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The morphological analysis represents the basic method used to determine the phenotype changes of the genetically modified mouse. In this way the observed phenotype changes are correlated to the genetic modification and enable the insight in the function of the investigated gene. The challenge of the morphological analysis increases with the increased complexity of the investigated tissues and organs. Therefore the most complex organ to be studied is the brain. In order to compare the brain neuroanatomy of the mutant and wild type mice, the brain could be studied at the gross-morphology level as a whole, at the light microscope level on histological sections, or at the ultrastructural level by electron microscopy (1). In order to compare the brain morphology at the ultrastructural level of the mutant and the wild type mouse, we used the mouse mutant Stam2Gt1Gaj, where due to the gene trap modification the function of Stam2 was hindered. Due to the brain size and the nature of electron microscopy analyisis only two brain regions were selected to be viewed by electron microscope, CA2 region of the hippocampus and the adjacent cerebral cortex. In order to compare the equivalent regions of the brain in the mutated and wild type mice the regions of interest were determined on vibratome sections, and the selected pieces of tissues were processed for electron microscopy. Adult, 3 months old mice were anesthetized with 1% avertin and perfusion fixation was performed. 0.1 M phosphate buffer was injected within the left heart ventricle in order to rinse the blood from the circulatory system. Afterwards, the animals were perfused with 12.5% glutaraldehyde, which resulted with initial fixation of the brain. Skull was opened with sharp scissors and the brain was removed and immersed in 6% glutaraldehyde for 1 hour. The brain was afterwards cut in 200 µ m thick sagital sections using vibratome. After determination of the brain regions of interest, the tissue was washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 hours. Subsequent to contrasting in uranyl acetate over night, the tissue was dehydrated in ethanol and embedded in Durcopan (Fluka). 1 µ m thick semithin sections were cut using the ultramicrotome and examined by light microscopy (figure 1). The selected and comparable regions of the semithin sections were trimmed and 70nm thick ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined by electron microscopy on transmission electron microscope Zeiss902A (2) (figure 2). The described procedure enabled the comparison of the brain ultrastructure of mice with different genetic background. The comparison was complemented with the photographing of the serial frames of the selected regions in order to increase the size of area for analysis and acquire the necessary data to be used in image analysis applications. Nevertheless, the variability of the tissue examined should be add to the variability among the mice belonging to the same genotype. The presented example highlights the difficulties of morphology analyisis of the genetically modified mice.

Endosomes are involved in the CNS in a variety of actions including formation of synaptic vesicles, axonal transport and receptor sorting. To elucidate function of Stam2 in the CNS, gene trap approach was applied. The expression pattern analysis showed that Stam2 was expressed in developing neural tube and heart. In addition, alternatively spliced variant of Stam2 was found.

STAM2 (Signal transducing adaptor molecule 2) is a signaling adaptor presumed to act in the intracellular cargo sorting for degradation at the level of the late endosome (1). Although its function was already assessed at the cellular level in permanent cell cultures, its function in mammalian organism could be revealed only through genetic modification at the level of the whole animal. In order to characterize Stam2 function in mouse, the gene trap method was applied. It comprises the genetic modification of embryonic stem cells with a non-homologous vector containing a splice acceptor and fused lacZ and neoR genes without their own promoters. In case the vector was inserted in an endogenous gene, the splice acceptor would be recognized and lacZ and neoR genes would be driven by the promoter of the endogenous gene. This enabled to screen the genetically modified embryonic stem cells and the corresponding mouse lines for lacZ activity, which mirrored the expression of the endogenous gene. In addition the vector insertion disturbed the proper protein translation 3’ to the gene trap vector. After the selection process, the mouse line carrying the gene trap mutation in Stam2 gene was chosen for further characterization. The molecular analysis defined the exact place of the insertion and its consequences on the mRNA and the protein level. This showed that the gene trap vector was between exon 2 and 3, and the STAM2 protein production in the homozygous mice was highly diminished (2). Stam2 expression pattern was assessed by monitoring lacZ activity in the investigated tissues. lacZ gives rise to beta-galactosidase, which changes the color of its substrate X-gal in blue. Stam2 expression started in 8.5-day embryos in the hind gut. In 9.5-day embryos it was present in the heart, while in 10.5-day embryos appeared in the ventral part of the neural tube (Figure 1). The expression in the heart persisted during embryo development and continued in the adults. The expression in the neural tube was continuous to the expression in the brain, in particular from 16.5-day embryos in the brain cortex, and from 18.5-day embryos in the hippocampus. In addition to the brain and the heart, Stam2 was expressed as well in the testis and developing adrenal and pituitary glands. In order to assess Stam2 function the phenotype of the homozygous carriers of gene trap mutation was compared with the wild type mice. The homozygotes were alive, fertile and did not show any major abnormalities. The functional studies of mouse behavior were combined with detailed morphological analysis of embryos and adult organs and tissues. The morphological studies involved the classical histology using the hematoxylin-eosin staining, the semithin sections stained by toluidine blue, immunohistochemistry of the selected markers, and finally the ultrastructural analysis using electron microscope (Figure 2). These studies are expected to reveal the fine differences in phenotypes of the wild type and the mutant mice, which would then reveal the Stam2 function in mouse, in particular in the mouse brain.

