animals : this prospective study includes client - owned companion dogs with 
 spinal cord injury located between t3-l7 vertebrae secondary to external trauma , and 
 paraplegia without dpp . the study protocol was approved by the ankara university animal 
 experimentations local ethics committee ( 2011 - 15 - 395 ) , and informed consents were obtained 
 from each dog owner . inclusion criteria : dogs with paraplegia , lacking dpp due to external 
 trauma , weighing < 20 kg , having physically intact spinal cord which is determined by 
 magnetic resonance imaging ( mri ) and confirmed in decompressive surgery ( n=2 ) , or just 
 during decompressive surgery ( n=11 ) , the cases with no signs of functional integrity of 
 spinal cord which was further confirmed by electrophysiology . animals that showed no 
 acceptable clinical improvement for at least 42 days ( required time for the stem cell 
 preparation ) after the spinal cord injury , despite conventional surgical and physical 
 treatments or only physical treatments ( in chronic cases ) , were included in the study . percutaneous intraspinal cell therapy applied for two times with 21 days intervals and 
 followed up for at least 2 months after second cell transplantation . neurological examination : texas spinal cord injury scale ( tscis ) which 
 includes the evaluation of gait , proprioception and nociception   was performed to evaluate the neurologic score just after received 
 spinal injury , as well as the outcomes ( just before the 1st cell transplantation , 3 weeks 
 after the 1st cell transplantation and once in 21 days after 2nd cell transplantation ) . neurological improvement was assessed by comparing the difference between the neurological 
 score at presentation and at the final examination . vertebral column radiography : vertebral column instability was evaluated 
 according to the three compartments theory . the dorsal compartment incorporates the 
 articular processes , laminae , pedicles and spinous processes ; the middle compartment 
 includes the dorsal longitudinal ligament , the dorsal aspect of the vertebral body and the 
 dorsal part of the annulus fibrosus ; the ventral compartment contains the ventral 
 longitudinal ligament , lateral and ventral parts of the annulus fibrosus , the nucleus 
 pulposus and remaining parts of the vertebral body . damage to any two of the three compartments indicates instability , and spinal 
 stabilization was carried out for those cases . however , involving one compartment was 
 accepted as stabile , and just decompression was performed . magnetic resonance imaging : all mri procedures were carried out under 40 
 g / kg intravenous ( iv ) medetomidine hydrochloride ( domitor , 
 pfizer ah , new york , ny , u.s.a . ) and 5 mg / kg intramuscular ( i m ) ketamine hydrochloride 
 ( ketasol , richter pharma ag , wels , austria ) anesthesia and under 
 spontaneous - breathing . the mri of two chronic cases ( case nos : 1 and 11 ) was obtained by 
 using a superconducting magnet 1.5 tesla mri unit ( magnetom , siemens ag , munich , germany ) 
 using spinal coil . t1 weighted images ( tr : 370700ms and te : 1520 ms ) and t2 weighted 
 images ( tr : 2,0004,000 ms , te : 90110 ms ) were acquired at 2 mm slice thickness , in three 
 views as sagittal , transversal and axial planes . gadolinium diethylene - triaminepentaacetic 
 acid ( magnevist , bayer pharma ag , berlin , germany ) was used as the paramagnetic contrast 
 medium and was intravenously administered at a dose of 0.2 mm / kg iv . electrophysiological examination : sep were studied under the same general 
 anesthesia protocol , which was provided for mri . briefly , tibial nerve was stimulated from 
 the popliteal fossa because of easy accessibility to the nerve in this area , and the 
 potentials were recorded from two segments cranial and two segments caudal of the lesion , by 
 a hypodermic stainless steel needle . the recording needles were placed as active electrode 
 inserted near to the arcuate ligament and the reference electrode inserted sub - fascially and 
 about 2 cm laterally over the paraspinal muscles on the ipsilateral side . the injury potential ratings 
 in posttraumatic sep recordings were evaluated as ; 0 : isoelectric line , 1 : major 
 deformation , uncertain response , 2 : complete injury potential , 3 : incomplete injury 
