RESUMEN
Repetitive transorbital alternating current stimulation (rtACS) improves vision in patients with chronic visual impairments and an acute treatment increased survival of retinal neurons after optic nerve crush (ONC) in rodent models of visual system injury. However, despite this protection no functional recovery could be detected in rats, which was interpreted as evidence of "silent survivor" cells. We now analysed the mechanisms underlying this "silent survival" effect. Using in vivo microscopy of the retina we investigated the survival and morphology of fluorescent neurons before and after ONC in animals receiving rtACS or sham treatment. One week after the crush, more neurons survived in the rtACS-treated group compared to sham-treated controls. In vivo imaging further revealed that in the initial post-ONC period, rtACS induced dendritic pruning in surviving neurons. In contrast, dendrites in untreated retinae degenerated slowly after the axonal trauma and neurons died. The complete loss of visual evoked potentials supports the hypothesis that cell signalling is abolished in the surviving neurons. Despite this evidence of "silencing", intracellular free calcium imaging showed that the cells were still viable. We propose that early after trauma, complete dendritic stripping following rtACS protects neurons from excitotoxic cell death by silencing them.
Asunto(s)
Supervivencia Celular , Dendritas/metabolismo , Estimulación Eléctrica , Neuronas/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Animales , Axones/metabolismo , Muerte Celular , Modelos Animales de Enfermedad , Terapia por Estimulación Eléctrica , Potenciales Evocados Visuales , Ratones , Microscopía Confocal , Plasticidad Neuronal , Traumatismos del Nervio Óptico/etiología , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/terapia , Ratas , Retina/citología , Retina/metabolismoRESUMEN
PURPOSE: Transcorneal alternating current stimulation (tACS) has become a promising tool to modulate brain functions and treat visual diseases. To understand the mechanisms of action a suitable animal model is required. However, because existing animal models employ narcosis, which interferes with brain oscillations and stimulation effects, we developed an experimental setup where current stimulation via the eye and flicker light stimulation can be applied while simultaneously recording local field potentials in awake rats. METHOD: tACS was applied in freely-moving rats (N = 24) which had wires implanted under their upper eye lids. Field potential recordings were made in visual cortex and superior colliculus. To measure visual evoked responses, rats were exposed to flicker-light using LEDs positioned in headset spectacles. RESULTS: Corneal electrodes and recording assemblies were reliably operating and well tolerated for at least 4 weeks. Transcorneal stimulation without narcosis did not induce any adverse reactions. Stable head stages allowed repetitive and long-lasting recordings of visual and electrically evoked potentials in freely moving animals. Shape and latencies of electrically evoked responses measured in the superior colliculus and visual cortex indicate that specific physiological responses could be recorded after tACS. CONCLUSIONS: Our setup allows the stimulation of the visual system in unanaesthetised rodents with flicker light and transcorneally applied current travelling along the physiological signalling pathway. This methodology provides the experimental basis for further studies of recovery and restoration of vision.
Asunto(s)
Córnea/fisiología , Terapia por Estimulación Eléctrica/métodos , Modelos Animales , Animales , Terapia por Estimulación Eléctrica/efectos adversos , Terapia por Estimulación Eléctrica/instrumentación , Potenciales Evocados , Párpados , Neuroestimuladores Implantables/efectos adversos , Estimulación Luminosa , Ratas , Colículos Superiores/fisiología , Corteza Visual/fisiología , Vías Visuales/fisiología , Percepción Visual/fisiologíaRESUMEN
The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.