Epileptogenic effects of NMDAR antibodies in a passive transfer mouse model

Abstract

Most patients with N-methyl D-aspartate-receptor antibody encephalitis develop seizures but the epileptogenicity of the antibodies has not been investigated in vivo. Wireless electroencephalogram transmitters were implanted into 23 C57BL/6 mice before left lateral ventricle injection of antibody-positive (test) or healthy (control) immunoglobulin G. Mice were challenged 48 h later with a subthreshold dose (40 mg/kg) of the chemo-convulsant pentylenetetrazol and events recorded over 1 h. Seizures were assessed by video observation of each animal and the electroencephalogram by an automated seizure detection programme. No spontaneous seizures were seen with the antibody injections. However, after the pro-convulsant, the test mice (n = 9) had increased numbers of observed convulsive seizures (P = 0.004), a higher total seizure score (P = 0.003), and a higher number of epileptic 'spike' events (P = 0.023) than the control mice (n = 6). At post-mortem, surprisingly, the total number of N-methyl D-aspartate receptors did not differ between test and control mice, but in test mice the levels of immunoglobulin G bound to the left hippocampus were higher (P < 0.0001) and the level of bound immunoglobulin G correlated with the seizure scores (R(2) = 0.8, P = 0.04, n = 5). Our findings demonstrate the epileptogenicity of N-methyl D-aspartate receptor antibodies in vivo, and suggest that binding of immunoglobulin G either reduced synaptic localization of N-methyl D-aspartate receptors, or had a direct effect on receptor function, which could be responsible for seizure susceptibility in this acute short-term model.

Keywords: NMDAR; antibody; mouse electroencephalography; seizures.

Figure 1. Experimental technique and epileptogenic effects of a single injection of NMDAR antibodies.

(A) Diagram of placement of subcutaneous wireless transmitter in mice. (B) Fluorescent beads (red) lining the lateral ventricle show successful needle placement of the IgG injection (hippocampal fields indicated by DAPI staining in blue). (C and D) Group ethograms of all healthy control IgG (C, n = 6), and NMDAR antibody IgG (D, n = 9) injected mice following PTZ injection showing the times of different seizure events over the 60-min period before culling (see Supplementary Videos 1 and 2 for examples of stage 2 and stage 3 seizures, respectively). (E) Stage 3 convulsive seizures but not stage 2 seizures were more frequent in the NMDAR antibody IgG-injected mice following PTZ (P = 0.003). (F) The average score calculated for the 60-min observation period following PTZ was higher for NMDAR antibody IgG- than healthy control IgG-injected mice (P = 0.003). Error bars are mean ± SEM. HC = Healthy control; NMDAR-Ab = NMDAR antibody.

Figure 2. EEG telemetry and analysis.

(A) 2D representation of the event library with corresponding examples of library events. One second epochs of EEG on the right are defined by six separate metrics (calculated characteristics of the EEG waveform), two of which are indicated on the axes. Each small square (red/light green/dark green) represents an event as defined by six separate metrics and confirmed by visual inspection of the EEG and accompanying video. Similar events cluster together when plotted. ‘Spike’ was seen with a convulsive seizure, ‘Shake large’ and ‘Shake small’ were both seen with headshakes and needed to be differentiated from epileptiform activity. This library was used to screen EEG data by automated computer detection. (B) A representative EEG of an NMDAR antibody-injected mouse post-PTZ shows a number of ‘spikes’ corresponding to convulsive seizures (upper trace), compared to the EEG of a healthy control IgG-injected mouse, which has minimal spike activity (lower trace). (C) When analysed using the computer-based event detection program and blinded observer verification, the number of spikes seen in the hour following PTZ injection was greater in the NMDAR antibody (n = 7; data from two mice were excluded because there was significant signal drop-out/loss during the PTZ seizure induction hour) compared to the healthy control IgG (n = 6) injected mice (P = 0.023, Mann-Whitney). Results are mean ± SEM.

Figure 3. Detection of bound human IgG and NMDAR expression in hippocampi ex vivo.

(A) Human IgG injected in vivo was detected post-mortem in NMDAR antibody IgG-injected mice with anti-human IgG merged with the nuclear stain DAPI. The typical pattern of NMDAR antibody in the molecular cell layer with sparing of the granule cell layer was found. (B) Only low levels of IgG were found in the hippocampi of healthy control IgG-injected mice. (C) Bound human IgG in the hippocampi, as determined by the mean fluorescence intensity analysis of brain sections, was higher in the NMDAR antibody IgG-injected mice than in healthy control IgG-injected mice in CA1, CA3 and dentate gyrus (DG). (D) For the NMDAR antibody animals (n = 5) there was a linear correlation between IgG binding and seizure score (R² = 0.8; P = 0.04). (E) Binding of commercial antibody to an intracellular epitope of the NR1 subunit detected only after permeabilization. (F) Binding of the commercial antibody was not different between NMDAR antibody IgG (n = 9) and healthy control IgG (n = 7) injected brains. (G). Total surface NMDAR expression in the hippocampus measured by binding of an NMDAR antibody-positive CSF and detected with anti-human IgG; note that the green fluorescence here reflects NMDAR with IgG already bound in vivo as well as the CSF NMDAR antibody binding. (H) Total NMDAR, IgG-bound NMDAR on adjacent sections, and non-IgG bound NMDAR. Non-IgG bound NMDAR was calculated by subtracting the IgG-bound fluorescence from that of the total for each pair of sections, and was reduced in the NMDAR antibody IgG-injected mice compared to the healthy control IgG group (P = 0.03, Mann-Whitney test). Examples are given in Supplementary Fig. 3. IgG binding (green), DPAI binding to nuclei (blue). The data are presented as mean ± SEM. Columns represent results from NMDAR antibody IgG- (red) and healthy control IgG- (blue) injected mice. Numbers below the columns represent (number of brains, number of sections analysed).