Axotomy and Nerve Injury Model
Objective: To assess protective effects of GDNF treatment on axotomy-induced changes in small diameter DRG neurons, including prevention of IB4 binding downregulation, TMP activity loss, somatostatin expression changes, conduction velocity slowing, and A-fiber sprouting
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Equipment1
Not applicable • Not applicable • Not applicable • Not explicitly stated
Materials3
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Protocol Steps
Identify target neurons
Identify small diameter DRG neurons that express GDNF receptor components (RET, GFRα-1, and GFRα-2) and can be labeled with IB4 lectin
Note: Virtually all IB4-labeled cells express RET mRNA, and majority (79%) express GFRα-1, GFRα-2, or both
View evidence from paper
“In lumbar dorsal root ganglia, virtually all IB4-labeled cells express RET mRNA, and the majority of these cells (79%) also express GFRα-1, GFRα-2, or GFRα-1 plus GFRα-2”
Perform axotomy
Perform surgical axotomy of DRG neurons to induce nerve injury
Note: Axotomy induces multiple changes in small diameter neurons including downregulation of IB4 binding, TMP activity, and somatostatin expression
View evidence from paper
“GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression”
Administer GDNF treatment
Apply GDNF treatment to axotomized neurons to assess protective effects
Note: GDNF prevents axotomy-induced changes; NGF does not provide similar protection
View evidence from paper
“GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons”
Assess IB4 binding
Measure IB4 binding to determine if GDNF prevents downregulation of this marker
Note: IB4 binding is normally downregulated after axotomy
View evidence from paper
“GDNF can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding”
Assess TMP activity
Measure thiamine monophosphatase (TMP) activity to determine if GDNF prevents its loss
Note: TMP activity is normally downregulated after axotomy
View evidence from paper
“GDNF can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression”
Assess somatostatin expression
Measure somatostatin expression to determine if GDNF prevents its downregulation
Note: Somatostatin expression is normally downregulated after axotomy
View evidence from paper
“GDNF can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression”
Measure conduction velocity
Assess conduction velocity in small diameter DRG cells to determine if GDNF prevents slowing
Note: Conduction velocity normally slows after axotomy in small diameter DRG cells
View evidence from paper
“GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells”
Assess A-fiber sprouting
Examine dorsal horn lamina II for A-fiber sprouting to determine if GDNF prevents this change
Note: A-fiber sprouting into lamina II normally occurs after axotomy
View evidence from paper
“GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells and the A-fiber sprouting into lamina II of the dorsal horn”