animals : this study used client - owned 96 dogs that met the following 
 criteria : suffering from spinal cord compression located between t3 and l3 vertebral 
 articulation for duration of more than 6 hr and less than 4 days of neurologic dysfunction 
 prior to mri and disk extrusion confirmed by mri and/or decompressive surgery . all dogs with 
 thoracolumbar hansen type i ivdh were admitted to kyoto animal referral medical center 
 ( kyoto ar ) between november 2010 and august 2012 . age , sex , breed , duration of clinical 
 signs and treatment history before admission were recorded for all dogs . neurological 
 dysfunction was classified into five categories at admission as previously reported with 
 some modifications : grade i , spinal 
 hyperesthesia only ; grade ii , ambulatory paraparesis ; grade iii , non - ambulatory paraparesis ; 
 grade iv , paralysis of the hind limb with intact deep pain sensation ; grade v , paralysis of 
 the hind limb without deep pain sensation . serum sample collection ; pnf - h measurement : approximately 1 
 ml of serum samples were obtained prior to anesthesia for each mri . peripheral blood was collected into a microsample tube ( fuji plain tube , fujifilm medical 
 co. , ltd . , pnf - h was 
 measured using a commercial elisa kit ( elisa kit for neurofilament subunit nf - h ver 2.1 , 
 encor biotechnology inc . , pnf - h is reported to 
 have extensive cross - species immunoreactivity ; 
 previous data proved that this kit could detect canine pnf - h using the mouse anti - pnf - h 
 antibody . mri : mri scans were performed using 0.3 t permanent magnetic equipment 
 ( airis vento , hitachi medical systems , tokyo , japan ) . t1w spin - echo images and t2w fast 
 spin - echo images were obtained in the sagittal and transverse planes , respectively . t1w 
 transverse ( tr 450 , te 2125 , slice thickness 3.0 mm ) , t1w sagittal ( tr 400 , te 25 , slice 
 thickness 3.0 mm ) , t2w transverse ( tr 3000 , te 100 , slice thickness 3.0 mm ) and t2w sagittal 
 ( tr 3000 , te 100 , slice thickness 3.0 mm ) images were acquired . tokyo , japan ) , t2w sagittal and transverse images were analyzed to 
 determine the presence of spinal cord t2w hyperintensity . image analysis was performed 
 individually by two investigators ( y.n . and t.u . ) , who were blinded to clinical information . when both investigators agreed that t2w hyperintensity was present on the sagittal mri , the 
 length of the hyperintensity was measured and divided by the length of the 2nd lumbar 
 vertebra to calculate a standardized ratio ( t2h / l2 ) as described previously . surgical procedures : all dogs received hemilaminectomy on the same day or 
 a day after mri at kyoto ar or at one of seven other referral veterinary hospitals . the length of the decompression was determined by the extent of 
 the dispersed disc material . postoperative care included opioid analgesics , bladder 
 management , passive range of motion and physical rehabilitation . administrations of corticosteroids and/or 
 nonsteroidal anti - inflammatory drugs ( nsaids ) before , at admission and during perioperative 
 periods were recorded . long - term follow - up : all surviving dogs with thoracolumbar ivdh were 
 assessed for voluntary ambulation 12 weeks after the first admission to this study . ambulatory status was determined by in - hospital examination or through a questionnaire sent 
 to referring veterinarians , which consisted of inquiries about the motor function of the 
 affected limb and the dog s ability to ambulate with or without assistance . functional 
 outcome was considered successful , if the dog regained the ability to walk with conscious 
 proprioception without assistance . dogs that regained motor function of the hind limbs but 
 were unable to walk without assistance were considered to have an unsuccessful outcome . dogs 
 that did not regain ambulation during the follow - up period or that were died because of 
 progressive myelomalacia were also classified as unsuccessful outcomes . statistical analysis : linear regression was used to assess the 
 associations of serum pnf - h concentration and t2h / l2 value . receiver - operating 
 characteristics ( roc ) curve analysis was performed to estimate the effectiveness of serum 
 pnf - h concentration or t2h the optimal cutoff value was 
 selected to maximize the youden index ( sensitivity + specificity  1 ) . to evaluate 
 prediction efficacy of concurrent examination of serum pnf - h concentration , t2h / l2 value and 
 deep pain sensation for unsuccessful surgical outcome sensitivity and specificity were calculated by a cross table made from the results of 
