Live Imaging of Dorsal Root Axons after Rhizotomy

Summary

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Abstract

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo¹. These mice have been used extensively for in vivo imaging of muscle^2-4 and brain^5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy^8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished¹⁰. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord¹¹.

In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

Protocol

1. Microscope set up and imaging preparation

1. Our imaging set up consists of a Leica MZ16 fluorescent stereomicroscope with a fast shutter and a cooled CCD camera controlled by Metamorph software.
2. Prepare a thermostatically controlled heating pad and adjust output to 32.5°C to maintain the animal's body temperature during and following surgery.
3. Warm sterile Ringer's solution or artificial cerebrospinal fluid (ACSF) to 32.5°C in advance for irrigation of spinal cord during surgery.
4. Anesthetize the animal with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (120 mg/kg) cocktail.
5. Shave the upper back with....

Discussion

Imaging DR regeneration directly in living mice is particularly challenging because it requires a substantial dorsal laminectomy to monitor axon growth over a wide area followed by multiple invasive surgical and anesthetic procedures in subsequent imaging sessions. Several strategies helped to overcome these challenges. First, successful imaging required reducing mouse mortality (approximately 25%) by minimizing the duration of anesthesia and bleeding, and by meticulous post-op care. The mortality was also reduced by usi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
H line thy1-YFP (2-4 months old, either sex)	Jackson Laboratory	003782
Xylazine (AnaSed injection, sterile solution)	Lloyd, Inc.	4811	8 mg/kg
Ketamine (Ketamine hydrochloride injection, USP)	Hospira Inc.	2051	120 mg/kg
Buprenorphine (Buprenex injectable) (0.05 mg/kg)	Reckitt Benckiser	7571
Small animal hair clippers	Oster Professional Products	76059-030
Hair removal lotion	Church & Dwight Co.	NAIR with Baby Oil
Gauze sponges	Fisher Scientific	22-362-173
Cotton-tipped swabs	Fisher Scientific	14-960-3Q
1 mL syringes	BD Biosciences	309602
Subcutaneous (Sub-Q) needles, 26ga.	BD Biosciences	305115
Spring scissors and forceps	Fine Science Tools
2.5-mm curved rongeurs	Fine Science Tools	16221-14
Lactated Ringer’s Injection USP	B. Braun Medical	BBR-L7502
Sterile saline solution	APP Pharmaceuticals	918610
Thin synthetic matrix membrane (Biobrane)	Bertek Pharmaceuticals	62794-096-251
Artificial dura	Gore Preclude MVP Dura Substitute, W.L. Gore and Associates	1MVP40
5-0 silk sutures	Ethicon Inc.	K-580
Wound clips	Perfect - Ets Bruneau, (Burnea, France)	A75
Fluorescent stereomicroscope	Leica Microsystems	MZ16
CCD camera	Hamamatsu Corp.	ORCA-Rx2
Temperature Controller	World Precision Instruments, Inc.	ATC 1000
Metamorph software	Molecular Devices
Photoshop	Adobe