The Use of Silica Microparticles to Improve the Efficiency of Optical Hyperthermia (OH).

Although optical hyperthermia could be a promising anticancer therapy, the need for high concentrations of light-absorbing metal nanoparticles and high-intensity lasers, or large exposure times, could discourage its use due to the toxicity that they could imply. In this article, we explore a possible role of silica microparticles that have high biocompatibility and that scatter light, when used in combination with conventional nanoparticles, to reduce those high concentrations of particles and/or those intense laser beams, in order to improve the biocompatibility of the overall procedure. Our underlying hypothesis is that the scattering of light caused by the microparticles would increase the optical density of the irradiated volume due to the production of multiple reflections of the incident light: the nanoparticles present in the same volume would absorb more energy from the laser than without the presence of silica particles, resulting either in higher heat production or in the need for less laser power or absorbing particles for the same required temperature rise. Testing this new optical hyperthermia procedure, based on the use of a mixture of silica and metallic particles, we have measured cell mortality in vitro experiments with murine glioma (CT-2A) and mouse osteoblastic (MC3T3-E1) cell lines. We have used gold nanorods (GNRs) that absorb light with a wavelength of 808 nm, which are conventional in optical hyperthermia, and silica microparticles spheres (hereinafter referred to as SMSs) with a diameter size to scatter the light of this wavelength. The obtained results confirm our initial hypothesis, because a high mortality rate is achieved with reduced concentrations of GNR. We found a difference in mortality between CT2A cancer cells and cells considered non-cancer MC3T3, maintaining the same conditions, which gives indications that this technique possibly improves the efficiency in the cell survival. This might be related with differences in the proliferation rate. Since the experiments were carried out in the 2D dimensions of the Petri dishes, due to sedimentation of the silica particles at the bottom, whilst light scattering is a 3D phenomenon, a large amount of the energy provided by the laser escapes outside the medium. Therefore, better results might be expected when applying this methodology in tissues, which are 3D structures, where the multiple reflections of light we believe will produce higher optical density in comparison to the conventional case of no using scattering particles. Accordingly, further studies deserve to be carried out in this line of work in order to improve the optical hyperthermia technique.

Slowdown intracranial glioma progression by optical hyperthermia therapy: study on a CT-2A mouse astrocytoma model.

Metallic nanorods are promising agents for a wide range of biomedical applications. We report an optical hyperthermia method capable of inducing slowdown tumor progression of an experimental in vivo CT-2A glioblastoma tumor. The tumor model used in this research is based on the transplantation of mouse astrocytoma CT-2A cells in the striatum of mice by intracranial stereotaxic surgery. Two weeks after cell implant, the resulting tumor is treated by irradiating intratumoral injected gold nanorods, biofunctionalized with CD133 antibody (B-GNRs), using a continuous wave laser. Nanoparticles convert the absorbed light into localized heat (reaching up to 44 °C) due to the effect of surface plasmon resonance. A significant slowdown in CT-2A tumor progression is evident, by histology and magnetic resonance imaging, at one (p = 0.03) and two weeks (p = 0.008) after irradiation treatment. A notable deceleration in tumor size (15%-75%) as compared to the control untreated groups, it is observed. Thus, laser irradiation of B-GNRs is found to be effective for the treatment of CT-2A tumor progression. Similarities between the pre-clinical CT-2A tumor model and the human astrocytoma disease, in terms of anatomy, metastatic behavior and histopathology, suggest that hyperthermic treatment by laser irradiation of B-GNRs administered into high-grade human astrocytoma might constitute a promising alternative treatment to limit the progression of this deadly disease.

Review: Could neurotransmitters influence neurogenesis and neurorepair after stroke?

Brain ischaemia and reperfusion produce alterations in the microenvironment of the parenchyma, including ATP depletion, ionic homeostasis alterations, inflammation, release of multiple cytokines and abnormal release of neurotransmitters. As a consequence, the induction of proliferation and migration of neural stem cells is redirected towards the peri-infarct region. The success of new neurorestorative treatments for damaged brain implies the need to describe with greater accuracy the mechanisms in charge of regulating adult neurogenesis, under both physiological and pathological conditions. Recent evidence demonstrates that many neurotransmitters, glutamate in particular, control the subventricular zone (SVZ), thus being part of the complex signal network that exerts a remarkable influence on the production of new neurones. Neurotransmitters provide a link between brain activity and SVZ neurogenesis. Therefore, a deeper knowledge of the role of neurotransmitters systems, such as glutamate and its transporters, in adult neurogenesis, may prove a valuable tool to be utilized as a neurorestorative therapy in this pathology.

