Spinal cord injuries often result in a grave prognosis for motor and sensory function. Common causes in dogs include vertebral fracture, IVDH, and ischemic myelopathy. In small animal practice, IVDH is the most common cause of SCI and is often detected in the thoracolumbar region.1 Unfortunately, it is difficult to identify the extent of spinal cord damage in dogs. A key criterion for use in assessing the severity of injury is evaluation of nociception in the pelvic limbs. In non-ambulatory dogs with IVDH, 86% to 96% of dogs with nociception regained the ability to walk after decompressive surgery2–5 compared with 41% to 62% of dogs that lacked nociception before surgery but regained the ability to walk after decompressive surgery6–10 Thus, evidence of nociception at the time of decompressive surgery can be a useful positive prognostic indicator. However, lack of nociception 2 weeks after decompressive surgery may not necessarily be a negative prognostic indicator. Investigators in one study8 found that only 1 of 10 dogs without nociception at 2 weeks after surgery recovered ambulation and nociception, and investigators in another study9 found that 8 of 17 dogs recovered ambulation despite a persistent lack of nociception. Ambulation observed in the latter report9 was believed to be spinal reflex walking that originated from the local spinal circuit, as opposed to the ambulation for dogs in the former report.8
Various kinds of cells have beneficial effects on the promotion of functional recovery and tissue repair in SCI. Cells examined include Schwann cells,11 neural stem cells,12 macrophages,13 olfactory ensheathing cells,14 and BMSCs.15 It has been reported that BMSCs, which comprise approximately 0.125% of the total marrow cells, can be isolated by exploiting their tendency to adhere to tissue culture plastics.16 Results of in vivo and in vitro experiments have indicated that BMSCs include a small number of mesenchymal stem cells.17
Several studies15,18–20 in rodents with experimentally induced contusions have revealed that BMSC transplantation improves functional recovery of animals with acute and chronic SCI. However, investigators in another study21 failed to detect similar benefits. In a study18 conducted by our research group, we found that BMSCs administered into the CSF promoted functional recovery in rats with SCI. Rats injected with BMSCs had much better gait performance than did control rats, with the Basso, Beattie, and Bresnahan score reaching a mean ± SEM of 13.9 ± 3.0 at 5 weeks after SCI in BMSC-injected rats; control rats had a mean score of 10.1 ± 2.1 at 5 weeks after SCI. The BMSCs that were transplanted into the CSF were conveyed via the CSF to the spinal cord (where most BMSCs attached to the spinal cord surface, with a few invading the lesion) and promoted tissue repair. These results indicated that infusion of BMSCs into the CSF could be applied in the murine species. The clinical administration of BMSCs via lumbar puncture to human patients with acute SCI has been reported by our research group.22 However, no studies have been conducted in dogs with regard to BMSC transplantation via lumbar puncture for treatment of paraplegia associated with SCI.
The objective of the study reported here was to determine whether injection of BMSCs into CSF could promote improvement of locomotor function with minimal complications in dogs with chronic SCI. Our hypothesis was that transplantation of autologous BMSCs into the CSF would promote locomotor and sensory function in dogs with chronic spinal cord injury.
Materials and Methods
Animals—Dogs that had been referred for treatment of severe IVDH to the Nakayama Veterinary Hospital between January 2005 and September 2008 were eligible for inclusion in the study. Dogs that remained paraplegic with no improvement in locomotion and a lack of nociception in the pelvic limbs for at least 1 month after decompressive surgery were included in the study. Miniature Dachshunds that weighed < 10 kg, had severe SCI caused by thoracolumbar IVDH (T10 through L3), and had no history of other diseases were included. All owners provided written consent for inclusion of the dogs in the study. The study was performed in accordance with the Japanese Regulations for Animal Welfare issued by The Ministry of Education, Culture, Sports, Sciences, and Technology of Japan.
