Platelet concentrates (PCs) collection

Allogeneic PCs were collected using apheresis with a MCS + platelet collection system (Haemonetics, Braintree, MA, USA) from volunteer regular healthy donors at the Taipei Blood Center (Guandu, Taiwan), with approval from the Institutional Review Board of Taipei Medical University (TMU-JIRB N201802052). These clinical-grade PCs, initially intended for transfusion, were suspended in 100% plasma, were not leucoreduced, and were anticoagulated with a citrate–phosphate-dextrose solution. They were stored at 22 ± 2 °C under mild agitation, following standard licensed procedures. Upon reaching their expiry date, five days after collection, the PCs were transported to the Taipei Medical University laboratory within 90 min under controlled ambient conditions. Upon arrival, the PCs were placed on a slow-speed platelet agitator at 22 ± 2 °C and processed either the same day or the next. Before processing, the sample was collected aseptically to determine the blood cell count using a ABC Vet blood cell counter from ABC Diagnostics (Montpellier, France).

Preparation of PEVs

PEVs were prepared from at least three PC donations (n = 3) and were later pooled for analysis. PEVs were obtained from the plasma supernatant of PC, as previously described [30] (Fig. 1). Firstly, PC was centrifuged at 3000×g for 30 min to pelletize the platelets. The supernatant underwent a second centrifugation at 6000×g for 10 min at 25 ± 2 °C to eliminate any residual cell debris. PEVs were isolated through high-speed centrifugation at 25,000×g for 90 min at 18 ± 2 °C. The recovered PEV pellet was washed, re-suspended in phosphate buffer saline (PBS) at a ratio of 0.01 mL per mL of the original PC volume, aliquoted, and stored at − 80 °C until further use.

Fig. 1

Preparation of PEVs. Diagram illustrating the process of preparing PEVs. PC was centrifuged at 3000×g for 30 min to pelletize the platelets and obtain PC supernatant followed by 6000×g for 30 min to remove the residual platelets. PEVs were isolated by high-speed centrifugation at 25,000×g for 90 min and resuspended in PBS. PCs: Platelet concentrates; PEVs: Platelet-extracellular vesicles

Biophysical and membrane markers characterization of PEVs

Dynamic Light Scattering (DLS): We used 100 µL samples for DLS size distribution measurements in a disposable low-volume cuvette, as described previously [22, 30]. Measurements were made using a Zetasizer Nano ZS from Malvern Instruments, UK.

Nanoparticle Tracking Analysis (NTA): NTA (NanoSight NS300, Malvern Instruments, UK) was used. PEVs were diluted 10,000-fold in 0.1 µm-filtered PBS. An 800 µL sample was injected via a syringe pump and data was collected over 60 s.

Tunable resistive pulse sensing (TRPS): TRPS analysis was done by qNano (Izon, New Zealand) with 400 nm pore size of nanopore membranes and 210 nm (CPC200) particle size calibrating beads. 40 µL of pre-diluted PEVs (10-fold) were introduced to the upper fluid cells. The particles rate was recorded for 10 min. The data were processed by qNano software.

PEV surface markers: The EV markers were detected using a Exo-Check Exosome antibody membrane array (EXORAY210B-8) from System Biosciences, following the manufacturer's instructions. 50 µg of PEVs sample was treated with lysis buffer, followed by the addition of 1 µL of labeling reagent, and a 30-min incubation with continuous mixing. Any excess labeling reagent was removed by using a provided separation column and several washing steps. After, 5 mL blocking buffer was added. The labelled PEVs lysate/blocking buffer mixture then was introduced to an antibody array membrane and incubated overnight at 4 °C on a shaker. Subsequently, the membrane was washed to eliminate free molecules and followed by the incubation of the detection reagent mixture for 30 min at room temperature. Following this step, streptavidin-HRP and chemiluminescent detecting solutions were added. Each location emitted light corresponding to the presence of antibodies bound to their respective immobilized capture sites on the membrane, which then was imaged using an Invitrogen iBright CL750 system. The blank served as an internal control. To quantify the intensity of the antibody-bound bands, the background intensity of the blank was subtracted from each band’s intensity.

