Evaluation of Serum Phosphorylated Neurofilament Subunit NF‐H as a Prognostic Biomarker in Dogs With Thoracolumbar Intervertebral Disc Herniation Hidetaka Nishida1,2, DVM, PhD, Masanari Nakayama1, DVM, PhD, Hiroshi Tanaka1, DVM, PhD, Hiroaki Kamishina3, DVM, PhD, Takeshi Izawa4, DVM, PhD, Shingo Hatoya2, DVM, PhD, Kikuya Sugiura2, DVM, PhD, Yoshihisa Suzuki5, MD, PhD, Chizuka Ide6, MD, PhD, and Toshio Inaba2, DVM, PhD 1
Nakayama Veterinary Hospital, Nara, Japan ,2 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan ,3 Faculty of Applied Biological Sciences, Department of Veterinary Medicine, Gifu University, Gifu, Japan ,4 Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan ,5 Department of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Research Institute, Osaka, Japan and 6 Faculty of Nursing and Rehabilitation, Department of Occupational Therapy, Institute of Regeneration and Rehabilitation, Aino University, Osaka, Japan
Corresponding Author Toshio Inaba, DVM, PhD, Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1‐58 Rinku Ourai Kita, Izumisano, Osaka, Japan. E‐mail: [email protected] Submitted December 2012 Accepted December 2013 DOI:10.1111/j.1532-950X.2014.12144.x
Objective: To investigate whether pNF‐H is a prognostic biomarker of spinal cord injury (SCI) in paraplegic dogs with thoracolumbar intervertebral disc herniation (IVDH). Study Design: Prospective, case‐control clinical study Animals: Dogs (n ¼ 60) with SCI from IVDH and 6 healthy dogs. Methods: Serum from 60 thoracolumbar IVDH dogs (Grade 4: 22 dogs; Grade 5: 38 dogs) collected 1–3 days after injury, and 6 control dogs, was analyzed using enzyme‐linked immunosorbent assay (ELISA) against a phosphorylated form of the high‐molecular‐weight neurofilament subunit NF‐H (pNF‐H). Serum pNF‐H levels were compared between different IVDH grades and their prognostic value was investigated. Results: pNF‐H levels were significantly greater in Grade 5 than Grade 4 dogs. There were significant differences in pNF‐H levels between dogs that regained voluntarily ambulation and those that did not. All 8 dogs that had high pNF‐H levels 1–3 days after injury did not regain the ability to walk after surgery. Conclusions: Serum pNF‐H levels might be a biomarker for predicting prognosis of canine SCI.
Severe spinal cord injury (SCI) is a major cause of permanent disability in dogs, with intervertebral disc herniation (IVDH) in the thoracolumbar region being the most common cause.1 Accurate prediction of SCI damage might guide veterinarians in choice of neuroprotective intervention. Clinical scales of SCI severity are a standardized tool, widely used to assess neurologic state and motor score in dogs with SCI2; however, they have limitations for predicting prognosis of SCI. Detection of specific markers in serum and cerebrospinal fluid (CSF) may indicate presence of particular diseases, allow disease monitoring, and provide accurate and rapid clinical prognosis. Small calcium‐binding protein S100b, neuron specific enolase (NSE), microtubule associated protein tau, and the phosphorylated form of the high‐molecular‐weight neurofilament subunit NF‐H (pNF‐H) have all been proposed Work done at the Nakayama Veterinary Hospital.
as biomarkers for various kinds of neuronal injury.3,4 S100b and tau are found in astrocytes or neurons, but also in other cell types.4,5 S100b and NSE have peak levels 6 hours after injury and return to baseline at 24 hours in rats with SCI.6 pNF‐H is found only in neurons and is quite resistant to proteases, so that pNF‐H released from damaged and diseased axons remains in CSF and serum in undegraded form.7,8 Serum pNF‐H levels correlate with severity of SCI and brain injury in a rat contusion model7,9; serum pNF‐H has an initial peak of expression at 16 hours after SCI in rats, and a second, usually larger, peak at 3 days.7 Thus, detection of this protein in serum might provide information about the extent of neuronal loss. We are unaware of any reports examining pNF‐H in dogs, so our purpose was to investigate whether serum pNF‐H levels in blood are correlated with prognosis of SCI in dogs. Our hypothesis was that dogs with high levels of pNF‐H between 1 and 3 days after injury would have a grave prognosis for ability to walk.
