The laminar organization of the motor cortex in monodactylous mammals: a comparative assessment based on horse, chimpanzee, and macaque.

The architecture of the neocortex classically consists of six layers, based on cytological criteria and on the layout of intra/interlaminar connections. Yet, the comparison of cortical cytoarchitectonic features across different species proves overwhelmingly difficult, due to the lack of a reliable model to analyze the connection patterns of neuronal ensembles forming the different layers. We first defined a set of suitable morphometric cell features, obtained in digitized Nissl-stained sections of the motor cortex of the horse, chimpanzee, and crab-eating macaque. We then modeled them using a quite general non-parametric data representation model, showing that the assessment of neuronal cell complexity (i.e., how a given cell differs from its neighbors) can be performed using a suitable measure of statistical dispersion such as the mean absolute deviation-mean absolute deviation (MAD). Along with the non-parametric combination and permutation methodology, application of MAD allowed not only to estimate, but also to compare and rank the motor cortical complexity across different species. As to the instances presented in this paper, we show that the pyramidal layers of the motor cortex of the horse are far more irregular than those of primates. This feature could be related to the different organizations of the motor system in monodactylous mammals.

Expression of aromatase P450(AROM) in the human fetal and early postnatal cerebral cortex.

Aromatase (P450(AROM)), the enzyme responsible for the conversion of testosterone (T) into 17-ß estradiol (E(2)), plays a crucial role in the sexual differentiation of specific hypothalamic nuclei. Moreover, recent findings indicate that local E(2) synthesis has an impact on other brain areas including hippocampus, temporal cortex and cerebellum, and may thus influence also cognitive functions. Numerous studies have described the expression and the distribution of P450(AROM) throughout ontogenesis and postnatal development of the central nervous system in several mammals, but data referring to humans are scarce. In the adult human brain, P450(AROM) has been detected in the hypothalamus, limbic areas, and in the basal forebrain, and described in glial cells of the cerebral cortex and hippocampus. In this study we report the expression, distribution and cellular localization of P450(AROM) in the human fetal and early postnatal cerebral cortex. In our series of fetal brains of the second trimester, P450(AROM) expression appeared at gestational week (GW) 17 and resulted limited to groups of cells localized close to the growing neuroepithelium in the ventricular and subventricular zones. At GWs 20-24, scattered P450(AROM) immunoreactive (-ir) neural cells were identified in the intermediate plate and subplate, and in the parietal cortical plate. In perinatal and early postnatal individuals the quantity of P450(AROM)-ir elements increased, and revealed the morphology typical of glial cells. Double labeling immunostaining with anti-GFAP and anti-P450(AROM) antisera, and subsequent confocal analysis, confirmed this observation. Our data show that the expression of P450(AROM) in the fetal cortex starts approx at the end of the fourth gestational month, but increases steadily only in the last trimester or in the early postnatal period. This temporal trend may suggest that P450(AROM) could act as a differentiation-promoting factor, based on timing of the steroid actions.

Manganese intoxication decreases the expression of manganoproteins in the rat basal ganglia: an immunohistochemical study.

Manganese (Mn) is a cofactor for some metalloprotein enzymes, including Mn-superoxide dismutase (Mn-SOD), a mitochondrial enzyme predominantly localized in neurons, and glutamine synthetase (GS), which is selectively expressed in astroglial cells. The detoxifying effects of GS and Mn-SOD in the brain, involve catabolizing glutamate and scavenging superoxide anions, respectively. Mn intoxication is characterized by impaired function of the basal ganglia. However, it is unclear whether regional central nervous system expression of manganoproteins is also affected. Here, we use immunocytochemistry in the adult rat brain, to examine whether Mn overload selectively affects the expression of GS, Mn-SOD, Cu/Zn-SOD, another component of the SOD family, and glial fibrillary acid protein (GFAP), a specific marker of astrocytes. After chronic Mn overload in drinking water for 13 weeks, we found that the number and immunostaining intensity of GS- and Mn-SOD-positive cells was significantly decreased in the striatum and globus pallidus, but not in the cerebral frontal cortex. In addition, we found that GS enzymatic activity was decreased in the strio-pallidal regions but not in the cerebral cortex of Mn-treated animals. In contrast, Cu/Zn-SOD- and GFAP-immunoreactivity was unchanged in both the cerebral cortex and basal ganglia of Mn-treated rats. Thus, we conclude that in response to chronic Mn overload, a down-regulation of some manganoproteins occurs in neurons and astrocytes of the striatum and globus pallidus, probably reflecting the vulnerability of these regions to Mn toxicity.