 logistic regression analysis . all analyses were performed 
 using commercially available software ( jmp , sas institute japan ltd . , since two dogs were excluded because of loss 
 of follow - up , 94 dogs with thoracolumbar ivdh was examined in this study . six dogs died due 
 to progressive myelomalacia ; functional outcome of these dogs was considered unsuccessful 
 and included in the statistical analyses . there were 25 sexually intact 
 females , 12 spayed females , 44 sexually intact males and 13 castrated males . breeds included 
 miniature dachshund ( 81 ) , french bulldog ( 3 ) , pekingese ( 3 ) , papillon ( 2 ) , welsh corgi ( 2 ) , 
 toy poodle ( 1 ) , beagle ( 1 ) and mixed breed ( 1 ) . the median duration of neurological signs 
 before admission was 2 days ( range , 14 days ) . after long - term follow - up , the functional outcomes of 74 dogs were 
 considered successful . all the dogs with non - ambulatory outcome were in grade iv or v. of 
 the 20 dogs with non - ambulatory outcomes , 6 dogs ( all were in grade v ) died because of 
 progressive myelomalacia , and 14 dogs ( 4 in grade iv and 10 in grade v ) did not regain 
 ambulation ( table 1table1.serum pnf - h concentration and sagittal length of t2w hyperintensity within 
 initial neurologic grade and surgical outcomeneurologic gradeserum pnf - h 
 ( ng / ml)t2h / l2surgical outcomenmean(sd)mean(sd)n#successunsuccesspmii121.9(1.1)0.10(0.34)11200iii202.9(4.5)0.11(0.49)12000iv382.6(3.3)0.43(0.85)143440v247.1(6.2)1.93(2.05)188106total943.5(4.3)0.71(1.38)3474146neurologic grade means neurologic grade at presentation . t2h / l2 , sagittal length of t2w hyperintensity ( mm ) divided by 2nd lumber 
 vertebral body length ( mm ) . compressive lesions were seen at 109 articulations ; t11 - 12 ( 10 ) , t12 - 13 
 ( 19 ) , t13-l1 ( 30 ) , l1 - 2 ( 28 ) and l2 - 3 ( 22 ) . the number of compressive lesions was observed 
 at one ( 80 ) , two ( 13 ) or three intervertebral disc spaces ( 1 ) . the long - term outcome was 
 investigated at 12 weeks after the first admission at kyoto ar ( 18/94 ) and through 
 questionnaires to veterinarians ( 76/94 ) . sixty - five dogs were administered corticosteroids , 
 12 were given nsaids , 2 were given both corticosteroids and nsaids , and 19 received 
 neither . t2h / l2 , sagittal length of t2w hyperintensity ( mm ) divided by 2nd lumber 
 vertebral body length ( mm ) . the mean serum pnf - h concentration in all dogs was 3.5  4.3 
 ng / ml . serum pnf - h concentration and t2h / l2 value for 
 each grade at the initial neurological examination are listed in table 1 . correlation analysis between serum pnf - h concentration and t2h / l2 value exhibited no linear 
 relationship ( r=0.046 , fig . 1fig . 1.correlation between serum pnf - h concentration and sagittal length of intramedullary 
 t2w hyperintensity ( t2h / l2 ) . dogs with serum pnf - h < 2.4 ng / ml had 
 successful outcome , except for one case . falsely diagnosed cases were in the area 
 ( shadows ) , when serum pnf - h was used for detection of intervertebral disc herniation 
 alone . linear relationship was not observed between t2h / l2 value and serum pnf - h 
 concentration ( r=0.046 ) . ) . all the dogs with serum pnf - h higher than 5.9 
 ng / ml or t2h / l2 value higher than 3.9 had unsuccessful 
 outcome ; with only one exception , all dogs with serum pnf - h lower than 2.4 
 ng / ml had successful outcome . correlation between serum pnf - h concentration and sagittal length of intramedullary 
 t2w hyperintensity ( t2h / l2 ) . dogs with serum pnf - h < 2.4 ng / ml had 
 successful outcome , except for one case . falsely diagnosed cases were in the area 
 ( shadows ) , when serum pnf - h was used for detection of intervertebral disc herniation 
 alone . linear relationship was not observed between t2h / l2 value and serum pnf - h 
 concentration ( r=0.046 ) . the roc curve analysis suggested that optimal serum pnf - h cutoff concentration was 2.6 
 ng / ml . the sensitivity and specificity for predicting 
 unsuccessful outcome at this value were 95% ( 95% ci ; 90.699.4% ) and 75.7% ( 95% ci ; 
 67.084.4 ) , respectively . the area under the roc curve was 0.91 ( 95% ci ; 0.840.98 ) , and the 
 overall ability of serum pnf - h concentration to predict unsuccessful long - term outcome was 
 significant ( p=0.010 , fig . 2afig . 2.receiver-operating characteristic ( roc ) curve to predict unsuccessful outcome by 
 serum pnf - h concentration ( a ) and t2h / l2 value ( b ) . a ) sensitivity and specificity of 
 serum pnf - h concentration at 2.6 ng / ml were 95% and 
 75.7 % , respectively . area under the roc curve was 0.91 , and the overall ability of 
