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