Bioluminescence Imaging of Neuroinflammation in a Transgenic Mouse

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 bioluminescence imaging of neuroinflammation in transgenic mice, showcasing luciferase-luciferin reactions in activated astrocytes. The protocol enables non-invasive quantification of astrocytic activation, aiding studies on CNS inflammation and therapeutic efficacy.

Protocol

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

Animal Model

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

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 amplitude set to 50%) on ice. To avoid contamination by cross-seeding. Use clean sonication probes to prepare different inoculum.

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.

Bioluminescence Imaging

1. Image Tg(M83+/-:Gfap-luc+/-) mice every 2-4 weeks using a bioluminescence imaging system.
2. Prior to imaging shortly anesthetize mice in an isoflurane/oxygen chamber with 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.
3. Shave and depilate the heads of the mice with a depilatory cream. Color the ears in black with a non-irritating marker to block unspecific bioluminescence.
4. Dilute D-luciferin potassium salt, the substrate of luciferase, in PBS to a 30 mg/mL stock solution. Store this in 1 mL aliquots at -20 °C. Protect the dissolved D-luciferin from exposure to light.
5. Weigh the animals prior to injection. Calculate the exact volume of D-luciferin for injection of 150 mg/kg body weight. Intraperitoneally inject D-luciferin and return the animal to the anesthesia chamber.
6. Start the imaging software. After the imaging system has reached its operating temperature initialize the system by clicking on the 'Initialize' button in the control panel.
7. Select the buttons 'Luminescent' and 'Photograph'. Set the exposure time to 60 s, the binning to 'Medium', the F/Stop to '1', and the EM gain to 'Off'.
8. Confirm that the excitation filter is set to 'Block' and the emission filter to 'Open', and set the subject height to 1.50 cm.
9. Ten min after injection with D-luciferin, place the animals onto the heating plate in the imaging chamber and ensure that their muzzles are correctly placed in the anesthesia outlet. Close the isoflurane/oxygen flow from the inhalation chamber and open the flow for the imaging chamber with a flow rate of 0.25 L/min and an evaporation of 2%. Close the door of the imaging chamber properly.
10. Click on the 'Acquire' button to measure the bioluminescence, which takes 60 s.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
27-gauge syringe	VWR	613-4900
Isoflurane	Piramal Healthcare	PZN 4831850
Depilatory cream	Veet
Secureline lab marker	Neolab	25040
D-luciferin potassium salt	Acris	LK10000	30 mg/mL stock solution
Thermomixer	Eppendorf	5776671
Sonopuls Mini20 sonicator	Bandelin	3648
IVIS Lumina II imaging system	PerkinElmer
Living Image 3.0 Software	PerkinElmer
Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice	The Jackson Laboratory	4479