Clinical procedures—The diagnosis of thoracolumbar IVDH was made on the basis of myelography and confirmed during decompressive surgery. Dogs were administered atropine (0.05 mg/kg, IV) and diazepam (0.2 mg/kg, IV). Anesthesia was induced with propofol (5.0 mg/kg, IV) and maintained by administration of isoflurane. Physical conditions were continuously monitored via an ECG and measurement of Paco2. Myelography was performed on anesthetized dogs by use of iotrolana (0.45 mL/kg) injected via lumbar puncture. Multiple myelographic views were obtained after the iotrolan injection for evaluation of compressive disk materials located circumferentially around the spinal cord in the affected area.23
Decompressive surgery was performed immediately after myelography. Methylpredonisolone sodium succinate (30 mg/kg, IV), cefazolin (30 mg/kg, IV), and fentanyl (3 to 5 mg/kg/h, IV) were administered before surgery. Hemilaminectomy was performed on the side ipsilateral to the spinal cord compression, as determined by the results of myelography. Extruded disk material was removed. Postoperative management included the administration of antimicrobials and analgesics, management of urinary incontinence, and nursing care to prevent decubital ulcers. All dogs were managed in the same way with respect to rehabilitation during the 6-month postoperative period.
One month after decompressive surgery, dogs that were paraplegic with no improvement in locomotion and a lack of nociception in the pelvic limbs were assigned to transplantation (n = 10 dogs) or control (13) groups such that there would be no differences in age, body weight, and site of disk herniation. Power analysis indicated that a sample size of 17 dogs in each group (34 total) would be needed to detect differences of 45% in recovery of motor function with a power of 0.80 and α of 0.05.b
Transplantation of BMSCs—Although all owners were informed about BMSC transplantation 1 month after decompressive surgery, the time of owner consent, and hence BMSC transplantation, varied. Therefore, dogs received BMSC transplants between 1 and 3 months after decompressive surgery.24 Control dogs did not receive sham lumbar punctures or injections because the clients did not agree to the sham treatments.
Isolation and culture of BMSCs—The bone marrow of the femur was aspirated by use of a perfusion method described in other studies.24,25 Briefly, 2 sterilized 13-gauge bone marrow biopsy needlesc were inserted into the proximal and distal ends of the femur. The needle in the proximal end of the femur was connected to a syringe that contained 50 mL of physiologic saline (0.9% NaCl) solution with heparin.d The needle in the distal end of the femur was connected via an extension tube to a 50-mL syringe to collect bone marrow fluid. The physiologic saline solution was infused gently into the bone marrow cavity to flush bone marrow cells toward the other end of the femur for collection. Extracted bone marrow cells were collected in a syringe. This process was repeated twice for each dog.
Bone marrow perfusate was centrifuged, and pelleted cells were suspended in 15 mL of Dulbecco PBS solution.e Cell suspensions were loaded onto a lymphocyte separation solutione (15 mL) and centrifuged at 400 × g for 30 minutes at 20°C. The buffy coat at the interface was collected, mixed with 20 mL of Dulbecco PBS solution, and centrifuged again at 300 × g for 5 minutes. Pelleted cells were washed twice with Dulbecco PBS solution. The bone marrow cells obtained were plated on three 25-cm2 tissue culture flasksf in Dulbecco modified Eagle mediume that contained 10% fetal bovine serumg and 1% antimicrobial-antimycotic solution.e Nonadherent cells were removed by replacing the medium 48 hours after initial plating.
