L5-L6 Level Research Articles

Innovative surgical and stress-stimulated rat model of ligamentum flavum hypertrophy

Background and purposeAnimal models of LFH are still in the exploratory stage. This study aimed to establish a reliable, efficient, and economical model of LFH in rats for the study of human ligamentum flavum (LF) pathological mechanisms, drug screening, development, improvement of surgical treatment, disease prevention, and other aspects.Methods and materialsForty rats were divided into an experimental group and a sham group of 20 rats. The experimental group (n = 20) was treated with an innovative operation combined with stress stimulation at the L5-L6 segments, the L5 and L6 spinous processes, transverse processes, and supraspinous ligaments were excised, along with removal of the paraspinal muscles at the L5-L6 level. One week after surgery, the rats were subjected to slow treadmill running daily. In the experimental group (n = 20), the spinous process, transverse process, supraspinous ligament and paraspinous muscle of L5 and L6 were excised. And for a week after the surgery, the rats ran on a treadmill at a slow pace every day. While the sham group (n = 20) was treated with sham operation only. Seven weeks later, MRI, immunohistochemistry (IHC), and western blot (WB) will be performed on the LF of the L5-6 segment in the two groups of rats.ResultsMRI results showed that the LF in the experimental group was significantly thicker than that in the sham group. Masson staining results indicated that LF thickness, collagen fiber area, and collagen volume fraction (CVF) were significantly higher in the experimental group than in the sham group. IHC and WB showed that the expression of TGF-β1, COL1, and IL-1β in the LF of the experimental group was significantly higher than that in the LF of sham group.ConclusionThrough innovative surgical intervention combined with stress stimulation, a relatively reliable, efficient, and convenient rat LFH model was established.

Attenuation of the degenerative effects of endothelin-1 on cartilaginous end plate cells by the endothelin receptor antagonist BQ-123 via the Wnt/β-catenin signaling pathway

Background ContextEndothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown. PurposeThe purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway. Study Design/SettingThe influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated. MethodsTo establish a model for CEP degeneration, three lumbar discs (L3–L4, L4–L5, and L5–L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/β-catenin signaling pathway was also investigated by Western blotting. ResultsAfter 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect. ConclusionsEndothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.

Enhancement of posterolateral lumbar spine fusion using recombinant human bone morphogenetic protein-2 and mesenchymal stem cells delivered in fibrin glue.

Mesenchymal stem cells have shown great potential for accelerating bone healing. In the present study, we evaluate the efficacy of fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 composite for posterolateral spinal fusion in a rabbit model. Forty adult rabbits underwent posterolateral intertransverse fusion at the L5-L6 level. The animals were randomly divided into four groups based on the implant material: fibrin glue, fibrin glue/mesenchymal stem cells composite, fibrin glue-recombinant human bone morphogenetic protein-2 (fibrin glue/recombinant human bone morphogenetic protein-2) composite, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 composite. After six weeks, the rabbits were euthanized for manual palpation, radiographic examination, biomechanical testing, and histology. Manual palpation results showed that the fusion rate for fibrin glue, fibrin glue/mesenchymal stem cells, fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 was 0, 0, 40%, and 70%, respectively. Moreover, fusion rate determined by radiographic examination for fibrin glue, fibrin glue/mesenchymal stem cells, fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 was 0, 0, 40%, and 80%, respectively. Gray analysis showed that fibrin glue/recombinant human bone morphogenetic protein-2 group had higher ossification area and density than fibrin glue group; and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group had higher ossification area and density than fibrin glue/recombinant human bone morphogenetic protein-2 group. Formation of continuous bone masses between L5 and L6 level in mesenchymal stem cells/recombinant human bone morphogenetic protein-2/fibrin glue group was further confirmed by computed tomography scanning and three-dimensional reconstruction. Biomechanical testing demonstrated that the fusion strength (flexion, extension, lateral bending, and axial rotation) in fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group is significantly higher than that in fibrin glue/recombinant human bone morphogenetic protein-2 group. The formation of mature bone tissues between transverse processes of the fused specimens from both fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 groups was confirmed by HE staining, and quantitative real-time polymerase chain reaction results showed the upregulation of CD31, type I collagen, osteocalcin, and osteonectin in the fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group. In conclusion, our findings show that mesenchymal stem cells delivered with recombinant human bone morphogenetic protein-2 using fibrin glue as carrier are more effective in enhancing spine fusion than recombinant human bone morphogenetic protein-2 without mesenchymal stem cells in the rabbit model.

