Neurobiology of the Regenerating Retina and Its Functional Reconnection With the Brain by Means of Peripheral Nerve Transplants in Adult Rats

Abstract

Axotomy-induced degradation of retinal ganglion cells (RGC) can be delayed if the destructive features of activated microglial cells are pharmacologically neutralized, and prevented if the axons are permitted to regrow into transplanted autologous peripheral nerve (PN) pieces. Axotomized central nervous system neurons, whose regenerating axons are guided to their natural target areas in the brain with the aid of PN grafts, are capable of establishing synaptic contacts with normal morphological and electrophysiological properties. This study was undertaken to 1) morphometrically characterize and classify the regenerating rat RGC, 2) examine target-dependent effects on survival of subsets of neurons, and 3) investigate whether reconnected neurons are capable of restoring visual functions.In analogy to the normal rat retina, as a first step, the retrogradely labeled, regenerating RGC were categorized into five classes which are morphologically distinct and reminiscent of normal RGC correlates (called types RI, RII, RIII, Rδ-cells, and displaced RGC). It appeared that all types of ganglion cells contributed proportionally to regeneration of axons. Transplantation of a PN graft which was not reconnected with a central target (blind-ending group) and monitoring of the extant neurons showed a progressive disappearance of the regenerating RGC, such that 6 months after surgery predominantly few, large cells survived. When the retinas were treated with macrophage/microglia inhibiting factor (MIF), and the regenerating axons were guided into the pretectum, predominantly large RGC of type RI survived. Guidance of the axons into their major natural target, the superior colliculus (SC), resulted in selective survival of many small, RII-like RGC. Calculation of the dendritec coverage factors for the major types of RGC revealed that dendrites of the most abundant, small cells of type RII overlapped uniformly and covered the retinal surface completely, whereas cells of types RI and RIII did not suffice for surface coverage. The results of this first part of the work suggest that combined suppression of axotomy-induced microglial activation and guidance of regenerating axons with a PN graft into central targets is a suitable technique to produce sufficient numbers of regenerating axons which may retrieve some functional properties. Target-specific neuronal contacts are likely involved in morphological stabilization and better survival of regenerating neurons.The second goal of this study was to analyze the functional significance of the reestablished synaptic contacts made by regenerated retinocollicular neurons. Adult rats were trained in a T- or Y-maze to obtain a food reward with the aid of visual cues. One of their optic nerves was transected and the regenerating axons were guided into the optic tract with a PN graft, to enable them to reinnervate the SC and thalamus. Postoperative testing of the animals showed a drastic improvement of visual perception. The protocol of denervation of the SC (prior to, simultaneous with, or with a delay with respect to fiber arrival) determined the performance of the animals. Rats belonging to the first two groups performed almost as well as they had before the transplantation. The functional integrity of the retina was assessed by electroretinography, which revealed typical rod spectral sensitivity at 380 and 500 nm but reduced responsiveness to illumination. In accordance, neuroanatomical assessment of the functionally relevant RGC revealed intact morphologies and multiple synaptic contacts both within the retina and within the SC. Neuroanatomical tracing of small contingents of axons throughout the regenerative pathway revealed a rough retinotopic arrangement within the graft and the area of termination. Thus, animals could discriminate between simplified vertical versus horizontal stripes, and visual evoked potentials were positive after grafting. These findings show that a restricted population of retinofugal axons can restore higher visual functions such as pattern-discrimination behavior in the adult rat. Prerequisites for the restoration of visual perception are first, the preservation of the intraretinal integrity, and second, the temporal matching of fiber arrival and denervation of postsynaptic neurons.