Primary motor cortex alterations in Alzheimer disease: A study in the 3xTg-AD model. / Alteraciones en la corteza motora primaria en la enfermedad de Alzheimer: estudio en el modelo 3xTg-AD.

INTRODUCTION: In humans and animal models, Alzheimer disease (AD) is characterised by accumulation of amyloid-ß peptide (Aß) and hyperphosphorylated tau protein, neuronal degeneration, and astrocytic gliosis, especially in vulnerable brain regions (hippocampus and cortex). These alterations are associated with cognitive impairment (loss of memory) and non-cognitive impairment (motor impairment). The purpose of this study was to identify cell changes (neurons and glial cells) and aggregation of Aß and hyperphosphorylated tau protein in the primary motor cortex (M1) in 3xTg-AD mouse models at an intermediate stage of AD. METHODS: We used female 3xTg-AD mice aged 11 months and compared them to non-transgenic mice of the same age. In both groups, we assessed motor performance (open field test) and neuronal damage in M1 using specific markers: BAM10 (extracellular Aß aggregates), tau 499 (hyperphosphorylated tau protein), GFAP (astrocytes), and Klüver-Barrera staining (neurons). RESULTS: Female 3xTg-AD mice in intermediate stages of the disease displayed motor and cellular alterations associated with Aß and hyperphosphorylated tau protein deposition in M1. CONCLUSIONS: Patients with AD display signs and symptoms of functional impairment from early stages. According to our results, M1 cell damage in intermediate-stage AD affects motor function, which is linked to progression of the disease.

Trophic factors intervention regenerates the nestin-expressing cell population in a model of perinatal excitotoxicity: Implications for perinatal brain injury and prematurity.

We previously showed that TSC1 (a combination of transferrin and IGF-1) is a potent inductor of myelinogenesis in myelin deficient rats and in demyelinated adult mice. More recently, we demonstrated that regeneration of oligodendrocyte progenitors and myelin are possible with a single dose of TSC1 in a mouse model of Premature birth. Here, using the same mouse model of perinatal white matter damage due to glutamate excitotoxicity (GME), we tested the hypothesis that regeneration of endogenous nestin-expressing neural progenitors improves the outcome of prematurity. Treatments: N-methyl-D-aspartate (NMDA), saline, NMDA+TSC1 together or NMDA followed byTSC1 3 days later, were stereotaxically delivered into the corpus callosum of P4 mouse pups. Fluorescence analysis showed an intense enrichment of nestin-expressing cells in groups injected with NMDA+TSC1 from which many were generated by proliferation. Moreover, when TSC1 was injected three days after the primary insult it was still able to reduce ventricular enlargement and extensively rescue nestin-expressing progenitors. Cells co-expressing the proliferation marker Ki67, CNPase and faint nestin label were more abundant in groups injected with MNDA+TSC1 at 35 days after injection. Stereological analysis showed that the number of nestin-expressing cells in the sub-ventricular zone correlated inversely with the volume of the ventricle. A delayed administration of TSC1 after excitotoxicity reduced ventriculomegaly but not as much as, when NMDA and TSC1 were injected simultaneously. Thus, the earliest TSC1 was administered, the more tissue was rescued as shown by reduced ventriculomegaly. Astrocytes responded to GME by upregulating the expression of estrogen receptor and this expression was attenuated in the presence of TSC1 suggesting a decreased inflammation and a lesser need for estrogen-mediated central nervous system (CNS) neuroprotection.

Activity increase in EpoR and Epo expression by intranasal recombinant human erythropoietin (rhEpo) administration in ischemic hippocampi of adult rats.

