Changes in NGF/c-Fos colocalization in specific limbic structures of juvenile and aged rats after open field stimulation.

Changes in NGF release during stressful events have been associated with the activation of neurons expressing NGF receptors. This study examined the influence of acute stress-induced stimulation on NGF/c-Fos colocalization in the following limbic regions: the paraventricular (PV) nucleus of the hypothalamus, medial (MeA) nucleus of the amygdala, and CA3 hippocampus. Juvenile (P21) and aged rats (P360) were exposed to a 15-minute acute open field (OF) test. Double immunofluorescence staining, used to detect NGF-ir and c-Fos-ir cells, revealed a higher percentage of NGF/c-Fos-ir neurons in the P21 control group than in the P360 control group. Under OF acute stimulation, a statistically significant (p < 0.05) increase of NGF/c-Fos level in CA3 of juvenile animals and in PV and CA3 of the aged rats was observed. These observations indicate that the investigated structures in both age groups show a different response to acute OF stimulation. Acute OF affects the levels of NGF/c-Fos more significantly in aged rats.

Fluoride alters type I collagen expression in the early stages of odontogenesis.

Fluoride alters the expression and post-translational modifications of extracellular matrix proteins in dentin. The aim of our study was to determine the effects of fluoride on type I collagen expression during the early stages of tooth germ development in rats. Pregnant dams were divided into three groups and fed a standard diet. From the fifth day of pregnancy the three groups received tap water with, respectively, trace amounts of fluoride (C), a low fluoride concentration (FL) or and a high fluoride concentration (FH). Changes in type I collagen expression and distribution were evaluated. The expression of type I collagen was restricted to the extracellular spaces of cells of mesenchymal origin. In the youngest animals the most intense immunoreactivity for type I collagen was detected in predentin of the FL group. Although the intensity of immunostaining increased in proportion to the age of the animals, the largest increase in the groups investigated was detected in the FL group. We concluded that a low concentration of fluoride can act as a stimulator of type I collagen deposition in the extracellular matrix of dentin, while high concentrations of fluoride have an opposite effect, acting as an inhibitor of type I collagen formation in dentin.

Immunoreactivity of c-Fos, NGF and its receptor TrkA in the periventricular zone of the rat hypothalamus after open field exposure.

The immunoreactivity (ir) for c-Fos, NGF and TrkA, following an acute and chronic open field stress, were studied in the periventricular zone of rat hypothalamus. Adult rats were divided into three groups: control, exposed to acute (single exposure--15 minutes) and chronic (multiple exposures--15 minutes daily for 21 days) open field stress. In the control rats neurons immunoreactive to c-Fos, TrkA and NGF were found. The number of TrkA- and NGF-ir cells was high, whereas this of c-Fos-ir ones was low. In animals exposed to acute open field stress the number of c-Fos-ir cells in the examined nuclei varied, however it was much higher than that in the control animals. The number of TrkA-ir neurons in all the studied nuclei was also higher than that in the control animals, but the increase of the number of NGF-ir neurons was not observed in supraoptic nucleus. In the animals exposed to chronic open field stress the number of c-Fos-ir cells was increased in comparison to that in the control rats. After chronic stress exposure the number of TrkA-ir neurons in supraoptic nucleus remained high in comparison to that in animals exposed to acute stress, whereas it was decreased in other studied nuclei. No significant differences in the number of NGF-ir cells were observed between the groups exposed to the acute and chronic stress. Observed decrease of c-Fos- and TrkA-ir in the studied nuclei in the animals suffering from chronic stress in comparison with the acute one may indicate the occurrence of habituation phenomenon. This phenomenon does not concern NGF-ir.

The immunoreactivity of c-Fos, NGF and its receptor TrkA after open-field exposure in the central and medial nuclei of the rat amygdala.

The amygdala is a critical component of the neuroanatomical stress circuit. It plays a role in the generation of responses to emotional stimuli. The central (CeA) and medial (MeA) amygdaloid nuclei are implicated in activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. The immunoreactivity (-ir) of c-Fos, NGF and its receptor, TrkA, following acute and chronic open-field stress were studied in the CeA and MeA nuclei of the amygdala. The material consisted of 21 male adult rats divided into three groups: non-stressed (control) animals, rats exposed to acute (once only lasting 15 min) and chronic (15 min daily over 21 days) aversive stimulation (open-field exposure). The brains were stained with the use of immunohistochemical methods for c-Fos, NGF or TrkA. In the control rats c-Fos-, TrkA- and NGF-ir cells were observed in the nuclei studied, but the quantity varied, being moderate or high (immunoreactive to TrkA and NGF) or low (immunoreactive to c-Fos). In the animals exposed to acute open-field stress the number of c-Fos-ir, NGF-ir and TrkA-ir cells in the nuclei under examination was differentiated but higher than that in the control animals. In the animals exposed to chronic open-field stress the number of c-Fos-ir cells in the nuclei studied was similar and was smaller than those in animals exposed to acute stress. The number of TrkA-ir neurons was also lower in comparison to that in animals exposed to acute stress. However, no significant differences in the number of NGF-ir cells were observed between the groups exposed to acute and chronic stress. Diverse expression of c-Fos protein following both acute and chronic stress stimulation may prove the functional heterogeneity of the amygdaloid nuclei investigated. The decrease observed in both c-Fos- and TrkA-ir in MeA (only TrkA in CeA) of animals exposed to chronic stress may indicate the phenomenon of habituation.

