Experimental design and housing conditions
Twenty adult male Sprague-Dawley rats 3-month-old (280-300 gr) were randomly assigned to the following groups: Saline group (n = 10) as control and 6-OHDA group (n = 10). After stereotaxic injection of toxin or saline solution the animals were housed for two weeks in monitored enriched environment cages (10 animals per cage) consisting of large cage (790 mm × 460 mm × 640 mm) with two floors, which were connected by a plastic ramp and an external running wheel. A 12 h light/12 h dark cycle was established with access to food and water ad libitum as previously described [15]. Current EE cages were supplied with an automatic recording system. This novel system consists, basically, of an infrared camera connected to a raspberry device programmed to collect pictures accurately every 30 seconds. In order to measure changes in the activity, all images were analyzed by an own designed customized program.
Two weeks after the intrastriatal injection the amphetamine test was performed for evaluating motor deficits and with the purpose of confirming that no rat presented more than 3 turns per minute (tpm). After amphetamine test, rats were sacrificed for histological and biochemical analysis.
All the experimental protocols were approved by the Ethical Committee and Animal Welfare (CEEA) of the University of the Basque Country (CEEA/M20/2016/176), and in accordance with Spanish Royal Decree RD 1201/2005, European Directive 2003/65/EC and the European Recommendation 2007/526/EC on the protection of animals used for scientific purposes. Furthermore, subclinical changes were daily evaluated throughout an observational protocol based in Morton-Griffiths ones [16]. This supervision protocol allowed us to assess the animal welfare by evaluating qualitatively the changes induced by this model regarding a sort of detectable variables.
6-OHDA lesions
Rats were lesioned as we previously described [14,15]. Briefly, rats were unilaterally injected into the striatum with 7.5 micrograms of 6-OHDA, but a short time of evolution (two weeks) was elapsed before sacrifice in order to achieve a preclinical lesion.
Monitoring analysis
An in-house customized automatic system based on image processing with a camera infrared Camera Module v2 (Pi NoIR) was used for the analysis of the rat group behavior. A schematic diagram of the working procedure for image processing is described in Fig. 1.
This process was divided in two phases: image pre-processing and image analysis.
1. In the first phase video sequences were acquired by a high-quality camera and pre-processed by an own toolbox in MATLAB to create series of binary image frames of the rat group activity. Thus, the activity areas (motion detection) were calculated based on a frame difference method. Frame absolute difference was calculated between two consecutive frames with a sampling period of 30s. Images were converted from RGB into grey scale, after that in binary images and then by a threshold filter extracted the main activity area of the system (rats, and objects moved by the rats). Finally, activity areas were defined applying morphological filters to reduce the noise and for smoothing the images.
2. During the second phase the trajectories of the animal group is generated by the system centroid (the average position of all the activity areas in the binary image) evolution. The centroid is estimated by k-means algorithm over the binarized frame series [17,18]. Thus, within each frame, the coordinates of the centers of every object were calculated, and K-means were applied to find the center of the entire group, that is the “centroid”. A centroid with two coordinates:
3. The group trajectory consists of the evolution of that centroid from the first frame to the last one.
Finally, the rat groups’ behavior was described and modelled by the following parameters:
1) Number of activity areas in the binary images.
2) Shannon entropy of the centroid trajectory. Shannon entropy is a main concept in information theory and is a measure of average uncertainty (information content). Entropy in biosignals gives information about the system evolution and behavior and can be applied to analyze pathological behaviors [17,18].
3) Velocity, speed and acceleration defined as:
being speed the absolute value of velocity. An acceleration:
Amphetamine-induced rotation test
Two weeks after intrastriatal injection the amphetamine test was developed following the previously described methodology for this behavioral test [19]. Briefly, D-amphetamine (5 mg/kg) in 0.9 % NaCl; Sigma-Aldrich, St. Louis, USA) was intraperitoneally administered and the animals were placed in an individual circular cage (rotameter). After 15 min of latency, the total number of full ipsilateral (IL) rotations was recorded during 90 min (Multicounter LE3806; Harvard Apparatus, Holliston, MA, USA) and data expressed as the number of rotations per minute.
Morphological analysis
Morphological evaluation was performed as we previously described [14,15,20].
Tissue processing for histological evaluation
After behavioral test, five rats from each group were transcardially perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). Brains removed and 50 mm serial coronal sections including striatum and substantia nigra (SN) were collected for further analysis.
TH immunostaining
Tyrosine hydroxylase (TH) immunohistochemical staining was developed on free-floating coronal slices. Briefly, sections were incubated with rabbit polyclonal anti-TH (Ref: AB-152, Millipore; 1:1000). Sections were processed with the avidin-biotin-peroxidase complex using a commercial kit (Ref. PK-6102, Elite ABC kit, Vector Laboratories, Burlingame, CA) and the reaction was shown by using 3,3-diaminobenzidine (DAB).