Erythropoietin in the nervous system is a potential neuroprotective factor for cerebral ischemic damage due to specific-binding to the erythropoietin receptor, which is associated with survival mechanisms. However, the role of its receptor is unclear. Thus, this work assessed whether a low dose (500UI/Kg) of intranasal recombinant human erythropoietin administered 3h after ischemia induced changes in the activation of its receptor at the Tyr456-phosphorylated site in ischemic hippocampi in rats. The results showed that recombinant human erythropoietin after injury maintained cell survival and was associated with an increase in receptor phosphorylation at the Tyr456 site as an initial signaling step, which correlated with a neuroprotective effect.

REST/NRSF-induced changes of ChAT protein expression in the neocortex and hippocampus of the 3xTg-AD mouse model for Alzheimer's disease.

AIMS: The cholinergic system is one of the neurotransmitter systems altered in Alzheimer's disease (AD), the most common form of human dementia. The objective of this work was to determine the REST/NRSF involvement in altered ChAT expression in the neocortex and hippocampus of an AD transgenic mouse (homozygous 3xTg-AD) that over-expresses 3 proteins, amyloid-ß precursor protein, presenilin-1, and tau, all of which are associated with AD and cause cellular degeneration. MAIN METHODS: Two groups (WT and 3xTg-AD) of 11-month-old female mice were analyzed and compared. Half of the brains of each group were used for ChAT immunohistochemistry, and Western Blot analyses of ChAT and REST/NRSF were performed on the other half. KEY FINDINGS: We observed significant decreases in the number of ChAT-immunoreactive cells in the Meynert nucleus and of fibers in the frontal motor cortex and hippocampal CA1 area in transgenic mice compared with control mice. An increased level of REST/NRSF protein and a reduction of ChAT protein expression in the 3xTg-AD mice compared with their controls were also found in both in the latter two cerebral regions. SIGNIFICANCE: The increased REST/NRSF expression reported here and its effect on the regulatory region for ChAT transcription could explain the decreased expression of ChAT in the 3xTg-AD mouse; these findings may be associated with the degeneration observed in AD.

Cholinergic markers in the cortex and hippocampus of some animal species and their correlation to Alzheimer's disease.

INTRODUCTION: The cholinergic system includes neurons located in the basal forebrain and their long axons that reach the cerebral cortex and the hippocampus. This system modulates cognitive function. In Alzheimer's disease (AD) and ageing, cognitive impairment is associated with progressive damage to cholinergic fibres, which leads us to the cholinergic hypothesis for AD. DEVELOPMENT: The AD produces alterations in the expression and activity of acetyltransferase (ChAT) and acetyl cholinesterase (AChE), enzymes specifically related to cholinergic system function. Both proteins play a role in cholinergic transmission, which is altered in both the cerebral cortex and the hippocampus due to ageing and AD. Dementia disorders are associated with the severe destruction and disorganisation of the cholinergic projections extending to both structures. Specific markers, such as anti-ChAT and anti-AChE antibodies, have been used in light immunohistochemistry and electron microscopy assays to study this system in adult members of certain animal species. CONCLUSIONS: This paper reviews the main immunomorphological studies of the cerebral cortex and hippocampus in some animal species with particular emphasis on the cholinergic system and its relationship with the AD.

[Morphological analysis of the hippocampal region associated with an innate behaviour task in the transgenic mouse model (3xTg-AD) for Alzheimer disease]. / Análisis morfológico de la región del hipocampo asociado a una tarea conductual innata en el modelo de ratón transgénico (3xTg-AD) para la enfermedad de Alzheimer.

