Ken Kadoya

Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.

Despite advances in promoting axonal regeneration after acute spinal cord injury (SCI), elicitation of bridging axon regeneration after chronic SCI remains a formidable challenge. We report that combinatorial therapies administered 6 weeks, and as long as 15 months, after SCI promote axonal regeneration into and beyond a midcervical lesion site. Provision of peripheral nerve conditioning lesions, grafts of marrow stromal cells, and establishment of NT-3 gradients supports bridging regeneration. Controls receiving partial components of the full combination fail to exhibit bridging. Notably, intraneuronal molecular mechanisms recruited by delayed therapies mirror those of acute injury, including activation of transcriptional activators and regeneration-associated genes. Collectively, these findings provide evidence that regeneration is achievable at unprecedented postinjury time points.

Efficient retrograde neuronal transduction utilizing self-complementary AAV1.

Adeno-associated virus (AAV) is frequently used for gene transfer into the central nervous system (CNS). Similar to adenovirus and rabies virus, AAV can be taken up by axons and retrogradely transported, resulting in neuronal gene expression distant from the injection site. We investigated the retrograde transport properties of self-complementary AAV (scAAV) serotypes 1-6 following peripheral injection. Injection of scAAV into either rat extensor carpi muscle or sciatic nerve resulted in detectable retrograde vector transport and reporter gene expression in spinal cord motor neurons (MNs). Serotype 1 resulted in the highest level of retrograde transport, with 4.1 +/- 0.3% of cervical MNs projecting to the extensor carpi transduced following intramuscular injection, and 7.5 +/- 3.1% of lumbar MNs transduced after sciatic nerve injection. In contrast to scAAV1, retrograde transduction with scAAV2 was undetectable following intramuscular injection, and was detected in only 0.81 +/- 0.15% of MNs projecting to the sciatic nerve following intranerve injection. Furthermore, sciatic injection of single-stranded AAV1 required injection of tenfold higher numbers of viral particles for detectable transgene expression compared to scAAV1, and then only 0.91 +/- 0.24% of lumbar MNs were transduced. Our data provide the basis for increased retrograde transduction efficiency using peripheral injections of scAAV1 vectors for therapeutic gene delivery to the spinal cord.Adeno-associated virus (AAV) is frequently used for gene transfer into the central nervous system (CNS). Similar to adenovirus and rabies virus, AAV can be taken up by axons and retrogradely transported, resulting in neuronal gene expression distant from the injection site. We investigated the retrograde transport properties of self-complementary AAV (scAAV) serotypes 1-6 following peripheral injection. Injection of scAAV into either rat extensor carpi muscle or sciatic nerve resulted in detectable retrograde vector transport and reporter gene expression in spinal cord motor neurons (MNs). Serotype 1 resulted in the highest level of retrograde transport, with 4.1 ± 0.3% of cervical MNs projecting to the extensor carpi transduced following intramuscular injection, and 7.5 ± 3.1% of lumbar MNs transduced after sciatic nerve injection. In contrast to scAAV1, retrograde transduction with scAAV2 was undetectable following intramuscular injection, and was detected in only 0.81 ± 0.15% of MNs projecting to the sciatic nerve following intranerve injection. Furthermore, sciatic injection of single-stranded AAV1 required injection of tenfold higher numbers of viral particles for detectable transgene expression compared to scAAV1, and then only 0.91 ± 0.24% of lumbar MNs were transduced. Our data provide the basis for increased retrograde transduction efficiency using peripheral injections of scAAV1 vectors for therapeutic gene delivery to the spinal cord.

Artificial intervertebral disc replacement using bioactive three-dimensional fabric: design, development, and preliminary animal study.