Quantitative Study of Parathyroid Hormone (1-34) and Bone Morphogenetic Protein-2 on Spinal Fusion Outcomes in a Rabbit Model of Lumbar Dorsolateral Intertransverse Process Arthrodesis

A posterolateral rabbit spinal fusion model was used to evaluate the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and teriparatide (PTH [1-34]) used individually and in combination on spinal fusion outcomes. To test the efficacy of parathyroid hormone on improving spinal fusion outcomes when used with BMP-2. Of the more than 250,000 spinal fusion surgical procedures performed each year, 5% to 35% of these will result in pseudarthrosis. Growing controversy on the efficacy and cost of rhBMP-2 for improving spinal fusion outcomes has presented a challenge for clinicians. Research into PTH as an adjunct therapy to rhBMP-2 for spinal fusion has not yet been investigated. Forty-eight male New Zealand white rabbits underwent bilateral posterolateral intertransverse process arthrodesis surgery at the L5-L6 level. Animals were divided into 6 groups. Two groups were treated with autograft alone or autograft and PTH (1-34), whereas the other 4 groups were treated with low-dose rhBMP-2 alone, high-dose rhBMP-2 alone, or either dose combined with PTH (1-34). All animals were euthanized 6 weeks after surgery. The L4-L7 spinal segment was removed and assessed using manual palpation, computed tomography (CT), and biomechanical testing. CT assessments revealed fusion in 50% of autograft controls, 75% of autograft PTH (1-34) animals, 87.5% in the 2 groups treated with low-dose rhBMP-2, and 100% in the 2 groups treated with high-dose rhBMP-2. CT volumetric analysis demonstrated that all groups treated with biologics had fusion masses that were on average significantly larger than those observed in the control group (P < 0.0001). Biomechanical data demonstrated no statistical difference between controls, PTH (1-34), and low-dose rhBMP-2 in any testing orientation. PTH (1-34) did not increase bending stiffness when used adjunctively with either low-dose or high-dose rhBMP-2. Although intermittent teriparatide administration results in increased fusion mass volume, it does not improve biomechnical stiffness over use of autograft alone. When delivered concurrently with high- and low-dose rhBMP-2, teriparatide provided no statistically significant improvement in biomechanical stiffness. N/A.

Assessment of in vivo spinal cord conduction velocity in rats in an experimental model of ischemic spinal cord injury

Experimental laboratory investigation of spinal cord conductivity alterations in a rat model of ischemic spinal cord injury (SCI). To observe the epidural spinal cord stimulation-induced electromyography responses, and to investigate the possible alterations of spinal cord conduction velocity (SCCV) and compound muscle action potentials (CMAPs) after ischemic SCI in rats. Adnan Menderes University, Institute of Health Science, Aydin, Turkey. SCI was induced by transient occlusion of the abdominal aorta in male Sprague-Dawley rats. Spinal cord histopathology was examined to determine neuronal damage and Tarlov scale was used to grade locomotor functions. Epidural electrical stimulation of spinal cord was performed by monopolar needle electrodes sequentially at L1-L2 and L5-L6 levels, and CMAPs were recorded from the left gastrocnemius muscle by surface electrodes. Amplitudes and durations of CMAPs were evaluated and SCCVs were calculated by analyzing the latency difference of CMAPs. Ischemia-induced SCI resulted in significant reduction of Tarlov scores and a significant decline in number of viable neurons. Similarly, a significant decrement was observed in SCCV following spinal cord ischemia. This study demonstrated that measurement of SCCV via epidural electrical stimulation is possible and displays a significant decline after spinal cord ischemia in rats. We suggest that this method can be beneficial to quantify neuronal damage after experimental ischemic SCI.

