Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

All procedures including animals were performed with approval of the animal protection committee of North Rhine-Westphalia State Environment Agency (LANUV). Animals were housed and cared for according to standard conditions with a 12 h light/dark cycle and free access to food and water.

1. Animal Model

1. Intercross hemizygous Tg(Gfap-luc^+/-) mice with hemizygous Tg(M83^+/-) mice to generate hemizygous bigenic Tg(M83^+/-:Gfap-luc^+/-) mice^15,16.
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^12,17.
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) as has been previously described¹⁴.
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 or 2 µg/µL for intraglossal 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. Intraglossal Injections

1. Anesthetize animals with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia. Use vet ointment on the animal's eyes to prevent dryness while under anesthesia.
2. Fix narcotized animals carefully with adhesive tape onto a heating plate in a dorsal recumbent position with their heads facing towards the investigator.
3. Fill a 27 G disposable hypodermic syringe with 5 µL of sonicated alpha-synuclein fibrils or PBS.
4. Use one blunt-nosed thumb forceps with serrated tips to hold the animal’s mouth open, and a second smaller pair of forceps to carefully pull out the tongue to make the bottom side of the tongue accessible for injection.
5. Inject the needle of the syringe into the right or left bottom side of the tongue in proximity to the hypoglossal nerve. Slowly inject the inoculum after 5 s. Slowly retract the needle after 5 s to ensure that the inoculum has penetrated the tissue and is not lost while retracting the needle.
6. Release the animal from its fixations and leave it on the heating plate under constant monitoring until complete recovery.

4. 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.

5. 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. Stop the isoflurane flow and monitor the animals until they have completely recovered after returning them back to their cages.
11. Use the imaging software to quantify the bioluminescence. Within the 'Tool Palette' select 'ROI Tools'. Under 'Type' within the 'ROI Tools' select 'Measurement ROI'.
12. Within the 'ROI Tools' click the "circle" tool and select the number of ROIs to draw from the pull down menu – ideally '3' for three mice.
13. In the image, right-click each ROI and under 'Properties' adjust the width and height to 1.25 cm. Position each ROI over the brain area that is quantified.
14. In the upper left of the image, set the units panel to 'radiance (photons)'.
15. Within the 'Tool Palette', select 'Image Adjust' and adjust the minimum and the maximum of the 'Color Scale', for instance, from 0.20e6 to 1.00e6.
16. Under 'File' save the data with 'Save'.

6. Biochemical Analysis

1. Isolate the brains and spinal cords according to established protocols^18,19.
2. Homogenize whole brain and whole spinal cord samples separately in PBS in the presence of protease and phosphatase inhibitors (diluted to 1x in PBS) in two 30 s cycles with 6,000 rpm in a tissue homogenizer. Ensure that the brain and spinal cord samples are homogenized to a final concentration of 20% (wt/vol).
3. Sonicate the samples twice while keeping them on ice with a constant pulse of 10 s and an amplitude set to 50%.
4. Adjust the homogenates to 10% in PBS and 750 mM NaCl by diluting them 1:1 with 1.5 M NaCl in PBS.
5. To separate the cellular debris, centrifuge the homogenates at 1,000 x g for 5 min at 4 °C. Keep the supernatants for the next steps and discard the pellets.
6. Measure the protein concentration in the homogenates using the bicinchoninic acid (BCA) assay according to the manufacturer's instructions. Incubate 1.0 mg of total protein from brain homogenates or 0.8 mg from spinal cord homogenates in N-laurylsarcosyl at a final concentration of 10% (wt/vol) for 15 min on ice.
7. Overlay the homogenates onto a 3 mL sucrose cushion of 10% (wt/vol) in distilled water and ultracentrifuge them at 465,000 x g for 1 h at 4 °C.
8. Resuspend the pellets in a buffer containing 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 192 mM glycine, 25 mM Tris, and 5% (wt/vol) sucrose.
9. Boil the samples at 100 °C for 5 min and load them onto SDS-polyacrylamide gels for electrophoresis in a morpholineethanesulfonic acid (MES) buffer system.
10. Transfer the separated proteins to polyvinylidene difluoride (PVDF) membranes in a semidry blotting system.
11. Incubate the membranes in 0.4% (vol/vol) formalin solution in PBS for 30 min at room temperature on a rotor with 35 rpm to cross-link the proteins with the membrane.
12. Block the membranes in TBS buffer containing 0.05% (vol/vol) Tween 20 and 5% (wt/vol) milk for 1 h at room temperature.
13. Incubate the blots with the primary antibody in blocking buffer overnight at 4 °C.
14. Wash the membranes three times in TBS buffer and incubate them with peroxidase- or fluorophore-conjugated secondary antibodies in TBS for 1 h at room temperature.
15. Visualize the bound secondary antibodies by chemiluminescence or fluorescence according to established protocols²⁰.

7. Immunofluorescence Analysis

1. Dehydrate the dissected brains and spinal cords in a tissue processing station and embed them into paraffin using a paraffin station.
2. Cut the paraffin-embedded tissues with a microtome in 8 µm thick coronal sections and mount the sections on glass slides.
3. Deparaffinize the tissue sections by incubating them in two separate xylol baths for 5 to 10 min and rehydrate them through a series of graded ethanol baths (100%, 90%, 70%, 50%) and finally with H₂O.
4. Incubate the sections in 0.01 M citrate buffer (pH 6.0) for 5 min at room temperature and additionally boil them for 10 min in a microwave oven.
5. Let the sections cool down to room temperature and incubate them with a 3% hydrogen peroxide solution for 30 min to inhibit endogenous peroxidases.
6. Block the tissue sections by incubation with 20% (vol/vol) normal goat serum, 1% (vol/vol) bovine serum albumin (BSA), and 0.5% Triton X-100 in PBS for 1 h at room temperature.
7. Incubate the sections with the primary antibody diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and 0.25% Triton X-100 in PBS overnight at 4 °C.
8. Wash the sections twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS.
9. Stain the sections with the corresponding fluorophore-conjugated secondary antibodies and the nuclear dye DAPI diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and PBS for 1 h at room temperature.
10. After washing twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS, coverslip the slides with embedding media and visualize the staining with a confocal laser scanning microscope.