Animals and experimental design
Sixty male adult C57BL/6 J mice at age of 12 weeks (purchased from Taconic Biosciences) were used in this study. Male C57BL6 mice were chosen as they develop a more severe phenotype on HFD when it comes to metabolic parameters . The animals were housed in a 12-hour (h) light/dark cycle with free access to food and water. All experimental procedures were carried out in accordance with the European Directive 2010/63/EU guidelines and approved by the Ethical Committee at Lund University. Mice were fed either HFD or a control diet (CTRL) for 7 weeks before 6-hydroxydopamine (6-OHDA) toxin or vehicle infusion into the medial forebrain bundle (MFB) and maintained on the same diet throughout the study. Behavioral assessments were conducted 8–9 weeks post-lesion, and glucose tolerance test (GTT) and blood sampling were performed 10 weeks post-lesion corresponding to 17 weeks after induction of diet. The mice were sacrificed 14 weeks after the lesion, that is 21 weeks after induction of diet.
Several mice had to be excluded from the study due to either unsuccessful PD lesion, illness, or death during experimental procedures before reaching the study end point. Twenty-five mice were included in the final analysis.
An HFD regime was used to induce insulin resistance mimicking DMT2 . After 1 week of acclimatization, the mice were split randomly into two groups and fed with either HFD containing 60% fat (D12492, Research Diets, USA, n = 30) or a CTRL diet with a matched formula containing 10% fat (D12450B, Research Diets, USA, n = 30). Mice were kept on the respective diet for a total of 21 weeks. The effect of the diet was measured by the glucose tolerance test (GTT) and insulin levels 17 weeks after the induction of the diet.
Glucose tolerance test (GTT) and insulin resistance index (HOMA-IR)
For the GTT, d-glucose (2 g/kg) was injected intraperitoneally (i.p.) in mice anesthetized with midazolam (0.4 mg/mouse; Dormicum®; Hoffmann-La Roche) and a combination of fluanison (0.9 mg/mouse) and fentanyl (0.02 mg/mouse; Hypnorm®; Janssen, Beerse, Belgium). Mice were fasted 4 h before the GTT and kept on a heating pad for the entire time to maintain the body temperature. Blood (30 μl) was drawn retro-orbitally at 0, 15, 30, 60, and 120 min after glucose injection. Plasma was stored at − 80 °C until analysis. Glucose and insulin levels were determined by Amplex red glucose/glucose oxidase assay kit (Invitrogen™) and insulin ELISA (Mercodia Mouse Insulin ELISA; Mercodia AB, Uppsala, Sweden), respectively. Fasting blood collected from the tolerance test at time point 0 was used to obtain an insulin resistance index (Homeostatic Model Assessment of Insulin Resistance (HOMA-IR); HOMA-IR = (Glucose0 in mmol/l) × (Insulin0 in μg/l)/22.5) . The cut-off value for insulin resistance was chosen based on the 90th percentile of the HOMA-IR values of the sham/CTRL diet group .
Seven weeks after initiation of the diet, animals on HFD and CTRL diet were randomized to receive either 6-OHDA (lesion group) or 0.9% sodium chloride (sham group). The number of animals per group is specified in the respective figure legends. The 6-OHDA (lyophilized powder including 0.025% ascorbic acid, Sigma) was reconstituted with 0.9% saline to obtain a concentration of 1 μg/μl and stored at − 20 °C and kept on ice during the surgery. 6-OHDA or saline was infused unilaterally into the MFB using stereotaxic surgery as previously described . Briefly, a Hamilton syringe was connected to a cannula (tip diameter equal 50 μm), and 1 μl of 6-OHDA solution or saline was infused into the MFB at a rate of 0.5 μl/min using the following coordinates: from Bregma: A/P = − 1.2, M/L = − 1.3, and D/V = − 4.75 . The cannula was left for 2 min before being slowly retracted. During surgery, mice were anesthetized with 4–5% isoflurane (IsoFlo vet, Apoteksbolaget, Sweden) in a carrier of oxygen and nitrous oxide mixture at a ratio of 2:1 for induction and then maintained at 2% isoflurane throughout the entire surgery. At the site of incision, 100 μL of Marcain (0.25 mg/mL, Apoteksbolaget, Sweden) was injected subcutaneously (s.c.) to provide local anesthesia. At the end of the surgery, the skin was sutured and mice were injected s.c. with 1 ml of saline.
