Astrocyte specific proteins content in the different pArts of the rAt And mongoliAn gerbil brAin during ontogenesis

Astrocyte-specific proteins are used as markers of astrocytes, particularly in the case of age-related brain dysfunctions and neurodegenerative disorders. However, the data on the content of these proteins in different parts of the brain during ontogenesis are insufficient. in this research, the content of astrocyte spe cific Ca 2+ -binding protein S100B and glial fibrillary acidic protein (GFAP) in cerebellum, thalamus, and hip pocampus of Wistar rats and mongolian gerbils during ontogenesis were determined. Animals were divided by age into four groups (n = 6-10): 1 – newborn (1-day-old); 2 – 30-day-old; 3 – 90-day-old; 4 – 180-day-old. Fractions of soluble and filamentous proteins from the different parts of the brain were obtained by differen tial centrifugation and solubilization with 4m urea. the quantitative levels of S100B and GFAP were deter mined with ELiSA. it was revealed that the content of S100B protein increased significantly by day 180 in all studied parts of the rat and gerbil brain. the content of GFAP both soluble and filamentous forms in the brain of 1 day-old animals was low, but raised significantly after 30 days in all studied parts of gerbil brain, and increased gradually up to 180 days in the rat brain. the magnitude of the increase in the content of the studied astrocyte-specific proteins was shown to be different and to depend on the brain part, postnatal development stage of the animals and their species.

D uring formation and maturation, the brain gradually develops and manifests astroglia. Astrocytes are the most common glial cells in the central nervous system (CNS) [1]. These glial cell types interact closely with neurons to provide structural [2], metabolic, and trophic [3] support and are actively involved in modulating the excitability of neurons and nerve impulses [4]. Astrocytes regulate neurotransmitter systems [5], synaptic processes [6], ion homeostasis [7], antioxidant protection [8], and energy metabolism [9]. According to current data, astrocytes also play a key role in conducting neuropathic pain [10]. Thus, functional changes in astrocytes can interact with surrounding cells, such as neurons and microglia [11]. Importantly, physio logic and pathologic properties of astrocytic metabolic plasticity bear translational potential in defining new potential diagnostic biomarkers and novel therapeutic targets to mitigate neurodegeneration and age-related brain dysfunctions [12].
The primary astrocyte proteins are calciumbinding protein S100B and glial fibrillary acidic protein(GFAP).GFAPisahistospecificcomponent oftheintermediatefilaments(IF)ofthecytoskeleton of astrocytes [13]. This protein provides structural support and tensile strength to the cytoskeleton of normal astrocytes [14]. The distribution of GFAP in differentpartsofthebrainisunevenanddependson the number of astroglial cells: it is known that the content of astroglial cells in adult rats is maximum in the medulla oblongata (the complex of lower oils) and minimum in the cerebral cortex [13]. GFAP, as part of IF, plays an integral role in the migration of astrocytes and maintenance of the stable morphology of their processes during the development of reactive astrocytosis [15]. The use of GFAP as a mo-lecular index of neurotoxicity and a marker of nerve tissue damage has been proposed [16,17]. Allegedly, this protein is involved in the molecular mechanisms of neuron-astrocyte interactions [18]. Due to its high specificity and early release from the CNS after traumatic brain injury, GFAP can be a very useful marker for early diagnosis [13]. The production of GFAP by astrocytes and the assembly of this protein into intermediate filaments interact with the Ca 2+ -regulated EF-hand proteins, S100B, in a dosedependent manner [19].
The S100B protein was discovered by Moore [20] and is considered one of the nodal molecular components of complex intracellular systems that ensure the functional homeostasis of brain cells by combining and integrating various calciumdependent metabolic processes. Quantitative changes in S100B are currently regarded as a marker of brain damage (cortical, ischemic, etc.), metabolic disorders inthebrain,orbeingundertheinfluenceofvarious factorsinthebody [20].Characteristically,fluctuations in the concentration of S100B in the brain are not always accompanied by a marked deterioration in the somatic condition of animals, but, at the same time,thesefluctuationscanleadtovariousdisorders of the integrative brain function, depending on the degree of hyperproduction of this protein.
Astrocyte dysfunction contributes to the develop ment of psychiatric and neurodegenerative disorders [19,20]. With age, the number of astrocyte-specificproteinsincreases;however,thereare no quantitative data on the dynamics of the levels of theseproteinsindifferentpartsofthebraininboth laboratory animals and humans. This study aimed to investigate the redistribution and quantitative indicatorsofastrocyte-specificproteins,S100BandGFAP, indifferentpartsofthebrainsofratsandMongolian gerbilsatdifferentstagesofontogenesis.