Veterinary Surgery 43 (2014) 289–293 © Copyright 2014 by The American College of Veterinary Surgeons
289
pNF‐H as a Prognostic Biomarker in Dogs With Spinal Cord Injury
MATERIALS AND METHODS Inclusion Criteria Dogs diagnosed with severe thoracolumbar IVDH (Grade 4 or 5), 1–3 days after injury, were prospectively enrolled through Nakayama Veterinary Hospital (April, 2009–December, 2012), affected dogs were excluded if they had other diseases. Control dogs were prospectively enrolled through Nakayama Veterinary Hospital and were eligible if they were healthy and had no IVDH history. All owners provided written consent for participation of their dogs in this study. Neurologic dysfunction was classified2 as: paraspinal hyperesthesia only (Grade 1), ambulatory paraparesis and ataxia (Grade 2), non‐ambulatory paraparesis (Grade 3), paraplegia with intact pain perception (Grade 4), paraplegia with no pain perception (Grade 5). Blood samples, collected before physical examination or surgery, were coagulated using a serum separator tube for 30 minutes and centrifuged at 3000 rpm for 10 minutes. Serum was frozen at 70°C and stored until assay. Diagnosis of thoracolumbar IVDH was made from CT scans and myelography, and was confirmed during decompressive surgery. Hemilaminectomy was performed on the side ipsilateral to the spinal cord compression and extruded disk material removed. Postoperative care included administration of cephalexin (22 mg/kg orally twice daily for 1 week) and buprenorphine (0.01–0.02 mg/kg subcutaneously once or twice daily for 1–3 days), management of urinary incontinence, and nursing care to prevent decubital ulcers.
Nishida et al.
standard curve was constructed by plotting mean absorbance of the standards against known concentrations of the standards on a logarithmic scale, using the 4 parameter algorithm. The concentration of each specimen was calculated from the standard curve. The limit of detection (23.5 pg/mL) was calculated from the mean absorbance of the blank (dilution buffer only) plus three standard deviations from the absorbance of the blank. We defined the limit of detection as 70 pg/mL because the sample was diluted threefold. Inter‐ and intra‐assay CVs for pNF‐H were
Nakayama Veterinary Hospital, Nara, Japan ,2 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan ,3 Faculty of Applied Biological Sciences, Department of Veterinary Medicine, Gifu University, Gifu, Japan ,4 Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan ,5 Department of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Research Institute, Osaka, Japan and 6 Faculty of Nursing and Rehabilitation, Department of Occupational Therapy, Institute of Regeneration and Rehabilitation, Aino University, Osaka, Japan
Corresponding Author Toshio Inaba, DVM, PhD, Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1‐58 Rinku Ourai Kita, Izumisano, Osaka, Japan. E‐mail: [email protected] Submitted December 2012 Accepted December 2013 DOI:10.1111/j.1532-950X.2014.12144.x
Objective: To investigate whether pNF‐H is a prognostic biomarker of spinal cord injury (SCI) in paraplegic dogs with thoracolumbar intervertebral disc herniation (IVDH). Study Design: Prospective, case‐control clinical study Animals: Dogs (n ¼ 60) with SCI from IVDH and 6 healthy dogs. Methods: Serum from 60 thoracolumbar IVDH dogs (Grade 4: 22 dogs; Grade 5: 38 dogs) collected 1–3 days after injury, and 6 control dogs, was analyzed using enzyme‐linked immunosorbent assay (ELISA) against a phosphorylated form of the high‐molecular‐weight neurofilament subunit NF‐H (pNF‐H). Serum pNF‐H levels were compared between different IVDH grades and their prognostic value was investigated. Results: pNF‐H levels were significantly greater in Grade 5 than Grade 4 dogs. There were significant differences in pNF‐H levels between dogs that regained voluntarily ambulation and those that did not. All 8 dogs that had high pNF‐H levels 1–3 days after injury did not regain the ability to walk after surgery. Conclusions: Serum pNF‐H levels might be a biomarker for predicting prognosis of canine SCI.
