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Abstract

Source: Breid, S. et al. Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils. J. Vis. Exp. (2017).

This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

Protocol

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

1. Animal Model

1. Intercross hemizygous Tg(Gfap-luc^+/-) mice (transgenic mice expressing firefly luciferase) with hemizygous Tg(M83^+/-) mice (transgenic mice expressing human alpha-synuclein with the familial A53T mutation)  to generate hemizygous bigenic Tg(M83^+/-:Gfap-luc^+/-) mice.
2. Genotype the progeny with real-time polyemerase chain reaction (PCR) for the presence of the transgene encoding human alpha-synuclein and with standard PCR for firefly luciferase.
3. Inoculate the animals at an age of six-to-eight weeks.

2. Inoculum Preparation

1. Prepare monomeric recombinant human alpha-synuclein with the A53T mutation in Tris-buffered saline (TBS) buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.2).
2. Prepare fibrillar alpha-synuclein for inoculations by agitating 3 µg/µL of monomeric recombinant human alpha-synuclein in an orbital shaker at 800 rpm and 37 °C for 5 days.
3. Dilute fibril assemblies in phosphate-buffered saline (PBS) to reach a final concentration of 1 µg/µL for intraperitoneal inoculations.
4. Fragment the alpha-synuclein fibrils with a rod sonicator for 1 min (40 pulses of 0.5 s duration with a one-second pause between each pulse and an amplitude set to 50%) on ice. To avoid contamination by cross-seeding, use clean sonication probes to prepare different inoculum.

3. Intraperitoneal Injections

1. For intraperitoneal injections, narcotize the animals shortly in an anesthesia chamber by inhalation with isoflurane/oxygen using a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.
2. Fill a 27 G disposable hypodermic syringe with 50 µL of sonicated alpha-synuclein fibrils or PBS.
3. Directly inject into the peritoneum of the mouse and avoid penetrating the small intestine or cecum located behind the abdominal wall by holding the animal in a dorsal position with the head facing away from the investigator and downward at approximately 45°. Monitor animals until they have recovered from the anesthesia.

Materials

Phosphate-buffered saline (PBS)	Invitrogen	14190169
27-gauge syringe	VWR	613-4900
Isoflurane	Piramal Healthcare	PZN 4831850
Sonopuls Mini20 sonicator	Bandelin	3648
Tg(M83+/-) mice or B6;C3-Tg(PrnpSNCA*A53T)83Vle/J mice	The Jackson Laboratory	4479