Origin Of The Sequestration-type Herniated Disc And Its Correlation With The Expression Of VEGF

1. BackgroundGenerally, sequestration-type herniated disc is been seen the absolute and relative indication of surgery, these patients occupy an percentage of 15~20 in all patients treated with surgery. The spontaneous disappearance or diminution of lumbar herniated discs in the spinal canal has been recognized in recent year, and this could be a possible explanation for relief of symptoms without surgery. And the spontaneous disappearance or diminution can be seen in sequestration-type herniated disc more often. Some authors believe that patients with sequestration-type herniated disc can be treat without surgery, if these patients can tolerate the symptoms for a period of time. Inflammatory cell infiltration, especially macrophages infiltration and proteolytic enzymes such as matric metalloproteinases secret in herniated disc are necessary for spontaneous disappearance or diminution. VEGF plays an important role in the initiation of angiogenesis through stimulation of the formation of new blood vessels, lumen formation, and endothelial migration.2. ObjectiveTo investigate the origin of sequestration-type herniated disc and the role of VEGF in the sequestrated tissue, and elucidate the correlation between the the origin and the expression of VEGF. 3. Materials and methods3.1 Clinical materialsThirty-seven patients with sequestration-type herniated disc who undewent operation at the 2nd Hospital of Medical School of Zhejiang University between 2000 and 2003 were selected. All subjects received simple anteroposterior and lateral radiographs, CT and MRI of the lumbar spine examinations. The operative levels were L4~5(19 patients), Ls~Si(17 patients) and L3~4(l patients). Ages of the subjects ranged from 35 to 76 years, with a mean of 49.7 years . The duration from onset of legsymptom to surgery ranged from 2 to 192 weeks, with a mean of 36.8 weeks.3.2 Pathological study39 tissue samples were surgically removed from 37 patients with sequestration-type lumbar disc herniation.3.2.1 All the tissues were performed with HE stain and immunohistochemical study. In HE stain, the origin of tissue was identified.3.2.2 In immunohistochemical study, anti-CD34 antibody for vascular endothelial cells, anti-CD68 for macrophages, and an anti-vascular endothelial growth factor antibody was used for vascular endothelial growth factor (VEGF).3.3 Imaging study3.3.1 HE stain images(4><10) were inputted personal computer (Dell, Round Rock,TX,USA). The distance of the surface and bottom of granulation tissue were measured as the depth of granulation tissue (Osiris Version4.09, Genevese ,Geneva university).3.3.2 The number of CD34+ cells was randomly counted in 10 fields in high-power field (40x10), and the mean number in each region was used as the representative value. CD68+ cells and VEGF + cells were also treated in the same manner. The number of CD34+, CD68+ cells and VEGF+ cells represent the number of new blood vessel, macrophages and VEGF + cells.3.3.3 Quantification of angiogenesis, macrophages infiltration and expression of VEGF in granulation tissue.The degree of angiogenesis = the depth of granulation tissue xthe number of new blood vesselThe degree of macrophages infiltration = the depth of granulation tissue xthe number of macrophagesThe degree of expression of VEGF = the depth of granulation tissue xthe number ofVEGF+ cells 3.4 StatisticsStatistical analysis was performed with the SPSS10.0 analysis software (SPSS Inc.,IL, USA). Analysis of variance (ANOVA/Scheffe s post hoc analysis) was used for comparisons among the three groups. The relationship between two variables was determined using Pearson's correlation coefficient ( r ). P Values less than 0.05 wereconsidered significant. 4. Results62% (24/39) of the sequestrated tissues were mainly originated from nucleus pulposus, 31%(12/39) mainly from endplate, and only 7%(3/39) mainly from anulus fibrosus.Granulation tissue was found in 92%( 36/39) of the sequestrated tissues. In the granulation tissue, many macrophages were found around new blood vessels, and many spindle-shaped cells expressing VEGF were observed.Furthermore, a positive correlation was demonstrated in extent between angiogenesis and macrophage infiltration(r=0.371, p=0.020). Also there was a positive correlation in extent between VEGF expression and angiogenesis, and between VEGF expression and macrophage infiltration(r=0.342, p=0.033;r=0.440, p=0.005).The sequestrated tissues were divided into 3 groups according to the content of nucleus pulposus: nucleus pulposus group, nucleus pulposus contained group and non-nucleus pulposus group. There were significant different among the3 groups in extent of angiogenesis, macrophage infiltration and VEGF expression (F=5.663, p=0.008;F=3.604, p=0.038;F=4.110, p=0.025). 5. ConclusionThe origin of sequestration-type herniated disc is mainly from nucleus pulposus. The vascular endothelial growth factor produced by the spindle-shaped cells acts to promote angiogenesis inside granulation tissue of the sequestrated tissue, and that new blood vessels play an important role as a passage for macrophages infiltration, which secret proteolytic enzymes such as matrix metalloproteinases and cause an absorption of that sequestrated tissue. Moreover, the extent of VEGF expression is mainly depended on the origin of the sequestrated tissue.