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Treadmill Training Modifies KIF5B Motor Protein in the STZ-Induced Diabetic Rat Spinal Cord and Sciatic Nerve

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Treadmill Training Modifies KIF5B Motor Protein in the STZ-Induced Diabetic Rat Spinal Cord and Sciatic Nerve 7UDLQLQJDQG$[RQDO0RWRU3URWHLQLQ'LDEHWHV Original Article 7UHDGPLOO7UDLQLQJ0RGL¿HV.,)%0RWRU3URWHLQLQWKH STZ-induced Diabetic Rat Spinal Cord and Sciatic Nerve Masoud Rahmati PhD15H]D*KDUDNKDQORX3K'2, Mansoureh Movahedin PhD3, Seyed Javad Mowla PhD4, Ali Khazani PhD5, Mary- am Fouladvand MSc6, Shiva Jahani Golbar MSc7 Abstract Background:3UHYLRXVUHVHDUFKKDVGHPRQVWUDWHGGLDEHWLFLQGXFHGD[RQDOWUDQVSRUWGH¿FLWV+RZHYHUWKHPHFKDQLVPRID[RQDOWUDQV- SRUWLPSDLUPHQWLQGXFHGE\GLDEHWHVLVSRRUO\XQGHUVWRRG.LQHVLQPRWRUSURWHLQVKDYHEHHQVKRZQWRWUDQVSRUWYDULRXVFDUJRVDORQJKLJKO\ SRODUL]HGQHXURQV,QWKHSUHVHQWVWXG\ZHLQYHVWLJDWHGWKHHIIHFWRIUHJXODUWUHDGPLOOH[HUFLVHRQ.,)%DQG6XQGD\'ULYHU 6<' P51$ OHYHOVLQVHQVRU\DQGPRWRUSDUWVRIVSLQDOFRUGDQG.,)%FRQWHQWLQVFLDWLFQHUYHVRIVWUHSWR]RWRFLQ 67= LQGXFHGGLDEHWLFUDWV Methods: Forty male Wistar rats were divided into four groups: (1) diabetic trained (DT: n = 10); (2) Non-trained diabetic (NTD: n = 10); (3) QRUPDOFRQWURO 1&Q DQG QRUPDOWUDLQHG 17Q 7ZRZHHNVDIWHU67=LQMHFWLRQ PJNJLS WKHUDWVZHUHVXEMHFWHGWR WUHDGPLOOH[HUFLVHIRUGD\VDZHHNRYHUZHHNV:HGHWHUPLQHGP51$OHYHOVDQGSURWHLQFRQWHQWE\5HDOWLPH3&5DQG(/,6$ Results:([HUFLVHWUDLQLQJGHFUHDVHGEORRGJOXFRVHOHYHOVLQWKH'7UDWV'LDEHWHVLQFUHDVHGWKH.,)%DQG6<'P51$LQERWKVHQVRU\ DQGPRWRUSDUWVDQG.,)%FRQWHQWLQVFLDWLFQHUYHVLQWKH17'0RUHRYHUH[HUFLVHWUDLQLQJPRGXODWHGWKH.,)%DQG6<'P51$DQG .,)%FRQWHQWWRQRUPDOOHYHOVLQWKH'7([HUFLVHWUDLQLQJLQ17UDWVLQFUHDVHG.,)%DQG6<'P51$LQVHQVRU\DQGPRWRUSDUWVDQG .,)%FRQWHQWLQVFLDWLFQHUYHV Conclusions:2XUUHVXOWVVXJJHVWWKDWGLDEHWHVVHHPVWRFKDQJHVSLQDOFRUG.,)%DQG6<'P51$DQGVFLDWLFQHUYHV.,)%FRQWHQWDQG H[HUFLVHWUDLQLQJPRGL¿HVLWZKLFKPD\EHDWWULEXWDEOHWRWKHWUDLQLQJLQGXFHGGHFUHDVHGK\SHUJO\FHPLD Keywords: Diabetes, exercise, kinesin, sunday driver Cite this article as: Rahmati M, Gharakhanlou R, Movahedin M, Mowla SJ, Khazani A, Fouladvand M, Jahani Golbar S. Treadmill Training Modifies KIF5B Motor Protein in the STZ-induced Diabetic Rat Spinal Cord and Sciatic Nerve. Arch Iran Med. 2015; 94 – 101. Introduction regulatory microtubules protein that serve as rail roads, adaptors and scaffold proteins that regulate cargoes attachment to motor mong the most prevalent complication of diabetes, neu- SURWHLQVDQGPHWDEROLFPRGL¿FDWLRQWKDWGLVUXSWHQHUJ\VXSSO\ ropathy develops as an irreversible complication in more of motor proteins.6-12 Among different regulators of axonal mo- Athan half of with diabetes type 1 or 2.1 Diabetes is associ- tor proteins, SYD as a scaffold protein can interacts with kine- ated with slowing of motor nerve conduction velocity and reduced sin and mediates the axonal transport of at least three classes of muscle contractile properties.2,3 In contrast, on the sensory side, vesicles.11,12 It was shown that SYD interacts directly with the tail there is not only slowing of sensory nerve conduction velocity but domain of kinesin heavy chain and activates it for microtubule- also atrophy of perikarya and axons associated with down-regula- based transport.13 Among the 45 