Anti-brain autoantibodies and dysfunctional excitatory neurotransmission in obsessive-compulsive disorder



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ANTI-BRAIN AUTOANTIBODIES AND DYSFUNCTIONAL EXCITATORY NEUROTRANSMISSION IN OBSESSIVE-COMPULSIVE DISORDER

SUPPLEMENTARY METHODS AND MATERIALS

Recruitment and Assessment of subjects

This study was conducted at the Department of Psychiatry, NIMHANS, Bangalore following a protocol approved by the departmental protocol review board. All volunteers were included into the study after they gave written informed consent for lumbar puncture. All patients above the age of 15 years, screened at the psychiatry outpatient service of NIMHANS and found to be having OCD and who have not been on any psychotropic agents, were administered the Structured Clinical Interview for DSM IV- clinician version (SCID-CV) (First, 1997). Patients fulfilling the diagnostic criteria, and not having any lifetime history of psychotic disorder or mental retardation were included and rated on the Yale-Brown Obsessive Compulsive Symptom Checklist (Y-BOCS) and Yale-Brown Obsessive Compulsive Severity Rating Scale (Goodman, et al 1989a; Goodman, et al 1989b). Twenty-three psychotropic drug-naïve OCD patients who consented to take part were included in the study.

Sex-matched control population was identified from patients scheduled for various operative procedures under spinal anaesthesia in a general hospital located in the same geographical area and also from staff working in the hospital who volunteered to be part of the study. The control probands included 7 normal volunteers and 16 patients who underwent operative procedures for various conditions under spinal anaesthesia (Inguinal hernia- 8, fractures of the femur and tibia- 4, fibroid uterus- 1, benign hypertrophy of prostate- 1, varicocoele-1 and anal fissure-1). They were included into the study after obtaining informed consent and administration of SCID-CV (First, 1997) to rule out lifetime history of any psychiatric illness and history of head injury or any other neurological illness. 23 consenting psychiatrically normal controls were included into the study.

Basic socio-demographic details of the consenting patients and controls were collected in a semi-structured format devised for the study. Physical examination of patients and control subjects ruled out presence of any movement disorders. None of the patients and controls had history suggestive of Rheumatic fever, chorea and any other neurological or major systemic illnesses other than that reported earlier.


Sample collection and storage


About 2 ml. of CSF was collected from patients diagnosed to have OCD, after overnight fasting, under aseptic conditions from L4- L5 intervertebral space, using disposable spinal needles. The same procedure was followed for collecting CSF from the 7 normal volunteers who were not undergoing any operative procedure. For the rest of the control group, 2 ml. of CSF removed to inject the anaesthetic for spinal anaesthesia for surgical procedure was collected aseptically in the operation theatre from the 16 volunteers who were admitted for various operative procedures as detailed earlier. The CSF sample was stored at -70 °C. The stored CSF was used subsequently for the estimation of autoantibodies directed against BG and thalamic homogenates using western blot technique as well as for the estimation of amino acids.

10 ml. of blood was collected from the antecubital vein from patients as well as controls. Serum was separated by centrifugation at 2500 rpm and stored in aliquots at -70 °C.

The brain regions used for the western blot study were obtained from the Human Brain Tissue Repository at the Department of Neuropathology, NIMHANS, Bangalore. The BG specimen included a thin slice of caudate, putamen and globus pallidus at the level of the mamillary body taken from a 7-year boy, and the thalamus specimen was taken from a 34-year old man. Both had died of road traffic accidents and did not suffer from any known neurological disease before death. Both of them had received only hypertonic mannitol and other anti-oedema measures, but no other medication, for 10 hours during their hospital stay. Their bodies were shifted to freezer within 1 hour after death and maintained at 4 °C. Time lapsed between the time of death and collection of the brain and freezing the anatomical areas, was 20 hours for the basal ganglia specimen and 15 ½ hours for the thalamus specimen. At autopsy, the brain specimens were oedematous, but there was no coning or evidence of diffuse axonal injury and ventricular systems, cerebral vessels, subarachnoid space and brain stem were normal. The specimen from the 7-year boy had a small haematoma in the left temporal lobe with laceration and perilesional oedema.
Western Blot analysis

