Pathogenesis of calcium crystal deposition in the ligamentum flavum correlates with lumbar spinal canal stenosis.

OBJECTIVE: To investigate the histological and immunohistochemical properties of degenerative changes and calcium crystal deposition in the lumbar ligamentum favum. METHODS: We examined the ligamentum flavum harvested from 119 surgical cases with symptomatic lumbar spinal stenosis. Sections of the ligament were examined by scanning electron microscopy (SEM), energy dispersive X-ray micro-analysis, and were immunostained for S-100 protein, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and CD34. The results were compared with those of ligament tissue harvested from 10 cases of lumbar disc herniation. RESULT: The elastic fibres of the ligamentum favum showed regular, or sometimes irregular, and fragmented fibre bundles. Large areas of fibrosis with reduced elastic component and increased collagenous tissue were frequently seen in the degenerated ligaments. Calcium crystal deposits were observed in these fibrous ligaments, associated with many hypertrophic chondrocytes, and with small blood vessel formation. These chondrocytes stained positively for S-100 protein, VEGF and bFGF Calcium pyrophosphate dihydrate crystals were identified in the calcium deposit area. CONCLUSION: We believe that rupture of elastic fibre bundles is the first change to occur in degeneration of the ligamentum favum. Calcium crystal deposition was seen within these fibrous and chondrometaplastic areas. Hypertrophic chondrocytes regulate crystal formation and tissue reconstruction by secreting cytokines.

Lordotic alignment and posterior migration of the spinal cord following en bloc open-door laminoplasty for cervical myelopathy: a magnetic resonance imaging study.

We investigated lordotic alignment and posterior migration of the spinal cord following en bloc open-door laminoplasty for cervical myelopathy. Fifty-five patients (32 men and 23 women) were studied, with an average follow-up of 2.4 years. Radiological examination included evaluation of lordosis of the cervical spine and spinal cord, degree of enlargement of bony spinal canal, and the magnitude of posterior cord migration. We also correlated these changes with neurological improvement. Postoperatively, there was an average of 5% loss of cervical spine lordosis (P > 0.01) on radiographs and 12% reduction in the lordotic alignment of the spinal cord (P > 0.05) on magnetic resonance imaging. Postoperatively, the size of the bony spinal canal increased by 48%. Posterior cord migration showed a significant correlation with the preoperative cervical spine and spinal cord lordosis (P < 0.05). Thirty-seven (67%) patients with neurological improvement exceeding 50% showed significant posterior cord migration following laminoplasty compared with those demonstrating less than 50% improvement (P = 0.01). Our results suggest that a significant neurological improvement is associated with posterior cord migration after cervical laminoplasty.

Plasticity of the spinal cord contributes to neurological improvement after treatment by cervical decompression. A magnetic resonance imaging study.

To investigate the relationship between morphological plasticity of the spinal cord and neurological outcome after surgery for compressive lesions, we correlated the transverse area of the cervical spinal cord measured by transaxial magnetic resonance imaging (MRI) obtained during the early postoperative period (1-6 months) with neurological function assessed at a median postoperative follow-up period of 2.5 years. Measurements on MRI in 56 patients (35 men and 21 women) included evaluation of the cross-sectional area of the cervical cord and the subarachnoidal space at the level of decompression. The transverse area of the cervical cord increased by 30 to 62% postoperatively and that of the subarachnoidal space by 57 to 95%. Neurological improvement was noted in all patients and averaged 63% in our assessment scale. Expansion of the cervical cord during the early postoperative period correlated significantly with the late postoperative neurological status (P = 0.009). Our results suggest that an increase in the cross-sectional area of the cervical spinal cord, representing spinal cord morphological plasticity, is a significant factor in determining the late neurological improvement following decompressive surgery.

Histological investigation of spinal cord lesions in the spinal hyperostotic mouse (twy/twy): morphological changes in anterior horn cells and immunoreactivity to neurotropic factors.