kinesin motor proteins that are tion of structural protein synthesis and loss of terminal epidermal involved in axonal and intracellular transport, in neuronal cells axons.4,5 KIF5B transport synaptic vesicle precursors, membrane organ- 6HYHUDOVWXGLHVKDYHGHPRQVWUDWHGWKDWD[RQDOWUDQVSRUWGH¿FLWV elles, and mitochondria.14,15 On the other hand, impairment of in many neurodegenerative diseases might be due to alterations anterograde axonal transport of some nerotrophic factors and syn- of molecular motor proteins that carry cargoes, structural and aptic proteins is demonstrated in neuronal cells of diabetic rats.16,17 Also, indirect evidence shows that axonal transport of mitochon- 18 $XWKRUV¶DI¿OLDWLRQV 1Department of Physical Education and Sport Sciences, dria may be decreased in diabetic neuropathy. )DFXOW\ RI /LWHUDWXUH +XPDQLWLHV /RUHVWDQ 8QLYHUVLW\ .KRUUDPƗEƗG ,UDQ Previous studies have shown that treadmill exercise training can 2Department of Physical Education and Sport Sciences, Faculty of Humanities, improve peripheral nervous tissue regeneration in non-diabetic Tarbiat Modares University, Tehran, Iran. 3Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 4Depart- rats and mice after nerve injury and in diabetic rats, improves ment of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, neuropathic pain and increases axonal regeneration after sciatic Tehran, Iran. 5Department of Physical Education and Sport Sciences, Faculty nerve transection.19–24 Exercise training is an interesting model of Humanities, Islamic Azad University, Ardabil, Iran. 6Young Researchers and Elite Club, Borujerd Branch, Islamic Azad University, Borujerd, Iran. 7Depart- with which increase activation of sensory and motor neurons, ax- ment of Physical Education and Sport Sciences, Tehran Kish International Uni- onal transport of proteins, and synaptic remodeling.25–28 We have versity, Tehran, Iran. previously demonstrated that the amount of CGRP anterogradely &RUUHVSRQGLQJDXWKRUDQGUHSULQWV Reza Gharakhanlou PhD, Department of Physical Education and Sport Sciences, Faculty of Humanities, Tarbiat Modares transported along axons by fast transport is increased in sciatic University, Tehran, Iran. Tel: +98-2182884646, Fax: +98-2182884646, E-mail: motoneurons of exercise-trained rats.26 These studies have proved [email protected]. Postal address: Tehran, Jallale Ale Ahmad, Tarbiat that exercise training increases the quantity of axonal proteins and Modares University, Faculty of Humanities, Department of Physical Education and Sport Sciences. axonal transport, but the effects of exercise training on motor pro- Accepted for publication: 31 December 2014 teins that transport these neurotrophic factors are not elucidated 94 Archives of Iranian Medicine, Volume 18, Number 2, February 2015 05DKPDWL5*KDUDNKDQORX00RYDKHGLQHWDO yet. Moreover, to our knowledge, no studies have been performed minutes of cool-down. Most animals ran for the majority of the to analyze the effect of diabetes on KIF5B motor proteins expres- exercise period without any encouragement. The animals did not sion in the sensory and motor neurons. In the present study, we appear excessively fatigued, which is an important consideration, investigated KIF5B changes in spinal cord and sciatic nerve fol- as