Treadmill training stimulates brain-derived neurotrophic factor mRNA expression in motor neurons of the lumbar spinal cord in spinally transected rats

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<ul><li><p>TREADMILL TRAINING STIMULATES BNEUROTROPHIC FACTOR mRNA EXPR</p><p>SP</p><p>more motor neurons that contained BDNF mRNA labeling</p><p>within processes in the ST rats that received the higher</p><p>ticity, neurotrophins, locomotion.</p><p>(Keeler et al., 2012). In this study, passive cycling ofthe hindlimbs was sucient to raise BDNF mRNAlevels in motor neurons, raising the possibility thatactivity during treadmill training would have a similareect. In addition to motor neurons, other cells in the</p><p>State University Drive, Los Angeles, CA 90032-8162, United States.Tel: +1-323-343-4855; fax: +1-323-343-6024.</p><p>E-mail address: (R. D. de Leon).Abbreviations: BDNF, brain-derived neurotrophic factor; DIG,Digoxigenin; HSP27, heat shock protein 27; ISH, in situ hybridization;mRNA, messenger ribonucleic acid; ST, spinally transected; ST100,</p><p>Neuroscience 224 (2012) 135144of spinal neurons to BDNF levels in the lumbar spinalcord, it was necessary to demonstrate that synthesis ofBDNF mRNA occurred in spinal neurons. Only onestudy to date has examined cellular expression ofBDNF mRNA expression in spinal cord-injured animals</p><p>*Corresponding author. Address: Department of Kinesiology andNutritional Science, California State University, Los Angeles, 5151amount of treadmill training. These ndings suggested that</p><p>motor neurons and other ventral horn cells in ST rats syn-</p><p>thesized BDNF in response to treadmill training. The nd-</p><p>ings support a mechanism by which postsynaptic release</p><p>of BDNF from motor neurons contributed to synaptic plas-</p><p>ticity. 2012 IBRO. Published by Elsevier Ltd. All rightsreserved.</p><p>Key words: body weight-supported treadmill training, plas-OF THE LUMBAR SPINAL CORD IN</p><p>M. S. JOSEPH, a N. J. K. TILLAKARATNE b ANDR. D. DE LEON c*aDepartment of Biological Science, California State University, Los</p><p>Angeles, CA, United States</p><p>bDepartment of Integrative Biology and Physiology, Brain</p><p>Research Institute, University of California, Los Angeles,</p><p>CA, United StatescSchool of Kinesiology and Nutritional Science, California</p><p>State University, Los Angeles, CA, United States</p><p>AbstractBrain-derived neurotrophic factor (BDNF)</p><p>induces plasticity within the lumbar spinal circuits thereby</p><p>improving locomotor recovery in spinal cord-injured</p><p>animals. We examined whether lumbar spinal cord motor</p><p>neurons and other ventral horn cells of spinally transected</p><p>(ST) rats were stimulated to produce BDNF mRNA in</p><p>response to treadmill training. Rats received complete</p><p>spinal cord transections as neonates (n= 20) and one</p><p>month later, received four weeks of either a low (100 steps/</p><p>training session; n= 10) or high (1000 steps/training ses-</p><p>sion; n= 10) amount of robotic-assisted treadmill training.</p><p>Using combined non-radioactive in situ hybridization and</p><p>immunohistochemical techniques, we found BDNF mRNA</p><p>expression in heat shock protein 27-labeled motor neurons</p><p>and in non-motor neuron cells was greater after 1000</p><p>steps/training session compared to the 100 steps/training</p><p>session and was similar to BDNF mRNA labeling in</p><p>untrained Intact rats. In addition, there were signicantly0306-4522/12 $36.00 2012 IBRO. Published by Elsevier Ltd. All rights reserve</p><p>spinally transected + treadmill training for 100 steps/session; ST1000,spinally transected + treadmill training for 1000 steps/session.</p><p>135RAIN-DERIVEDESSION IN MOTOR NEURONSINALLY TRANSECTED RATS</p><p>INTRODUCTION</p><p>The neurotrophin, brain-derived neurotrophic factor(BDNF), improves the ability of the lumbar spinal cord togenerate locomotion in spinal cord-injured animals(Boyce et al., 2007, 2012; Ying et al., 2008). Themechanisms are unknown, but it is believed that BDNFtriggers changes in the spinal circuits by raising theexcitability of spinal neurons (Boyce et al., 2012) andstrengthening synaptic connections within the circuitry(Ying et al., 2005). The benecial eects can beproduced by the delivery of exogenous BDNF to theinjured spinal cord (Boyce et al., 2012) but BDNF levelsare also increased by hindlimb exercise (Hutchinsonet al., 2004; Ying et al., 2005; Macias et al., 2009;Sandrow-Feinberg et al., 2009; Cote et al., 2011; deLeon et al., 2011). Treadmill training in particular iseective in raising BDNF levels (Hutchinson et al., 2004).Thus, stimulating the production of endogenous BDNFpromotes plasticity within the spinal circuits and leads toimproved locomotor recovery after spinal cord injury.