RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1G93A rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+ . First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1G93A rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1G93A spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1G93A oligodendrocytes. In addition, the Ba2+ -sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1G93A spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Canales de Potasio de Rectificación Interna , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras , Oligodendroglía , Canales de Potasio de Rectificación Interna/genética , Ratas , Médula EspinalRESUMEN
Alzheimer's disease (AD) involves selective loss of basal forebrain cholinergic neurons, particularly in the nucleus basalis (NB). Similarly, Parkinson's disease (PD) might involve the selective loss of pedunculopontine tegmental nucleus (PPT) cholinergic neurons. Therefore, lesions of these functionally distinct cholinergic centers in rats might serve as models of AD and PD cholinergic neuropathologies. Our previous articles described dissimilar sleep/wake-state disorders in rat models of AD and PD cholinergic neuropathologies. This study further examines astroglial and microglial responses as underlying pathologies in these distinct sleep disorders. Unilateral lesions of the NB or the PPT were induced with rats under ketamine/diazepam anesthesia (50 mg/kg i.p.) by using stereotaxically guided microinfusion of the excitotoxin ibotenic acid (IBO). Twenty-one days after the lesion, loss of cholinergic neurons was quantified by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and the astroglial and microglial responses were quantified by glia fibrillary acidic protein/OX42 immunohistochemistry. This study demonstrates, for the first time, the anatomofunctionally related astroglial response following unilateral excitotoxic PPT cholinergic neuronal lesion. Whereas IBO NB and PPT lesions similarly enhanced local astroglial and microglial responses, astrogliosis in the PPT was followed by a remote astrogliosis within the ipslilateral NB. Conversely, there was no microglial response within the NB after PPT lesions. Our results reveal the rostrorostral PPT-NB astrogliosis after denervation of cholinergic neurons in the PPT. This hierarchically and anatomofunctionally guided PPT-NB astrogliosis emerged following cholinergic neuronal loss greater than 17% throughout the overall rostrocaudal PPT dimension.
Asunto(s)
Lesiones Encefálicas/patología , Neuronas Colinérgicas/patología , Neuroglía/metabolismo , Análisis de Varianza , Animales , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/complicaciones , Antígeno CD11b/metabolismo , Desnervación/métodos , Modelos Animales de Enfermedad , Agonistas de Aminoácidos Excitadores/toxicidad , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Iboténico/toxicidad , Masculino , Neuroglía/efectos de los fármacos , Núcleo Tegmental Pedunculopontino/lesiones , Núcleo Tegmental Pedunculopontino/patología , Ratas , Ratas WistarRESUMEN
Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.
Asunto(s)
Astrocitos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Neuronas , Canales de Potasio de Rectificación Interna , Animales , Masculino , Ratones , Astrocitos/metabolismo , Conducta Animal , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/fisiopatología , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Compelling evidence supports the use of a moderate static magnetic field (SMF) for therapeutic purposes. In order to provide insight into the mechanisms underlying SMF treatment, it is essential to examine the cellular responses elicited by therapeutically applied SMF, especially in the nervous system. The Na(+)/K(+) pump, by creating and maintaining the gradient of Na(+) and K(+) ions across the plasma membrane, regulates the physiological properties of neurons. In this study, we examined the expression of the Na(+)/K(+) pump in the isolated brain-subesophageal ganglion complex of the garden snail Helix pomatia, along with the immunoreactivity and current of the Na(+)/K(+) pump in isolated snail neurons after 15 min exposure to a moderate (10 mT) SMF. Western blot and immunofluorescence analysis revealed that 10 mT SMF did not significantly change the expression of the Na(+)/K(+) pump α-subunit in the snail brain and the neuronal cell body. However, our immunofluorescence data showed that SMF treatment induced a significant increase in the Na(+)/K(+) pump α-subunit expression in the neuronal plasma membrane area. This change in Na(+)/K(+) pump expression was reflected in pump activity as demonstrated by the pump current measurements. Whole-cell patch-clamp recordings from isolated snail neurons revealed that Na(+)/K(+) pump current density was significantly increased after the 10 mT SMF treatment. The SMF-induced increase was different in the two groups of control snail neurons, as defined by the pump current level. The results obtained could represent a physiologically important response of neurons to 10 mT SMF comparable in strength to therapeutic applications.