materials and methods
This research was performed on 24 Wistar rats and 24 Mongolian gerbils. Animals were divided into four groups by age (n = 6-10): 1 -newborn animals (1-day-old), 2 -30-day-old, 3 -90-day-old, 4 -180-day-old. The rats and Mongolian gerbils were exposed to standard conditions with a natural change in lighting and adherence to the general diet. All animals had free access to food and water. The experiment was conducted in accordance with the "Regulations on the use of animals in biomedical experiments" [21].
The amount of total protein in the obtained fractions was determined as described by Bradford [23] and expressed in mg/100 mg of tissue.
Statistical processing of the obtained data was performed using application packages Microsoft® Excel 2000 (Microsoft®) and STATISTICA® for Windows 6.0 (StatSoft Inc.). Statistical analysis was carried out with one-way analysis of variance, ANOVA . Data at P < 0.05 were considered statisticallysignificant.

results and discussion
In the first part of the experiment, the total amounts of proteins in the cytosolic and 4 M urea extracted cytoskeletal fractions obtained from different parts of the brains of rats and gerbils during postnatal development were investigated.
The total protein content in the cytosolic fractionsfromthedifferentpartsofthebrainofnewborn rats was 1.93-2.21 mg/100 mg of tissue ( Fig. 1, a).
In the cerebellum and thalamus, total protein contents gradually increased during the developmen-tal period until day 90: 2.77 ± 0.19 mg/100 mg of tissue in the cerebellum and 3.07 ± 0.21 mg/100 mg of tissue in the thalamus. However, on the 180 th day of postnatal development, total cytosolic protein contentinthesebrainareassignificantlydecreased, compared to levels in 90-day-old rats, however, it wasnotdifferedcomparetotheotherstudiedtime of postnatal development. The dynamics of the to- tal protein content in the hippocampus differed from those of the other brain parts, decreasing to 1.89 ± 0.12 mg/100 mg of tissue in 30-day-old rats and 1.49 ± 0.09 mg/100 mg of tissue in 180-day-old rats, compared to newborns. In newborn gerbils, the level of total protein content in the cytosolic fractions ranged from 1.21 to 2.41 mg/100 mg of tissue, with the highest

Cerebellum
Hippocampus Thalamus rate observed in the hippocampus and the lowest in the thalamus (Fig. 1, B).Similartothefindings in rats, on day 30 of postnatal development, total protein content increased in the cerebellum (2.41 ± 0.12 mg/100 mg of tissue) and thalamus (1.80 ± 0.15 mg/100 mg of tissue) of gerbils but decreased in the hippocampus. The increase in the cerebellum and thalamus is probably due to the continuing anabolic processes in the direction of formation and maturation of certain structures in these parts of the brain after birth, unlike in the hippocampus, where structures are formed during embryogenesis [25]. In the thalamus, the total amount of cytosolic proteins reached its apex on day 30 of postnatal development and remained constant until day 180. In the cerebellum, this rate increased up to the 90 th day of postnatal development and stayed at 2.76 ± 0.05 mg/100 mg of tissue until the 180 th day. Therewerenosignificantchangesinthehippocampus of gerbils during ontogenesis. Studies of the total pool of proteins in the cytoskeletal fractions obtained from different parts of the brain of rats showed that levels in 1-day-old animals ranged from 0.58-0.94 mg/100 mg of tissue ( Fig. 2, a).
The total protein content in the cerebellum of 30-day-old rats increased significantly to 1.03 ± 0.13 mg/100 mg of tissue and doubled in the hippocampus to 1.91 ± 0.12 mg per 100 mg of tissue. In the thalamus, an increase in the total protein content was started from the 30 th day of postnatal development, but these data were not reliable because thetotalproteincontentfluctuatedinawiderange.
The total cytoskeletal protein content decreased as in the cerebellum and hippocampus of 180-dayold rats compare to the 30-day-old. However, no significantchangesweredetectedinthesecontents onday90andday180.Inthethalamus,thisfigure increasedsignificantlyonthe90 th day of postnatal development to 1.27 ± 0.12 mg/100 mg of tissue and remained constant at the developmental phase.