INTRODUCTION: Different animal models for Alzheimer disease (AD) have been designed to support the hypothesis that the neurodegeneration (loss of neurons and synapses with reactive gliosis) associated with Aß and tau deposition in these models is similar to that in the human brain. These alterations produce functional changes beginning with decreased ability to carry out daily and social life activities, memory loss, and neuropsychiatric disorders in general. Neuronal alteration plays an important role in early stages of the disease, especially in the CA1 area of hippocampus in both human and animal models. METHODS: Two groups (WT and 3xTg-AD) of 11-month-old female mice were used in a behavioural analysis (nest building) and a morphometric analysis of the CA1 region of the dorsal hippocampus. RESULTS: The 3xTg-AD mice showed a 50% reduction in nest quality associated with a significant increase in damaged neurons in the CA1 hippocampal area (26%±6%, P<.05) compared to the WT group. CONCLUSIONS: The decreased ability to carry out activities of daily living (humans) or nest building (3xTg-AD mice) is related to the neuronal alterations observed in AD. These alterations are controlled by the hippocampus. Post-mortem analyses of the human hippocampus, and the CA1 region in 3xTg-AD mice, show that these areas are associated with alterations in the deposition of Aß and tau proteins, which start accumulating in the early stages of AD.

5-HT denervation of the adult rat prefrontal cortex induces changes in the expression of α4 and α7 nicotinic acetylcholine receptor subtypes.

INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout several brain regions. Formation of the α4ß2 and α7 subtypes in particular is involved in the organisation of different types of memory. Furthermore, due to their location, these receptors can control the release of various types of neurotransmitters and contribute to synaptic plasticity. METHODS: Rats were divided into three groups, an experimental group (E), a sham-operated group, (S) and an intact group (T). In group E, stereotactic guidance was used to induce a chemical lesion with 1 µ/µL of 5,7-dihydroxytryptamine (5,7-DHT) in the anteroventral part of the dorsal raphe nucleus (DRN). In the sham-operated group (S), animals underwent surgery including delivery of the same excipient solution to the same site. The intact group (T) received no treatment whatsoever. Twenty days after surgery, animals in all groups were euthanised by decapitation to evaluate the expression of α4 and α7 nAChRs by means of molecular biology techniques. RESULTS: 5-HT denervation of the rat PFC differentially modified the expression of α4 and α7 receptors: while α4 receptor expression increased, α7 expression decreased. CONCLUSION: Expression differences observed between the two subtypes may be due to their separate locations. The α4 subtype is found in postsynaptic locations and may be related to adaptive changes in postsynaptic cells, while the location of α7 is presynaptic. This explains why the lesion and the elimination of 5-HT fibres in the CPF would cause a decrease in α7 expression.

Scanning electron microscopy of the orbital Harderian gland in the male Atlantic bottlenose dolphin (Tursiops truncatus).

The ultrastructure of the Harderian gland of Atlantic bottlenose dolphin (Tursiops truncatus) was examined by scanning electron microscopy (SEM). We found the following surface features: the typical round appearance of the ascinar glandular unit with a finely granular surface, a thin cortex and immediately below two types of cells: type I cells (characterized by small lipid vacuoles) and type II cells (characterized by large lipid vacuoles). It has been suggested that different cells forms represent a single cell type in varying activity states. Additionally, a coalescent tubular complex, a small balloon-like structures and large globular structures were observed. These structures may be reservoirs of secretion products.

Changes in hippocampal NMDA-R subunit composition induced by exposure of neonatal rats to L-glutamate.

Overactivation of NMDA-Rs may mediate excitotoxic cell death associated with epileptic seizures, and hypoxic-ischemic conditions. We assessed whether repeated subcutaneous administration of l-glutamate to neonatal rats affects the subunit composition of NMDA-Rs. Accordingly, cortical and hippocampal tissue from 14-day-old rats was analyzed by Western blotting and RT-PCR to quantify the protein and mRNA expression of different NMDA-R subunits. In addition, tissue sections were Nissl stained to assess the cell damage in this tissue. Early exposure of neonatal rats to L-glutamate differentially affects the expression of mRNA transcripts for NMDA-R subunits in the cerebral cortex and hippocampus. In the cerebral cortex, a decrease in NR2B subunit mRNA expression was observed, as well as a loss of NR1 and NR2A protein. By contrast, neonatal L-glutamate administration augmented the transcripts encoding the NR1, NR2B, and NR2C subunits in the hippocampal formation. The expression of mRNA encoding the NR2A subunit was not affected by neonatal L-glutamate administration in either of the brain regions examined. This differential expression of NMDA-R subunits following neonatal exposure to L-glutamate may represent an adaptive response of the glutamate receptors to overactivation in order to reduce the effect of high L-glutamate during the early period of life when the animal is more vulnerable to excitotoxicity.

