Animals and surgeries
Thirty-six adult male Wistar rats (200–300 g, 2.5 months) were used for this study. Rats were housed in Plexiglas cages (33 x 44 x 20 cm) at the animal housing facilities (n = 5 per cage) under a 12/12 h light/dark cycle (light on at 08:00) at room temperature (RT) with water and food ad libitum. All experimental procedures were performed according to the International Guide for the Care and Use of Laboratory Animals (National Institute of Health, 1999), register # ICS-2017-007 from ethical committee of Instituto de Ciencias de la Salud from Universidad Veracruzana, and the NOM-062-ZOO-1999 and NOM-087-ECOL-SSA1-2002 from Mexican legislation. All efforts were made to minimize animal discomfort during the study.
Two sets of experiments were planned according to the different time window between the intranigral injection of LPS and the euthanasia. Rats were injected either with an intracranial injection of LPS or saline in the SNpc. The groups of animals were then perfused three or seven days after the LPS.
Three groups of animals (n = 6) were used for each set of experiments: group 1: sham (dura mater removed); group 2: saline (saline solution intranigral injection) and group 3: LPS (intranigral LPS injection). Animals were injected intranigrally with 10 µg/2 µl of LPS or saline solution at the same volume.
Intracranial surgery was done as follows. Rats were anesthetized with a single intraperitoneal dose of ketamine/xylazine (10 mg/kg/8 mg/kg) and received an intranigral injection of LPS from Escherichia Coli 055: B5) (Sigma-Aldrich; St. Louis, MO, USA) or saline. We used the following coordinates: AP + 3.0 mm, from the interaural point; ML + 2.8 mm from the intraparietal suture and DV -6.9 mm from the dura mater. The injection flow was 0.2 µL/min maintained by a perfusion micropump (Stoelting, Wood Dale, IL, USA) according to previous procedure [21]. Once the LPS or saline solution was injected, according to each case, the needle was left inside five minutes to allow complete diffusion, then was withdrawn slowly after which the animals were sutured.
Perfusion. Three or seven days after surgery, animals were deeply anesthetized with 100 mg/kg i.p. of sodium pentobarbital (SedalPharma®, Laboratorios Pet's Pharma México), and perfused transcardially with 200 mL of 0.1 M PBS (from 1 M PBS, 8.1 mM Na2HPO4, 1.2 mM KH2PO4, 138 mM NaCl, 2.7 mM of KCl, filtered, pH 7.4) and with 100 mL of 4% paraformaldehyde in 0.1 M PBS using a peristaltic pump. During the perfusion, only the head of the animal was favored, so it was necessary to mechanically occlude the descending aortic artery.
Extraction of brain tissue. Once the perfusion was completed, the brains were immediately removed and post-fixed for 24 hours at 4°C in 4% paraformaldehyde. After this, they were placed in 30% sucrose solution in 0.1 M PBS under the same storage conditions. Subsequently, brains were cut in 35 µm-thick serial sections in a cryostat (Leica CM1520, Leica Inc, Germany) in coronal plane to obtain a representative sample of all levels of the SNpc. The 35 µm slices were collected starting from the anterior-most portion of the SNpc and sequentially placed with a soft-bristled brush inside a sterile 24-well box (Nalgene), previously filled with 0.1 M PBS. Sections were placed one by one in six series; this allowed 9–10 representative sections of SNpc to be obtained for each well. The tissue was stored in cryoprotective solution at − 20°C until used.
Immunofluorescence. Tissues were washed three times for 10 minutes with PBS containing 0.13 M NaCl, 0.010 M Na2HPO4 and 0.002 M NaH2PO4 at pH 7.4. Next, they were immersed in citrate buffer at 60–80°C for 20 minutes and rinsed again with PBS for five minutes and after with TBS-Triton 0.05%. Subsequently, they were incubated for one hour in 10% horse serum + 0.1% sodium azide for blocking nonspecific sites, then washed five minutes with horse serum 1% + sodium azide 0.1% and incubated with primary antibodies [anti-TH made in rabbit, (1:1000) for identification of dopaminergic neurons; anti-OX42 made in mouse (1:500) and Iba-1 (1:500) produced in rabbit for microglial cells; anti-NeuN made in mouse (1:300), to detect neuronal nuclei] in 1% horse serum solution + 0.1% sodium azide for 48 hours at room temperature (RT). After that, three 10-minute washes were done with TBS-Triton 0.05% and tissues were incubated with fluorescent secondary antibodies: Alexa Fluor 488 made in goat anti-rabbit IgG (H + L), Alexa Fluor 488 made in goat anti-mouse IgG (H + L) and Alexa Fluor 555 made in goat anti-mouse IgG (H + L), for four hours (at a concentration of 1:1000 in horse serum 1% + sodium azide 0.1%) and protected from light. Next, three washes were done with PBS for 10 minutes and tissues were incubated with DAPI (1:1000) for 30 minutes to label the cell nuclei. They were rinsed with PBS three times for 10 minutes and finally were mounted on non-gelatinized slides and protected with antifading reagent (Pro-Long Gold) and coverslips.
Image Capture And Quantifications
TH and Iba-1 immunoreactivity were visualized under the multichannel epifluorescence microscope (Eclipse 90i, Nikon), adapted to a high-resolution camera (Nikon DXM1200F), using software to systematically capture images (ACT-1). Representative mosaic images of the SNpc were also taken with a high-resolution camera with a microscope with an automated stage (Eclipse TE2000-E, Nikon) covering the section with systematic images according to x and y coordinates. Image mosaics were built using a stitching tool plugin (Image J). The number of cells was quantified using a specialized software (Image J) and considering the physical dissector as stereological criteria.