 potential , 4 : morphological change and 5 : normal response ( fig . motor - evoked potentials were studied after magnetic stimulation two segments caudally and 
 two segments cranially from the injury site using double 70 mm remote control coil ( magstim 
 200 , the magstim co. , ltd . , the recordings were carried out as active 
 electrode inserted subcutaneously over the epimysium of gastrocnemius muscle and the 
 reference electrode inserted subcutaneously over the achilles tendon , and ground electrode 
 was placed between stimulation and recording points . the presence or absence of the potentials after the stimulation from two 
 segments cranially from the injured area was taken into account in the evaluation .. surgical techniques : according to the presence of compression and 
 instability , hemilaminectomy or laminectomy was carried out for decompression within 24 hr 
 of admission time in acute cases . just decompression was performed in chronic cases within 
 three days after admission to clinic . the cases with spinal instability were treated using 
 screw+ poly - methyl methacrylate ( pmma ) as usual . sublaminar or transverse lamina wiring , or 
 transarticular process pinning and tension wiring were also carried out , and combination of 
 those techniques was demonstrated in fig . 2.the employed spinal stabilization ; transversal wiring , sublaminar wiring , 
 transarticular process pinning and tension wiring . the details of the employed treatment method are presented in table 1table 1.clinical summary data for paraplegic dogs without nociception due to spinal 
 trauma transplanted with autologous neurogenically - induced bone marrow - derived 
 mesenchymal stem cellscase nosignalment of the dogneurologic examination at the admission 
 timex - ray / mrisurgical procedureneurologic examinationat the time of first cell trans . 15 m mi mix breedhbcat : 1wkg0 , p0 , n0sca ( 4)scr ( 0)mcr ( )mca ( + ) t13 proc . artc . and lamina fracturemri : hyperintense 
 lesion in t2w images , and right side dorsolateral compressiont13 right hlg1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca(+)g1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) there was 
 no change at 12 . month22y fi huskyhbcat : 1dg0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) t12 - 13 fracture and luxation , 2/3 compressiont12 - 13 proc . . stabilized with pin and tension 
 wire , and hl , lw between t11-l1g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) 36 m fi mix breedhbcat : 1dg0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) l1 burst fracturehl in t13-l1 and lw including t12 - 13-l1 - 2 - 3g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) 47 m mi mix breedhbcat : 1dg0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) t12 - 13 luxation 1/3 compression , l5 ( 1/3 compression ) 
 vertebral body fracturet12 dorsal lt12 - 13 and l5 - 6 proc . pin tension 
 wireg0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) 58 m f mix breedhbcat : 1dg0 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) l2 ( 2/3 compression ) and l7 ( 2/3 ) vertebral body 
 fracturel on l2 - 3 , and stabilization with screw and pmma , and 
 proc . screw fixation of l7-s1g1 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g2 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g2 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) g2 , p0 , n0sca ( 4 ) , scr ( 1)mcr (  ) , mca ( + ) 62y fi mix breedgunshot woundat : 2dg0 , p0 , n0sca ( 2 ) , scr ( 0)mcr (  ) , mca ( )pellet on t11 level at the left sidepellet had taken off with hlg0 , p0 , n0sca ( 2 ) , scr ( 0)mcr (  ) , mca ( )g1 , p0 , n0sca ( 2 ) , scr ( 0)mcr (  ) , mca ( )g1 , p0 , n0sca ( 2 ) , scr ( 0)mcr (  ) , mca ( )72.5 m fi mix breedhbcat : 2dg1 , p0 , n0sca ( 4 ) , scr ( 2)mcr (  ) , mca ( + ) l5 burst fracturel4 - 5 - 6 sublaminar wiring , l4 - 5 left side hlg2 , p0 , n0sca ( 4 ) , scr ( 2)mcr (  ) , mca ( + ) g4 , p1 , n1sca ( 4 ) , scr ( 3)mcr ( + ) , mca ( + ) g4 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) g4 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) g4 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) 88 m fi mix breedhbcat : 2dg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) t12 - 13 luxation , 1/3 compressiont12 - 13 proc . artc . pin and tension wire stabilisationt12 - 13 left side mini hl and disk fenestrationg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) 91y mi pointerhbcat : 2dg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) t9 - 10 luxation 1/3 compressiont9 - 10 proc . pin and tension wire and left side 