[Structural and functional characteristics of glutamate transporters: how they are related to epilepsy and oxidative stress]. / Caracteristicas estructurales y funcionales de los transportadores de glutamato: su relacion con la epilepsia y el estres oxidativo.

AIMS: The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress. DEVELOPMENT: Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because they participate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growing body of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to the pathological disorders that are found in epilepsy. CONCLUSIONS: A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future.

The orbital Harderian gland of the male atlantic bottlenose dolphin (Tursiops truncatus): a morphological study.

The ultrastructure of the Atlantic Bottlenose dolphin Harderian gland (HG) has been described but some questions remain unanswered. The purpose of this work was to define the gland's structure, ultrastructure and the differences between cells (types I and II) of the male dolphin using optic, fluorescence and electron transmission microscopy. Three different cells were observed under optic and fluorescence microscopic examination, while only two cell types (types I and II) were distinguished by electron transmission microscopy. Type I (oval nuclear envelope) exhibited three different cell populations and type II (indented nuclear envelope) exhibited two different cell populations. Although, we observed both types of vesicles in both types of cells they differed, principally, in quantity. The glands also possessed prominent duct systems, with three orders of complexity. The dolphin orbital HG appears to function as a mixed heterologous gland with two types of cells that exhibit both types of vesicles and other distinguishable differences.

Hyperthermia-induced seizures modify the GABA(A) and benzodiazepine receptor binding in immature rat brain.

Effects of hyperthermia-induced seizures (HS) on GABA(A) and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-days-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA(A) and BDZ receptor binding. GABA(A) binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA(A) and BDZ binding in immature brain. HS-induced GABA(A) and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.

Prenatal-through-postnatal exposure to moderate levels of ethanol leads to damage on the hippocampal CA1 field of juvenile rats: a stereology and Golgi study.

Experimental paradigms conducted to assess the neurotoxic effects of ethanol exposure on hippocampus development have yielded controversial findings. Hippocampal CA1 population and some cytoarchitectural parameters of pyramidal cells were studied after exposure to ethanol during early development, in rats. Examination of 30-day-old offspring of rats exposed to moderate levels of ethanol during gestation through lactation showed an increased volume of the hippocampal CA1 field compared to untreated or pair-fed control pups, as well as a reduced number of pyramidal neurons. In addition, the number of spines from surviving CA1 pyramidal neurons was reduced. Furthermore, stubby and wide spines were proportionally increased, while the proportion of mushroom and ramified spines was reduced; no variation in the proportion of thin spines was observed. Because alcoholic women usually drink alcohol before, during, and after pregnancy, a broad-range experimental model of alcohol exposure was used in this study. The present findings show that experimental exposure to moderate levels of ethanol, resembling the human situation in alcoholic mothers, leads to loss of hippocampal CA1 pyramidal neurons, along with several pathological and plastic events in the dendritic arborization of these neurons. Some ethanol-induced excitotoxicity-related mechanisms, which may be underlying these effects, are discussed.

Ultrastructural analysis of guided nerve regeneration using progesterone- and pregnenolone-loaded chitosan prostheses.

