Neuroprotective effects of NGF, BDNF, NT-3 and GDNF on axotomized extraocular motoneurons in neonatal rats

Abstract

Neurotrophic factors delivered from target muscles are essential for motoneuronal survival, mainly during development and early postnatal maturation. It has been shown that the disconnection between motoneurons and their innervated muscle by means of axotomy produces a vast neuronal death in neonatal animals. In the present work, we have evaluated the effects of different neurotrophic factors on motoneuronal survival after neonatal axotomy, using as a model the motoneurons innervating the extraocular eye muscles. With this purpose, neonatal rats were monocularly enucleated at the day of birth (postnatal day 0) and different neurotrophic treatments (NGF, BDNF, NT-3, GDNF and the mixture of BDNF+GDNF) were applied intraorbitally by means of a Gelfoam implant (a single dose of 5 μg of each factor). We first demonstrated that extraocular eye muscles of neonatal rats expressed these neurotrophic factors and therefore constituted a natural source of retrograde delivery for their innervating motoneurons. By histological and immunocytochemical methods we determined that all treatments significantly rescued extraocular motoneurons from axotomy-induced cell death. For the dose used, NGF and GDNF were the most potent survival factors for these motoneurons, followed by BDNF and lastly by NT-3. The simultaneous administration of BDNF and GDNF did not increase the survival-promoting effects above those obtained by GDNF alone. Interestingly, the rescue effects of all neurotrophic treatments persisted even 30 days after lesion. The administration of these neurotrophic factors, with the exception of NT-3, also prevented the loss of the cholinergic phenotype observed by 10 days after axotomy. At the dosage applied, NGF and GDNF were revealed again as the most effective neuroprotective agents against the axotomy-induced decrease in ChAT. Two remarkable findings highlighted in the present work that contrasted with other motoneuronal types after neonatal axotomy: first, the extremely high efficacy of NGF as a neuroprotective agent and, second, the long-lasting effects of neurotrophic administration on cell survival and ChAT expression in extraocular motoneurons.

Keywords: 3,3′-diaminobenzidine tetrahydrochloride; ALS; ANOVA; BDNF; BSA; CNS; ChAT; ChAT down-regulation; DAB; GDNF; HRP; NGF; NRS; NT-3; NT-4/5; P; PBS; PBS-T; SEM; abducens; amyotrophic lateral sclerosis; analysis of variance; bovine serum albumin; brain-derived neurotrophic factor; central nervous system; choline acetyltransferase; glial cell-line-derived neurotrophic factor; horseradish peroxidase; lesion-induced cell death; nerve growth factor; neurotrophic factor; neurotrophin-3; neurotrophin-4/5; normal rabbit serum; oculomotor; phosphate-buffered saline; phosphate-buffered saline with 0.1% Triton X-100; postnatal day; postnatal development; standard error of the mean.