The results of the assessment of the total protein content in the cytoskeletal fraction of gerbils were ranging from 0.41-0.51 mg/100 mg of tissue for allstudiedbrainsectionsonthefirstdayofpostnatal development (Fig. 2, B). Then, this content gradually, butinsignificantly,decreasedbyday30.In90-dayold gerbils, there was a clear decreasing pattern in the cerebellum and thalamus, in contrast to the stable level in the hippocampus. On day 180 of postnatal development, the total protein content in all studied parts of the brain increased, with the highest rate in the hippocampus at 0.65 ± 0.05 mg/100 mg of tissue.
It was earlier investigated the intensity of the S100B protein biosynthesis during ontogenesis in humans and animals, revealing that the protein S100Bappearsatweeks10-15indifferentpartsof the brain (cerebellum, pons, brainstem, midbrain, and spinal cord, and so on) of the human embryo [20]. By the 30 th week, the S100B protein accumulates in all parts of the CNS, except the frontal lobe, where the increase in protein content coincides with the appearance of the bioelectrical activity of the brain.
The accumulation of the S100B protein at the differentstagesoftheontogenesisofchickens,mice, and rats has also been studied in detail [26]. For example , in the brain of 3-to 15-day-old mice, the level of the S100B protein remains relatively low, then sharply increases from the 16th to the 22 nd day of postnatal development: it increases during this period by about 4-fold. A similar pattern has also been observedinthebrainofrats,withtheonlydifference being that the protein most intensely accumulates between the 16 th and 24 th days of postnatal development of the animal. The intensity of the S100B protein synthesis in the brain correlates with the level of mRNA in the corresponding parts of the brain.
In this study, we aimed to comparatively de-terminethelevelsoftheS100Bproteinindifferent parts of the brains of rats and gerbils (Fig. 3, a), as previous data relate only to the total content of S100B in the brain.
Hiden et al. have demonstrated that the most intense biosynthesis of this protein occurs in the pyramidal cells of the hippocampus [27]. There is still a growing interest in the functional role of the S100B protein. The content of this protein increases during education and training of animals, which is confirmedbythefactthatthereisamoreintensive inclusion of labeled amino acids in the S100B protein of the brain during the training period. It has also been shown that mice of inbred lines have a higher amount of the S100B protein in the brain, and these animals learn faster than control animals, in which the protein level in the brain is lower. Without a doubt, the S100B protein contained in neurons is primarily concentrated in the synaptic membranes and nucleoli of neurons.
Our studies of S100B in the cerebellum, hippocampus, and thalamus of gerbils during postnatal development showed that the dynamics of the content of this protein in all studied brain regions over 30 days were similar to those in rats (Fig. 3, B), but the content of the protein was itself lower in gerbils than in rats.
The content of S100B in the cerebellum of new-borngerbilswas0.08±0.003μg/100mgoftissue,in thehippocampus,itwas0.05±0.001μg/100mgof tissue,andinthethalamus,itwas0.06±0.005μg/100 mg of tissue. In 30-day-old gerbils, the level of this protein in the cerebellum increased to 0.31 ± 0.01 μg/100mgoftissue,inthehippocampus,itroseto 0.25±0.02μg/100mgoftissue,andinthethalamus, itwas0.29±0.01μg/100mgoftissue.Nosignificant changes were detected in the concentration of S100B in all three observed regions during the next 30 days of postnatal development. However, on day 180, there were substantial increases in the protein contents in all studied parts of the gerbil brain -in thehippocampus,itwas0.56±0.03μg/100mgtissue,inthethalamus,itwas0.56±0.11μg/100mg oftissue,andinthecerebellum,themostsignificant changewasregisteredat1.69±0.11μg/100mgof tissue. ThefindingsindicateanincreaseinS100Bcontents during 180 days of postnatal development. Literature data indicate that the level of this protein also increases with age. Brain aging is associated with the increased expression of the S100B gene and its mRNA in rats [28], as well as in the brains of neurologically healthy individuals [29]. However, available quantitative data on S100B in the aging brain are contradictory [28,29]. Moreover, according to some studies, the content of S100B and its mRNA, as well as the density of S100B-positive astrocytes in the hippocampus of mice, do not change with age [30]. One study revealed that the level of S100B in thehumancerebrospinalfluidalsodoesnotdifferin healthy young and elderly people [31]. The increase in this protein in various parts of the brain at the later stages of ontogenesis is still an open question and requires a detailed study. In addition, as noted above [19], this protein is one of the regulators of the filamentousformsofthecytoskeletonandmayhave impact on GFAP contents.