 hl , and t8 t11 screw + pmmag0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca(+)g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) 106 m m mix breedhbcat : 1dg0 , p0 , n0sca ( 1 ) , scr ( 0)mcr (  ) , mca ( + ) l5 vertebral body fracture1/3 compressionl4 - 5 right side h , vertebral body reconstruction plate 
 andl4 - 5 proc . pin tension wire , g1 , p0 , n0sca ( 4 ) , scr ( 3)mcr (  ) , mca ( + ) g3 , p0 , n1sca ( 4 ) , scr ( 3)mcr (  ) , mca(+)g3 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) g3 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) g3 , p1 , n2sca ( 4 ) , scr ( 4)mcr ( + ) , mca ( + ) 118y f mix breed , hbc and operated 1 year ago due to 
 scrinal traumaat : 1 yearg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) mri : epidural fibrosis on t12 levelhyperintense 
 lesion on t2 w 17 mm long at the scrinal cord hlg1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca(+)g2 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g2 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g2 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) 124 m fi mix breedhbcat : 2dg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) l3 - 4 luxation2/3 compressionl and screw+pmma in l2 - 5g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) 134y m mix breedhbcat : 1 monthg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) t12 - 13 fracture and luxation : 1/3 compression lg0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g0 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) g1 , p0 , n0sca ( 4 ) , scr ( 0)mcr (  ) , mca ( + ) artc . : articularis ; at : admission time ; fi : female intact ; g : gait score ; hbc : hit by 
 car , hl : hemilaminectomy ; l : laminectomy ; lt : left ; lw : laminotransversal wiring ; mca : 
 mep caudal ; mi : male intact ; mcr : mep cranial ; n : nociception ; p : proprioception , 
 pmma : polymethylmethacrylate ; proc . : processus ; rt : right ; sca : sep caudal ; scr : sep 
 cranial ; trans . : transplantation ; y : years .. the employed spinal stabilization ; transversal wiring , sublaminar wiring , 
 transarticular process pinning and tension wiring . artc . : articularis ; at : admission time ; fi : female intact ; g : gait score ; hbc : hit by 
 car , hl : hemilaminectomy ; l : laminectomy ; lt : left ; lw : laminotransversal wiring ; mca : 
 mep caudal ; mi : male intact ; mcr : mep cranial ; n : nociception ; p : proprioception , 
 pmma : polymethylmethacrylate ; proc . : processus ; rt : right ; sca : sep caudal ; scr : sep 
 cranial ; trans . : transplantation ; y : years . in the cases that were admitted during acute - stage ( < 48 hr ) , surgical management 
 ( decompression or decompression + stabilization ) and collection of the bone marrow were 
 carried out during the same anesthesia . however , in the cases which were admitted in chronic 
 stages ( cases 1 , 11 and 13 ) , just the bone marrow was collected at the admission time for 
 processing . in the former cases , all cases underwent physical therapy and rehabilitation at the 
 interval between bone marrow collection and cell transplantation , intra - operative analgesia provided with a 0.02 mg / kg / hr iv constant - rate infusion of 
 fentanyl ( fentanyl 50 g / ml , johnson & 
 johnson , diegem , belgium ) and administration of epidural morphine with 0.1 mg / kg ( morfin 
 hcl 0.01 g / ml , osel , istanbul , turkey ) at the decompression 
 defect prior to closure of the incision . analgesia at discharge was achieved with carprofen 
 ( rimadyl , pfizer ) with 2 mg / kg , po , q 12 hr for 57 days . isolation and culture of bm - mscs : bone marrow was collected from the iliac 
 crest and transported to the laboratory within an hour . bm - mscs were cultured according to 
 standard methods [ 6 , 27 ] . under sterile conditions , ~510 ml of bone marrow was 
 suspended in -mem ( hyclone , ge life sciences , logan , ut , u.s.a . ) containing 1% 
 penicillin  streptomycin ( pen / strep ) and 2 mm l - glutamine ( sigma - aldrich , munich , germany ) 
 and was washed three times in 0.1 m phosphate buffer saline ( pbs ) with sequential 
