A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia

Abstract

BackgroundThe cell bodies of sensory neurons, which transmit information from the external environment to the spinal cord, can be found at all levels of the spinal column in paired structures called dorsal root ganglia (DRG). Rodent DRG neurons have long been studied in the laboratory to improve understanding of sensory nerve development and function, and have been instrumental in determining mechanisms underlying pain and neurodegeneration in disorders of the peripheral nervous system. Here, we describe a simple, step-by-step protocol for the swift isolation of mouse DRG, which can be enzymatically dissociated to produce fully differentiated primary neuronal cultures, or processed for downstream analyses, such as immunohistochemistry or RNA profiling.FindingsAfter dissecting out the spinal column, from the base of the skull to the level of the femurs, it can be cut down the mid-line and the spinal cord and meninges removed, before extracting the DRG and detaching unwanted axons. This protocol allows the easy and rapid isolation of DRG with minimal practice and dissection experience. The process is both faster and less technically challenging than extracting the ganglia from the in situ column after performing a dorsal laminectomy.ConclusionsThis approach reduces the time required to collect DRG, thereby improving efficiency, permitting less opportunity for tissue deterioration, and, ultimately, increasing the chances of generating healthy primary DRG cultures or high quality, reproducible experiments using DRG tissue.

Satellite glial cells represent a population of developmentally arrested Schwann cells

“…Cervical DRGs from female Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were isolated from 90-day-old animals (P90, adult DRG; Malin, Davis, & Molliver, 2007;Sleigh, Weir, & Schiavo, 2016). The epineurium and nerve roots were removed and the DRGs were transferred to 5 mg/ml collagenase type 2 (Worthington).…”

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post‐inflammatory rat model for irritable bowel syndrome

“…DRGs T13-L2 (splanchnic colonic afferents) and L6-S1 (pelvic colonic afferents) were harvested bilaterally. Briefly, the spinal column was dissected and cut down the midline, and after removal of the spinal cord and meninges, DRGs of interest were extracted as previously described (Sleigh et al, 2016). Samples were snap-frozen in liquid nitrogen and stored at À80°C.…”

Calcium imaging of primary canine sensory neurons: Small‐diameter neurons responsive to pruritogens and algogens

“…Positioning in canine cadavers is easier in sternal recumbency to access the spinal column due to their quadruped anatomy compared with dorsal recumbency as done in human cadavers (Valtcheva et al, ). Although the dorsal laminectomy approach was determined to be a less rapid method for isolation in rodents (Sleigh, Weir, & Schiavo, ), this approach is most efficient in dogs due to their larger size, increased musculature, and the increased size and density of their skeleton. Additionally, the rapid processing of DRG tissue developed in this study increased neuronal cell viability and improved reproducible cultures of predominantly sensory neurons (i.e., small‐diameter neurons).…”

“…Although the dorsal laminectomy approach was determined to be a less rapid method for isolation in rodents (Sleigh, Weir, & Schiavo, 2016), this approach is most efficient in dogs due to their larger size, increased musculature, and the increased size and density of their skeleton. Additionally, the rapid processing of DRG tissue developed based on known pruritogens and algogens with a focus on specific agonists for receptors known to be expressed on canine DRG (Rossbach & Bäumer, 2014) and agonists used in previous murine calcium imaging studies (Akiyama et al, 2010b(Akiyama et al, , 2012Fukuyama, Ganchingco, & Bäumer, 2017a;Fukuyama, Ganchingco, Mishra, et al, 2017b;Fukuyama et al, 2018;Liu et al, 2016;Schemann et al, 2012;Wilson et al, 2011Wilson et al, , 2013.…”