Cervical Axotomy with Neurotrophic Factor Infusion — Nao R. Kobayashi | ReplicateScience
Experiments/Cervical Axotomy with Neurotrophic Factor Infusion
Source Paper
BDNF and NT-4/5 Prevent Atrophy of Rat Rubrospinal Neurons after Cervical Axotomy, Stimulate GAP-43 and Tα1-Tubulin mRNA Expression, and Promote Axonal Regeneration
Nao R. Kobayashi, Da-Peng Fan, Klaus M. Giehl, Annie M. Bedard, Stanley J. Wiegand et al.
Cervical Axotomy with Neurotrophic Factor Infusion
behavioralratnot specified
Objective: To test whether infusion of recombinant neurotrophic factors (BDNF, NT-4/5, NGF, or NT-3) prevents atrophy of axotomized rubrospinal neurons, stimulates regeneration-associated gene expression (GAP-43 and Tα1-tubulin mRNA), and promotes axonal regeneration into peripheral nerve grafts
Materials & Equipment Checklist
8 items
Gather these items before starting the experiment. Check off items as you prepare.
Equipment1
not specified • not specified • not specified • not mentioned
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Protocol Steps
View Abstract
Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Tα1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors. In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Tα1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and Tα1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and Tα1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.
1
Cervical axotomy of rubrospinal neurons
Perform surgical cervical axotomy to axotomize rubrospinal neurons
not specifiednot specified
Note: This is the initial injury procedure that triggers the experimental timeline
View evidence from paper
“Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy”
2
Analyze trk receptor expression in axotomized RSNs
Perform in situ hybridization to determine expression patterns of trk family neurotrophin receptors in rubrospinal neurons
not specifiednot specified
Note: Full-length trkB receptors expressed in virtually all RSNs; expression declined 7 days after axotomy. Full-length trkC mRNA expressed at low levels. TrkA mRNA not detected. p75 expression restricted to small subpopulation
View evidence from paper
“In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy”
3
Perform RT-PCR analysis of trkC isoforms
Use RT-PCR to detect mRNAs encoding trkC isoforms with kinase domain inserts
not specifiednot specified
Note: trkC isoforms with kinase domain inserts present at levels comparable to unmodified receptor
View evidence from paper
“Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor”
4
Begin neurotrophic factor infusion
Infuse recombinant human BDNF, NT-4/5, NGF, NT-3, or vehicle control into the vicinity of axotomized rubrospinal neurons
Between days 7 and 14 after axotomynot specified
Note: Infusion period spans from day 7 to day 14 post-axotomy, during the period when atrophy typically occurs
View evidence from paper
“infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy”
5
Assess neuronal cell size
Measure cell body size of rubrospinal neurons to determine if atrophy was prevented
Measured at 2 weeks after termination of BDNF treatmentnot specified
Note: BDNF or NT-4/5 treatment fully prevented atrophy; effect still evident 2 weeks after treatment termination. Vehicle, NGF, or NT-3 had no significant effect
View evidence from paper
“infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment”
6
Analyze GAP-43 and Tα1-tubulin mRNA expression
Measure expression levels of regeneration-associated genes GAP-43 and Tα1-tubulin mRNA in treated and control neurons
not specifiednot specified
Note: BDNF or NT-4/5 treatment stimulated expression of both genes and maintained trkB expression. Vehicle, NGF, or NT-3 had no significant effect
View evidence from paper
“BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Tα1-tubulin mRNA and maintained the level of trkB expression”
7
Assess axonal regeneration into peripheral nerve graft
In a separate experiment, quantify the number of axotomized rubrospinal neurons that regenerated into a peripheral nerve graft
not specifiednot specified
Note: BDNF infusion increased the number of axotomized RSNs that regenerated into peripheral nerve grafts
View evidence from paper
“infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft”
Subjects / Specimens
Species
rat
Strain
not specified
Age
not specified
Sex
unknown
Weight
not specified
Rubrospinal neurons (RSNs) were the target neurons for study