Dipeptide L-сarnosine (β-alanyl-L-histidine) — nervous tissue cryoprotector non-hybernate animals

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In this work, the cryoprotective properties of dipeptide L-carnosine (β-alanyl-L-histidine) were studied on slices of the olfactory cortex of the brain of rats. Changes in the activity of N-methyl-D-aspartate receptors were analyzed as the most vulnerable to the effect of cryopreservation (CP), for this purpose, extracellular NMDA potentials were recorded. Slices were incubated with L-carnosine (20 mM) in the medium and frozen at a slow rate (0.1°C/min) down to –10°C and after CS (30 days) they were heated at the same rate (0.1°C/min) to +37°C. The effectiveness of cryoprotection of L-carnosine was determined by changes in the amplitudes of NMDA potentials after CP compared to before CP. The dipeptide restored the pH of the freezing medium 6.9 (without L-carnosine) to the optimum pH 7.3—7.4, promoted dehydration of free water from slices after CP, inhibited the development of glutamate excitotoxicity in slices. The data obtained prove that L-carnosine exhibits the properties of a non-toxic effective cryoprotector in the nervous tissue of warm-blooded non-hibernating animals.

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A. A. Mokrushin

Institute of Physiology, I. P. Pavlov, Russian Academy of Sciences

Author for correspondence.
Email: mok@inbox.ru
Russian Federation, Saint Petersburg

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2. Fig. 1. Study of cryoprotective properties of L-carnosine on slices of the olfactory cortex of the rat brain (a) when measuring the amplitudes of NMDA potentials (b, c). a - scheme of a tangential slice of the olfactory cortex of the rat brain with the main morphological structures and localisations of stimulating and recording electrodes: LOT - lateral olfactory tract, SE - stimulating electrode, PC - piriform cortex, RE - recording electrode, b - focal potential, in slice on electrical stimulation of LOT with indication of postsynaptic components: early AMPA potential and late NMDA potential (μV), c - zoomed in NMDA potential - indicator of activity of NMDA-dependent mechanisms. Only modifications of NMDA potentials during the action of L-carnosine during CW were investigated in this work. Dotted line, isoline - slice potential at rest; vertical arrow indicates the method of measuring NMDA potential amplitudes at the time point 8 ms from the stimulation artefact. Calibration - as indicated

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3. Fig. 2. Effect of L-carnosine application at different concentrations on the amplitude of NMDA potentials in rat olfactory cortex slices. The abscissa axis is scale conditional. Different concentrations of L-carnosine were tested on a separate group of slices (n = 12). Changes in the amplitudes of NMDA potentials in relation to the values before CS (control) were evaluated by nonparametric Wilcoxon-Mann-Whitney U-criterion, p ≤ 0.05 (*)

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4. Fig. 3. Effects of preincubation of slices in control without and with L-carnosine (20 mM) on acid-base level (pH) of ICR before and after the end of CW (a), grey background - optimal pH ranges (pH 7.2-7.4), at which NMDA potential amplitudes are maintained, n = 7. The effects of preincubation of slices in control without L-carnosine and with L-carnosine (20 mM) on the modification of NMDA potential amplitudes before and after CW (b), reliability of differences in pH values of freezing solution (L-carnosine 20 mM after CW) in comparison with the values before CW (control before CW without L-carnosine) were determined by nonparametric Wilcoxon-Mann-Whitney U-criterion, n = 7

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5. Fig. 4. Changes in free water content (swelling - slice weight, mg) in slices under the influence of L-carnosine (20 mM) before and after CW (a), n = 5. Effects of slice swelling under the influence of L-carnosine (20 mM) on changes in NMDA potential amplitudes before and after CW (b). Differences of NMDA potential amplitudes compared to the values before CW (‘control before CW’) and after CW (‘without carnosine’ and after CW with ‘carnosine, 20 mM’) were determined by nonparametric Wilcoxon-Mann-Whitney U-test, n = 5

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6. Fig. 5. Testing of L-carnosine (20 mM) for the occurrence of excitotoxicity in brain slices during warming after CS. Abscissa axis - solution temperatures at which NMDA potential amplitudes were measured, scale is irregular, n = 9. The rate of slices heating is 0.1 oC/min. Other notations are shown in the figure. Differences of NMDA potential amplitudes in relation to the values before CS (control) were determined by nonparametric Wilcoxon-Mann-Whitney U-criterion, p ≤ 0.05 (*)

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