Injection of BMSCs—Autologous BMSCs were injected into the CSF 3 times at 1-week intervals, as described in another study.24 Briefly, BMSCs were obtained 1, 2, and 3 weeks after the start of culture; cells were removed from culture plates via incubation with 0.25% trypsin and 1mM EDTAe at 37°C. The number of cells was counted by use of a hemacytometer. Number of transplanted BMSCs ranged from 0.3 × 105 cells to 3.0 × 106 cells (median, 1.3 × 106 cells) at each time point. The total number of transplanted BMSCs ranged from 1.4 × 106 cells to 5.6 × 106 cells (median, 4.7 × 106 cells). Dogs were treated with atropine and diazepam, and anesthesia was induced with propofol and maintained by administration of isoflurane. A 23-gauge spinal needle was inserted by use of fluoroscopic monitoring into the subarachnoid space between L5 and L6. Intrathecal placement of the needle was ensured by outflow of CSF. The BMSCs were suspended in 0.5 mL of physiologic saline solution and injected into the CSF slowly over a 5-minute period, and then were theoretically conveyed through the CSF to the lesion.
Clinical evaluation and TSCIS—Physical and neurologic examinations of dogs were performed every month for ≥ 6 months; the endpoint of the study was 6 months. Time 0 for the control group was the day of the decompressive surgery, whereas time 0 for the transplantation group was the day of the last of the 3 BMSC injections. The referring veterinarians, who were unaware of the treatment group for each dog, video recorded the dogs and evaluated the behaviors and assessed proprioception and nociception. Dogs were identified as serial numbers for the investigators, who also were unaware of the treatment group for each dog. The investigators, who were not the personnel who cultured the BMSCs, also scored the dogs. Successful recovery was defined as a dog that could walk without assistance.
Investigators also evaluated improvement of function by use of the TSCIS.26 This system involves evaluation of each pelvic limb separately and has 3 components: gait, proprioceptive positioning, and nociception. All components were based on standard clinical neurologic examination techniques for dogs.27 Gait scores for pelvic limbs ranged from 0 to 6, as determined on the basis of clinically relevant movement of the limbs. Dogs were categorized as grade 0 (no voluntary movement), grade 1 (protraction with no ground clearance), grade 2 (protraction with inconsistent ground clearance), grade 3 (protraction with ground clearance in > 75% of steps), grade 4 (ambulatory with moderate paresis-ataxia), grade 5 (ambulatory with mild paresis-ataxia), or grade 6 (normal gait). Proprioceptive positioning (also referred to as knuckling) was used as a postural reaction test (score of 0 to 2). Lack of a response was assigned a score of 0. Dogs that corrected limb positions after a prolonged period (> 2 seconds) were referred to as a delayed response and assigned a score of 1. Dogs were judged to be clinically normal and assigned a score of 2 if they were able to correct the limb positions immediately. Nociception was assessed by applying a painful stimulus (clamping a hemostat on the distal aspect of a limb or on a nail bed of a digit) and observing the dog for physiologic (tachycardia, tachypnea, and mydriasis) or behavioral (orientation toward the stimulus, vocalization, and licking) responses. Dogs were scored as having normal nociception (score, 2) or no nociception (score, 0). Dogs were assigned scores of 1 to 10 for each pelvic limb; thus, the maximum combined limb score was 20.
Statistical analysis—Distributions of age and body weight of dogs were compared between the 2 groups by use of a Student t test. Recovery rate of locomotor function was analyzed by use of a Fisher exact test. Scores of the TSCIS were compared between the 2 groups by use of a Mann-Whitney U test. Values of P < 0.05 were considered significant.
Results
The transplantation group consisted of 10 dogs (3 males and 7 females) that were 4 to 7 years old (median, 4.5 years) and weighed 3.2 to 8.8 kg (median, 5.2 kg). The sites of disk herniation ranged from T10–11 to L1–2 (Figure 1). The control group consisted of 13 dogs (11 males and 2 females) that were 3 to 9 years old (median, 5 years) and weighed 3.3 to 10.0 kg (median, 5.5 kg). The sites of disk herniation ranged from T10–11 to L2–3. There was no significant difference in age (P = 0.88) or body weight (P = 0.77) between the transplantation and control groups.
