A total of 61 male eight-week-old healthy naïve C57Bl/6 mice (Charles River, UK) were used in this study. 27 mice were used within the full lesion study and 34 within the partial lesion study. All 61 mice were housed in standard cages of 5 to 6 mice within a specific pathogen-free dedicated facility and 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 adhere to the ARRIVE guidelines for pre-clinical animal studies (Supplementary file 1), are in accordance with the UK Animals Scientific Procedures Act (1986) and were approved by King’s College London Animal Welfare and Ethical Review Body. The experimenter was blinded to the experimental groups for all surgical, behavioural and histological procedures.
Unilateral 6-hydroxydopamine (6-OHDA) lesioning and chondroitinase ABC (ChABC) treatment
All surgeries were conducted in a dedicated surgical procedure room between 9am and 5pm using a randomised block design to reduce bias between the blinded treatment groups. Fully lesioned animals were assigned to ChABC treatment (n=14) or saline control (n=14). Partially lesioned animals were assigned to ChABC treatment (n=17) or saline control (n=17). Final animal numbers were determined by previously conducted power calculations in order to reduce and refine animal usage. One of the 14 fully lesioned mouse from the ChABC group was euthanised prematurely due to poor health and therefore was not included within this study (final n=13 for this group). Inhalation anaesthesia was used in this study to facilitate both rapid induction and recovery from anaesthesia. 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. In order to generate full and partial 6-OHDA lesions, the toxin was injected into the region of the SNc and striatum, respectively . Doses of 6-OHDA administered were selected based on on previous pilot studies conducted in this same laboratory.
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; 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).
Immediately after surgery, animals were administrated buprenorphine (Vetergesic; 0.1 mg/kg; s.c.) for analgesia and 1ml of warmed Hartmann’s solution (Aqupharm 11; s.c.) for rehydration. Once animals regained consciousness, they were returned to their home cages and continued to receive 1ml of warmed Hartmann’s solution twice a day for one week.
Motor dysfunction was assessed between 10am and midday using two behavioural tests that were performed outside of the home cages. Animals were assessed in the order in which they were lesioned, retaining the randomised and blinded design. The cylinder test measured the degree of forelimb ability as described previously . 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 terminally anaesthetised following a 1ml injection (i.p.) of sodium pentobarbital (200mg/ml; 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 . Previous studies have reported no difference in outcomes when comparing stereological analysis with manual cell counting in the 6-OHDA lesion model . 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) 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 .
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.
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 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.