Dbd00

A twenty-seven-gauge butterfly needle connected to a manometer was inserted in the cisterna cerebellomedularis of a marmoset, a manometer was held up and then the CSF pressure was measured by recording the height meniscus of CSF in the manometer tube^{62 (link)}. CSF collection was performed as previously reported^{63 (link)}. During the collection, the marmoset was placed in a prone position, and the back of the head and the neck were kept at level. The BDNF concentration in the CSF was measured using an assay kit DBD00 (R&D, Minneapolis, MN, USA) according to the manufacturer’s protocol.

In order to clarify the residual particles of cell membranes indirectly, specific platelet cell surface antigens, CD41 (human integrin alpha-IIb ELISA kit, CUSABIO, College Park, MD) and CD61 (human integrin beta-3 ELISA kit, CUSABIO), were measured using commercially available ELISA kits in 12 PL production samples (9 sPL and 3 mPL) according to the manufacturer's instructions. Optical densities were measured by using a spectrophotometer (model 550 reader; Bio-Rad, Hercules, CA). All samples and standards were run in triplicate. The growth factor level was extrapolated from a standard curve. If any obtained data were under the mean minimum detectable dose, they were considered as nondetectable (ND) in the analysis.
Concentrations of PDGF-BB (DBB00, R&D Systems, Minneapolis, MN), TGF-β1 (DB100B, R&D Systems), and BDNF (DBD00, R&D Systems) in 12 PL production samples (9 sPL and 3 mPL) were also measured using commercially available ELISA kits according to the manufacturer's instructions. When the concentrations were measured, PL samples were diluted to 1 : 20 in PDGF-BB or 1 : 100 in TGF-β1 and BDNF.

All the subjects underwent a peripheral venous blood draw between 08:00 and 10:00 am. The samples were collected in anticoagulant-free tubes and immediately centrifuged for 20 min at a speed of 3000 r/min to obtain patients’ plasma. Plasma samples were then separated and stored at −80 °C until future analysis. ELISA kits were used to measure plasma levels. The details of all the ELISA kits were as follows: mBDNF (DBD00—R&D Systems, Minneapolis, MN, USA); tPA (DTPA00—R&D Systems, Minneapolis, MN, USA); and IL-1β (ab214025—Abcam, Cambridge, MA, USA). Plasma proBDNF, TrkB, TNF- $\mathsf{\alpha }$ , and IL-6 levels were estimated using a DuoSet human ELISA kit (proBDNF: DY3175; TrkB: DYC397; TNF- $\mathsf{\alpha }$ : HSTA00E; IL-6: HS60DC—R&D Systems, Minneapolis, MN, USA) combined with a DuoSet ELISA Ancillary Reagent kit (DY008—R&D Systems, Minneapolis, MN, USA). Based on the manufacturers’ instructions, we determined the concentration of factors in each sample. The results are exhibited in pg/mL. All the experiments were performed in duplicate.

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure BDNF protein concentration in serum (DBD00; R&D Systems, USA). Protocols were conducted according to the manufacturer’s instructions and as described in our previous work (Schroter et al. 2019 ) (for details, see Supplementary Material).

Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure serum protein concentrations of tPA, PAI-1, BDNF, proBDNF, TrkB and p75NTR. The human tPA, PAI-1 and BDNF levels were measured by ELISA kits [tPA (DTPA00; R&D Systems; USA); PAI-1 (DSE100; R&D Systems; USA); BDNF (DBD00, R&D Systems; USA)] and proBDNF, TrkB and p75NTR levels were assessed using DuoSet human ELISA Development System (proBDNF: DY3175, TrkB: DYC397-2, p75NTR: DY367 and Ancillary Reagent: DY008; R&D Systems; USA). Protocols were performed according to the manufacturer’s instructions. In order to minimize assay variance, each protein concentrations of all subjects were measured on the same day. All experiments were performed in duplicate. The sensitivity of tPA, PAI-1 and BDNF assay were 1.40-16.1 pg/mL, 0.014-0.142 ng/ml and less than 20 pg/mL, respectively; while the sensitivity of proBDNF, TrkB and p75NTR assay were not given exactly in the specification. No significant cross-reactivity or interference was observed in each assay.

10 ml of venous blood was taken from subjects in the morning after overnight fast. Glucose, TG, HDL-C levels were measured using enzymatic methods by DADE Dimension AR®. Leptin and insulin levels were measured using a radioimmunoassay kit from Linco Research (St Louis, USA). Serum BDNF levels were determined using an ELISA protocol, according to the manufacturer’s instructions (DBD00; R & D Systems, Europe).