Animals
27 and 34 eight-week-old male C57Bl/6 mice (Charles River, UK) were used within the full lesion and partial lesion studies, respectively. All 61 mice were maintained on a 12:12 hour light/dark cycle (07:00am lights on) with food and water ad libitum. Room temperature and humidity were kept at 22 ± 2°C and 55 ± 2% respectively. All in vivo studies were performed in accordance with the UK Animals Scientific Procedures Act (1986) and were approved by King’s College London Animal Welfare and Ethical Review Body. All surgical, behavioural and histological procedures were performed whilst blinded to the experimental groups.
Unilateral 6-hydroxydopamine (6-OHDA) lesioning and chondroitinase ABC (ChABC) treatment
All surgeries were conducted in a randomised block design to reduce bias between the blinded treatment groups. Fully lesioned animals were assigned to ChABC treatment (n=13) or saline control (n=14). Partially lesioned animals were assigned to ChABC treatment (n=17) or saline control (n=17). Anaesthesia was induced with a 5% isoflurane/oxygen mixture then maintained at 3% isoflurane/oxygen. Body temperature was monitored and maintained at 37°C with a homeothermic heating blanket (Harvard Apparatus). The surgical site was sterilised with 0.4% chlorhexidine (Hibiscrub) before a midline incision was made along the scalp. The skull was then cleaned and dried with cotton swabs.
To establish a full unilateral 6-OHDA lesion, fine-bore holes (Ø 0.5 mm) were drilled at coordinates AP: -3.0 mm and ML: +1.2 mm (relative to bregma and skull surface). A blunt-ended 30G needle was then inserted supranigrally to DV: -4.5 mm (relative to bregma and skull surface) before 8 μg 6-OHDA.HBr (Sigma-Aldrich) in 1 μl 0.02% ascorbate/saline was administered unilaterally at a rate of 0.5 μl/min. The needle was left in place for 4 min to allow for toxin diffusion.
To establish a partial unilateral 6-OHDA lesion, fine-bore holes (Ø 0.5 mm) were drilled at coordinates AP: +0.5 mm and ML: +2.0 mm (relative to bregma and skull surface). A blunt-ended 30G needle was then inserted intrastriatally to DV: -3.5 mm (relative to bregma and skull surface) before 4 μg 6-OHDA.HBr in 1 μl 0.02% ascorbate/saline was administered unilaterally at a rate of 0.5 μl/min. The needle was left in place for 4-min to allow for toxin diffusion.
5-min after injection of 6-OHDA, animals from both studies received two intracerebral injections of either saline or ChABC (10 U/ml in saline; Seikagaku) into the same 6-OHDA injected hemisphere. In order to achieve digestion of CSPGs along the entire nigrostriatal tract, 1 μl ChABC was administered into the SNc (AP: -2.3 mm; ML: +1.0 mm and DV: -4.2 mm; relative to bregma and skull surface) and 1 μl ChABC into the striatum (AP: +0.02 mm; ML: + 2.2 mm and DV: -3.5 mm; relative to bregma and skull surface). These coordinates were determined by a previously run pilot study that showed these to provide most effective CSPG digestion along the entire nigrostriatal tract (data not shown).
After the surgery, all animals were administered buprenorphine (Vetergesic; 0.1 mg/kg; s.c.) for analgesia. All animals additionally received 1 ml of warmed Hartmann’s solution (Aqupharm 11; s.c.) twice a day for one week to maintain hydration.
Behavioural assessments
Motor dysfunction was assessed using two behavioural tests. The cylinder test measured the degree of forelimb ability as described previously [48]. Briefly, taking the lesion as day 0, on days -4, -1, 3, 10, 17, 24 and 31 for the full lesion study and on days -3, -1, 14, 21, 28 and 35 for the partial lesion study, mice were placed individually within 2 litre glass beakers (Ø 12 cm) and their forepaw preference was monitored during exploratory rearing behaviour captured through 5-min video recordings. Touches by the forepaw ipsilateral-to-the-lesion, contralateral-to-the-lesion or by both forepaws simultaneously were measured. An asymmetry score was then calculated to indicate the proportional use of each forepaw: a score of 50% indicated no bias and <50% denoted impairment of the injured (contralateral) forepaw.