Asunto(s)
Caracoles Helix/enzimología , Potenciales de la Membrana/fisiología , Sistema Nervioso/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Técnica del Anticuerpo Fluorescente , Caracoles Helix/efectos de los fármacos , Caracoles Helix/fisiología , Campos Magnéticos , Potenciales de la Membrana/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Neuronas/citología , Neuronas/fisiología , Ouabaína/farmacología , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
In multiple sclerosis (MS), glial cells astrocytes interact with the autoreactive immune cells that attack the central nervous system (CNS), which causes and sustains neuroinflammation. However, little is known about the direct interaction between these cells when they are in close proximity in the inflamed CNS. By using an experimental autoimmune encephalomyelitis (EAE) model of MS, we previously found that in the proximity of autoreactive CNS-infiltrated immune cells (CNS-IICs), astrocytes respond with a rapid calcium increase that is mediated by the autocrine P2X7 receptor (P2X7R) activation. We now reveal that the mechanisms regulating this direct interaction of astrocytes and CNS-IICs involve the coupling between P2X7R, connexin-43, and ß3-integrin. We found that P2X7R and astroglial connexin-43 interact and concentrate in the immediate proximity of the CNS-IICs in EAE. P2X7R also interacts with ß3-integrin, and the block of astroglial αvß3-integrin reduces the P2X7R-dependent calcium response of astrocytes upon encountering CNS-IICs. This interaction was dependent on astroglial mitochondrial activity, which regulated the ATP-driven P2X7R activation and facilitated the termination of the astrocytic calcium response evoked by CNS-IICs. By further defining the interactions between the CNS and the immune system, our findings provide a novel perspective toward expanding integrin-targeting therapeutic approaches for MS treatment by controlling the cell-cell interactions between astrocytes and CNS-IICs.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Astrocitos , Receptores Purinérgicos P2X7 , Integrina beta3 , Calcio , Comunicación CelularRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood-brain barrier (BBB), are enriched in two proteins, aquaporin-4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1(G93A) rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole-cell patch-clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir-specific Cs(+)- and Ba(2+)-sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Acuaporina 4/biosíntesis , Astrocitos/metabolismo , Tronco Encefálico/metabolismo , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Acuaporina 4/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Potasio de Rectificación Interna/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Superóxido Dismutasa-1RESUMEN
Liquid-liquid phase separation (LLPS) is emerging as a major principle for the mesoscale organization of proteins, RNAs, and membrane-bound organelles into biomolecular condensates. These condensates allow for rapid cellular responses to changes in metabolic activities and signaling. Nowhere is this regulation more important than in neurons and glia, where cellular physiology occurs simultaneously on a range of time- and length-scales. In a number of neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), misregulation of biomolecular condensates leads to the formation of insoluble aggregates-a pathological hallmark of both sporadic and familial ALS. Here, we summarize how the emerging knowledge about the LLPS of ALS-related proteins corroborates with their aggregation. Understanding the mechanisms that lead to protein aggregation in ALS and how cells respond to these aggregates promises to open new directions for drug development.
RESUMEN
We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.
Asunto(s)
Microscopía , Constricción , Técnicas de Placa-ClampRESUMEN
Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.
Asunto(s)
Astrocitos/metabolismo , Glutamina/metabolismo , Hipocampo/fisiología , Reconocimiento en Psicología , Transmisión Sináptica , Animales , Cognición , Colorantes Fluorescentes/química , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glutamina/química , Hipocampo/citología , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Sondas Moleculares , Neuronas/metabolismo , Rodaminas/química , Técnicas EstereotáxicasRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder affecting upper and lower motoneurons. The transgenic ALS rat model (hSOD-1(G93A)) was used for magnetic resonance imaging (MRI) study using a low field wide bore magnet. T2-weighted hyperintensities were observed in the brainstem, rubrospinal tract and vagus motor nuclei with prominent lateral ventricle and cerebral aqueduct enlargements. These changes could be observed already in presymptomatic animals. T2*-weighted MRI with magnetically labeled antibodies (against CD4) revealed lymphocyte infiltration in the brainstem-midbrain region corresponding to the areas of dilated lateral ventricles. Confocal imaging revealed reactive astroglia in these areas. Thus, with the use of wide bore MRI new sites of neurodegeneration and inflammation were revealed in the hSOD-1(G93A) rat model.
Asunto(s)
Esclerosis Amiotrófica Lateral/complicaciones , Encefalopatías/diagnóstico , Encefalopatías/patología , Supervivencia Tisular , Esclerosis Amiotrófica Lateral/patología , Animales , Encefalopatías/complicaciones , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Imagen por Resonancia Magnética , Microscopía Confocal , Ratas , Ratas Sprague-DawleyRESUMEN
Over 150 mutations in the SOD1 gene that encodes Cu/Zn superoxide dismutase (SOD1) cause 20-25% of familial ALS, albeit without a known gain-of-function mechanism. ALS is also non-cell-autonomous, the interactions between motor neurons and their glial neighbours being implicated in disease progression. The aim here was to investigate the biophysical effects of the exogenous human mutant SOD1-G93A on rat astrocytes in culture. Primary cortical astrocyte cultures were treated with recombinant human apo- mSOD1-G93A vs. wild-type control (wtSOD1) and recorded by patch-clamp and calcium imaging. Results showed that exogenous mSOD1 as well as wtSOD1 induced a decrease of membrane resistance, the effect being persistent (up to 13 min) only for the mutant form. Similarly, whole-cell inward currents in astrocytes were augmented by both wt and mSOD1, but the effect was twice larger and only progressed continuously for the latter. Both forms of SOD1 also induced a rise in intracellular Ca(2+) activity, the effect being dependent on external Ca(2+) and again only persisted with mSOD1, becoming significantly different from wtSOD1 only at longer times (14 min). In conclusion, this study points to membrane permeability and Ca(2+) signalling as processes affected by SOD1-G93A that presents the humoral factor triggering the role of astrocytes in ALS pathophysiology.
Asunto(s)
Astrocitos/citología , Calcio/metabolismo , Citosol/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Superóxido Dismutasa/farmacología , Animales , Animales Recién Nacidos , Biofisica , Células Cultivadas , Corteza Cerebral/citología , Citosol/metabolismo , Estimulación Eléctrica , Humanos , Mutación/genética , Ratas , Ratas Wistar , Superóxido Dismutasa/genética , Factores de TiempoRESUMEN
Recently neuroinflammation has gained a particular focus as a key mechanism of ALS. Several studies in vivo as well as in vitro have nominated immunoglobulin G (IgG) isolated from ALS patients as an active contributor to disease onset and progression. We have shown that ALS IgG affects astroglial Ca(2+) excitability and induces downstream activation of phosphatidylinositol 3-kinase. These studies were hampered by a lack of knowledge of the pathway of entry of immune factors in the CNS. Our MRI data revealed the blood-brain barrier BBB leakage and T cell infiltration into brain parenchyma in ALS G93A rats. Since astrocyte ensheathes blood vessel wall contributing to BBB stability and plays an important role in ALS pathogenesis, we have studied astrocytic membrane proteins water channel aquaporin-4 and the inwardly rectifying potassium channel. In this review, we will summarize data related to BBB disruption with particular emphasis on impaired function of astrocytes in ALS. We will discuss implication of membrane proteins expressed on astrocytic endfeet, aquaporin-4, and inwardly rectifying potassium channel in the pathology of ALS. In addition to ALS-specific IgGs, these membrane proteins are proposed as novel biomarkers of the disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Biomarcadores/sangre , Barrera Hematoencefálica/metabolismo , Inmunidad Humoral , Inflamación/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Acuaporina 4/sangre , Astrocitos/metabolismo , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Inflamación/sangre , Inflamación/patología , RatasRESUMEN
Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain rhythmic activity and survival of the animal.
Asunto(s)
Caracoles Helix/fisiología , Hibernación/fisiología , Neuroglía/fisiología , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Conductividad Eléctrica , Fenómenos Electrofisiológicos/fisiología , Neuroglía/ultraestructura , Neuronas/fisiología , Serotonina/farmacologíaRESUMEN
MRI was employed to follow the neurodegenerative foci and the localization of inflammatory cells by magnetically labeled CD4+ or CD8+ lymphocytes in the ischemia/reperfusion long-lived rats (9 and 13 months after 10 min of cardiac arrest). MRI of ischemic rats showed: (1) blood-brain barrier (BBB) leakage in the area of the dorsal hippocampus and brainstem-hindbrain level in basal cerebellum, (2) unlike anti-CD8 magnetic antibodies anti-CD4 ultra small paramagnetic iron oxide particles (USPIO) antibodies revealed hypointense areas in the brainstem-interbrain region and caudoputamen not found in animals that were not injected with USPIO antibodies, and (3) dilation in the retrosplenial area. Immunocytochemistry revealed microglial activation in the hippocampus and striatum, with indications of activation in thalamic lateral dorsal nuclei and the subventricular zone. In the CA1 and CA3 regions, it was noted that OX42- and ED1-positive granules appear in neuronal somata. Immunostaining of lymphocytes with TCR confirmed the T-cell presence in ischemic brain parenchyma of the hippocampus and striatum. The above observations thus point to a persistent dysfunction of BBB that in long-term may still lead to infiltration of T cells that are predominantly of helper (CD4+) type. Such inflammatory processes are backed by microglial activity even up to 1 year after ischemia/reperfusion. Moreover, in these animals an augmented expression of neurogenesis markers and neuroblast migration was also revealed in the subventricular zone. Thus, a balance of degenerative processes and inflammatory surveillance with neurogenesis could determine the long-term outcome of global ischemia survival or the previously proposed formation of amyloid plaques and Alzheimer's-type dementia.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Antígeno CD11b/metabolismo , Displasia Ectodermal Anhidrótica Tipo 1/metabolismo , Encefalitis/fisiopatología , Neurogénesis/fisiología , Daño por Reperfusión/fisiopatología , Animales , Biomarcadores/metabolismo , Barrera Hematoencefálica/fisiopatología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Encefalitis/metabolismo , Encefalitis/patología , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Imagen por Resonancia Magnética , Modelos Animales , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Rombencéfalo/metabolismo , Rombencéfalo/patología , Tasa de Supervivencia , Factores de TiempoRESUMEN
Onset of motoneuron death characterizing amyotrophic lateral sclerosis (ALS) is closely linked to modified astrocytic and glial environments. Here, we show that in the spinal cord from transgenic rat overexpressing mutated human SOD1, aquaporin-4 mRNA and protein are specifically overexpressed in the gray matter at end stage of disease. Immunohistochemistry and double immunofluorescence allowed to detect, in the spinal cord gray matter of the ALS rat, increased aquaporin-4 surrounding both vessel and motoneuron perikarya. The use of pre-embedding immunohistochemistry at electron microscopic level confirmed such localization associated with swollen astrocytic processes surrounding the vessels. The AQP4 immunohistochemical labeling surrounding several motoneuron perikarya was only seen in ALS rats. Identification of this AQP4-positive cellular type remains unclear.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Acuaporina 4/genética , Acuaporina 4/metabolismo , Médula Espinal/metabolismo , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Astrocitos/metabolismo , Astrocitos/patología , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutagénesis Sitio-Dirigida , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Médula Espinal/irrigación sanguínea , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Magnetic resonance imaging (MRI) is the only noninvasive technique that provides structural information on both cell loss and metabolic changes. After reviewing all the results obtained in clinical studies, reliable biomarkers in neurological diseases are still lacking. Diffusional MRI, MR spectroscopy, and the assessment of regional atrophy are promising approaches, but they cannot be simultaneously used on a single patient. Thus, for further research progress, reliable animal models are needed. To this aim, we have used the clinical MRI to assess neurodegenerative processes in the hSOD-1(G93A) ALS rat model and in the trimethyltin (TMT)-treated model of Alzheimer's-like disease. T2-weighted (T2W) hyperintensive neurodegenerative foci were found in the brainstem of the ALS rat with apparent lateral ventricle dilation (T1W-hypointensity vs. T2W-hyperintensity). Degenerative processes in these areas were also confirmed by confocal images of GFAP-positive astrogliosis. MRI after i.v.i. of magnetic anti-CD4 antibodies indicated an accumulation of inflammatory cells near dilated ventricles. TMT-treated rats also revealed the dilation of lateral ventricles. Expected deterioration in the hippocampus was not observed by clinical MRI, but immunocytochemistry could reveal significant redistribution of macro- and microglia in this structure. In both models, Gd-DTPA contrast revealed a compromised blood brain barrier that may serve as the passage for inflammatory immune cells in the vicinity of dilated lateral ventricles. Moreover, in both models the midbrain region of the dorsal hippocampus was the target of BBB compromise, thus revealing a potentially vulnerable point that can be the primary target of neurodegeneration in the central nervous system.