A Murine Model to Study Epilepsy and SUDEP Induced by Malaria Infection

Abstract

One of the largest single sources of epilepsy in the world is produced as a neurological sequela in survivors of cerebral malaria. Nevertheless, the pathophysiological mechanisms of such epileptogenesis remain unknown and no adjunctive therapy during cerebral malaria has been shown to reduce the rate of subsequent epilepsy. There is no existing animal model of postmalarial epilepsy. In this technical report we demonstrate the first such animal models. These models were created from multiple mouse and parasite strain combinations, so that the epilepsy observed retained universality with respect to genetic background. We also discovered spontaneous sudden unexpected death in epilepsy (SUDEP) in two of our strain combinations. These models offer a platform to enable new preclinical research into mechanisms and prevention of epilepsy and SUDEP.

Figure 1. Histological characterization of acute cerebral malaria.

(A) Examples of the appearance of sequestration of red blood cells (RBC) and white blood cells (WBC), as well as hemorrhages (HEM) in hippocampus, primary somatosensory (S1) and entorhinal cortex (Ent.), in infected (Infect.) versus control uninfected animals (Cont.). In sequestration, the blood cells are accumulating within blood vessels, whereas in hemorrhage the blood cells are extravascular. No cerebral vessel congestion or hemorrhage was observed in control mice. Magnification 100X, scale bar 150 μm. (B) Quantitative brain histological characteristics from different mouse-strain and parasite combinations from animals sacrificed at peak CM infection. Each block represents mean and standard error of the mean of values averaged over cohorts of 10 animals. The top row is the fraction of RBCs infected with parasite within the brain (parasitemia, Para, in %), with the peripheral parasitemia level indicated with gray. The second row is the ratio of brain to peripheral parasitemia known in human CM pathology as the sequestration index (SI). SI ratios greater than 1 indicate trapping of iRBCs within the brain, a hallmark of human CM. Note that most strain combinations have evidence of sequestration, but that it varies by brain region. The blood cell densities in rows 3–5 are all normalized by region and strain-specific control values, so control values appear at 1. The third row details the total blood cell (BC) density, a composite of WBC and RBC densities in rows four and five, normalized by the individual mouse strain control values. In the WBC/RBC ratio (plotted on log scale in the sixth row) we find unusually elevated ratios consistently equal or greater than 1 for strain/parasite combinations C57BL/6-PbNK65 and CBA-PbNK65. Because such high densities have not been reported in human histology from CM, these two strain combinations were eliminated from further study. Note that all control WBC/RBC ratios were less than 1. For each mouse strain, shipments of 30 animals were distributed evenly and randomly among each of 3 inoculation groups (PbANKA, PbNK65, Control) so that controls included littermates of infected animals. Bars indicate ± 1 s.e.m.

Figure 2. Parasitemia, Behavioral Signs and Survival curves from chronic monitoring cohorts.

(A) Shown in the upper panels are the peripheral parasitemia levels (average •, maxima *) for each mouse/strain combination, along with the survival probability (red line) through the infection (purple shading) and treatment (green shading) phases. Shown in the lower panel on the same time frame are the behavioral scale (BS) values, adapted from Caroll, et al.. The green dots mark individual treatment times. BS values include: Norm – Normal behavior; Ruff - Poor grooming including observation of ruffled hair; Slow - Slow movement, including hunched body posture; Ataxia - Tendency to roll over on stimulation, ataxia, evidence of hemi- or para-plegia, ~10% body weight loss; Coma - Comatose, convulsions, >20% body weight loss. Note that within 1 day of treatment, infected animals’ parasitemia drops by approximately half and their behavior returns nearly to normal. (B) Shown are Kaplan-Meir curves for survival by mice with CM by animal-parasite strain combinations for all animals inoculated for chronic monitoring. Those sacrificed according to protocol, malarial recrudescence, or associated with surgery were censored. Note that the mouse strain SW demonstrated the shortest survival rates when infected with PbANKA (log-rank test p < 0.05), in contrast to the longest survival rates for SW infected with PbNK65.

Figure 3. Summary time courses for each cohort and animal.

