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Feng et al. Growth cone collapse in adult sensory neurons

the injury site is a major mechanical barrier to axonal or after the addition of SEMA3A. The objective of
regeneration. Studies have found that the glial scar this study was to provide evidence that neurotrophic
contains many molecules that inhibit axonal growth. [4-8] factors are involved in mediating axonal regeneration
Semaphorin 3A (SEMA3A) is one of these molecules and neurite outgrowth as well as shed light on the
and is highly expressed in the glial scar. [9-11] SEMA3A roles of specific neurotrophic factors in mediating
is part of the Semaphorin family of inhibitory guidance neurite outgrowth.
cues specific to vertebrae, and it is normally present
during the development of the central and peripheral METHODS
nervous systems. [12-14] Semaphorins signal through
complex neuronal receptors that contain neuropilin-1 Enteric glia extraction and culture
and plexins, which are significantly expressed in All experiments were performed in accordance with the
axotomized dorsal root ganglion (DRG). This requirements of the Animals for Research Act of Ontario,
[13]
indicates that SEMA3A is a key mediator of axon Canada and the Guidelines of the Canadian Council on
retraction and growth cone collapse. [13,15] To date, Animal Care and were approved by the Animal Research
multiple studies have observed the effects of SEMA3A Ethics Board at our institution. Both EG extraction from
on DRG-derived neurons in animal models. [16-21] Two of the small intestines of adult female Wistar rats and EG
them employed growth cone collapse assays adapted identification confirmation by staining for glial fibrillary
from studies on embryonic neurons. [16,18] acidic protein and myelin protein zero (MPZ) were
performed using previously described methods [29] as
Enteric glia (EG) are the support cells of the enteric adapted in Hansebout et al. The presence of glial
[24]
nervous system (the nervous system of the gut) and fibrillary acidic protein, in conjunction with the absence
share a number of characteristics with central nervous of MPZ, is considered to be indicative of EG. [21,24]
system (CNS) astrocytes. It has been previously EG were cultured as per in Dulbecco’s modified
[16]
reported that in vivo locally transplanted EG facilitate eagle medium (DMEM/F) 12 1:1 (Invitrogen D8437)
ingrowth of transected dorsal root axons toward their supplemented with 10% fetal bovine serum (FBS)
targets through the spinal cord and across the non- and 1% penicillin/streptomycin (P/S). The medium
permissive peripheral/CNS boundary. [22] These EG was changed every 2-3 days, and the cells were then
induce the regeneration of neurofilament-positive subcultured using 0.25% trypsin-ethylene diamine
dorsal root axons into the injury site of rats given tetraacetic acid (Invitrogen 25200-056).
spinal cord crushes (using the clip-compression
model). [23] Additionally, there is published data that DRG extraction and culture
suggests EG secrete nerve growth factor (NGF), DRG were extracted from six- to sixteen-week old
brain-derived neurotrophic factor (BDNF), glial adult male Wistar rats and cultured as per Hall and
[30]
cell line-derived neurotrophic factor (GDNF), and as described by Hansebout et al., plating 3 × 10
[24]
4
neurotrophin-3 (NT-3) in culture and that these growth cells/well in a 6-well tissue culture plate. Non-neuronal
factors at least partially mediate neurite outgrowth cells were reduced with a 4 h incubation in dispase/
in DRG neurons in response to EG. [24] Studies have collagenase. They were then plated on a standard
shown that all of these growth factors imbue some petri dish. Neurons generally have difficulty adhering
resistance to SEMA3A-induced growth cone collapse to the petri dish surface, and the neurons mixed in
in cultured embryonic DRG neurons, [25,26] although solution were subsequently removed and replated. As
collapse sensitization varies with developmental per Reza et al., each well was coated with 3 mL of
[16]
stage. [26-28] A study by Wanigasekara et al. [18] is of 0.01% poly-L-lysine 48 h before extraction. The poly-
particular importance to our study, as it indicated L-lysine was replaced with 2 ng/mL laminin in HBSS
that NGF, GDNF, and NT-3 inhibit SEM3A-induced 24 h before extraction to facilitate DRG attachment.
collapse in adult DRG-derived sensory neurons when The neurons were acclimatized to culture conditions
administered 1 h before collapse. for 4 days before each experiment. Half of the 3 mL of
supplemented neurobasal medium was replaced with
We hypothesized that incubation with EG-conditioned fresh medium every other day.
medium would inhibit and perhaps reverse SEMA3A-
mediated growth cone collapse in cultured DRG Generation of enteric glial-conditioned medium
neurons, and theorized that inhibiting any one of the The EG medium was conditioned as per
neurotrophic factors in the EG-conditioned medium Hansebout et al. EG derived from adult male Wistar
[24]
would decrease its overall efficacy at preventing rats were seeded at 4 × 10 cells/well into a six-well
4
SEMA3A-mediated collapse, regardless of whether plate coated with rat tail collagen, with tissue culture
the conditioned medium was applied before, during, inserts covered in 3T3 mouse embryonic fibroblasts.
Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ August 31, 2016 181