Study Design. A new artificial intervertebral disc was developed, and its intrinsic biomechanical properties, bioactivity, and the effectiveness as a total disc replacement were evaluated in vitro and in vivo. Objectives. To introduce a new artificial intervertebral disc and to evaluate the in vitro mechanical properties, fusion capacity to bone, and segmental biomechanics in the total intervertebral disc replacement using a sheep lumbar spine. Summary of Background Data. The loss of biologic fusion at the bone–implant interface and prosthetic failures have been reported in previous artificial discs. There have been no clinically applicable discs with detailed experimental testing of in vivo mechanics and interface fusion capacity. Methods. The artificial intervertebral disc consists of a triaxial three-dimensional fabric (3-DF) woven with an ultra-high molecular weight polyethylene fiber, and spray-coated bioactive ceramics on the disc surface. The arrangement of weave properties was designed to produce mechanical behavior nearly equivalent to the natural intervertebral disc. Total intervertebral disc replacement at L2–L3 and L4–L5 was performed using 3-DF disc with or without internal fixation in a sheep lumbar spine model. The segmental biomechanics and interface histology were evaluated after surgery at 4 and 6 months. Results. The tensile-compressive and torsional properties of prototype 3-DF were nearly equivalent to those of human lumbar disc. The lumbar segments replaced with 3-DF disc alone showed a significant decrease of flexion–extension range of motion to 28% of control values as well as partial bony fusion at 6 months. However, the use of temporary fixation provided a nearly physiologic mobility of the spinal segment after implant removal as well as excellent bone–disc fusion at 6 months. Conclusion. An artificial intervertebral disc using a three-dimensional fabric demonstrated excellent in vitro and in vivo performance in both biomechanics and interface histology. There is a potential for future clinical application.

Clinical outcome of posterolateral endoscopic surgery for pyogenic spondylodiscitis: results of 15 patients with serious comorbid conditions.

Study Design. Clinical results of posterolateral endoscopic debridement and irrigation followed by percutaneous drainage for pyogenic spondylodiscitis were analyzed. Objectives. To report clinical results of transforaminal endoscopic surgery for pyogenic spondylodiscitis and to evaluate the effectiveness of this procedure in treatment of pyogenic spinal infections. Summary of Background Data. Pyogenic spinal infections have been increasing due to the development of medical treatment for patients with comorbid medical problems. Common treatments for spinal infections are administration of antibiotics or surgical debridement with bone grafts. There have been no reports, however, regarding the clinical outcome of posterolateral endoscopic treatment for pyogenic spinal infections. Methods. Fifteen consecutive patients with pyogenic spondylodiscitis in the thoracic or lumbar spine were enrolled. Preoperative antibiotic treatment had failed in all the patients. The procedures consisted of posterolateral endoscopic debridement and irrigation followed by percutaneous drainage through single portal under the combination of local and intravenous anesthesia. Pain response using visual analog scale (VAS, 0–100 mm), inflammation parameters, and duration of antibiotic therapy were investigated. Radiologic evaluation focused on bony fusion, local kyphosis, disc height reduction, and abscess formation. Results. All patients showed immediate pain reduction after surgery. Averaged VAS for pain was 86 before surgery and 25 at postoperative 1 week. Average of CRP was 4.00 mg/dL before surgery and 1.88 mg/dL at postoperative 1 week. Averaged duration of antibiotics therapy was 3.7 weeks. Spinal fusion was obtained in 13 patients. Two patients with neurologic deficits due to epidural abscess returned to normal. Preoperative psoas abscess in 6 patients disappeared after surgery on MRI. Conclusions. Posterolateral spinal endoscopic debridement and irrigation brought immediate pain reduction and good clinical results to patients who had comorbid medical problems and had pyogenic spondylodiscitis.