Confocal Microscopy
TH/OX42 and TH/NeuN positive cells were visualized using a high-resolution confocal microscope (Zeiss LSM 700) with a 20x objective and capturing systematic images of the SNpc with specialized software (ZEN 2010, Zeiss). For quantifications, TH+/OX42+ or TH+/NeuN+ immunofluorescence images were captured in 3D stacks configured with several optical sections with a 1-µm optical interval and analyzed with specialized software. In addition, DAPI was also captured as counterstaining. For numerical quantifications, including number of cells and the number phagocytic events, we used object counter tools (Image J) and optical dissector stereological criteria. To quantify volumes and visualize phagocytosis events, we used different three-dimensional rendering software (Imaris, Bitplane and Illucida FX, Los Angeles) to create isosurfaces and blend views. To visualize Voronoi partition and crystallization, we also used specialized software applications (ImageJ and Pixelmator). Analysis and visualization of relative florescence with 5 ramps was also done with specific application software (Image J).
Cell Culture Experiments
Cell lines
PC12 and BV2 cells were obtained from the INc-UAB institutional repository. BV2 is an immortalized murine microglia cell line, which has been widely used and successfully evaluated in response to LPS and IFN-γ [22]. Both cell lines were maintained at 37 C with 5% CO2 in their corresponding media, which for the PC12 cell line was DMEM supplemented with 7% fetal bovine serum, 7% horse serum and 0.2% penicillin-streptomycin and for the BV2 cell line was RPMI supplemented with 10% fetal bovine serum and 0.1% penicillin- streptomycin. For the different experiments, PC12 and/or BV2 were placed in a 24-well culture plate and grown for 24 hours then treated with different compounds such as LPS at different concentrations, IFN-γ, or co-cultured with PC12. Twenty-four hours after the treatment, the supernatant was collected to conduct the Griess assay or to treat subsequent cultures. At the end of the experiments, cells were fixed with 4% paraformaldehyde in PBS for immunocytofluorescence.
Immunocytofluorescence
The immunocytofluorescence was performed on cell cultures to simultaneously detect TH and CD11b. Cells were washed with PBS, then permeabilized with 0.02% saponin/PBS at RT and later rewashed with PBS1X. Nonspecific sites were first blocked with PBS1X, 0.01% saponin and 0.075% glycine and later nonspecific sites were again blocked with PBS1X, 0.01% saponin, 0.075% glycine and 5% BSA. Coverslips containing the cells were placed in a humid chamber with 12 µL of PBS1X, 0.01% saponin, 1% BSA and the primary antibody overnight. To detect TH and CD11b, the primary antibodies were anti-TH (sheep, 1:500) (Merck; Darmstadt, Germany) and anti-CD11b (mouse, 1:1000) (Abcam; Cambridge, United Kingdom), respectively. The following day coverslips were placed again in the 24-well plate, cells were washed with PBS 1X before being placed again in a humid chamber with PBS1X, 0.01% saponin, 1% BSA and the secondary antibody for 45 minutes. In order to detect TH, the secondary antibody was donkey anti-sheep (1:1000) in green fluorescence (Alexa Fluor 488) (Life Technologies; Carlsbad, CA, USA), and for CD11b detection the secondary antibody was goat anti-mouse (1:500) in red fluorescence (Alexa Fluor 555) (Bio-Rad Company; Berkeley, California, USA). Coverslips were later returned to the 24-well plate to be washed with PBS1X before adding 400 µL per well of the nucleus marker, DAPI (1:1000) (Life technologies; Carlsbad, CA, USA) in PBS1X. One more wash with PBS 1X was done before the coverslips were mounted on a glass slides using antifade reagent (Prolong Gold, Life Technologies; Carlsbad, CA, USA).
For every experimental condition, a secondary antibody control was also performed, leaving one coverslip per condition without any primary antibodies.
Immunocytofluorescence Quantification
To quantify the number of cells in the co-culture and its controls, we used an image analysis protocol. Each coverslip was imaged using a fluorescence microscope (Nikon Eclipse 90i) attached to a DXM 1200F digital camera and version 2.70 of the ACT-1 software (Nikon Corporation). With this system, we obtained 30 images per condition at 20x. Two quantification methods were used for the immunocytofluorescence labeling. One consisted of counting the number of cells, either PC12 or BV2, following stereological criteria and using a specialized software (Image J version 1.47, NIH, USA). The other method consisted of measuring the area of BV2 (CD11b) to estimate the activation, also using the same software (Image J version 1.47, NIH, USA).
Determination Of Nitrites By Griess Assay
The determination of nitrites by Griess assay is based on a chemical reaction in which nitric oxide in the presence of an aromatic amine produces different compounds in sequence, resulting in a pink-colored compound, easily detectable by spectrophotometry. First, the calibration curve was prepared with known concentrations of NaNO2 (100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, 1.5625 µM and 0.78125 µM), and 100 µL of each solution was added into a 96-well plate for the calibration curve, as was 100 µL of the experiment samples. Next, 100 µL of the Griess reagent (0.1 g in 2.5 mL milli-Q water) was added to every well. Duplicates were done for each condition. The plate was then incubated in darkness at RT for 15 minutes. Then the plate was read with specialized microplate reader software (KC Junior, Kansas City, MO, USA) and the nitrite ion equivalents of the samples of interest were calculated according to the calibration curve prepared in the same 96-well plate.
Statistics
The results were expressed as mean ± standard error of the mean (SEM), which were calculated with suitable software (Sigma STAT or Excel, Microsoft Office MSO, Redmond, Washington, USA). All the data were statistically analyzed with suitable software (R commander package, R software; version 3.5.2, Free Software Foundation’s GNU General Public License), using one-way analysis of variance (ANOVA) in pairwise comparisons of means mode. The value of p < 0.05 was the criterion to establish differences between means.