Effect of Shear Force on Intervertebral Disc (IVD) Degeneration: An In Vivo Rat Study

Mechanical stress on the intervertebral disc (IVD) may contribute significantly to IVD degeneration, although its pathomechanism has not been fully understood. The purpose of this study was to test the hypothesis that sustained application of static shear force would result in IVD degeneration with minimum injury. We applied shear force on the rat lumbar spine (L5-L6) using a custom-designed loading device for 1 or 2 weeks. Degenerative changes such as nucleus pulposus cavity loss and border disruption were observed from the histology sections, indicating that the application of sustained dorsoventral shear force on the L6 vertebra induced degeneration of the IVDs in L5-L6 and adjacent levels of motion segment in 1 and 2 weeks. The findings of the present study could be useful for gaining a more relevant understanding of the biomechanical load factors of IVD degeneration not only for enabling better therapeutic interventions but also reducing the risk of low back injury.

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty-four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5-L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66-4.05) compared with 3.26 ccm (2.97-3.55) in the control group (p<0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p<0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer.

The effect of methylene blue on peridural fibrosis formation after laminectomy in rats: an experimental novel study

Background context Despite progress in surgical techniques, some patients still face postoperative recurrence of pain, although the rate of successful outcomes is estimated to be approximately 70% and 86% after primary decompression spinal surgery. Recently, attention has been focused on peridural fibrosis (PF), which may be responsible for recurrent pain after laminectomy or discectomy. Methylene blue (MB) has been shown to prevent fibrosis formation in various tissues. Purpose The aims of this study were to investigate the effects of MB and assess the effects of different doses on the prevention of postlaminectomy fibrosis formation in a rat model. This preclinical model is a potential platform for future clinical trials to identify an effective agent for the prevention of clinically important epidural scar formation. Study design An established bilateral L5–L6 rat laminectomy model was used to evaluate postlaminectomy PF with macroscopic and microscopic analyses. Patient sample Seventy-five male adult white Sprague-Dawley rats that underwent laminectomy at the L5–L6 levels were divided into five groups of 15 rats each. Outcome measures Dissected specimens were evaluated macroscopically and microscopically by examiners who were unaware of the group assignment to record the presence or absence of PF formation. Methods Groups A and B served as controls and Groups C, D, and E received treatment. Group A (sham) underwent laminectomy, and Group B was treated with normal saline at the laminectomy site. Rats in Groups C, D, and E received 0.1 mL MB at concentrations of 0.5%, 1%, and 2%, respectively, at the laminectomy site. All rats were killed 4 weeks after laminectomy. The results were compared statistically with the nonparametric Kruskal–Wallis test and Poisson regression. Results Peridural fibrosis was found in five rats (33%) in control Groups A and B and in two rats (10%) in MB-treated laminectomy Groups C and D. The difference between control and MB groups was not statistically significant (p=.27). The preventive effect of MB on PF was not seen at the highest dose of MB (2%) in Group E. Severity of fibrosis was lower in Groups C (MB 0.5%) and D (MB 1%) than in Group E (MB 2%) (p<.01). Wound healing was not affected, and there was no cerebrospinal fluid leakage. No neurological deficits were seen. Conclusion Low doses of MB may be an effective agent in preventing PF formation after lumbar laminectomy in rats. Clinical significance and safety in human use are currently undetermined.