We examined the morphology of spinal accessory motoneurons and immunoreactivity to neurotrophins, brain-derived neurotropic factor (BDNF) and neurotrophin (NT)-3, as well as the presence of reactive astrocytosis in 70 tiptoe walking Yoshimura (twy) mice that develop calcification at C1-C2 vertebral level compressing the spinal cord. At the level of compression, the area of neuronal soma and total length of dendrites of wheat germ agglutinin-horseradish peroxidase (WGA-HRP)-labelled accessory motoneurons in the medial cell pool decreased significantly with decrement in motoneuron population, relative to the control. In contrast, at sites rostral to the compressive lesion, a significant enlargement of the neuron soma and dendritic elongation were noted, associated with increased motoneuron population and decreased transverse area of the cord at the level of compression. At this site, enhanced BDNF and NT-3 immunoreactivities were evident in the anterior horn cells. In mice with a more severe degree of compression, astrocyte-like cells showing BDNF immunoreactivity became abundant and axons in the anterior column demonstrated a marked NT-3 immunoreactivity. Our results suggest increased functional activity of anterior horn cells at levels rostra] to the site of compression. We speculate that the presence of BDNF and NT-3 in neurons and astrocyte-like cells is proportionate to the severity of chronic mechanical compression and may contribute to the heterotropic neuronal reserve and survival.

Simplified determining procedure for routine residue monitoring of sulphamethazine and sulphadimethoxine in milk.

A simplified determining/identifying method for residual sulphamethazine (SMZ) and sulphadimethoxine (SDM) in milk by using a high-performance liquid chromatography (HPLC) with a photo-diode array detector was presented. Both sulphonamides in cow's milk samples were extracted by only stirring with ethanol followed by an Ultrafree-MC/Biomax as a centrifugal ultra-filtration unit. For determination/identification of SMZ and SDM, a Mightysil RP-18 GP Aqua column and a mobile phase of 25% (v/v) ethanol solution (in water) with a photo-diode array detector was used. Average recoveries from spiked SMZ and SDM (10-1000 ng/ml each drug) were > or = 83% with the relative standard deviations between 1.4 and 3.7%. The limit of quantitation (LOQ) were calculated to be 5 ng/ml for SMZ and 10 ng/ml for SDM, respectively. The values were below the MRL/tolerance (SMZ, 25 ng/ml; SDM, 10 ng/ml). The total time and solvent required for the analysis of one sample were <35 min and <2 ml of only ethanol, respectively. No toxic solvents were used. The developed procedure was harmless to the human and environment.

Rapid liquid chromatographic determination of oxytetracycline in milk.

A simple method for the determination of residual oxytetracycline (OTC) in milk by high-performance liquid chromatography (HPLC) was developed. The sample preparation could be made without complex extraction and clean-up procedures. A LiChrospher 100 RP-8 end-capped column and a mobile phase of acetonitrile-acetic acid-water (28:4:68, v/v/v) with a photo-diode array detector was used. The average recoveries from spiked OTC (0.1, 0.5 and 1.0 microgram/ml) were in excess of 89.8% with coefficients of variation between 0.6 and 4.1%. The limit of detection was 0.05 microgram/ml. The total time required for the analysis of one sample was below 10 min.

Simultaneous high-performance liquid chromatographic determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in foods of animal origin.

A rapid and sensitive method for the determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in beef, pork, chicken and eggs by high-performance liquid chromatography (HPLC) was developed. The extraction of these compounds was performed using a mixture of 90% (v/v) acetonitrile solution and hexane (5:4, v/v) to minimize the fat content followed by purification by alumina column chromatography. These extracts contained sulphonamide analytes which were free from interfering compounds when examined by HPLC using a LiChrosorb RP-18 column. The average recoveries from spiked meat and egg were in excess of 80% with relative standard deviations between 0.4 and 5.0%. The practical limits of detection were 0.01 ppm for all samples.

Matrix solid-phase dispersion extraction and high-performance liquid chromatographic determination of residual sulfonamides in chicken.

