Technique for Intranasal Administration of α-Synuclein Aggregates

Parkinson disease was proposed to develop and progress through the prion-like propagation of alpha synuclein aggregates. In the current study, we are trying to associate the origin of alpha synuclein pathology in the olfactory bulb in Parkinson's disease. Recently, alpha synuclein pathology has been believed to propagate in a prion-like manner, starting from the olfactory bulb or the dorsal nucleus of the bigger synapse.

Although the origin alpha synuclein aggregate in the olfactory bulb remains unclear, recent studies suggest that the propagation may alternate from the olfactory mucosa. We showed that into another administration of alpha synuclein aggregates in the mouse model induced alpha synuclein pathology in the olfactory bulb. This simple method provide another way to evaluate alpha synuclein propagation from the olfactory mucosa to the olfactory bulb without a surgery technique.

Intranasal administration of alpha synuclein aggregate is a very simple and straightforward method. To begin, wear personal protective equipment, such as disposable gloves, masks, and safety gobbles. Prepare mouse alpha synuclein fiber solution in PBS at a concentration of four milligrams per milliliter and wrap the tube with a transparent film to prevent contamination.

Then, fill the ultrasonic bath of a sonicator with water and add ice cubes to cool it to four degrees Celsius. Sonicate 200 microliters of the fibril solution to generate alpha synuclein preformed fibrils for five minutes. Place an anesthetized mouse in a supine position on a paper towel, with its head slightly tilted downward.

Next, draw one microliter of the four milligrams per milliliter alpha synuclein solution into a P10 pipette and place the pipette tip near the mouse's nose to form a round drop at the end of the pipette tip. Now, intranasally administer the solution to the unilateral nostril. Wait for 30 seconds to one minute to let the mouse naturally inhale the solution.

Repeat this process until 20 microliters of alpha synuclein is administered. After alpha synuclein administration, clean the bench with commercial detergents if any contamination with the solution occurs and discard the gloves, then administer atipamezole at three milligrams per kilogram by intraperitoneal injection to facilitate recovery. After full recovery, return the mouse to the cage.

To begin, administer alpha synuclein preformed fibrils intranasally to the mice. After euthanizing the mouse, make a two-centimeter incision in the scalp with a scalpel. Using scissors, incise the cranial bone laterally from the base to the nose.

To obtain the olfactory bulbs, completely remove the cranial bone covering the olfactory bulbs, then, turn over the head and sever the brain nerves. Carefully remove the brain with forceps, keeping the olfactory bulbs intact. To preserve the bulbs, carefully cut the olfactory nerves as they attach to the skull bone.

Place the brain samples into 4%paraform aldehyde prepared in PBS and store them at four degrees Celsius overnight. Alpha synuclein pathology was not observed in the olfactory bulb on the treated side after one in three months, but Lewy neurite-like P alpha synuclein aggregates were seen in the glomeruli on the treated side after six and 12 months. These aggregates were absent in the bulbs on the control side.

The number of Lewy neurite-like alpha synuclein aggregates significantly increased in the olfactory bulbs on the treated side over 12 months, while the control side had very few such aggregates.