Cryo-electron microscopy (cryo-EM): Samples were prepared for cryo-EM on a FEI Vitrobot by pipetting 4 μL onto 200 mesh holey carbon film with glow-discharged 20 s (Electron Microscopy Sciences), blotted for 3 s in 100% humidity at 4 °C, and stored in liquid nitrogen until imaging. Images were taken at 50,000-fold magnification a dose of 2417 e/nm2s operating at 200 kV on a FEI Tecnai F20 at Cryo-EM facility, Academia Sinica (Nangang, Taiwan).

Proteins, growth factors quantification, proteomics, and bioinformatic analysis

The total protein content was measured using a bicinchoninic acid (BCA) assay. PEVs was initially diluted 10-fold. A 25-µL sample then was mixed with 200 µL of BCA working reagent (1:50 ratio) in a 96-well plate and shaken for 30 s. After incubating for 30 min at 37 °C, the purple color that developed was measured at 562 nm. Bovine serum albumin (BSA) was used as a standard, with concentrations ranging from 25 to 2000 µg/mL.

The levels of growth factors present in PEVs were quantified in triplicate using a DuoSet ELISA kit, following the manufacturer's instructions (R&D Systems, DY248, DY222, DY795, DY236, DY293, Minneapolis, MN, USA), as done previously [31]. PEVs were diluted 1000-, 10-, and 5-fold to determine the concentrations of PF4, PDGF-AB, and BDNF, respectively. EGF and VEGF were measured without dilution. Absorbance was measured and analyzed with a BioTek EPOCH 2 microplate reader (Santa Clara, CA, USA).

The Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) procedure for proteomic analysis was performed as previously described [7]. PEVs samples were treated overnight with acetone pre-cooled to − 20 °C at a sample-to-acetone volume ratio of 1:4. The mixture was then centrifuged at 15,000×g for 10 min at 4 °C. The resulting pellet was washed twice using a 1:4 ratio of cold acetone to water, followed by centrifugation at 13,000×g for 10 min at 4 °C. The supernatant was discarded, and the pellet was air-dried and re-suspended in 6 M urea. Protein content was quantified using the BCA protein assay, and 20 μg of protein was used. Data Analysis: For each raw data file generated by MS, peak lists were prepared using Data Analysis version 4.3 (LC-QTOF; Bruker Daltonics, Billerica, MA, USA) and Proteome Discoverer versions 2.2 or 2.4 (LTQ Orbitrap; ThermoFisher Scientific). An human UniProt Swiss-Prot database (comprising 20,431 annotated proteins, release 2019.07) was used for analysis. The false discovery rate (FDR) for spectrum and protein matching was set at 1%. We have included HPPL sample in this proteomic analysis simultaneously with PEVs to assess the similarity of their protein lists. Furthermore, the PEVs proteins were identified using Vesiclepedia and ExoCarta, the two main manually curated databases that compile identified EV cargo. The functional enrichment analyses were performed using an open-access software, Functional Enrichment analysis tool (FunRich, FunRich version 3.1.4).

For western blot (WB) analysis, 30 mg of PEVs’ protein were suspended in Bio-rad LDS samples buffer 4X and heated for 5 min at 100 °C. Samples were loaded onto GenScript 12% SDS-PAGE gels and separated for 30 min at 200 V. The proteins were then electrotransferred for 50 min at 100 V onto a hydrophobic polyvinylidene difluoride membrane (PVDF) (Pall, USA). After 45 min of blocking in 5% BSA, the membranes were incubated with the primary antibody for an additional night at 4 ± 2 °C. The monoclonal antibodies were used as follow: anti-rabbit CD41 (1/1000, ab134131, Abcam), anti-rabbit CD61 (1/1000, ab7166, Abcam), anti-rabbit CD42a (1/1000, ab133573, Abcam), CD62P (1/1000, ab255822, Abcam), anti-mouse CD9 (1/500, sc-13118, Santa cruz), and anti-mouse CD63 (1/500, sc-5275, Santa cruz). The membranes were then washed three times with 1 × Tris-NaCl-Tween-20 (TNT) for 15 min before the incubation with horseradish peroxidase (HRP)-labeled secondary antibodies for 45 min at room temperature. Immuno-reactivity was detected using the ECL kit (RPN2106, GE Healthcare), and visualized with a UVP system (Level, 115 V ~ 60 Hz, Upland CA, USA).