Simultaneous determination of the six sulfonamides (SAs) sulfadiazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline in chicken using matrix solid-phase dispersion (MSPD) with neutral aluminium oxide as an MSPD sorbent and high-performance liquid chromatography (HPLC) is presented. In the present MSPD, six SAs could be isolated by only one step, elution with a 70% (v/v) aqueous ethanol solution, without the sorbent conditioning and the sorbent-tissue matrix washing. For the HPLC determination, a LiChrospher 100 RP-8 and a mixture of 1% acetic acid solution (pH 3.0, in water)-acetonitrile-N,N-dimethylformamide (78:22:5, v/v/v) as the mobile phase with a photodiode array detector were used. Average recoveries were greater than 87.6% with relative standard deviations between 0.5 and 8.6%. The total time and amount of solvent required for the analysis of one sample were <1.5 h and <12 ml, respectively.

Simple and rapid extraction method of total egg lipids for determining organochlorine pesticides in the egg.

A simple and rapid extraction method of total egg lipids for determination of organochlorine pesticides (OCPs) in the egg was developed. After being mixed with anhydrous sodium sulphate, the extraction of lipids in egg yolk was performed using n-hexane-acetonitrile (2:1, v/v). Troublesome emulsions did not occur. Using the present method, an average of 3.03 g of egg lipids was collected from 10 g of egg yolk. Compared with classical methods, the present method is handy; needs much shorter analysis time and less requirement of solvents and has higher efficiency of egg lipid extraction and higher recoveries of OCPs.

Calcium crystal deposition in the ligamentum flavum of the lumbar spine.

OBJECTIVE: To investigate the histological and immunohistochemical properties of the lumbar ligamentum flavum with calcium crystal deposition. METHODS: We examined the histological characteristics of the ligamentum flavum of the lumbar spine containing calcium deposits, obtained from 16 surgical cases with symptomatic lumbar spinal stenosis. Sections of the ligaments were also immunostained for elastase, chymotrypsin and S-100 protein, and examined by energy dispensive X-ray microanalysis and scanning electron microscopy. The results were compared with those of ligaments without calcium deposits. RESULTS: The elastic fibres of ligaments with calcium deposits showed marked degeneration (irregular arrangement and fragmentation of the fibre bundles) and nodular granulomatous lesions. Calcium crystal deposits were present in this area and in the nodular granulomatous areas. The calcified areas were surrounded by neutrophils and new small blood vessels. Immunostaining for elastase and chymotrypsin was positive in these areas, and S-100 protein-containing chondrocytes were detected around and within the calcified areas. Calcium pyrophosphate dihydrate crystals were identified in the calcified areas in all patients. CONCLUSION: Nodular degeneration of the elastic fibres of the ligament is probably caused by the action of proteolytic enzymes. Degeneration results in small granulomatous areas that form the nidus for calcium crystal deposition. Accumulated neutrophils, and S-100 protein-containing chondrocytes appear to precipitate the deposition of calcium crystals in the granulomatous lesions of the degenerated lumbar ligamentum flavum.

Postoperative bone re-growth in lumbar spinal stenosis. A multivariate analysis of 48 patients.

STUDY DESIGN: Forty-eight patients who underwent posterior decompressive surgery for lumbar spinal stenosis were studied. The effect of bone re-growth after posterior decompression in lumbar spinal stenosis on clinical outcome and the factors promoting the bone re-growth were investigated with a multivariate analysis model. METHODS: Bone re-growth at the sites operated upon was evaluated as a percentage of re-growth of the original laminectomy site based upon plain radiographs. The degree of bone re-growth was classified into four groups: 1) no significant re-growth (Group I, bone regrowth rate 10% or less); mild (Group II, 11% to 40%), moderate (Group III, 41% to 70%), and marked (Group IV, 71% to 100%). The average follow-up period was 4.5 years (range, 2 to 7 years). RESULTS: No significant bone re-growth was shown in 6% of the patients. Mild re-growth was shown in 50%, moderate re-growth in 29%, and marked re-growth in 15%. A multivariate analysis indicated that a total block in the preoperative myelogram, a follow-up period of more than 5 years, decompression at more than three spinal levels, and age under 60 years were associated with moderate or marked bone re-growth. Spinal instability accelerated the bone re-growth mainly in the mid and later follow-up intervals. Spinal levels adjacent to a fusion showed more bone re-growth. Patients with moderate and marked bone re-growth had poorer clinical outcomes than those with no significant and mild bone re-growth. CONCLUSION: Bone re-growth in a surgical defect will occur in most patients after posterior decompression. Moderate and marked postoperative bone re-growth are possibly related to recurrence of neurologic symptoms in the middle of and later on in follow-up periods.

