Effects of Intrahypothalamic Administration of microRNA Inhibitors and Mimetics on Blood Plasma Biomarkers in the Rat Aging.

The effect of let-7a-5p, miR-9a-3p, miR-132-3p, miR-218a-5p microRNA inhibitors and mimetics, when administered into the dorsomedial nucleus of the hypothalamus (DMN), on the markers of age-related changes in blood plasma in 3-month-old and 24-month-old male rats was studied. In the 24-month-old control rats, the content of C-reactive protein (CRP) increased, and the level of myoglobin decreased compared to the 3-month-old animals. After te administration of miRNA inhibitors, the level of CRP significantly increased, and the content of myoglobin decreased, and after the administration of miRNA mimetics, opposite changes were observed. We found no significant differences in the content of somatotropic hormone and testosterone between the control and experimental groups, as well as between the 3-month-old and 24-month-old animals.

[Age-related changes in AKT and mTOR expression in mediobasal hypothalamus neurons with aging.]

The hypothalamus is a regulatory center that controls homeostasis, reproduction, circadian rhythms, and the endocrine system. It is also involved in the regulation of aging. Expression of protein kinase B (AKT), the mammalian target of rapamycin (mTOR), and phosphorylated forms of AKT (pAKT) and mTOR (pmTOR) was studied by Western blotting in 3-, 12-, and 24-month-old male rats in the arcuate (ARN), dorsomedial (DMN) and ventromedial (VMN) nuclei of the hypothalamus. The results showed that the components of AKT/mTOR signaling change differently in the mediobasal hypothalamic nuclei of rats with age. The expression of AKT and pAKT decreased in the ARN, DMN, and VMN with aging; the expression of mTOR and pmTOR increased in the ARN, but decreased in the DMN and VMN in aged rats. The results obtained can serve as a basis for future studies of the mechanisms of age-related diseases and their pharmacological treatment.

Changes in the Expression of Calbindin and Calretinin in Interneurons of the Spinal Dorsal Horns Under Conditions of Antiorthostatic Suspension in Mice.

In female C57Bl/6 mice subjected to antiorthostatic suspension of the hind limbs for 30 days, calbindin- and calretinin-containing interneurons of the dorsal horns of the upper thoracic segments of the spinal cord were studied using immunohistochemical methods. In mice of the experimental group, cross-sectional area of calbindin- and calretinin-containing interneurons decreased in laminae I, II, and III and increased in laminae IV and V and in the region of the medial edge of the dorsal horn. After antiorthostatic suspension, expression of calretinin decreased in interneurons of laminae I and II and calbindin expression increased in the interneurons of laminae III, IV, and V. The total number of interneurons in laminae of the spinal cord detected by Nissl staining in the control and experimental groups remained unchanged. After antiorthostatic suspension and in control group, number of interneurons immunoreactive for calbindin and calretinin was maximum in lamina II and minimum in laminae IV and V and in the region of the medial edge of the spinal dorsal horn.

Changes in the Neurochemical Composition of Motor Neurons of the Spinal Cord in Mice under Conditions of Space Flight.

Expression of choline acetyltransferase, 200-kDa neurofilament protein, 28-kDa calbindin, neuronal NO synthase, caspase 3, and Ki-67 in the motor neurons of spinal cord segments T3-T5 in male C57Bl/6 mice after 30-day space flight in the Bion-M1 biosatellite was studied by immunohistochemical methods. Under conditions space flight, the size of motoneurons increased, the number of neurons containing choline acetyltransferase and neurofilaments, decreased, and the number of calbindin-positive neurons increased; motoneurons, expressing neuronal NO synthase and caspase 3 appeared, while Ki-67 was not detected. Fragmentation of neurons with the formation structures similar to apoptotic (residual) bodies was observed in individual caspase 3-positive motoneurons.

[CHANGES OF SPINAL MOTOR NEURONS IN MICE AFTER А SPACE FLIGHT].