Procoagulant assays

Microparticle (MP)-activity assay: The assessment of pro-thrombogenic activity associated with the presence of PEVs expressing functional PS was conducted using an Zymuphen MP-activity assay (Hyphen BioMed, Paris, France), as previously described [31]. 100 µL of PEVs were pre-diluted at 2 × 103 and 3 × 104-fold respectively, in the sample diluent. These were then added to an Annexin-precoated microplate and incubated for 60 min at 37 °C. Activation of prothrombin to thrombin was initiated by adding 100 µL of factor Xa-Va in combination with calcium, and 50 µL of prothrombin, followed by a 10-min incubation at 37 °C. After five washing steps with 300 µL of a washing solution, 50 µL of a chromogenic substrate was introduced. The subsequent formation of a chromogenic substance following a 3-min incubation at 37 °C was halted by addition of 50 µL of 2% citric acid. Absorbance was then measured at 405 nm. Platelet pellet lysate (PPL) and its heat treated fraction (heated-PPL or HPPL), which are prepared from isolated platelets and are enriched in platelet factors and contain PEVs [9, 31], were included as controls using the same volume as the PEVs.

STA-procoagulant-phospholipid assay-(PPL): The STA-PPL assay (Diagnostica, Stago, Asnières, France) was used to evaluate the procoagulant activity associated with PEVs, as described [32]. Undiluted PEVs, PPL and HPPL (25 µL) were combined with 25 µL of citrated human plasma depleted of phospholipids. This mixture was incubated at 22 ± 2 °C for 120 s. The coagulation process was triggered upon adding 100 µL of activated factor X (FXa), and the clotting time was then recorded using an STA compact automatic coagulometer. Both positive and negative controls, as provided by the kit, were tested following the manufacturer's guidelines.

Assessment of neuroprotective activity of PEVs in vitro in LUHMES cells

LUHMES cell culture and treatment: LUHMES cells were seeded at a density of 4 × 106 in a flask pre-coated with 50 μg/mL poly-l-ornithine (P3655, Sigma-Aldrich) and 1 μg/mL fibronectin (F1141, Sigma-Aldrich). They were cultured in Advanced DMEM/F12 medium (12634010, Thermo Fisher), supplemented with N2 (17502-048, Thermo Fisher), 2 mM l-glutamine (25030-124, Thermo Fisher), and 40 ng/mL recombinant bFGF (4114-TC, R&D Systems). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 to facilitate proliferation. For differentiation, 2.5 × 106 cells were seeded in a T75 flask containing the proliferation medium and incubated for 24 h. The medium was then switched to a differentiation medium, composed of Advanced DMEM/F12, N2 supplement, 2 mM l-glutamine, 1 mM dibutyryl-cAMP (D0627, Sigma-Aldrich), 1 μg/mL tetracycline (T-7660, Sigma-Aldrich), and 2 ng/mL recombinant human BDNF (DY248, R&D Systems). After two days, the cells were transferred to 24-well plates to complete the differentiation process over an additional three days. On the fifth day of differentiation, LUHMES were treated with 5% (v/v) PEVs and, subsequently, a dose of 1 µM erastin (E7781, Sigma-Aldrich) was added for neurotoxic stimulation through induction of ferroptosis. Cell viability was evaluated 24 h later.

Cell viability assay: 50 μL of CCK-8 solution (96992, Sigma-Aldrich) containing WST-8 was added to each well containing 500 μL of cell medium. The amount of formazan produced is proportional to the number of viable cells. The plate was incubated for 4 h at 37 ± 1 °C in a 5% CO2 humidified incubator. After incubation, absorbance at 450 nm was measured using a Tecan Infinity M200 microplate reader. Viability was calculated as a percentage of untreated control cells, using the formula: [(O.D. of sample − O.D. of blank)/(O.D. of control − O.D. of blank)] × 100.

PEVs impact on differentiation and restoration of human SH-SY5Y neuroblastoma cells

