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
Aim. Evidence-backed execution summary for 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 from 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.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Isolation and Culture of SHEDs
reagent used in the protocol.
- Use
- For the isolation of EVs SHEDs from third to fifth passages were grown until the cultures reached subconfluence, then standard medium was changed to the serum-free medium MSC NutriStem XF (Biological Industries, Kibbutz Beit Haemek, Israel).
Materials and Methods
reagent used in the protocol.
- Use
- The following chemicals were purchased from Sigma-Aldrich, St. Louis, MO: Apomorphine (A4393-1G), 3,3′-diaminobenzidine (DAB, D5905), ExtrAvidin Peroxidase Staining Kit (EXTRA2), Bovine serum albumine (BSA, A2058) and anti-TH antibody (T2928), Triton X-100 (X100), and 6-OHDA...
Isolation and Culture of SHEDs
reagent used in the protocol.
- Use
- 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 incubat...
Isolation of EVs
reagent used in the protocol.
- Use
- 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 successi...
Experimental Design (In Vivo)
reagent used in the protocol.
- Use
- 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.
Experimental Design (In Vivo)
reagent used in the protocol.
- Use
- Overall, during 15 days each rat received a total of 180 µl EV solution containing approximately 43 × 10 8 EVs. After brain removal, immunohistochemical assessments were performed.
Immunohistochemistry
reagent used in the protocol.
- Use
- The next day after the apomorphine rotation test, rats were deeply anesthetized with a ketamine/xylazine (100/10 mg/kg i.p.) mixture, transcardially perfused with ice-cold saline and fixed with cold 4% paraformaldehyde (PFA) solution. The brains were removed and postfixed in 4% PFA for 24 hours. After fi...
Immunohistochemistry
reagent used in the protocol.
- Use
- For each brain, 30 µm thick coronal slices were obtained using a cryotome at -23°C (CM1850, Leica Biosystems, Nussloch, Germany): 6 sections each from both the corpus striatum (AP plane -0.36 mm to -0.60 mm from bregma) and SN (AP plane -4.80 mm to -5.0...
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 successi...
- Use
- 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 successi...
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,...
- Use
- 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,...
Experimental Design (In Vivo)
Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). (A): Transmission electron microscopy of EVs isolated from SHEDs ( × 30,000 magnification). A magnified image of EV is shown on the left panel ( × 120,000 magnification)....
- Use
- Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). (A): Transmission electron microscopy of EVs isolated from SHEDs ( × 30,000 magnification). A magnified image of EV is shown on the left panel ( × 120,000 magnification)....
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 stereota...
- Use
- 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 stereota...
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...
- Use
- 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...
Quantification
The mounted brain sections were scanned using a Pannoramic Midi II Scanner (3DHISTECH, Budapest, Hungary) at × 200 magnification. The optical density of neuronal (TH) staining was determined in the corpus striatum and SN regions. An observer blinded to the treatment of the animals performed all measurements in d...
- Use
- The mounted brain sections were scanned using a Pannoramic Midi II Scanner (3DHISTECH, Budapest, Hungary) at × 200 magnification. The optical density of neuronal (TH) staining was determined in the corpus striatum and SN regions. An observer blinded to the treatment of the animals performed all measurements in d...
Transmission Electron Microscopy of EVs
EV samples for transmission electron microscopy have been prepared according to the previously published protocol with some modifications. Briefly, EVs in PBS were fixed by adding 4% PFA to a final concentration of 2% and incubated for 40 minutes on ice. To absorb the sample, Formvar-carbon coated copper...
- Use
- EV samples for transmission electron microscopy have been prepared according to the previously published protocol with some modifications. Briefly, EVs in PBS were fixed by adding 4% PFA to a final concentration of 2% and incubated for 40 minutes on ice. To absorb the sample, Formvar-carbon coated copper...
Protein Isolation and Western Blot Analysis
For preparation of total cell lysates, cell monolayers were washed twice with cold PBS, pH 7.3, and lysed in Pierce RIPA buffer supplemented with 1 × Halt protease inhibitor cocktail for 15 minutes on ice. Samples were centrifuged at 14,000 g for 30 minutes at 4°C. Supernatants derived after cen...
