Study of histopathological results of idiopathic retinal membrane

Nội dung text: Study of histopathological results of idiopathic retinal membrane

1. Journal of military pharmaco-medicine STUDY OF HISTOPATHOLOGICAL RESULTS OF IDIOPATHIC RETINAL MEMBRANE Nguyen Van Cuong*; Dang Tran Dat**; Nguyen Dinh Ngan* SUMMARY Objectives: To describe histopathological results of idiopathic retinal membrane (iERMs) and the association between these results. Subjects and methods: A cross-sectional decriptive study which used standard vitrectomy to obtain epiretinal membrane in 35 iERMs in Vietnam National Institute of Ophthalmology. Results: Six different cell types were discovered based on morphology including glial cell (35/35), fibroblast (23/35), myofibroblast (23/35), macrophage (13/35), lymphocyte (5/35) and neutrophil (2/35). The number of cell type in one sample ranged from 1 to 5 types (average 2.85 ± 1.28 cell types in a sample). There were statistically significant difference between the number of cell type in sparse density (3.0 ± 1.2 cell types in a sample) and in dense density group (2.2 ± 1.3 cell types in a sample) in dense density group (p = 0.01, test t-student). The inflammatory cells (macrophage, lymphocyte, neutrophil) just appeared in few samples. Conclusion: There were 6 types of cells in iERM samples and glial cell is the most popular. Cell density and number of cell types were correlated. The inflammatory cells (macrophage, lymphocyte, neutrophil) just appeared in few samples with high number of cell types. * Keywords: Idiopathic epiretinal membrane; Histopathology. INTRODUCTION ocular pathology were found and its Epiretinal membrane (ERM) is contractile pathophysiology has remained unclear. membrane that occurs on the inner Until now, many studies about histopathology surface of the retina and can lead to of the membrane were done to investigate significant visual impairment when located the pathology of this disease. There were at the central retina. It is caused by the 2 components detected: the proliferative proliferation of avascular cellular sheets cells (origin or deprivation), and extracellular on the inner retinal surface and along the matrix (collagen, fibronectin). In particular, internal limiting membrane (ILM), which proliferative cells are the most important possesses contractile properties and as a component that forms extracellular matrix. result, leads to variable visual symptoms The types of cells in iERM were quite and visual impairments, primarily due to different in studies because of different the mechanical distortion of the macular methods of histopathology (including area [1, 2]. Idiopathic ERM is an ERM, in chemical staining such as hematoxylin which no underlying causative factors or eosin (HE), periodic acid schiff (PAS); * 103 Military Hospital ** National Institue of Ophthalmology Corresponding author: Nguyen Dinh Ngan (ngan.ophthal@gmail.com) Date received: 26/06/2017 Date accepted: 11/08/2017 180
2. Journal of military pharmaco-medicine immune marker staining such as 2. Methods. immunocytochemistry, immunohistochemistry * Design: Cross-sectional descriptive and detecting by transmission electron study. microscopy). However, the frequent types * Size of sample: of cells in iERM were as follows: glial Size of sample calculated using the cells, hyalocytes, pigment epithelium cells, descriptive study formula: 34 patients. We macrophages, fibroblasts, myofibroblasts, carried out the study in 35 patients. and inflammatory cells [1, 2, 3, 6, 7, 10, 12]. Until now, there has not been study of * Surgical procedure: iERM pathology in Vietnam, so we carried Patients were performed vitrectomy out this study with the aim: To describe standard with 3 ways through pars plana histopathological results of idiopathic trocar with BIOM system. Posterior retinal membrane and the association vitreous detachment procedure was taken between histopathological results. in case of posterior vitreous attachment to retina. The epiretinal membrane was SUBJECTS AND METHODS stained by Trypan blue (0.5 mL, 0.25%; 1. Subjects. luoron GmbH, Neu-Ulm, Germany) to clearly observe. Then, the membrane was The iERM patients were indicated for peeled off by intraocular forceps. After vitrectomy and membrane peeling in the taking out of eye ball, the epiretinal Department of Vitreous and Retina, Vietnam membrane was fixed immediately in 0.9% National Insitute of Ophthamology (VNIO) saline solution to bring to histopathological from 7 - 2016 to 11 - 2016. laboratory. * Inclusion criteria: * Stain and analysis: - Patients with iERM were diagnosed - Stain procedure (HE staining on clinical and OCT and indicated for method): The specimens were flattened surgery at stage 1 or 2 (according to on the antiseptic glass slide and dried in Pournaras C.J (2011) [9]). alcohol solution several times with - The specimens after iERM peeling decreasing alcohol concentration (100%, were at least 2 x 2 mm in size and 90%, 80%, 70%). Afterward, they were contained central area (according to stained with hematoxylin within 3 minutes Marie Ueki (2016) [8]). and then washed until blue in about 5 - Patients agreed to participate in the minutes. Then, the specimens were study. separated in 1% alcoholic acid (1% HCl in 70% ethanol) for 5 minutes and washed * Exclusion criteria: with distilled water until they are green - Contraindicated surgery due to severe again. Next, the specimens were stained systemic disease, such as high blood with eosin Y 1% solution in 10 minutes pressure, high glycaemia. and re-washed with distilled water within 5 - Patients did not agree to participate minutes. The specimens then were fixed in the study. on microscope slides with paraffin. 181