Effect of Honey on Peridural Fibrosis Formation after Laminectomy in Rats: A Novel Experimental Study

Despite progress in surgical techniques, some patients still face postoperative recurrence of pain. Recently, more attention has been focused on peridural fibrosis (PF), which may be responsible for recurrent pain after laminectomy or discectomy. Honey has been shown to exert anti-inflammatory effects on exposed tissues besides its well-known antibacterial properties. The aim of this study were to investigate the effects of honey on the prevention of postlaminectomy fibrosis formation in a rat model. A controlled blinded study was performed in 45 male adult white Sprague-Dawley rats that underwent laminectomy at the L5-L6 levels. They were divided into 3 groups (A, B, and C) of 15 rats each. Group A (sham) underwent laminectomy and group B was treated with normal saline at the laminectomy site. Rats in group C received 0.1 mL honey at the laminectomy site. All rats were killed 4 weeks after laminectomy. PF was found in 5 rats (33%) of control groups A and B, and in 2 rats (10%) in honey-treated laminectomy group C. The difference was not statistically significant. Wound healing was not affected, and there was no cerebrospinal fluid leakage. Although honey appears to be safe, it cannot cause a significant reduction of PF formation after lumbar laminectomy in rats.

The gastrin-releasing peptide system in the spinal cord mediates masculine sexual function

The lumbar spinal segments are of particular interest because they are sexually dimorphic and contain several neuronal circuits that are important in eliciting male sexual responses such as erection and ejaculation. Gastrin-releasing peptide (GRP) is a member of the bombesin-like peptide family first isolated from the porcine stomach. A collection of neurons in the lumbar spinal cord (L3-L4 level) of male rats projects to the lower lumbar spinal cord (L5-L6 level), releasing GRP onto somatic and autonomic centers known to regulate male sexual reflexes. All these target neurons express and localize specific receptors for GRP. This system of GRP neurons is sexually dimorphic, being prominent in male rats but vestigial in females. The system is completely feminine in genetically XY rats with a dysfunctional androgen receptor gene, demonstrating the androgen-dependent nature of the dimorphism. Pharmacological stimulation of GRP receptors in this spinal region remarkably restores sexual reflexes in castrated male rats. Exposure of male rats to a severe traumatic stress decreases the local content and the axonal distribution of GRP in the lumbar spinal cord and results in an attenuation of penile reflexes in vivo. Administration of a specific agonist for GRP receptors restores penile reflexes in the traumatic stress-exposed male rats. This review summarizes findings on this recently identified spinal GRP system, which may be vulnerable to stress, that controls male reproductive function. The identification of a male-specific neuronal system regulating sexual functions offers new avenues for potential therapeutic approaches to masculine reproductive dysfunction.

Biomimetic calcium phosphate coatings as bone morphogenetic protein delivery systems in spinal fusion

Background context Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to enhance spinal fusion rates. Case reports of soft-tissue swelling, ectopic bone formation, and osteolysis have recently surfaced. It is hypothesized that incorporation of rhBMP-2 within a calcium phosphate (CaP) coating may help to localize delivery and mitigate these complications. Purpose To compare the characteristics of posterolateral fusion between rabbits receiving rhBMP-2 delivered via physical adsorption to a collagen sponge or rhBMP-2 incorporated within the physical structure of a CaP coating on a collagen sponge. Study design/setting New Zealand white rabbit model of posterolateral lumbar fusion at L5–L6. Methods Eighteen (18) New Zealand white rabbits underwent posterolateral spinal fusion at L5–L6. Rabbits received bilateral collagen sponges that were either coated with CaP (n=3), coated with CaP and dipped in rhBMP-2 (n=3), coated with a hybrid CaP–rhBMP-2 film (n=6), or coated with a hybrid CaP-rhBMP-2 film and dipped in rhBMP-2 (n=6). Animals were followed weekly with radiographs and were sacrificed at 6 weeks. Fusion masses were further characterized by manual palpation, computed tomography, and histology. Results Radiographic evaluation showed that animals in Group 3 (incorporated BMP) fused at 4 weeks, whereas animals in Group 2 (adsorbed BMP) and Group 4 (incorporated and adsorbed BMP) fused by 6 weeks. Animals that received rhBMP-2 physically adsorbed to the collagen sponge showed extension of the fusion mass beyond the L5–L6 level in 56% of cases and bone resorption in 78%. Histology of fusion masses showed mature bone formation in animals belonging to Groups 2, 3, and 4 and extensive osteoclast recruitment in animals belonging to Groups 2 and 4. Conclusions Delivery of rhBMP-2 via incorporation within CaP coatings results in increased rates of radiographic fusion. The burst release profile of rhBMP-2 adsorbed to surfaces, although effective in achieving fusion, may result in increased osteoclast recruitment.

