Comparative Analysis On Immunohistochemistry Of Inflammatory Marks In Erector Spinae With Ankylosing Spondylitis

[Objective]1. To compare observation the positive expression for various kinds of the inflammatory marks (CD4, CD8, CD20, CD68, MHC-I) in muscles with ankylosing spondylitis (AS) and polymyositis (PM), and to study the number of inflammatory infiltrating types.2. To analytically contrast the sensibility expression for the kinds of the inflammatory marks in the erector spinae with AS, in order to obtain the relevance of four marks.[Methods]1. Patients collection1.1Source of patients The6cases with AS come from The Nan Fang Hospital of outpatients and4cases with AS come from the Hospitals of traditional Chinese and Western medicine, and8males,2females, their age are range from20to28years, durations are4±0.6years; The10cases with PM come from The Nan Fang Hospital of inpatient at neurology medical ward, and including3males,7females, their age are range from42to50years, durations are2±0.4years; And10cases with lumbar intervertebral disc protrusion (LIDP) as controls, all of them come from the spinae surgery ward in Guangzhou Orthpaedic Trauma Hospital,7male,3females, their age are range from51to60years, durations are6±1.2years. 1.2Inclusion criteria1.2.1AS patients with inclusion criteria (five are necessary)(1) Consistent with the1984New York standard of AS patients;(2) Aged more15, male or female;(3) Lumbar activities limited;(4) X-ray showed the sacroiliac joints display sclerosis or the ilium of the sacroiliac joints display burr-like changes.(5) Erythrocyte sedimentation rate, C-reactive protein, pain score and other indicators suggest that patients had been in a stable condition period.1.2.2PM patients with inclusion criteria:the diagnosis standards of DM/PM use standards of Bohan/Peter in1975(1) Progressive symmetrical myasthenia;(2) muscle biopsy show:necrosis, regenerating fibres, infiltration of inflammatory cells in muscle, or/not with muscle atrophy;(3) raised serum creatine kinase levels;(4) electromyography show injure from muscle.(5)If the doctor is satisfied with curative effect after immunosuppressive agent treated, despite without definite diagnosis, we will study these patients in groups.1.2.3LIDP patients with inclusion criteria:All patients had been made a definite diagnosis for LIDP, meanwhile including (1) the patients need surgical operation treatment;(2) erythrocyte sedimentation rate (ESR), rheumatoid factor, somatic antibody (’O’antibody) are normal.1.3Exclusion criteria1.3.1AS patients with exclusion criteria (one of them)(1) Aged less than15years; pregnancy or breast-feeding women or the frail patients;(2) Combined with cardiovascular and other serious underlying diseases, the mentally diseases, severely abnormal liver and kidney function, or obvious skin lesions;(3) Persons who informed consent, not signed the study treatment, for exact diagnose through traumatic biopsy.1.3.2PM patients with exclusion criteria (1) it wasn’t made a definite diagnosis through the muscle biopsy, and after immunosuppressive agent treatement it is unsatisfactory therapeutic efficacy. We will exclude these patients in groups;(2) the patients with PM who had other illness e.g. tumor or overlap syndrome(OS) be excluded;(3) The patients with PM were treated immunosuppressive agent over4 weeks, we wouldn’t adopt these patients.1.3.3LIDP patients with exclusion criteria:Ruled out the clear heart, brain, lung, liver, kidney diseases, a history of other major organs.2. Collection and fixture muscle2.1Collection of specimens of AS Routinely disinfect skin of the erector spine biopsy with the muscle about5cm from the midline at the level of L3/4. And sectioning cutis by scalpel biopsy technique with asepsis and under local anaesthetic of2%lidocaine. Cut the muscle about3×5×10mm3. Then Close a wound, and bath the wound with iodine and to cover the wound with antiseptic gauze of mupirocin ointment.10Muscle samples were fixed immediately and snap frozen in liquid nitrogen cooled isopentane. Then the samples were conserved under ultra low temperature refrigerator (-80℃Type:USA ULT1786-4-V41). Cryostat sections were later prepared and stained with haematoxylin and eosin and histochemical sections prepared using both the marks of CD4, CD8, CD20, CD68and MHC-I.2.2The samples of patients with PM were collected from their biceps, and the remaining approaches were the same as those of AS.2.3The samples of patients with LIDP were collected from their erector spinae on the operation, and the remaining approaches were the same as those of AS, too.3. Made frozen section and staining The samples of three groups(AS, PM, LIDP) cut into slices in permanent cold case by freezing microtome (Type:Germany Leica CM1850), and their thickness is about4um, about200slids. Then Cryostat sections were divide between HE and immunohistochemistry staining (by ABC technology). Every group were marked.3.1HE staining The sections were stained by the hematoxylin, then used a little water to wash away them, Closing behind they were differentiated by the1%hydrochloric acid in ethanol. And were slightly washed by0.5%eosin dyed, distilled with the water, were dehydrated