Prenatal Hypoxia Impairs Olfactory Function in Postnatal Ontogeny in Rats.

Analysis of the age-related dynamics of olfactory behavior in the odor preference and food search testsshowed that all male Wistar rats, regardless of age, preferred valerian essential oil, whose components have the properties of pheromones in rodents, when given a selection of eight essential oils; young rats displayed better food-seeking results than adult and old animals. Acute prenatal hypoxia (PH) on E14 (7% O2 for 3 h) led to impairment of the valerian odor preference at all ages studied and to decreased productivity of food searches. Neurodegenerative processes were seen in the piriform cortex after PH, with reductions in the number of neurons and increases in glial elements. We have previously observed these changes in the entorhinal cortex and hippocampus, but not in the olfactory bulbs. This suggests that PH-induced decreases in olfactory function in rats may result from impairments to the formation of the central elements of the analyzer during the first months of postnatal ontogeny.

Maternal Hyperhomocysteinemia Induces Neuroinflammation and Neuronal Death in the Rat Offspring Cortex.

Maternal hyperhomocysteinemia is one of the common complications of pregnancy that causes offspring cognitive deficits during postnatal development. In the present work, we evaluated the effect of prenatal hyperhomocysteinemia on structural and ultrastructural organization, neuronal and glial cell number, apoptosis (caspase-3 content and activity), inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1ß), and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation in the offspring brain cortex in early ontogenesis. Wistar female rats received methionine (0.6 g/kg body weight) by oral administration during pregnancy. Histological and biochemical analyses of 5- and 20-day-old pups' cortical tissue were performed. Lysosome accumulation and other neurodegenerative changes in neurons of animals with impaired embryonic development were investigated by electron microscopy. Neuronal staining (anti-NeuN) revealed a reduction in neuronal number, accompanied by increasing of caspase-3 active form protein level and activity. Maternal hyperhomocysteinemia also elevated the number of astroglial and microglial cells and increased expression of interleukin-1ß and p38 MAPK phosphorylation, which indicates the development of neuroinflammatory processes.

Regulation of Neprilysin Activity and Cognitive Functions in Rats After Prenatal Hypoxia.

The amyloid-degrading enzyme neprilysin (NEP) is one of the therapeutic targets in prevention and treatment of Alzheimer's disease (AD). As we have shown previously NEP expression in rat parietal cortex (Cx) and hippocampus (Hip) decreases with age and is also significantly reduced after prenatal hypoxia. Following the paradigms for enhancement of NEP expression and activity developed in cell culture, we analysed the efficacy of various compounds able to upregulate NEP using our model of prenatal hypoxia in rats. In addition to the previous data demonstrating that valproic acid can upregulate NEP expression both in neuroblastoma cells and in rat Cx and Hip we have further confirmed that caspase inhibitors can also restore NEP expression in rat Cx reduced after prenatal hypoxia. Here we also report that administration of a green tea catechin epigallocatechin-3-gallate (EGCG) to adult rats subjected to prenatal hypoxia increased NEP activity in blood plasma, Cx and Hip as well as improved memory performance in the 8-arm maze and novel object recognition tests. Moreover, EGCG administration led to an increased number of dendritic spines in the hippocampal CA1 area which correlated with memory enhancement. The data obtained allowed us to conclude that the decrease in the activity of the amyloid-degrading enzyme NEP, as well as a reduction in the number of labile interneuronal contacts in the hippocampus, contribute to early cognitive deficits caused by prenatal hypoxia and that there are therapeutic avenues to restore these deficits via NEP activation which could also be used for designing preventive strategies in AD.

[ROLE OF CASPASE-3 IN REGULATION OF THE CONTENT OF THE AMYLOID-DEGRADING NEUROPEPTIDASE NEPRILYSIN IN THE CORTEX OF RATS AFTER HYPOXIA].