Sites of disk herniation in 23 dogs with thoracolumbar IVDH resulting in paraplegia and loss of pelvic limb nociception that persisted for at least 1 month after decompressive surgery. Dogs were assigned to 2 groups (10 dogs that received BMSC transplantation [black bars] and 13 control dogs that received no additional treatment [white bars]). Two dogs in the transplantation group and 3 dogs in the control group had herniated disks at 2 sites.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1118
Sites of disk herniation in 23 dogs with thoracolumbar IVDH resulting in paraplegia and loss of pelvic limb nociception that persisted for at least 1 month after decompressive surgery. Dogs were assigned to 2 groups (10 dogs that received BMSC transplantation [black bars] and 13 control dogs that received no additional treatment [white bars]). Two dogs in the transplantation group and 3 dogs in the control group had herniated disks at 2 sites.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1118
Sites of disk herniation in 23 dogs with thoracolumbar IVDH resulting in paraplegia and loss of pelvic limb nociception that persisted for at least 1 month after decompressive surgery. Dogs were assigned to 2 groups (10 dogs that received BMSC transplantation [black bars] and 13 control dogs that received no additional treatment [white bars]). Two dogs in the transplantation group and 3 dogs in the control group had herniated disks at 2 sites.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1118
Transplantation of BMSCs was performed between 1 and 3 months (mean, 51.5 days) after decompressive surgery. The BMSC group was evaluated for 6 to 35 months (median, 23 months) after BMSC transplantation. The control group was evaluated for 7 to 36 months (median, 24.5 months) after decompressive surgery.
Dogs were paraplegic with loss of nociception in pelvic limbs at the time of BMSC transplantation. Six of 10 dogs in the transplantation group regained the ability to walk, which was significantly (P = 0.037) higher than the proportion of dogs in the control group (2/13) that regained the ability to walk. The 6 dogs in the transplantation group that regained the ability to walk were 4 to 7 years old, whereas the 2 dogs in the control group that regained the ability to walk were 3 and 5 years old. There was no association between age and functional recovery. In the transplantation group, dogs regained the ability to walk 0.5 to 4 months (mean, 2.1 months) after BMSC transplantation. Two of those 6 dogs had further improvement of coordination between the thoracic and pelvic limbs. In contrast, the 2 dogs in the control group regained the ability to walk at 2.5 and 3 months after decompressive surgery; however, they did not have further improvements in coordination between the thoracic and pelvic limbs. There was a significant (P = 0.026) difference in TSCIS score between the transplantation and control groups at the endpoint of the study (6 months after BMSC transplantation or after decompressive surgery for the transplantation and control groups, respectively; Table 1).
Mean (range) TSCIS scores of 23 dogs with thoracolumbar IVDH resulting in paraplegia and loss of pelvic limb nociception that persisted for at least 1 month after decompressive surgery.
| Time (mo) | Control group* | |
|---|---|---|
| 0† | 0 (0) | 0.8 (0–2) |
| 0.5 (0–2) | 3.2 (0–8) | |
| 2 | 1.2 (0–2) | 5.0 (2–12) |
| 3 | 2.3 (0–6) | 5.4 (2–14) |
| 4 | 2.3 (0–6) | 5.6 (2–14) |
| 5 | 2.3 (0–6) | 5.6 (2–14) |
| 6‡ | 2.3 (0–6) | 5.6 (2–14) |
The control group comprised 13 dogs that received no additional treatment, whereas the transplantation group comprised 10 dogs that received BMSC transplantation (0.3 × 105 BMSCs to 3.0 × 106 BMSCs [median, 1.3 × 106 BMSCs]) 3 times at 1-week intervals beginning 1 to 3 months after decompressive surgery.
Time 0 for the control group was the day of the decompressive surgery, whereas time 0 for the transplantation group was the day of the last of the 3 BMSC injections.
End point of the study.
All dogs had fecal and urinary incontinence. Only 1 dog (transplantation group) recovered nociception. No complications, including signs of pain, aggravation of neurologic function, or tumorigenesis, were detected in association with injection of BMSCs for > 6 months after transplantation.