Recently, numerous guide chambers for the treatment of injured nerves made up of different biomaterials have been designed, capable of hosting living cells or carrying neurotrophic or neuroactive substances to be directly released to the injured tissue. In this study, chitosan prostheses containing neurosteroids (progesterone and pregnenolone) were used for bridging a 10-mm gap in the rabbit facial nerve. Gas chromatography was used to quantify neurosteroid content in the prostheses prior to and after subcutaneous implantation at different periods of up to 60 days. The regeneration of the nerve fibers were evaluated at 15 and 45 days after axotomy by means of ultrastructural morphometric analysis. Different nerve fibers regenerative patterns were seen depending the groups studied and the analyzed stages. At 15 days after axotomy, the newly regenerating tissue revealed Schwann cells holding nonmyelinated nerve fiber bundles in an incipient and organized regenerative pattern. At 45 days, the regenerating tissue showed myelinated nerve fibers of different sizes, shapes, and myelin sheath thickness. Although the regeneration of the nerve fibers under neurosteroid treatment showed statistically significant differences in comparison with vehicle regenerated tissue, progesterone-loaded chitosan prostheses produced the best guided nerve regeneration response. These findings indicate that chitosan prostheses allowed regeneration of nerve fibers in their lumen, and when containing neurosteroids produced a faster guided nerve regeneration acting as a long-lasting release delivery vehicle.

Neurogenesis in the subventricular zone following transcranial magnetic field stimulation and nigrostriatal lesions.

Neurogenesis continues at least in two regions of the mammalian adult brain, the subventricular zone (SVZ) and the subgranular zone in hippocampal dentate gyrus. Neurogenesis in these regions is subjected to physiological regulation and can be modified by pharmacological and pathological events. Here we report the induction of neurogenesis in the SVZ and the differentiation after nigrostriatal pathway lesion along with transcranial magnetic field stimulation (TMFS) in adult rats. Significant numbers of proliferating cells demonstrated by bromodeoxyuridine-positive reaction colocalized with the neuronal marker NeuN were detected bilaterally in the SVZ, and several of these cells also expressed tyrosine hydroxylase. Transplanted chromaffin cells into lesioned animals also induced bilateral appearance of subependymal cells. These results show for the first time that unilateral lesion, transplant, and/or TMFS induce neurogenesis in the SVZ of rats and also that TMFS prevents the motor alterations induced by the lesion.

Scanning electron microscopy of the superficial pineal gland of the 15-day-old rat (Rattus norvegicus).

The presence of a cortex and medulla in the superficial pineal gland has been a controversial point in the morphology of this structure in mammals. The published reports indicate contradictory data especially in rodents. In this study the pineal gland of 15-day-old male rats (Rattus norvegicus) were studied, using scanning electron microscopy, in an attempt to determine whether or not a cortex and medulla are apparent in the pineal gland of young rats. The superficial pineal gland of the 15-day-old rat exhibited both a cortex and a medulla; these areas exhibited different structural organizations. The cortex had a thickness of 40-80 microm and the cells did not show a particular arrangement. The center of the gland was composed of a medulla, which had a width of 1000-1200 microm, and consisted of cells arranged in cords; its morphology was distinctly different from that of the cortex.

[The effects of the social environment on the brain]. / Efectos cerebrales del medio ambiente social.

AIMS: This work analyses the main studies dealing with the mechanisms by which the brain is altered by chronic stress and the impact of social stimuli on the activation of these mechanisms, which can lead to behavioural disorders and cognitive impairment in communities of mammals. DEVELOPMENT: The physiological and hormonal responses triggered as a response to stress are linked to alterations in certain areas of the brain and more particularly in the hippocampus. These mechanisms include hyperactivity of the hypothalamus-pituitary-adrenal axis, raised levels of corticosteroids and excitatory amino acids, neurotoxicity due to intracellular accumulation of calcium, apoptosis and a number of factors having to do with the immunological system. Most of these studies have involved the exogenous application of supraphysiological levels of corticosteroids or challenging the individual with stimuli that do not properly belong to their natural surroundings. Nevertheless, it is also possible that these mechanisms are triggered by aversive social stimuli from the natural environment, such as confrontation, establishing hierarchies, neglect and social evaluation. It has been proved that social stress has important effects on conduct and health, especially with regard to the structural and functional integrity of the brain. CONCLUSIONS: Social stress can trigger important alterations in the nervous system of individuals exposed to it and these changes can manifest themselves as varying types of disorders affecting conduct and the cognitive skills. Nevertheless, not all natural surroundings give rise to these adverse effects, as balanced communities offer their members support, protection and a series of other advantages.

