Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.
Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin-horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer is administered in dried form. An alternative method is described for anterograde labelling of cortical motor neurons using biotinylated dextran. This tracer may be applied by either microinjection of 10% biotinylated dextran or implanting small globules of the dried tracer into the motor cortex. While more laborious, microinjection results in better anterograde labelling than implantation of dried biotinylated dextran. A procedure is also described for preparing serial coronal sections through the entire spinal cord and thaw-mounted on a minimum number of slides. The labelled nerve processes in these tissue sections can be visualised in the spinal cord under a fluorescent microscope following incubation with cy3-streptavidin complex. Permanent labelling of the biotinylated nerve processes is achieved by incubation of tissue sections with streptavidin-horseradish peroxidase conjugate followed by stringent washes and staining with tetramethylbenzidine. Use of tetramethylbenzidine allows resolution of a greater number of finer labelled processes than diaminobenzindine and allows clear visualisation of individual regenerating corticospinal tract processes. Using these procedures, we demonstrate that the corticospinal tract is completely lesioned by a standardised contusion spinal cord injury produced by the New York University weight-drop device.
Ferguson
I A
IA
Department of Human Physiology and Centre for Neuroscience, Flinders University School of Medicine, GPO Box 2100, SA 5001, Adelaide, Australia. i.ferguson@flinders.edu.au
Xian
C
C
Barati
E
E
Rush
R A
RA
D000818:Animals; D001370:Axonal Transport / Q000187:drug effects* / Q000502:physiology; D019771:Axotomy; D001560:Benzidines; D001710:Biotin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D003911:Dextrans / Q000493:pharmacokinetics*; D005260:Female; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D006651:Histocytochemistry; D008845:Microinjections; D008867:Microtomy; D015335:Molecular Probes / Q000493:pharmacokinetics*; D009044:Motor Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009416:Nerve Regeneration / Q000502:physiology; D009445:Neuroanatomy; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011712:Pyramidal Tracts / Q000293:injuries / Q000473:pathology* / Q000503:physiopathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000473:pathology* / Q000503:physiopathology; D019809:Streptavidin; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate / Q000493:pharmacokinetics*
D001560:Benzidines; D003911:Dextrans; D005456:Fluorescent Dyes; D015335:Molecular Probes; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; C076397:biotinylated dextran amine; C021758:3,3',5,5'-tetramethylbenzidine; D001710:Biotin; D019809:Streptavidin