 centrifugation ( at 25c and 1,000 rpm ) . subsequently , the cells were seeded in tissue 
 culture flasks containing -mem supplemented with pen / strep , l - glutamine and 10% fetal 
 bovine serum ( biowhittaker - lonza , basel , switzerland ) at 37c with humidified 5% 
 co2 . the non - adherent cells were removed by replacing the medium at the second 
 day of subculture . the cells were cultured up to passage 2 under the same conditions , with 
 medium changes every other day . at this point , ~5.0  10 bm - mscs the remaining bm - mscs were 
 cultured likewise for the subsequent dose administration . in vitro neurogenic differentiation of bm - mscs : bm - mscs were induced to 
 differentiate into the neurogenic lineage by a two - step protocol . briefly , cells were placed in tissue culture flasks coated with poly 
 hydroxyethyl methacrylate ( biowhittaker - lonza ) and cultured in the b-27 and n-2 ( serum - free 
 chemically - defined neurogenic supplements ) containing neurobasal medium ( mesenpro with 10% 
 mesenpro supplement , pen / strep and l - glutamine ; invitrogen ) , supplemented with 100 
 ng / ml human recombinant epidermal growth factor ( egf ) 
 and 10 ng / ml human recombinant basic fibroblast growth 
 factor ( bfgf ) ( both recombinant dna expressed in yeast ( s. cerevisiae ) ; 
 merck - millipore , darmstadt , germany ) . the resulting neurospheres were stained to confirm 
 positivity to nestin ( santa cruz biotechnology , heidelberg , germany ) . at the second step , the acquired neurospheres were disaggregated by accutase ( stempro 
 accutase , gibco , grand island , ny , u.s.a . ) , and the cell suspension was plated in tissue 
 culture flasks which were coated with poly ornithine ( sigma - aldrich ) . the 
 neurosphere - derived cell suspension was then cultured for six days in the neurobasal medium 
 supplemented with 100 ng / ml nerve growth factor ( ngf ) 
 ( from mouse submaxillary gland ; merck - millipore , darmstadt , germany ) and 10 
 ng / ml brain - derived neurotrophic factor ( bdnf ) ( human 
 recombinant ; merck - millipore ) for further induction into the neurogenic lineage . immunohistochemistry revealed that these cells were positive to glial fibrillary acidic 
 protein ( gfap ) and anti-23-cyclic nucleotide-3-phosphodiesterase ( cnpase ) , as well as to 
 beta iii tubulin and microtubule - associated protein-2 ( map-2 ) ( abcam , cambridge , ma , 
 u.s.a . ) . at ~80% confluence , the cells were removed from the surface by 0.05% trypsin/0.53 
 mm edta solution ( sigma - aldrich ) and washed three times with pbs . one ml of 
 cell suspension was transferred into a syringe and was delivered to the surgery room within 
 ~30 min . cell transplantation procedure : three spinal needles ( 22g90 mm quincke 
 tip , egemen , izmir , turkey ) were inserted percutaneously through the hemilaminectomy or 
 laminectomy defect intraspinally into three different points under general anesthesia with 
 the same protocol which is used in mri . the location of the needle tip was confirmed by 
 observing cerebrospinal fluid leakage from the needle hub . subsequently , the needle tip was 
 directed into the spinal cord parenchyma percutaneously , and a slow injection was carried 
 out . about 5.0  10 nibm - mscs suspended in 1.0 ml of pbs were 
 injected by needles in equal volume . the cell transplantation procedure ( ~5.0  
 10 nibm - mscs ) was repeated after 21 days . the neurological and 
 electrophysiological examinations of the cases were performed at the 42nd day ( time of the 
 first cell transplantation ) , at the 63rd day ( time of the second cell transplantation ) , and 
 2 , 5 , 7 and 12th months after the second cell transplantation . in most of the cases , bm - mscs expanded quite well in - vitro demonstrating 
 fusiform , fibroblastoid phenotype starting from the first passage ( fig . 3afig . formation of colonies ( arrow ) is 
 typical in confluent msc cultures ( cfu - f ) . ( b ) formation of nestin - positive 
 neurospheres ( inner photo ) after the first neurogenic induction step . ( c , d ) 
 disaggregated cells are directed into the second neurogenic induction stage yielding 
 neurogenic phenotypes . ) . at the first neurogenic induction step , the cells formed neurospheres with a 
 diameter of ~100150 m within 3 days ( fig . 3b ) . at this stage , > 95% of the neurosphere cells were positive to nestin . 
