Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Abstract

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin+ and calcitonin gene-related peptide (CGRP)+ sprouts peaking at day 14, matching both large, neuropeptide+ subpopulations of axotomized facial motoneurons, but with CGRP+ sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin+ sprouts revealed a stronger response following cut compared with crush (day 7–14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12–48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting. J. Comp. Neurol. 518:699–721, 2010. © 2009 Wiley-Liss, Inc.

A combined electrophysiological and morphological study of neuropeptide Y–expressing inhibitory interneurons in the spinal dorsal horn of the mouse

“… 32 The galanin antibody was raised against the synthetic peptide, and staining is absent in the brains of galanin-knockout mice. 35 The nNOS antibody was raised against purified recombinant rat nNOS and labels a band of 155 kDa in Western blots of rat hypothalamus, while immunostaining is abolished by preabsorption with nNOS. 20 The parvalbumin antibody is directed against the mouse protein and recognises a band of 13 kDa on Western blots of mouse brain homogenates.…”

Functional differences between neurochemically defined populations of inhibitory interneurons in the rat spinal dorsal horn

“…We have reported that dorsal horn immunostaining with the galanin and NPY antibodies can be abolished by pretreatment with the corresponding peptides [51,59], and staining of neurons with the galanin antibody is absent from the brains of galanin knockout mice [34]. The nNOS antibody labels a band of 155 kDa in Western blot tests of rat hypothalamus, and staining is abolished by preincubation with nNOS [17].…”

Neurochemical Characterisation of Lamina II Inhibitory Interneurons that Express GFP in the PrP-GFP Mouse

“…The nNOS antibody labels a band of 155 kDa in Western blots of rat hypothalamus, and staining is abolished by pre-incubation with nNOS [ 49 ]. We have reported that dorsal horn immunostaining with the galanin and NPY antibodies can be abolished by pre-treatment with the corresponding peptides [ 50 , 51 ], and staining of neurons with the galanin antibody is absent from the brains of galanin knock-out mice [ 52 ]. The rabbit and goat parvalbumin antibodies were raised against mouse and rat parvalbumin, respectively, and recognise a protein band of the appropriate size on Western blots.…”