by various concentrations of ethanol dehydration:80%ethanol,95%ethanol I, and95%ethanol II, finally by the dehydrated alcohol, were blocked with the neutral resin cementing.Immunohistochemistry staining The sections were fixed by the ice-acetone. hydrogen peroxide3%was used to inhibit the endogenous peroxidase. After two rinses in TBS of5min each. Normal goat serum (NGS) was applied for10min to block non-specific binding; Primary anti-body was applied and incubated overnight at4℃in a moist chamber, followed by secondary antibody, and secondary antibody were incubated at room temperature for30min; Followed by tertiary antibody of the avidin-biotin-peroxidase complex (ABC); After two rinses in TBS of5min each; With two rinses of5min in TBS between each. Finally, a solution of equal volumes of diaminobenzidine-tetrahydro-chloride(DAB)0.1%in TBS. Frozen sections cut from the muscle of PM formed the positive control for the inflammatory marks. Meanwhile, the muscle of LIDP formed the negative control; Negative controls included omission of the primary antibody.4. Pictures acquisition and analysis Photomicrography under fixed light, then select10fields of view in camera on every section. Followed measure the Area, Max density, IOD (Integrated Optical Density) of the positive expression area and calculate the average of each index and Rate(Area value/View area) with image pro-plus6.0software. Area result identity in IOD by calculating four index, IOD value is the best indicator of the positive expression Area and Density. Therefore, we select IOD.5. Statistical analysis The statistical tests were performed using the original measurement data by SPSS13.0software. The intensity of positive expression on each primary antibodies between AS and PM, all antibodies between AS and LIDP, and differences among the sensibility of four antibodies in AS group were analyzed using one-way ANOVA. There were statistically significance by double-sides P<0.05.[Result]1. Before analyzing, these sections of each group were observed by two pathologists. CD20is no significant positive expression in three groups. Therefore, CD20were not adopted in the comparison research process.2. HE staining AS group:There are the scattered fibrosis with central migration of nuclei within the muscle fibers; Meanwhile the phenomenon of muscle cell fibrosis which appear occur in the place where is infiltrated by the inflammatory cell. There are focal, mild monocyte infiltrates, aggregates and the muscle cells membrane ruptured and increased variability in fibre size in the muscles; and show that a part of myofibers atrophy. Especially the macrophages collect and ingest the damaged cells around endomysial blood vessels.PM group:The necrosis and regeneration of muscle fibers was scattered or segmental in each section, including the inflammatory cell infiltrates. And inflammation was more obvious in the endomysium. The necrosis was more marked in the muscle bundle.LIDP group:The muscle cells are fairly uniform and regular polygons. There is no necrosis, regeneration of muscle fibers and the inflammatory cell infiltrates in the muscle tissue.3. Result of IOD The positive expressions were significantly different in three antibodies(CD4, CD8, CD68, MHC-I) between muscle of AS and LIDP (all P<0.05), the positive expression(mean IOD) were more intensive AS than LIDP. And the positive expressions were no obvious changes in two antibodies(CD8, MHC-I) between muscle of AS and PM(all P>0.05); The positive expressions were significantly different in two antibodies(CD4, CD68) between the muscle of AS and PM(all P<0.05) and intensity of the positive expression were PM>AS by means.4. The sensitivity of four antibodies in AS The sensitivity expressions were significantly different in four antibodies(CD4, CD8, CD68, MHC-I) in muscle with AS (F=70.144, P<0.05), MHC-Ⅰ>CD4>CD68>CD8by their means.5. Result of ImmunohistochemicalAS group:The CD4T lymphocytes and the CD8lymphocytes are weakly positive expression in vascular region of inflammatory cell infiltration(CD4more strong CD8). The CD4T lymphocytes and CD68macrophages invade the local inflammatory cell and fibrous muscle tissue. But there are negative around the normal muscles. Expression of CD68and MHC-Ⅰ are strongly positive on the membrane surface; Also, the MHC-I expression was present on endangium of the blood vessels, inflammatory cells and its’endochylema.PM group:Four marks were present on the all inflammatory cells, moreover, it’s very strongly. CD68is the most sensitive in the PM group.[Conclusion]Both AS groups and PM are being inflammatory cell infiltration in the muscle cells. However, there are mononuclear cells infiltrate mainly in AS, and lymphocyte cells infiltrate mainly in PM. Positive expression of various inflammatory markers in the muscle cells are more obviously stronger AS than LIDP, but less weaker than PM. The inflammation had mainly invaded the blood vessels and connective tissue around the muscle cells with PM. Though both AS groups and PM are variability in fibre size, PM is more larger and more stronger than AS. Finally it is most significant that the MHC-I expression is present on the endochylema inflammatory cells.