Biomechanical and Morphologic Evaluation of a Three-Dimensional Fabric Sheep Artificial Intervertebral Disc : In Vitro and In Vivo Analysis

Study Design. We have developed a new artificial intervertebral disc consisting of triaxial three-dimensional fabric for the sheep lumbar spine. To clarify the characteristics of the new implant, a series of biomechanical tests and morphologic evaluations were conducted. Objectives. To investigate the static, viscoelastic, and fatigue properties of the three-dimensional fabric disc in comparison with natural sheep disc and to evaluate their biomechanical and morphologic alteration in vivo. Summary of Background Data. In its human dimensions the three-dimensional fabric disc revealed mechanical properties similar to a natural human disc. Methods. The disc-body units from sheep spine and the sheep three-dimensional fabric discs underwent tensile-compressive (200 N), torsional (5 Nm), and creep-recovery tests (30 minutes–30 minutes, 200 N). After fatigue loading (2 million, compressive 200 N) the biomechanical changes and the debris were investigated. For in vivo evaluation after placing in the sheep psoas muscles for 6 months, the surface of the three-dimensional fabric disc was evaluated using macroscopy and scanning electron microscopy, followed by previous biomechanical tests. Results. The behavior of the sheep three-dimensional fabric disc was similar to that of natural sheep disc in tensile-compressive and creep-recovery tests. In torsional testing the behavior of natural sheep disc was more rigid than that of the sheep three-dimensional fabric disc. After fatigue loading there was no biomechanical change and no debris detected. Six months after surgery no morphologic deterioration was observed nor were there changes in biomechanical parameters. Conclusions. The sheep three-dimensional fabric disc exhibited biomechanical and morphologic biostability, appropriate viscoelasticity, and excellent fatigue properties. The three-dimensional fabric disc has a potential for clinical application of human intervertebral disc replacement.

Biomechanical evaluation of Anterior spinal instrumentation systems for scoliosis: In vitro fatigue simulation

Study Design. A biomechanical study was designed to assess the bone–screw interface fixation strength among five anterior spinal instrumentation systems for scoliosis before and after a fatigue simulation. Objectives. The objectives of the current study were twofold: 1) evaluate the static (initial) strength at the bone–screw interface and 2) evaluate dynamic (post fatigue) strength of the bone–screw interface after a fatigue simulation to investigate a possible mechanism for postoperative loss of correction. Summary of Background Data. Although the recent advancement of anterior instrumentation for scoliosis has permitted shorter fusion segments and improved surgical correction, the loss of correction over the instrumented segments still has been reported in one-rod systems. Little is known about the mechanism for loss of correction. Methods. Twenty-five fresh-frozen calf spines (T6–L6) were used. A total of five instrumentation systems included the following: Anterior ISOLA (ISOLA), Bad Wildungen Metz (BWM), Texas Scottish Rite Hospital system (TSRH), Cotrel–Dubousset Hoph (CDH), and Kaneda Anterior Scoliosis System (KASS). Screw pullout and rotational tests in the sagittal plane using a single vertebra were performed to investigate bone–screw interface fixation strength before and after a fatigue simulation. To simulate cyclic loading that the spine could undergo in vivo, a fatigue simulation using compressive–flexion loading up to 24,000 cycles was carried out. Results. Mean maximum tensile pullout force decreased in the following order: KASS > CDH > BWM > TSRH > ISOLA (F = 29.91, P < 0.0001). KASS blunt tip screw was 26% stronger in pullout force than KASS sharp tip screw (P < 0.05). The one-rod system demonstrated a positive correlation between pullout force and both bone mineral density and screw insertional torque. For fatigue analysis the rotational strength at the most cephalad and caudal segments significantly decreased after a fatigue simulation in the one-rod system (P < 0.05). The two-rod system showed no significant decrease after a fatigue simulation. Conclusions. Simulating the cyclic loading to the construct, screw loosening at the bone–screw interface was produced in the one-rod system. This screw loosening may elucidate one mechanism for loss of correction in the one-rod system. The two-rod system may have the potential to minimize the risk of loss of correction.

Axonal growth and connectivity from neural stem cell grafts in models of spinal cord injury.

Spinal cord injury (SCI) damages both gray matter and white matter, but white matter damage is responsible for the vast majority of the subsequent functional loss. Neural stem cells (NSCs) have been investigated as a means of improving outcomes after SCI, either through neuroprotective properties that limit secondary damage or by direct cell replacement. This review will focus on cell replacement strategies, and the ability of multipotent NSCs to form new functional synaptic relays across sites of even severe SCI. The ability of these early stage neurons to extend axons from the lesion site in large numbers and over long distances constitutes an important mechanism underlying their potential to promote neural repair.