The expression of choline acetyltransferase (ChAT), neurofilament (NF) protein 200 kDa, calbindin (CAB) 28 kDa, neuronal NO-synthase (nNOS), caspase 3, Ki-67 was studied in motor neurons from TIII­TV segments of the spinal cord in C57/ BL6 male mice by immunohistochemical methods 12 h after a 30 days-long space flight on the Bion-M1 biosatellite. Mice living under standard vivarium conditions served as a control. The motoneurons of experimental animals demonstrated the reactive changes that were manifested by the increase of their size, decrease in the number of subpopulations expressing ChAT and NF, increase of subpopulations containing CAB, appearance of motor neurons expressing nNOS, caspase-3, and the absence of Ki-67. The lack of proliferating gliocytes, the preservation of nucleolar component indicate the absence of necrobiotic changes characteristic of necrosis. The cytoplasmic vacuolization was observed in thionine-stained CAB-immunoreactive neurons. The disintegration of motor neurons into fragments resembling the apoptotic bodies was observed in single cells that contained caspase-3.

[Subpopulation of calbindin-immunoreactive interneurons in the dorsal horn of the mice spinal cord].

In the dorsal horn of the spinal cord in the plates I-IV on the thoracic and lumbar levels different subpopulations of interneurons immunoreactive for calbindin 28 kDa (CAB IR), which are specific to each plate. In the area of the medial edge of the dorsal horn, we have found a special subpopulation of CAB IR interneurons whose morphometric characteristics differ from CAB IR interneurons subpopulations of said plates. The number of CAB IR interneurons was maximal in the plate II at all levels of the spinal cord. Leveled differences are more CAB IR interneurons and larger area of the cross sections at the lumbar level.

[Calbindin-containing neurons of the ventral horn of murine spinal cord gray matter].

The study was performed in 4 C57black/6 mice to examine the neurons located in T(II), L(IV), L(V) and L(VI) segments of the spinal cord (SC) ventral horn, containing 28 kD calbindin (CAB) and 200 kD neurofilament (NF) proteins. To demonstrate immunoreactive neurons, the cells were labeled with antibodies against CAB and double labeled with antibodies against CAB and NF. The total cell population was demonstrated using NeuroTrace Red Fluorescent Nissl Stain. Results have shown that CAB-immunopositive neurons were identified in ventromedial area of the ventral horn at all SC levels and were represented by Renshaw cells. CAB-positive interneurons located in the medial area of the ventral horn were present only in SC lumbar segments. CAB-positive motorneurons that were identified in the medial area of the ventral horn, were present in one SC segment (L(IV)) and were also found to contain a NF protein.

[Age-dependent changes of neurochemical properties of sensory neurons].

Sensory neurons represent various groups of neurons differ on their morphological, immunohistochemical and receptor characteristics. The most of large neurons with myelinated Adelta fibers contain neurofilament 200 kD (NF200), some small afferent neurons can bind the isolectin B4 (IB4). Also, sensory neurons may include different types of tyrosine kinases (trkA, B and C) and neurotransmitters. Neuropeptides are generally located in small and medium-sized neurons. The proportion of neurons which contain trkA decreases and the percentage of NF200-, IB4-,substance P- and CGRP-positive neurons increases during the early development. Development of different types of sensory neurons fulfill under control of neurotrophins.

Age-associated changes in sympathetic neurons containing neurofilament 200 kDa during chemical deafferentation.

Neurofilament with a molecular weight of 200 kDa is detected in the rat sympathetic ganglia since birth. The percentage of neurons containing this neurofilament decreases during the first 20 days of life. Just solitary neurofilament-positive neurons are detected in rats at the age of 180 and 360 days. Chemical deafferentation by capsaicin, used as a model of age-associated neuron degeneration, leads to a significant reduction of the level of neurofilament-200-imminopositive neurons in comparison with the control starting from day 10 of life. Presumably, part of the sympathetic ganglionic neurons are capsaicin-positive and their function is afferent.

[Topography and morphometric characteristics of NF200(+)-neurons of the spinal cord gray matter after deafferentation with capsaicin].

The study was performed in 80 female Wistar rats aged 3, 10, 20, 30, 60, 180 and 360 days to examine the effect of capsaicin deafferentation on the topography of neurons containing 200 kD neurofilaments (NF200(+)-neurons) in the gray matter of the spinal cord II thoracic segment. The deafferentation was modeled by a single subcutaneous administration of 150 mg/kg of capsaicin to 2-day-old rats. Intact rats (n = 40) served as a control. To demonstrate NF200(+)-neurons the cells were labeled with antibodies against NF200. To count the proportion of immunoreactive neurons, the total neuronal population was marked with NeuroTrace Fluorescent Nissl stain. The results of the work have shown that NF200(+)-neurons were found in the ventral horn, intermediate zone and the dorsal horn of the spinal cord and differed from each other in their morphometric characteristics. In capsaicin-treated animals, the numbers of medial and lateral NF200(+)-motoneurons and their cross-sectional area were similarly reduced in the spinal cord (plate IX). In the intermediate zone (plate VII) and in the dorsal horn (plates III-IV) the number of NF200(+)-neurons in capsaicin-treated animals was not changed but their cross-sectional area exceeded normal values.