Late radiographic findings after anterior cervical fusion for spondylotic myeloradiculopathy.

A retrospective study was performed to evaluate the radiographic changes that occurred at spinal levels adjacent to fused vertebrae after anterior cervical fusion. One hundred six patients with cervical spondylotic myeloradiculopathy (88 men, 18 women) were followed for an average of 8.5 years. The average age at follow-up was 64 years. Forty-two patients underwent a single-level fusion, 52 had a two-level fusion, and 12 had three levels fused. Seventeen patients who underwent additional surgery after anterior fusion also were reviewed, with an average follow-up period of 2.9 years. Postoperatively, cervical flexion-extension resulted in significantly increased movement about the vertebral interspace at the upper adjacent level. An increment of posterior slip of the vertebra immediately above the fusion level, with associated spinal canal compromise of less than 12 mm, significantly affected neurologic results. Patients with multilevel fusions notably exhibited these radiographic abnormalities at adjacent levels. Spinal canal stenosis, when associated with dynamic spinal canal stenosis in the vertebra above the fusion level, affected late neurologic results. Results of salvage laminoplasty were not satisfactory. Unnecessarily extended longer fusion must be avoided.

Calcium crystal deposition in the ligamentum flavum of the cervical spine.

This article reports eight patients who underwent posterior decompressive surgery for myeloradiculopathy caused by calcium crystal deposition in the ligamentum flavum of the cervical spine. All were women with an average age of 72 years and showed neurologic improvement postoperatively. Four patients had diabetes mellitus, and knee meniscus calcification on radiographs were noted in four patients. The ligamentum flavum of C5-6 and C6-7 were sites frequently involved. Crystallographic examination confirmed resected deposits as calcium pyrophosphate dihydrate crystal in all patients and additional hydroxyapatite crystal in two. Histology confirmed marked degeneration in elastic fibers about the calcium deposits. This pathologic condition is possibly spinal manifestation of systemic calcium crystal deposition disease.

Potential usefulness of 18F-2-fluoro-deoxy-D-glucose positron emission tomography in cervical compressive myelopathy.

STUDY DESIGN: This case study describes the usefulness of high-resolution 18F-2-fluoro-deoxy-D-glucose (18FDG)-positron emission tomography (PET) for metabolic neuroimaging of the cervical spinal cord in patients with compressive myelopathy. OBJECTIVE: To examine whether 18FDG-PET imaging could visualize deterioration of cervical spinal cord function associated with a variable degree of compression and to determine its potential usefulness during assessment of myelopathy. SUMMARY OF BACKGROUND DATA: A few studies have described the use of 18FDG-PET imaging in cervical cord diseases, but visualization of the cervical spinal cord before and after surgical decompression for compressive myelopathy has not been reported. The potential usefulness of 18FDG-PET imaging for assessment of the function of compressed cervical cord has not been discussed previously. METHODS: An 18FDG-PET scan was performed before and after surgery in seven patients with cervical compressive myelopathy. The correlation between the metabolic rate of glucose of the cervical spinal cord and neurologic scores was evaluated. The metabolic rate of glucose in different vertebral levels was also measured. RESULTS: Preoperative metabolic rate of glucose was high in two patients but low in the other five. At the time of the second postoperative examination, metabolic rate of glucose was higher in six of the seven patients, and the increase was associated with neurologic improvement. Use of 18FDG was not related to changes in signal intensities on T2-weighted magnetic resonance images. The metabolic rate of glucose decreased at the affected vertebral level in four patients, increased in two, and did not change in one, relative to the unaffected levels. CONCLUSIONS: High-resolution 18FDG-PET neuroimaging may provide clinically useful qualitative and quantitative estimation of impaired metabolic activity of the compromised cervical spinal cord in compressive myelopathy. 18FDG-PET images may also offer additional information related to neuronal dysfunction induced by mechanical compression.

Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production.

STUDY DESIGN: Surgically obtained cervical herniated intervertebral discs were examined histologically and immunohistochemically. The production of nitric oxide (NO) in the local tissue was examined using the electron spin resonance (ESR) method. OBJECTIVES: To investigate the local histologic and immunohistochemical changes in cervical disc herniation, including NO production, and to compare such changes with those in autopsy cases. SUMMARY OF BACKGROUND DATA: Very little is known about the histopathologic processes of cervical disc herniation. In addition, no information is available on the level of in vivo NO production in cervical disc herniation. METHODS: Thirty-six herniated cervical discs obtained from 31 patients were immunohistochemically examined for localization of blood vessels, matrix metalloproteinase (MMP)-3, and inducible NO synthetase (iNOS). We also compared the production of NO, measured by the ESR method, in eight specimens with that of five control discs obtained from fresh cadavers. RESULTS: The presence of herniated discs correlated with the degeneration of cartilaginous endplate and torn anulus fibrosus. Formation of new blood vessels around the herniated discs was detected, using von Willebrand factor antibody, in seven uncontained hernias and 20 contained hernias. Immunohistochemical studies showed the presence of cells positive for MMP-3 (chondrocytes), iNOS (chondrocytes and granulation tissue) in cervical disc hernias. ESR analysis showed a significantly higher NO production in herniated cervical discs than in disc samples of fresh cadavers. CONCLUSIONS: Herniated cervical intervertebral disc is characterized by the presence of an inflammatory process associated with neovascularization and increased expression of MMP-3. Production of NO was markedly high in both contained- and uncontained-type hernias.

Normal-phase high-performance liquid chromatographic determination of spiramycin in eggs and chicken.

A precise method is presented for determination of residual spiramycin (SP) in chicken eggs and tissues by high-performance liquid chromatography (HPLC). The sample preparation was performed by homogenizing with a mixture of acetonitrile and n-hexane (5:4, v/v) to minimize the fat amount followed by ultra-filtration using a MolCutII(R). The extracts containing SP were free from interfering compounds when examined by the normal-phase HPLC using a LiChrosorb(R) NH(2) column and a mobile phase of acetonitrile-water (85:15, v/v) with a photo-diode array detector. The average recoveries from spiked SP (0.1, 0.5 and 1.0 ppm) were in excess of 89.0% with coefficients of variation between 1.4 and 2.4%. The limit of detection was 0.1 ppm.

Mutagenicity and DNA-damaging activity of decomposed products of food colours under UV irradiation.

Five synthetic food colours Food Red Nos 3, 40 and 102 and Food Blue Nos 1 and 2, and their UV irradiated products were tested for mutagenic activity by means of the Ames test using Salmonella typhimurium strains TA98 and TA100. Food colours were irradiated with UV light for 14 days. Food Red Nos 3, 40 and 102 and Food Blue No. 1 were non-mutagenic before and after irradiation. UV irradiated products of Food Blue No. 2 were mutagenic in TA98 with or without S-9 mix. The mutagenic activity increased with increasing irradiation period, reached maximum potency on day 6, and then decreased. Moreover, Food Blue No. 2 showed DNA-damaging activity after 14 days of irradiation in rec-assay using Bacillus subtilis strains H17 and M45. The capillary electrophoresis was applied for the analysis of UV irradiated products of Food Blue No. 2. The original peak of Food Blue No. 2 was decomposed into seven peaks after UV irradiation.