Analysis of the effect of a caspase-3 inhibitor on the content of the amyloid-degrading neuropeptidase neprilysin (NEP) in the cortex of rats subjected to prenatal hypoxia (7% O2, 3 h) on the 14-th day of the embryonic development (E14) was performed. It was found that rats subjected to prenatal hypoxia on days 20-30 after birth have an increased content and activity of caspase-3 with reduced levels of NEP and of the C-terminal fragment of the amyloid precursor protein (AICD) regulating NEP expression. In hypoxic animals 3 days after a single injection of a caspase inhibitor (i. v., Ac-DEVD-CHO, P20) the content of AICD and NEP was found to be increased up to the levels observed in control rats. The data obtained suggest that the increase of caspase-3 enzyme activity could affect NEP expression via proteolytic degradation of its transcription factor AICD. These data for the first time demonstrate the role of caspases in AICD-dependent regulation of NEP production in the brain of mammals under hypoxic conditions.

[Morphofunctional changes in field CA1 of the rat hippocampus after pentylenetetrazole and lithium-pilocarpine induced seizures].

Animal models of epilepsy are very diverse and are used to elucidate the mechanisms underlying epileptogenesis and seizures. A single administration of pentylenetetrazole (PTZ) induces seizures, however it does not increase risk of further development of epilepsy. Pilocarpine immediately after injection evokes status epilepticus and after a latent period spontaneous convulsions develop in animals, i. e., the drug initiates the process of epileptogenesis. Assuming that in the PTZ model morphofunctional changes are mainly transient whereas changes in the lithium-pilocarpine (PC) model can indicate development of the brain epileptizationm, we compared morphological and functional characteristics in field CA1 of the hippocampus in a control and two groups of experimental animals 24 h after administration of convulsants. We revealed changes specific to the PC model and indicating the process of neurodegeneration: a decrease of the cell density, an altered NeuN expression, and an increase of the proapoptotic protease caspase-3 activity. A characteristic feature of the PTZ model was appearance of hyperchromic neurons with normal viability. In both models expression of the excitatory amino acids carrier EAAT1 increased by about 40% as compared to control. These morphofunctional correlates of reversible changes in the nervous tissue, caused by the convulsive state, and the early disturbances leading to the long-term brain epileptization can be used as indicators for evaluating therapeutic potential of novel anticonvulsive drugs.

[Effects of repeated short-term immobilization stress on plastisity of brain cortical division and cognition in adult rats with normal and disturbed embryogenesis].

The aim of the work was the analysis of the changes in a number of labile synaptopodin-positi- ve dendritic spines in parietal cortex and the CA1 field of the hippocampus, which characterize plasticity of intracellular interaction, and of the memorization after short-term repeated immobili- zation stress (daily for 5 minutes, 10 days), both in control rats and in rats subjected to prenatal hypoxia (E14, 7% of O2, 3 hours). There were observed deterioration of short-term and long-term memory, decrease in number labile spines in the CA1 field of the hippocampus (for 17.3 ± 10.4%; p ≤ 0.05) and their increase in a molecular layer of brain parietal cortex (for 36.9 ± 9.2%) at the adult rats with normal embryogenesis after immobilization stress in comparison with control intact animals. At the rats subjected to a prenatal hypoxia, regardless of that, they were ex- posed to an immobilized stress at an adult stage or not, was noted both violation of short-term and long-term memory, and decrease in number labile spines in the CA1 field of the hippocampus (for 22.9 ± 10.5%) and parietal cortex (for 28.1 ± 9.3%). The obtained data allow to conclude that the increase of plasticity providing adaptive behavior of animals, takes place in neocortical neuronal networks as a reply to a short-term repeating stress only at normal brain formation during embryo- genesis, while, violation of embryogenesis leads to decrease in plasticity and adaptive opportuni- ties of the nervous system during further ontogenesis.

[The ability of NMDA glutamate receptor blockers to prevent a pentylenetetrazole kindling in mice and morphological changes in the hippocampus].