Quantitative analysis of influence of sevoflurane on the reactivity of microglial cells in the course of the experimental model of intracerebral haemorrhage.

BACKGROUND: Microglial cells play an important role in the pathophysiology of intracerebral haemorrhage. We have examined the possible influence of sevoflurane on the reactivity of microglial cells during intracranial haemorrhage. METHODS: Forty adult male rats were divided into two groups. All animals were anaesthetized with fentanyl, dehydrobenzperidol and midazolam. In the experimental group animals additionally received sevoflurane 2.2 vol% end-tidal concentration. Intracranial haemorrhage was produced through infusion of blood into the striatum. The microglial cell population (numerical density of immunoreactive cells and their distribution) was assessed on days 1, 3, 7, 14 and 21 after producing a haematoma using antibodies OX42 and OX6. RESULTS: In the control group significant differences in the density of OX42-ir cells between 3rd and 7th (81.86 vs. 129.99) (95% CI: -77.99 to -18.25, P = 0.0035) and between 14th and 21st (105.36 vs. 63.81) (95% CI: 13.21 to 69.89, P = 0.006) survival days were observed. However, significant increase of percentage of amoeboid OX42-ir cells between 3rd and 7th (0.98 vs. 48.71) (95% CI: -52.17 to -43.30, P = 0.0001) and between 7th and 14th (48.71 vs. 58.47) (95% CI: -13.96 to -5.55, P = 0.0002) and then their decrease - between 14th and 21st (58.47 vs. 31.74) (95% CI: 22.52 to 30.93, P = 0.0001) days of observation were noted. In the sevoflurane groups OX42-ir cells were not found. On the 3rd day the density of OX6-ir cells in the sevoflurane group was significantly lower than that in the control group (12.39 vs. 34.57) (95% CI: -49.78 to -2.96, P = 0.02). The percentage of an amoeboid form of OX6-ir cells was significantly lower in the sevoflurane group than that in the control group (27.31 vs. 82.03) (95% CI: -72.52 to -36.92, P = 0.0001) (58.76 vs. 82.37) (95% CI: -38.81 to -8.41, P = 0.003) (42.87 vs. 81.55) (95% CI: -53.23 to -24.10, P = 0.0001) respectively for 3rd, 7th and 14th days of survival. CONCLUSION: Administration of sevoflurane during anaesthesia in animals with intracerebral haemorrhage evoked a decrease of activation of the microglial cells.

Influence of the "open field" exposure on calbindin D28K, calretinin, and parvalbumin containing cells in the rat midbrain - developmental study.

The aim of our study was to analyze the influence of the open field (OF) exposure on: 1. Distribution of c-Fos positive nuclei in: ventral tegmental area, substantia nigra, periaqueductal gray. 2. Appearance of calbindin-D28k, calretinin and parvalbumin in midbrain neurons that are engaged in the stress response. 3. Changes of c-Fos and calcium-binding proteins expression during maturation. The material consisted of Wistar rats of age between 0 and 90 days. The OF exposure was applied throughout 10 min and 90 min before the death of the animals. The brain sections were double stained using the antibodies against c-Fos, CB, CR or PV. Our results showed that in all studied nuclei age-related increase of c-Fos expression (without changing of its distribution properties) was found. PV didn't show any co-localization with c-Fos in neurons of studied regions at any ages, however some PV-immunoreactive (PV-ir) basket-like structures around c-Fos-immunoreactive (c-Fos-ir) neurons were observed. In the youngest group of rats c-Fos-ir cells and cells immunoreactive for CB and CR constituted separate neuronal populations. During maturation increases in the level of their co-localization with c-Fos was observed. We may conclude that in adult rat midbrain structures CB-immunoreactive (CB-ir) and CR-immunoreactive (CR-ir) cells (probably projection neurons) are mainly activated in the stress response following OF exposure. In the contrary PV-ir cells has only an indirect (modulatory) influence upon the c-Fos-ir cells.

The influence of sevoflurane on the reactivity of astrocytes in the course of the experimental intracerebral haemorrhage in rat.