 at the second step of neurogenic induction , the acquired nestin - positive neurospheres were 
 disaggregated , and the acquired cells were differentiated further into the subsequent stages 
 forming neural tube - like structures , as well as multiple branching processes resembling 
 early steps of development ( fig . the 
 population of cells were positive to gfap , cnpase , beta iii tubulin and map-2 ( fig . gfap ( glial fibrillary acidic protein ) ; cnpase ( anti-23-cyclic 
 nucleotide-3-phosphodiesterase ) , -tubulin iii , map-2 ( microtubule - associated 
 protein-2 . ) . immunohistochemistry revealed that differentiated cells were highly positive to 
 map-2 ( ~90% ) and partially positive to beta iii tubulin ( ~37% ) , cnpase ( ~12% ) and gfap 
 ( ~10% ) markers , indicating the existence of a mixed population of mature and immature 
 neurogenic cells , for the most part of neuronal cells . formation of colonies ( arrow ) is 
 typical in confluent msc cultures ( cfu - f ) . ( b ) formation of nestin - positive 
 neurospheres ( inner photo ) after the first neurogenic induction step . ( c , d ) 
 disaggregated cells are directed into the second neurogenic induction stage yielding 
 neurogenic phenotypes . gfap ( glial fibrillary acidic protein ) ; cnpase ( anti-23-cyclic 
 nucleotide-3-phosphodiesterase ) , -tubulin iii , map-2 ( microtubule - associated 
 protein-2 . mean age during admission was 19.3 months ( ranging from 
 2.5 months to 8 years ) , and the mean body weight of the dogs was 11 kg ( range : 619 kg ) . subjected breeds were mixed breed ( n=12 ) , siberian husky ( n=1 ) and pointer ( n=1 ) . ten dogs 
 were presented within 48 hr of onset of clinical signs and operated at the same day . the 
 rest of three dogs were presented one week , one month and one year after the trauma for 
 each , and they were operated after stem cells preparation . at the admission time , all cases 
 were paraplegic due to spinal trauma , and neuroanatomic localization was between t3-l7 . spinal trauma dispersed according to the radiographic images of the cases as ; vertebral body 
 fracture ( n=3 ) , fracture and luxation ( n=2 ) , laminar fracture ( n=1 ) , burst fracture ( n=2 ) , 
 luxation ( n=3 ) , gunshot wound ( n=1 ) and epidural fibrosis due to a former trauma ( n=1 ) . spinal cord injury was further clarified by mri at the admission time only in 2 chronic 
 cases , in which the hyper - intense lesions ( it may be myelomalacia or glial reaction ) were 
 seen at the spinal cord in t2 weighted imaging in the injured area of the spinal cord 
 parenchyma . one case ( case no : 1 ) had compression at the right dorsolateral side of t13 
 spinal cord segment in transversal images and a 24 mm - length hyper - intense lesion in 
 sagittal and dorsal planes of mri . the other case ( case no : 11 ) had narrowing epidural space 
 at t12 level , causing a 17 mm - long hyper - intense lesion in both dorsal and sagittal planes 
 of mri , and the lesion was interpreted as epidural fibrosis . in the remaining cases , traumatic lesions were obvious in survey radiography , and further advanced diagnostic 
 imaging was declined . spinal instability was determined according to the three compartments 
 theory , and stabilization was carried out in 9 
 cases as follows : sublaminar wire ( n=1 ) ; pin - tension wire at the articular processes in both 
 side ( n=2 ) ; screw on vertebral bodies and pmma ( n=1 ) ; pin - tension wire at the articular 
 processes+ screw and pmma ( n=1 ) ; pin - tension wire at the articular processes and 
 laminotransversal wiring ( n=1 ) ; laminotransversal wiring ( n=1 ) ; screw on vertebral 
 bodies+pmma and lag screw on articular processes ( n=1 ) ; vertebral body plate and pin - tension 
 wire at the articular processes ( n=1 ) . spinal trauma which did not cause instability was 
 treated by laminectomy ( n=1 ) and hemilaminectomy ( n=3 ) . in the case with the gunshot wound , 
 the bullet was located dorsally at the vertebral body in epidural space . the color changes , 
 which are reddish to brown , of dura mater were seen in all cases during the surgery . in 2 of 
 the chronic cases ( case nos . : 11 and 13 ) , the spinal cord was covered by extensive fibrous 
 tissue and was thinner in diameter . the gait score for tscis at the time of admission was  0  in all cases except for 1 case 
 with  1  , however , the proprioception and nociception scores were also  0  in all cases . somatosensory - evoked potentials were flat - line at the cranial area of injury in 8 cases ,  1  
