Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats methods
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Isolation and Culture of SHEDs
Cells were obtained from human exfoliated deciduous teeth of a child, whose parents had signed an informed consent. Material was collected under the approval of the Lithuanian Bioethics committee (Nr. 6B-08-173). Briefly, the collected tooth was washed in phosphate-buffered saline (PBS) and incubated in low glucose (LG; 1 mg/ml) Dulbecco's modified Eagle's medium (DMEM; Biochrom) with 200 U/ml penicillin, 200 µg/ml streptomycin and 2.5 µg/ml of amphotericin B (all from Biochrom, Berlin, Germany). Pulp tissue was scraped out and placed in collagenase type I (Gibco, Invitrogen, Grand Island, NY) solution, the latter of which consisted of 0.2% collagenase in DMEM with 1% bovine serum albumin (BSA; Applichem, Darmstadt, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin and incubated for 1 hour at 37°C in an orbital shake...
Isolation of EVs
Isolation of EVs was performed using differential centrifugation according to the described protocol with some modifications. All centrifugation steps were performed at 4°C. Supernatants collected from SHEDs cultivated in serum-free medium MSC NutriStem XF (Biological Industries) were centrifuged successively at increasing speeds (300 g for 10 minutes, 2,000 g for 10 minutes, then at 20,000 g for 30 minutes). The final supernatants were ultracentrifuged at 100,000 g for 70 minutes in Sorvall LYNX 6000 ultracentrifuge, with rotor T29-8x50 in oak ridge centrifuge tubes with sealing caps (all from Thermo Fisher Scientific, Rochester, NY), then the pellets were washed in 40 ml PBS and ultracentrifuged again at 100,000 g for 70 minutes. Final pellets of EVs (exosomal fraction) were resuspended in sterile PBS and stored at -20°C.
Isolation of EVs
Nanoparticle tracking analysis (NTA) was performed with NanoSight LM10 (Malvern Panalytical, Malvern, UK). NTA analyses revealed that EV fractions contained vesicles of approximately 100 nm in size (Fig. A- C). EV fractions were also positive for the characteristic markers of exosomes (Syntenin 1, HSP70, MFG-E8; Fig. C). All preparations of EVs were derived from the same SHED line. Before the experiment, all EV preparations were pooled and divided into the single dose aliquots (10 µl). According to the NTA measurements single dose of EV contained 2.85 × 10 8 vesicles.
Animals
Male Wistar rats (280 ± 20 g ) were obtained from the State Research Institute Centre for Innovative Medicine Laboratory animals breeding house, Vilnius, Lithuania. Experiments on animals were performed under the approval of the Lithuanian Laboratory Use Ethics Committee under the State Food and Veterinary service (No. G2-51). All efforts were made to minimize animal suffering and reduce the number of animals used. The experiments were conducted in accordance with the EU Directive 2010/63/EU and local laws and policies on the protection of animals used for scientific purposes. The animals were housed in a controlled laboratory environment (temperature 22°C ± 2°C, humidity 55%-65%, 12-hour light/dark cycle), five animals per polypropylene cage with food and water provided ad libitum.
Experimental Design (In Vivo)
EVs were administered using a small pipette. Solution of 5 µl was administered in each nostril with 2 minutes interval. Each daily dose contained 2.85 × 10 8 vesicles/10 µl.
Unilateral 6-OHDA Lesion
Thirty minutes before the induction of general anesthesia, rats received imipramine (20 mg/kg) to protect adrenergic neurons against the development of 6-OHDA-induced lesions. The animals were anesthetized with isoflurane (3%-3.5% for induction and 2% for maintenance) and placed on a stereotaxic frame (Stoelting Inc.). On experimental day 15, unilateral 6-OHDA (20 µg in 3 µl of 0.2% ascorbic acid) or aCSF (control, 3 µl) were injected intra-MFB during stereotaxic surgery using the following coordinates: -2.2 mm anteroposterior, +1.5 mm mediolateral, and -8.0 mm dorsoventral relative to the bregma, using a 27-gauge needle attached to a 50 µl microsyringe (Hamilton). Injection flow was controlled using an electronic pump (World Precision Instruments, Sarasota, FL) at a rate of 1 µl per min. The mi...
Apomorphine-Induced Rotation Behavior
On experimental day 23, apomorphine-induced rotational behavior was used to evaluate the dopaminergic neuron lesion induced by 6-OHDA. Apomorphine was dissolved in saline and injected subcutaneously at the dose of 0.2 mg/kg. After 5 minutes, the rotations were monitored for 30 minutes. The number of contralateral rotations (to the nonlesioned side) was recorded by examiner blinded to the groups.
CatWalk Gait Test
The CatWalk XT quantitative gait analysis system (Noldus, Wageningen, The Netherlands) is a nonintrusive, accurate tool to determine gait ability in rodents. This method is described elsewhere,. In brief, it is based on rodent voluntary movement through an enclosed 1.3 m long glass walkway that is illuminated with green fluorescent lighting the walkway from the side and reflecting internally after the rodent comes in contact with the glass floor. High-speed video camera was located under the walkway and was used to obtain footprint images. The rats were trained twice: 7 days prior to 6-OHDA injection and on experimental day 7. Before each training session, the animals were placed on the walkway and allowed to habituate for 2 minutes. Testing was performed on experimental day 20 between 11:00 and 14:00, at least 1 hour after intranasal EVs administration. Testing was succ...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
from paperStatistical comparison
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
inferred from protocolSource and audit
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Evidence quotes (8)
Cells were obtained from human exfoliated deciduous teeth of a child, whose parents had signed an informed consent. Material was collected under the approval of the Lithuanian Bioethics committee (Nr. 6B-08-173). Briefly, the collected tooth was washed in phosphate-buffered saline (PBS) and incubated in low glucose (LG; 1 mg/ml) Dulbecco's modified Eagle's medium (DMEM; Biochrom) with 200 U/ml penicillin, 200 µg/ml streptomycin and 2.5 µg/ml of amphotericin B (all from Biochrom, Berlin, Germany). Pulp tissue was scraped out and placed in collagenase type I (Gibco, Invitrogen, Grand Island, NY) solution, the latter of which consisted of 0.2% collagenase in DMEM with 1% bovine serum albumin (BSA; Applichem, Darmstadt, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin and incubated for 1 hour at 37°C in an orbital shaker platform. After digestion, the cell suspension was diluted in PBS and centrifuged at 250 g for 5 minutes. The supernatant was discarded, cells resuspended in LG DMEM supplemented with 10% fetal bovine serum (Gibco), glutamine and antibiotics and plated. Cultures were maintained at 37°C in a h...