40 adult Wistar rats were divided into two groups depending on the applied anaesthesia. In both groups animals were generally anaesthetized with fentanyl, dehydrobenzperidol administered intraperitoneally and midazolam given intramuscularly. In the second group (SEVO) animals received sevoflurane of 2.2 vol% end-tidal concentration. Intracerebral haematoma was produced through infusion of 100 microl of autologous blood into the striatum. Each group was divided into five subgroups depending on the length of survival period: 1, 3, 7, 14, 21 days. The astrocytic population was studied by means of anti-GFAP staining. Stereological analysis was applied to estimate the numerical density of immunoreactive cells and the distribution of their types. On 7th day of observation the density of GFAP-immunoreactive astrocytes in SEVO was lower (p<0,05) than that in the control group. In the control group, the increase (p<0.05) of per cent of activated astrocytes between the 1st and 3rd survival day was noted, which remained at this level till the end of observation. In SEVO group, the increase (p<0.05) of per cent of activated astrocytes between the 3rd and 7th day and the decrease (p<0.05) between the 14th and 21st survival day were observed. During days of observation the per cent of activated astrocytes was lower (p<0.05) in the SEVO group than that in the control group. Administration of sevoflurane during anaesthesia to animals with intracerebral haemorrhage has evoked not only the delay of the activation of astrocytes but also decrease in its level.

Open field stress and neurons containing calcium-binding proteins in the piriform cortex of the rat.

In the present study we wanted to check whether the expression of the c-Fos protein (the marker of cellular activity) appears in cells containing calcium-binding proteins (CaBPs) in animals exposed to the open field test. Eight adult Wistar rats were examined. In the first step the open field test was applied throughout 10 minutes. After perfusional fixation brains were frozen and cut on the cryostat in the coronal plane and stained with the standard immunohistochemical method. Sections were double stained for c-Fos and CaBPs: parvalbumin (PV), calbindin (CB), calretinin (CR). c-Fos positive cells were localized predominantly in layers II and III of the piriform cortex (PC). The double labeling study showed that neurons containing CaBPs are rarely c-Fos-immunoreactive. Often PV-positive and CB-positive fibers surround c-Fos-positive neurons in layers II and III in a form of a basket. It seems that cells containing CaBPs are not directly involved in the response to aversive stimuli but cells containing those calcium-binding proteins might influence directly c-Fos positive neurons of PC.

Apoptosis in the course of experimetal intracerebral haemorrhage in the rat.

Intracerebral haematoma was produced in 25 adult rats by infusion of 100 microl of autologous blood into the striatum. The animals' brains were removed at 1, 3, 7, 14 and 21 days after production of the haematoma. The TUNEL method was used to detect DNA fragmentation and TUNEL-positive cells were qualified. TUNEL-positive cells were already found on the first day of observation and were present for three weeks after haematoma production. These results provide evidence that programmed cell death is associated with intracerebral haemorrhage.

The relationships between neurons containing dopamine and nitric oxide synthase in the ventral tegmental area.

Ventral tegmental area (VTA) is a heterogeneous group of dopaminergic cells which contains interfascicular (IF), parabrachial (PBP) and rostral linear (RLi) nuclei. Neurons of this area are involved in the regulation of motor and motivational aspects of behavior and reveal high neuronal plasticity. Among many various neurotransmitters and neuromodulators, nitric oxide (NO) is localized in this region. In the present study, we investigated morphology and distribution of nitric oxide synthase (NOS)-positive neurons in VTA and their colocalization with dopaminergic neurons. The study was performed on six adult Wistar rats. After perfusional fixation, the brains were cut, immunostained for tyrosine hydroxylase (TH) and NOS and studied by confocal laser microscopy. In each of the three studied nuclei of VTA we investigated three different neuronal populations. Numerous TH-immunoreactive (TH-ir) and NOS-immunoreactive (NOS-ir) neurons are present in the studied region. Among them, a considerable number showed coexistence of both neurotransmitters. The populations of TH-ir and NOS-ir neurons interact with each other as manifested by the presence of NOS-ir endings on TH-ir neurons and vice versa. Taking the above into account, it may be suspected that NO is involved in the modulation of dopaminergic transmission.

The developmental pattern of c-fos expression in the rat thalamus following open-field stress stimulation.

The thalamic nuclei with their defined set of input-output connections are the primary channel for information flow to the cerebral cortex. Several data suggest that neurons of that area are involved in the response to various aversive stimulations. However the pattern of activation seems to depend on the stress model as well as the stage of maturation. In the present study we would like to check which nuclei of the thalamus show expression of c-fos in the response to the "open field test", and how this response pattern changes during the maturation process. 30 rats of age ranged from P0 to P120 (P-postnatal day) were studied. The experimental group was exposed to the "open field test" for 10 minutes. After perfusion and fixation, brains were cut and stained for c-fos with immunohistochemical method. Our results showed that during development the pattern of c-fos activity in the thalamic nuclei after stress stimulation undergoes significant changes. Distinct c-fos expression was observed in the paraventricular nucleus, intergeniculate leaflet and ventral lateral geniculate nucleus. These findings suggest that these nuclei may play a direct role in the stress reaction involved in the response to the "open field test".