 in 4 cases and  2  in 1 case . no motor - evoked potential was obtained from the gastrocnemius 
 muscle after stimulating the spinal cord from the 2 segments cranial injured area . at the 42nd day which is average time for preparing cells for each case after the 
 operation or physical therapy : the gait score had improved one score for each in 
 5 cases . the sep were improved in one case at the cranial part of injured 
 area with one score , and one case showed improvement in both caudal and cranial areas with 3 
 score . all cases received two doses of autologous 5.0  10 nibm - mscs suspension at the 
 post - operative 42nd and 63rd days , through the decompression defect intraspinally , except 
 for one case in which injection was performed subarachnoidally from the l5 - 6 intervertebral 
 space by lumbar punction due to inaccessibility caused by pmma ( case no . injection was achieved 
 by palpating the laminectomy or hemilaminectomy defect by the absence of spinous process or 
 articular process , respectively , and insertion of spinal needles into the spinal cord . appearance of the cerebrospinal fluid was accepted as landmark , the tip of needle was 
 progressed into the spinal cord parenchyma , and injection was carried out . employed stabilization techniques patients with pmma had seroma formation at the operation site , however , seroma was 
 drained , and the operation site was recovered uneventfully . from the first to the second cell transplantation , 4 cases improved 1 gait score , and 2 
 cases improved 2 gait score . one of these cases which showed 2 gait score improvement , 
 progressed 1 score for each in proprioception , nociception and proximal sep , and also 
 proximal mep changed negative to positive . two months after the second cell transplantation , 
 2 cases exhibited 1 score improvement in gait score . two cases showed 1 score improvement in 
 both nociception and proximal sep , also one of these cases improved 1 score in 
 proprioception , and proximal mep changed negative to positive in this case . all progresses 
 were observed until 2nd month after the second cell transplantation , however , there was no 
 improvement after this period . in general , improvement of gait , nociception and 
 proprioception after cell transplantation was observed in just 2 cases , and only gait score 
 improvement was seen in 6 cases , and no improvement was recorded in 5 cases . sep and mep 
 results had improved in both of the cases , which showed improvement nociception and 
 proprioception . as a result , overall tscis score did not change after the cell 
 transplantations in 5 cases , one score increased in 6 cases and five score in 2 cases . to the authors knowledge , there is not an existing report on percutaneous nibm - mscs 
 transplantation for the treatment of paraplegia due to external trauma in dogs . the present 
 study reports promising results in 2 cases and also some improvement of the locomotion in 6 
 cases . even though spontaneous improvement in this poorest neurologic grade has been 
 reported , it is not so common [ 11 , 29 ] . these hopeless cases have been selected for this 
 study on purpose , and minus gains are acceptable . according to the clinical and electrophysiological results , there was no 
 remarkable difference between the time of admission and prior to cell transplantation ( 42nd 
 day ) . when the results of data were recorded before cell transplantation with the end point 
 of observation period , there are barely positive developments in clinical signs . this study 
 does not exclude the possibility of spontaneous improvement entirely , yet presents 
 acceptable positive results open to further investigation . there is a wide range of studies regarding spinal stabilization techniques , involving the 
 use of pins or screws and polymethymetacrylate , vertebral body plating , vertebral stapling , 
 external skeletal fixation , veterinary string of pearls plates , pedicle screw  rod fixation , 
 lubra plates and tension bands [ 17 , 22 , 23 , 39 , 41 , 43 , 45 ] . the 
 surgical stabilization applied in this case series was acceptable , and we report no implant 
 failures in any case . some modifications applied in the cases reported here , such as 
 articular processes pining and tension wiring , and sublaminar wiring and/or subtransversal 
 wiring , were also found versatile , cost effective and easy applicable techniques for 
 providing spinal stabilization . for preventing further and repeated trauma due to spinal 