Severe spinal cord injury (SCI) is a major cause of permanent disability in dogs, with intervertebral disc herniation (IVDH) in the thoracolumbar region being the most common cause.1 Accurate prediction of SCI damage might guide veterinarians in choice of neuroprotective intervention. Clinical scales of SCI severity are a standardized tool, widely used to assess neurologic state and motor score in dogs with SCI2; however, they have limitations for predicting prognosis of SCI. Detection of specific markers in serum and cerebrospinal fluid (CSF) may indicate presence of particular diseases, allow disease monitoring, and provide accurate and rapid clinical prognosis. Small calcium‐binding protein S100b, neuron specific enolase (NSE), microtubule associated protein tau, and the phosphorylated form of the high‐molecular‐weight neurofilament subunit NF‐H (pNF‐H) have all been proposed Work done at the Nakayama Veterinary Hospital.
as biomarkers for various kinds of neuronal injury.3,4 S100b and tau are found in astrocytes or neurons, but also in other cell types.4,5 S100b and NSE have peak levels 6 hours after injury and return to baseline at 24 hours in rats with SCI.6 pNF‐H is found only in neurons and is quite resistant to proteases, so that pNF‐H released from damaged and diseased axons remains in CSF and serum in undegraded form.7,8 Serum pNF‐H levels correlate with severity of SCI and brain injury in a rat contusion model7,9; serum pNF‐H has an initial peak of expression at 16 hours after SCI in rats, and a second, usually larger, peak at 3 days.7 Thus, detection of this protein in serum might provide information about the extent of neuronal loss. We are unaware of any reports examining pNF‐H in dogs, so our purpose was to investigate whether serum pNF‐H levels in blood are correlated with prognosis of SCI in dogs. Our hypothesis was that dogs with high levels of pNF‐H between 1 and 3 days after injury would have a grave prognosis for ability to walk.
Veterinary Surgery 43 (2014) 289–293 © Copyright 2014 by The American College of Veterinary Surgeons
289
pNF‐H as a Prognostic Biomarker in Dogs With Spinal Cord Injury
MATERIALS AND METHODS Inclusion Criteria Dogs diagnosed with severe thoracolumbar IVDH (Grade 4 or 5), 1–3 days after injury, were prospectively enrolled through Nakayama Veterinary Hospital (April, 2009–December, 2012), affected dogs were excluded if they had other diseases. Control dogs were prospectively enrolled through Nakayama Veterinary Hospital and were eligible if they were healthy and had no IVDH history. All owners provided written consent for participation of their dogs in this study. Neurologic dysfunction was classified2 as: paraspinal hyperesthesia only (Grade 1), ambulatory paraparesis and ataxia (Grade 2), non‐ambulatory paraparesis (Grade 3), paraplegia with intact pain perception (Grade 4), paraplegia with no pain perception (Grade 5). Blood samples, collected before physical examination or surgery, were coagulated using a serum separator tube for 30 minutes and centrifuged at 3000 rpm for 10 minutes. Serum was frozen at 70°C and stored until assay. Diagnosis of thoracolumbar IVDH was made from CT scans and myelography, and was confirmed during decompressive surgery. Hemilaminectomy was performed on the side ipsilateral to the spinal cord compression and extruded disk material removed. Postoperative care included administration of cephalexin (22 mg/kg orally twice daily for 1 week) and buprenorphine (0.01–0.02 mg/kg subcutaneously once or twice daily for 1–3 days), management of urinary incontinence, and nursing care to prevent decubital ulcers.
Nishida et al.
standard curve was constructed by plotting mean absorbance of the standards against known concentrations of the standards on a logarithmic scale, using the 4 parameter algorithm. The concentration of each specimen was calculated from the standard curve. The limit of detection (23.5 pg/mL) was calculated from the mean absorbance of the blank (dilution buffer only) plus three standard deviations from the absorbance of the blank. We defined the limit of detection as 70 pg/mL because the sample was diluted threefold. Inter‐ and intra‐assay CVs for pNF‐H were
