Participants and Sera
Sera were collected from 8 children with acute SC and from 8 age-matched controls (mean age 11.1 ± 5 and 11.9 ± 3.8 years respectively; 2 tailed t-test p value=0.74) (table 1). The study was approved by Shaare Zedek Medical Center’s Helsinki committee and all parents signed an informed consent. Serum samples were stored in -70°C.Gama-immunoglobulin (IgG) fraction was isolated from the pool of SC patients sera and from the pool of the normal controls sera (4 ml containing 0.5 ml from each of the participants) by ammonium sulfate (10%) precipitation for 1 hour on ice followed by centrifugation (10,000 rpm at 10°C for 15 min). The precipitates were dissolved in 1 ml PBS, dialyzed 3X against PBS, concentrated by ultrafiltration (Vivaspin 2 MWCO 10000, Sartorius Stedim Biotech) and filtered to remove aggregates. The resulting IgG concentration was 935 mg/L for the SC sera pool and 874 mg/L for the control pool (the normal range for IgG values in serum is 340-1240 mg/L).
Western BlotAnalysis
Western blot was performed on cell extracts from three cell types: rat striatum, a human neuroblastoma cell line (SH-SY-5Y) and rat liver cells. Total protein was extracted by suspending the harvested cells in lysis buffer containing 10 mM Tris Base, 5 mM EDTA, 140 mM sodium chloride, 10 mM sodium fluoride, 0.5% NP 40 and 1 µM phenylmethylsulfonyl fluoride (PMSF). Following 30 min incubation on ice, the mixture was centrifuged (14,000 rpm, 10 min) and supernatants were collected. Protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL). Fifty μg protein of each sample was separated by 12% SDS-PAGE gels, and transferred to a nitrocellulose membrane. The membranes were blocked in 5% non-fat milk for 1 hour at room temperature and then were incubated overnight at 4°C with IgG extract (concentrated 2-fold) from either the SC patient (8 year old female) or from age matched control (7 year old female) (diluted 1:10 in milk), followed by biotinilated anti-human antibody (1:2,000; Sigma-Aldrich) and avidin conjugated to horseradish peroxidase (HRP; 1:25,000 Sigma-Aldrich). The membranes were developed with the ECL Plus detection system (Amersham Pharmacia Biotech).
Animals and Stereotactic Injection
Wistar male rats (Harlan, Israel) weighing ~220 g at the beginning of the experiment were used. Rats were placed under a 12 hour light/12 hour dark cycle in IVC plexiglas cages with ad libitum access to food and water. Six μl of the IgG fraction from the SC or control sera pools were injected using a stereotactic frame (Stoelting, USA) under chloral hydrate anesthesia. The injection targets were three different locations of the rats' left striatum: AP +1.7 Lat 2.5 Ven. -4.5, AP +0.2 Lat 3.5 Ven. -5.5 and AP -0.9 Lat 4.0 Ven. -4.5 (coordinates relative to the bregma). A total of 2 µl was injected per site at a rate of 1 µl/min. The needle was left for an additional 5 min at the location before it was slowly removed. Eight rats were injected with the SC IgG (6 µg/rat) and eight with the control, IgG (6 μg/rat), by the same investigator (OS). Previous experiments using stereotactic injection of 10 μL led to a macroscopic brain lesion in the injected area. All experimental protocols were approved by the Ethics Committee for Animal Use for Research and Education of Tel Aviv University.
Behavioural tests in rats
Amphetamine- and apomorpine-induced rotational behavior. On day 10, rats were injected with D- amphetamine (2.5 mg/kg, IP) which releases synaptic dopamine, and on day 17 with apomorphine (1 mg/kg, SC), a dopamine agonist. The rotational behavior (number and direction) was measured automatically by using a Rotameter (San Diego Instruments), for 60 min after D-amphetamine injection and for 45 min after apomorphine injection. Spontaneous exploratory forelimb use test. We assessed the forelimb use during explorative activity in a transparent plexiglas cylinder for 5 minutes. Forelimb-use asymmetry was determined by independent use of the left or right forelimb for contacting the wall during rearing and score was calculated by ipsilateral minus contralateral forelimb use.1, 2
Immuohistochemistry
Naïve rats: Wistar male rats weighting approximately 250 grams were sacrificed after intracardial infusion of ice cold PBS followed by 4% paraformaldehyde. Brains were removed and immersed in 4% paraformaldehyde for 24 hours followed by cryoprotection for 48 hours in 30% sucrose. Frozen tissues were creosectioned (10 micron). Sections were blocked in PBS containing 5% goat serum (Biological Industries, Beit Haemek, Israel), 1% BSA (Sigma-Aldrich, St. Louis, USA) and 0.5% triton X100 solution for 1h in RT. Following blocking, sections were exposed to IgG from SC or control serum (1:10 in a diluted blocking buffer 1:1 in PBS, overnight at 4°C). Sections were then exposed to biotinilated anti-human IgG antibody (1:200, Sigma-Aldrich) and then to 488 alexa-conjugated streptavidin (1:500, Invirtogen).
Stereotacticly injected rats: Rats were sacrificed on day 17 after injection, brains were removed and creosectioned as described above. Following blocking, sections from 3 animals per group were exposed to biotinilated anti-human IgG antibodies and then to 488 alexa-conjugated streptavidin as described above. For thyrosine hydroxylase and GAD65/67 staining, sections were microwave-boiled in citric buffer for antigen unmasking. Prior to blocking, peroxidase quenching was performed by exposing the sections to H2O2 (3%) for 10 minutes. Sections were incubated with the primary antibody overnight at 4°C (anti-tyrosine hydroxylase (TH), 1:200, Sigma-Aldrich or anti GAD65/67, 1:300, Millipore). After washing with PBS, sections were incubated with a anti mouse biotinilated secondary antibody (Invitrogen, Carlsbad, CA) for one hour followed by DAB staining using Vector ABC kit (Vector, Peterborough, UK).
1.Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 2000;39(5):777-787.
2.Kells AP, Henry RA, Connor B. AAV-BDNF mediated attenuation of quinolinic acid-induced neuropathology and motor function impairment. Gene Ther 2008;15(13):966-977.