NMDA and AMPA receptor expression and cortical neuronal death are associated with p38 in glutamate-induced excitotoxicity in vivo.

Early overstimulation of ionotropic glutamate receptors (iGluRs), such as the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, produces excitotoxicity in several brain regions. The molecular composition of those receptors and their regulation by intracellular signaling systems could be determinants in the development of progressive neurodegenerative mechanisms in the central nervous system (CNS). Studies of p38 mitogen-activated protein kinase (MAPK) activation, morphologic changes including cell number, and the expression of the NR1 and GluR2 subunits, by reverse transcriptase-PCR were evaluated at early postnatal ages (postnatal day [PD]8-14) in cerebral cortex of rats treated with monosodium glutamate (MSG; 4 mg/g body weight) administered subcutaneously on PD1, 3, 5, and 7. An important increase in p38 activity at PD8 and loss of cortical cell number were observed from PD8-14 in animals treated with MSG, together with significant morphologic changes characterized by cell shrinkage, nuclear hyperchromatism, and cytoplasmic vacuolation. These morphologic changes were prevented by SB203580, an inhibitor of p38 signaling, at PD8-14. No change in cerebral cortex thickness was observed among experimental or control rats. A significant increase in NR1 subunit expression was observed in response to MSG from PD8-14. GluR2 expression increased from PD8-12, but at PD14, its expression was reduced to 54% with respect to controls. SB203580 prevented alone the decreased in GluR2 expression induced by MSG. These results suggest that initial neuronal death (at PD8 and 10) in cerebral cortex may be due to an excessive Ca2+ influx through NMDA receptors, whereas the further damage process could be mediated by AMPA receptors through p38 signaling. This could represent a determinant mechanism to decide whether nerve cells survive or die.

Second female case of Myhre syndrome.

Myhre syndrome is a rare disorder characterized by low birthweight, short stature, mental retardation, facial dysmorphism (blepharophimosis, midfacial hypoplasia, prognathism), heart anomalies, muscle hypertrophy, decreased joint mobility and deafness. To date 11 male cases and only one female case have been reported. This paper describes the second female case and compares the clinical and radiological findings between female and male patients.

Facial nerve regeneration through progesterone-loaded chitosan prosthesis. A preliminary report.

Biodegradable nerve guides have represented new treatment alternatives for nerve repairing. They are gradually biodegradable, exert biological effects directly to the injured nerve, and act as drug- or cell-delivery devices. Furthermore, progesterone (PROG) has been demonstrated to promote injured peripheral nerve regeneration. In this study, it was hypothesized that PROG delivered from chitosan prostheses provides better facial nerve regenerative response than chitosan prostheses with no PROG. As there are no reports on the use of the former as nerve-guide material in the regeneration of injured nerves, this is the main objective of the present work. Chitosan prostheses containing PROG were used to bridge 10-mm gaps in rabbit facial nerves. The regenerated nerves were evaluated 45 days after implantation in animals with the use of light microscopy and morphometric analysis. Gas chromatography was used in order to quantify PROG content in prosthesis prior to and after implantation in subcutaneous tissue at different periods of up to 60 days. In addition, the prosthesis walls were evaluated with histological techniques in order to assess their integrity and the surrounding tissue reaction. Chitosan prostheses allowed PROG release during the time needed for nerve regeneration. At 45 days myelinated nerve fibers were observed in both the proximal and distal stumps. This parameter and the N ratio were higher in the progesterone-treated group when compared to that of the vehicle control. Findings indicate that chitosan prostheses were useful in nerve regeneration, acting as a long-lasting PROG delivery device a faster nerve regeneration.