 serum pnf - h concentration in this analysis to predict unsuccessful long - term outcome 
 was significant ( p=0.010 ) . b ) sensitivity and specificity of t2h / l2 
 value at 0.84 were 65 % and 86.5 % , respectively . area under the roc curve was 0.78 , 
 and the overall ability to predict unsuccessful long - term outcome was significant 
 ( p=0.043 ) . the sensitivity of serum pnf - h was higher than those of 
 t2h / l2 , although the specificity of t2h / l2 was higher than that of pnf - h . ) . on the other hand , the sensitivity and specificity of t2h / l2 were 65% ( 95% ci ; 
 55.474.6% ) and 86.5% ( 95% ci ; 79.693.4% ) , respectively . the cut - off value of t2h / l2 was 
 0.84 based on the roc analysis . the area under the roc curve was 0.78 ( 95% ci ; 0.660.90 ) , 
 and the overall ability of t2h / l2 to predict unsuccessful long - term outcome was also 
 significant ( p=0.043 , fig . receiver - operating characteristic ( roc ) curve to predict unsuccessful outcome by 
 serum pnf - h concentration ( a ) and t2h / l2 value ( b ) . a ) sensitivity and specificity of 
 serum pnf - h concentration at 2.6 ng / ml were 95% and 
 75.7 % , respectively . area under the roc curve was 0.91 , and the overall ability of 
 serum pnf - h concentration in this analysis to predict unsuccessful long - term outcome 
 was significant ( p=0.010 ) . b ) sensitivity and specificity of t2h / l2 
 value at 0.84 were 65 % and 86.5 % , respectively . area under the roc curve was 0.78 , 
 and the overall ability to predict unsuccessful long - term outcome was significant 
 ( p=0.043 ) . the sensitivity of serum pnf - h was higher than those of 
 t2h / l2 , although the specificity of t2h / l2 was higher than that of pnf - h . an increase in serum pnf - h concentration , t2h / l2 and deep pain sensation by one unit 
 conferred an increase in the odds ratio ( or ) of unsuccessful outcome at long - term follow - up 
 ( or : 2.6 , 95% ci : 1.35.2 , or : 1.9 , 95% ci : 1.13.6 and or : 2.3 , 95% ci : 0.315.5 , 
 respectively ) . in addition , serum pnf - h concentration and t2h / l2 value were significantly 
 correlated to long - term surgical outcome ( p=0.009 and 0.032 , respectively ) ; 
 however , deep pain sensation was not ( p=0.41 ) . cross table based on 
 logistic regression analysis indicated that the combination of serum pnf - h concentration and 
 t2h / l2 value revealed a sensitivity of 90% and a specificity of 97.5% in predicting an 
 overall long - term unsuccessful outcome . the results of this study exhibited that the sensitivity and specificity for predicting 
 long - term outcome by combination of serum pnf - h concentration and t2h / l2 value were 90% and 
 97.5% , respectively . these were sufficiently reliable and showed an improvement in 
 specificity , compared to using each parameter separately . the sensitivity to predict 
 unsuccessful surgical outcome of serum pnf - h concentration was superior to other 
 conventional factors , such as t2w hyperintensity alone or deep pain sensation . however , the 
 specificity of t2h / l2 was superior compared with those of serum pnf - h concentration . this 
 implies that prediction with serum pnf - h alone would lead to inaccurate prognosis as was 
 seen in a case with serum pnf - h concentration of 2.0 ng / ml 
 revealed unsuccessful outcome . deep pain sensation had been used as the standard marker to predict functional 
 outcome , though it was not significantly associated with unsuccessful outcome in this study 
 based on the result of logistic regression analysis ( p=0.41 ) . however , dogs 
 without deep pain sensation had increased risk of nonambulatory outcome ( or : 2.3 , 95% ci : 
 0.315.5 ) . the sensitivity to predict unsuccessful surgical outcome by serum pnf - h concentration was 
 sufficient enough , although a wide range of serum pnf - h levels , ( more than 2.4 and lower 
 than 5.9 ng / ml ) were obtained in this study . there has 
 been no report about blood pnf - h level in patients with spinal cord injury , although 
 increased serum pnf - h in dogs with thoracolumbar ivdh in grades iv and v has been reported 
 within 3 days after the onset of clinical signs . pnf - h is thought to be released from damaged axons and then enters blood circulation 
 directly or through damaged barrier . pnf - h had 
 an initial peak of expression at 16 hr , and the second peak at 3 days in experimentally 
 induced spinal cord injury in rats . thereafter , 
 pnf - h levels returned to the baseline over the following few days . since serum samples were collected 1 to 4 days after the onset of 
 the clinical signs , pnf - h was released into blood circulation at a significantly high level . 