Discussion
To our knowledge, there have been no reports about BMSC transplantation for the treatment of paraplegic dogs with chronic SCI. The study reported here revealed that improvement of locomotor function can be achieved by use of BMSCs injected into the CSF of dogs with chronic SCI. No adverse effects of BMSC transplantation were detected during the study.
In the present study, 1 of 10 dogs that received BMSCs recovered nociception, whereas none of the 13 control dogs recovered nociception. This is consistent with the observation that BMSC transplantation in rats had no effect on sensory function.28 Although no significant differences in recovery of nociception were detected between the transplantation and control groups, it is possible that use of a larger population may have enhanced our ability to detect whether BMSCs would impact nociceptive recovery. Power analysis indicated that a sample size of 200 dogs in each group (400 dogs total) would be needed to detect differences of 10% in recovery of sensory function (power of 0.80 and α of 0.05). It is clear that a larger cohort would be needed.
It has been reported8 that the prognosis is poor when nociception does not return within 2 weeks after injury. Therefore, dogs that were paraplegic with no sign of improvement in pelvic limb locomotion and nociception for at least 1 month after decompressive surgery were used in the present study. Two of 13 dogs in the control group regained the ability to walk < 3 months after decompressive surgery. In another study9 with a population of 64 dogs with IVDH that initially lacked nociception, 9 were euthanatized within 3 weeks after surgery, 37 regained nociception and the ability to walk, and 18 survived but never regained nociception. Of those 18 dogs, 7 eventually regained the ability to walk after decompressive surgery. The interval until those 7 dogs regained the ability to walk ranged from 16 to 72 weeks (mean, 37.6 weeks). The recovery rate for dogs that lived and never regained nociception (7/18) in that study9 was higher than the recovery rate for the control group of the present study (2/13) and that for dogs that could walk (1/10) in another report.8 It is difficult to compare recovery rates among studies because of the numerous variables, such as inclusion criteria, type of surgery performed, assessment of nociception, euthanasia, and the endpoint used for the outcome. In the present study, no dogs were euthanatized after surgery. Monitoring the dogs for a longer period may have changed the outcomes in the present study. The most likely explanation for the conflicting findings is a small number of dogs in each study population.
In the present study, all dogs were managed in the same way with respect to rehabilitation. Training on a treadmill, which is a more aggressive form of rehabilitation, enhances the recovery of motor function in rodents with experimentally induced contusions.29 Cell transplantation plus training on a treadmill may promote more substantial beneficial effects of functional recovery in dogs.
Recovery of locomotor function in dogs without nociception may be a result of development of spinal reflex walking. Spinal reflex walking is believed to be attributable to the central pattern generator of the spinal cord.30 In one report,31 dogs walking apparently via spinal reflex became nonambulatory following transection of the spinal cord proximal to the site of the original injury, which suggests that ambulation depends, at least in part, on intact axons crossing the region of the original spinal cord lesion. In the study reported here, 2 dogs in the transplantation group were able to walk and had coordination of the thoracic and pelvic limbs, which suggested some contribution of axonal connections within the injured spinal cord. The preservation of as few as 5% to 10% of the descending axons is adequate to drive the local circuits responsible for basic locomotion.32
Various techniques, including evaluation of motor-evoked potentials and magnetic resonance imaging,33,34 have been used to objectively assess SCI. Magnetic resonance imaging and evaluation of sensory-evoked potentials can be performed in lightly sedated dogs, but the obtained variables do not necessarily reflect actual functional improvement.34–36 Therefore, direct functional evaluation is considered more practical and useful. The TSCIS is a method for assessment of gait, proprioceptive positioning, and nociception. The TSCIS score has been associated with magnetic resonance signal characteristics within the spinal cord of dogs with disk herniation, and inter-rater agreement is high.26 In the present study, the TSCIS was used for evaluation of functional improvement. However, a technique for computerized gait analysis in dogs has been reported.37 This method may be used to detect more subtle changes in gait than would the TSCIS and could provide a continuous, rather than an ordinal, measure.
Mesenchymal stem cells are readily obtained from the bone marrow by aspiration; they can then be cultured to large quantities and used as autologous transplants for the treatment of SCI. They have no harmful immunologic reactions. Currently, they are considered to be one of the cells most useful for the clinical treatment of SCI.16 Other cellular treatments are currently being investigated and may hold equal or greater promise.
Transplantation of BMSCs improves motor function in rats with acute and chronic paraplegia.15,19,20,38 Bone marrow stromal cells could differentiate into neurons and glial cells following transplantation and promote motor function.20,38 In another study18 conducted by our research group in rats with SCI, BMSCs administered into the CSF were conveyed through the CSF to the spinal cord (where most BMSCs attached to the spinal cord near the surface, with a few invading the lesion) and promoted tissue repair and functional recovery. However, BMSCs on the spinal cord as well as within the lesion disappeared by 3 weeks after injection.18 Bone marrow stromal cells did not differentiate into neural cells in the host tissue as determined by results of immunohistochemical analysis.15 Bone marrow stromal cells protect and repair damaged neurons in vitro via production of growth factors.39 These facts suggest that the effects of BMSCs may be caused, at least in part, by trophic factors released from BMSCs that rescue neuronal tissue from degeneration and promote axonal regeneration. Identifying compounds that BMSCs release into the CSF will be important in further elucidating the mechanisms by which BMSCs act at the SCI site.
Analysis of results of the present study suggests that BMSC transplantation via the CSF is effective even in dogs with chronic paraplegia. It required a long time (approx 1 month) before the effects became manifested after BMSC transplantation in the present study. This may have been the result of delayed tissue repair in dogs with chronic SCI. A 3-injection regimen (1 injection/wk for 3 weeks), which was used in the present study, may yield beneficial effects in dogs with chronic paraplegia. Engrafting of cells directly into the lesion may risk additional damage to the spinal cord. Unfortunately, we do not have evidence that the BMSCs reached the spinal cord lesion. It is indicated that BMSCs delivered via lumbar puncture, which is the same as our BMSC injection, reached the lesion and promoted tissue repair in rodents with experimentally induced contusions.40 Cell injection into the CSF via lumbar puncture may be a safer and more effective method of cell transplantation.
Because the present study used client-owned dogs, there were limitations. Histologic as well as immunohistochemical data were not available; therefore, the behavior of regenerating axons within the lesion and the final outcome of engrafted BMSCs are unknown. Additional limitations include the study design (small single-center population, and investigators were aware of the treatments received by each dog) and the lack of a sham-control group. The recovery rate of motor function in a sham-control group (ie, dogs injected with saline solution) may have been higher than that for the control group used in the present study. Performing lumbar puncture and transiently changing CSF flow dynamics could affect syrinxes and influence outcome.41 Although the effect of repeated anesthesia on dogs was not determined, it has been reported in another study42 that treatment with propofol does not significantly affect CSF dynamics in rabbits.
To our knowledge, the study reported here is the first in which treatment of SCI via injection of BMSCs into the CSF has been evaluated. No complications were detected that might have been caused by the administration of BMSCs into the CSF. Transplantation of BMSCs into the CSF could be a useful treatment to improve locomotor function in dogs with chronic SCI.
ABBREVIATIONS
| BMSC | Bone marrow stromal cell |
| IVDH | Intervertebral disk herniation |
| SCI | Spinal cord injury |
| TSCIS | Texas Spinal Cord Injury Scale |
Isovist, 240 mg of iodine/mL, Schering, Osaka, Japan.
StatMate, GraphPad Software Inc, San Diego, Calif.
13-gauge Jamshidi needle, Allegiance Healthcare Corp, McGaw Park, Ill.
Novo Heparin 1000, Hoechst Marion Roussel Co Ltd, Tokyo, Japan.
Nakarai Tesque Inc, Kyoto, Japan.
Asahi Techno Glass, Chiba, Japan.
ICN Biomedicals Inc, Aurora, Ohio.
References
- 2.↑
Davis GJ, Brown DC. Prognostic indicators for time to ambulation after surgical decompression in nonambulatory dogs with acute thoracolumbar disk extrusions: 112 cases. Vet Surg 2002; 31: 513–518.
- 3.
Ferreira AJ, Correia JH, Jaggy A. Thoracolumbar disc disease in 71 paraplegic dogs: Influence of rate of onset and duration of clinical signs on treatment results. J Small Anim Pract 2002; 43: 158–163.
- 4.
Necas A. Clinical aspects of surgical treatment of thoracolumbar disc disease in dogs. A retrospective study of 300 cases. Acta Vet Brno 1999; 68: 121–130.
- 5.
Ruddle TL, Allen DA, Schertel ER, et al. Outcome and prognostic factors in non-ambulatory Hansen type I intervertebral disc extrusions: 308 cases. Vet Comp Orthop Traumatol 2006; 19: 29–34.
- 6.
Duval J, Dewey C, Roberts R, et al. Spinal cord swelling as a myelographic indicator of prognosis: a retrospective study in dogs with intervertebral disc disease and loss of deep pain perception. Vet Surg 1996; 25: 6–12.
- 7.
Ito D, Matsunaga S, Jeffery ND, et al. Prognostic value of magnetic resonance imaging in dogs with paraplegia caused by thoracolumbar intervertebral disk extrusion: 77 cases (2000–2003). J Am Vet Med Assoc 2005; 227: 1454–1460.
- 8.↑
Laitinen OM, Puerto DA. Surgical decompression in dogs with thoracolumbar intervertebral disc disease and loss of deep pain perception: a retrospective study of 46 cases. Acta Vet Scand 2005; 46: 79–85.
- 9.↑
Olby N, Levine J, Harris T, et al. Long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: 87 cases (1996–2001). J Am Vet Med Assoc 2003; 222: 762–769.
- 10.
Scott HW, McKee WM. Laminectomy for 34 dogs with thoracolumbar intervertebral disc disease and loss of deep pain perception. J Small Anim Pract 1999; 40: 417–422.
- 11.↑
Martin D, Robe P, Franzen R, et al. Effects of Schwann cell transplantation in a contusion model of rat spinal cord injury. J Neurosci Res 1996; 45: 588–597.
- 12.↑
Ogawa Y, Sawamoto K, Miyata T, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 2002; 69: 925–933.
- 13.↑
Knoller N, Auerbach G, Fulga V, et al. Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: phase 1 study results. J Neurosurg Spine 2005; 3: 173–181.
- 14.↑
Collazos-Castro JE, Muñetón-Gomez VC, Nieto-Sampedro M. Olfactory glia transplantation into cervical spinal cord contusion injuries. J Neurosurg Spine 2005; 3: 308–317.
- 15.↑
Wu S, Suzuki Y, Ejiri Y, et al. Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord. J Neurosci Res 2003; 72: 343–351.
- 16.↑
Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal cells: nature, biology, and potential applications. Stem Cells 2001; 19: 180–192.
- 17.↑
Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71–74.
- 18.↑
Ohta M, Suzuki Y, Noda T, et al. Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation. Exp Neurol 2004; 187: 266–278.
- 19.
Hofstetter CP, Schwarz EJ, Hess D, et al. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A 2002; 99: 2199–2204.
- 20.
Zurita M, Vaquero J. Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation. Neuroreport 2004; 19: 1105–1108.
- 21.↑
Nandoe Tewarie RD, Hurtado A, Ritfeld GJ, et al. Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation into the contused adult rat thoracic spinal cord. J Neurotrauma 2009; 26: 2313–2322.
- 22.↑
Saito F, Nakatani T, Iwase M, et al. Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report. J Trauma 2008; 64: 53–59.