To indicate the degree of the nigrostriatal lesion, behavioral tests were performed 8 weeks after the surgical intervention corresponding to 15 weeks on the respective diet.
To assess the rotational behavior, mice were placed in a glass bowl (diameter 50 cm) and harnessed to an automated rotometer to count the number and direction of the rotation as described . Mice were injected with d-amphetamine (Sigma, 5 mg/kg; dissolved in sterile 0.9% saline) and allowed to acclimatize for 5 min before recording rotations. Data were obtained for 90 min and presented as the number of full-body turns per min with a positive value for ipsilateral rotation and a negative value for the contralateral rotations.
The corridor test was performed to assess the lateralized sensorimotor integration of the mice . In short, the mice were first habituated for 10 min on two consecutive days before the test. The test was performed in a corridor (60 cm long, 4 cm wide, and 15 cm high) containing sucrose tablets that were scattered randomly along the floor. On the day of testing, mice were habituated for 5 min in an empty corridor and then immediately transferred to an identical corridor that contained 10 pots on each side, each containing 2–3 sucrose tables. The number of explorations made by each mouse at the ipsilateral or contralateral side was counted until a total of 20 counts was reached or 5 min had passed. The mice were put on food restriction during the habituation and the test with maintaining at 85% free-feeding body weight. The data are presented as the percentage of contralateral explorations out of the number of total explorations.
The cylinder test was performed to assess the spontaneous forelimb lateralization. The mice were placed in a Perspex cylinder (height 20 cm, diameter 19 cm) with a mirror behind to allow a clear view. They were captured digitally by videotaping for 3 min for later analysis by a blinded examinator. The data are expressed as the percentage of weight-bearing contralateral paw touches of the cylinder wall out of the total number of touches .
For immunohistochemistry, mice were deeply anesthetized with sodium pentobarbital (Apoteksbolaget, Sweden) and transcardially perfused with 0.9% saline for 3 min followed by 4% paraformaldehyde (PFA) for 5 min. Brains were collected, kept in 4% PFA solution for 4 h, and then stored in a 25% sucrose solution for several days. Brains were cut coronally at 30-µm-thick sections.
For immunohistochemical staining, sections were quenched with a peroxidase solution (3% H2O2, 10% methanol), diluted in phosphate-buffered saline (PBS) for 10 min before blocking for 1 h at room temperature (RT) in 3% serum diluted with 0.25% Triton X-100-PBS (PBS-TX) (Alfa Aesar). Primary antibodies diluted with 1% serum in PBS-TX were incubated overnight (O/N) at RT for rabbit anti-tyrosine hydroxylase (TH; 1:1000, Millipore) and anti-CD11b (1:200, Bio-Rad) or for 2 h at RT followed by O/N incubation at 4 °C for rat anti-dopamine transporter (DAT, 1:1000, Millipore).
Sections were then incubated for 2 h at RT with the corresponding biotinylated secondary antibodies (1:200, Vector Laboratories), followed by 1-h incubation with an avidin–biotin kit (Vectastain Elite ABC kit, Vector Laboratories), and the staining was revealed using chromogen 3,3-diaminobenzidine (DAB, Peroxidase Substrate Kit, Vector Laboratories).