Western blotting was carried out to detect the presence of specific antibodies against BG (including caudate, putamen & globus pallidus nuclei) and thalamic homogenate using the procedure similar to Singer and colleagues (Singer, et al 1998). Frozen BG and thalamic tissues homogenized in cold 50 mM Tris-HCl, pH 7.4, containing triton-X, 1 µg/ ml aprotinin, 10 µg/ ml leupeptin, 10 µg/ ml pepstatin and 1 mM phenylmethylsulfonyl fluoride, were centrifuged at 15,000 rpm, and the pellets resuspended in storage buffer. The samples in storage buffer were assayed for protein and stored in aliquots at –70 °C. About 85 μg of protein from BG and thalamic homogenate were individually subjected to electrophoresis in 7.5 % acrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes. The nonspecific binding sites in the PVDF membrane were blocked for 12 hours with PBS containing 5% skimmed milk and 0.1% Tween-20 and then exposed to the primary antibody for 2 hours at room temperature. During an initial period of standardisation, dilutions of sera and CSF used in the final experiments were determined. Different dilutions of sera (1:100, 1:150, 1:200, 1:250 and 1:300) and CSF (1:25, 1:50, 1:100, 1:150) were initially used on the same gels during the standardisation phase to determine the optimal dilutions. The 1:250 dilution for sera and 1:50 dilution for CSF finally used in the western blotting analyses were found to be the optimal dilutions for maximising sensitivity and minimizing background staining. The difference in the optimal dilutions of sera and CSF required for this experiment was not unusual as we expected a higher non-specific background staining from sera containing higher levels of immunoglobulins compared to CSF. The dilution of sera in our study is similar to the dilutions used in some of the other studies investigating antineuronal and/ or anti-basal ganglia antibodies in tic disorder and OCD (10, 11). Paired sera or CSF samples (i.e. one OCD patient and one control sample) were used as primary antibody, to run on the same gel. The western blotting steps were carried out by SB who was blind to the status of the individual samples (i.e. whether from patient or control) that constituted each set of paired sample. The primary antibody solutions were prepared with serum and CSF as below, after initial standardisation to reduce non-specific background labelling:



  1. Serum: Sera from patient or control were diluted 1: 250 in PBS containing 3% skimmed milk powder solution.

  2. CSF: CSF samples from patient or control were diluted 1: 50 in PBS containing 3% skimmed milk powder solution.

Subsequently the PVDF membranes were washed and exposed to secondary antibody, goat antihuman horseradish peroxidise-linked IgG (1: 3000) in PBS containing 3% skimmed milk powder, for 1 hour at room temperature. After washing thrice, the reaction was visualised using enhanced chemiluminescence reagents and protocol according to Amersham and exposing to Kodak Xomat film. Molecular weights of the protein bands highlighted were determined by matching with the bands of molecular weight marker run simultaneously on the PVDF membrane. Two investigators (SKS, AM) blind to subject diagnosis read presence of bands on Western blot and their relative position with reference to molecular weight markers. Another investigator not involved in reading the gels interpreted the results.

Amino acid estimation

CSF amino acid (Glutamate, GABA, Glycine, Taurine) estimation was carried out by KC, who was blind to subject status, using an isocratic high performance liquid chromatography (HPLC)- electrochemical detection (ECD) system involving the use of pre-column o-pthaladehyde (OPA)- sulphite derivatisation method (Rowley, et al 1995) in a subsample of subjects for whom adequate CSF was available (22 OCD patients and 21 Controls). CSF Glutamate measured using an enzymatic technique from some of these subjects has been previously reported (4). Physiological standard amino acid solutions (L-Glutamic Acid, Glycine, Taurine, γ-Amino-n-Butyric Acid) solutions were prepared in polyethylene vials with crystalline amino acids (Sigma, St. Louis, USA) dissolved in double deionized water at a standard concentration of .001M and frozen in aliquots at -20o C for 5 days. Working solutions were prepared daily by thawing the stock solutions and later diluting the same.

Briefly, the steps of amino acid estimation included preparation of the CSF and standard samples by treating them with 1M perchloric acid (PCA) (1:9) (Qualigens, India) followed by centrifugation at 12000 rpm for 15 minutes at -4C and filtration through a 0.2μ Millipore filter. PCA treated CSF was then added to an equal volume of mobile phase (unchanged pH 4.80 approx.) while the standards were diluted with mobile phase (unchanged pH 4.80 approx.) depending on the concentration required. Both the treated CSF samples and the standards were reacted with freshly prepared derivatising agent based on a method described by Smith and Sharp (Smith and Sharp, 1994). The derivatisation agent was prepared by dissolving 22 mg of OPA in 0.5ml of sodium sulphite (1M) to which was added 0.5ml of absolute ethanol (Hayman Limited, England) and 0.9ml of sodium tetraborate buffer (0.1M) adjusted to pH 10.4 with 5M sodium hydroxide. The reagent was prepared in a darkened vial and remained stable throughout the working day. All chemicals other than absolute ethanol were obtained from Sigma, St Louis, USA. 1 ml of the amino acid standard or CSF sample was reacted at room temperature with 20 μl of freshly prepared derivatising agent for 10 min in a darkened polyethylene vial before injection onto the column. 20 μl of the reacted mixture was then injected.

The HPLC experiments were performed using a Supelco LC-18 analytical column, 25cmX4.6mm, 5μm, reverse-phase column protected by a guard column. Compounds were eluted isocratically with a mobile phase delivered at 0.65ml/min using a Shimadzu pump (model LC-10AD). The mobile phase consisted of 0.1M monosodium phosphate and 0.5 mM EDTA with 25% methanol (v/v) and water adjusted to pH 5 with 1M sodium hydroxide. Water and methanol (Qualigens, India) were HPLC grade. Mobile phase was filtered through 0.45μM Millipore filters and degassed for 30 minutes. Old mobile phase was replaced with fresh as necessary. An electrochemical detector (Shimadzu, Model L-ECD-6A) with a glassy carbon working electrode combined with an Ag/AgCl reference electrode was operated at +0.85V. A Rheodyne injection valve with a 20 μl sample was used to manually inject the samples. The HPLC analysis was done under ambient temperature conditions. Quantification of the amino acids and processing of the data obtained were done using Shimadzu class LC-10 software package.



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First MB, Spitzer RL, Gibbon M, Williams JBW (1996). Structured Clinical



Interview for DSM-IV Axis I Disorders- Clinician Version (SCID-CV). American Psychiatric Press: Washington, DC.

Goodman WK, Price LH, Rasmussen SA, Mazure C, Delgado P, Heninger

GR, Charney DS (1989a). The Yale-Brown Obsessive Compulsive Scale. II. Validity. Arch Gen Psychiatry 46:1012-1016.

Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL,

et al (1989b). The Yale-Brown Obsessive Compulsive Scale. I. Development, use, and reliability. Arch Gen Psychiatry 46:1006-1011.

Singer HS, Giuliano JD, Hansen BH, Hallett JJ, Laurino JP, Benson M,

Kiessling LS (1998). Antibodies against human putamen in children with Tourette syndrome. Neurology 50:1618-1624.

Rowley HL, Martin KF, Marsden CA (1995). Determination of in vivo amino

acid neurotransmitters by high-performance liquid chromatography with o-phthalaldehyde-sulphite derivatisation. J Neurosci Methods 57:93-99.

Smith SE, Sharp T (1994). An investigation of the origin of extracellular GABA



in rat nucleus accumbens measured in vivo by microdialysis. J Neural Transm Gen Sect 97:161-171.

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