[Characterization of afferent neurons of spinal ganglions sensitive to capsaicin].

Morphological features of TRPV1(+)-, SP(+)-, CGRP(+)-, NF200(+)-neurons has been studied in thoracic spinal ganglions in 3-month-old rats under chemical deafferentation. The results have shown that from 6.5 up to 41.3% of ganglionic neurons of control group had markers mentioned above. The heterogeneity of nociceptive neurons in control group was kept in capsaicin-treated animals. In both groups, TRPV1(+)-neurons were prevailed, populations of SP(+)-, CGRP(+)- and NF200(+)-neurons formed smaller groups. Sensitivity to capsaicin was shown in largest neurons in each population; neurons in experimental group had smaller cross-sectional area, particularly in group of TRPV1(+)-neurons.

[Substance P-immunopositive neurons in rat sensory ganglion of the spinal nerve in postnatal development].

Afferent neurons containing substance P (SP) were studied immunohistochemically in the sensory ganglion of the spinal nerve in 30 rats aged 10-90 days. The results obtained indicated that SP-immunoreactive neurons are present in thesel ganglia from the moment of birth. During the development, the percentage of SP-containing neurons decreased till day 10. SP-immunoreactive neurons were represented by the cells of very small or small size.

[Age-related development of calbindin-immunopositive neurons of rat sympathetic ganglia].

Neurons of cranial cervical, stellate and celiac sympathetic ganglia containing calbindin (CALB) were studied in rats (n = 60) aged 3-90 days using immunohistochemical method. The results obtained indicate that the largest population of CALB-immunopositive neurons was located in the stellate ganglion. The proportion of CALB-containing neurons in sympathetic para- and prevertebral ganglia decreased during the development. Final formation of CALB-immunopositive group of neurons was observed by the end of the first month of life.

[Age-related changes in sensory neurons containing calcitonin gene-related peptide under conditions of afferentation deficit in rats].

Morphological features of calcitonin gene-related peptide (CGRP)-immunoreactive neurons were studied in the sensory ganglia of the vagus and thoracic nerves in 3-, 10-, 20-, 30-, 60-, 90-, and 120-day-old rats under conditions of chemically-induced deafferentation. We found that, in rats, CGRP-containing neurons appeared in both ganglia immediately after they were born and their number decreased with aging. Most of CGRP-immunoreactive neurons were small in size, i.e., up to 600 microm2. Administration of capsaicin modified age-related changes in the number of CGRP-immunopositive neurons. In the thoracic nerve ganglion, the mean square of these cells and their number substantially decreased, whereas, in the vagus nerve ganglion, positive cells were not observed.

[Peculiarities of age changes of NF200(+)-neurons in the sensory ganglia of different segmental levels after chemical deafferentation].

Morphological features of the neurons containing neurofilaments with molecular mass of 200 kD (NF200+), were studied in the sensory ganglia of thoracic and lumbar spinal nerves in rats (n = 80) during the first year of their life. Capsaicin treatment (150 mg/kg) of the newborn animals resulted in the change of age dynamics of NF200+ neurons. This was reflected by a reduction of NF200+ neuron numbers and their cross-sectional areas in both ganglia. Segmental differences included greater reduction of NF200+ neuron number in the sensory ganglion of lumbar spinal nerve in both early and late developmental periods.

[Morphometric and histochemical characteristic of cluster organization of the neurons of rat spinal nerve sensory ganglion and of sympathetic trunk cervicothoracic ganglion].

The topographic pattern of neurons in spinal nerve sensory ganglion and of sympathetic trunk cervicothoracic ganglion was investigated at adult rats (n = 12). Results of the work have shown, that these organs are characterized by a heterogeneity of the neurons according to their morphometric (Nissl's stain) and histochemical (acetylcholinestherase and NADPH-diaphorase activity) parameters. The common feature of ganglia organization was demonstrated by the clustered pattern of neuron topography. Clusters were formed on a principle of heterogeneity, uniting from 3-4 to 9-10 neurons. The application of a combined histochemical method has allowed to identify the histo-functional charactristics of neurons within the clusters of the ganglia studied, which were also detected by standard histological methods.