We investigated in mice the relationship between convulsions and morphological changes of hippocampal neurons that arise in the development of pentylentetrazol (PTZ)-induced kindling. The kindling was caused by of 35 mg/kg PTZ i.p. 3 times a week for a month. By the end of this period, 70% of the mice responded to the injections of PTZ with pronounced clonic or tonic-clonic seizures. The hippocampal slices (layer stratum pyramidale, CA1, Nissl's stain) obtained from mice exhibiting seizures revealed a large number of modified cells (24.7 +/- 2.1%). These hyperchromic neurons have been characterized by a decrease of the size cell body, there was a loss of turgor, the body cells shrink, and dendritic spines curl. Part of the cells took the shape of elongated neck. Such modified the dark type of neurons contained only 2.3 +/- 2.3% in the hippocampus of intact mice, and 30% of the mice resistant to the convulsive action ofPTZ during the period of observation. The expression of protein NeuN (Fox3) in hippocamal neuron including the hyperchromic once suggests that neurons on the whole did not die and were relatively viable. Preventive administration of NMDA receptor blockers (0.5 mg/kg, memantine 0.1 mg/kg or IEM-1958 1 mg/kg, s.c.) 30 minutes prior to PTZ reduced the proportion of mice which exhibited PTZ kindling from 70% to 40%. The modified neurons were observed in which the PTZ kindling due to the blocker presence did not develop, i.e., the same as in intact mice. Contrary, 24.0 +/- 5.6% of hyperchromic neurons were found in the hippocampal slices from mice manifested seizures, despite the co-administration of NMDA blockers. The data obtained indicate that modified neurons are the result of seizures suffered by the animals in the course of PTZ kindling, and that the blockade of NMDA glutamate receptors can suppress manifestations of seizures and the accompanying morphological changes of hippocampal neurons.

Molecular properties of lysine dendrimers and their interactions with Aß-peptides and neuronal cells.

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer's, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aß-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aß-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aß. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs.

[Study of distribution of protein of the spine apparatus synaptopodin in cortical brain parts of rats submitted to hypoxia at different periods of embryogenesis].

A comparative study of the nervous tissue and distribution of the spine apparatus protein synaptopodin was performed in all layers of the brain sensorymotor cortex and hippocampal CA1 area in control rats and in the rats submitted to hypoxia at E14 and E18. It was found that beginning from the 20th day of postnatal development, in rats submitted to hypoxia both at E14 and E18 there was observed a statistically significant decrease of the mean number of labile synaptopodin-positive spines in the stratum radiatum molecular of the hippocampus area CA1. The decrease of the number of labile spines in the sensorymotor brain cortex was revealed only in the I layer beginning from the 20th day after birth in the rats submitted to hypoxia at E14. Maximal differences in the studied brain areas were observed in adult rats (exposed to hypoxia at E14: in the neocortex--a decrease by 23 +/- 10%, in hippocampus--by 24 +/- 8%, respectively). In adult animals, the increased degeneration of neuzons was not detected. It is suggested that disturbances in cognitive functions and in the capability for learning observed in rats after prenatal hypoxia can be due to a decrease of the amount of the labile synaptopodin-positive spines, which leads to a change of the structural-functional properties of neuronal networks and to a decrease of their plasticity.

[Change of the neocortex nervous tissue in rat ontogenesis after hypoxia at various terms of embryogenesis].

The performed study has shown that in rats submitted to hypoxia (3 h, 7% O2) at the 14th day of embryogenesis (E14) as compared with control animals, density of disposition of cells in the brain cortex decreased for the first month of postnatal ontogenesis (maximally by 40.8% by P20). In dying neurons, swelling of the cell body, lysis of organoids, and disturbance of the cytoplasmic membrane intactness were observed. Two waved of neuronal death by the mechanism of caspase-dependent apoptosis were revealed; the first involved large pyramidal neurons of the V layer (P10-20), the second--small pyramidal and non-pyramidal neurons of the II--III layers (P20-30). In neuropil of molecular layer, a decrease of the mean amount of labile synaptopodin-positive dendrite spines was observed, as compared with control. In rats exposed to hypoxia at E18, no changes of cell composition and structure of the nervous tissue were found in the studied brain cortex areas. Thus, formation of the cortex nervous tissue in postnatal ontogenesis of rats submitted to hypoxia at the period of neuroblast proliferation-migration is accompanied not only by a change of the cell composition of various cortex layers in early ontogenesis, but also by a decrease of the number of the synaptopodin-positive spines in molecular layer, the decrease being preserved in adult animals.