Most of the work that has been carried out on thestudyofGFAPcentersarounditsfilamentous form, while in the investigation on the nature of changesintheratiooffibrillatedandsolubleforms of GFAP, their polypeptide heterogeneity remains unclear [32]. GFAP is absent in the brain of newborn rats and unmyelinated white matter of the brain of infants,butwithgrowth,itissynthesizedandphosphorylated in glial cells after stimulation by hormones (norepinephrine) and various growth factors [14,33].
In newborns, radial glia and immature astrocytes mostly express vimentin. However, during the maturation of nerve tissues in the early stages of development, vimentin is gradually replaced by GFAP indifferentiatedastroglialcells [34].
In this research, we evaluated the level of two forms(solubleandfilamentous)GFAPinthecerebellum, hippocampus, and thalamus of rats and gerbils ( Fig. 4, a, B).
A small amount of soluble GFAP was detected in newborn rats in all experimental areas of thebrain.Duringthefirstmonthofthelifeofrats, there was a significant increase in the amount of sGFAP in the cerebellum (2.12 ± 0.45 μg/100 mg oftissue)andhippocampus(0.80±0.05μg/100mg oftissue),butthemostsignificantincreasewasobservedinthethalamus(4.46±0.70μg/100mgof tissue). In 3-month-old experimental animals, we observed that the content of sGFAP in the cerebellum increased 2.5-fold compared with the amount in 1-month-old rats. sGFAP also increased in the hippocampus and thalamus compared with 1-month-old rats; however, these data were not reliable because the content of sGFAP fluctuated in a wide range. For 180-day-old animals, a significant increase in sGFAP in all parts of the brain of rats was observed; the content of sGFAP in the cerebellum was 11.19±0.30μg/100mgoftissue,inthehippocampus,itwas2.27±0.26μg/100mgoftissue,andin thethalamus,itwas7.20±0.90μg/100mgoftissue.
Like in rats, low levels of the soluble form of GFAP were found in all studied parts of the brain of newborn gerbils, but these levels increased marked ly within 30 days (Fig. 4, B). The content of sGFAP in the cerebellum of 30-day-old gerbils was 1.12±0.03μg/100mgoftissue,butin90-day-old animals,itincreasedto1.67±0.11μg/100mgoftissue and remained constant until 180 days. In contrast to the cerebellum, sGFAP in the hippocampus and thalamus of adult animals reached peak levels on day 30ofpostnataldevelopment,at1.51-1.68μg/100mg oftissuebutdidnotchangesignificantlybyday180.
There were low contents of this form in the cerebellum, hippocampus, and thalamus of newborn rats (Fig. 5, a).
In 30-day-old animals, fGFAP content increased considerably in all studied parts of the brain: in the cerebellum,itwas12.90±2.61μg/100mgoftissue, inthehippocampus,itwas4.81±1.01μg/100mgof tissueandinthethalamus,itwas5.40±1.01μg/100 mgoftissue.fGFAPalsoincreasedsignificantlyin the hippocampus and thala mus of 90-day-old rats compared to their 1-month-old counter parts. The pattern in the cerebellum of 90-day-old rats was also that of an increase, compared to 1-month-old animals, but the data, in this case, were not reliable becausethecontentoffGFAPfluctuatedinawide range.
Similar to the content in rats, fGFAP levels in all studied parts of the brain of newborn gerbils were also minimal (Fig. 5, B), which is consistent with the generally accepted fact that the process of the activation of the expression of the GFAP gene begins only after the birth of mammals [33].
Age is directly related to numerous neurodegenerative brain disorders, and most of them are associated with astroglial dysfunctions [35]. Our findingsshowthatchangesoccurinboththetotal proteincontentandthecontentofastrocyte-specific proteins,GFAPandS100B,indifferentpartsofthe brains of gerbils and rats depending on the period of postnatal development. Reportedly, during maturation and formation, the brain gradually develops and manifests astroglia. The results of this investigation suggest that the most intense development of astrocytes and the biosynthesis of GFAP occur in the cerebellum by the 90 th day of postnatal development of gerbils and rats, and in the thalamus and hippocampus, these processes occur earlier.
Conclusions. There are specific changes in both the total protein content and the content of astrocyte-specificproteins,solubleandfilamentous forms of GFAP and calcium-binding protein S100B, inthedifferentstudiedpartsofthebrainsofgerbils and rats, and these changes depended on the term of postnatal development, brain`s area and animals' specie.