Microinfusion of antineuronal antibodies into rodent striatum: Failure to differentiate between elevated and low titers

https://doi.org/10.1016/j.jneuroim.2005.02.018Get rights and content

Abstract

An autoimmune-mediated mechanism has been proposed for several pediatric movement disorders. In a three-center (Brown, Yale, and Johns Hopkins) collaborative effort, serum antineuronal antibodies (ANAb) were measured by use of ELISA or immunohistochemical techniques on 35 children (mean age 11.4 years) with Tourette syndrome, attention deficit hyperactivity disorder, and/or obsessive compulsive disorder. Eight sera, 4 containing the highest and 4 the lowest levels of ANAb, were identified at each institution. Selected sera (total of 9 with elevated and 7 with low ANAb) were re-encoded and sent to each center for infusion into the ventrolateral striatum of 16 male Sprague–Dawley rats. Animals were observed for behavioral abnormalities for 3 days before the start of infusion, during infusion on days 2–4, and for 2 days after infusion. Combined stereotypy scores increased after antibody infusion, but there was no significant effect based on serum titer (p = 0.85). Scores differed among centers, but analyses based on individual institutional data again failed to show an effect based on elevated or low ANAb values (Brown, p = 0.95; Yale and Johns Hopkins, p = 0.81). Post hoc studies with sham surgery and infusion of phosphate-buffered saline support suggestions of nonspecific behavioral effects unrelated to antibody titer. This report emphasizes that any conclusions about antibody-mediated movement disorders that are based upon results from the rodent infusion model must be considered with caution.

Introduction

Antineuronal antibodies (ANAb) have been proposed as the pathophysiological mechanism for several movement disorders including, Sydenham chorea (SC), Tourette syndrome (TS), pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), paroxysmal dyskinesias, dystonia, and myoclonus (Dale et al., 2002a, Dale et al., 2002b, DiFazio et al., 1998, Swedo et al., 1998, Swedo et al., 1993). In some of these conditions, it is proposed that antibodies formed against group A beta-hemolytic streptococcus cross-react with specific brain neurons, causing unique changes in cellular function that give rise to movement disorders. For example, in SC, antibody-mediated signaling has been shown to involve specific induction of calcium/calmodulin-dependent protein kinase II activity (Kirvan et al., 2003). In other disorders, such as Tourette syndrome, elevated levels of antineuronal antibodies are found in individuals without evidence of a postinfectious etiology (Church et al., 2003, Morshed et al., 2001, Singer et al., 1998).

Documentation of an association between a movement disorder and the presence of serum antineuronal antibodies does not in itself establish pathogenicity. For example, many control subjects have nonpathogenic ANAb in their sera (Avrameas, 1991, Dighiero et al., 1983, Lacroix-Desmazes et al., 1998, Nobrega et al., 1993, Seigneurin et al., 1988, Wendlandt et al., 2001). Thus, in order to confirm a causal effect, the disorder must be inducible in healthy subjects/animals by passive transfer of autoantibodies from affected subjects. Several noninflammatory (no disruption of the blood-brain barrier) antibody-mediated animal models have been developed to provide a mechanism to accomplish this goal. One approach has been the direct infusion of antibodies from patients with movement disorders into regions of the animal brain that are known to be associated with the production of specific types of abnormal movements (Hallett et al., 2000, Taylor et al., 2002). For example, stereotypic movements can be reliably elicited in response to pharmacologic manipulation of the rodent lateral striatum (Canales and Graybiel, 2000, Dickson et al., 1994, Kelley et al., 1988).

To date, three studies have evaluated the effect of infusing sera or IgG containing elevated levels of ANAb from individuals with Tourette syndrome (non-PANDAS) into rodent striatum. Hallett and colleagues compared the effect of dilute serum (1:6) infused into the ventral striatum, from 5 TS patients and controls (Hallett et al., 2000). Sera from TS subjects, with no reported association with streptococcal infections, selected on the basis of antibody titers against a solubilized neuroblastoma cell membrane fraction produced a significant increase in stereotypic behaviors in Fischer 344 rats (e.g., licks and forepaw shakes) as well as episodic utterances. Abnormal behaviors were identified during the 3-day period of microinfusion and on days 8–10 after microinfusion. Taylor and colleagues infused sera from TS patients, ranked for autoantibody levels by use of an immunofluorescent technique against rat striatum, into the ventrolateral striatum of male Sprague–Dawley rats (Taylor et al., 2002). In that study, behavioral observations were reported during the 5 days of serum infusion. Results showed a significant increase of oral stereotypies in 12 rats receiving higher titer sera as compared to 12 rats infused with lower titer patient sera and 12 controls. No differences were found across the three groups for levels of antistreptolysin-O and anti-deoxyribonuclease-B titers. Lastly, Loiselle and coworkers microinfused serum from 5 subjects with TS, each with elevated ANAb titers against fresh human postmortem putamen, and 5 children with PANDAS into Fischer 344 ventral striatum or ventrolateral striatum, bilaterally (Loiselle et al., 2004). In that study, despite infusion of patient sera at the same coordinates used in the Hallett and Taylor protocols, there was no significant increase in stereotypic behaviors and no rat developed any audible abnormality.

