European Review for Medical and Pharmacological Sciences 2018; 22: 7607-7613 Expression of lamin A/C protein in degenerated human intervertebral disc C.-Z. WU1, D.-Q. OU2, L.-M. RONG1, Y.-C. XU1, J.-W. DONG1, L. FAN1, Q.-Y. WANG1 1Department of Spine Surgery, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China 2Department of Orthopedics, First People’s Hospital of Shunde, Foshan, China Cheng-Zhi Wu and Ding-Qiang You contributed equally to this work Abstract. – OBJECTIVE: This study aimed to the nuclear intima and plays an important role evaluate the expression characteristics of lamin in maintaining nuclear shape and structure1. The A/C proteins in intervertebral disc degeneration hydrolysis of lamina proteins in cell apoptosis (IVD) specimens from patients with different de- has been elucidated2. Since cell apoptosis plays generation grades. Lamin A/C proteins have an essential role in intervertebral disc degener- been shown to result in age-related changes in 3-5 the osteoarticular system. However, the expres- ation (IVD) , we were interested in elucidating sion characteristics of these nuclear proteins in the connection between lamin A/C hydrolysis degenerated human IVD tissues have not been and IVD. explored previously. 6 PATIENTS AND METHODS: Several studies have confirmed that lamin Degenerated hu- A/C is linked to bone and cartilage metabolism. man IVD tissues were obtained during spinal surgery. Articular cartilage samples after total In bone research, some scholars reported that knee replacement surgery were used as con- bone intensity and quantity are significantly low- trols. Sections of these tissues were stained er in lamin A/C knockout mice than in normal with hematoxylin and eosin, Masson, safranin mice. In cartilage metabolism, damage to or in- O, and immunostained using lamin A/C anti- terference with lamin A can lead to cartilage cell body. Western blot was performed to evaluate 7 lamin A/C expression in IVD tissues. Lamin A/C death . Further bone and cartilage studies showed expression was analyzed based on different de- that lamin A/C expression is significantly dif- generation grades. ferent in young and old mice, and its expression RESULTS: In patients with IVD degeneration, decreases with increasing age8. Lamin A/C plays mild or moderate degenerative discs contained an important role in bone and joint systems. How- high amounts of lamin A/C proteins. Lamin A/C ever, the expression of lamin A/C in degenerated expression was primarily localized in the nucle- ar envelope of IVD cells, and associated with human IVD, which is similar to cartilage have not apoptosis in cell nuclei, as determined by immu- been studied in detail before. This study aimed to nostaining and TUNEL assay. detect the expression characteristics of the lamin CONCLUSIONS: This paper is the first to re- A/C in degenerated human IVDs. port that lamin A/C proteins are present in IVD tissues and its expression may be related to disc degeneration. Patients and Methods Key Words: Lamin A/C, Osteoarticular system, Intervertebral Patient Selection and Sample Collection disc degeneration (IVD), Spinal surgery. This study was approved by the institutional Ethics Committee. Informed written consent was obtained from each participating patient. The Introduction general clinical data for the cohort are shown in Table I. Degenerative IVD were obtained from Lamin A/C is a member of the nucleoprotein patients subjected to spinal fusion surgery. All family that belongs to the type V intermediate patients underwent routine lumbar magnetic res- filament. Lamin A/C mainly distributes below onance imaging (MRI) examination. The IVDs Corresponding Author: Qi-You Wang, MD; e-mail: [email protected] 7607 C.-Z. Wu, D.-Q. Ou, L.-M. Rong, Y.-C. Xu, J.-W. Dong, L. Fan, Q.-Y. Wang Table I. Detail of patients. were classified according to the modified Pfirr- ing (thickness 50-70 nm). Finally, uranium-lead mann evaluation. IVD samples classified in the double staining was performed (2% uranyl ace- degeneration Grades I to III were grouped togeth- tate staining for 30 min; lead citrate staining for er. Samples in Grades VI to VIII were placed into 5 min). The sections were observed under a JEM another group (Table I) 1400 TEM (Jeol, Japan). Three specialists assessed the IVD degener- ation grades. The opinion of two doctors was Histology sufficient to classify the samples; when the three IVD samples were fixed in 4% polyformal- doctors disagreed on the grade of degeneration, dehyde for 24 h, decalcified in 10% EDTA, and the specimens were excluded from this study. All embedded in paraffin. Four serial sections (5 the samples were collected in our department from µm thick) per sample were cut with a microtome June 2014 to October 2014. Samples were rinsed in (Leica, Wetzlar, Germany). To observe cell mor- phosphate-buffered saline (PBS) and sheared into phology, density, and collagen expression, three pieces under a