Biomechanical Analysis of a Disc Prosthesis Distal to a Scoliosis Model

Biomechanical study of bovine spines. The purpose of this study was to perform a biomechanical test to analyze intervertebral deflections following placement of both 1 and 2 semiconstrained TDRs in the subjacent segments of a long fusion. Long-term sequela of long lumbar fusion for scoliosis include adjacent segment disease and flatback syndrome. Total disc replacement (TDR) is a viable option for the treatment of these conditions. Little data has been published regarding the placement of a TDR distal to a scoliosis fusion. Six thoracolumbar bovine spines (T12-S1) were instrumented from T12 to L5, with bilateral pedicle screw fixation at each level. L5-L6 and L6-S1 served as the test levels. One TDR (FlexiCore, Stryker Spine, Allendale, NJ) was initially performed adjacent to the fusion, followed by a subsequent TDR insertion at the last spinal segment. The applied load, total specimen deflection, and local transducer deflections were recorded before and after a TDR at both levels. The results were expressed as a percentage of the intact specimen. Flexion, extension, lateral bending, and torsional deflections were recorded. There were no significant differences (P > 0.05) in sensor deflection observed at the L5-L6 and L6-S1 levels in the anterior and lateral transducers when compared to intact spines specimens. A similar effect was observed at the L5-L6 and L6-S1 levels in the anterior and lateral transducers when compared to intact or prior L5-L6 and intact L6-S1 constructs. This study has shown that using the FlexiCore system at 1 and/or 2 intervertebral disc spaces caudal to a scoliosis fusion model did not significantly change the sensor deflection at the 2 segments adjacent to a scoliosis fusion construct. Future research will continue to define the clinical setting and patients best suited for management by TDR systems.

Parathyroid Hormone (1-34) Augments Spinal Fusion, Fusion Mass Volume, and Fusion Mass Quality in a Rabbit Spinal Fusion Model

The posterolateral rabbit spinal fusion model was used to assess the effect of intermittent parathyroid hormone on spinal fusion outcomes. To test the hypothesis that intermittent parathyroid hormone (PTH) improves spinal fusion outcomes in the rabbit posterolateral spinal fusion model. Spinal fusion is the definitive management for spinal deformity or instability, yet despite current technology, 5% to 40% of lumbar fusions result in pseudarthrosis. Animal studies have demonstrated enhanced fracture healing with the use of PTH, but the effect of PTH on spinal fusion is poorly described. Forty-four male New Zealand white rabbits underwent bilateral posterolateral spine fusion (L5-L6 level). Twenty-two rabbits received daily subcutaneous injections of PTH (1-34) (10 microg/kg) and 22 received an injection of saline fluid. All were killed 6 weeks after surgery. L5-L6 vertebral segments were removed and analyzed with manual bending, faxitron radiography, microCT, and histomorphometry. Manual bending identified fusion in 30% (control) versus 81% (PTH) animals (P < 0.001). A radiographic scoring system ("0" = no bone formation, "5" = full fusion) resulted in an average score of 3.36 (control) versus 4.51 (PTH) (P < 0.001). MicroCT analysis demonstrated a median mass of 3.5 cc (control) (range, 2.25-5.40 cc) versus 6.03 cc (PTH) (range, 4.34-10.58 cc) (P < 0.001). Histology showed a median percentage bone area of 14.3% (control) (n = 12) versus 29.9% (PTH) (n = 15) (P < 0.001). The median percentage cartilage was 2.7% (control) (n = 5) versus 26.6% (PTH) (n = 5) (P < 0.01). Osteoclast quantification revealed median values of 140.5 (control) (n = 6) and 345.0 (PTH) (n = 8) (P < 0.001) respectively, and the percentage of osteoblasts revealed a median value of 31.4% (control) (n = 6) versus 64.4% (PTH) (n = 8) (P < 0.001). Intermittent PTH administration increased posterolateral fusion success in rabbits. Fusion bone mass and histologic determinants were also improved with PTH treatment. PTH has promise for use as an adjunctive agent to improve spinal fusion in clinical medicine.