Recommendations guide for experimental animal models in stroke research.

INTRODUCTION: The progress of effective therapies for stroke has become a challenging task for both researchers and clinicians. Some pitfalls in clinical trials might have their origins in the pre-clinical experimental ischaemic models for the evaluation of potential neuro-protective agents. METHODS: We aim to standardise the methods for the development of stroke animal models throughout Spain, to produce document with appropriate recommendations and best practice in order to improve experimental methods in the field of stroke research. RESULTS: Members of several experienced stroke research groups prepared a guide with recommendations in the application of focal cerebral ischaemic models. The main features of this guide are based on the selection of the most appropriate animal model, taking in account the objective of the study, the species, strain, age, sex of animals, as well as risk factors. The experimental design must include a sham control group and the sample size calculation. Animal randomisation and blind analysis, masked assessment of outcomes, monitoring of body temperature and cerebral blood flow, and the reporting of reasons for excluding animals from the study, as well as the mortality rate, are other main points to fulfil in the application of stroke models. CONCLUSIONS: Standardised methods are essential to increase the success of the pre-clinical findings in the stroke neuroprotection field to be able to translate to the clinical practice.

Whole-body periodic acceleration reduces brain damage in a focal ischemia model.

Stroke is the second most common cause of death and major cause of disability worldwide. Actual treatment involves surgery and/or thrombolytic drugs, but there is an urgent need for new approaches. Periodic acceleration, a rocking headward to footward movement of the whole body, is a non-invasive method to induce pulsatile shear stress on the vascular endothelium eliciting an enhanced production and secretion of endothelium-derived products such as nitric oxide, prostacyclin, prostaglandin E2, tissue plasminogen activator (tPA), and adrenomedullin. All these products have been shown to protect the brain from ischemic injuries. A rat model of focal brain ischemia was treated with application of periodic acceleration for 3 h immediately after the onset of ischemia. Controls remained static for the same period of time. Brain damage was assessed by magnetic resonance imaging (MRI) and biochemical markers. A significant reduction in brain damage was observed, 7 days post-ischemia, in rocked rats when compared with the static controls, through MRI. Furthermore, rocked animals had significantly lower levels of Beclin 1 and fractin than their static counterparts, and some isoforms of nitric oxide synthase were regulated by periodic acceleration. Our results show that periodic acceleration may provide a novel, affordable, non-invasive therapeutic option for the treatment of stroke.

Distribution and expression pattern of the nitrergic system in the cerebellum of the sheep.

The nitrergic system produces nitric oxide as an atypical neurotransmitter in the nervous system. Nitric oxide is produced from l-arginine through specific enzymes known as nitric oxide synthases. Of these, the more abundant form in neurons is the constitutive neuronal nitric oxide synthase, although the inducible isoform can be expressed as well, especially following stress or other injuries. The excessive formation of nitric oxide results in protein nitration, particularly at tyrosine residues, thus the presence of nitrotyrosine can be used as a marker of nitric oxide production. In previous studies we have shown the distribution of the components of the nitrergic system in the cerebellum of rodents, where neuronal nitric oxide synthase immunoreactivity was present in stellate and basket cells, and occasionally in granule cells. Here, we present evidence that in the sheep, as a model of larger mammals, most cerebellar neurons display an intense immunostaining for neuronal nitric oxide synthase, including unipolar brush cells, and Lugaro and Golgi neurons, which are not immunoreactive in rodents. In addition, weak immunoreactivity for inducible nitric oxide synthase and nitrotyrosine was found in particular cell types, indicating a basal expression for these markers. Our results suggest a larger dependence on the nitrergic system for the cerebella of larger mammals. Since this increase happens in both activating and inhibitory neurons of the cerebellar circuitry, we propose that in these animals there is a higher steady-state regulation of the cerebellum based on nitric oxide.

Cholinergic somata and terminals in the rat substantia nigra: an immunocytochemical study with optical and electron microscopic techniques.

The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN). Scarce, large (20-30 microns in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat--for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4). Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH). Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons. Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells. Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Light and electron microscopic study of galanin-immunoreactive nerve fibers in the rat posterior thalamus.

Light and electron microscopic immunocytochemistry was used to study certain cell groups in the posteromedial thalamus which contain galanin-immunoreactive (GAL-IR) fibers. The nuclei subparafascicularis pars parvicellularis (SPFpc) and parafascicularis (PF) contain a dense network of GAL-IR fibers which form basketlike structures around unstained cells. The periventricular area also contains numerous GAL-IR fibers and these also occasionally form basketlike structures. The GAL-IR terminal fields continue caudally in the mesodiencephalic junction and merge with other GAL-IR fibers in the dorsal aspects of the substantia nigra and around the dorsolateral tip of the medial lemniscus. Ultrastructural analysis of the GAL-IR basketlike structures revealed that GAL-IR terminals make numerous synapses with the cell bodies and proximal dendrites of SPFpc neurones. These results suggest that the activity of cells in the SPFpc and PF nuclei may be strongly influenced by galanin-containing nerve fibers probably originating in the spinal cord.

Distribution of the inositol 1,4,5-trisphosphate receptor, P400, in adult rat brain.

The distribution of the inositol 1,4,5-trisphosphate receptor protein, P400, was investigated in adult rat brain by immunocytochemistry with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactive neuronal cell bodies were detected in the cerebral cortex, the claustrum, the endopiriform nucleus, the corpus callosum, the anterior olfactory nuclei, the olfactory tubercle, the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the hippocampal formation, the dentate gyrus, the caudate-putamen, the fundus striatum, the amygdaloid complex, the thalamus, the caudolateral part of the hypothalamus, the supramammillary nuclei, the substantia nigra, the pedunculopontine tegmental nucleus, the ventrotegmental area, the Purkinje cells in the cerebellum, the dorsal cochlear nucleus, the subnucleus oralis and caudalis of trigeminal nerve, and the dorsal horn of the spinal cord. Immunoreactive fibres were found in the medial forebrain bundle, the globus pallidus, the stria terminalis, the pyramidal tract, the spinal tract of trigeminal nerve, and the ventral horn of spinal cord. Nerve fibres forming a dense plexus ending in terminal-like boutons were detected in relation to nonimmunoreactive neurons of the dentate, interpositus, and fastigial nuclei of the cerebellum and around neurons of the vestibular nuclei. This receptor protein binds a specific second messenger, inositol 1,4,5-trisphosphate, which produces a mobilization of intracellular Ca2+ and a modulation of transmitter release.

Subcellular localization of nitric oxide synthase in the cerebral ventricular system, subfornical organ, area postrema, and blood vessels of the rat brain.

The distribution of neuronal nitric oxide synthase (nNOS) has been studied in the more rostral portion of the lateral ventricle, subfornical organ, area postrema and blood vessels of the rat central nervous system. nNOS was located by means of a specific polyclonal antibody, by using light and electron microscopy. Light microscopy showed immunoreactive varicose nerve fibers and terminal boutons-like structures in the lateral ventricle, positioned in supra- and subependimal areas. The spatial relationships between immunoreactive neuronal processes and the wall of the intracerebral blood vessels were studied. Electron microscopy showed numerous nerve fibers in the wall of the lateral ventricle; many were nNos-immunoreactive and established very close contact with ependymal cells. Immunoreactive neurons and processes were found in the subependymal plate of the ventricular wall, the subfornical organ, the area postrema, and the circularis nucleus of the hypothalamus. In these last three areas, the immunoreactive neurons were found close to the perivascular space of fenestrated and nonfenestrated blood vessels. The nNOS immunoreactivity was localized to the endoplasmic reticulum, cisterns, ribosomes, neurotubules, and in the inner part of the external membrane. In the terminal boutons, the reaction product was found surrounding the vesicle membranes. This distribution showed nNOS as a predominantly membrane-bound protein. The nitrergic nerve fibers present in the wall of the ventricular system might regulate metabolic functions as well as neurotransmission in the subfornical organ, area postrema and circularis nucleus of the hypothalamus.

Lesion-induced expression of low-affinity nerve growth factor receptor-immunoreactive protein in Purkinje cells of the adult rat.

Normal adult cerebellar Purkinje cells in the rat rarely express low-affinity nerve growth factor receptor immunoreactivity. However, intense anti-low-affinity nerve growth factor receptor immunostaining was observed as early as one day after a lesion of the cerebellar cortex. Low-affinity nerve growth factor receptor immunoreactivity was confined to a selected group of Purkinje cells, the number of which reached a maximum at three days postlesion, and, in some neurons, persisted up to 10 days after damage. The intensity of Purkinje cell immunolabeling decayed abruptly with distance from the lesion site. Reactive Purkinje cells exhibited deposition of immunoreaction product in the cell soma, dendrites and axons. Characteristically, most Purkinje cell axons exhibiting intense low-affinity nerve growth factor receptor immunoreactivity had beaded, varicose morphology. Varicose fibres with the appearance of recurrent collaterals of Purkinje cell axons were also low-affinity nerve growth factor receptor-positive. Our results indicate that adult rat Purkinje cells increase low-affinity nerve growth factor receptor-immunoreactive protein in response to injury, suggesting that, in the cerebellum, low-affinity nerve growth factor receptor or low-affinity nerve growth factor receptor-like molecules may be involved in regulating neuronal plasticity during adulthood.

Immunocytochemical localization of cholinergic terminals in the region of the nucleus basalis magnocellularis of the rat: a correlated light and electron microscopic study.

The cholinergic circuitry in the nucleus basalis magnocellularis of the rat was investigated in a correlated light and electron microscopic study by using monoclonal antibodies against the acetylcholine-synthesizing enzyme, choline acetyltransferase, following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. After the immunocytochemical approach, large cholinergic cells and a few immunoreactive fibres exhibiting a varicose appearance, were detected by light microscopy in portions of the nucleus basalis magnocellularis located within the anatomical limits of the globus pallidus, mostly in its ventromedial part. Cholinergic neurons and fibre-like structures were also found within the substantia innominata on the edge of globus pallidus. The same material studied by light microscopy was analysed with the electron microscope. At the ultrastructural level, the immunopositive neurons showed the same cytological characteristics and pattern of synaptic input as cholinergic basal forebrain cells. Additionally, scarce immunoreactive preterminal axons and terminal boutons were detected in the region. The immunoreactive terminals were scattered or formed occasional clusters and appeared as heavily immunostained vesicle-filled boutons making exclusively axodendritic synaptic contacts principally with immunonegative distal dendrites. Both symmetric and asymmetric synaptic contacts established between these structures were detected, although the symmetric contacts were the more numerous. The surface of postsynaptic immunonegative dendrites in asymmetric synaptic contact with immunoreactive terminals was generally covered by terminals that lacked detectable immunoreactivity. In contrast, those in symmetric synaptic contact with labelled terminals showed much sparser input from immunonegative terminals, suggesting that they may belong to interneurons. Very rarely, cholinergic terminals were detected in asymmetric synaptic contact with dendrites which also contained positive immunoreaction product. Asymmetric contacts were frequently characterized by the presence of subjunctional dense bodies. The detection of cholinergic terminals in the region of the nucleus basalis magnocellularis of the rat indicates that this region not only contains cholinergic projecting neurons, but receives a cholinergic input itself. Results of this study provide evidence of the existence of a cholinergic transmission in the basal forebrain of the rat, and also that acetylcholine might play a role in the regulation of the extrinsic cortical cholinergic innervation. The possible sources of this innervation are discussed.

Insulin-like growth factor I-immunoreactive peptide in adult human cerebellar Purkinje cells: co-localization with low-affinity nerve growth factor receptor.

It has been proposed that Insulin-like growth factor I is involved in the development, growth and maintenance of the central nervous system possibly interacting with other trophic factors. High levels of insulin-like growth factor I have been detected in the cerebellum during development and adulthood suggesting a specific role for insulin-like growth factor I in this brain area. While there is ever increasing data regarding the cell types containing endogenous insulin-like growth factor I in the rat brain, no information on the human brain is yet available. In the present study we sought to analyse the precise location of insulin-like growth factor I peptide in the adult human cerebellum using a specific antiserum against recombinant human insulin-like growth factor I. After immunocytochemistry, numerous Purkinje cells exhibited intense positive staining occupying the cell soma, dendrites and dendritic spines as well as axons. Occasionally, immunoreactive Purkinje cell axons were arciform and exhibited bulbous dilatations along their proximal length. Putative recurrent collaterals of Purkinje cell axons were also insulin-like growth factor I reactive. Double-staining immunocytochemistry in the same sections consistently showed, as expected, co-expression of insulin-like growth factor I and calbindin, although a few calbindin containing Purkinje cells lacked insulin-like growth factor I immunostaining suggesting there are insulin-like growth factor I positive Purkinje cell subsets in the human cerebellum. In addition, co-expression of insulin-like growth factor I and low-affinity nerve growth factor receptor-immunoreactive protein was found in a subpopulation of insulin-like growth factor I positive Purkinje cells. The results of this study prove the presence of insulin-like growth factor I immunoreactivity in a Purkinje cell subpopulation of the adult human cerebellum suggesting that insulin-like growth factor I may participate in paracrine or autocrine regulatory systems in the adult human brain.

Localization of C-PON immunoreactivity in the rat main olfactory bulb. Demonstration that the population of neurons containing endogenous C-PON display NADPH-diaphorase activity.

