Abstract
In order to explore the role of a specific gene/protein in the specific segment of the spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, lentivirus containing shRNA for CXCL13 were injected into the dorsal horn of the spinal lumbar enlargement segment (Jiang et al., 2016). This technique allows the study of the role of CXCL13 in the ipsilateral dorsal horn in neuropathic pain without affecting DRG or contralateral dorsal horn.
Background
The dorsal horn of spinal cord is a well-organized and stratified neuronal complex, which transmits the sensory information from the body surface and deep tissues to the brain. The activity of neurons in the spinal dorsal horn can be regulated by primary afferents, descending fibers, as well as spinal glial cells such as astrocytes and microglia. Understanding how the specific protein and gene in spinal neurons or glial cells contribute to the mechanism of physiological and pathological pain is of increasing interest. In order to explore the role of a specific gene/protein in the spinal cord, the technique of intrathecal injection is commonly used to deliver agonist, antagonist, siRNA, or miRNA into the subarachnoid space. The knockout and conditional deletion of a specific gene is another exhaustive and expensive approach. However, the limitation of these methods is lack of spinal segment- and cell type-specific deletion or overexpression. This protocol shows that intraspinal injection can deliver specific viral vectors into specific segmental spinal dorsal horn to investigate the function of a gene in a region- and cell type-specific manner.
Materials and Reagents
Equipment
Procedure
Notes:
Data analysis
To test the infection of CXCL13 shRNA lentivirus vectors in the spinal cord, the double staining of immunofluorescence is used. Four days after intraspinal injection, animals are deeply anesthetized with isoflurane. When lacking the corneal reflex, they are perfused through the ascending aorta with PBS followed by 4% paraformaldehyde with 1.5% picric acid in 0.01 M PBS. After the perfusion, the lumbar spinal cord segments are removed and postfixed in the same fixative overnight. Spinal cord sections (30 μm, free-floating) are cut in a cryostat and processed for immunofluorescence as we described previously (Jiang et al., 2016). To examine the cell types that expressed the GFP, the sections are immunostained with GFAP, ionized calcium binding adaptor molecule 1 (IBA-1), and NeuN. Throughout the dorsal horn of spinal cord, GFP is primarily localized in NeuN-positive neurons or GFAP-positive astrocytes and some IBA-1-positive microglia (Jiang et al., 2016).
Acknowledgments
The intra-spinal injection protocol was funded by the National Natural Science Foundation of China (NSFC 81571070, 31371121). This intra-spinal injection protocol was developed and used in articles published by Jiang et al. (2016), Lu et al. (2014), and Zhang et al. (2012).
References
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