Neprimjerena aktivacija mikroglije implicirana je u patogenezi Alzheimerove bolesti (AD), ali nije poznato kako dovodi do disfunkcije neurona i spoznajnog urušavanja. Da bismo bolje razjasnili ulogu upalnih procesa u nastanku i progresiji AD-a, pomoću ELISA i multipleks ELISA metode mjerili smo koncentracije imunosnih medijatora i biljega aktivacije mikroglije (sTREM2) i inflamasoma (ASC) u cerebrospinalnoj tekućini (CSF) i plazmi uzoraka izuzetih od osoba s AD-om, blagim spoznajnim poremećajem (MCI) i kognitivno zdravih kontrolnih ispitanika (HC). Dodatno, korištenjem metoda imunohistokemijskog bojenja postmortalnog tkiva mozgova osoba s AD-om i HC-a analizirali smo aktivaciju mikroglije i izraženost inflamasoma. Rezultati su pokazali jaču aktivaciju mikroglije i NLRP1 inflamasoma u hipokampalnoj formaciji osoba s AD-om. Analizom likvorskih biljega utvrđene su veće koncentracije sTREM2 i ASC u bolesnika s AD-om, ako i njihova pozitivna korelacija s biljezima neurodegenerativnih i neurofibrilarnih promjena. Likvor bolesnika s AD-om imao je jasan proupalni profil i povećane koncentracije medijatora stečene imunosti. Zaključno, ova je studija pokazala snažniji imunosni odgovor u bolesnika s AD-om, veću uključenost mehanizama stečene imunosti i visoku korelaciju između aktivacije imunološkog sustava i razvoja neurofibrilarnih promjena., Aberrant microglial activation has been implicated in the pathogenesis of Alzheimer’s disease (AD), but how it leads to neuronal dysfunction and cognitive decline is not known. To better clarify the role of inflammatory processes in the development and progression of AD, using ELISA and multiplex ELISA method we measured concentrations of the immune mediators and markers of microglial (sTREM2) and inflammasome (ASC) activation in cerebrospinal fluid (CSF) and plasma samples of AD, mild cognitive impairment, and cognitively healthy control subjects (HC). We also analysed microglial and inflammasome activation using immunohistochemical staining methods in postmortal human brain tissue of the AD and HC subjects. The results have shown stronger microglial and NLRP1 inflammasome activation in the hippocampal formation of the AD subjects. Analysis of the CSF markers has shown higher concentrations of sTREM2 and ASC in patients with AD and their positive correlations with markers of neurodegenerative and tau pathological changes. The CSF of AD patients has a pro-inflammatory profile and increased concentrations of mediators of adaptive immunity. Taken together, this study has shown a stronger immune response in AD patients, higher involvement of adaptive immunity mechanisms, and a high correlation between the activation of the immune system and the development of neurofibrillary changes.