A total of 96 adult male Sprague-Dawley rats (250±20 g) were obtained from the laboratory animal center of Shahid Beheshti University of Medical Sciences, Tehran, Iran. The animal experiments were approved by the ethics committee of the university (IR.SBMU.MSP.REC.1395.243). The animals were kept under standard conditions (temperature 22-24°C) in a 12:12 h light/dark cycle with humidity (50-70%) and free access to water and food.
The animals were divided into eight groups randomly, each consisting of 12 animals: normal control with no surgical or injection procedure (group I); 6-OHDA (4 µg/kg, group II); 6-OHDA+cell transplantation (group III); 6-OHDA+cell transplantation+ melatonin (group IV); 6-OHDA+melatonin (20 mg/kg; group V); vehicle (group VI); melatonin sham (group VII); and 6-OHDA sham (group VIII).
Simulation of the experimental model of PD
The animals were anesthetized intraperitoneally with a mixture (1 ml/kg), containing 9 mg/kg of xylazine and 90 mg/kg of ketamine. Induction of PD in animals was performed by a unilateral single-dose injection of 6-OHDA (4 µg; Sigma, USA) in 2 µL of physiological saline (containing 0.02% ascorbic acid) into the right SN, using a Hamilton micro syringe (Hamilton, Reno, NV, USA) (Jeon et al. 1995). Coordinates were set according to the Atlas of Paxinos (Paxinos and Watson 2006): anteroposterior (AP): 4.3 mm; lateral (L): 1.6 mm; and dorsoventral (DV): 8.2 mm.
Apomorphine-induced turning behavior
In order to ensure proper induction of PD, apomorphine-induced turning behavior was induced in all animals. Accordingly, the animals received a subcutaneous neck injection of apomorphine hydrochloride (0.05 mg/kg; Sigma, USA), in 1% ascorbic acid and 0.9% NaCl, and were placed on metal testing bowls for 30 minutes. Next, the number of contralateral rotations was recorded and analyzed.
Differentiated cells were collected at approximately 2×104 cell μL for transplantation. Cell transplantation was performed, using a unilateral stereotaxic injection with a Hamilton microsyringe. Coordinates were set according to the atlas of Paxinos (9): anteroposterior (AP): 9.2 mm; lateral (L): 3 mm; and dorsoventral (DV): 4.5 mm. Each animal received a slow infusion of cell suspension (2.5 μL) for 3-5 minutes.
Melatonin (sigma, USA) was dissolved in 2% DMSO and 98% Miglyol® 812N to preparing 10 mg/mL concentration; it was protected from light and prepared fresh each time. The total administration dose was 20 mg/kg/day. It was initiated three days after 6-OHDA treatment and continued for seven days.
Assessment of motor coordination
For evaluation of balance, coordination, and motor control of rats, the rotarod test was used, based on a linear accelerating protocol (4-40 rpm in 300 seconds). Three trials were carried out for each rat per day, and the average latency to fall was considered for each day. The test was performed one week after 6-OHDA injection, and continued for four consecutive weeks. The rats were sacrificed after the tests, and their brains were extracted for laboratory analyses.
The rats were deeply anesthetized and sacrificed with sodium pentobarbital (60 mg/kg, Eutasil) intraperitoneally. Afterwards, trascardial perfusion was performed using normal saline containing 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). All animal brains were exposed by a midline incision along the skull,next, they were dissected. Four brains from each group were immediately stored at -80°C for molecular tests, while others were immediately immersed in a fresh 10% formalin solution for one week. Subsequently, the samples were embedded in paraffin blocks. In addition, complete serial sections (5-10 μm) were prepared using a microtome for stereological study. Every 10 sections were sampled. Next, 8-10 tissue sections were obtained from each animal in a systematic random sampling with a random number between1-10. Finally, the selected sections were stained with haematoxylin and eosin (H&E) and analyzed.
Western blot assays
Following behavioral tests, Western blotting was carried out to investigate caspase-3 expression in the striatum and SNpc. The rats were sacrificed under ketamine/xylazine anesthesia after decapitating their brains (striatum and SNpc). Tissue samples were homogenized in tissue lysis buffer (1:10 w/v; Sigma). Next, protein concentrations were determined based on Bradford assay. Protein samples (30 lg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking the membrane in 10% milk with TBS-T buffer (10 mM Tris–HCl, 120 mM NaCl, and 0.1% Tween-20; pH: 7.4) for one hour at room temperature, it was incubated with caspase-3 primary antibodies (GeneTex Cat# GTX22300, RRID:AB_367945) (1:1000) at 4°C overnight. It was then washed three times in TBST buffer, incubated with 1:10000 dilutions of HRP-conjugated anti-rabbit/goat IgG at room temperature for one hour. Visualization was carried out using an Excellent Chemiluminescent Substrate (ECL) kit (GE Healthcare, Bucks, UK). Density of the bands on Western blots was also quantified through densitometric analysis of scanned blots in ImageQuant software.
Measurement of GSH assay
Reduced glutathione (GSH) reacts with 5-5–dithio-bis-2(nitrobenzoic acid) (DTNB) to form a yellow dianion of 5′thio-2-nitrobenzoic acid (TNB) which is measured by its absorbance at 412 nm.
Estimation of striatum and SN volume
The rats’ striatum and SN tissue samples were fixed in 4% PFA for one week. Following tissue processing, serial coronal sections (25 mm) were prepared and stained with Cresyl violet 0.1%. Cresyl violet was used to demonstrate the Nissl substance in neurons. For volume estimation, 10-12 sections per block were selected through systematic uniform random sampling. Stereological studies were carried out using a projection microscope. To measure the total volume of striatum and SN at 25× magnification, the Cavalieri's Principle was used based on the following formula (Mayo et al. 1999):
V (striatum and SN) = (a/p) ×ΣP (striatum and SN)×d
Moreover, distance between the sampled sections (d) was calculated. Also, the section area was estimated using the point-counting method. The area per point (a/p) was 0.36 mm2, and on average, 500 points were counted per animal.
Estimation of neuron and glial cell count
The total number of neurons and glial cells in the striatum was determined using the optical disector technique. Microscopic fields were selected at equal intervals of stage movement, based on systematic uniform random sampling (SURS). A microcator was used for measuring the Z-axis movement of the microscope stage. An unbiased counting frame with inclusion and exclusion borders was superimposed on the images, viewed on the monitor. The nucleus count was determined if the nuclei were placed completely or partially within the counting frame and did not reach the exclusion line. Numerical density (Nv) was also calculated based on the following formula:
where “ΣQ” represents the number of nuclei, “ΣP” denotes the total number of unbiased counting frames in all fields, “h” is the height of the dissector, “a/f ” is the frame area, “t” is the real section thickness measured in every field by the microcator, and “BA” is the block advance of microtome set at 10 μm. The total number of neurons and glial cells was estimated by multiplying the numerical density (Nv) by the total volume.
Ntotal Nv V
Estimation of coefficient error(CE)
For volume estimations, CE was calculated using the following formula: Pi2
where “B” and “A” represent the mean section boundary length and mean section area, respectively. For estimating the total neuron and glial cell count, CE was derived from CE (V) and CE (Nv), based on the following formula
(Gundersen and Jensen 1987):
CE(N) = [ CE2 (Nv) + CE2 (v)]1/2
Statistical analysis between the groups was examined using ANOVA and Tukey’s post hoc test. All statistical analysis was performed in SPSS version 24 (SPSS Inc., Chicago, USA), and P<0.05 was considered significant.