[Formation of structural and ultrastructural organization of striatum rat postnatal ontogenesis upon changes in their embryonal development].

By light microscopy (by Nissl and Golgi), electron microscopy, and immunohistochemistry methods, formation of structure of the brain striatum dorsolateral part from birth to the 3-month age was studied in rats submitted to acute hypoxia at the period of embryogenesis. It has been established that hypoxia at the 13.5th day (E13.5) leads to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, while the majority of large neurons at this period are degenerated by the type of chromatolysis with swelling cell body and processes and lysis of cytoplasmic organoids. By the end of the 3rd week, shrunk hyperchromic or picnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was detected in neuropil around degenerating neurons. By the 30th day as well as in adult rats there was observed destruction of mitochondrial apparatus, an increase of the number of lysosomes, and the appearance of bladed nuclei - signs of apoptotic cell death, which was also confirmed by an increased expression of proapoptotic p53 protein and its colocalization with caspase-3 in a part of neurons. Morphometrical analysis has shown a decrease of density of striatum cell arrangement and a change of ratio of different cell types in the rats submitted to hypoxia as compared with control group. At early stages of postnatal ontogenesis there was the greatest decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons with the area more than 80 microm2. After 3 weeks of postnatal development the number of middlesize neurons (30-95 microm2) decreased (by 11.8-19.2%) as compared with control. The obtained data show that a change of conditions of embryogenesis (hypoxia) at the period of the most intensive proliferation of the forebrain neuroblasts leads to disturbances of the process of formation of the striatum nervous tissue. This can be the cause of delay of development and disturbances of behavior and learning observed in rats submitted to prenatal hypoxia.

Formation of the structural and ultrastructural organization of the striatum in early postnatal ontogenesis of rats in altered conditions of embryonic development.

Light (Nissl and Golgi methods) and electron microscopy methods were used to study the formation of the structure of the striatum during the first two weeks after birth in rats subjected to acute hypoxia at different times of embryogenesis. The dynamics of the physiological development of the same population of rats were studied in parallel. Hypoxia at day 13.5 of embryogenesis was found to lead to delayed neurogenesis (delayed establishment of elements of the neuropil and differentiation of cells) and abnormalities in the structure of the striatum (degeneration, particularly chromatolysis, of neurons and the appearance of glial nodes). Morphometric analysis demonstrated a decrease in the total number of cells in the striatum; small changes in large neurons were seen. Hypoxia at day 18.5 of embryogenesis produced no significant changes. Structural abnormalities were accompanied by changes in the process of the animals' physiological development. The data obtained here show that changes in the conditions of embryogenesis (hypoxia) during the period of the most intense proliferation of neuroblasts in the forebrain lead to impairment of the process of formation of striatal nervous tissue and the body as a whole in the period of early postnatal ontogenesis.

[Formation of striatum structural and ultrastructural organization in the early postnatal ontogenesis of rats subjected to altered conditions of their embryonic development].

Formation of the structure of striatum during two postnatal weeks in rats subjected to acute hypoxia during various periods of their embryonic development was studied using light microscopic (Nissl's stain and Golgi's silver nitrate impregnation) methods and electron microscopy. This study was supplemented by a simultaneous investigation of physiological development of the same population of rats. The data obtained demonstrated that prenatal hypoxia on day 13.5 of embryonic development (E13.5) led to a delayed neurogenesis (retardation in the development of neuropil elements and cell differentiation) as well as to the malformation of the structure of striatum (degeneration, in particular, chromatolysis of neurons and glial nodule formation). Morphometric analysis demonstrated that prenatal hypoxia on E13.5 resulted in a statistically significant decrease in cell number in the striatum, these changes being especially pronounced in large neurons. Prenatal hypoxia on E18.5, however, caused no significant changes in striatum. Structural changes in the striatum were shown to be accompanied by significant changes in the physiological development of animals. The data obtained demonstrated that the alteration of the conditions of embryogenesis (hypoxia) during the period of most intensive proliferation of forebrain neuroblasts resulted in the disturbances of the formation of both striatum nervous tissue of the organism as a whole during early postnatal ontogenesis.

