| Objectives To study the effects of intradiscal injected collagenase on herniated lumbar disk, the effects of epidural injected collagenase on autografts of lumbar disk tissue transplanted into epidural space, the effects of collagenase on phospholipase A2 (PLA2) activity in vivo disease models in animals.Methods Experiment 1: 54 lumbar disks of 9 adult sheep were randomly divided into control group and model group. Left anterolateral annulus fibrosus of intervertebral disks in model group were partial resected with 2.5mm-diameter-hole saw in depth of 5mm.Disk height index of intervertebral disks on radiographs, areas and average T2 relaxation time of high intensity zone of disks on T2-weighted MRI and histological score on photomicrographs (HE staining, Masson staining and safranin O-fast green staining) were measured at 2 months, 4 months and 6 months after operation. The morphology of intervertebral disk herniation at the injured sites was also observed on axial T2-weighted MRI and photomicrographs. Experiment 2: 108 lumbar disks of 18 adult sheep were divided into 6 groups according disk type (normal or model disk) and treatment (control, saline, and collagenase).Lumbar intervertebral disks were surgically injured to induce disk herniation and degeneration 10 weeks before intradiscal injections. Indices in experiment 1 were studied 1 day, 1 week, 2 weeks, 4 weeks, and 12 weeks after intradiscal injection of drugs.Experiment 3: Autografts of intervertebral disk tissue of 9 adult sheep was taken out through anterior approach and transplanted into epidural space in lumbar spinal canal through interspaces between left L3-4 lamina and right L6-7 lamina. Collagenase (300U/0.3ml) or 0.3ml saline was randomly epidural injected into lumbar spinal canal and infiltrated around autografts. Histological changes of autografts were studied 1 day, 1 week, 2 weeks after collagenase injection. Experiment 4: 42 Wistar rats were randomlyassigned to one of three treatment groups: 1) saline, 2) dexamethasone, 3) collagenase. The animals were tested for mechanical hyperalgesia with von Frey fibres on Day 0(preoperation), Day 7(pretreatment), and Day 14(prior to death).3 animals randomly selected from three groups (each animal from each group) were killed on Day 0 to determine the baseline of PLA2 activity within unmanipulated rat sciatic nerve; other 39 animals underwent operation according Maves-modified Bennet-Xie model on right sciatic nerves while Sham operations were performed on left sciatic nerves. On Day 7, 3 animals from each group were killed to determine PLA2 activity prior to treatment. The remainders were given a single infusion of saline, dexamethasone or collagenase around the inflamed nerve. On Day 14, the remaining animals were sacrificed and their sciatic nerves were removed to determine PLA2 activity.Results Experiment 1: Disk height index of model intervertebral disks on radiographs, areas and average T2 relaxation time of high intensity zone of model disks on T2-weighted MRI decreased significantly (P<0.01,vs control); histological score of model disks on photomicrographs increased significantly (P<0.01,vs control) at 2 months, 4 months and 6 months after operation. Above-mentioned indices aggravated gradually during 2-6 months after operation, but had no significant difference among 2 months, 4 months and 6 months after operation. The outermost 1/3 annulus fibrosus defect showed the ability to heal, the defect induced by the hole saw led initially to deformation and bulging of the collagen bundles, eventually to inner extension of the tear and complete failure, nucleus pulposus protruded into the inner 2/3 defect. Experiment 2: Collagenase has chemonucleolysis effects on degenerative intervertebral disk tissue similar to normal disk tissue. Collagenase can dissolve nucleus pulposus in the center of model disks and protrusion sites simultaneously while only central portion and inner annulus fibrosus was dissolved in normal disks. Endplates in normal disks were destroyed more severe than in model disks after collagenase injection.Experiment 3: Collagenase injected autografts of nucleus pulposus were dissolved significantly 1 day after injection, infiltrated with lots of macrophagocytes after 1 week with structure of nucleus pulposus destroyed, replaced by granulation tissue after 2 weeks. Saline injected autografts of nucleuspulposus didn't show significant changes 1 day after injection; lots of lymphocytes infiltration was found after 1 week, fibroblasts and macrophagocytes infiltration was found after 2 weeks. Annular fibrosus was only minimal dissolved on the verge of autografts by collagenase.Experiment 4: All animals developed behavioral changes consistent with inflammatory mononeuropathy 24 to 72 hours postoperatively, include gait disturbance, flexion deformity and hyperalgesia of the involved hindlimb. The degree of mechanical hyperalgesia was comparable between groups at Day 7. By Day 14, the mechanical hyperalgesia was less evident in the dexamethasone- and collagenase-treated groups than in the saline-treated controls (P<0.01). The level of PLA2 activity was lower in the dexamethasone- and collagenase-treated groups than in the saline-treated controls (P<0.01). There was no significant difference between the dexamethasone- and collagenase-treated groups (P>0.05).Conclusions A novel sheep model of intervertebral disk herniation can be developed by partial resection of anterolateral annulus fibrosus with 2.5mm-diameter-hole saw in depth of 5mm quantitatively, which is simple, rapid, effective and reproducible. Collagenase can induce chemonucleolysis in model disks and dissolve nucleus pulposus in the center and protrusion sites. Epidural injected collagenase can directly dissolve autografts of nucleus pulposus transplanted into lumbar spinal canal and also stimulate inflammatory response to accelerate disorganization and absorption of autografts of nucleus pulposus. Collagenase exhibits anti-inflammatory properties in vivo, reducing PLA2 activity and ameliorating mechanical hyperalgesia in this model of inflammatory sciatic neuropathy.
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