METHOD FOR DETECTING MICROTOPOGRAPHY OF NEURONS IN THE CENTRAL NERVOUS SYSTEM Russian patent published in 2023 - IPC G01N33/50 G01N30/00 

FIELD: medicine; neuromorphology.

SUBSTANCE: invention can be used to detect the microtopography of neurons in the central nervous system in representatives of various mammalian species. The method includes the following: euthanizing the animal, subsequent perfusion, fixation, obtaining the studied brain blocks, preparing sections, placing them in a dye solution, washing the sections, layering them with a brush on a glass slide, passing them through alcohols and a balm. At the same time, solutions of NaH₂PO₄×2H₂O are prepared separately in distilled water — 58 mg in 2l of H₂O and NaOH — 12.6 g in 0.5l of H₂O; phosphate buffer consisting of a mixture of solutions of 660 ml of NaH₂PO₄ and 340 ml of NaOH, at pH=7.2. A perfusate is then prepared, consisting of 5 g of NaCl dissolved in 0.5l of H₂O. 0.4l of polyglucin and 2.0 g of heparin are added to it, and the temperature of the solution should be 37°C. Next, a solution consisting of 0.5 mg of horseradish peroxidase (HRP) is prepared, dissolved in 4 ml of 2% dimethyl sulfoxide (DMSO), 0.1 ml of which, using a stereotaxic stage, hydraulically, a graduated glass electrode with a tip diameter of 4–6 µm. The solution is slowly injected with the help of a perfusator into the brain object under study, which is then stored in a thermostat at a temperature of 18–20°C and after 24–48 hours, depending on the object of the study. After the injection of HRP and DMSO, the brain object is perfused with mixtures of the following solutions: 1% of paraformaldehyde and 2.5% of glutaraldehyde in phosphate buffer at pH 7.2. Then it is fixed in this mixture for 4 hours and prepare frozen sections 40 μm thick, they are placed for 40–50 minutes in a 30% sucrose solution in phosphate buffer, previously diluted 1:1 with distilled water. Then the sections are further processed according to the method of M.M. Mesulam.

EFFECT: invention provides increased sensitivity of the analyzed cortical-cortical and cortical-subcortical connections of the brain, localization of neurons at the level of light microscopy.

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