Several possibilities have been put forward to explain the variability among the aforementioned studies, including methods used to quantify antineuronal antibodies, concentrations of sera infused, strains of rodents, timing of observations, and methods for recording activity. In order to establish a model useful for evaluating autoimmune hypotheses, the present study was a three-center collaborative effort among the previously reporting institutions (Brown, Yale, and Johns Hopkins), designed to clarify discrepancies in prior results. Sera from children with TS were first assayed under blinded conditions for antineuronal antibodies at each of the three institutions. Selected samples were then returned for infusion into an animal model by using a unified protocol. It was hypothesized that TS sera containing elevated levels of ANAb would induce a significantly increased amount of stereotypic behavior as compared to that induced by infusion of sera with lower titers.

Section snippets

Overview

ANAb titers were measured on 35 sera, with each center using a different method. An independent coordinator then identified sera with “elevated” titers and with “lower” titers. Selected samples were re-encoded and sent to the three centers where the investigators were unaware of the titers. Sixteen male Sprague–Dawley rats had cannulas stereotactically placed in their ventrolateral striatum, bilaterally. One week after surgery, undiluted sera were infused into the striatums for 4 days. Sera,

Antineuronal antibody titers

Antineuronal antibody titers were measured by three different methods on the same serum samples. The titers determined by ELISA (with either fresh human putamen or neuroblastoma cell membrane) were highly correlated (ρ = 0.81, p = 0.0001), but titers determined by an immunohistochemical technique against rat striatum did not correlate with either ELISA performed with neuroblastoma membrane (ρ = − 0.09, NS) or ELISA performed with human putamen (ρ = − 0.09, NS) (Fig. 1).

Samples selected for infusion

Samples containing elevated

Discussion

In this collaborative effort performed in three institutions with studies on the same sera, there was no significant difference in stereotypic behaviors induced by sera from neuropsychiatric patients containing either elevated or low concentrations of ANAb. This finding was consistent across all individual centers, as well as when analyzed as total mean values. These results differ from two prior publications, one reporting increased stereotypies in rats infused with elevated-compared to

Acknowledgments

The authors thank Dr. Pamela Talalay for the helpful discussion during the preparation of the manuscript and John Hong for technical support. This research was supported in part from grants from the Tourette Syndrome Association and the NIH MH49351, KO2 MH01527 and RO1 MH52711.

References (27)

  • J.J. Canales et al.

    A measure of striatal function predicts motor stereotypy

    Nat. Neurosci.

    (2000)
  • A.J. Church et al.

    Tourette's syndrome: a cross sectional study to examine the PANDAS hypothesis

    J. Neurol. Neurosurg. Psychiatry

    (2003)
  • A.J. Church et al.

    Anti-basal ganglia antibodies: a possible diagnostic utility in idiopathic movement disorders?

    Arch. Dis. Child.

    (2004)
  • Cited by (46)

    • Tourette syndrome

      2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 2
    • Tourette Syndrome and Tic Disorders

      2016, Handbook of Behavioral Neuroscience
    • Animal models of tic disorders: A translational perspective

      2014, Journal of Neuroscience Methods
      Citation Excerpt :

      In an attempt to demonstrate that TS pathogenesis may be linked to the presence of autoantibodies, serum from TS patients has been injected into the striatum of rats. This treatment has been shown to lead to motor and oral stereotypies, as well as episodic vocalizations and increased genital grooming (Hallett et al., 2000; Taylor et al., 2002; Singer et al., 2005), in association with IgG deposits (Hallett et al., 2000). Rats treated with serum from TS patients were also found to exhibit increased levels of dopamine and reduced DAT expression (Jijun et al., 2010).

    • Tourette Syndrome

      2014, Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease: Fifth Edition
    • Behavioral and neural effects of intra-striatal infusion of anti-streptococcal antibodies in rats

      2014, Brain, Behavior, and Immunity
      Citation Excerpt :

      We (Brimberg et al., 2012) and others (Hoffman et al., 2004) have found that exposure of rodents to GAS antigen leads to behavioral alterations, fulfilling Criterion D. Others (Ben-Pazi et al., 2012; Doyle et al., 2012; Hallett et al., 2000; Loiselle et al., 2004; Singer et al., 2005; Taylor et al., 2002; Yaddanapudi et al., 2010) have attempted to demonstrate that passive transfer of Immunoglobulin G (IgG) alters behavior (Criterion E). Some have been successful (Doyle et al., 2012; Hallett et al., 2000; Taylor et al., 2002; Yaddanapudi et al., 2010) while others have not (Ben-Pazi et al., 2012; Loiselle et al., 2004; Singer et al., 2005). The aim of the present study was to extend our previous findings (Brimberg et al., 2012) and test whether intra-striatal passive transfer of antibodies to animals would lead to the induction of a behavioral syndrome similar to those reported (Criterion E).

    View all citing articles on Scopus
    1

    Tel.: +1 585 275 2808.

    2

    Tel.: +1 410 955 7212; fax: +1 410 614 2297.

    3

    Tel.: +1 203 785 7971.

    4

    Tel.: +1 401 729 2582.

    View full text