 decreased serum pnf - h in unsuccessful dogs might be due to proteolysis , because proteolysis 
 of neurofilament in ischemic tissue has been reported . in this study , although t2w hyperintensity is easily examined , additional 
 parameters are required for accurate prediction of outcomes , because of relatively low 
 sensitivity of t2w for predicting poor functional outcome . length of t2w hyperintensity has 
 been associated with poor functional outcome with cervical spinal cord injury in human 
 patients [ 8 , 18 ] and dogs with thoracolumbar ivdh [ 13 , 
 15 ] . the usefulness of such signal characteristics 
 on mri might be limited without histopathological information . t2w hyperintensity includes 
 various pathological changes , such as edema , inflammation or hemorrhage [ 2 , 9 , 10 , 20 ] . intraparenchymal hemorrhage has been of greater prognostic value in human patients with 
 spinal cord injury compared to those without hemorrhage [ 3 , 18 , 19 , 27 , 30 ] . in veterinary medicine , detection of intraparenchymal hemorrhage by mri has a 
 limitation due to the body size of the patients and relatively lower - field magnetic strength 
 of equipment . t2 star weighted image is used to depict intraparenchymal hemorrhage in 
 various tissues and lesions ; however , t2 star 
 weighted images had difficulty detecting spinal cord intraparenchymal hemorrhage in dogs . thus , t2w is the sole imaging parameter for detecting spinal cord intraparenchymal 
 hemorrhage in dogs at present . the supposition was that serum pnf - h concentration and t2w hyperintensity would reflect 
 spinal cord injury independently , since each of them represents different histopathological 
 events . our findings revealed that correlation between these two parameters was 
 significantly low ; however , histopathological events of dogs with increased serum pnf - h 
 concentration and higher t2h / l2 value might occur concurrently . primary injury produces 
 intraparenchymal hemorrhage and edema , which results in expression of pnf - h . secondary demyelination including swelling and 
 degeneration of myelin sheath results in continuous , and higher expression of pnf - h . this secondary demyelination was reported to occur 
 in canine ivdh - associated injury . in our present 
 study , 0.3 t low - field strength mri was used , and t2w imaging might have not completely 
 detected lesions associated with demyelination . the sensitivities of 3 t and 1 t instruments 
 in detecting t2w spinal cord hyperintensity were not significantly different from a previous 
 study . however , the results of our study may not 
 directly apply to all imaging studies , especially those obtained using higher magnetic field 
 units . though serum pnf - h was measured as a prognostic biomarker in this study , human pnf - h 
 in cerebrospinal fluid had been studied previously . a serum biomarker may 
 provide quick and simple clinical diagnosis / prognosis in animals with spinal injuries , if 
 this assay becomes commercially available . in addition , repeated measurements of a serum 
 biomarker allow monitoring of disease progression without anesthesia . further studies using 
 higher field strength of mri equipment and consecutive examination of pnf - h in dogs with 
 spinal cord injury are needed in order to apply these combinatory measurements for accurate 
 prognosis . from the study reported here , a combinatory analysis of serum pnf - h concentrations and especially , serum pnf - h concentration of 
 > 2.6 ng / ml and t2h / l2 value of > 0.84 can be 
 associated with unsuccessful long - term outcome .