Early phenomena following cryogenic lesion of rat brain : a preliminary study

The cerebrovascular laminin becomes detectable following lesions, whereas the lamina basalis-receptor β-dystroglycan disappears. These alterations may be indirect markers of a glio-vascular detachment which may result in the impairment of blood-brain-barrier. The present study estimates the correlations between the post-lesion exudation and the aforementioned phenomena. Lesions were performed in ketamine-xylazine anaesthesia with a copper rod cooled with dry ice. Immediately, or in 5 or 10 min brains were fixed in buffered 4% paraformaldehyde. Immunohistochemical reactions were performed in floating sections. Exudation was estimated with immunohistochemical detection of plasma-fibronectin and immunoglobulins. Glio-vascular connections were investigated with immunohistochemistry (GFAP, S100, glutamine synthetase), and electron microscopy. Laminin immunoreactivity appeared already at immediate fixation. Exudate was found only around the vessels. β-dystroglycan was still detectable. At five-ten minutes the territory of exudate became confluent and dystroglycan disappeared. Some but not all vessels were free of astrocytes. Electron microscopy demonstrated wide perivascular ’spaces’. ’In vivo’ monitoring was attempted with a multiphoton microscope in the Department of Biophysics of Semmelweis University. Astrocytes were labeled supravitally with sulforhodamine 101 so glio-vascular connections were visible. However, neither in the intact brain nor in 30-min post-lesion period astrocyte motility was observed

Cyclophilin D regulates lifespan and protein expression of aging markers in the brain of mice

Abstract Cyclophilin D (cypD) modulates the properties of the permeability transition pore, a phenomenon implicated in the manifestation of many diseases including aging. Here, we examined the effects of partial or complete deletion of cypD on i) lifespan, ii) forebrain protein expression of 18 aging markers as well as regional expression of GFAP, mGluR1, and alpha-synuclein, and iii) behaviour of aged (> 24 month) male and female mice. Both male and female cypD heterozygous but not KO mice exhibited increased lifespans compared to WT littermates, associated with alterations in the protein expression of some markers, albeit without exhibiting changes in behaviour

Elevated Glucagon-like Peptide-1 Receptor Level in the Paraventricular Hypothalamic Nucleus of Type 2 Diabetes Mellitus Patients

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have been approved for the treatment of type 2 diabetes mellitus (T2DM); however, the brain actions of these drugs are not properly established. We used post mortem microdissected human hypothalamic samples for RT-qPCR and Western blotting. For in situ hybridization histochemistry and immunolabelling, parallel cryosections were prepared from the hypothalamus. We developed in situ hybridization probes for human GLP-1R and oxytocin. In addition, GLP-1 and oxytocin were visualized by immunohistochemistry. Radioactive in situ hybridization histochemistry revealed abundant GLP-1R labelling in the human paraventricular hypothalamic nucleus (PVN), particularly in its magnocellular subdivision (PVNmc). Quantitative analysis of the mRNA signal demonstrated increased GLP-1R expression in the PVNmc in post mortem hypothalamic samples from T2DM subjects as compared to controls, while there was no difference in the expression level of GLP-1R in the other subdivisions of the PVN, the hypothalamic dorsomedial and infundibular nuclei. Our results in the PVN were confirmed by RT-qPCR. Furthermore, we demonstrated by Western blot technique that the GLP-1R protein level was also elevated in the PVN of T2DM patients. GLP-1 fibre terminals were also observed in the PVNmc closely apposing oxytocin neurons using immunohistochemistry. The data suggest that GLP-1 activates GLP-1Rs in the PVNmc and that GLP-1R is elevated in T2DM patients, which may be related to the dysregulation of feeding behaviour and glucose homeostasis in T2DM

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display

Abstract The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs