RESUMEN
Heterozygous mutations in the GBA1 gene - encoding lysosomal glucocerebrosidase (GCase) - are the most common genetic risk factors for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model used constitutive overexpression of wild-type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. Although increased aSyn pathology and grip strength reductions were observed in this model, the nigrostriatal system remained largely intact. The second model involved injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V knock-in mouse model. The GBA1 D409V mutation did not exacerbate the pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Ratones , Mutación/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.
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Glucosilceramidasa/metabolismo , Glicoesfingolípidos/metabolismo , Animales , Encéfalo/metabolismo , Femenino , Técnicas de Sustitución del Gen , Glucosilceramidasa/genética , Immunoblotting , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Parkinsonianos/enzimología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Mutación Puntual/genéticaRESUMEN
Visual system toxicity may manifest anywhere in the visual system, from the eye proper to the visual brain. Therefore, effective screening for visual system toxicity must evaluate not only ocular structures (ie, eye and optic nerve) but also multiple key brain regions involved in vision (eg, optic tract, subcortical relay nuclei, and primary and secondary visual cortices). Despite a generally comparable pattern across species, the neuroanatomic organization and function of the visual brain in rodents and rabbits exhibit appreciable differences relative to nonrodents. Currently recognized sampling practices for general toxicity studies in animals, which are based on easily discerned external neuroanatomic landmarks and guided by extant stereotaxic brain atlases, typically will permit histopathologic evaluation of many brain centers involved in visual sensation (eg, optic chiasm, optic tract, dorsal lateral geniculate nucleus, primary and secondary visual cortices) and often some subcortical brain nuclei involved in light-modulated nonvisual activities needed for visual attention and orientation (eg, rostral colliculus in quadrupeds, termed the superior colliculus in bipeds; several cranial nerve nuclei). Pathologic findings induced by toxicants in the visual brain centers are similar to those that are produced in other brain regions.
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Cuerpos Geniculados , Neuroanatomía , Animales , Encéfalo , Mamíferos , Conejos , Retina , Colículos SuperioresRESUMEN
BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/farmacocinética , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/inmunología , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/líquido cefalorraquídeo , Ácido Aspártico Endopeptidasas/inmunología , Encéfalo/metabolismo , Femenino , Infusiones Intraventriculares , Macaca fascicularisRESUMEN
Multiple sclerosis (MS) causes demyelinating lesions in the white matter and increased iron deposition in the subcortical gray matter. Myelin protons have an extremely short T2* (<1ms) and are not directly detected with conventional clinical magnetic resonance (MR) imaging sequences. Iron deposition also reduces T2*, leading to reduced signal on clinical sequences. In this study we tested the hypothesis that the inversion recovery ultrashort echo time (IR-UTE) pulse sequence can directly and simultaneously image myelin and iron deposition using a clinical 3T scanner. The technique was first validated on a synthetic myelin phantom (myelin powder in D2O) and a Feridex iron phantom. This was followed by studies of cadaveric MS specimens, healthy volunteers and MS patients. UTE imaging of the synthetic myelin phantom showed an excellent bi-component signal decay with two populations of protons, one with a T2* of 1.2ms (residual water protons) and the other with a T2* of 290µs (myelin protons). IR-UTE imaging shows sensitivity to a wide range of iron concentrations from 0.5 to ~30mM. The IR-UTE signal from white matter of the brain of healthy volunteers shows a rapid signal decay with a short T2* of ~300µs, consistent with the T2* values of myelin protons in the synthetic myelin phantom. IR-UTE imaging in MS brain specimens and patients showed multiple white matter lesions as well as areas of high signal in subcortical gray matter. This in specimens corresponded in position to Perl's diaminobenzide staining results, consistent with increased iron deposition. IR-UTE imaging simultaneously detects lesions with myelin loss in the white matter and iron deposition in the gray matter.
Asunto(s)
Encéfalo/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador , Hierro/análisis , Imagen por Resonancia Magnética , Esclerosis Múltiple/diagnóstico por imagen , Vaina de Mielina/patología , Adulto , Anciano , Cadáver , Estudios de Factibilidad , Femenino , Sustancia Gris/diagnóstico por imagen , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Fantasmas de Imagen , Agua , Sustancia Blanca/diagnóstico por imagenRESUMEN
Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP â PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.
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Archaea/metabolismo , Bacterias/metabolismo , Metabolismo Energético/fisiología , Hongos/metabolismo , Péptido Sintasas/química , Fosforribosil Pirofosfato/química , Secuencia de Aminoácidos , Archaea/enzimología , Bacterias/enzimología , Hongos/enzimología , Humanos , Fosforribosil Pirofosfato/biosíntesis , Fosfotransferasas (Aceptor del Grupo Fosfato) , Estructura Secundaria de Proteína , Ribosamonofosfatos/químicaRESUMEN
We used controlled cortical impact in mice to model human traumatic brain injury (TBI). Local injury was accompanied by distal diaschisis lesions that developed within brain regions anatomically connected to the injured cortex. At 7 days after injury, histochemistry documented broadly distributed lesions, particularly in the contralateral cortex and ipsilateral thalamus and striatum. Reactive astrocytosis and microgliosis were noted in multiple neural pathways that also showed silver-stained cell processes and bodies. Wisteria floribunda agglutinin (WFA) staining, a marker of perineuronal nets, was substantially diminished in the ipsilateral, but less so in the contralateral cortex. Contralateral cortical silver positive diaschisis lesions showed loss of both phosphorylated and unphosphorylated neurofilament staining, but overall preservation of microtubule-associated protein (MAP)-2 staining. Thalamic lesions showed substantial loss of MAP-2 and unphosphorylated neurofilaments in addition to moderate loss of phosphorylated neurofilament. One animal demonstrated contralateral cerebellar degeneration at 7 days post-injury. After 21 days, the gliosis had quelled, however persistent silver staining was noted. Using a novel serial section technique, we were able to perform electron microscopy on regions fully characterized at the light microscopy level. Cell bodies and processes that were silver positive at the light microscopy level showed hydropic disintegration consisting of: loss of nuclear heterochromatin; dilated somal and neuritic processes with a paucity of filaments, tubules, and mitochondria; and increased numbers of electron-dense membranous structures. Importantly the cell membrane itself was still intact 3 weeks after injury. Although the full biochemical nature of these lesions remains to be deciphered, the morphological preservation of damaged neurons and processes raises the question of whether this is a reversible process.
Asunto(s)
Lesiones Traumáticas del Encéfalo/patología , Encéfalo/patología , Encéfalo/ultraestructura , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
The Advanced Topographic Laser Altimeter System (ATLAS) Instrument has been in integration and testing over the past 18 months in preparation for the Ice, Cloud and Land Elevation Satellite - 2 (ICESat-2) Mission, scheduled to launch in 2017. ICESat-2 is the follow on to ICESat which launched in 2003 and operated until 2009. ATLAS will measure the elevation of ice sheets, glaciers and sea ice or the "cryosphere" (as well as terrain) to provide data for assessing the earth's global climate changes. Where ICESat's instrument, the Geo-Science Laser Altimeter (GLAS) used a single beam measured with a 70 m spot on the ground and a distance between spots of 170 m, ATLAS will measure a spot size of 10 m with a spacing of 70 cm using six beams to measure terrain height changes as small as 4 mm.[1] The ATLAS pulsed transmission system consists of two lasers operating at 532 nm with transmitter optics for beam steering, a diffractive optical element that splits the signal into 6 separate beams, receivers for start pulse detection and a wavelength tracking system. The optical receiver telescope system consists of optics that focus all six beams into optical fibers that feed a filter system that transmits the signal via fiber assemblies to the detectors. Also included on the instrument is a system that calibrates the alignment of the transmitted pulses to the receiver optics for precise signal capture. The larger electro optical subsystems for transmission, calibration, and signal receive, stay aligned and transmitting sufficiently due to the optical fiber system that links them together. The robust design of the fiber optic system, consisting of a variety of multi fiber arrays and simplex assemblies with multiple fiber core sizes and types, will enable the system to maintain consistent critical alignments for the entire life of the mission. Some of the development approaches used to meet the challenging optical system requirements for ATLAS are discussed here.
RESUMEN
Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.
Asunto(s)
Proteínas Asociadas a Microtúbulos/deficiencia , Trastornos Parkinsonianos/fisiopatología , Fenotipo , Proteínas Quinasas/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Envejecimiento , Animales , Animales Modificados Genéticamente , Encéfalo/patología , Encéfalo/fisiopatología , Dopamina/metabolismo , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/fisiología , Técnicas de Inactivación de Genes , Genes Recesivos , Masculino , Proteínas Asociadas a Microtúbulos/genética , Actividad Motora/fisiología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Proteína Desglicasa DJ-1 , Proteínas Quinasas/genética , Ratas Long-Evans , Serotonina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Neurological dysfunction is common in humans and animals with lysosomal storage diseases. ß-Mannosidosis, an autosomal recessive inherited disorder of glycoprotein catabolism caused by deficiency of the lysosomal enzyme ß-mannosidase, is characterized by intracellular accumulation of small oligosaccharides in selected cell types. In ruminants, clinical manifestation is severe, and neuropathology includes extensive intracellular vacuolation and dysmyelination. In human cases of ß-mannosidosis, the clinical symptoms, including intellectual disability, are variable and can be relatively mild. A ß-mannosidosis knockout mouse was previously characterized and showed normal growth, appearance, and lifespan. Neuropathology between 1 and 9 months of age included selective, variable neuronal vacuolation with no hypomyelination. This study characterized distribution of brain pathology in older mutant mice, investigating the effects of two strain backgrounds. Morphological analysis indicated a severe consistent pattern of neuronal vacuolation and disintegrative degeneration in all five 129X1/SvJ mice. However, the mice with a mixed genetic background showed substantial variability in the severity of pathology. In the severely affected animals, neuronal vacuolation was prominent in specific layers of piriform area, retrosplenial area, anterior cingulate area, selected regions of isocortex, and in hippocampus CA3. Silver degeneration reaction product was prominent in regions including specific cortical layers and cerebellar molecular layer. The very consistent pattern of neuropathology suggests metabolic differences among neuronal populations that are not yet understood and will serve as a basis for future comparison with human neuropathological analysis. The variation in severity of pathology in different mouse strains implicates genetic modifiers in the variable phenotypic expression in humans.
RESUMEN
We evaluated the acute (up to 24 h) pathophysiological response to primary blast using a rat model and helium driven shock tube. The shock tube generates animal loadings with controlled pure primary blast parameters over a wide range and field-relevant conditions. We studied the biomechanical loading with a set of pressure gauges mounted on the surface of the nose, in the cranial space, and in the thoracic cavity of cadaver rats. Anesthetized rats were exposed to a single blast at precisely controlled five peak overpressures over a wide range (130, 190, 230, 250, and 290 kPa). We observed 0% mortality rates in 130 and 230 kPa groups, and 30%, 24%, and 100% mortality rates in 190, 250, and 290 kPa groups, respectively. The body weight loss was statistically significant in 190 and 250 kPa groups 24 h after exposure. The data analysis showed the magnitude of peak-to-peak amplitude of intracranial pressure (ICP) fluctuations correlates well with mortality rates. The ICP oscillations recorded for 190, 250, and 290 kPa are characterized by higher frequency (10-20 kHz) than in other two groups (7-8 kHz). We noted acute bradycardia and lung hemorrhage in all groups of rats subjected to the blast. We established the onset of both corresponds to 110 kPa peak overpressure. The immunostaining against immunoglobulin G (IgG) of brain sections of rats sacrificed 24-h post-exposure indicated the diffuse blood-brain barrier breakdown in the brain parenchyma. At high blast intensities (peak overpressure of 190 kPa or more), the IgG uptake by neurons was evident, but there was no evidence of neurodegeneration after 24 h post-exposure, as indicated by cupric silver staining. We observed that the acute response as well as mortality is a non-linear function over the peak overpressure and impulse ranges explored in this work.
Asunto(s)
Traumatismos por Explosión/fisiopatología , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/fisiopatología , Presión del Aire , Animales , Fenómenos Biomecánicos , Traumatismos por Explosión/patología , Barrera Hematoencefálica/fisiopatología , Lesiones Encefálicas/etiología , Presión Intracraneal , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Electrophysiological and behavioral studies have demonstrated that muscimol administered through the cranial meninges can prevent focal neocortical seizures. It was proposed that transmeningeal muscimol delivery can be used for the treatment of intractable focal neocortical epilepsy. However, it has not been proved that muscimol administered via the transmeningeal route can penetrate into the neocortex. The purpose of the present study was to solve this problem by using combined autoradiography-histology methods. Four rats were implanted with epidural cups over the parietal cortices. A 50 µL mixture of [³H] muscimol and unlabeled muscimol with a final concentration of 1.0mM was delivered through each cup on the dura mater. After a 1-hour exposure, the muscimol solution was removed and replaced with formalin to trap the transmeningeally diffused molecules. Then the whole brain was fixed transcardially, sectioned, with the sections subjected to autoradiography and thionine counterstaining. Results showed that (1) [³H] muscimol diffused through the meninges into the cortical tissue underlying the epidural cup in all rats. (2) [³H] muscimol-related autoradiography grains were distributed in all six neocortical layers. (3) [³H] muscimol-related autoradiography grains were localized to the cortical area underneath the epidural delivery site and were absent in the cerebral cortical white matter and other brain structures. This study provided evidence that muscimol can be delivered via the transmeningeal route into the neocortical tissue in a spatially controlled manner. The finding further supports the rationale of using transmeningeal muscimol for the treatment of intractable focal neocortical epilepsy.
Asunto(s)
Meninges/química , Meninges/metabolismo , Muscimol/metabolismo , Neocórtex/química , Neocórtex/metabolismo , Animales , Autorradiografía , Difusión , Masculino , Meninges/diagnóstico por imagen , Neocórtex/diagnóstico por imagen , Radiografía , Ratas , Ratas Long-EvansRESUMEN
Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration â¼ 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rat's head. The insult produced â¼ 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.
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Axones/patología , Traumatismos por Explosión/patología , Lesión Axonal Difusa/patología , Animales , Axones/metabolismo , Traumatismos por Explosión/fisiopatología , Encéfalo/patología , Encéfalo/fisiopatología , Lesión Axonal Difusa/etiología , Lesión Axonal Difusa/fisiopatología , Modelos Animales de Enfermedad , Masculino , Ropa de Protección , Ratas , Ratas Sprague-DawleyRESUMEN
Adequate tissue sampling is known to reduce the likelihood that the toxicity of novel biomolecules, chemicals, and drugs might go undetected. Each organ, and often specific structurally and functionally distinct regions within it, must be assessed to detect potential site-specific toxicity. Adequate sampling of the brain requires particular consideration because of the many major substructures and more than 600 subpopulations of generally irreplaceable cells with unique functions and vulnerabilities. All known neurotoxicants affect specific subpopulations (usually neurons) rather than damaging a certain percentage of cells throughout the brain; thus, all populations should be independently assessed for lesions. Historically, the affected neural cell subpopulation has not been predictable, but it is now clear that sampling selected populations (e.g., cerebral cortex, hippocampus, cerebellar folia) cannot forecast the health of other populations. This article reviews the neuroanatomical domains affected by several model neurotoxicants to illustrate the need for more comprehensive neurohistological evaluation during nonclinical development of novel compounds. The article also describes an easily executed, cost-effective method that uses a set number of evenly spaced coronal (cross) sections to accomplish this comprehensive brain assessment during nonclinical safety studies performed in rodents, dogs, and nonhuman primates.
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Encéfalo/anatomía & histología , Encéfalo/fisiología , Enfermedades del Sistema Nervioso/patología , Neuroanatomía/métodos , Animales , Muerte Celular , Estudios de Evaluación como Asunto , Técnicas Histológicas/métodos , Humanos , Modelos Animales , Enfermedades del Sistema Nervioso/inducido químicamente , Síndromes de Neurotoxicidad/patología , Proyectos de InvestigaciónRESUMEN
Many new therapeutics for Alzheimer's disease delay the accumulation of amyloid-ß (Aß) in transgenic mice, but evidence for clearance of preexisting plaques is often lacking. Here, we demonstrate that anti-Aß immunotherapy combined with suppression of Aß synthesis allows significant removal of antecedent deposits. We treated amyloid-bearing tet-off APP (amyloid precursor protein) mice with doxycycline to suppress transgenic Aß production before initiating a 12 week course of passive immunization. Animals remained on doxycycline for 3 months afterward to assess whether improvements attained during combined treatment could be maintained by monotherapy. This strategy reduced amyloid load by 52% and Aß42 content by 28% relative to pretreatment levels, with preferential clearance of small deposits and diffuse Aß surrounding fibrillar cores. We demonstrate that peripherally administered anti-Aß antibody crossed the blood-brain barrier, bound to plaques, and was still be found associated with a subset of amyloid deposits many months after the final injection. Antibody accessed the brain independent of plasma Aß levels, where it enhanced microglial internalization of aggregated Aß. Our data support a mechanism by which passive immunization acts centrally to stimulate microglial phagocytosis of aggregated Aß, but is opposed by the continued aggregation of newly secreted Aß. By arresting the production of Aß, combination therapy allows microglial clearance to work from a static amyloid burden toward a significant reduction in plaque load. Our findings suggest that combining two therapeutic approaches currently in clinical trials may improve neuropathological outcome over either alone.
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Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Hipocampo/patología , Inmunización Pasiva/métodos , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Análisis de Varianza , Animales , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hipocampo/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Resultado del TratamientoRESUMEN
This session at the 2010 joint symposium of the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP) explored modern neuropathology methods for assessing the neurotoxicologic potential of xenobiotics. Conventional techniques to optimally prepare and evaluate the central and peripheral neural tissues while minimizing artifact were reviewed, and optimal schemes were set forth for evaluation of the nervous system during both routine (i.e., general toxicity) studies and enhanced (i.e., specialized neurotoxicity) studies. Stereology was introduced as the most appropriate means of examining the possible impact of toxicants on neural cell numbers. A focused discussion on brain sampling took place among a panel of expert neuroscientists (anatomists and pathologists) and the audience regarding the proper balance between sufficient sampling and cost- and time-effectiveness of the analysis. No consensus was reached on section orientation (coronal sections of both sides vs. a parasagittal longitudinal section with several unilateral hemisections from the contralateral side), but most panelists favored sampling at least 8 sections (or approximately double to triple the current complement) in routine toxicity studies.
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Enfermedades del Sistema Nervioso/inducido químicamente , Enfermedades del Sistema Nervioso/patología , Sistema Nervioso/anatomía & histología , Pruebas de Toxicidad/métodos , Xenobióticos/toxicidad , Animales , Congresos como Asunto , Estudios de Evaluación como Asunto , Humanos , Síndromes de Neurotoxicidad/patología , Sociedades CientíficasRESUMEN
Although prion diseases are most commonly modeled using the laboratory mouse, the diversity of prion strains, behavioral testing and neuropathological assessments hamper our collective understanding of mouse models of prion disease. Here we compared several commonly used murine strains of prions in C57BL/6J female mice in a detailed home cage behavior detection system and a systematic study of pathological markers and neurotransmitter systems. We observed that mice inoculated with RML or 139A prions develop a severe hyperactivity phenotype in the home cage. A detailed assessment of pathology markers, such as microglial marker IBA1, astroglial marker GFAP and degeneration staining indicate early striatal pathology in mice inoculated with RML or 139A but not in those inoculated with 22L prions. An assessment of neuromodulatory systems including serotonin, dopamine, noradrenalin and acetylcholine showed surprisingly little decline in neuronal cell bodies or their innervations of regions controlling locomotor behavior, except for a small decrease in dopaminergic innervations of the dorsal striatum. These results implicate the dorsal striatum in mediating the major behavioral phenotype of 139A and RML prions. Further, they suggest that measurements of activity may be a sensitive manner in which to diagnose murine prion disease. With respect to neuropathology, our results indicate that pathological stains as opposed to neurotransmitter markers are much more informative and sensitive as markers of prion disease in mouse models.
