| Sažetak | Korteks čini najveći dio volumena mozga te se ovdje procesuira većina pristiglih informacija. Istražujući tijek razvoja mozga, nailazi se na veliki broj neodgovorenih pitanja. Bolje razumijevanje tog perioda dalo bi potpuniju sliku o funkcijama i mehanizmima koji se događaju u mozgu, a nova saznanja o neuralnim matičnim stanicama mogu se pokazati iznimno korisna u terapiji neuroloških bolesti i ozljeda. Jedan od načina identifikacije neuralnih matičnih stanica, kao i zrelih stanica, je pomoću imunohistokemijskih markera. U ovom istraživanju testiramo markere SOX2, SOX9, GFAP, NeuN, MBP, Iba1, Ki67 na korteksima novorođenih oposuma (Monodelphis domestica). Testiranje je provedeno koristeći dvije metode, imunohistokemiju (IHC) te izotropni frakcionator (IFR). U sklopu IHC metode, korišteni su tanko narezani poprečni presjeci korteksa, dok je za IFR korištena homogena smjesa jezgri stanica korteksa. U obje metode izvršena su imunooznačavanja sa spomenutim markerima te vizualizacija pomoću fluorescentnog mikroskopa. SOX2+ i SOX9+ stanice kolokalizirane su u području ventrikula. Postoji velika razlika u broju SOX2+ i SOX9+ stanica u ostatku korteksa, u korist SOX2+ stanica. Ta se razlika s vremenom smanjuje, što upućuje na početak astrogeneze. GFAP+ stanice su jasno vidljive tek kod nešto starijih oposuma te su uglavnom koekspimirane sa SOX9, dok kod mlađih oposuma to nije bio slučaj. Mlađi oposumi imali su veliki broj NeuN+/SOX2+ stanica što bi upućivalo da se radi o mladim neuronima kod kojih dolazi po sporijeg utišavanja ekspresije SOX2 kao moguća posljedica produljene neurogeneze oposuma. MBP nije dao informacije o prisutnosti oligodendrocita, također, marker proliferacije, Ki67, nije se pokazao dovoljno specifičan. Suprotno tome, Iba1 marker jasno je označio mikroglije već kod najranije testirane dobi oposuma. IHC metodom je ispitana valjanost markera koji su pomogli u identifikaciji i lokalizaciji stanica korteksa mladih oposuma. IFR metoda je dodatno trebala pomoći u kvantifikaciji stanica, no dobiveni podaci nisu pouzdani. Nema studija koje bi potvrdile točnost tih podataka. |
| Sažetak (engleski) | The cerebral cortex makes up the largest part of the brain's volume, and most of the information received is processed here. Exploring the course of brain development, one encounters a number of unanswered questions. A better understanding of this period would give a more complete picture of the functions and mechanisms occurring in the brain, and new insights into neural stem cells may prove extremely useful in the treatment of neurological diseases and injuries. One way to identify neural stem cells, as well as mature cells, is by using immunohistochemical markers. In this study, we test the markers SOX2, SOX9, GFAP, NeuN, MBP, Iba1, Ki67 on cortices of newborn opossums (Monodelphis domestica). Testing was performed using two methods, immunohistochemistry (IHC) and isotropic fractionator (IFR). As part of the IHC method, thinly sliced cross sections of the cortex were used, while for IFR a homogeneous mixture of cortex cell nuclei was used. In both methods, immunolabeling with the mentioned markers and visualization using a fluorescent microscope were performed. SOX2+ and SOX9+ cells are colocalized in the ventricular area. There is a large difference in the number of SOX2+ and SOX9+ cells in the rest of the cortex, in favor of SOX2+ cells. This difference decreases over time, suggesting the onset of astrogenesis. GFAP+ cells are clearly visible only in slightly older opossums and are mostly coexpressed with SOX9, while in younger opossums this was not the case. Younger opossums had a large number of NeuN+/SOX2+ cells, which could indicate that these are young neurons that have a slower silencing of SOX2 expression as a possible consequence of prolonged neurogenesis of the opossum. MBP did not provide information on the presence of oligodendrocytes, also, the proliferation marker, Ki67, did not prove to be specific enough. In contrast, the Iba1 marker clearly marked microglia already at the earliest tested age of the opossum. The validity of markers that helped in the identification and localization of cortex cells of young opossums was examined by the IHC method. The IFR method was supposed to further aid in cell quantification, but the data obtained is not reliable. There are no studies to confirm the accuracy of this data. |
