RESUMEN
Ocular surface diseases are among the most frequent ocular pathologies, with prevalence ranging from 20% of the general population. In addition, ocular pain following corneal injury is frequently observed in clinic. The aim of the study was to characterize the peripheral and central neuroinflammatory process in the trigeminal pathways in response to cornea alteration induced by chronic topical instillations of 0.2% benzalkonium chloride (BAC) in male C57BL/6J mice. In vitro BAC induced neurotoxicity and increases neuronal (FOS, ATF3) and pro-inflammatory (IL-6) markers in primary mouse trigeminal ganglion culture. BAC-treated mice exhibited 7days after the treatment reduced aqueous tear production and increased inflammatory cell infiltration in the cornea. Hypertonic saline-evoked eye wipe behavior was enhanced in BAC-treated animals that exhibited increased FOS, ATF3 and Iba1 immunoreactivity in the trigeminal ganglion. Ocular inflammation is associated with a significant increase in IL-6 and TNF-α mRNA expression in the trigeminal ganglion. We reported a strong increase in FOS and Iba1 positive cells in particular in the sensory trigeminal complex at the ipsilateral interpolaris/caudalis (Vi/Vc) transition and Vc/upper cervical cord (Vc/C1) regions. In addition, activated microglial cells were tightly wrapped around activated FOS neurons in both regions and phosphorylated p38 mitogen-activated protein kinase was markedly enhanced specifically in microglial cells during ocular inflammation. Similar data were obtained in the facial motor nucleus. These neuroanatomical data correlated with the increase in mRNA expression of pro-inflammatory (TNF-α, IL-6, CCL2) and neuronal (FOS and ATF3) markers. Interestingly, the suppression of corneal inflammation 10days following the end of BAC treatment resulted in a marked attenuation of peripheral and central changes observed in pathological conditions. This study provides the first demonstration that corneal inflammation induces activation of neurons and microglial p38 MAPK pathway within sensory trigeminal complex. These results suggest that this altered activity in intracellular signaling caused by ocular inflammation might play a priming role in the central sensitization of ocular related brainstem circuits, which represents a significant factor in ocular pain development.
Asunto(s)
Encefalitis/etiología , Lesiones Oculares/complicaciones , Neuritis/etiología , Neuralgia del Trigémino/etiología , Animales , Antiinfecciosos Locales/toxicidad , Compuestos de Benzalconio/toxicidad , Córnea/patología , Modelos Animales de Enfermedad , Lesiones Oculares/inducido químicamente , Movimientos Oculares/efectos de los fármacos , Movimientos Oculares/fisiología , Lateralidad Funcional/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas v-fos/metabolismo , Factores de Tiempo , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 µm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.
Asunto(s)
Córnea/inervación , Enfermedades de la Córnea/diagnóstico , Imagenología Tridimensional , Microscopía/métodos , Neuronas Aferentes/ultraestructura , Sensación , Ganglio del Trigémino/ultraestructura , Animales , Enfermedades de la Córnea/fisiopatología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/fisiología , Ganglio del Trigémino/fisiologíaRESUMEN
Consumption of certain substances during pregnancy can interfere with brain development, leading to deleterious long-term neurological and cognitive impairments in offspring. To test whether modulators of adenosine receptors affect neural development, we exposed mouse dams to a subtype-selective adenosine type 2A receptor (A2AR) antagonist or to caffeine, a naturally occurring adenosine receptor antagonist, during pregnancy and lactation. We observed delayed migration and insertion of γ-aminobutyric acid (GABA) neurons into the hippocampal circuitry during the first postnatal week in offspring of dams treated with the A2AR antagonist or caffeine. This was associated with increased neuronal network excitability and increased susceptibility to seizures in response to a seizure-inducing agent. Adult offspring of mouse dams exposed to A2AR antagonists during pregnancy and lactation displayed loss of hippocampal GABA neurons and some cognitive deficits. These results demonstrate that exposure to A2AR antagonists including caffeine during pregnancy and lactation in rodents may have adverse effects on the neural development of their offspring.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/embriología , Cafeína/farmacología , Feto/efectos de los fármacos , Feto/embriología , Antagonistas de Receptores Purinérgicos P1/farmacología , Envejecimiento/patología , Animales , Animales Recién Nacidos , Encéfalo/patología , Movimiento Celular/efectos de los fármacos , Trastornos del Conocimiento/patología , Susceptibilidad a Enfermedades , Femenino , Feto/patología , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Glutamatos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Haplorrinos/embriología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Hipocampo/patología , Ratones , Red Nerviosa/efectos de los fármacos , Embarazo , Ratas , Receptores de Adenosina A2/metabolismo , Convulsiones/embriología , Convulsiones/patología , Telencéfalo/efectos de los fármacos , Telencéfalo/embriología , Telencéfalo/patologíaRESUMEN
In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.