Statins are lipid-lowering drugs that act by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in cholesterol biosynthesis. Animal studies have shown neuroprotective effects of statins in cerebral stroke. However, the underlying mechanisms are not fully understood. The nuclear factor-kappa B (NF-κB) transcription factor is involved in the regulation of apoptosis in stroke. Different dimers of NF-κB regulate the gene expression of proteins involved in both neurodegeneration and neuroprotection. We aimed to determine whether simvastatin improves stroke outcome via inhibition of the RelA/p65-containing subunit and downregulation of stroke-induced pro-apoptotic genes or via activation of NF-κB dimers containing the c-Rel subunit and upregulation of anti-apoptotic genes during the acute stroke phase. Eighteen-month-old Wistar rats, subjected to permanent MCAO or sham surgery, were administered simvastatin (20 mg/kg b.w.) or saline for 5 days before the procedure. Stroke outcome was determined by measuring cerebral infarct and assessing motor functions. The expression of NF-κB subunits in various cell populations was investigated using immunofluorescence/confocal microscopy. RelA and c-Rel were detected by WB. The NF-κB-DNA binding activity was investigated using EMSA, and expression of Noxa , Puma , Bcl-2 , and Bcl-x genes was analyzed by qRT-PCR. Results showed a 50% infarct size reduction and significant motor function improvement in the simvastatin-treated animals which correlated with a decrease in RelA and a transient increase in the c-Rel level in the nucleus, normalization of the NF-κB-DNA binding activity, and downregulation of the NF-κB-regulated genes. Our results provide new insights into the statin-mediated neuroprotective action against stroke based on NF-κB pathway inhibition.

The biology of three amelanotic melanoma cell lines (Ab, B16F10, and A375) of different species origin was analyzed during in vitro induced melanization in these cells. Melanin production was induced by DMEM medium characterized by a high level of L-tyrosine (a basic amino acid for melanogenesis). The biodiversity of amelanotic melanoma cells was confirmed by their different responses to melanogenesis induction; Ab hamster melanomas underwent intensive melanization, mouse B16F10 darkened slightly, while human A375 cells did not show any change in melanin content. Highly melanized Ab cells entered a cell death pathway, while slight melanization did not influence cell biology in a significant way. The rapid and high melanization of Ab cells induced apoptosis documented by phosphatidylserine externalization, caspase activation, and mitochondrial energetic state decrease. Melanoma cell type, culture medium, and time of incubation should be taken into consideration during amelanotic melanoma cell culture in vitro. L-tyrosine, as a concentration-dependent factor presented in the culture media, could stimulate some amelanotic melanoma cell lines (Ab, B16F10) to melanin production. The presence of melanin should be considered in the examination of antimelanoma compounds in vitro, because induction of melanin may interfere or be helpful in the treatment of amelanotic melanoma.

The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)-known as proteasome inhibitors-have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly, DMSO administration also caused damage to some of the ependymal cells, but the aggresome-like structures were not formed. Our results indicate that all of the studied classes of proteasome inhibitors are detrimental to ependymal cells to some extent, and may cause severe changes in the ventricular system. The safety implications of their usage in therapeutic strategies to attenuate intracerebral hemorrhagic injury and in brain cancer treatment will require further studies.

The proteins' ubiquitination and their further degradation by proteasomes are crucial for cell cycle regulation, transcription and DNA replication, inflammatory response, and apoptosis. Proteasome inhibitors have recently become considered as a promising method in cancer and inflammatory disease therapy In this study, utilizing the rat model, we try to establish the influence of proteasome inhibitor MG-132: (1) on the basis of spontaneous and evoked locomotor activity and (2) on the condition of nigrostriatal projections eight weeks after MG-132 intraperitoneal administration. We also discuss the current status of knowledge about intraperitoneal administration of MG-132, a laboratory method that is being used more and more. Our results revealed a lack of motor abnormalities, but significant loss (20%) of substantia nigra pars compacta dopaminergic neurons after systemic MG-132 administration. This loss was accompanied by a corresponding decrease (8%) of density of dopaminergic terminals in dorsolateral striatum. Moreover, evidence of very limited but ongoing fibre degeneration within the dorsal striatum suggests that MG-132 severely disturbed the nigrostriatal pathway.In summary, intraperitoneal application of proteasome inhibitor MG-132, despite the encouraging results of experimental treatment and prevention of many pathological processes, should be used with caution because of the potential adverse effects on the structure of the central nervous system, especially elements of the nigrostriatal pathway