 instability , spinal stabilization is crucial for the successful rehabilitation of paraplegic 
 cases . these techniques are investigated in detail for both dogs and cats by the authors 
 ( ob , pc , unpublished data ) . bone marrow - derived stem cells can differentiate into glial cells or neurons which may 
 improve the effects of regenerative therapy in spinal cord injury [ 24 , 35 ] . the best therapeutic 
 outcomes can be achieved with optimal dosage , timing and optimal administration route . bone marrow stromal cells do not differentiate into 
 neural cells in the host tissue as determined by immunohistochemical analysis . thus , we carried out a serum - free msc 
 differentiation protocol which yielded a mixed population of partially mature neurogenic 
 cells that were positive to gfap , cnpase , beta iii tubulin and map-2 . mscs may prove to be 
 beneficial for treating sci as they can be expanded and differentiated into the neurogenic 
 lineage through neurospheres in vitro . additionally , the insufficient clinical results of spinal cord injury cases , 
 motivates us to investigate autologous nibm - mscs in paraplegic dogs without dpp , due to 
 external trauma . the percutaneous transplantation technique   preferred in this study is minimally invasive , reliable and easily applicable , 
 and does not require advanced level of experience . an experimental canine model utilizing neural - differentiated allogeneic msc transplantation 
 with matrigel for the treatment of spinal cord injury , revealed functional improvement 1 
 week upon sci . findings were attributed to the neurotrophic effects , including increase in 
 neurotrophin expression , decreased inflammation and astrogliosis , as well as increased 
 neuronal extension and regeneration . however , 
 bm - mscs in the spinal cord were not observable by the third week of injection . multiple 
 administration of bm - mscs leads to improved cell grafting compared to a single application 
 of bm - mscs in spinal cord injury [ 26 , 28 , 35 ] . there was 
 no chance to make histopathologic or immunohistochemical investigation of the injured spinal 
 cord areas that may explain the mechanism of improvement in our cases , but increased 
 neurotrophin expression or neuronal extension might be provided an improvement in the 
 presented study . in the present study , we preferred to transplant the cells 2 times with 21 days interval to 
 sustain the positive effect for a longer period as it has been reported before . we observed the beneficial effects of cell therapy 
 in chronic spinal cord injury cases ( more than 42 days ) at the second month , but there was 
 no improvement at the later periods . the use of bm - mscs for sci has provided re - myelination 
 and neuroprotection from releasing cytokines in experimentally - induced spinal cord injuries 
 in rat models [ 2 , 12 ] . even though autologous mscs transplantation has shown more beneficial 
 effects than allogeneic mscs transplantation , allogeneic mscs transplantation has also been 
 found useful in improving functional recovery following sci [ 16 , 26 , 46 ] . as the time required for preparing autologous nibm - mscs is relatively long , 
 it is not possible to deliver these cells at the acute stage of spinal cord injury . there 
 may be a possibility to shorten the time span between bone marrow collection and cell 
 transplantation to around 2830 days ( i.e. 1921 days of msc expansion + 9 days of 
 neurogenic induction ) , which will necessitate subtraction of the second dose . however , this 
 was not the option in our study ; and longer culture time was needed to obtain the required 
 number of stem cells for the second transplantation . use of banked allogenic stem cells can 
 be a possibility to shorten the time span , however , potential drawbacks of using allogeneic 
 source need to be evaluated in additional studies . providing a suitable environment for 
 cells assures better outcomes in chronic spinal cord injury . researchers aim to solve the 
 time limitation and support the therapy with additional modalities . the results of cell injections into the csf in dogs with intervertebral disc disease ( ivdd ) 
 are as follows : 6 of 10 cases regained walking ability , but only a single case showed 
 improvement in nociception . the same group 
 reports their results of intraspinal injection of bone marrow stromal cells to paraplegic 
 dogs without dpp due to external trauma as : 2 out of 7 cases showed improvement in walking 