exhausting exercise training may alter the levels of lactic acid lowing the induction of diabetes and treadmill training. and cause fatigue that may increase diabetic complications.32 Materials and Methods Tissue preparation Twenty four hours after the last training session, animals were Animal characteristics anesthetized with a mixture of Ketamine (75mg/kg-1) and Xyla- zine (5 mg/kg-1) administered intraperitoneally. The lumbar en- Experiments were performed on 300 to 320 g male Wistar rats largement (L4–L6) was isolated and sensory and motor (or frontal (Razi institute Animal Center, Karaj, Iran). The rats were housed and dorsal) parts were separated using the central canal as a refer- at a constant room temperature of 22 ± 2°C under a 12-h light/ ence and tissues were stored at -70°C until analyzed.33,34 dark cycle (lights on at 6:00 AM), with access to food and water ad libitum in the Animal Center of Tarbiat Modares University. Analysis of gene expression by Real-Time PCR The experimental protocols to perform this study were approved Motor and sensory neuron tissues were transferred to 1 mL lysis by the Ethics Committee on the use of animals of Tarbiat Modares reagent (Qiazol; Qiagen) and were homogenized (Tissue-Tearor; University, Tehran, Iran. All efforts were made to minimize dis- Biospec, Bartlesville, OK). RNA extraction was performed iden- comfort of the animals and reduce the number of experimental WLFDOO\IRUERWKWLVVXHVXVLQJDSXUL¿FDWLRQNLW 51HDV\0LQL.LW animals. All procedures conformed to the ethical guidelines for Qiagen) in accordance with the manufacturer’s instructions. Mo- the care and use of laboratory animals, published by the National tor and sensory parts mRNA was pooled by successively adding Institutes of Health. ethanol-precipitated RNA (RNeasy; Qiagen). Concentrations of RNA were determined by UV spectrophotometry (Eppendorff, Groups and design Germany). Total cDNA synthesis was performed using ABI The rats were randomly assigned into 4 groups: (1) diabetic PRISM cDNA arhive kit in 0.2 mL microfuge tubes. The reaction trained (DT), (2) diabetic control (DC), (3) healthy trained (HT), PL[WXUHRIȝ/FRQWDLQHGȝJWRWDO51$î57EXIIHU and (4) healthy control (HC). Two weeks after inducing diabetes, îG173PL[WXUHîUDQGRPSULPHUV0XOWL6FULEH57 8 6 weeks of endurance training protocol ensued. For the purpose ȝ/ DQG51DVHIUHHZDWHU7KHF'1$V\QWKHVLVUHDFWLRQVZHUH of familiarization, rats in all groups were adapted to the treadmill carried out for 20 minutes at 42°C, followed by denaturation for 5 by running for 5 days. This protocol was designed as once a day minutes at 95°C and 5 minutes at 4°C. for 10 min/session at a speed of 10 m/min at a slope of 0 degree. Real-time PCR assays were performed in 48-well plates in ABI 7300 real-time PCR instrument (Applied Biosystems). The prim- Induction of diabetes ers and probes were purchased from Applied Biosystems, Foster Following an overnight fast, diabetes was induced in the hyper- City, California, USA. The forward and reverse primers for glycemic groups by a single intraperitoneal (i.p.) injection of STZ .,)% JHQH ZHUH ƍ*$7*7$$$*&$$&&**$**** DQG 29,30 (45mg/kg; Sigma, St. Louis, MO). STZ solution was prepared ƍ7*77***$*$7$&*$$*&7** 7KH IRUZDUG
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