Amphetamine-induced rotations were counted on day 35 (full) or day 37 (partial) post-lesion. Animals were placed within cylindrical arenas (Ø 40 cm) in which the motion-tracking tool Ethovision XT6 was used for recording. A custom optimised calibration file was loaded, which allowed the recording of full 360° rotations about the animal's midpoint. Ipsiversive and contraversive rotations were then individually measured before calculating net ipsiversive rotations. Following a 20-min habituation period, mice were administered with D-amphetamine hemisulphate (Tocris; 5 mg/kg in saline; i.p.) and rotations were recorded for 90-min thereafter.
Histological assessment of lesion severity
On day 42 post-lesion (full lesion study) or day 39 post-lesion (partial lesion study) all animals were humanely killed following sodium pentobarbital terminal anaesthesia (200mg; Sigma; i.p.). Animals were then formalin-perfused and their brains post-fixed in 4% paraformaldehyde/PBS before being embedded in paraffin wax blocks. 7 µm thick coronal sections were cut with a microtome (Thermo Scientific) at three rostrocaudal levels of the SNc (rostral: -2.92 mm, medial: -3.16 mm and caudal: -3.52 mm AP; relative to bregma) and three levels of the striatum (rostral: +1.0 mm, medial: +0.5 mm and caudal: -0.22 mm AP; relative to bregma). Sections were then mounted on Poly-L-lysine coated slides (VWR). Three sections from each of the three levels of the striatum (9 in total) were incubated with rabbit polyclonal anti-tyrosine hydroxylase (TH) primary antibody (AB152, Millipore) overnight before being washed in TBS twice and incubated in biotinylated goat anti-rabbit secondary antibody (BA-1000, VectorLabs) for 2h at room temperature. Following a further two TBS washes, the sections were incubated with streptavidin-biotinylated horseradish peroxidase conjugate (PK6100, VectorLabs) for 30-min at room temperature. Slides were then immersed in diaminobenzidine tetrachloride for 10-min and mounted.
Cells of the SNc and TH-positive fibres of the striatum, in both the lesioned and intact hemispheres, were imaged with 100X and 50X magnification, respectively (Axioskop, light-field compact microscope). For the purpose of side-to-side comparisons where the intention is to quantify percentage loss between the intact and lesioned hemispheres in a given animal, we adopted manual cell counting [20]. Previous studies have reported no difference in outcomes when comparing stereological analysis with manual cell counting in the 6-OHDA lesion model [21]. ImageJ software was used to count the number of viable (i.e. intact round cells with a clear nucleus and cytoplasm) A9 TH-positive SNc cells and to measure mean grey value (MGV) of striatal TH-positive fibres in the dorsal and ventral striatum. In all cases, the operator was blinded to treatment throughout the analysis. An average of all three analysed levels of the SNc/striatum was produced for both the saline- and ChABC-treated groups. In fully lesioned mice, no differences between levels were noted for either striatum or SNc, so data were pooled across the entire rostrocaudal extent, while for partially lesioned mice the levels were analysed independently. Data are expressed as number of cells (SNc) or fibre density (striatum and rostral SNc [in partial lesion study]) in the lesion side as a percentage of the respective intact side.
Confirmation of CSPG digestion by chondroitin-4-sulphate stub histology
Immunohistochemistry was used to stain for the C4S-stub antigen that remains following ChABC-mediated digestion. Staining for C4S (mouse monoclonal; 1:500; MPBio #636511) was completed using sections adjacent to the TH-stained sections whilst using the same protocol as previously described for TH [29].
To confirm whether the extent of ChABC-mediated digestion of CS-GAGs along the nigrostriatal pathway in vivo was maximal, the effect of subsequent ChABC exposure ex vivo was assessed. Briefly, 7 µm SNc brain sections from saline- and ChABC-treated mice from both the full and partial lesion studies (n=3 for each group) were incubated with ChABC (10 U/ml; Seikagaku) or tris-buffered saline for 3 h at 37°C. C4S-stub immunoreactivity was then stained for, as described above.
Statistical analysis
All quantitative data are expressed as mean ± standard error of the mean (S.E.M.). Statistical analyses were conducted with GraphPad Prism (version 7) software. Behavioural data were analysed using two-way repeated measures ANOVA, SNc cell count data and TH density were analysed by unpaired Student t-tests and striatal TH-positive fibre MGV data were analysed by two-way ANOVA. Post-hoc tests were applied when appropriate as detailed in figure legends.