Calbindin-D28k (CB), parvalbumin (PV) and calretinin (CR) are calcium-binding proteins (CaBPs) considered to be markers for certain subpopulations of neurons in the central nervous system. The aim of this study was to describe the pattern of distribution of CB-, PV- and CR-immunoreactive elements in the rabbit corticomedial amygdaloid complex during the postnatal period. The time course of changes in CaBPs expression during maturation of the selected nuclei indicates their diversity. During the first month after birth, CaBPs expression stabilizes earliest in the anterior cortical and then in the medial nuclei. Later, during the second month of postnatal life, the posteromedial and posterolateral cortical nuclei maturate. The central nucleus requires a considerably longer time to reach maturity - about three months are needed to stabilize CaBPs expression in all its subdivisions. This nucleus also shows the most differentiated, time-dependent distribution of CaBPs immunoreactivity (especially CB), distinct in its divisions. The differences in the CaBPs immunoreactivity confirm previous reports concerning dissimilar origin and development, and also reflect the diversity of connectivity of the amygdaloid body - the collection of nuclei, considered as one functional integrity.

__________________________________________________________________________ Unsatisfactory cerebral ischaemic stroke treatment make understanding of pathophysiology of this process and developing effective therapies the focus of interest of many scientific disciplines. Both these goals can be achieved in the course of basic and clinical research. A large body of information concerning this issue is controversial. Animal ischaemic models seem to be a very useful tool for biochemical and pathophysiological research; however their employment in new drug development research is widely questioned. In this report we discuss the most widely used experimental models of the ischaemic stroke, their advantages and disadvantages, and possible reasons for discrepancy in results of basic and clinical research. Interdisciplinary Problems of Stroke; 11 (2): 70–79

There is large body of evidence suggesting distinct susceptibility to ischemia in various brain regions. However, the reason for this remains unexplained. Comparative studies of programmed cell death (PCD) pathways indicate their differentiated evolutional origin. The caspase-independent pathway is regarded as an older, whereas the caspase-dependent--as more advanced. In our study we address the question of whether there are any characteristic differences in the activation and course of PCD in phylogenetically and morphologically distinguished brain structures after transient focal ischemia. Using Western blot, we studied changes in expression of caspases: 3, 8, 9, and AIF in the frontoparietal neocortex, archicortex (CA1 and CA2 sectors of the hippocampus) and striatum, during reperfusion after 1 h occlusion of the middle cerebral artery. The caspase and AIF expression were differentiated between the studied structures. The activation of only the caspase-dependent pathway was observed in the neocortex. In the archicortex and striatum both caspase-dependent and caspase-independent pathways were activated, although in the latter the extrinsic apoptotic pathway was not activated. In summary, it is conceivable that structures of different evolutionary origin undergo cell-death processes with the participation of phylogenetically distinguished mechanisms. The previously reported unequal susceptibility to ischemia may co-exist with activation of different cell death pathways.

The role of fibroblast growth factor receptors (FGFR) in normal brain development has been well-documented in transgenic and knock-out mouse models. Changes in FGF and its receptors have also been observed in schizophrenia and related developmental disorders. The current study examines a transgenic th(tk-)/th(tk-) mouse model with FGF receptor signaling disruption targeted to dopamine (DA) neurons, resulting in neurodevelopmental, anatomical, and biochemical alterations similar to those observed in human schizophrenia. We show in th(tk-)/th(tk-) mice that hypoplastic development of DA systems induces serotonergic hyperinnervation of midbrain DA nuclei, demonstrating the co-developmental relationship between DA and 5-HT systems. Behaviorally, th(tk-)/th(tk-) mice displayed impaired sensory gaiting and reduced social interactions correctable by atypical antipsychotics (AAPD) and a specific 5-HT2A antagonist, M100907. The adult onset of neurochemical and behavioral deficits was consistent with the postpubertal time course of psychotic symptoms in schizophrenia and related disorders. The spectrum of abnormalities observed in th(tk-)/th(tk-) mice and the ability of AAPD to correct the behavioral deficits consistent with human psychosis suggests that midbrain 5-HT2A-controlling systems are important loci of therapeutic action. These results may provide further insight into the complex multi-neurotransmitter etiology of neurodevelopmental diseases such autism, bipolar disorder, Asperger's Syndrome and schizophrenia.

A growing body of evidence indicates the common origin of the claustrum, endopiriform nucleus, and the basolateral nuclear complex of amygdala from the lateral and ventral parts of the pallium, as the claustroamygdaloid complex. It seems very probable that at least some of the claustral interneurons derive from subcortical sources. The postnatal development of neuropeptide Y-, somatostatin- and vasoactive intestinal polypeptide-containing interneurons was studied during the 4 postnatal months (P0-P120; P, postnatal day). The study was conducted on 45 Wistar rats of both sexes. Our results indicate that neuropeptide-containing interneurons are not morphologically mature at the moment of birth. The characteristic features of neuronal bodies and the relatively long period of postnatal development may indicate their migration from the subcortical neurogenetic centers. Morphological changes in the neuropil are also reported. Although developmental patterns differ between various neuropeptide-containing neuronal subpopulations, two phases of development can be distinguished in each of them: the early phase (P0-P4) during which undifferentiated neurons and neuropil dominate, and the late phase (P7-P28) during which the characteristic features of an adult-like structure gradually appear. Later these observed developmental changes are terminated. The postnatal development of neuropeptide-containing interneurons is completed after 4 weeks of life. This period, which is important for the structural and functional development of the claustrum, must be taken into account in future studies on this structure.