Adjacent Segment Degeneration

The adjacent discs of 13 goats, originally used in a lumbar spinal fusion model study, were analyzed for symptoms of intervertebral disc degeneration by means of magnetic resonance imaging (MRI), macroscopy, and histology. These goats were followed for 6 months and the results were compared with 6 control goats. To evaluate the development of adjacent segment disc degeneration in vivo in a goat lumbar spinal fusion model. There is ongoing debate on whether adjacent segment degeneration (ASD) develops through increased biomechanical load on discs adjacent to fusion sites, or by the natural process of pre-existing degenerative disease. Animal models offer an opportunity to separate these factors by evaluating the development of ASD in nondegenerated animal spines. In a spinal fusion model study 2 segments (L3-L4 and L1-L2) were fixated and followed for 3 months (n = 6) and 6 months (n = 7) in 13 skeletally mature goats. Two adjacent discs (T13-L1 and L4-L5), 1 interjacent disc (L2-L3) and a control disc (L5-L6) were analyzed by means of magnetic resonance imaging, macroscopy, and histology. These results were compared with the discs of 6, nonoperated "normal" goats. No differences were observed in the adjacent and interjacent intervertebral discs after both follow-up periods. However, severe degenerative changes were observed in the L5-L6 level, originally included as controls. Large animal fusion models offer an excellent opportunity to study ASD in vivo, as pre-existing degenerative disc disease is not present and biomechanical effects of the fusion can be studied more isolated. Our results suggest that adjacent disc degeneration does not develop in our spinal goat fusion model. There is, however, an increased risk of disc degeneration in the L5-L6 level through an unclear mechanism.

Intraspinal adenosine induces spinal cord norepinephrine release in spinal nerve-ligated rats but not in normal or sham controls.

Intrathecal adenosine is antinociceptive under conditions of central sensitization, but not in response to acute stimuli in normals. The reasons for this selective circumstance of action remain unclear, but some evidence links adenosine's antinociceptive effects to release of norepinephrine by terminals in the spinal cord. The purpose of this study was to test whether spinal adenosine induces norepinephrine release selectively in settings of hypersensitivity. Rats randomly assigned to spinal nerve ligation, sham operation, or no operation were anesthetized. A microdialysis fiber was implanted in the spinal cord dorsal horn at the L5-L6 level and perfused with artificial cerebrospinal fluid. After washout and a baseline sample period, adenosine at various concentrations was infused through the fiber for 150 min, and samples were collected every 15 min. In ligated, but not in sham or normal animals, adenosine perfusion increased norepinephrine in spinal cord microdialysates in a concentration-dependent manner. The effects of adenosine plateaued after 75 min and remained stable until the end of the experiment. Intravenous injection of selective adenosine A1 and A2 receptor antagonists revealed that adenosine's effect on spinal norepinephrine release was A1 receptor mediated. This is the first study to provide direct evidence that adenosine is able to release norepinephrine in spinal cord dorsal horns in living animals. However, this effect was only seen in animals after spinal nerve ligation. These data are consistent with behavioral studies demonstrating that adenosine's antinociceptive effects in rats after spinal nerve ligation is totally dependent on intact spinal noradrenergic terminals and can be blocked by spinal alpha 2-adrenergic receptor antagonists.