 ability , but none had nociception . this is 
 consistent with the observation that bm - msc transplantation in rats had no effect in sensory 
 function . in a case series   about ivdd without 
 dpp , walking ability was regained in 57.81% ( 37/64 ) , and 9 dogs were euthanized within 3wk , 
 and 18 of them survived but never regained nociception . however , 7 dogs out of 18 regained 
 the ability to walk . the results of our study are in line with the previous studies , and improvement in gait score is higher than the 
 nociception and proprioception scores presented by other groups . in another study conducted 
 by our research group in dogs with ivdd and lacking dpp , in eight months - follow up , 4 cases 
 were evaluated by the same parameter ; gait score had improved in 3 cases , proprioception 
 improved in 2 cases , and nociception improved in 3 cases . some recent studies [ 7 , 26 ] show that the results of ivdd are better than 
 external trauma even though the preferred cell types were different in the studies . the success rate after external spinal trauma is lower ( % 0 ) [ 4 , 17 ] than that of ivdd ( % 076 ) 
 [ 3 , 8 , 10 , 29 , 36 ] , as it involves the treatment of many structures 
 ( e.g. bone , ligaments ) , for overcoming instability , and also the possibility of iatrogenic 
 trauma . comparing recovery rates of different studies is complicated , due to many variables , 
 such as case selection criteria , the duration of dpp absence or loss of motor function , and 
 the treatment modality of choice . the rate of success and the results of the presented study 
 are not dramatically different compared to the data in the literature [ 4 , 29 ] . however , the improvement in 
 nociception and proprioception can be accepted as a critical point for carrying out further 
 investigations with higher number of cases and including new criteria . among previous reports with cases without dpp after external trauma , the success rate was 
 also poor even with combined surgical or medical treatments [ 11 , 17 , 37 ] . nevertheless , improvement in gait score in 6 of the cases , and improvement in 
 proprioception and nociception in 2 cases can be accepted as encouraging results . the main limitation of this study can be listed as ; lack of histological and 
 immuno - histochemical examinations , lack of a control group and being a single - center study 
 design . randomized multi - center clinical studies are required to clarify the contribution of 
 nibm - mscs transplantation in dogs with sci . however , it is possible to conclude that 
 percutaneous intraspinal injection of autologous nibm - mscs provides some clinical benefits 
 in dogs with paraplegia , lacking dpp after external spinal trauma .
the aim of this study was to investigate the effects of percutaneous transplanted 
 autologous neurogenically - induced bone marrow - derived mesenchymal stem cells ( nibm - mscs ) 
 in paraplegic dogs without deep pain perception ( dpp ) secondary to external spinal trauma . 
 thirteen client owned dogs that had failed in improvement neurologically at least 42 days 
 after conservative management , decompression and decompression - stabilization were included 
 in the study . 
 each dog received two doses of autologous 5.0  106 nibm - mscs 
 suspension , which were positive to 2,3-cyclic - nucleotide-3-phosphodiesterase ( cnpase ) 
 and microtubule - associated protein 2 ( map-2 ) , as well as to glial fibrillary acidic 
 protein ( gfap ) and beta iii tubulin . 
 the cells were injected into the spinal cord through 
 the hemilaminectomy or laminectomy defects percutaneously with 21 days interval for 2 
 times . 
 the results were evaluated using texas spinal cord injury scale ( tscis ) , 
 somatosensory evoked potentials ( sep ) and motor evoked potentials ( mep ) at the admission 
 time , cell transplantation procedures and during 2 , 5 , 7 and 12th months after the second 
 cell transplantation . 
 improvement after cell transplantation in gait , nociception , 
 proprioception , sep and mep results was observed in just 2 cases , and only gait score 
 improvement was seen in 6 cases , and no improvement was recorded in 5 cases . 
 all 
 progresses were observed until 2nd month after the second cell transplantation , however , 
 there was no improvement after this period . in conclusion , 
 percutaneous transplantation of 
 autologous nibm - mscs is a promising candidate modality for cases with spinal cord injury 
 after spinal trauma and poor prognosis .