Claustrum is a telencephalic structure integrating information of various modalities. Proper functioning of this structure depends on the presence of a network of intrinsic connections. This includes GABA-ergic neuronal populations that also contain calcium-binding proteins (CaBPs). The goal of this study was to analyze qualitative and quantitative the 5-HT-containing fibers in the rat claustrum and to assess the relationships between these fibers and the populations of claustral neurons expressing CaBPs. We used the methods of immunocytochemistry and morphometry. The serotonergic fibers in the claustrum are heterogeneous, both with respect to their morphology and spatial distribution. Thin varicose fibers are more numerous and are homogeneously distributed within the claustrum. Remaining fibers were thicker and possessed larger varicosities. They were present mainly in the ventral part of the claustrum. Although the serotonergic fibers are found in the vicinity of claustral cells containing CaBPs, direct contacts between these fibers and cells are rare. Other mechanisms, including volume transmission, may possibly mediate serotonergic influences.

The developmental changes of 25-kDa synaptosomal-associated protein (SNAP-25) expression in the rat striatum and cerebral cortex were examined using Western- blotting and densitometric scanning of immunoblots. Analysis of the striatum extracts from postnatal day 0 (P0) to postnatal day 120 (P120) demonstrated that SNAP-25 is poorly expressed until P14. From this point the expression level gradually increases to reach a maximum on P60 and then decreases. The pattern of SNAP-25 expression in the rat cerebral cortex is different. Two peaks are observed, the first on P10 and the second on P60, after which the expression level decreases. These results appear to confirm the role of SNAP-25 protein in axon outgrowth and synaptogenesis in the nervous system.

A total of 14 patients of various ages diagnosed with schizophrenia and, as an age-matched control group, 12 healthy subjects were examined using the MRI method of neuro-imaging. The volume of the following structures was evaluated in the right and left hemispheres: the superior temporal gyrus, the basolateral temporal area (the region including the middle temporal gyrus, inferior temporal gyrus and fusiform gyrus), the parahippocampal gyrus, the hippocampal head, the amygdaloid body and the inferior horn of the lateral ventricle. In schizophrenia a significant increase in the volume of the amygdaloid body on both the left and right sides was observed. In the patients, as in the control group, we noticed significant asymmetry between the left and right sides in the volume of the structures studied. The left amygdaloid body was significantly larger than the right, whereas the left hippocampal head and the temporal horn of the lateral ventricle were smaller than the right. Our findings suggest that in the early stages of schizophrenia, despite the increased volume of the amygdaloid body, the asymmetry between the structures of the temporal lobe is still present. However, the changes observed in the temporal lobe could be related to the functional disturbances observed in this disease.

In our study we used c-Fos protein (as a marker of cellular activity) to identify whether cells containing parvalbumin (PV) in the piriform cortex (PC) are engaged in the response to stress stimulation and to discover how this expression changes during maturation. The material consisted of Wistar rats of postnatal (P) ages between 0 and 120 days divided into 9 groups: P0, P4, P7, P10, P14, P21, P30, P90, P120. Each group consisted of 5 experimental and 3 control animals. Rats of the experimental groups were exposed to the "open field test" throughout 10 minutes. The control animals were kept in a home cage. Our results showed that c-Fos activity in the open field test was observed in layers II and III of PC after birth. It then increased and stabilised on P30. In the second week of life PV-positive cells were also observed in those layers. These achieved maturity in the 4th week of life. After this time basket-like structures appeared but the level of PV/c-Fos co-localisation was low. Only small differences were observed between the anterior and posterior parts of PC. In the anterior part a higher number of PV-positive neurons, neuropil threads, and basket-like structures and a larger degree of PV/c-Fos co-localisation were observed. Our results suggested that during maturation PV cells are not directly activated in response to stress stimuli but PV neurons via their numerous endings influence the activation of c-Fos-positive cells predominantly in the anterior part of PC.

Our intention in the present study was to ascertain whether NO-producing cells in the basolateral complex (BLC) and paracapsular intercalated nerve cell groups (Ip) of the amygdala are activated in the open field (OF) test. The material consisted of 8 adult rat brains. The OF test 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 (marker of neuronal activation) and against nitric oxide synthase (NOS -- marker of NO-producing cells). The neurons containing NOS and those revealing c-Fos activity constituted distinct populations within both the BLC and Ip but NOS-immunoreactive fibres often surrounded the c-Fos-immunoreactive neurons. Our results suggest that (1) neurons of the basolateral complex of the amygdala and paracapsular intercalated islands are involved but probably not crucial for the open field stress response and (2) NOS-immunoreactive cells in the BLC and Ip are not activated after OF exposure.

