α-シヌクレイン線維の末梢接種後のトランスジェニックマウスにおける神経炎症の生物発光イメージング

The overall goal of this bioluminescence imaging method is to quantify neuroinflammation in the central nervous system of a Parkinson's disease mouse model after peripheral injection of alpha-synuclein fibrils. This method can help determine when and to what degree neuroinflammation occurs in mouse models of neurodegenerative diseases. The main advantage of this technique is the neuroinflammation can be noninvasively and repeatedly monitored until any mice develop neurologic illness.

The implication of this technique is 10th hour diagnosis of neurological disease because an increase in bioluminescence due to nerve inflammation not only accompanies but often preceded neurological disease. Begin by properly anesthetizing the animal for surgery. Administer a toe-pinch and confirm that the animal does not withdraw the limb to indicate a surgical plane of anesthesia.

Apply ophthalmic ointment to prevent dryness while under anesthesia. Use adhesive tape to carefully fix the animal onto a heating plate in a dorsal recumbent position with the head towards the investigator. Then fill a 27 gauge disposable hypodermic syringe with five microliters of sonicated alpha-synuclein fibrils.

Next, use a pair of blunt nosed thumb forceps to hold the animal's mouth open and use the smaller pair of forceps to carefully pull out the tongue to make the underside of the tongue accessible for injection. Insert the needle of the syringe into the right or left bottom side of the tongue in proximity to the hypoglossal nerve. After five seconds, slowly inject the inoculum.

Allow the needle to remain in place for a further five seconds to ensure that the inoculum penetrates the tissue, and then carefully withdraw the needle. After the injection, release the animal and allow it to remain on the heating plate under constant monitoring until complete recovery. For intraperitoneal injections, briefly narcotize the animals in an anesthesia chamber with isoflurane.

Again, pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia. Fill a 27 gauge disposable hypodermic syringe with 50 microliters of sonicated alpha-synucelin fibirls, or PBS. Hold the animal in a dorsal position with the head facing away from the investigator and downward at approximately 45 degrees.

Directly inject into the peritoneum of the mouse without penetrating the small intestine or cecum located behind the abdominal wall. Monitor animals until they have recovered from the anesthesia. Prior to imaging, shave and depilate the heads of isoflurane anesthetized mice.

Color the ears in black with a non-irritating marker to block unspecific bioluminescence. Weigh the animals prior to injection. Then calculate the exact volume of D-Luciferin for injection at 150 milligrams per kilogram of body weight.

Intraperitoneally inject the calculated volume of D-Luciferin, and then return each animal to the anesthesia chamber. 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.

Then select the buttons Luminescent and Photograph. Set the exposure time to 60 seconds, the Binning to medium, the F/Stop to one, and the EM gain to off. Select Block and then Open.

And then under the tab subject height, choose 1.50 from the dropdown menu. Ten minutes after injection with D-Luciferin, place the animals onto the heating plate in the imaging chamber, and ensure that their muscles are correctly placed in the anesthesia outlet. Switch the isoflurane flow from the inhalation chamber to the imaging chamber, and properly close the door of the imaging chamber.

Click on the acquire button to measure the bioluminescence, which takes 60 seconds. Stop the isoflurane flow, and then return the animals to their cages. Use the imaging software to quantify bioluminescence by opening the tool palette menu and selecting ROI Tools.

Under type, select Measurement ROI. Within ROI Tools, click the circle tool and select the number of ROIs to draw from the pull-down menu. Ideally three for three mice.

In the image, right-click each ROI and under properties, adjust the width and height to 1.25 centimeters. Position each ROI over the brain area that is quantified. In the upper left of the image, set the units panel to radiance in photons.

Then open the tool palette menu. Select image adjust and adjust the minimum and the maximum of the color scale. For instance, from 200, 000 to one million.

Lastly, under file, click save to save the data. As shown here, after intraperitoneal injection with alpha-synuclein fibrils, four of the five A53T synuclein GFAP luciferous reporter transgenic mice developed neurologic disease within 229 days as indicated by the purple squares. However, only one of five interglossally injected mice developed neurologic illness within 285 days as indicated by the green circles.

Bioluminescence imaging revealed that the transgenic mice emitted elevated bioluminescence from their brains and spinal cords, shortly before they developed signs of neurologic disease. This bioluminescence was more pronounced after intraperitoneal injection and absent after PBS injection suggesting an absence of astrocytic gliosis. This figure shows the bioluminescence in the brains of transgenic mice over time after intraperitoneal injection of alpha-synuclein fibrils.

As shown by the blue, green, brown, and orange circles. Increased levels of bioluminescence above a threshold of 2 million photons per second per square centimeter per steradian were observed some weeks before these four individual mice developed neurologic signs of disease. One of five animals also showed elevated levels of bioluminescence shortly before it died 285 days after intraglossal injection with alpha-synuclein fibrils.

In contrast, for healthy PBS injected control mice, and one animal that did not develop disease after intraperitoneal injection, with alpha-synuclein fibrils indicated by the red circles, the bioluminescence signal remained below threshold within the period of observation. Immunofluorescence staining of brain sections of diseased animals with antibodies against GFAP and Iba1 confirmed neuroinflammation with microgliosis and reactive astrocytes in regions with alpha-synuclein deposits. Healthy animals that did not accumulate deposits of alpha-synuclein showed little staining for GFAP and Iba1.

Once mastered, this technique can be done in less than one hour if it's performed properly. After its development, this technique paved the way for researchers in the field of neurodegeneration to noninvasively explore neuroinflammation in the central nervous system of mice. After watching this video, you should have a good understanding of how to peripherally inject mice with alpha-synuclein fibrils and how to perform bioluminescence imaging to monitor CNS neuroinflammation.

The overall goal of the procedure is to peripherally induce neurologic illness by intraglossal or intraperitoneal injection with alpha-synuclein fibrils and to noninvasively image the induction of neuroinflammation over time.