3. Journal of military pharmaco-medicine - Analysis: The fixed specimens were identified as the number of cells more checked by optical microscope with than 10 cells in a field (40x). different magnifications (10x, 20x, 40x). - Type of cells: Basing on the cells' The macroscopic properties were identified shapes and sizes; the color, size, shape on 10x objective lens. The cell density was of the cell nucleus; characteristics of the cytoplasm and the fiber cytoplasm. determined on the 20x objective lens. The cell types were identified on the 40x - The number of cell types in a sample. objective lens. * Statistical analysis: The results are processed according to * Data collection: statistical method medicine by Excel and - The cell density of sample: "sparse" SPSS 16.0 program (SPSS, Inc., Chicago, was identified as the number of cells less IL, USA). In the analysis, the value of than 10 cells in a field (40x), "dense" was p < 0.05 as statistically significant. RESULTS 1. Histopathological results. * The cell density: 34% a b 66% Dense density Spare density Chart 1: The distribution of cell density . Image 1: (a) Sparse density; (b) Dense density. Lei Chi Wang (2015) used HE staining groups: dense (9 - 83 cells/field 40x) (12 method to detect the number of cells in samples), sparse (1 – 11 cells/field 40x) the idiopathic and secondary ERM and (11 samples) [12]. Mari Ueki (2016) using also found the low density (< 10 cells/field, HE staining method, cell density was 40X) samples made up about 1/3 samples divided into 2 groups: dense density as (13/35 samples). They divided the cell above 100 cells/field 40x (1 sample) and density into 3 levels: dense (20 cells/40x sparse density as less than 100 cells/field field) (12 samples), medium (10 - 20cells/ 40x (10 samples) [6]. There were 12 field 40x) (10 samples), low (< 10 cells/ cases with sparse density and 23 cases field 40x) (13 samples) [4]. Chong Shao with dense density in our study. These Bu (2015) studied on iERM causing macular results of cell density were quite similar hole, the cell density was divided into 2 with other previous studies [4, 6, 12]. 182
4. Journal of military pharmaco-medicine * Number of cell types in a sample: No.of samples 29% 22% 20% 20% 9%10 7 7 8 3 No. of cell types 1 2 3 4 5 Chart 2: Distribution of number of cell types. In our study, the number of cell types ranged from 1 to 5 types of cells in one sample (average 2.85 ± 1.2 types of cells). Most of samples (21/35) had more than 2 different types of cells. Other previous studies pointed the same results. Kampik’s study (1981) showed from 1 to 3 cell types in one sample in his study, Ganderfor (2012) (1 - 3 cell types), Lei Chi Wang (2015) (1 - 4 cell types), Marie Ueki (2016) (2 - 4 cell types) [3, 6, 12]. However, these studies were evaluated the cell types in quite small number of samples (from 15 to 35 cases). On the other hand, they had used some modern methods to classify the cell types (such as: electron microscope - Kampik, Gandorfer; some immune markers - Lei Chi Wang, Marie Ueki) so they may categorize more exactly the cell types as well as the origins of these cells [3, 4, 6]. * Distribution of cell types: No.of samples 35 23 23 13 5 2 GLA FIB MFB MAC LPC NEU Cell types Chart 3: Distribution of cell types. (GLA: Glial cell; FIB: Fibroblast; MFB: Myofibroblast; MAC: Macrophage; LPC: Lymphocyte; NEU: Neutrophil) 183
5. Journal of military pharmaco-medicine Glial cell was the most popular cell in iERM, it was detected in all specimens. All other studies (Marie Ueki [2016], Sao Chong Bu [2014], Kampik [1981], Gandorfer [2012], Lei Chi Wang [2015]) also found this cell in their specimens [3, 6, 12]. Sebag (2004), Foos (1997) explained that glial cells may be the first cell migrate on the surface of retinal after PVD and they were become the bridge for other cells to migrate and proliferate on the surface of retina. These studies also suggested that Muller glial cell with their growth factors played the important role in supporting the development of iERM. Image 2: Fibroblast. Image 3: Myofibroblast . Image 4: Macrophage. Image 5: Neutrophil. Image 6: Lymphocyte . Image 7: Glial cell. Fibroblast was the second frequent included cases in stage 2 (77%), so the type of cell with the appear in 23 incidence of myofibroblast may be higher specimens (65.7%). These cells may be than other studies. Myofibroblast was differentiated from astrocyte and formed proven as the main factors creating the fiber of basic substrate of iERM. substrate collagen formation, secretion