The Effect of Simvastatin on the Dynamics of NF-κB-Regulated Neurodegenerative and Neuroprotective Processes in the Acute Phase of Ischemic Stroke.

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.

Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors.

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.

Nigrostriatal pathway degeneration in rats after intraperitoneal administration of proteasome inhibitor MG-132.

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.

Expression of the calcium-binding proteins in the central, medial and cortical nuclei of the rabbit amygdaloid complex during postnatal development.

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.

The molecular mechanisms of cell death in the course of transient ischemia are differentiated in evolutionary distinguished brain structures.

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.

Serotonergic hyperinnervation and effective serotonin blockade in an FGF receptor developmental model of psychosis.

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.

Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat.

The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin. We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0-P120; P: postnatal day). Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons. In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period.

NPY-, SOM- and VIP-containing interneurons in postnatal development of the rat claustrum.

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.

The anatomical relationships between the serotonergic afferents and the neurons containing calcium-binding proteins in the rat claustrum.

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.

Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit.

The present paper describes parvalbumin, calbindin-D28k and calretinin immunoreactivity in the claustrum and endopiriform nucleus of adult rabbits. Studied neuronal populations are characterized by morphological heterogeneity. Four types were identified in each subpopulation of cells containing calcium binding proteins on the basis of the number of processes and their branching pattern. There were no spatial differences in the distribution of cells containing either parvalbumin or calbindin-D28k in the claustrum and endopiriform nucleus. Well documented presence of the various projective zones in the rabbit claustrum did not reflect the specific distribution of neurons containing calcium binding proteins, except those containing calretinin. Their localization may correspond with the limbic zone. We have found that the rabbit claustrum and endopiriform nucleus have different pattern of parvalbumin and calretinin immunoreactivity. The former was more intense in the claustrum and the distribution of cell types was significantly different from that in the endopiriform nucleus. Calretinin-positive cells were observed in the claustrum, while in the endopiriform nucleus they were scarce. The distribution of neither calbindin-D28k-ir neurons nor fibers allowed differentiation of claustrum and endopiriform nucleus. Significant differences between the claustrum and endopiriform nucleus observed in the rabbit might be related with ontogenetic as well as other (functional) factors.

Developmental expression of SNAP-25 protein in the rat striatum and cerebral cortex.

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.

Neuropeptide-containing neurons in the endopiriform region of the rat: morphology and colocalization with calcium-binding proteins and nitric oxide synthase.

The endopiriform nucleus, further divided into dorsal and ventral parts, and the neighbouring pre-endopiriform (pEn) nucleus form a region of highly heterogeneous structure involved in numerous physiological and pathological processes. Nonpyramidal neurons of this region containing three neuropeptides-somatostatin (SOM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP)-were examined in this study. Their colocalization with three calcium-binding proteins-parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and with nitric oxide synthase (NOS), was investigated by qualitative and quantitative methods. The results are summarized as follows: (1) all studied substances are distributed in neurons of the entire region, (2) SOM-ir neurons constitute the most numerous neuropeptide-containing population, whereas NOS-ir neurons make up the largest population of all studied, (3) colocalizations are found in the endopiriform region (Enr) (SOM with CB, PV and NOS; VIP with CR; NPY with NOS and NOS with CR), (4) heterogeneity of the endopiriform region appears in the differences of cells' shape distributions of single-labeled (SOM-, CR-PV-ir) and double-labeled (SOM/CB-, SOM/PV-, NPY/NOS- and NOS/CR-ir) neurons, as well as in differentiated percentage values of SOM/NOS, NPY/NOS and VIP/CR double-labeled neurons in three studied parts; additionally, differences in distribution of immunoreactive neuropil elements between parts of the region are observed. Numerous regional differences concerning neuronal morphology and immunocytochemical characteristics justify further division of the endopiriform region into distinguished parts. Some immunocytochemical features of the neurons in studied region may contribute to the role in epileptogenesis.

Distribution of immunoreactivity of calcium-binding proteins in the rabbit piriform cortex.

The piriform cortex has been extensively studied due to its possible role in epileptogenic activity. Neurones containing calcium-binding proteins (CaBPs), as a component of inhibitory circuitry, seem to be critically involved in this pathological process. The aim of the present study was to characterise the pattern of distribution of CaBPs-immunoreactivity in the piriform cortex of the adult rabbit. It comprises labelled cells, fibres (often with varicosities) and terminals. It varies among the layers. Moreover, the distribution of the parvalbumin- and calretinin-immunoreactive fibres and terminals allows even further subdivision of the layer I into two sublayers. Calretinin-ir neurones are located in subpial (Ia) layer, while parvalbumin - as well as calbindin-D28k-ir ones are mainly located in the second and third layer. Cajal-Retzius-like neurones containing calretinin, Chandelier cells containing parvalbumin and basket cells containing calbindin D28k and parvalbumin can be distinguished among labelled subpopulations of CaBPs neurones. In general, the pattern of PV- CR- and CB-immunoreactivity is similar to that previously characterised in other mammals, i.e., rats, guinea pigs, hedgehog, and tenrecs. The pattern is organised in topographic fashion confirming the complexity of regulatory circuits in the rabbit piriform cortex.

Parvalbumin containing neurons of the piriform cortex in open field stress -- a developmental study in the rat.

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.

The influence of open field exposure on neurons containing nitric oxide synthase in the basolateral complex and paracapsular intercalated nerve cells of the rat amygdala.

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.

Do the asymmetry and the size of the structures of the temporal lobe persist in early stages of schizophrenia?

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.

Morphometric analysis of the small intestine in wild type mice C57BL/6L -- a developmental study.

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.

A magnetic resonance volumetric study of the temporal lobe structures in depression.

Depression is one of the most common psychiatric disorders and is associated with considerable morbidity. In recent years structural-imaging technology has provided an opportunity to examine the brain anatomy in patients with the psychiatric illness. 10 patients of various ages and, as the control group, 16 healthy subjects were examined using the MRI method of neuroimaging. The volumes of the following structures were evaluated in the right and left hemispheres: the superior temporal gyrus, the basolateral temporal area (the region including middle temporal gyrus, inferior temporal gyrus and fusiform gyrus), the parahippocampal gyrus, the hippocampal head, the amygdaloid body and the lateral ventricle. The significant difference between the control group and the group with depression concerned the volume of the temporal horn of the lateral ventricle of both hemispheres. In depressed patients the left temporal horn was 49.8% and the right 38.4% larger in comparison with the control group. In the control group there were significant differences between the left and right hemispheres in the volume of all the structures studied, whereas in the group with depression these difference in volume between the hemispheres concerned only the amygdaloid body and the lateral ventricle.