Effect of aluminum consumption on the vestibulo-ocular reflex.

The effects of chronic exposure (90 days) to Aluminum chloride (AlCl(3)) were analyzed in 3, 10 and 24 month old male rats (n=270) by investigating the function of the VOR (vestibulo-ocular reflex) in correlation with Aluminum (Al) concentrations in blood and brain. The VOR was chosen and tested in basal conditions (pre-exposure measures) and during the continuous administration of three different concentrations of AlCl(3) (0.5, 1, 2 g/l in drinking solution): the control animals being exposed to NaCl (0.125, 0.25 and 5 g/l in drinking solution). Results showed that LOAEL (Lowest Observed Adverse Effect Level) was 43.1+/-11.4 mg Al/kg-day. At this concentration the Al induced significant VOR impairment in all exposed rats, regardless of animal age. Neuroanatomical analysis showed that VOR impairment was not dependent on astrocyte damage nor evidences of amyloid deposits in the exposed rats was found. Significant changes of Al concentrations were observed in nervous tissue, while its concentration in whole blood was unaffected. Furthermore, results show that it is possible to identify an individual neurotoxic threshold for each animal and therefore hypothesize the clinical use of the VOR test for the evaluation of individual risk toxicity to chronic Al exposure.

Metallothionein-I-II and GFAP positivity in the brains from frontotemporal dementia patients.

Frontotemporal dementia regards a group of presenile progressive neurodegenerative form of dementias which includes Pick's disease, corticobasal degeneration, frontotemporal dementia with motor neuron disease, frontal lobe degeneration, dementia-parkinsonism-amyotrophy complex, familial non-specific dementia mapping to chromosome 3, non-Alzheimer degenerative dementia lacking distinctive histological features as well as a number other infrequent syndromes with dementia and focal neurological signs. The aim of this study was to investigate the regional distribution of metallothionein-I-II, an ubiquitary group of buffering proteins, in cases of frontotemporal dementia. The aim of the present study was to study the metallothionein-I-II expression in relationship to the expression in astrocytes of glial fibrillary acidic protein (GFAP) as we have already done in previous studies of Alzheimer's and Binswanger's diseases [31,32]. Our findings indicate that metallothionein-I-II expression in the most affected areas is likely to be regionally distinct and layer-dependent, in that it is highest in the deep layers of the frontotemporal cortex and the allocortex (hippocampus) while insignificantly immunopositive in the occipital cortex. In addition, the potential use of metallothionein-I-II as a new pharmacological approach to contrast some deleterious aspects of this disease has been also discussed.

Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment.

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.

In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase.

Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimer's disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases.

[Carcinosarcoma of the larynx: immunohistochemical study, clinical considerations, therapeutic strategies]. / Il carcinosarcoma della laringe: studio immunoistochimico, considerazioni cliniche, strategie terapeutiche.

Carcinosarcoma is a rare laryngeal neoplasm accounting for less than 1% of all malignancies involving this organ. Given its double epithelial, mesenchymal nature, this neoplasm is described in the literature in many ways: sarcomatoid carcinoma, variants of squamous cell carcinoma with fused cells, pseudoscarcoma, pleumorphous carcinoma, polypoid carcinoma, metaplastic carcinoma. Although not invalidating, cytokeratin positive immunohistochemistry is fundamental for diagnosis. Given the different histopathological pictures, the rareness and heterogeneous natural history of such tumors, it is not surprising there is as yet no agreement as to the treatment of choice. An accurate analysis of the literature indicates that both the therapeutic approach and prognosis are the same as those found for normal squamous cell carcinoma. The authors describe a case of laryngeal carcinosarcoma, analyzing the clinical-therapeutic aspects, histopathological features and immunohistochemistry of this rare neoplasm.