For double immunofluorescence staining, sections were washed 3 times for 10 min with PBS, blocked with 5% serum in PBS-TX for 1 h at RT, and incubated with the following primary antibodies diluted with 3% serum in PBS-TX O/N at RT: goat anti-CD31 (1:200, R&D Systems) rat anti-CD13 (1:200, Bio-Rad), rabbit anti-fibrinogen (1:400, Abcam), rat anti-CD11b (1:200, Bio-Rad), or rabbit anti-mouse immunoglobulin G (IgG) (1:200, Dako).
Sections were then incubated for 1 h at RT with the respective fluorophore-tagged secondary antibodies: CY3-conjugated donkey anti-rat IgG or Alexa Fluor 647 donkey anti-rabbit diluted with 3% serum in PBS-TX (1:500; Jackson ImmunoResearch) or with an anti-goat biotinylated secondary antibody (1:200, Vector Laboratories) followed by fluorophore-conjugated streptavidin (Alexa Fluor 647, 1:500; Jackson ImmunoResearch). Sections were incubated with DAPI (4′,6-diami-dino-2-phenylindole) for 10 min, then washed and mounted on a gelatinized slide, and coverslipped with mounting medium (PVA/DABCO).
Oil red O staining and quantification
Oil red O (ORO) staining was used to stain the lipids in the liver. A frozen liver sample was sliced to a 10-μm thickness using a cryostat (Leica CM3050E). The sections were then immediately fixed with 4% PFA solution for 10 min followed by washing with distilled water for 5 min. The slides were dipped in 60% isopropanol solution and immersed in a staining jar containing a filtered ORO dye (Sigma life science) diluted with distilled water in a ratio of 50:50 for 15 min. After that, the slides were dipped again in 60% isopropanol then passed in a double washing with distilled water for 10 min. Finally, PVA/DABCO mounting media was used to coverslip the slides. High-resolution images were obtained at 10 × magnification (Olympus BX53 microscope). The ORO+ lipid density was quantified using the area fraction measurement tool of ImageJ software (NIH, USA). The density was expressed as the percentage of the ORO+ fraction area of the image area.
Confocal microscopy and image analysis
Confocal images were sampled from striatal sections according to AP + 0.62 to + 1.18 relative to bregma , using a Leica DMi8 confocal microscope. The collected images were obtained from a z-stack size of 10 µm and 1 µm step size. For maximum image projection, ImageJ software (NIH, USA) was used for z-stack image reconstruction. The same acquisition settings were applied for each image.
Quantification of vessel density and branching points
Vessels stained with CD31 were analyzed in 2–3 striatal sections obtained from the dorsolateral striatum, whereby 2–3 images per section were acquired (image size 630 µm × 630 µm). The obtained z-stack images were reconstructed for maximum projection. Quantification of vessel density was performed using the measurement tool of the ImageJ software (NIH, USA). The density was expressed as the percentage of CD31+ area of the image area. Branching points of the CD31+ vessels were counted manually per image by counting vessel intersections on the maximum projection images that were verified to be vessel branches and not vessel overlay by reviewing the corresponding z-stacks image.
Quantification of pericyte density
Image analysis of CD13+ pericytes was performed on 2–3 striatal sections whereby 2–3 images (image size 630 μm × 630 μm) per section were obtained from the dorsolateral striatum. The maximum projection image of CD13+ was analyzed using the area fraction measurement tool of ImageJ software (NIH, USA). The density was expressed as the percentage of CD13+ area of the image area. The ratio of CD13+ pericyte density to CD31+ vessel density was calculated by dividing the CD13+ pericyte density by the CD31+ vessel density.
Extravascular fibrinogen and IgG quantification
To quantify vascular leakage, extravascular fibrinogen and immunoglobulin G (IgG) were analyzed. CD31 staining was used to delineate vessels and to identify and exclude intravascular fibrinogen or IgG. The area covered by extravascular fibrinogen or IgG was analyzed and reported as the percentage of the total image area analyzed using ImageJ (NIH, USA).