SH-SY5Y cells were grown in DMEM (11965092, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin. The cells were incubated at 37 ± 1 °C in a humidified 5% CO2 atmosphere. They were kept in T75 flasks until reaching 80–90% confluence. For sub-culturing, the cells were washed with PBS, treated with trypsin–EDTA for 2–3 min until detached, and then re-suspended in fresh medium. Depending on the experiment, they were either re-seeded in T75 flasks or plated at a specific density. To determine the ability of PEVs to stimulate cell maturation, undifferentiated SH-SY5Y cells were seeded at 24 well-plate and after one day of incubation, they were treated using 10 μM retinoic acid (RA, RM2625, Sigma-Aldrich) as a differentiation agent, 2% (v/v) PEVs, or 2% (v/v) HPPL. RA and HPPL served as a positive control and some cells were maintained in a medium containing 0.5% FBS without any treatment, used as a negative control group. The culture medium was refreshed every three days, and the cells were examined on the seventh day of the experiment. To assess the promotion of SH-SY5Y cell differentiation, fluorescence labelling with β-III tubulin (Ab18207, Abcam, 54 kDa, 1:500 dilution) was used. The culture medium was removed, and the cells were rinsed with PBS before fixation in 2% paraformaldehyde (PFA), at RT for 30 min. Following this, the cells were permeabilized for 20 min at RT using 0.2% PBS-Triton X-100, and non-specific binding was blocked for 1 h with 1% BSA in PBS. The primary anti-β-III tubulin (Ab18207, Abcam) was added and incubated overnight at 4 °C. After, the wells were washed with PBS, and the cells were incubated for 1 h with Alexa Fluor-488-conjugated goat anti-rabbit IgG antibody (A32723, Invitrogen), the nuclei were stained using DAPI. Fluorescence images were captured using a Leica DMi8 fluorescence microscope (Sage Vision, West Chester, PA, USA). The fluorescence intensity of β-III tubulin was quantified using Image J software (1.6, NIH, Bethesda, MA, USA) by measuring the integrated fluorescence density for each treatment in three separate trials. Furthermore, the quantification of neurite extensions was done by FIJI plugin Simple Neurite Tracer (SNT. V4.2.1). From the captured images of B-III tubulin fluorescence, the length of each neurite extension was measured individually (Figure S1). The ratio of neurite length in treated cells to that in untreated cells was then calculated. Moreover, to investigate the neurorestorative properties of PEVs, a scratch assay was conducted on SH-SY5Y cells that were differentiated by RA treatment. Following this, a 100 µL tip was used to create a wound in the cell monolayer. Subsequently, we added 5% (v/v) of PEVs and HPPL at the same dose as a positive control. The wounded zone free of cells was examined under microscopy (Leica DMi8 microscope, Wetzlar, Germany) over two days. The results are expressed as a wound-healing index, determined by the formula: (initial wound area − final wound area)/initial wound area.

Assessment of anti-inflammatory activity of PEVs in BV-2 microglia cells

BV-2 microglia cell culture: We used immortalized mouse microglial BV-2 cells to assess the ability of PEVs to modulate microglial activation. BV-2 cells were cultured in T75 flasks using DMEM medium (SH30243, Thermo Fisher) supplemented with 10% FBS and 100 U/mL of penicillin. The cells were incubated at a controlled temperature of 37 ± 1 °C in a 5% CO2 humidified incubator. When the cell confluence reached 80–90%, the cells were passaged. A total of 2 × 104 cells were grown per well in 24-well plates to be used for our experiments. The cells were supplemented with 10% FBS until confluent. Then, they were pre-treated with PEVs at a concentration of 5% (v/v) medium and 5% (v/v) HPPL, which was used as a non-inflammatory control. After 1 h, 100 ng/mL of lipopolysaccharide (LPS, L4130, Sigma-Aldrich) was added to trigger inflammation. The cells lysate and supernatant were collected after a 24-h incubation period. The upregulation of Tnf-\(\alpha\) gene expression was assessed in the cell lysate by using quantitative reverse transcription PCR (RT- qPCR). Mouse TNF-α (DY410, R&D System) IL-6, and IL-1β ELISA kits (431304, 432604, BioLegend) were used following the manufacturer's instructions to determine the levels of inflammatory markers in the cell supernatant.

Assessment of neuroprotective activity of PEVs in TBI and PD animal models

Study of PEVs diffusion in the mice brain: PEVs, as well as HPPL used as a control, were labelled with Alexa Fluor 568 dye (Thermo Fisher Scientific, San Jose, CA, USA) following the supplier's instructions and as described in our recent study [33]. A total volume of 60 μL of fluorescently-labelled PEVs or HPPL were administered intranasally, 2–3 μL at a time, alternating the nostrils. The mice were anaesthetized 7 h after the final administration using Zoletil-50 (66F4, Virbac, France) and Rompun (PP1523, Bayer, Switzerland) and perfused with 0.9% cold NaCl. The brains were subsequently fixed in 4% PFA and transferred to a cryoprotective solution. Sagittal cryosections with 30 μm thickness were prepared and imaged using a fluorescent slide scanner (ImageXpress® Pico) for observation.

