A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria

Abstract

Background: Cerebral malaria (CM) is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM) models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies.

Methodology/principal findings: A quantitative, rapid murine coma and behavior scale (RMCBS) comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment.

Conclusions/significance: Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field). The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

Figure 1. Mean RMCBS and day of infection curves.

These data represent the typical trajectory of disease in the MCM model, as highlighted by the RMCBS. Lightly colored lines represent individual subjects, and the two darker lines represent the mean scores from the experimental mice (red dashed line, n = 16) and the control mice (blue dotted line, n = 3). Comparing the mean RMCBS curves was significant, p<0.0001, and the t-test to assess the difference in RMCBS at day 8 of infection was also significant, p = 0.04.

Figure 2. Parasitemia levels in experimental and control mice.

Mice inoculated with PbA, as well as non-inoculated controls, were followed daily for parasitemia levels. Mean parasitemia on day 5 was 3.5%, with a peak of 11.8% on day 8 (including an outlier of 37%). Light gray lines represent individual subjects, and the two darker lines represent the mean levels from the experimental mice (dark dashed line, n = 16) and the control mice (dark dotted line, n = 3).

Figure 3. Overall outcome data.

A and B. Compared cumulative parasitemia data from 5 experiments involving symptomatic MCM versus asymptomatic infected mice, p = 0.21 and 0.11, with (A) and without (B) outliers of parasitemia >20%, respectively. C. Cumulative RMCBS data from the same 5 experiments. Pair-wise comparison of symptomatic MCM mice versus control: mean difference −14.6 (95%CI −16.4 to −12.7, p<0.0001); the symptomatic MCM versus asymptomatic: mean difference −11.3 (95% CI −13.9 to −8.7, p<0.0001); and the asymptomatic MCM versus control mice: mean difference −3.3 (95% CI −6.3 to −0.3, p = 0.027). Tukey-Kramer's method was used to adjust for multiple comparisons.

Figure 4. Increased frequency in surveillance via RMCBS.

Mice were rapidly assessed at frequent intervals on day 6 of infection via the RMCBS, allowing the operator to closely follow the trajectory of disease progression.

Figure 5. Variable RMCBS outcomes.

Mean RMCBS curves for MCM-susceptible mice (C57BL/6, n = 3), an asymptomatic C57BL/6 mouse (n = 1), infected MCM-resistant mice (balb/c, n = 4), and non-infected control C57BL/6 (n = 2) and balb/c mice (n = 2). Comparing slopes of mean RMCBS curves: infected C57BL/6 versus infected balb/c (p = 0.01); infected C57BL/6 versus non-infected C57BL/6 controls (p = 0.004); symptomatic infected C57BL/6 versus asymptomatic infected C57BL/6 (p = 0.06, marginally significant); infected balb/c versus non-infected control balb/c (p = 0.48); and infected balb/c versus asymptomatic infected C57BL/6 and non-infected control C57BL/6 (p = 0.70).

Figure 6. Parasitemia curves.

Mean parasitemia curves for symptomatic MCM-susceptible mice (C57BL/6), MCM-resistant mice (balb/c), an asymptomatic MCM mouse (C57BL/6), and non-infected control mice (C57BL/6 and balb/c).

Figure 7. Intracerebral hemorrhage in a symptomatic mouse.

PbA-infected mouse with final RMCBS of 5, H&E at 1000×, bar = 80-micrometers.

Figure 8. Correlation between RMCBS and number of hemorrhages.

A. Mice were serially sacrificed on days 2, 4, 6, 8, 9 and 10, demonstrating a strong negative correlation between the number of intracerebral hemorrhages and the RMCBS score on the day of sacrifice, r = −0.64 (p = 0.0001, experimental n = 22, control n = 6). B. Comparing the total number of hemorrhages in cohorts of mice that scored between 16–20 versus 0–15 (p<0.0001, Mann-Whitney U). Red crosshairs = mean RMCBS.

Figure 9. Hemorrhages and day of sacrifice.

Intracerebral hemorrhage tally in experimental mice versus day at which subject was sacrificed, demonstrating a positive correlation. Spearman Correlation = 0.776, p<0.0001.

Figure 10. Outcome of chloroquine treatment of symptomatic mice with RMCBS≤12.

Mean RMCBS scores of Non-Infected Controls (NIC, n = 6); Experimental Viable Treated (EVT, n = 5, peak parasitemia range 4.2%–5.9%, hemorrhage range 23–108 [mean 51]); Infected Non-Treated Controls (INTC, n = 15, peak parasitemia range 1.9%–12.1%, hemorrhage range 1–243 [mean 42]). An EVT animal received treatment on day 7 of infection (at RMCBS of 11), clinically worsened (RMCBS 7 on day 8), but made a full recovery.

Figure 11. Hemorrhage in recovered EVT mouse.

Only three small intracerebral hemorrhages were found on post-mortem examination of the EVT mouse that recovered (Figure 10), one of which is demonstrated here (black arrow). H&E, 1000×, bar = 80-micrometers.