Materials
Prosaposin PS18 was purchased from the Elabscience (Texas, USA). Bovine serum albumin, fetal bovine serum, L-glutamate, NMDA, paraformaldehyde, polyethyleneimine, and Triton X-100, were purchased from the Sigma (St. Louis, USA). Alexa Fluor 488 (secondary antibody), B27 supplement, Dulbecco’s modified Eagle’s medium, Neurobasal Medium, and trypsin were purchased from Invitrogen (Carlsbad, USA). TH antibody was purchased from Millipore (Burlington, USA). In Situ Cell Death Detection Kit was purchased from Roche (Indianapolis, USA).
Adult male and time-pregnant Sprague-Dawley rats were purchased from BioLASCO, Taiwan. The use of animals was approved by the Animal Research Committee of the National Health Research Institutes of Taiwan (NHRI-IACUC- 109097-M1). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).
Primary cultures of rat ventral mesencephalon cells
Primary cultures were prepared from embryonic (E15) ventral mesencephalon (VM) tissues obtained from fetuses of timed-pregnant Sprague-Dawley rats. The whole brain was removed aseptically, and a small piece of tissue comprising the VM was dissected. After removing the blood vessels and meninges, pooled VM tissues were trypsinized (0.25%; Invitrogen, Carlsbad, CA) with gentle mixing for 15 min at 37°C. After rinsing off trypsin with pre-warmed DMEM/F-12 (Invitrogen), cells were dissociated by trituration, counted, and plated into 96-well (6.0 × 104/well) cell culture plates pre-coated with poly-D-lysine (Sigma-Aldrich). The culture plating medium consisted of Dulbecco’s modified Eagle medium/F12 supplemented with 10% heat-inactivated fetal bovine serum, 1 mM L-glutamine, and 2% B27 (Invitrogen). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The cultures were fed by exchanging 50% of media with feed media (Neurobasal medium, Invitrogen) with 0.5 mM l-glutamate and 2% B27 with antioxidants supplement on DIV (days in vitro) 3 and 5.
Immunocytochemistry and quantitation
After removing the PFA solution, cells were washed with PBS, and the fixed cultures were treated for 1 hour with blocking solution (2% BSA, 0. 1% Triton X-100 and 5% goat serum in PBS). The cells were incubated for 1 day at 4°C with specific mono/polyclonal antibodies (i.e., TH) and then rinsed three times with PBS. The bound primary antibody was visualized using Alexa Fluor 488 secondary (Invitrogen). Images were acquired using a monochrome camera Qi1-mc attached to Nikon TE2000-E inverted microscope.
Terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick -end labeling (TUNEL)
Cultures were examined for DNA fragmentation using a TUNEL-based method (In Situ Cell Death Detection Kit; Roche, Indianapolis, IN). Briefly, 4% PFA fixed cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. To label damaged nuclei, 50 µL of the TUNEL reaction mixture was added to each sample and kept at 37° C in a humidified chamber for 60 min. Procedures for positive and negative controls were carried out as described in the manufacturer’s manual (Roche). Controls were consisted of not adding the label solution (terminal deoxynucleotidyl transferase) to the TUNEL reaction mixture. Nikon TE2000 inverted microscope equipped with fluorescence was used to examine apoptosis.
Drug administration
Animals were anesthetized with 3% isoflurane. PS18 (2 mM/20 µL) or vehicle (saline, 20 µl) was administered intracerebroventricularly (AP, − 0.8 mm; LV, − 1.5 mm; DV, − 3.5 mm) at 15 min before 6-OHDA lesioning through a Hamilton microsyringe. The speed of injection was controlled by a syringe pump (Micro 4, WPI, Sarasota, FL).
6-OHDA lesioning
Animals were anesthetized with 3% isoflurane. 6-OHDA (3 µg/µl × 2.5 µl dissolved in 0.1% ascorbic acid) was stereotactically injected into the 2 sites of left striatum (AP, 1 mm; LV, 3.2 mm; DV, -6.1 mm below the skull and AP, 1 mm; LV, 2.6 mm; DV, − 5.5 mm below the skull) at 0.25 µl/min over a 10 min period.
Behavioral Test:
(1) Locomotor activity: Locomotor activity was examined on day 14 after 6OHDA lesioning. Rats were individually placed in 42 × 42 × 31 cm Plexiglas activity chambers containing horizontal and vertical infrared sensors (Accuscan, Columbus, OH) placed 2.5 cm apart. Three variables were measured: (i) horizontal activity (HACTV, the total number of beam interruptions that occurred in the horizontal sensors in one hour), (ii) vertical activity (VACTV, the total number of beam interruptions that occurred in the vertical sensor in one hour), and (iii) total distance travelled (TOTDIST, the distance, in centimeters, traveled by the animals in one hour).
(2) Rotational behavior: Rotational behavior was evaluated using an 8-channel rotometer system (RotoMax, AccuScan Instruments, Inc). Meth (2.5 mg/kg)-induced rotation per hour was counted by a computer, as we previously described 26.
Quantitative reverse transcription-PCR
Nigra tissues from the lesioned and non-lesioned side hemispheres were collected. Total RNAs were isolated using TRIzol Reagent (ThermoFisher, #15596-018, Waltham, MA, USA), and cDNAs were synthesized from 1 µg total RNA by use of RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, #K1631, Waltham, MA, USA). cDNA levels for PERK, ATF6, BIP, IRE1, actin, and GAPDH were determined by specific universal probe library primer-probe sets or gene-specific primers (Table 1). Samples were mixed with TaqMan Fast Advanced Master Mix (Life Technologies, #4444557, Carlsbad, CA, USA) or SYBR (Luminaris Color HiGreen Low ROX qPCR Master Mix; ThermoScientific, Waltham, MA, USA). Quantitative real-time PCR (qRT-PCR) was carried out using the QuantStudio™ 3 Real-Time PCR System (ThermoScientific, Waltham, MA, USA). The expression of the target genes was normalized relative to the endogenous reference gene (beta-actin and GAPDH averages) with a modified delta-delta-Ct algorithm. All experiments were carried out in duplicate.
Statistical analysis
Data are presented as the mean ± SEM. Unpaired t-test, one- or two-way ANOVA, and post-hoc Newman–Keuls (NK) test were used for statistical comparisons, with a significance level of p < 0.05.