Ethical approval for animal study of CCI mouse model of TBI: The study adhered to ethical guidelines for the welfare of animals and were conducted following the animal use protocol from Taipei Medical University (TMU, Taipei, Taiwan; application no. LAC 2020-0042). Male C57/BL6 mice (8-week-old, 20–30 g weight) were obtained from the Taiwan National Laboratory Animal Center (Nangang, Taipei, Taiwan). The mice were kept in groups of 4–5 per cage (cage size: L29.3 × W18.9 × H12.9 cm ± 3%) in a controlled environment with constant temperature (19–24 °C) and humidity (60–70%) on a 12-h light/dark cycle at TMU Laboratory Animal Center.

CCI method: The mice were randomly allocated into three groups: a sham group, and two treatment groups receiving PEVs and PBS (as a control, n = 7–10 mice/group). Anesthesia and Surgery: Mice were anesthetized via intraperitoneal injection with a mixture of Zoletil (10 mg/kg) and xylazine (10 mg/kg) given at a dosage of 10 μL/g body weight. Following anesthesia, the surgical area was shaved, and the mouse was placed in a stereotaxic device with its head immobilized by ear bars. Surgical sites were cleansed by using cotton tips soaked in iodine and ethanol. A midline incision exposed the skull, where a 4-mm diameter hole was made between the bregma and lambda on the right hemisphere. Mild injury was inflicted using an impactor (eCCI-6.3, Custom Design & Fabrication, Sandston, VA, USA) with specific parameters (3-mm tip at a velocity of the actuator of 3 m/s, a deformation depth of 0.2 mm, and a dwell time of 250 ms) as before [7]. The injury was initiated by hitting the surface of the cortex perpendicularly. Sham-operated (Sham) mice underwent a hole-drilling procedure without any brain impaction. The wound was then closed, and antibiotic ointment was applied. Mice were placed in a heated cage for recovery. Treatment Protocol: Approximately 2 h post-injury, samples of 60 μL or 1.2 × 108 number of PEVs and PBS (as a control) were administered intranasally using a pipette by alternating the nostrils (2-3-μl at a time per nostril) and maintaining 5-min intervals between each 20-μL administration. This treatment was repeated on 3 consecutive days, with each mouse receiving 180 μL in total. On day 7, mice were sacrificed by cervical dislocation, the brains were quickly collected and rinsed in cold PBS, and the injured area of the ipsilateral cortex was collected using a 4.0-mm biopsy punch. Samples were then frozen in liquid nitrogen until further gene expression analysis by RT-qPCR.

Genes expressions analysis: RNA extraction from the collected tissues was performed using a RNeasy Lipid Tissue Mini Kit (cat. no. 74804, Qiagen) following the manufacturer's protocol. 1000 μL of Qiazol was added to each frozen sample and homogenized using a tissue ruptor. After 5 min, 200 μL of chloroform was added and shaken vigorously. The mixture was centrifuged at 12,000×g, for 15 min at 4 °C, then, the upper aqueous phase containing the RNA was transferred to a new tube, and an equal volume of 70% ethanol was added and vortexed. 700 μL of each sample was next assigned to a RNeasy Mini Spin column sitting on a 2-mL collection tube and centrifuged at 8000×g for 15 s. The flow-through was discarded, and the spin column was put back on the tube. Then 700 μL of RW1 was added to the spin column and centrifuged at 8000×g for 15 s. Again, the flow-through was discarded, and 500 μL of RPE was added to the spin column. The column was centrifuged at 8000×g for 15 s. The flow-through was then discarded, and this step was repeated. Finally, the spin column was transferred to a new 1.5 mL Eppendorf tube, and the total RNA was eluted by adding 50 μL of RNase-free water and centrifugation at 8000×g for 1 min. NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA) was next used to quantify the total RNA concentration. RT-qPCR for inflammatory markers and oxidative stress. We used 1µg of total RNA to synthesize a complementary DNA (cDNA) using an Applied Biosystems High-Capacity cDNA reverse transcription kit (ref 4368814). Afterward, the RT was run with the following program at a thermal cycler (StepOneTM Real-Time PCR System): 10 min at 25 °C, 120 min at 37 °C, and 85 °C, 5 min. The obtained cDNA was stored at − 20 °C before using in qPCR. Validated primers (supplementary Table 1) were used to perform qPCR. Reactions were prepared using 5 μL of Power SYBR Green PCR Master Mix (cat.4367659, ThermoFisher Scientific, Watham, MA, USA), 0.1 μL of forwarding primer, 0.1 μL of reverse primer, 2 μL of cDNA pre-diluted 20 times, and 2.8 μL of RNase-free water for each sample. StepOneTM Real-Time PCR System (ThermoFisher Scientific) was used with an amplification profile of 50 °C, 2 min, 95 °C for 10 min, followed by 40 cycles of (95 °C for 15 s, 60 °C, 25 s, 95 °C for 15 s), melting curve at 60 °C for 1 min. Inflammatory markers targeting astrocytic markers, microglial markers, cytokines and chemokines, and chemokines receptors, including Gfap, Cd68, Trem2, Ccl4, Tlr2, and Tnf-\(\alpha\), were screened.