Recently the increasing prevalence of gastrointestinal diseases, including neoplasm, has resulted in the necessity of characterising not only the tumours, but also healthy mucosa. Research into the morphological changes of healthy mucosa under different experimental conditions, including drugs, special diets and the use of probiotic bacteria, is greatly facilitated by the availability of animal models. In spite of the widespread use of mice in gastrointestinal research, there is a lack of information on the qualitative and quantitative histological characteristics of the intestinal mucosa of the mouse. The aim of this study was to assess the morphological characteristics and the postnatal development of the small intestine of wild type mice -- C57BL/6J. The mice were aged either 5 weeks or 12 weeks. The 12-week-old mice had been weaned at the age of 5 weeks. After dissection the small intestine was divided into 5 equal portions and randomly chosen microscopical sections from each were stained with haematoxylin and eosin. The parameters describing the morphology of the small intestine (villus height, depth of the crypt, villus width near the crypt, width of the villus connective tissue near the crypt, thickness of the muscular layer and the height of the enterocytes and their nuclei) were evaluated under a light microscope. In both age groups the height and width of the villi decreased, while the thickness of the muscular layer increased in the distal direction. The height of the enterocytes decreased and the height of the enterocyte nucleus increased towards the colon in both age groups. The depth of the crypts was greater in the younger animals than in the older ones. Our data provides the baseline morphological description of the small intestinal mucosa in wild type mice, strain C57BL/6J, which can be used as a reference for testing the influence of drugs, toxins, nutrients and inborn mutations on the mouse intestine.

The aim of the present study is to follow topographical and morphological changes in the development of the amygdaloid basolateral complex (BLC) in the rabbit. The material consists of 35 brains of New Zealand rabbits of both sexes, divided into 7 age groups (P2-P90). In cresyl violet preparations BLC is already well visible on P2 and is composed of the lateral (divided into dorsolateral and ventromedial divisions), basolateral and homogenous basomedial nuclei. On about the 7th postnatal day it is possible to divide the basomedial nucleus (BM) into dorsal (Bmd) and ventral (BMv) divisions. The topography and subdivisions set on P7 are maintained in further periods of life. The morphology of neurons (shape, dendrites, staining) changes significantly until P21 in all BLC nuclei. Our results indicate that BLC achieves morphological maturity relatively late, which is probably connected with a long creation of emotional memory and regulation of emotional behaviour.

The morphometric analysis of changes occurring in the rat and rabbit ventroposterolateral (VPL) nucleus of the thalamus during the postnatal development was performed using unbiased stereological methods. The materials used in the study included 30 Wistar rats and 32 New Zealand rabbits aged from P0 to P180 (P-postnatal day), which were divided into six and eight age groups, respectively. The following stereological parameters of VPL nucleus on the cresyl violet stained sections were determined: volume of the nucleus, numerical density and total number of neurons. The total number of neurons indicated that the development of VPL nucleus in both species ended within the third week of postnatal life. The volume of VPL nucleus increased gradually (by about 2.2 and 5 times in rats and rabbits, respectively) in comparison with the volume of the cerebral hemisphere during the development from P0 to adulthood. The numerical density of VPL neurons decreased rapidly at the beginning of postnatal life and stabilized by the end of the third week. In both species, the gradual increase in the volume of VPL nucleus and the simultaneous decrease in the neuronal density in the first week of postnatal life were mainly caused by changes in the neuropil volume. The total number of cells did not change remarkably during the first postnatal week. However, it decreased significantly during the second week. This decrease was probably due to the naturally occuring cell death. These results show that the most prominent qualitative and quantitative changes in VPL nucleus and its neurons occur during the first two weeks of postnatal life of rats and rabbits. Also, because the thalamocortical relay neurons completely acquire their physiological features, this the most critical period of time for their morphological maturation.

An immunocytochemical double-staining method was applied in order to study the co-localisation of nitric oxide synthase (NOS) with three calcium-binding proteins, calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) in the claustrum of the rat during the first 4 months of life (postnatal days: PO-P120). The co-localisation of NOS/PV and NOS/CB is reported. These neurons fall into the category of non-pyramidal cells. Double-labelled NOS/CB neurons are observed in the claustrum starting from P4, whereas double-labelled NOS/PV neurons are observed from P14 onwards. The percentages of double-labelled neurons increase in relation to the age. Double-labelled NOS/CB and NOS/PV neurons, although they do not constitute a numerous population, play an important role in the process of maturation of the claustrum. This is confirmed by the occurrence of these types of neurons at definite stages of maturation and by the increase in their number.