Zinc-bound metallothioneins as potential biological markers of ageing.

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

Localization of metallothionein I-II immunoreactivity in bovine pituitary gland.

Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper). Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland. Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland. Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis. While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of alpha-MSH by POMC-cells located in the pars intermedia of the adenohypophysis.

Aluminum inhibits the lysosomal proton pump from rat liver.

Lysosomes are cytoplasmatic organelles, delimitated by a single lipoprotein membrane, that contain several enzymes mostly belonging to the hydrolases in that they function mainly for intracellular digestion. Lysosomal internal pH is characteristically acidic and it is maintained around pH 4.5 by a proton pump, an ATPase, that uses energy from ATP hydrolysis to translocate H+ ions into lysosomes. In the presence of Al3+ the proton pump activity is markedly reduced compromising acidic vesicles functionality. Among different species utilized, Al2(SO4)3 and AlF3 were the most effective. Aluminum effect was not observed when the delta pH was produced artificially by nigericin.

Activation of monoamine oxidase type-B by aluminum in rat brain homogenate.

Monoamine oxidase type B (MAO-B) activity is elevated in certain neurological diseases such as Alzheimer's and Parkinson's disease with respect to age-matched controls; the cause of this elevation is unknown. The documented accumulation of aluminum in certain neurodegenerative diseases prompted us to test the effect of Al3+ on the activity of MAO-B in rat brain homogenate. Results showed that the metal ion significantly increased MAO-B enzymatic activity in a dose-dependent manner, yielding a K(M) of 5.69 microM compared with 34.45 microM in the absence of the metal ion. The Vmax of 45.34 micromol/min was unchanged in the presence of the metal ion.

Histochemical localization of glycoconjugates on microglial cells in Alzheimer's disease brain samples by using Abrus precatorius, Maackia amurensis, Momordica charantia, and Sambucus nigra lectins.

Four lectins (Abrus precatorius (APA), Maackia amurensis (MAA), Momordica charantia (MCA) and Sambucus nigra (SNA)) have been used to identify glycohistochemically the microglial cells (MGC) activation in autoptic brain samples from Alzheimer's disease (AD) subjects. Three of these lectins (APA, MAA and MCA) have utilized as microglial cell markers for the first time. The identification of new markers for the study of MGC is very important to better understand the role of these type of cells in the metabolic/dismetabolic control of betaA4 in AD which still represents a vexata questio.

Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease.

One of the neuropathological characteristics of Alzheimer's disease is the presence of a large number of reactive astrocytes, often, but not always, associated with senile plaques. The factors responsible for such an activation are as yet totally unknown. Other characteristic features of this disease such as betaA4 amyloid accumulation, senile plaques and neurofibrillary tangles represent well known pathological phenomena. Some studies suggest that betaA4 plays a major role in the reactive astrocytosis characteristic of Alzheimer's disease. In the normal human brain, metallothionein isoforms I and II are expressed in astrocytes but not in neurons. In the present study, we used anti-metallothionein antibodies to detect cells expressing metallothioneins isoforms I and II in normal and Alzheimer's disease (AD) brain sections. Results showed that expression of these proteins in the cortex, cerebral white matter and cerebellum is a relevant anatomopathological characteristic of Alzheimer's disease. Analysis of Alzheimer's disease brain sections revealed high expression of metallothioneins I/II in astrocytes and microcapillaries, and in the granular but not the molecular layer of the cerebellum. Furthermore, metallothionein expression can be used as a marker to identify subtypes of astrocytes.

Aluminum alters intracellular calcium homeostasis in vitro.