</p><p>Interestingly, the cells that synthesize and releaseBDNF in spinal cord-injured animals are unknown. Weand others have shown that treadmill training increasedBDNF protein expression in motor neurons of spinallytransected (ST) rats (Macias et al., 2009; de Leonet al., 2011). These ndings suggest that BDNF wassecreted by motor neurons in response to treadmilltraining. However, it was not clear whether the motorneurons themselves synthesized BDNF or if the BDNFwas derived from other cells such as muscle bers(Gomez-Pinilla et al., 2002). Large increases in BDNFmRNA levels in hindlimb muscles were stimulated by asingle bout of hindlimb exercise in ST rats (Dupont-Versteegden et al., 2004), suggesting target hindlimbmuscles synthesized BDNF then retrogradelytransported BDNF to motor neurons (Koliatsos et al.,1993). In order to understand the possible contributionventral horn, e.g. interneurons, glial cells, may produce</p><p>d.</p></li><li><p>of BDNF mRNA. We used in situ hybridization (ISH) andimmunohistochemical techniques (Tillakaratne et al.,</p><p>EXPERIMENTAL PROCEDURES</p><p>roscienExperimental design</p><p>Twenty female SpragueDawley rats received a complete mid-thoracic spinal transection at ve days of age. After weaning(21 days old), a robotic device was used to assess the ability ofthe rats to perform hindlimb stepping on a treadmill. The ratswere distributed into two experimental groups that werebalanced according to their locomotor performance during thebaseline tests. One group (n= 10) received daily treadmilltraining that consisted of 100 steps/session while the othergroup (n= 10) performed 1000 steps/training session andthese rats will be referred to as the spinally transected +treadmill training for 100 steps/session (ST100) and spinallytransected + treadmill training for 1000 steps/session (ST1000)rats respectively. Training was performed ve days/week forfour weeks. The animals were perfused with 4%paraformaldehyde, the spinal cords were removed andprocessed for the histology, ISH and immunohistochemicalexperiments. All procedures with rats were carried out inaccordance with NIH guidelines and the protocols wereapproved by the Institutional Animal Care and Use Committeeat California State University, Los Angeles.</p><p>Spinal cord transection</p><p>The spinal cords of the rats were transected at a mid-thoraciclevel as previously described (Cha et al., 2007). Briey, thepups were anesthetized using isourane (1%). A dorsal mid-line skin incision was made over the mid-thoracic vertebra and2002) to study the expression of BDNF mRNA in heatshock protein 27 (HSP27)-labeled motor neurons andother ventral horn cells in rats that received a completemid-thoracic spinal cord transection at ve days of age.Previous studies have shown that BDNF mRNA istargeted to dendrites of postsynaptic neurons and locallytranslated into protein (An et al., 2008; Chiaruttini et al.,2009). Thus, we included an analysis of BDNF mRNA inmotor neuron processes. A robotic treadmill system wasused to train the ST rats to perform either 100 or 1000steps/training session and the kinematic data from thisstudy have previously been reported (Cha et al., 2007).Here, we hypothesize that imposing a higher amount oftreadmill training would result in a greater synthesis ofBDNF by motor neurons and aect its subcellularlocalization. The ndings were consistent with thishypothesis and support a mechanism in whichpostsynaptic release of BDNF from motor neuronscontributed to plasticity within the lumbar spinal circuitscontrolling locomotion.BDNF that inuenced enhanced plasticity, but theircontribution to the BDNF pool has not yet beenexamined in exercised spinal cord-injured animals.</p><p>In the present study, we examined whether treadmilltraining in ST rats inuenced motor neuronal expression</p><p>136 M. S. Joseph et al. / Neuthe overlying fascia and muscles were retracted to expose thedorsal surface of the vertebrae. A partial laminectomy wasperformed at the mid-thoracic level to expose the spinal cord.The spinal cord was then lifted with a curved probe andcompletely transected. Afterwards, the skin incision was closedwith sutures. The entire surgical procedure took about 1015 min.</p><p>Following surgery, the rats were allowed to recover in a warmincubator. The temperature was maintained at 37 C. Theneonatal pups were placed in the incubator until fully alert (1030 min) and then returned to the mothers. After the rats wereweaned (21 days old), the rats were housed in spacious cages,23 rats per cage. The bladders and colons of the rats werechecked daily.</p><p>Robotic-assisted treadmill training</p><p>A commercially available robotic device (Rodent Robot 3000,Robomedica Inc.) was used to train treadmill stepping in therats. It consisted of two robotic arms that were attached to theankles of the rat, a motorized body weight support system anda treadmill (Cha et al., 2007). A thin, padded strip of neoprenewas placed around the rats ankle. A metal clip at the end ofthe robotic arm held the two ends of the neoprene striptogether to form a loop around the ankle. A soft vest wasplaced over the shoulders of the rat and was attached to amechanical arm, which raised the rats body above thetreadmill and controlled the amount of weight exerted on thehindlimbs. The robotic device was used to count the number ofsteps performed by the rats as previously described (Heng andde Leon, 2009). Briey, a step was detected whenever therobotic arm was displaced by 1 mm in the horizontal direction.A training session was completed when the total number ofsteps performed by both hindlimbs was 100 or 1000 steps.