Images were visualized, captured and analyzed at 4x and 40x magnification by an Olympus BX-50 photomicroscope.
Double immunofluorescence staining of Caspase-3 and NeuN
Coronal sections were incubated with rabbit anti-caspase 3 (H-277) (Ref: sc-7148, Santa Cruz Biotechnology Inc; 1:50) and monoclonal mouse anti-NeuN (Ref: MAB377, Chemicon International, Inc.; 1:100). Sections were subsequently incubated with secondary antibodies conjugated to Alexa Fluor-488 (Ref: A11029; Invitrogen; 1:400) and Alexa Fluor-568 (Ref: A11036 Invitrogen; 1:400). Immunostained sections were further reincubated with Hoechst for nuclear counterstaining. Images were finally analyzed at 20x magnification using Olympus Fluoview FV500 confocal microscope.
Stereological analysis
The stereological analysis was performed as previously described [14,15,20] by using a computerized image analysis system (Mercator Image Analysis system, Explora Nova, La Rochelle, France) connected to an Olympus BX-50 photomicroscope.
In brief, in order to evaluate the TH-immunoreactivity (ir) in the IL striatum (lesioned striatum) the volume of preserved striatum was calculated by delimiting negative areas and the entire striatum and multiplying these measurements by the thickness of the slices and the intersectional distance. Values were expressed as a percentage of the TH-negative volume versus the entire volume of the IL striatum.
Changes in the density of dopaminergic neurons and axodendritic network were evaluated through quantifying the TH-ir neuronal density in the entire SN and TH-ir axodendritic network density (ADN) in the SN reticulata (SNr). Data were expressed as the percentage of neurons or ADN presents on the lesioned side versus the non-lesioned hemisphere.
The study of the topological distribution was performed following the previously described approach [20,21].
Biochemical analysis
Western blotting
After behavioral test, 5 rats of each group were decapitated to obtain fresh brain tissue. Ipsi- and contralateral (CL) striatum and SN were collected by microdissection and quickly frozen. Proteins were isolated and the concentration of soluble proteins was quantified by the Bio-Rad Protein Assay (Ref: 500-0006, Bio-Rad Laboratories) based on Bradford’s method.
As previously described [14,15], for each sample 20 mg of proteins was loaded into polyacrylamide CRITERION TGX 12% gels (Bio-Rad Laboratories Inc., Spain) for electrophoresis and then transferred to a PVDF membrane in a Trans-Blot Turbo Transfer System (Bio-Rad, USA) for 7 min. Membranes were incubated with the following primary antibodies: rabbit anti-Phospho-AKT (Ser 473) (1:1000), rabbit anti-AKT (1:1000), rabbit anti-Phospho-p44/42 MAPK (extracellular signal-regulated protein kinases 1 and 2 [ERK 1/2]) (Thr202/204) (1:1000), rabbit anti-P44/42 MAPK (ERK 1/2) (1:1000) (all of them from Cell Signaling Technology Inc. USA), rabbit anti-caspase 3 (H-277) (1:1000) (Santa Cruz Biotechnology Inc., Spain), rabbit anti-β-Actin (1:2000) (Sigma-Aldrich, Spain) and rabbit anti-Beta-Tubulin (1:1000) (Novus Biologicals, USA) at 4 °C overnight. Afterwards they were incubated with anti-rabbit IgG peroxidase conjugated secondary antibodies (1:2000) (Sigma-Aldrich, Spain) for 2 hours at RT and immunoblots were developed with an enhanced chemiluminescence kit (GE Healthcare Life Science, UK). The luminescence of the reaction product was detected in a personal scanner, LI-COR C-DiGit (LI-COR, Bonsai Advanced Technologies SL, Spain), and visualized bands were analyzed with Image Studio Lite 4.0 software (LI-COR, Bonsai Advanced Technologies SL, Spain). β-Actin and β-tubulin were used as loading controls.
Statistical analysis
All results were expressed as the mean ± SE (standard error). Statistical analysis was developed with GraphPad Prism (v 5; GraphPad Software, Inc., USA) and SPSS Statistics (v 20; IBM Corporation, Armonk, NY, USA). Prior to the analysis, the Shapiro–Wilk test was used to assess the normal distribution of the samples, and Levene’s test was used to determine the homogeneity of variance. Mann-Whitney U-test was performed to assess differences between groups and within groups in the monitoring analysis. The ROC (Receiver Operating Characteristics) curve was performed and the AUC (Area Under the Curve) was calculated in order to measure the accuracy of the model which are a metrics for checking classification models, in this case: Saline group and 6-OHDA group. The behavioral data, stereology and densitometry results were analyzed by means of the two-tailed unpaired Student's t test to compare differences between groups. A one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparisons test was used to test the differences between rostro-caudal gradients within each experimental group. The correlation was examined by Pearson product-moment correlation coefficient. P values < 0.05 were considered statistically significant.