DA neurons and fiber quantification
DA neurons labeled with TH in the substantia nigra (SN) were identified and counted at 20 × magnification at three SN sections per mouse located approximately at Bregma − 3.16 mm, − 3.40 mm, and − 3.64 mm [33, 37] using the Olympus BX53 microscope and CellSens software. The data were presented as a percentage of TH+ cells remaining in the ipsilateral side compared to the mean of the TH+ cells in the contralateral intact hemisphere of sham/CTRL diet mice corresponding to 100%.
The density of TH+ and DAT+ fibers was evaluated in 3 striatal sections per mouse located between AP + 0.62 to + 1.18 relative to bregma . High-resolution images were obtained at 4 × magnification (Olympus BX53 microscope). The optical density of the striatal fibers was analyzed by ImageJ (NIH, USA) and normalized to the corpus callosum for each picture. The data is presented as percentage of fiber density in the ipsilateral side compared to the mean of the fiber density in the contralateral intact hemisphere of sham/CTRL diet mice corresponding to 100%.
Multiplex sandwich ELISA
Leptin levels were quantified from plasma samples collected before mice were sacrificed using a mouse multiplex sandwich ELISA (Meso Scale Discovery, Metabolic hormones combo1, Gaithersburg, MD), according to the manufacturer instructions. Each sample (50 μl) was measured in triplicate, and the mean calculated. The coefficient of variation was below 20% for the assay.
CD11b+ cell analysis
CD11b was used for labeling the microglial/phagocytic cells in the striatum. We analyzed the number, activation state, and localization of the microglial cells in relation to vessels.
CD11b+ cell count
Using unbiased stereology counting, the number of CD11b+ cells was estimated in the dorsal lateral striatum. The cells were counted in every 12th striatal section (section sampling fraction ssf = 12). A total of 3 sections of each mouse were used between AP + 0.62 to + 1.18 relative to bregma . The count was performed in a Leica microscope equipped with a digital camera (Lecia MPS52) and connected to MicroBrightField stereological investigator software (Stereo Investigator MBF Bioscience V2019, US). Using a similar area for each section, the region of interest was delineated in the dorsolateral striatum using a 5 × magnification and counted at 100 × magnification. The counting frame (size 150 μm × 150 μm) was distributed randomly over grid (size 400 μm × 400 μm). The total count was calculated using optical fractionator population estimates. The accepted Gunderson coefficient of error for each animal was set at 0.08.
Microglial activation was quantified based on the morphological changes of the cell. Activated microglia were identified by a larger cell body and shorter process and branches . Modified from the previous method , ImageJ (NIH, USA) was used to measure cell branch density and cell body density. The ratio of branch density/cell body density was calculated as an index of microglial activation, whereby the lowest value represents the most activated microglia.
Distribution of microglia with respect to their perivascular location was presented in images showing microglial cells within a 20-μm distance from the vessel border by using the Distance Map plugin of ImageJ (NIH, USA). The communication between the vessels and perivascular microglia was quantified by measuring the density of interaction points between CD11b+ cells and CD31+ vessels using the colocalization plugin tool of ImageJ (NIH, USA) which was used to define and highlight the interaction points. The data is expressed as the ratio of the interaction point density to the CD31+ vessel density per image.
All data were analyzed by GraphPad Prism version 8. Data were first examined for normal distribution using the Shapiro–Wilk normality test. The non-normally distributed data were transformed before the analysis: HOMA-IR, insulin, and GTT (AUC) data were transformed to log values, while fibrinogen and IgG leakage data were transformed to sqrt values. All data were analyzed by two-way ANOVA followed by a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli test as multiple comparison test for all means . For comparison of the TH+ cell count between the CTRL diet and HFD, a two-tailed t-test was used. Data were presented as mean ± standard error of the mean (SEM). Significance was considered at a p-value < 0.05. Correlations were performed using a simple linear regression analysis with R2 coefficient. Graphs were plotted with curves indicating a 95% confidence interval. The equation slope was considered different from zero with a p-value < 0.05.