Abstract
When injected into the motor cortex of rats, anterograde tracers label fibers of the associated descending corticospinal tract (CST) that originate from pyramidal neurons in the tracer-injected cortex. These fibers can be assessed at the level of the spinal cord to determine the integrity of the descending CST and the spatial distribution of axons in the spinal grey matter. Here we provide detailed methods on the minimally invasive stereotaxic injection of anterograde tracers into the forelimb sensorimotor representation in the rat cortex. In addition, we detail the fixing and processing of spinal tissue for assessment of CST integrity and branching into spinal grey matter.
Keywords: Cortical spinal tract, Anterograde tracer, Spinal cord, Animal model
Background
The motor cortex contains upper motor neurons which give rise to descending axons that form the CST and terminate in the spinal cord. Anterograde tracers injected into the motor cortex of rats label these descending fibers of the CST allowing their terminals to be assessed at the level of the spinal cord (Figure 1). In rodent models, anterograde labeling of the CST is widely used to assess CST integrity in a range of spinal cord injury models including lesions (Thallmair et al., 1998; Vavrek et al., 2006) and contusions (Hill et al., 2001). Further, anterograde cortical tracers have been used to assess spared fibers and plasticity of the CST in various models of brain injury such as cortical stroke (Wahl et al., 2014; Wiersma et al., 2017), traumatic brain injury (Ueno et al., 2012) and pyramidotomy (Starkey et al., 2012). Here we provide detail methods on injection of anterograde tracers into motor cortex corresponding the forelimb. We describe how to create burr holes without inducing damage to the cortex and the use of pulled glass pipette tips for minimally invasive injection. Since the CST is widely assessed in both brain and spinal cord injury models, we offer a systematic method of assessing the CST and branching fibers in the surrounding spinal grey matter that accounts for variability due to inter-animal differences in tracer efficacy or uptake.Figure 1. Use of anterograde cortical tracers to label the corticospinal tract in rats. A. Tracer injection sites in the rat cortex (shown as green circle) and anterograde tracer labeled descending fibers (green line). B. Coronal sections of the brain at tracer injection sites. C. Transverse slices of the spinal cord (C4) with tracer labeled fibers of the CST shown in green.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
To determine if the number of labeled fibers in the CST is significantly different between two groups, use Student’s unpaired two-tailed t-test. To analyze statistical differences between groups in fiber count and length, use two-way ANOVA testing followed by post hoc Holm–Sidak’s testing.
Notes
Recipes
Acknowledgments
This work was supported by Alberta Innovates Health Solutions, Heart and Stroke Foundation of Canada, Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. Grants from the Canada Foundation for Innovation and Province of Alberta Small Equipment Grants were used to acquire confocal microscopy equipment. This protocol and figures are adapted from previous work: Wiersma, A. M., Fouad, K. and Winship, I. R. (2017). Enhancing spinal plasticity amplifies the benefits of rehabilitative training and improves recovery from stroke. J Neurosci 37(45): 10983-10997. The authors have no conflicts of interest to declare.
References
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