Asunto(s)
Hipercinesia/complicaciones , Hipercinesia/patología , Neostriado/patología , Enfermedades por Prión/complicaciones , Enfermedades por Prión/patología , Priones/patogenicidad , Animales , Conducta Animal , Colina/metabolismo , Gliosis/complicaciones , Gliosis/patología , Homeostasis , Hipercinesia/fisiopatología , Locus Coeruleus/metabolismo , Locus Coeruleus/patología , Locus Coeruleus/fisiopatología , Longevidad , Mesencéfalo/patología , Mesencéfalo/fisiopatología , Ratones , Neostriado/fisiopatología , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/patología , Inhibición Neural , Neuronas/metabolismo , Neuronas/patología , Parvalbúminas/metabolismo , Enfermedades por Prión/fisiopatología , Serotonina/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Carbonyl sulfide (COS) is an odorless gas that produces highly reproducible lesions in the central nervous system. In the present study, the time course for the development of the neurotoxicological lesions was defined and the gene expression changes occurring in the posterior colliculus upon exposure to COS were characterized. Fischer 344 rats were exposed to 0 or 500 ppm COS for one, two, three, four, five, eight, or ten days, six hours per day. On days 1 and 2, no morphological changes were detected; on day 3, 10/10 (100%) rats had necrosis in the posterior colliculi; and on day 4 and later, necrosis was observed in numerous areas of the brain. Important gene expression changes occurring in the posterior colliculi after one or two days of COS exposure that were predictive of the subsequent morphological findings included up-regulation of genes associated with DNA damage and G1/S checkpoint regulation (KLF4, BTG2, GADD45g), apoptosis (TGM2, GADD45g, RIPK3), and vascular mediators (ADAMTS, CTGF, CYR61, VEGFC). Proinflammatory mediators (CCL2, CEBPD) were up-regulated prior to increases in expression of the astrocytic marker GFAP and macrophage marker CSF2rb1. These gene expression findings were predictive of later CNS lesions caused by COS exposure and serve as a model for future investigations into the mechanisms of disease in the central nervous system.
Asunto(s)
Encefalopatías/inducido químicamente , Encéfalo/metabolismo , Daño del ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Degeneración Nerviosa/metabolismo , Óxidos de Azufre/toxicidad , Administración por Inhalación , Animales , Apoptosis/efectos de los fármacos , Encéfalo/patología , Encefalopatías/genética , Encefalopatías/metabolismo , Encefalopatías/patología , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Inflamación/metabolismo , Factor 4 Similar a Kruppel , Masculino , Necrosis , Degeneración Nerviosa/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Óxidos de Azufre/administración & dosificaciónRESUMEN
SUMMARY: DNA-binding repressor proteins that govern transcription initiation in response to end products generally regulate bacterial biosynthetic genes, but this is rarely true for the pyrimidine biosynthetic (pyr) genes. Instead, bacterial pyr gene regulation generally involves mechanisms that rely only on regulatory sequences embedded in the leader region of the operon, which cause premature transcription termination or translation inhibition in response to nucleotide signals. Studies with Escherichia coli and Bacillus subtilis pyr genes reveal a variety of regulatory mechanisms. Transcription attenuation via UTP-sensitive coupled transcription and translation regulates expression of the pyrBI and pyrE operons in enteric bacteria, whereas nucleotide effects on binding of the PyrR protein to pyr mRNA attenuation sites control pyr operon expression in most gram-positive bacteria. Nucleotide-sensitive reiterative transcription underlies regulation of other pyr genes. With the E. coli pyrBI, carAB, codBA, and upp-uraA operons, UTP-sensitive reiterative transcription within the initially transcribed region (ITR) leads to nonproductive transcription initiation. CTP-sensitive reiterative transcription in the pyrG ITRs of gram-positive bacteria, which involves the addition of G residues, results in the formation of an antiterminator RNA hairpin and suppression of transcription attenuation. Some mechanisms involve regulation of translation rather than transcription. Expression of the pyrC and pyrD operons of enteric bacteria is controlled by nucleotide-sensitive transcription start switching that produces transcripts with different potentials for translation. In Mycobacterium smegmatis and other bacteria, PyrR modulates translation of pyr genes by binding to their ribosome binding site. Evidence supporting these conclusions, generalizations for other bacteria, and prospects for future research are presented.