Static and dynamic analysis of five anterior instrumentation systems for thoracolumbar scoliosis

Study Design. A nondestructive biomechanical investigation among five anterior spinal instrumentation systems for scoliosis. Objectives. The purpose of this study is to analyze the static and dynamic biomechanical stability of five different systems. Summary of Background Data. Although a variety of anterior spinal instrumentation systems for scoliosis are available, very few attempts have been made at comparative biomechanical studies. Methods. Thirty calf spines were underwent static biomechanical tests, including flexion–extension, axial rotation, and lateral bending loading modes in the multisegmental spinal model. Five anterior instrumentation systems included: 1) Texas Scottish Rite Hospital system; 2) Bad Wildungen Metz; 3) anterior ISOLA; 4) Cotrel–Dubousset Hoph; and 5) Kaneda Anterior Scoliosis System. The initial and postfatigue stability after a cyclic loading test were analyzed by measuring the range of motion at instrumented segments compared to the intact within the same specimen (% to intact). Results. Two-rod systems showed a significant decrease in range of motion compared to one-rod systems in flexion–extension (P < 0.001) and axial rotation (P < 0.05). In lateral bending, all systems demonstrated a significant decrease in range of motion of less than 40% to the intact (P < 0.001). After cyclical loading test, all systems increased in range of motion. In flexion–extension, one-rod systems depicted a significant increase in range of motion, compared to two-rod systems (P < 0.05). Conclusions. In the initial stability analysis, two-rod systems are superior to one-rod systems. For one-rod systems, repeated physiologic loading may result in reduced stability in flexion–extension.

Comprehensive Monosynaptic Rabies Virus Mapping of Host Connectivity with Neural Progenitor Grafts after Spinal Cord Injury

Summary Neural progenitor cells grafted to sites of spinal cord injury have supported electrophysiological and functional recovery in several studies. Mechanisms associated with graft-related improvements in outcome appear dependent on functional synaptic integration of graft and host systems, although the extent and diversity of synaptic integration of grafts with hosts are unknown. Using transgenic mouse spinal neural progenitor cell grafts expressing the TVA and G-protein components of the modified rabies virus system, we initiated monosynaptic tracing strictly from graft neurons placed in sites of cervical spinal cord injury. We find that graft neurons receive synaptic inputs from virtually every known host system that normally innervates the spinal cord, including numerous cortical, brainstem, spinal cord, and dorsal root ganglia inputs. Thus, implanted neural progenitor cells receive an extensive range of host neural inputs to the injury site, potentially enabling functional restoration across multiple systems.

Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts

Neural progenitor cell (NPC) transplantation has high therapeutic potential in neurological disorders. Functional restoration may depend on the formation of reciprocal connections between host and graft. While it has been reported that axons extending out of neural grafts in the brain form contacts onto phenotypically appropriate host target regions, it is not known whether adult, injured host axons regenerating into NPC grafts also form appropriate connections. We report that spinal cord NPCs grafted into the injured adult rat spinal cord self-assemble organotypic, dorsal horn-like domains. These clusters are extensively innervated by regenerating adult host sensory axons and are avoided by corticospinal axons. Moreover, host axon regeneration into grafts increases significantly after enrichment with appropriate neuronal targets. Together, these findings demonstrate that injured adult axons retain the ability to recognize appropriate targets and avoid inappropriate targets within neural progenitor grafts, suggesting that restoration of complex circuitry after SCI may be achievable.Understanding how transplanted cells interact with the host nervous system will be important for cell based neural regeneration approaches. Here, the authors study the sensory fate of neural progenitor cell grafts transplanted to the injured spinal cord, and show that host axons retain the ability to distinguish appropriate and inappropriate graft targets.