Postnatal physiological development of rats after acute prenatal hypoxia.

The aim of the present work was to identify the characteristics of the physiological development of the brain and the formation of behavior in rats subjected to hypoxia on day 13.5 of embryogenesis. These animals showed delayed development and changes in nerve tissue structure in the sensorimotor cortex, along with disturbances to the processes forming normal movement responses during the first month after birth. These changes were partially compensated with age, though adult animals subjected to acute prenatal hypoxia were less able to learn new complex manipulatory movements. Alterations in nerve tissue structure and changes in the neuronal composition of the sensorimotor cortex correlated with the times of appearance of behavioral impairments at different stages of ontogenesis. Thus, changes in the conditions in which the body is formed during a defined period of embryogenesis lead to abnormalities in the process of ontogenetic development and the ability to learn new movements.

[Postnatal physiological development of rats after acute prenatal hypoxia]. / Postnatal'noe fiziologicheskoe razvitie krys posle ostroi prenata'noi gipoksii.

The aim of study was to investigate the physiological development of the brain and behaviour in rats subjected to prenatal hypoxia on the 13.5th day of embryogenesis. We have found that such rats manifested a delayed physiological development and a change in nervous tissue of the sensorimotor cortex, as well a disturbed formation of motor responses during the first month of postnatal ontogenesis. During maturation these modifications were in part compensated, however we observed a decrease of the rats' ability to learn new forepaw movements. The destruction of the brain tissue and the modification of neurons composition in the sensorimotor cortex correlated with changes of behaviour at different stages of ontogenesis. Thus, changes of the conditions under which an organism develops during embryogenesis, predetermine a disturbance in ontogenesis and the learning ability.

Fine structure of the dorsal lateral geniculate nucleus of the turtle, Emys orbicularis: a Golgi, combined HRP tracing and GABA immunocytochemical study.

The afferent and efferent cortical projections of the dorsal lateral geniculate nucleus (GLD) of adult specimens of the turtle Emys orbicularis were investigated after intraocular or intracortical injections of horseradish peroxidase (HRP), and the distribution of gamma aminobutyric acid (GABA) immunoreactivity in the nucleus was carried out by immunocytochemical techniques, both techniques being combined with light and electron microscopy. In addition, some specimens were prepared for double-labeling of HRP and GABA immunoreactivity, and additional samples impregnated by a rapid Golgi technique. On purely morphological grounds, four types of neurons can be distinguished by light microscopy: two types of large cells in the cell plate which project to the cortex, and two types of smaller cells in the neuropil and optic tract which do not. The small cells are consistently GABA-immunoreactive, while the former are, with extremely rare exceptions, immunonegative for GABA. The supposition that the small neurons of the neuropil are interneurons is supported by electron microscopic observations; these strongly GABA-immunoreactive cells have large plicated nuclei surrounded by a thin layer of cytoplasm poorly endowed with organelles. The dendrites of these cells may contain pleomorphic synaptic vesicles (DCSVs) and appear to be presynaptic to other dendritic profiles. These DCSVs are occasionally contacted by GABA-immunoreactive axon terminals, and more frequently by retinal terminals consistently immunonegative for GABA. The latter, frequently organized in glomeruli, also make synaptic contacts with immunonegative dendrites arising from corticopetal neurons of the cell plate. Two major categories of GABA-immunoreactive axon terminals can be distinguished, and we are led to the conclusion that one of these represents an intrinsic GABAergic innervation of the GLD, while the second is tentatively interpreted as an extrinsic source of GABA to the nucleus, possibly from ventral thalamic structures. The fine structure of the dorsal lateral geniculate nucleus of Emys orbicularis thus shows many similarities with that of mammals.