Microglial and astroglial cells in the rat paraclaustral reservoir during postnatal development: an immunohistochemical study.

A immunohistochemical study of postnatal development of the paraclaustral reservoir of migrating cells in the rat brain was performed using anti-GFAP (for astroglia), ED1 and OX-42 (for microglia) antibodies. From birth to the 4th day of postnatal life most GFAP-positive cells in the paraclaustral reservoir are similar to transitional astroglia. From the end of the first postnatal week they have the morphology of mature astrocytes, although during the next week, their density was a slightly higher than in neighboring structures. On the 21st day, the morphology and density of astroglial cells in the ventral part of the external capsule did not differ from the surrounding regions. ED1/OX-42- positive microglial cells present in the paraclaustral reservoir during the first postnatal week represented ameboid microglia; their density was clearly higher than in the neighboring structures. During the second week they began to transform into ramified microglia and from the 21st day on, only OX-42 positive resting microglial cells were observed in the ventral part of the external capsule. We suggest that the paraclaustral reservoir is a place of accumulation of astroglia and microglia during brain development and may possibly serve as source of glial cells for neighboring structures. Alternatively, these glial populations may perform local developmental functions.

Cholinergic endings on various neurones containing calcium binding proteins and glutamic acid decarboxylase in the hippocampus of the rat.

Immunohistochemical study of the cholinergic innervation of the hippocampal cells containing glutamic acid decarboxylase (GAD) and calcium binding proteins: parvalbumin (PV), calbindin D28k (CB) and calretinin (CR) was conducted on 5 adult rat brains. Analysis of sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and respectively either GAD or PV, CB, CR, using confocal laser-scanning microscope shows that the intensive cholinergic innervations receive GAD, PV and CB-positive hippocampal cells. Cholinergic afferentiations of the CR-positive neurones are considerably fewer.

Immunohistochemical study of microglial and astroglial cells during postnatal development of rat striatum.

The present study investigates the development of microglial and astroglial cells in the postnatal rat striatum, using immunohistochemical methods with panel antibodies that recognize macrophage antigens of unknown function--ED 1, complement type 3 receptor--OX-42 (for microglia) and glial fibrillary acidic protein (for astrocytes). On the day of birth, ED1/OX-42- immunoreactive microglial cells present in the striatum represent ameboid microglia. Between P0 and P10 we could observe the migration of ameboid microglial cells from neuroepithelial ventricular zone through internal and external capsules into the striatum. During the second postnatal week (P10, P14) a considerable decline of ameboid ED1-immunoreactive microglial cells and an increase of the number of OX-42 positive ramified cells was observed. At P21 only OX-42 positive ramified cells were observed in the whole striatum. On the day of birth, only a few GFAP-positive cells resembling radial glia were observed in the striatum. During the first postnatal week, the number of GFAP-positive cells increased significantly; they showed typical morphology of the astrocytes present in the adult animals. After P21 the final striatal population of astroglia was formed.

Expression and distribution of various antigens of developing microglial cells in the rat telencephalon.

The distribution of microglia during the early stages of postnatal development in the rat was studied on rat brain from day of birth to postnatal day 90 (P90), using immunohistochemical methods with a panel of monoclonal antibodies that recognized the complement type 3 receptor (OX-42), macrophage antigen of unknown function (ED1), and the major histocompatibility complex (MHC) class I (OX-18) or class II (OX-6) antigens. Starting from the day of birth, ameboid microglia can be differentiated with positive immunoreactivity to OX-42, OX-18, and ED1. Labeled cells were localized mainly in the developing white matter. After P21, only positive reaction to OX-42 was present, and those cells had the typical morphology of the resting microglial cells that were located either in the white or grey matter. The changes in the appearance of different antigens are correlated with the morphological differentiation and transformation of ameboid microglial cells that are to become ramified microglia, present in the adult animals.

Postnatal development of the rat striatum--a study using in situ DNA end labeling technique.

We have examined the development of rat striatum for evidence of cells dying in the process of physiological cell death. In present study we have indicated apoptotic cells in sections stained with cresyl violet (cell death characterized by pyknosis) or with DNA end labeling assay (TUNEL method). Our results demonstrated that cell loss during maturation of the rat striatum had the characteristics of apoptosis rather than necrosis. The greatest number of TUNEL-positive and pyknotic cells in the striatum were found during the first postnatal days; after 7th day of postnatal life a rapid decrease of its number was observed after the second postnatal week no TUNEL-positive cells were observed in the striatum. Our analysis suggests that apoptotic cell death occurring during the development of striatal neuronal population takes place during the first week of postnatal life.