- 23.↑
Tanaka H, Nakayama M, Takase K. Usefulness of myelography with multiple views in diagnosis of circumferential location of disc material in dogs with thoracolumbar intervertebral disc herniation. J Vet Med Sci 2004; 66: 827–833.
- 24.↑
Harada Y, Nakayama M, Tanaka H, et al. Clinical use of bone marrow derived mesenchymal stromal cells auto-transplantation for delayed union cases of a cat and a dog. Jpn J Anesth Surg 2006; 37: 79–84.
- 25.
Kushida T, Inaba M, Ikebukuro K, et al. A new method for bone marrow cell harvesting. Stem Cells 2000; 18: 453–456.
- 26.↑
Levine GJ, Levine JM, Budke CM, et al. Description and repeatability of a newly developed spinal cord injury scale for dogs. Prev Vet Med 2009; 89: 121–127.
- 27.↑
de Lahunta A. General somatic afferent system-GSA. In: de Lahunta A, ed. Veterinary neuroanatomy and clinical neurology. Philadelphia: WB Saunders Co, 1983; 166–169.
- 28.↑
Himes BT, Neuhuber B, Coleman C, et al. Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord. Neurorehabil Neural Repair 2006; 20: 278–296.
- 29.↑
Heng C, de Leon RD. Treadmill training enhances the recovery of normal stepping patterns in spinal cord contused rats. Exp Neurol 2009; 216: 139–147.
- 30.↑
Bregman BS, Kunkel-Bagden E, Reier PJ, et al. Recovery of function after spinal cord injury: mechanisms underlying transplant-mediated recovery of function differ after spinal cord injury in newborn and adult rats. Exp Neurol 1993; 123: 3–16.
- 32.↑
Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 1996; 139: 244–256.
- 33.
Sylvestre AM, Cockshutt JR, Parent JM, et al. Magnetic motor evoked potentials for assessing spinal cord integrity in dogs with intervertebral disk disease. Vet Surg 1993; 22: 5–10.
- 34.
Shepard MJ, Bracken MB. Magnetic resonance imaging and neurological recovery in acute spinal cord injury: observations from the National Acute Spinal Cord Injury Study 3. Spinal Cord 1999; 37: 833–837.
- 35.
Poncelet L, Michaux C, Balligand M. Somatosensory potentials in dogs with naturally acquired thoracolumbar spinal cord disease. Am J Vet Res 1993; 54: 1935–1941.
- 36.
Poncelet L, Michaux C, Balligand M. Study of spinal cord evoked injury potential by use of computer modeling and in dogs with naturally acquired thoracolumbar spinal cord compression. Am J Vet Res 1998; 59: 300–306.
- 37.↑
Hamilton L, Franklin RJ, Jeffery ND. Development of a universal measure of quadrupedal forelimb-hindlimb coordination using digital motion capture and computerized analysis. BMC Neurosci 2007; 8: 77.
- 38.
Chopp M, Zhang XH, Li Y, et al. Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport 2000; 11: 3001–3005.
- 39.↑
Hokari M, Kuroda S, Shichinohe H, et al. Bone marrow stromal cells protect and repair damaged neurons through multiple mechanisms. J Neurosci Res 2008; 86: 1024–1035.
- 40.↑
Paul C, Samdani AF, Betz RR, et al. Grafting of human bone marrow stromal cells into spinal cord injury: a comparison of delivery methods. Spine 2009; 34: 328–334.
- 41.↑
Higuchi H, Adachi Y, Kazama T. Effects of epidural saline injection on cerebrospinal fluid volume and velocity waveform: a magnetic resonance imaging study. Anesthesiology 2005; 102: 285–292.
- 42.↑
Momota Y, Artru AA, Powers KM, et al. Posttreatment with propofol terminates lidocaine-induced epileptiform electroencephalogram activity in rabbits: effects on cerebrospinal fluid dynamics. Anesth Analg 1998; 87: 900–906.
