| Sažetak | Objectives: The objective of this study was to analyze the tissue characteristics in retinal surface disorders in four groups of patients including iERM, macular rupture, retinal detachment and diabetic retinopathy. It was hypothesised that VEGF is more expressed in less differentiated cells which should also show an overexpression of NESTIN.
Materials and methods: ERM samples were collected over 4 years from 157 patients whose age group ranged between 12 and 82 years. The median age was 71 years. 85 out of the 157 patients were male and 72 were female. The inclusion criteria for the study was the diagnosis of ERM that were confirmed with optical coherence tomography (OCT) and visual acuity of 0.5 or less. The ERM tissues were collected during the Pars Plana Vitrectomy (PPV) surgery. The samples were embedded in paraffin blocks before they were cut and stained using immunohistochemistry. NESTIN was the primary antibody used and VEGF was the secondary antibody. Sections were observed and images were captured using a BX51 microscope (Olympus, Tokyo, Japan) equipped with a DP71digital camera (Olympus, Tokyo, Japan). Images were then processed with CellA Imaging Software for Life Sciences Microscopy (Olympus Tokyo, Japan). Microscopic imaging with Hematoxylin and eosin stain were also performed. Tsanou et al. scoring system was used to evaluate the expression of VEGF and NESTIN. The intensity of staining was scored 0 to 3 (0 = negative, 1 = weak, 2 = intermediate and 3 = strong staining) and was semiquantitatively scored as +, ++ and +++ corresponding to small, moderate and high number of positive cells, respectively.
Results: We found that the expression of NESTIN was mostly seen in ERMs of retinal detachments and VEGF was equally expressed in Groups 1, 3 and 4. Neither of the proteins were expressed in Group 2 samples which represented iERMs. It was also discussed that during future studies, it would be ideal to use a confocal microscope which will allow better tissue preservation and the expression of Ki67 and GFAP would also be analyzed.
Conclusions: It was concluded that the exact mechanism in the development and pathogenesis of some retinal surface disorders such as iERM formation is still unclear. We found NESTIN positive tissue in our samples which suggests their proliferation capacity and their immaturity. Positive VEGF expression in these tissues shows their potential for angiogenesis especially in patients with retinal detachments and diabetic retinopathy. The results from this study confirmed the hypothesis that VEGF is more expressed in less differentiated cells which also show an overexpression of NESTIN. |
| Sažetak (hrvatski) | Ciljevi: Cilj ovog istraživanja je analiza karakteristika tkiva u poremećajima površine mrežnice u četiri skupine bolesnika, uključujući iERM, rupturu makule, odvajanje mrežnice i dijabetičku retinopatiju. Pretpostavljeno je da je VEGF izraženiji u manje diferenciranim stanicama, što bi također trebalo pokazati prekomjernu ekspresiju NESTIN-a.
Materijali i metode: Uzorci ERM-a prikupljeni su tijekom 4 godine od 157 pacijenata bolesnika čija se dobna skupina kretala između 12 i 82 godina. Srednja dob bila je 71 godina. Od ukupnog broja bolesnika 85 ih je bilo muškog spola, a 72 ženskog. Kriteriji za uključivanje u studiju bili su dijagnoza ERM-a koja je potvrđena optičkom koherentnom tomografijom (OCT) i oštrinom vida od 0,5 ili manje. ERM tkiva prikupljena su tijekom operacije Pars Plana Vitrektomije (PPV). Uzorci su ugrađeni u parafinske blokove prije nego što su izrezani i imunohistokemijski obojeni. NESTIN je primarno korišteno protutijelo, a VEGF je sekundarno antitijelo. Promatrane su sekcije i snimljene su slike pomoću mikroskopa BX51 (Olympus, Tokyo, Japan) opremljenog digitalnim fotoaparatom DP71 (Olympus, Tokyo, Japan). Slike su zatim obrađene pomoću CellA Imaging Software-a za mikroskopiju Life Sciences (Olympus Tokyo, Japan). Također su provedena mikroskopska snimanja s Hematoxylin i eosin bojama. Tsanou i sur. Sustav bodovanja korišten je za procjenu ekspresije VEGF-a i NESTIN-a. Intenzitet bojenja je bodovan 0-3 (0 = negativno, 1 = slabo, 2 = srednje i 3 = jako) i bio je polu-kvantitativno ocjenjen kao +, ++ i +++ što odgovara malom, umjerenom i velikom broju pozitivnih stanica.
Rezultati: Otkrili smo da se ekspresija NESTIN-a uglavnom vidi u ERM-u retinalnih izdvajanja, a VEGF je jednako izražen u skupinama 1, 3 i 4. Nijedan od proteina nije eksprimiran u uzorcima skupine 2 koji su predstavljali iERM. Također je raspravljano da bi tijekom budućih studija bilo idealno koristiti konfokalni mikroskop koji će omogućiti bolje očuvanje tkiva i analizirati ekspresiju Ki67-a i GFAP-a.
Zaključci: Zaključeno je da je točan mehanizam u razvoju i patogenezi nekih poremećaja površine mrežnice kao što je iERM formacija još uvijek nejasan. Našli smo NESTIN pozitivno tkivo u našim uzorcima što sugerira njihovu sposobnost proliferacije i njihovu nezrelost. Pozitivna ekspresija VEGF-a u tim tkivima pokazuje njihov potencijal za angiogenezu, posebno u bolesnika s retinalnim odvajanjima i dijabetičkom retinopatijom. Rezultati ove studije potvrdili su hipotezu da je VEGF izraženiji u manje diferenciranim stanicama koje također pokazuju prekomjernu ekspresiju NESTIN-a. |