The presence of the neuropeptide C-terminal flanking peptide of neuropeptide-Y, C-PON, has been investigated in the main olfactory bulb of the rat using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. The distribution of immunoreactive structures to C-PON was examined in both horizontal and coronal sections. Endogenous C-PON was localized within two types of short-axon cells including (1) superficial short-axon cells in the glomerular layer and (2) deep short-axon cells lying in the deepest portion of the granule cell layer and in the adjacent white matter. In addition, varicose immunoreactive processes were detected in all layers, although they were more numerous in the deepest portion of the granule cell layer. Immunoreactive cell bodies and processes were also observed in the nucleus olfactorius anterior and in the intrabulbar portion of the anterior commissure. Nevertheless, immunoreactive structures were not localized in the lateral olfactory tract. The indirect immunofluorescence technique to detect endogenous C-PON in combination with the enzyme histochemical demonstration of NADPH-diaphorase activity, in single sections, showed that the NADPH-diaphorase procedure is a reliable marker for these C-PON positive cells. Also, indirectly, that, in the rat main olfactory bulb, C-PON and neuropeptide-Y are contained in the same cell types. Many glomeruli were stained following the NADPH-diaphorase procedure, but they were not C-PON immunoreactives. Results of this study provide evidence suggesting that C-PON may influence polysynaptically the function of mitral cells and, therefore, the olfactory bulb output.

Subcellular localization of nerve growth factor receptors in identified cells of the rat nucleus basalis magnocellularis: an immunocytochemical study.

The subcellular location of nerve growth factor receptor in the ventromedial portion of rat globus pallidus was investigated with affinity-purified monoclonal 192-IgG following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. At the light microscopic level, punctate immunoreaction product was observed in the perinuclear region and in the plasma membrane of large, probably cholinergic neurons. Examination in the electron microscope of these neurons confirmed that nerve growth factor receptor-stained cells were basal forebrain cholinergic neurons. Within these cells, immunostaining occurred in the Golgi apparatus, in multivesicular bodies and, occasionally, in rough endoplasmic reticulum cisternae and the nuclear envelope. Moreover, patches of immunoreactivity were observed associated with the outer surface of the plasma membrane of the soma and their proximal dendrites and also with the plasma membrane of distal dendrites showing scarcity of synaptic input. Positive immunostaining was never observed in synaptic clefts, but filled the space between the plasma membranes of immunoreactive neurons and those of thin glial processes in their vicinity. The location of membrane nerve growth factor receptor in close apposition to membranes of neighbouring astrocytes rather than near synaptic complexes, suggests that glial cells may be a physiological source of nerve growth factor.

Nitric oxide in the cerebral cortex of amyloid-precursor protein (SW) Tg2576 transgenic mice.

Changes in the amyloid-peptide (Abeta), neuronal and inducible nitric oxide (NO)synthase (nNOS, iNOS), nitrotyrosine, glial fibrillary acidic protein, and lectin from Lycopersicon esculentum (tomato) were investigated in the cerebral cortex of transgenic mice (Tg2576) to amyloid precursor protein (APP), by immunohistochemistry (bright light, confocal, and electron microscopy). The expression of nitrergic proteins and synthesis of nitric oxide were analyzed by immunoblotting and NOS activity assays, respectively. The cerebral cortex of these transgenic mice showed an age-dependent progressive increase in intraneuronal aggregates of Abeta-peptide and extracellular formation of senile plaques surrounded by numerous microglial and reactive astrocytes. Basically, no changes to nNOS reactivity or expression were found in the cortical mantle of either wild or transgenic mice. This reactivity in wild mice corresponded to numerous large type I and small type II neurons. The transgenic mice showed swollen, twisted, and hypertrophic preterminal and terminal processes of type I neurons, and an increase of the type II neurons. The calcium-dependent NOS enzymatic activity was higher in wild than in the transgenic mice. The iNOS reactivity, expression and calcium-independent enzymatic activity increased in transgenic mice with respect to wild mice, and were related to cortical neurons and microglial cells. The progressive elevation of NO production resulted in a specific pattern of protein nitration in reactive astrocytes. The ultrastructural study carried out in the cortical mantle showed that the neurons contained intracellular aggregates of Abeta-peptide associated with the endoplasmic reticulum, mitochondria, and Golgi apparatus. The endothelial vascular cells also contained Abeta-peptide deposits. This transgenic model might contribute to understand the role of the nitrergic system in the biological changes related to neuropathological progression of Alzheimer's disease.

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons.

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase-antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.

Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease.

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.

Adrenomedullin in the central nervous system.

Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.

Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS-immunoreactive multipolar neurons found in layers II-VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS-positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II-VI of aged rat cortex. While no iNOS-immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine-positive cells outnumbered total NOS-positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine-positive cells. Western blots of brain extracts showed a several-fold increase in both nNOS- and iNOS-immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine-containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation.