Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport

Abstract

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival.

Keywords: DRG; axonal transport; equine; mitochondria; neurodegenerative diseases.

FIGURE 1

Dissection of horse cervical DRGs prior to dissociation. Extracted ganglia (black stars) are first cleaned of non‐nervous tissue and dura mater (a) and then trimmed of nerve roots before dissociation (b) Scale bars: 1 cm. (c) Histological section of a horse cervical DRG stained with haematoxylin and eosin. The small satellite cells (long arrow) tightly surround the large sensory neurons (short arrow). Scale bar: 100 μm.

FIGURE 2

Dissociated horse DRG cultures in vitro. DRG neurons (short arrows) and neurite outgrowth is shown at indicated times in culture (a–f). Neurons are viable and all developed neurites. Glial cells also developed processes in cultures (long arrows). Histogram summarizing the range of neuronal soma diameters in DRG cultures (n = 118) (g). Scale bar: 100 μm.

FIGURE 3

Immunofluorescence characterisation of dissociated DRG culture at 14 days in vitro. Neurons (short arrow) are labelled with anti‐NeuN (red, a, b) and glial cells (long arrows) are stained with GFAP (green, a, b) and S100 (green, c). Glial cells also stained positive for NeuN (a, b). Cell nuclei counterstained with DAPI are shown in blue.

FIGURE 4

Morphology of glial cells in culture. Immunostaining of the cytoskeletal protein βIII‐tubulin (red) demonstrated extensive growth of the processes and branching at 21 DIV. All the cultures were grown in the same conditions. (a). Larger cells exhibited a higher number of processes compared to the smaller sized glial cells (b and c [phase contrast image]; arrows and short arrows respectively).

FIGURE 5

Mitochondrial morphology in horse DRG neuronal cultures 7 DIV. Mitochondria were labelled with Mitotracker red CMXRos (Invitrogen). Glial cell mitochondria (arrow) with an elongated morphology (a) compared to those of neuronal mitochondria (arrow, b); were significantly (****p < 0.0001; paired t‐test) longer than those in neurons (c). Each data point represents the mean value obtained for 25–50 mitochondria measured in each animal. Horizontal bar indicates mean and error bars indicate standard error of the mean. n = 8 animals per group.

FIGURE 6

Mitochondrial transport in DRG neurons and glial cells. Representative straightened cellular processes, kymograph and kymograph of tracked particles of mitochondrial transport from glial cells at 14 DIV (a) and neurons at 7 DIV (b). The straightened processes represent the first frame of the time lapse recording (total 180 frames; frame rate 1 fps) that was used to generate the original kymograph. Moving mitochondria were tracked using the ImageJ Difference Tracker (Andrews et al., 2010) set of plugins and another kymograph generated to show successfully tracked particles. Vertical lines indicate stationary mitochondria while lines deflecting to the right or left represent anterograde or retrograde moving mitochondria. Quantification of the maximum speed of mitochondrial movement with cell types indicated (C). Each data point represents the mean value obtained from mitochondria for each animal. Horizontal bar indicates mean and error bars indicate standard error of the mean. Statistically significant difference between groups is indicated (***p < 0.001, paired t‐test). n = 8 per group. Scale bar: 10 μm.