[Changes the TRPV1-immunoreactive neurons of the rat spinal nerve sensory ganglia induced by capsaicin].

Morphological features of the TRPV1-immunoreactive neurons were studied in thoracic spinal nerve sensory ganglion in 10-, 20-, 30-, 60- and 90-day-old rats under normal conditions and after chemical deafferentation caused by capsaicin (100 mg/kg) subcutaneous injection at postnatal day 2. TRPV1-immunoreactive neurons differed in their morphometric characteristics: positive neurons had very small and small sizes, negative cells had the small, medium and large sizes. Capsaicin treatment of newborn rats was shown to change age dynamics of TRPV1-positive neurons resulting in the reduction of the average cell cross-sectional area and cell numbers during the whole period of observation.

[NADPH-diaphorase-positive structures in the spinal cord and spinal ganglia].

Using the histochemical reaction with the various incubation medium pH, the localization of NADPH-diaphorase was studied in the spinal cord and spinal ganglia of the adult rat. The data obtained indicate, that the application of the incubation medium with pH 7.4 allows to demonstrate the neurons with various NADPH-diaphorase activity, to define not only the density of enzyme distribution, but also to calculate the numbers of NADPH-diaphorase-positive neurons. It was established, that both in the spinal cord and spinal ganglia, NADPH-diaphorase activity was found not in all the neurons: high activity was found in small and medium neurons of the spinal ganglia, while low activity was detected in motoneurons. Other cellular structures of the spinal cord also possessed NADPH-diaphorase activity and were found to be the interneurons.

[NADPH-diaphorase activity in the motor neurons of different spinal cord segments of albino rat under normal conditions and after deafferentation].

Age changes of NADPH-diaphorase activity were studied histochemically in the ventral horn motor neurons at different segmental levels of the spinal cord of rats aged 3-90 days both under normal conditions and in the model of deafferentation (by intraperitoneal capsaicin injection). Wave-like age changes of motor neuron enzyme activity were detected at the level of T(II), L(IV) and S(II) spinal segments with its increase by day 60 followed by a significant decrease to day 90. Age dynamics of NADPH-diaphorase activity development in the spinal cord motor neurons of intact rats characterizes the constructive processes in neurons, while the changes found after the deafferentation are indicative of the motor neuron damage and are manifested by an abrupt increase of the enzyme activity at the age of 90 days.

Development of nNOS-positive neurons in the rat sensory and sympathetic ganglia.

Neurochemical features in sympathetic and afferent neurons are subject to change during development. Nitric oxide (NO) plays a developmental role in the nervous system. To better understand the neuroplasticity of sympathetic and afferent neurons during postnatal ontogenesis, the distribution of neuronal NO synthase (nNOS) immunoreactivity was studied in the sympathetic para- and prevertebral, nodose ganglion (NG) and Th2 and L4 dorsal root ganglia (DRG) from female Wistar rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old, 6-month-old, 1-year-old, and 3-year-old). nNOS-positive neurons were revealed in all sensory ganglia but not in sympathetic ones from birth onward. The percentage of nNOS-immunoreactive (IR) neurons increased during first 10 days of life from 41.3 to 57.6 in Th2 DRG, from 40.9 to 59.1 in L4 DRG and from 31.6 to 38.5 in NG. The percentage of nNOS-IR neurons did not change in the NG later during development and senescence. However, in Th2 and L4 DRG the proportion of nNOS-IR neurons was high in animals between 10 and 30days of life and decreased up to the second month of life. In 2-month-old rats, the percentage of nNOS-IR neurons was 52.9 in Th2 DRG and 51.3 in L4 DRG. We did not find statistically significant differences in the percentage of nNOS-IR neurons between Th2 and L4 DRG and between young and aged rats. In NG and DRG of 10-day-old and older rats, a high proportion of nNOS-IR neurons binds isolectin B4. In newborn animals, only 41.3%, 45.3% and 28.4% of nNOS neuron profiles bind to IB4 in Th2, L4 DRG and NG, respectively. In 10-day-old and older rats, the number of sensory nNOS-IR neurons binding IB4 reached more than 90% in DRG and more than 80% in NG. Only a small number of nNOS-positive cells showed immunoreactivity to calcitonin gene-related peptide, neurofilament 200, calretinin. The information provided here will also serve as a basis for future studies investigating mechanisms of the development of sensory neurons.