Isolation of EVs was performed using differential centrifugation according to the described protocol with some modifications. All centrifugation steps were performed at 4°C. Supernatants collected from SHEDs cultivated in serum-free medium MSC NutriStem XF (Biological Industries) were centrifuged successively at increasing speeds (300 g for 10 minutes, 2,000 g for 10 minutes, then at 20,000 g for 30 minutes). The final supernatants were ultracentrifuged at 100,000 g for 70 minutes in Sorvall LYNX 6000 ultracentrifuge, with rotor T29-8x50 in oak ridge centrifuge tubes with sealing caps (all from Thermo Fisher Scientific, Rochester, NY), then the pellets were washed in 40 ml PBS and ultracentrifuged again at 100,000 g for 70 minutes. Final pellets of EVs (exosomal fraction) were resuspended in sterile PBS and stored at -20°C.
Nanoparticle tracking analysis (NTA) was performed with NanoSight LM10 (Malvern Panalytical, Malvern, UK). NTA analyses revealed that EV fractions contained vesicles of approximately 100 nm in size (Fig. A- C). EV fractions were also positive for the characteristic markers of exosomes (Syntenin 1, HSP70, MFG-E8; Fig. C). All preparations of EVs were derived from the same SHED line. Before the experiment, all EV preparations were pooled and divided into the single dose aliquots (10 µl). According to the NTA measurements single dose of EV contained 2.85 × 10 8 vesicles.
Male Wistar rats (280 ± 20 g ) were obtained from the State Research Institute Centre for Innovative Medicine Laboratory animals breeding house, Vilnius, Lithuania. Experiments on animals were performed under the approval of the Lithuanian Laboratory Use Ethics Committee under the State Food and Veterinary service (No. G2-51). All efforts were made to minimize animal suffering and reduce the number of animals used. The experiments were conducted in accordance with the EU Directive 2010/63/EU and local laws and policies on the protection of animals used for scientific purposes. The animals were housed in a controlled laboratory environment (temperature 22°C ± 2°C, humidity 55%-65%, 12-hour light/dark cycle), five animals per polypropylene cage with food and water provided ad libitum.
EVs were administered using a small pipette. Solution of 5 µl was administered in each nostril with 2 minutes interval. Each daily dose contained 2.85 × 10 8 vesicles/10 µl.
Thirty minutes before the induction of general anesthesia, rats received imipramine (20 mg/kg) to protect adrenergic neurons against the development of 6-OHDA-induced lesions. The animals were anesthetized with isoflurane (3%-3.5% for induction and 2% for maintenance) and placed on a stereotaxic frame (Stoelting Inc.). On experimental day 15, unilateral 6-OHDA (20 µg in 3 µl of 0.2% ascorbic acid) or aCSF (control, 3 µl) were injected intra-MFB during stereotaxic surgery using the following coordinates: -2.2 mm anteroposterior, +1.5 mm mediolateral, and -8.0 mm dorsoventral relative to the bregma, using a 27-gauge needle attached to a 50 µl microsyringe (Hamilton). Injection flow was controlled using an electronic pump (World Precision Instruments, Sarasota, FL) at a rate of 1 µl per min. The microsyringe needle was left in the injection site for 5 minutes after each injection to avoid liquid reflux.
On experimental day 23, apomorphine-induced rotational behavior was used to evaluate the dopaminergic neuron lesion induced by 6-OHDA. Apomorphine was dissolved in saline and injected subcutaneously at the dose of 0.2 mg/kg. After 5 minutes, the rotations were monitored for 30 minutes. The number of contralateral rotations (to the nonlesioned side) was recorded by examiner blinded to the groups.
The CatWalk XT quantitative gait analysis system (Noldus, Wageningen, The Netherlands) is a nonintrusive, accurate tool to determine gait ability in rodents. This method is described elsewhere,. In brief, it is based on rodent voluntary movement through an enclosed 1.3 m long glass walkway that is illuminated with green fluorescent lighting the walkway from the side and reflecting internally after the rodent comes in contact with the glass floor. High-speed video camera was located under the walkway and was used to obtain footprint images. The rats were trained twice: 7 days prior to 6-OHDA injection and on experimental day 7. Before each training session, the animals were placed on the walkway and allowed to habituate for 2 minutes. Testing was performed on experimental day 20 between 11:00 and 14:00, at least 1 hour after intranasal EVs administration. Testing was successful if the animal crossed the walkway without stopping and at least 3-4 each paw placements were recorded. Three complete runs across the walkway were recorded for each animal. CatWalk XT software was used to automatically analyze the video images of the runs. Data analysis was performed wi...
Machine-readable layer
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"datePublished": "2019",
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