</p><p>Tissue preparation</p><p>Beginning two hours after the last training session, the rats wereanesthetized with isourane (1%) followed by intracardiacperfusion with 4% paraformaldehyde in Sorensen phosphatebuer (Tillakaratne et al., 2002). The spinal cord was dissectedand was post-xed in 30% sucrose solution for a period of 4872 h for cryoprotection and then embedded in Tissue-Tekcompound, as spinal cord blocks. Transverse sections (18-lmthick) of the spinal cords were cut using a cryostat andcollected as free-oating sections in PBS. After washing inPBS, adjacent sections were processed for ISH andimmunohistochemistry. The tissue sections used to comparethe experimental groups were processed simultaneously. Tominimize tissue damage that may occur with tissue handling,free-oating sections were processed in net-wells (75-lmmesh; Costar, Cambridge, MA). Spinal cord sections in net-wells were transferred sequentially to net-well trays containingappropriate solutions. Incubation with cRNA probes, antibodies,and ribonuclease A (Sigma, St. Louis, MO) and color reactionswere performed in 24-well plates.</p><p>Non-radioactive ISH and immunohistochemistry</p><p>In order to localize BDNF mRNA in the spinal cord, ISH usingDigoxigenin (DIG)-labeled RNA probes was performed. DIG-labeled BDNF riboprobe was prepared from a cDNA templatecoding for the full-length rat BDNF gene (kindly provided by Dr.Amelia Russo-Neustadt). A pBluescript KS 700 bp fragment,linearized with XbaI, and transcribed with T3 RNA polymerase,generated the anti-sense cRNA. Linearization with HindIII andtranscription with T7 RNA polymerase generated the sensecRNA. For non-radioactive ISH, a mix of unlabeled and DIGlabeled Uracil (Roche Applied Sciences, Indianapolis, IN) was</p><p>ce 224 (2012) 135144used as described previously (Tillakaratne et al., 2002). Theconcentration of DIG-labeled ribo-probe was then quantiedusing known amounts of a control labeled probe provided in theRNA detection kit (Roche Applied Sciences, Indianapolis, IN).</p></li><li><p>containing the secondary antibody (anti rabbit IgG, conjugatedwith Fluorescein-isothiocynate FITC, 1:500 (Jackson</p><p>correlation (r) was calculated. All statistical analyses were</p><p>from the motor neuron cell body (see arrowheads in</p><p>roscienImmunoResearch Lab, West Grove, PA) diluted in PBS andincubated for one hour at room temperature. Following theincubation, sections were again washed in PBS three times andmounted on microscope slides and cover-slipped with Vectashieldmounting media with DAPI (Vector Laboratories, Burlingame, CA)for visualization and protection from photo bleaching.</p><p>Image analysis</p><p>A semi-quantitative analysis was performed to measure BDNFmRNA in motor neurons and other surrounding cells in theventral horn. Three-ve spinal cord sections from each animalwere analyzed. Microscopic images were acquired underuniform conditions for all spinal cord sections using C-Imagingsoftware (Compix Inc., PA) under Leica DMLA microscopeequipped with a Hamamatsu Digital color camera. A region ofinterest (ROI) was drawn around the ventral horn using thedorsal edge of the central canal as the vertical border. Objects(cells) with BDNF mRNA label (red) and/or HSP27 label(green) were identied based on intensity values relative to athreshold level. Motor neurons were dierentiated from non-motor neuron objects based on size of the object and HSP27label. After background labeling was subtracted from theimages, the mean red (intensity per pixel) value correspondingto BDNF mRNA label was subsequently measured within eachidentied motor neuron and non-motor neuron object. Motorneurons with processes expressing BDNF mRNA wereidentied by carefully inspecting the HSP27-positive processesemanating from the soma. A motor neuron process wasconsidered to contain BDNF mRNA only if BDNF mRNA label(red) could be unambiguously detected within the outline of theHSP27-positive (green) process. For each rat, the total numberof motor neurons (Fig. 3A), total number of non-motor neuronobjects (Fig. 5A) and total number of motor neurons with BDNFmRNA labeled processes were calculated by summing theFollowing the quantication of the probe, the proper workingconcentration was optimized for the spinal cord tissues. The rathippocampus was used as a positive control.</p><p>Five spinal cord sections from each rat representing lumbarsegments L2L5 were selected for ISH. Each hybridization wellcontained 20 lg labeled probe/100 ll hybridization solutionconsisting of 50% dextran sulfate, 250 lg/ll salmon spermDNA, 50% formamide, 5 hybridization salt 1 Denhardtssolution, and DEPC water as described before (Tillakaratneet al., 2002). Hybridization was carried out overnight in a 52 Cincubator. Antibody against dig conjugated to a peroxidase(anti-DIG-POD) (Roche Applied Scientic, Indianapolis, IN) wasused to detect DIG-labeled hybrids. Next, the Tyramide SignalAmplication (TSA) assay (PerkinElmer, Waltham, MA) withCyanin 3 was used to amplify the uorescent signal in ISH.</p><p>Following the BDNF mRNA labelin...</p></li></ul>


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