- Use
- For preparation of total cell lysates, cell monolayers were washed twice with cold PBS, pH 7.3, and lysed in Pierce RIPA buffer supplemented with 1 × Halt protease inhibitor cocktail for 15 minutes on ice. Samples were centrifuged at 14,000 g for 30 minutes at 4°C. Supernatants derived after cen...
Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Behavioral and immunohistochemical results are presented as the mean ± SEM. The behavioral data from apomorphine rotation test were analyzed by Student's t test. CatWalk data were analyzed by two-way analysis of variance (ANOVA) followed by Fisher's LSD post-test and quantitative immunoh...
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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?
Nanoparticle tracking analysis (NTA) was performed with NanoSight LM10 (Malvern Panalytical, Malvern, UK). NTA analyses revealed that EV fractions contained vesicles of approxim...
- 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
Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). (A): Transmission electron microscopy...
- 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
On experimental day 23, apomorphine-induced rotational behavior was used to evaluate the dopaminergic neuron lesion induced by 6-OHDA. Apomorphine was dissolved in s...
- 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
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 desc...
- 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
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
Behavioral and immunohistochemical results are presented as the mean ± SEM.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Nanoparticle tracking analysis (NTA) was performed with NanoSight LM10 (Malvern Panalytical, Malvern, UK). NTA analyses revealed that EV fractions contained vesicles of approxim...; Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). (A): Transmission electron microscopy...; On experimental day 23, apomorphine-induced rotational behavior was used to evaluate the dopaminergic neuron lesion induced by 6-OHDA. Apomorphine was dissolved in s...; 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 desc....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Behavioral and immunohistochemical results are presented as the mean ± SEM. The behavioral data from apomorphine rotation test were analyzed by Student's t test...; We did not observe gait impairments in the CatWalk training on day 7 after 6-OHDA injection (data not shown), whereas significant impairments were observed on postlesion d...; In the apomorphine rotation test, EV-treated 6-OHDA group rats demonstrated a significant decrease (2.2-fold) in the number of contralateral rotations compared...; Significant differences in TH density were observed between groups in the striatum ( F 3,12 = 21.33, p ≤.0001) and SN ( F 3,14 = 5....
from paperReporting output
Report representative outputs alongside summary comparisons for Nanoparticle tracking analysis (NTA) was performed with NanoSight LM10 (Malvern Panalytical, Malvern, UK). NTA analyses revealed that EV fractions contained vesicles of approxim..., Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). (A): Transmission electron microscopy..., On experimental day 23, apomorphine-induced rotational behavior was used to evaluate the dopaminergic neuron lesion induced by 6-OHDA. Apomorphine was dissolved in s..., 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 desc....
inferred from protocolStructured statistical methods
Behavioral and immunohistochemical results are presented as the mean ± SEM. The behavioral data from apomorphine rotation test were analyzed by Student's t test...; We did not observe gait impairments in the CatWalk training on day 7 after 6-OHDA injection (data not shown), whereas significant impairments were observed on postlesion d...; In the apomorphine rotation test, EV-treated 6-OHDA group rats demonstrated a significant decrease (2.2-fold) in the number of contralateral rotations compared...; Significant differences in TH density were observed between groups in the striatum ( F 3,12 = 21.33, p ≤.0001) and SN ( F 3,14 = 5....
source structuredSource and audit
What supports the facts on this page?
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
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "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",
"description": "Evidence-backed execution summary for 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 from 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.",
"totalTime": "PT3442M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Isolation and Culture of SHEDs",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Isolation of EVs",
"text": "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."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Isolation of EVs",
"text": "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."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Animals",
"text": "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."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Experimental Design (In Vivo)",
"text": "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."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Unilateral 6-OHDA Lesion",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Apomorphine-Induced Rotation Behavior",
"text": "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."
},
{
"@type": "HowToStep",
"position": 8,
"name": "CatWalk Gait Test",
"text": "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..."
}
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"tool": [
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"@type": "HowToTool",
"name": "Isolation of EVs"
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