Comparison of methods for determining presence and extent of anterior lumbar interbody fusion

Purpose of study: The purpose of this study was to compare radiographic, computed tomography (CT) imaging and histologic results for presence and extent of bony fusion of a titanium anterior lumbar interbody fusion device placed in a nonhuman primate model.Methods used: Anterior lumbar interbody fusions were performed at the L5–L6 level in 33 adult pigtail macaque monkeys using a titanium device and autogenous iliac crest bone graft. The presence of fusion (bridging trabeculations) and percent of anterior-posterior (AP) diameter healed (extent of fusion) were determined using serial AP and lateral radiographs, CT images and quantitive histology using identical evaluation methods. Matched pair nonparametric t tests were used to determine differences in results for the three techniques used.of findings: Plain film radiographs tended to overestimate the presence of bony fusion compared with both CT imaging and histology. In only 39% of cases radiographic grades were equivalent to CT and histology grades, whereas in 55% of the remaining cases the radiographic grades were greater. The difference was not, however, statistically significant. Although presence of fusion grades were equivalent in 82% of CT and histology evaluations, in only 15% of cases was the extent of fusion grade equivalent for the two techniques. CT imaging overestimated the percent AP diameter healed compared with actual histologic measurement in 73% of comparisons. This difference was statistically significant (p < .0002).Relationship between findings and existing knowledge: The accuracy of plain film radiographs for determining anterior lumbar fusion is often debated with wide ranges in bony fusion reported. CT imaging is believed to be a more definitive technique. However, only through histologic evaluation can the actual presence and extent of fusion be determined and the various techniques validated.Overall significance of findings: Plain film radiographs tended to overestimate the presence of anterior interbody fusion. Although CT imaging accurately predicted the presence of fusion, it significantly overestimated the extent of bony fusion present. The lack of extensive fusion in patients with anterior interbody fusions may affect long-term stability and contribute to clinical failure.Disclosures: No disclosures.Conflict of interest: Stephen Cook, consultant; Laura Patron, grant research support; Kirk Bailey, other support; Paul Glazer, consultant, EBI.

Long-term follow-up of posterolateral lumbar arthrodesis with recombinant human growth/ differentiation factor—5/mineralized collagen matrix in sheep model

Purpose of study: In recent development of bone graft substitutes, experiments using bone morphogenetic proteins with osteoconductive carrier have been studied for the clinical practice. The goal of this study is to evaluate the efficacy of a novel osteoinductive growth factor (rhGDF-5)/osteoconductive matrix (Healos) combination to induce and facilitate bone formation in posterolateral lumbar fusion in sheep model.Methods used: Twelve skeletally mature sheep underwent bilateral posterolateral spinal fusion and transpedicular screw fixation (Silhouette, Sulzer Spine-Tech) at the L3–L4 and the L5–L6 level. Autologous iliac crest bone or Healos/recombinant human growth/differentiation factor–5 (rhGDH-5) was used either on the L3–L4 or L5–L6 level alternatively. The animals were grouped into one of two survival time period groups: 6 months and 12 months. After sacrifice, internal organs of sheep were examined, and lumbar spine was harvested for gross inspection, radiographic study, microradiography and histologic evaluation.of findings: Both graft materials revealed solid fusion mass at 6 months and maintained well for 12 months. Bone fusion area (sectional fusion area on axial computed tomography scan) by Healos/rhGDF-5 was larger than that of autologous bone graft (241.8 ± 56.1 mm2 vs. 197.2 ± 61.9 mm2). But bone trabecular formation assessment showed no significant differences between Healos/rhGDF-5 and autograft at 12 months. There were no neurological deficits and no ectopic bone or mass formations.Relationship between findings and existing knowledge: These results show that posterolateral spinal arthrodesis using Healos/rhGDF-5 is effective and compatible to autograft in the sheep model.Overall significance of findings: This study demonstrated Healos was an effective carrier in the biologic and mechanical environment, and revealed successful fusion coupled with rhGDF-5.Disclosures: Device or drug: Healos. Status: approved device or drug: rhGDF-5. Status: investigational.Conflict of interest: No conflicts.