MPTP mice model of PD: All experiments were authorized by the National Ethical Committee in Animal Experimentation (Comité d'éthique en Expérimentation Animale Nord-Pas de Calais CEEA no.75) as well as the French Ministry of Education and Research (agreement number: 2018060818218219 v4) and were carried out in strict accordance with European Union Directive 2010/63/EU. The research was reported in compliance with the ARRIVE criteria for reporting animal experiments. 5-month-old male C57BL/6 mice (Janvier Le Genest St Isle, France) with a weight between 26 and 30 g from were used. The mice were housed in the animal laboratory of Département de Pharmacologie Médicale, University of Lille, France, with 9–10 mice of each group in large cages, at a temperature of 19–24 °C, 60–70% humidity, on a 12-h light/dark cycle. The animals were given a 7-day period to acclimate to the laboratory environment prior to conducting any experiments. They were randomly divided into 4 groups: sham group (n = 7) and MPTP groups with at least 17 mice in each. MPTP/PBS group was used as control. PEVs at the number 4 × 1010 was delivered intranasally approximately 15 h before MPTP intoxication. Mice were injected with 20 mg/kg (MPTP) (reference 199915, Sigma-Aldrich) or received saline by intraperitoneal injection at the day 0 (sham). The open field behavior test was performed at day 7 and after the mice were sacrified, and the brain tissues were collected for further analysis.

Open field test: The open field test was utilized in conjunction with an open-field infrared actimeter to evaluate spontaneous locomotor activity. Actimetry device (Panlab, Barcelona, Spain), an open field apparatus made from transparent Plexiglas a with a size (45 cm × 45 cm × 35 cm) integrated with two frames of infrared beams, was used. This test is based on mice natural tendency to explore a new environment. The activity was recorded over a 10-min. The major result was the total distance walked (in cm), mean velocity and rearing numbers, which were captured by two rows of infrared photocells and analyzed using the Actitrack software.

Mice brain tissue preparation for immunohistochemistry (IHC): Mice were anesthetized by dolethal via intraperitoneal injection (200 mg/kg), and perfused intracardially with cold saline containing heparin (5 mg/mL), followed by 4% PFA. Whole mice brain was collected and immediately stored in PFA overnight at 4 °C. After, they were transferred into 30% sucrose overnight before freezing at − 80 °C for cryo-sectioning. Frozen brains were cut in coronal sections with 20 μm of thickness and collected in coated glass slides.

Tyrosine hydroxylase (TH) staining: IHC to stain the TH marker of dopaminergic neurons was performed as described below. Substantia nigra (SN) were incubated with rabbit polyclonal anti-TH antibody (1/1000, AB152, Merck Millipore) overnight, followed by anti-rabbit goat secondary antibody (1/500, BA-1000, Vector), and avidin-biotinylated horseradish peroxidase HRP complex (PK-6100, Vectastain, Elite), and 3,3’Kdiaminobenzidine (DAB). Slices were imaged on a multi slide scanning microscope (ZEISS Axio Scan Z.I slide scanner (20× objective) and Zen software Blue edition (Zeiss, Oberkochen, Germany). The total numbers of TH-stained neurons in SN were bilaterally counted in seven sections between bregma – 2.92 mm and – 3.88 mm. For each animal, the neuron counts in each section were summed.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version 10.2.3 (La Jolla, CA, USA), and data are expressed as a mean ± standard deviation (SD) or standard error of the mean (SEM). Following confirmation of normal distribution, a one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test was performed for comparison, and differences were considered significant at p < 0.05. Number of independent experiments is described in the figure legends.