microscope. Samples were fixed in sections from each specimen were stained with 4% paraformaldehyde or 2.5% glutaraldehyde for 3 hematoxylin and eosin, safranin O-fast green and h. Cartilage for the control group was selected from Masson. patients undergoing knee replacement surgery. Immunostaining for LMNA Transmission Electron Microscopy (TEM) For immunohistochemistry analysis, paraffin The IVD block (1 mm3 volume) was fixed for sections were de-paraffinized with xylene and 3 h using 2.5% neutral glutaraldehyde and rinsed serially rehydrated. After treatment with EDTA six times using PBS for 30 min each time. Sub- (pH 9.0) in a microwave oven for 15 min for anti- sequently, 1% osmic acid was used for fixation gen retrieval, endogenous peroxidase activity was for 1.5-2 h. PBS was used to rinse the samples blocked with 3% H2O2 for 10 min at room tem- for three times. The above steps were carried out perature. A 1 h blocking step was conducted with at 4°C. After fixation, the samples underwent 3% goat serum and 1% bovine serum albumin routine gradient dehydration, permeabilization at (BSA) in PBS. Rabbit anti-human lamin A/C at room temperature for 3-4 h with resin mix (100% 5 µg/mL (Abclonal Technology, Rocky Hill, CT, acetone: Epon 812 = 1:1), and encapsulation in the USA) was applied overnight at 4°C, following by mold. After routine consolidation in the oven, a washing with PBS. Goat anti-rabbit secondary Leica UC7 ultramicrotome was used for section- antibody (1:5000 dilution) was applied for 1 h at 7608 Expression of lamin A/C protein in degenerated human intervertebral disc room temperature. After washing, a chromogenic followed by detection using an Odyssey Infrared reaction was performed using a Histostain ABC Imaging System (LI-COR, Bioscience, San Jose, kit (Boster, Wuhan, China) according to the man- CA, USA). This experiment was repeated four ufacturer’s instructions. times. Protein bands were analyzed using the LI-COR imaging software and exported to Mi- Cell Death Assay crosoft Excel. Signal intensities among different The terminal deoxynucleotidyl transferase degrees of degeneration were compared using dUTP nick end labeling (TUNEL) experiment-in- the nonparametric test (Kruskal-Wallis test) and cluded routine de-waxing of paraffin sections, Dunnett’s test. protease K antigen repair, and incubation at room temperature for 10 min, with 50-100 µL of mem- Statistical Analysis brane rupture liquid. According to the Roche SPSS 19.0 software (IBM, Armonk, NY, USA) TUNEL kit (St. Louis, MO, USA) appropriate was used for statistical analysis. All quantitative amounts of reagent 1 (TdT) and reagent 2 (dUTP) data were expressed as mean ± standard devia- were mixed at a ratio of 1:9 and added in the cir- tion. Comparison between groups was done using cle to cover the tissue. The sections were placed One-way ANOVA test followed by Post-Hoc test flat in a wet box and then incubated in a 37°C (Least Significant Difference). p-values < 0.05 water bath for 60 min. PBS (pH 7.4) was added were considered statistically significant. three times to wash cells on a shaking rotator. Subsequently, 3% hydrogen peroxide solution prepared with methanol was added, and samples Results were incubated in the dark at room temperature for 20 min to block endogenous peroxidase ac- Lamin A/C Expression in IVD tivity. Finally, an appropriate amount of reagent Figure 1 shows the immunohistochemical re- 3 (converter-POD) was added to all sections to sults of lamin A/C in degenerated human IVD. cover the tissues. The sections were laid flat in Lamin A/C accumulated around the nucleus of the wet box and incubated for 30 min in a 37°C the nucleus pulposus tissue (Figure 1 A1-A2, water bath pot. The sections were placed in PBS solid black arrows). Lamin A/C expression was (pH 7.4) on the shaking rotator and washed three lower in the annulus fibrosis than in the nucleus times. Routine diaminobenzidina (DAB) coloring pulposus (Figure 1 A1-A2, dashed black arrow). and hematoxylin nuclear counterstaining were The immunohistochemical results showed that performed. The cell apoptosis of sections was the expression of lamin A/C in mild degenerative observed under a microscope. IVD patients was significantly higher than in the control group (p < 0.05) (Figure 1 A1-A2). Carti- Quantitative Western Blot Analysis lage from human osteoarthritis after knee arthro- IVD samples were placed on ice immediate- plasty was used as positive control group (Figure ly after surgery and rinsed twice with PBS. To 1C). The negative control group (without primary isolate nuclear protein, tissues were lysed using antibody incubation) did not show non-specific a nuclear and cytoplasmic protein extraction kit staining (Figure 1C). (Boster, Wuhan, China) according to the man- ufacturer’s instructions, followed by centrifu- Apoptosis in IVD Tissue gation at 12,000 rpm to extract the final su- TUNEL detection reflects the rupture of nucle- pernatant.