The present paper reports data regarding the influence of aluminum, at micromolar concentrations, on intracellular calcium homeostasis. Al3+ modifies Ca2+ uptake in the endoplasmic reticulum (ER), accelerates Ca2+ release from mitochondria and strongly inhibits Ca2+-ATPase activity with a consequent high-level calcium accumulation inside the cell. These results suggest that Al3+ neurotoxicity may be related to an alteration of the intracellular calcium regulatory system.

Evaluation of MAO activities on murine neuroblastoma cells upon acute or chronic treatment with aluminium (III) or tacrine.

Monoamine-oxidase catalyses the oxidative deamination of various primary amines such as norepinephrine, serotonin, dopamine and others. Such an enzyme exists under two well known isotypes, A and B. In the brain monoamine-oxidase activity increases with ageing. The present paper reports data regarding the evaluation on the activity of monoamine-oxidase A and B in murine neuroblastoma cells carried out by a radioassay upon acute or chronic treatment of cells by aluminium or tacrine. Our data show that aluminium has a pronounced activatory effect on both enzymatic isoforms either in acute or long-term adaptation treatment. Conversely, tacrine produces an inhibitory effect of both monoamine oxidase isotypes after an acute treatment, while an inhibitory effect on the isoform A and an activation of the isoform B in long-term adaptation was observed. Such effects are discussed in terms of aluminium neurotoxicity and therapeutic implications for tacrine.

Effects of the aluminium speciation on the morphology of rabbit erythrocytes: a toxicological model.

The great majority of papers dealing with aluminium (Al) experimental toxicology in vivo and/or in vitro, do not consider the relevance of the metal chemical speciation as a conditio sine qua non to a correct interpretation of aluminium toxicological data. In fact, different aluminium compounds such as salts, stable, metastable, lipophilic or hydrophilic complexes with different thermodynamics and kinetics properties are indifferently utilized by most laboratories. In this connection, the molecular bases that explain Al toxicity are largely uninterpretable. The aim of this study, therefore, is to demonstrate how relevant is the issue of aluminium speciation to the understanding of the toxic properties of the metal ion. In our experimentation we used, as a heuristic model, rabbit erythrocytes (RBC) (Sheetz and Singer, 1974), because these animals are well known for their sensitivity to Al intoxication (Wisniewski and Sturman, 1989). Results reported herein show how the metal chemical speciation can paradigmatically modify the RBC morphology in a metal speciation-dependent manner. Finally, a new toxicological protocol that, by simplifying the methodology of aluminium solution preparation, could standardize the study of aluminium toxicity is also proposed.

Aluminum (III) induces alterations on the physical state of the erythrocytic membrane: an ESR evaluation.

The action of aluminum [Al(III)] as Al(acac)3 on erythrocytes causes biophysical effects such as osmotic fragility and echino-acanthocytes formation. In this paper, we present these effects in terms of variation of membrane fluidity, together with findings regarding conformational modifications of membrane proteins consequent to Al(III) exposure, as well as the effects on the mobility of the membrane protein bound sialic acid. To this end, we utilized ESR measurements of rabbits and humans erythrocytic ghosts after probing or labeling with suitable stable radicals used as spin probes or labels. Our results show that the lipophilic, hydrolytically stable toxicant Al(acac)3 causes a remarkable reduction of membrane fluidity in rabbit erythrocytes, an appreciable structural compacting effect on cytoskeletal and transmembrane proteins, as well as a reduction of rotational mobility of cell-surface sialic acid of human erythrocytes.

Rabbit spinal cord infarctuated upon aluminum (III)-liposomes intravenous treatment.

The present paper describes toxic effects of liposomes containing aluminum lactate (AlLac3) (Al-liposomes) on rabbits spinal cord after intravenous treatment of 42 days with 60 micrograms per day of metal-ion as AlLac3 at neutral pH as compared with an aqueous solution of the same metastable Al compound. Among the tissues examined, spinal cord appear to be the most injured one with large infarctual zones where Al is accumulated. Differently, the aqueous solution of AlLac3 is ineffective. Toxic aspects of Al lipophilic forms are herein discussed in terms of pharmacological implications.