was put at the bottom of the track to act as film. The model rat’s hind limb was painted with black ink and allowed to walk down the track. On each piece of paper, several footprints could be seen. Footprint was measured for length (PL), the distance from the first to the fifth toe (TS) and the distance from the second to the fourth toe (IT). These measurements were performed for both the experimental and normal sides and coded with the prefixes E for experimental and N for the normal. The data (NPL, EPL, NSF, ESF, NIT and EIT) were fed into the computer. The following formula was derived by Bain et al^[43], who calculated the sciatic function index as follows:

Sciatic function index = –38.3 (EPL – NPL)/NPL + 109.5 (ETS – NTS)/NTS + 13.3 (EIT – NIT)/NIT – 8.8

A score of “0” using these indices indicated normality, whereas a score of “–100” corresponded to total loss of function.

Wet weight of rat gastrocnemius muscle

After an overdose of pentobarbital anesthesia (120 mg/kg), animals were perfused through the aorta with PBS (pH 7.6), followed by 300–500 mL of fixative composed of 4% paraformaldehyde in PBS (pH 7.4). Wet weight of gastrocnemius muscle was recorded for each group at 12 months after surgery. Gastrocnemius muscle was harvested without the tendon and gently blotted with absorbent paper to remove any blood or serum, and promptly weighed. The results were expressed as a percentage of the normal contralateral muscle.

Immunofluorescence staining to determine the number of myelinated axons and Schwann cells in sciatic nerve of allograft rats

After rats were sacrificed with a fatal dose of pentobarbital sodium (2%) injected in the abdominal cavity, rats were fixed in the dorsal position and their sciatic nerves taken. Nerve tissues were postfixed in 4% glutaraldehyde in 0.1 M PBS at pH 7.4 for 12 hours at room temperature. After that, each sample was immersed in graded sucrose solution (from 15% to 30% in 0.1 M PBS) for 12 hours and kept in a refrigerator at 4°C.

After this embedding, a series of 5 µm thick transected serial sections were cut on a cryostat microtome (Frigocut, Germany) at –20°C. The sections were for immunochemistry (dried in room temperature for 24 hours), and were fixed with freezing acetone for 20 minutes, then washed with 0.01 M PBS containing 0.3% Triton X-100 three times, 5 minutes each time. Subsequently, sections were incubated in 10% normal goat serum for 1 hour at room temperature and incubated with rabbit anti-S100 polyclonal (Chemicon) and mouse anti-rat neurofilament 200 monoclonal antibodies (Sigma) at room temperature (1:200) in 0.01 M PBS for over 8 hours in a humidity chamber. Sections were washed three times with PBS and 0.3% Triton X-100, whereupon they were incubated in fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (1:200; Sigma) and Cy3-conjugated rabbit anti-mouse IgG (1:200; Sigma) in PBS for 1 hour at room temperature. After the same washing procedure, sections were cover slipped. The number of Schwann cells (S-100 positive cells^[15]) and myelinated axons (neurofilament 200 positive expression^[16]) in sciatic nerve tissue samples from limbs of transplanted rats was observed using fluorescence microscopy (Nikon, Japan, × 400).

Hematoxylin-eosin staining of changes in rat gastrocnemius muscle appearance

The gastrocnemius muscle tissues of rats were taken and fixed in 10% buffered formalin. Transected serial sections (5 µm) were cut and stained with hematoxylin and eosin for light microscopy (Nikon, Japan).

FK506和骨髓间充质干细胞治疗后肢异体移植大鼠

Use of FK506 and bone marrow mesenchymal stem cells for rat hind limb allografts

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