of Myofibroblasts were detected in 23 proteins stretched, so this cell was the cases (65.7%). Many other studies such main reason for the formation of retinal as Marie Ueki (2016), Sao Chong Bu traction. These characteristics of the cell (2014), Kampik (1981), Gandorfer (2002) also played an important role in many detected this cell [3, 6, 12]. Chi Wang Lei other pathological fibrosis formations such (2015) also found 73.3% of samples with as in cardiomyopathy, thus myofibroblasts myofibroblast cells [4]. This type of cell inhibitor agents for this kind diseases can be differentiated from some different should necessarily to research and develop. cells such as hyalocyte, Muller cells and Macrophage was detected in 13 cases retinal pigment epithelial cells in late (37.2%). Some other studies also detected stage of ERM [8, 9]. Our study mainly this cell type in iERM based on morphology 184
6. Journal of military pharmaco-medicine [3, 12]. Although our ERM cases were study (1981), Gandorfer (2012) have idiopathic, so the inflammatory factors discovered many samples with were eliminated, we had found macrophage, lymphocytes in their studies [3, 12]. In our the cell usually appeared in the tissue opinion, the studies do not find related to inflammation, in one third lymphocytes because they used samples. So the inflammatory mechanism immunohistochemistry method to classify may have played a role in the formation of type of cells and they did not use specific iERM. Malav Joshi et al (2013) likewise markers for lymphocyte. These cells gave them same comment about iERM usually appear in chronic inflammatory pathophysiology [5]. The macrophage place, so it was one more time proven secretes many inflammatory factors, such that inflammation contributed to iERM as interleukin-1, interleukin-6, interleukin-8, formation. Lymphocytes in iERM may be TNF-alpha thereby stimulating fibroblast, related to the secretion chemotaxis during fibroblast secretion mechanism. Therefore, the development of iERM. Chemotaxis the appearance of this cell may make the factors were also found in the vitreous macular edema situation more severe due chamber of iERM cases [2]. to membrane contraction. Neutrophil was detected in only 2 cases Lymphocyte was detected only in 5 in our study. All the previous studies had cases (16.7%). Some previous studies did not found this type of cell in their sample not mention to this type of cell (Sao Chong of ERM. In our study, these cells in the Bu (2014) [6], Lei Chi Wang (2015) [12], samples may suppose be from blood Marie Ueki (2016) [4]), meanwhile Kampik vessel damage in the surgical process. 2. Association between histopathological results. * Distribution of number of cell types by cell density : No.of cell 4.5 type 4 3.5 3 2.5 2 1.5 1 0.5 0 Dense Spare Density group Chart 4: Distribution of cell types by cell density . 185
7. Journal of military pharmaco-medicine Mean of number of cell types was 3.0 ± 1.2 in dense density group, mean of number of cell types was 2.2 ± 1.3 in sparse density group, the difference was statistically significant (p = 0.01, test t-student). The data showed that the number of cell types was closely related to cell density. In our opinion, the reason for this issue may be the interaction between different types of cells. In progress of iERM development, some types of cells may stimulate each other to migrate and proliferate. * Distribution of number of cell types by cell type: Table 1: Distribution of number of cell types by cell type. Number of cell types 1 2 3 4 5 Cell type (n = 7) (n = 7) (n = 8) (n = 10) (n = 3) Glial cell 7 7 8 10 3 Fibroblast 0 6 6 8 3 Myofibroblast 0 6 7 8 2 Macrophage 0 0 2 9 2 Lymphocyte 0 0 0 2 3 Neutrophil 0 0 0 0 2 Table 1 showed the cells of connective tissue (glial cell, fibroblast, myofibroblast) were the main part of iERM. These cells were detected in almost samples with different number of cell types. Glial cell was the most popular cell type (detected in 35/35 samples) which proved the important role of this cell in the mechanism of iERM. Otherwise, the inflammatory cells (macrophage, lymphocyte, neutrophil) just appeared in few samples with high number of cell types (from 3 cell types in a sample). This data proved that the inflammatory mechanism may contribute to create the iERM athough we could not clinically find the inflammation. However, this mechanism was appeared just in a few of iERM formation. CONCLUSSION of cell types in a sample range 1 to 5, mean 2.85 ± 1.28. We have evaluated the histopathology - The number of cell types in dense of 35 iERM cases using HE dying, the density group was statistically significant results showed: difference with the number of cell types in - There were 6 different cell types sparse density group (p = 0.01, test based on morphology, including glial cell t-student). The inflammatory cells (35/35), fibroblast (23/35), myofibroblast (macrophage, lymphocyte, neutrophil) just (23/35), macrophage (13/35), lymphocyte appeared in few samples with high (5/35) and neutrophil (2/35). The number number of cell types. 186
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