Pain behavior: Von Frey and hot plate tests

The threshold of mechanical pain was measured in acclimated rodents via the up-down method using von Frey filaments on the plantar surface. Briefly, the rats were placed individually in small cages with a mesh for the von Frey test, and a monofilament (Bio-VF-M; Bioseb, Vitrolles, France) was applied perpendicular to the plantar surface of the hindpaw until the paw buckled. A score ranging between 1.5 and 90 g was assigned in four consecutive positive responses to filaments with decreasing force or three consecutive negative responses to filaments with increasing forces. Nociceptive behaviors, including brisk paw withdrawal, licking, or shaking of the paw, in two trials were considered a positive response. The 50% paw withdrawal threshold (PWT) was determined as described previously (Zhou and Luo, 2015).

The hot plate trial was performed 1 day after the von Frey test. Rats were acclimatized for at least 30 min in the test compartment. A heat source (55°C) was positioned on the plantar surface of the hindpaw. Three hot plate trials were conducted, and the mean paw withdrawal latency was recorded. A 20 s cutoff time was used to prevent tissue damage.

At weeks 8 ~ 9, the rats in the PD and PD-rVM groups (n = 6/group) were anesthetized by intravenous injection of 70.0 mg/kg α-chloralose (dissolved in pre-warmed 0.9% saline and 10% polyethylene glycol) for fMRI on a 4.7-T spectrometer (Biospec 47/40; Bruker, Bremen, Germany). A 72 mm volume coil was employed as the radio-frequency transmitter, with a quadrature surface coil on the head as the receiver. Superparamagnetic iron oxide (SPIO; Resovist, Schering, Berlin, Germany) was intravenously administered at 15 mg Fe/kg as the contrast agent to acquire steady-state CBV-weighted fMR images, using previously described settings (Shih et al., 2009, 2012; Chen et al., 2013). Enhanced neural activity increases the influx of SPIO nanoparticles, which leads to a lower signal intensity and represents an increase in regional CBV (Mandeville et al., 2004). A time-series of one hundred gradient-echo images in axial-view were obtained for CBV-weighted fMRI using the following parameters: repetition time, 150 ms; echo time, 15 ms; flip angle, 22.5°; field of view, 2.56 × 2.56 cm; slice thickness, 1.5 mm; acquisition matrix, 128 × 64 (zero-filled to 128 × 128); temporal resolution, 16 s. A total of 100 time-series images were separated equally into five phases (20 fram es for each phase) corresponding to the “off,” ‘on,’ “off,” “on,” and “off” phases of nociceptive electrical stimulation; the electrical stimulation paradigm is described below.

As shown in Figure 3A, a pair of needle electrodes were used to deliver nociceptive electrical stimulation (10 mA intensity, 3 Hz square wave, 0.5 ms pulse) to the right forepaws of the animals using an A-M Systems model 2,100 constant-current stimulator (Carlsborg, WA, United States). Figures 3B,​,CC presents an example of the correlation map between the CBV signals and the stimulation paradigm. Correlation maps were created by plotting the correlation coefficient (CC) for the CBV signal changes and off–on–off–on–off electrical stimulation paradigm using the cross-correlation method on a voxel-by-voxel basis (Bandettini et al., 1993; Chen et al., 2013). As the experimental conditions affect this correlation, a cut-off point of r = ± 0.2 was empirically selected; this threshold effectively identifies spatial clusters in the striatum and primary somatosensory cortex (S1).

Representative fMRI scan of a PD rat and the positions of the ROIs in the 6-OHDA-lesioned and non-lesioned hemispheres. (A) Nociceptive electrical stimulation was applied to the right forepaw of the PD rat via a pair of needle electrodes. (B) The time course of CBV signal intensities during nociceptive stimulation in the left (intact: black) and right (lesioned: red) striata of the PD rat. (C) The ROIs for the left non-lesioned striatum are shown in black, and in red for the right 6-OHDA-lesioned striatum.

Furthermore, to assess the therapeutic effects of rVM tissue transplantation on DA innervation of the striatum, steady-state CBV weighted fMRI was conducted after intravenous administration of the dopamine D2 receptor antagonist eticlopride (S-(−)-Eticlopride hydrochloride, E101-100MG, Sigma-Aldrich; St. Louis, United States) at a dose of 1.0 mg/kg.

At the end of the study period, 6 weeks after cell transplantation, the PD and PD-rVM groups were euthanized for IHC analysis, as described previously (Weng et al., 2017). Briefly, rats were terminally anesthetized with chloral hydrate, perfused with normal saline followed by 4% paraformaldehyde (Sigma-Aldrich), and the brains were excised, post-fixed in 4% paraformaldehyde overnight at 4°C, cryprotected by immersion in 20% sucrose in 0.1 M PBS for 2 days followed by 30% sucrose in 0.1 M PBS for 2 days, and 30-μm-thick coronal sections were obtained using a Leica CM 3050 Cryostat Microtome (Leica Microsystems, Wetzlar, Germany). The sections were rinsed in phosphate-buffered saline (PBS), incubated with 1% hydrogen peroxide (Calbiochem, Torrey Pines, CA, United States) in PBS for 30 min, placed in blocking solution prevents non-specific binding of antibodies to tissue (0.5% Triton X-100 and 3% normal goat serum [Vector, Burlingame, CA, United States] in PBS), and incubated with a primary rabbit recombinant monoclonal anti-TH antibody (1:2,000; Millipore Corporation, Billerica, MA, United States; 4°C overnight), followed by secondary goat anti-rabbit biotinylated IgG antibody (1:200; Vector; 1 h), then avidin-biotin complex (1:200; Vectastain ABC kit, Vector; 60 min), developed in 3,3-diaminobenzidine (0.05%; Sigma-Aldrich) for 6 min, washed thrice with PBS, and mounted on gelatin-coated slides.

To access the number of TH-ir cells, 30 μm striatal micrographs were taken with color CCD camera attached to the confocal microscopy [OPTIPHOT-2 (10×), MICROPHOT-FXA (100× and 200×), Nikon, Tokyo, Japan or Zeiss LSM 880 confocal microscope; ZEISS]. Graft areas were measured by ImageJ, and the density of TH-ir within was expressed as the cell number/mm³.

For semi-quantitative measurement of TH-ir signals, 10× or 100×/200× images in the target and the reference regions (corpus callosum) were taken under the OPTIPHOT-2 microscope or MICROPHOT-FXA microscope (Nikon, Tokyo, Japan), respectively, and converted to 8-bit grayscale images (0–255 gray levels; Mausset-Bonnefont et al., 2003). The optical density (OD) of TH immunoreactivity were semi-quantitively scored using Image-Pro Plus v. 6.0 (Media Cybernetics, Inc., Bethesda, MD, United States), as follows:

OD ratio = (OD of target region − OD of corpus callosum)/OD of corpus callosum.