Intramuscular Injections Along the Motor End Plates to Shuttle Tracers Directly into Rat Motor Neurons

Abstract

Source: Mohan, R., et al. Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons. J. Vis. Exp. (2015)

This video demonstrates the targeted delivery of a fluorescent neuronal tracer into motor neurons via intramuscular injection at the motor end plates (MEPs). It outlines the steps involved in preparing the rat, injecting the tracer, and assessing labeled motor neurons for neurotherapeutic potential.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intramuscular Injections at the Motor End Plates

1. Pull glass micropipettes with a micropipette puller (the use of graded micropipettes with plungers is recommended). With the help of a dissecting microscope, break the tips of the micropipettes with a pair of forceps such that the internal diameter of the lumen of the micropipette is approximately 0.5 mm.
2. Ensure that all surgical instruments used are freshly autoclaved and that the surgical area is sterile.
3. Fill the micropipettes with Fluoro-Gold (5% in distilled water).
4. Induce anesthesia in the animal using Isoflurane (4% in O₂) in an induction chamber. Check for the righting reflex and ensure its absence before removing it from the induction chamber. Secure a nose cone over the snout of the rat and deliver Isoflurane (2% in O₂) for maintenance of anesthesia. Pinch the animal’s toes and gently touch the animal’s eye to ensure that both the pedal withdrawal and the corneal reflexes are absent.
   NOTE: A mixture of ketamine and xylazine can also be used (80 and 10 mg/kg, respectively, delivered intraperitoneally). Ketamine is a Schedule 8 (“Controlled”) drug, and necessary protocols associated with the purchase, use, storage, and disposal of the drug will have to be adhered to.
5. Apply eye lubricant to prevent drying of the eyes during the course of the procedure.
6. Shave the targeted limb and use gauze to wipe the shaved area with three alternating scrubs of chlorhexidine and 70% alcohol. Transfer the animal to the surgical area.
7. Place the animal on a clean underpad and position it appropriately to ensure good access to the targeted muscle(s).
8. Use a pair of forceps with tooth grips to lift the skin over the targeted muscle away from the underlying musculature and make an incision in the skin with surgical scissors. Ensure that the incision is large enough to completely expose the muscle(s) of interest. Ensure that there is minimal disruption to the fascia.
9. Use the motor end plate (MEP) photographs to mentally transpose the location and shape of the MEP region onto the muscle(s).
   NOTE: A fine felt marker could assist in reproducing the MEP region from the photograph onto the muscle(s) of interest.
10. Perform multiple injections along the full length of the MEP region (for Fluoro-Gold, 3 to 4 injections of 1-2 µl each should be sufficient). Gently wipe the muscle(s) to remove any seepage.
11. Bring the two ends of the incised skin close together with blunt forceps and close the wound with surgical clips. Infiltrate 0.1 ml Bupivacaine (0.5% in water) (or other local anesthetics) along the entire span of the wound.
12. Turn the anesthetic machine off and monitor the animal until it has fully recovered from anesthesia.
13. Wait for 14 days between the administration of intramuscular injections and the perfusion of the animals.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluoro-Gold	Fluorochrome, LLC	Nil	Diluted to 5%
Drummond PCR Micropipets 1-10 μl	Drummond Scientific	5-000-1001-X10	accompanied with plungers
Isothesia	Provet	ISOF00	1000 mg/g Isoflurane inhalation vapour
Autoclip 9 mm Wound Clips	Texas Scientific Instruments, LLC	205016