Changes in the amino acid pool of cerebral hemispheres in rats with total cerebral ischemia

Introduction 
Amino acids (AA) are important in metabolism and brain function. This is due not only to theircrucial role as the sources of synthesis of various biologically important compounds (proteins, mediators, lipids, biologically active amines),but also to their involvement in synaptic transmission as neurotransmitters and neuromodulators (glutamate, aspartate, glycine, GABA, taurine). Some AA are in charge of the formation of nervous system mediators: e.g., methionine (acetylcholine, DOPA, dopamine), tyrosine (catecholamines), serine and cysteine (taurine), tryptophan (serotonin), histidine (histamine), L-arginine (NO), glutamic acid (glutamate) [1, 2, 3].

Major cerebral circulatory disorders, which are modeled by total cerebral ischemia (TCI), lead to severe brain dysfunction. TCI affects the normalratioof AA playing an important role in the functioning of the nervous system. AA and their compositions can act as therapeutic agents for cerebral ischemia. To develop new AA preparations, it is necessary to study in detail how their ratios and quantities change during cerebral ischemia of varying severity, including TCI [4–7].

The objective of our study was to assess changes in the pool of AA in rats with TCI.

Materials and Methods

The experiments were performed on 16 outbred white male rats weighing 260±20 g in compliance with the requirements of the Directive of the European Parliament and Council #2010/63/EC of 22 September 2010 on the protection of animals used for scientific purposes (Protocol #1 of 5 January 2022).

The simulation was carried out under intravenous thiopental anesthesia (40-50 mg/kg).TCI was modeled by decapitation of animals. Brain samples were collected 1 hour after decapitation. After this time, according to the results of our previous studies, significant histological disorders of neurons occur (wrinkling and hyperchromia) [4–7]. At the same time, it was of interest to determine changes in the AApool corresponding to these disorders. This model was developedby the authors.

The control group consisted of decapitated rats; their heads were immediately immersed in liquid nitrogen, and the AA composition of the brain was examined after short-term cooling.

Method for studying the amino acid pool of the brain

After the brain was removed, fragments of the parietal lobe cortex and hippocampus weresampled and frozen in liquid nitrogen.Preparation of samples for the study included homogenization in a 10-fold volume of 0.2 M perchloric acid, centrifugation for 15 minutes at 13,000 g at 4°C, followed by collection of the supernatant. AA were analyzed by reverse-phase chromatography with pre-column derivatization using o-phthalaldehyde and 3-mercaptopropionic acid in sodium borate buffer on an Agilent 1100 chromatograph.

To avoid systematic measurement errors, we examined brain samples from the compared control and experimental groups of animals under the same conditions.

Continuous data were obtained. Since our experiment involved small samples following a non-normal distribution, the analysis was performed by nonparametric statistics using the licensed softwareSTATISTICA 10.0 for Windows (StatSoft, Inc., USA). All data are presented as Me (LQ; UQ), where Me is the median, LQ is the lower quartile value,and UQ is the upper quartile value. Differences between groups were considered significant at p=0.046 (non-parametric Games-Howell post-hoc test).

Results

The main pool of AA in the cerebral cortex was characterized by the parameters identified for the control group (Table).

Table. Amino acid pool in cerebral hemispheres of rats with total cerebral ischemia (TCI), nmol/g; Me (LQ/UQ)

* p=0.046, compared with the control group.

Compared with the values characteristic for the control group, 1 hour after decapitation, the TCI group animals exhibited in their parietal lobe (PL) and hippocampus (HC) samples the following trends: an increase in the content of aromatic AA(precursors of biogenic monoamines): tyrosine (by 43%, p=0.047, and 40%, p=0.047, respectively) and tryptophan (by 24%, p=0.049, and 23%, p=0.049, respectively).

Also, in TCI group vs. the control animals, the level of methionine increased by 32% in PL (p=0.046) and by 27% in HC (p=0.056). Methionine is a precursor of other sulfur-containing AA (serine, cystathionine, cysteine). In addition, we observed elevated levels of other sulfur-containing AA (cysteine, taurine), with the exception of cystathionine in PL and cysteine sulfinic acid in HC. In the TCI group, we noted an increase in the content of L-arginine in both PL and HC (by 20%, p=0.038 and 33%, p=0.038, correspondingly).

In contrast to the HC, in the PLof the TCI animals, the content of the citrulline, which is a precursor of L-arginine, increased by 22%, p=0.046.

As for the group of branched-chain amino acids (BCAA) (valine, isoleucine, leucine), the changes in TCI were multidirectional:specifically,we observed an increase in the isoleucinelevel by 12% in PL (p=0.046) and a decrease in valine content by 15 % in HC (p=0.054). Levels of other BCAAdid not change (p=0.057).

The ratio of the combined total content of BCAA to the combined content of aromatic AA in TCI significantly declined from 1.4 to 1.0 in PL (p=0.046) and from 1.6 to 1.0 in GC (p=0.039). We observed an increase in methionine content by 33% (p=0.046) in PL and an increase in tryptophan content by 24% (p=0.046) in HC.

The content of the inhibitory neurotransmitter glycine in TCI tended to increase in PL and HC (by 15% and 13%, respectively,p=0.049), while the content of excitatory neurotransmitters (aspartate and glutamate) did not change (p=0.058).

Changes in the content of essential amino acids (valine, isoleucine, leucine, methionine, lysine, histidine, threonine, tryptophan, phenylalanine) in TCI animals were multidirectionalboth in PL and HC. In PL, the methionine content increased by 33% (p=0.046), and in HC, tryptophan level increased by 24% (p=0.047).

However, the ratio of nonessential to essential AA in the TCI group did not change (p=0.053).

Discussion

The increase in methionine content in TCI reflects the identified lack of activation of oxidative processes in this grade of cerebral ischemia [2, 3, 8–11].

An increase in the content of sulfur-containing AAechoes actively occurring transsulfuration and dioxygenase reactions in the context of the pathway of cysteine transformations, including the synthesis of taurine [2, 10].

An increase in the level of L-arginine during cerebral ischemia may be associated with the low activity of reactions of its utilization due to oxygen deficiency, among which the formation of nitrogen monoxide plays a significant role. In turn, NO performs mediator functions, participates in the regulation of cerebral blood flow as a vasodilator, and is capable of exhibiting antioxidant, antiplatelet and anti-inflammatory properties [7–9, 12, 13].

An increase in the level of L-arginine and citrulline reflects the active course of reactions associated with the cytoplasmic part of the ornithine cycle, with the exception of thoseinvolving arginine [2, 3, 7].

Conclusion

One-hour TCIcaused the following changes in the AA pool: an increase in the content of aromatic AA(tyrosine and tryptophan) and methionine; an increase in the content of L-arginine; and also, an increase in the concentration of the inhibitory neurotransmitter glycine.

Changes in the AA pool in the PL and HC were similar, which made it possible to exclude a significant increase in methionine levels in the PL as a reflection of active oxidative effects in this part of the brain in combination with more pronounced morphological changes in the PLof rats with PCI.

Conflict of interest. The authors declareno conflicts of interest.