Genotoxicity of gardenia yellow and its components.

Gardenia fruit (Gardenia jasminoides ELLIS) is widely used as a natural food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. "Gardenia yellow" is a natural food colorant which is extracted by ethanol from gardenia fruit. The purpose of this study was to evaluate the genotoxicity of gardenia yellow. Genotoxicity of gardenia yellow and its components, crocetin, gentiobiose (a component of crocin), geniposide and genipin (formed by hydrolysis of geniposide), was studied by Ames test, rec-assay, and sister chromatid exchange (SCE) using V79 cells. Gardenia yellow and its components were found not to be mutagenic in the Salmonella reverse mutation assay. Gardenia yellow and genipin caused damage of DNA in rec-assay. Gardenia yellow induced a significant dose-dependent increase of SCE frequency (8.6 times at 1000 microg/ml as the value for the solvent control). Only genipin induced SCEs significantly among the components of gardenia yellow. Moreover, genipin induced a significant increase of tetraploids at all doses tested (95% at 8 microg/ml). Gardenia yellow preparation was analyzed by capillary electrophoresis (CE), and geniposide was detected. However, genipin was not observed. In conclusion, we have shown that genipin possesses genotoxicity. Furthermore, there were unidentified genotoxicants in gardenia yellow.

Characteristics and mechanism of the ossification of posterior longitudinal ligament in the tip-toe walking Yoshimura (twy) mouse.

To elucidate the mechanism of ossification of the posterior longitudinal ligament (OPLL), we examined the serial changes in the intervertebral disc of tip-toe walking Yoshimura (twy) mouse. At the age of 6 weeks, the volume of the nucleus pulposus increased in all intervertebral discs causing anterior and posterior herniation. Secondary to this herniation, the cartilagineous tissue of the annulus fibrosus was disrupted and showed regenerative proliferation with PCNA-positive cartilagineous cells. These cells were S-100 positive and the matrix was positive for chondroitin-4-sulfate proteoglycan, indicating the development of calcification. At the age of 15 weeks, the regenerative cartilagineous tissue of the annulus fibrosus reached the posterior longitudinal ligament together with neovascularization and appearance of PCNA-positive proliferating primitive mesenchymal cells. These cells were considered to be osteoblasts since they were positive for alkaline phosphatase and the matrix contained type I collagen. Using electron microscopic X-ray analysis, vesicles present in the matrix of regenerative cartilagineous cells of the annulus fibrosus were confirmed to contain calcium phosphate crystals, also indicating the development of calcification. In conclusion, our serial analysis indicates that ossification of posterior longitudinal ligament in twy mouse was triggered by enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilagineous tissues. Enchondral ossification of the new annulus fibrosus cartilagineous cells and membraneous ossification by primitive osteoblasts proceed to the final ossification of the posterior longitudinal ligaments.

Potential role of streptozotocin in enhancing ossification of the posterior longitudinal ligament of the cervical spine in the hereditary spinal hyperostotic mouse (twy/twy).

We investigated the effects of streptozotocin-induced diabetes mellitus on ossification of the posterior longitudinal ligament (OPLL) in the murine cervical spine. A genetically-bound spinal hyperostotic mouse, the tip-toe walking Yoshimura (twy) mouse, was used in these experiments. Histological examination showed that streptozotocin enhanced membranous and enchondral ossification of the posterior longitudinal ligament of the cervical spine, particularly in the area of the ligamentous enthesis. It also increased the number of alkaline phosphatase-positive osteoblast-like mesenchymal cells particularly around the enthesis, while the number of such cells was less in control twy mice and ICR mice treated with streptozotocin. The area of OPLL subsequently increased in size in streptozotocin-treated twy mice. We suggest that streptozotocin-induced diabetes enhances OPLL in the genetically-bound spinal hyperostotic mouse (twy/twy).