Thursday, October 31, 2001 4:18–4:44 pm Focused Review: Disc Metabolism: 4:18 Epidural glutamate from herniated disc material as a nociceptive stimulus in lumbar radiculopathy

Purpose of study: To determine whether levels of free glutamate previously measured from human herniated disc specimens could lead to physiological or behavioral symptoms consistent with a hyperesthetic state in glutamate-infused rats.Methods used: Sprague-Dawley rats were unilaterally infused with epidural glutamate at the L5–L6 levels for 72 hours by a subcutaneously implanted miniosmotic pump. Because herniated disc material shows an average concentration of 0.5 mM, we infused with concentrations of 0.002 to 2.0 mM. Animals were studied behaviorly by 49 C hot plate withdrawal tests, Von Frey fiber withdrawal tests for left-right hindpaw reactivities and 53 C tail flick assays. These were performed 24 hours before and 24, 72 and 144 hours after onset of glutamate infusion. Other animals were terminated 72 hours after onset of infusion and were evaluated immunohistochemically for NMDA, AMPA and kainate receptor expression in dorsal horn regions by quantitative means.of findings: Hot plate withdrawal showed significance at 72 hours and concentrations of 0.002, 0.02 and 0.2 mM. Tail flick tests showed similar differences in latencies compared with saline controls (p<.02; two-tailed t test). Von Frey fiber tests showed decreased latencies on the ipsilateral side to the infusion in glutamate-infused animals compared with saline controls (p<.05; two-tailed t test). Immunohistochemical evaluation showed increasing levels of glutamate expression over saline-infused controls ipsilateral to the side of glutamate infusion at 2.0 mM concentration. Results at lower infusion levels are pending (will be available at North American Spine Society meeting in October 2002).Relationship between findings and existing knowledge: These findings suggest that the concentrations of glutamate found in human disc material in the epidural space are large enough to have biologic effects on nociceptive responses.Overall significance of findings: Lumbar radiculopathy resulting from disc herniation may be amenable to treatment with glutamate antagonists. Cartilage may promote pain whenever degraded cartilage is in the vicinity of glutamate receptors in other joints as well.Disclosures: No disclosures.Conflict of interest: No conflict.

Low-intensity pulsed ultrasound improves spinal fusion

Background context: Increasing the incidence of solid bony fusion is a primary goal in spine surgery. Daily low-intensity pulsed ultrasound therapy has been shown to improve and accelerate the bone healing process. Purpose: The purpose of this study was to evaluate the efficacy of daily low-intensity pulsed ultrasound therapy to improve the rate and quality of spinal fusion. Study design: Canine fusion model prospective study. Patient sample: Fourteen adult male dogs were used. Outcome measures: Radiographic grading of plain films, computed tomography (CT) and magnetic resonance imaging (MRI), gross palpation, torsional stiffness and histologic grading were used to determine the presence or absence of fusion. Methods: Posterior noninstrumented bilateral fusions were evaluated at the L2–L3 and L5–L6 levels. Treatment with low-intensity pulsed ultrasound for 20 minutes per day over the fusion site (stimulated) was compared with fusion sites that received no stimulation (nontreated controls) at 6 and 12 weeks after surgery. Plain film radiographs, CT and MRI, mechanical torsion testing and histologic examination were performed. Results: At 6 weeks, ultrasound treated sites were more frequently fused compared with nontreated controls, although the difference in fusion rate was not statistically significant. At 12 weeks after surgery complete radiographic and histologic fusion occurred in 100% of ultrasound-treated sites. In the nontreated control sites 78% had achieved complete radiographic fusion and 44% had complete histologic fusion. Compared with control sites, the histological and mechanical fusion rate was significantly greater in ultrasound-treated sites ( P<.05) at 12 weeks. A statistically significant increase in mechanical stiffness in ultrasound-treated sites was also found at 12 weeks after surgery. Conclusions: Low-intensity pulsed ultrasound therapy may be a useful means to ensure successful spine fusion.