Estrogen and Progesterone Synthesis with Cellular Response in a C57BL/6 Mouse Model of Cuprizone-Induced Demyelination

Background: Demyelination refers to the degradation or loss of myelin sheath. In demyelination model studies, it has been reported that demyelination is regressed by giving steroid hormones such as estrogen and progesterone. However, there are not many studies investigating the synthesis of these two hormones by the brain during demyelination and remyelination. Neurosteroids are steroid hormones synthesized by the brain independently from peripheral tissues. In this study, it was aimed to have knowledge about the synthesis of these two hormones by the brain in experimentally formed demyelination process in brains of C57BL/6 mice and their role in the cellular response formed in the region.Materials, Methods & Results: In the study, 36 C57BL/6 mice were used: 12 mice were fed normal diet for 12 weeks as control group (Group I); 12 of them were fed 0.2% cuprizone diet for 12 weeks (Group II) and 12 mice were fed normal diet for 4 weeks after feeding cuprizone diet for 8 weeks (Group III). At the end of the experiment, mice were perfused with 4% paraformaldehyde and brain tissues were blocked in paraffin. 6 μm-thick section was taken from each block. Sections were stained histologically with LFB staining and immunohistochemically with MBP staining in order to determine the demyelination in sections. All sections were also immunohistochemically stained with GFAP to detect astrocytes, with NG2 to detect young OPCs, with aromatase for estrogen synthesis and with 3βHSD antibodies for progesterone synthesis. At the end of the study, complete myelination was observed in group I, while severe demyelination was determined in group II as a result of blind evaluation of LFB and MBP staining by two pathologists. In group III, demyelination was found to be mild. In immunostaining with GFAP and NG2 antibodies, the number of GFAP and NG2 positive cells in Group II was found to be increased compared to the control group. The difference between these two groups was statistically significant (P < 0.01). In group III, the number of GFAP and NG2 positive cells were found to be increased compared to the control group; however, it was found to be lower than that in experimental group II (P < 0.01). In immunohistochemical staining with aromatase and 3βHSD antibody, there was no staining observed in the control groups. While an intense staining was observed in experimental group II, fewer glial staining was noticed in experimental group III when compared to the experimental group II. The difference between these two groups was found to be statistically significant (P ˂ 0.01).Discussion: Aromatase is an enzyme that converts testosterone into estrogen. On the other hand, 3βHSD is an enzyme that converts pregnenolone to progesterone. Expression of aromatase from tissues refers to the synthesis of estrogen and expression of 3βHSD refers to progesterone synthesis. In previous demyelination studies carried out with cuprizone, it has been reported that demyelination is regressed by giving estrogen and progesterone during demyelination. In the presented study, we observed that enzyme levels that catalyze the synthesis of estrogen and progesterone increased during demyelination. In the study, it was determined that estrogen and progesterone levels were increased in the region by enzymes released from the glial cells of the brain as a response to damage formed during demyelination. Interestingly, during the period in which cuprizone was excluded from the diet, it was observed that remyelination began to be formed again and that enzyme levels synthesizing these hormones started to decrease. These results suggested that estrogen and progesterone may be synthesized in the brain after a damage and may contribute to remyelination by initiating a number of cell to cell signaling steps

Nitric Oxide Synthase Expression in Naturally Infected Sheep Brain with Listeria monocytogenes and Relationship with Cell Death

Background: Nitric oxide is synthesized from L-arginine and catalyzed by a family of NOS. There are three different NOS isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Nitric oxide is an important apoptosis regulator in mammalian system that can induce and prevent apoptosis depending on levels of NO production and environmental conditions of the cell. NOS expression and its relationship with apoptosis has not been well elucidated in listerial meningoencephalitis in sheep. The aim of this study was to investigate eNOS and iNOS expressions in the brain of sheep with natural listeriosis and to compare them with apoptosis which is shaped in the region.Materials, Methods & Results: In the study, formalin fixed and paraffin embedded brainstem tissue from 25 sheep naturally infected with LM were used from archives. Five μm-thick section was taken from each block. Histopathologically, sections were stained with H&E. Five normal sheep brain tissues were used as control. At the end of the study, Histopathologically in brainstem tissue infected with LM, multifocal microabscesses in different sizes mixed with neutrophils and macrophages were detected and perivascular mononuclear cell infiltration and meningitis characterized by mononuclear cell infiltration were found. All sections were also immunohistochemically stained with LM, eNOS and iNOS antibodies. In addition, TUNEL method was used to determine apoptosis in brain tissues. As a result of immunostaining, listeria immunoreactivity was observed in microabscesses. The Listeria antigens were detected mainly in the cytoplasm of the neutrophils and macrophages and located extracellulary in microabscesses. Both eNOS and iNOS immunoreactivity were observed in very few neurons and glial cells in normal control sheep. Neurons and glial cells in brain tissues of infected animals stained with eNOS and iNOS. But, eNOS and iNOS expressions in listeriosis animals more higher than in control and the this difference was statistically significant (P < 0.05). At the same time, TUNEL immunopositivity was observed mainly in the nuclei of neuron and glial cells and this findings was found to be significantly increased compared to the control group (P < 0.05).Discussion: The pathogenesis of listerial meningoencephalitis depends on many factors and is still a subject of research. NO is a pluripotent regulator of diverse cellular functions. NOS can trigger cellular damage. However, there are studies suggesting that it can prevent oxidative stress. eNOS and iNOS expressions may vary depending on disease and animal species. Increased levels of NO have been reported to induce apoptosis in many diseases. However, there are studies indicating that NO is able to prevent apoptosis according to the isoform in which it is synthesized. eNOS and iNOS have not been reported together expressions and with their relationship apoptosis in sheep brain with listerial meningoencephalitis. This is the first study of eNOS and iNOS expression and and its relation to cell death in sheep brain infected with LM. Our findings are consistent with previous studies suggesting that NO expression is effective on the pathogenesis of some disease in the central nervous system. However, there are studies that do not match the findings of the present study. This suggests that the role of NO synthesis in the pathogenesis of diseases may vary according to the amount of expression, type of disease and tissue, or animal species. In the present study showed that the expression of both eNOS and iNOS with increased TUNEL positive cells was statistically significant in the listeriosis compared the control brain tissue. These results suggests that eNOS and iNOS can be expressed by diverse brain cells in the pathogenesis of listeriosis in sheep. In addition this, synthesis of NO can induce the apoptosis in sheep brain with listerial meningoencephalitis