(A) Typical time course of an experiment from inoculation through to seizure associated (SA) death. (B) Time courses of all recorded animals, with columns for experimental strain combinations and control cohorts, and row for individual animals. The time courses begin with inoculation (magenta triangle) at day 0, and the period of infection is marked in purple. Treatment is indicated with green markers, the time of electrode implantation indicated with blue markers, and the periods of recording marked with blue background. The time of seizure occurrence over 10 s is indicated by red markers, with height representing duration in minutes. Time of death is indicated by the transition to grey (from investigator sacrifice, non-SA death) or pink (SA death) shading. Spontaneous deaths from the SA-death cohort forms the group further analyzed for possible SUDEP. Lettering within (A–E) correlate subjects with the seizure example panels in Fig. 4. (C) Cumulative time of recordings in grey and uncensored survival curves for each strain combination. Total recording time represented is 2751 days. (D) Cumulative number of seizures for each strain combination, with seizure origins indicated as hippocampal, cortical, and unknown. The cumulative number of seizures (>10 s long) marked is 786, with none observed from control animals. (E) Total number of seizures by category for each animal. Multiple seizure types are frequently seen in individual animals in the postmalarial epilepsy cohorts, while no seizures were recorded in any of the control animal cohorts.

Figure 4. Examples of seizure types and SUDEP.

Seizures were classified using the 2010 International League against Epilepsy seizure classification system depending on the mode of onset and semiology using video-EEG recordings. (A.a) Seizure of cortical onset with secondary generalization. In compressed time-scale below, (A.b), note direct current (DC) potential changes in cortical and hippocampal electrodes consistent with propagating spreading depression (SD) following seizure termination (vertical broken green line). In (B) is shown a focal hippocampal seizure, and in C a focal cortical seizure, both from the same animal. (D) Illustrates an example of a primary generalized seizure preceded by a series of pre-ictal generalized spikes. In (E) is shown an example of a sudden death during seizure. The cortical focal seizure shown in (E.a) is punctuated by the animal becoming behaviorally quiet, and is followed by propagating depolarizations consistent with SD, shown in (E.b). Following the seizure, the muscle activity is quiet enough to reveal the EKG reflected in the EMG lead which demonstrates progressive bradycardia leading to asystole shown in (E.c–E.g). EEG montage: EAL, EEG anterior left (frontal); EPL, EEG posterior left (somatosensory); DL, depth hippocampus left; DR, depth hippocampus right; EAR, EEG anterior right, EPR, EEG posterior right. Filter settings for traces shown: Seizure traces bandpass 1–50 Hz; SD traces low-pass below 1 Hz; EMG/EKG traces 0.1–55 Hz.

Figure 5. Seizure Origin and Evolution.

Seizure characterizations by origin within the brain and evolution of focal versus generalized seizure. Subdivisions within bars represent different animals. Note that each color represents a strain combination, and the individual counts are normalized by total number of seizures, so within each color-coded cohort the first 3 columns, and the last 3 columns, of a given color add to 1.

Figure 6. Methodology.

(A–C) Stereological Cell Counting Methods. (A) Brain regions of interest analyzed including the hippocampal sub-regions of the dentate gyrus (DG) outlined in blue, the CA3 outlined in white, and the CA1 outlined in black; somatosensory cortex outlined in green; and entorhinal cortex outlined in red. In B is shown a hematoxylin and eosin stained specimen with a superimposed optical fractionator counting frame (45 μm × 45 μm). Cells touching the purple boundary or inside the box are counted, but not if they touch an orange line. In C is shown a representative histogram demonstrating the number of cells counted along each plane of a single microscope slide. The top of the slide represents the first plane to come into focus. Note that a 2 μm guard zone was placed at the top and bottom of the 18 μm optical dissector depth. Scale bar for image A is 1000 μm and for image B is 15 μm. (D–H) Recording System Components. (D) Functional Schematic and photographs of custom 8-channel recording amplifier that includes a 24-bit digitizing analog front end, a microcontroller and power conditioning middle, and electrical isolation for power and USB. (E) Custom low-torque 4-circuit commutator that allows the recording amplifier to hang below it and permit free rotational motion of the cabled animal. (F) Custom headmount connector. (G) Micro-reaction chamber (μRC) electrodes created from hollowed out 50 μm gold coated stainless steel (type) wires internally deposited with iridium oxide to create very low electrical impedance electrodes. These μRC electrodes have reduced recording noise, and maintain quality recordings over the long periods of time required to perform these chronic experiments. (H) Custom designed animal housing cages that permit long-term video and electronic recordings from implanted animals.