Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway

Abstract

The in utero environment profoundly impacts childhood neurodevelopment and behaviour. A substantial proportion of pregnancies in Africa are at risk of malaria in pregnancy (MIP) however the impact of in utero exposure to MIP on fetal neurodevelopment is unknown. Complement activation, in particular C5a, may contribute to neuropathology and adverse outcomes during MIP. We used an experimental model of MIP and standardized neurocognitive testing, MRI, micro-CT and HPLC analysis of neurotransmitter levels, to test the hypothesis that in utero exposure to malaria alters neurodevelopment through a C5a-C5aR dependent pathway. We show that malaria-exposed offspring have persistent neurocognitive deficits in memory and affective-like behaviour compared to unexposed controls. These deficits were associated with reduced regional brain levels of major biogenic amines and BDNF that were rescued by disruption of C5a-C5aR signaling using genetic and functional approaches. Our results demonstrate that experimental MIP induces neurocognitive deficits in offspring and suggest novel targets for intervention.

Fig 1. In utero exposure to EMIP induces a persistent neurocognitive phenotype in offspring but is not associated with regional volumetric anatomical changes determined by MRI.

(a) Maternal parasitaemia (day one to seven post infection, n = 5) expressed as percent of infected red blood cells (iRBCs) per total red blood cells counted. (b) Offspring weight from one to six weeks of age in unexposed (n = 15) and malaria exposed offspring (n = 13). (c) Testing performance (preference ratio) and (d) total exploration time of unexposed (UE, n = 15) and malaria exposed (EX, n = 13) offspring (6 weeks of age) in the NOR test. (e) Performance of UE (n = 15) and EX (n = 15) offspring (6 weeks of age) in the TST test. (f) Testing performance (preference ratio) and (g) total exploration time of unexposed (UE, n = 11) and malaria exposed (EX, n = 12) offspring tested at 20 weeks of age in the NOR test. (h) Performance of UE (n = 11) and EX (n = 12) offspring in the TST test at 20 weeks of age. (i) Correlation between entorhinal cortical volume and performance (preference ratio) in the NOR test of unexposed and malaria exposed offspring (n = 24, spearman rho = 0.4912, P = 0.0044). (j) Depiction of average MRI, generated from all scans and all experimental groups, and outline of area used to define the entorhinal cortex. **P < 0.01, ***P < 0.005; T-Test. Data are presented as mean +/- SD.

Fig 2. Micro-CT images of fetal cerebral vasculature at gestational day 18.

Maximum intensity projection renderings of the axial (a) and sagittal (b) view of vasculature from wild-type unexposed offspring, axial (c) and sagittal (d) view of vasculature from wild-type malaria exposed offspring, axial (e) and sagittal (f) view of vasculature from C5ar-/- unexposed offspring and axial (g) and sagittal (h) view of vasculature from C5ar-/- malaria exposed offspring.

Fig 3. Quantitative analysis of fetal cerebral vasculature using a vessel segmentation algorithm determined (a) the number of vessel segments and (b) the total length of all vessel segments (mm) of wild-type unexposed (WT UE, n = 8), wild-type malaria exposed (WT EX, n = 7), C5a receptor knockout unexposed (C5ar-/- UE, n = 7) and C5a receptor knockout malaria-exposed (C5ar-/- EX, n = 7) offspring.

*P < 0.05; ANCOVA and post-test. Box plots depict median and interquartile range.

Fig 4. In utero exposure to EMIP is associated with localized changes in tissue levels of major biogenic amines in offspring at 8 weeks of age.

Tissue levels of (a) dopamine, and (b) serotonin in the frontal cortex, (c) norepinephrine in the temporoparietal cortex, (d) serotonin in the striatum, (e) serotonin and (f) norepinephrine in the cerebellum of unexposed (UE, n = 15) and malaria exposed (EX, n = 15) offspring. * P < 0.05, **P < 0.01, ***P < 0.005; T-Test. Box plots depict median, 95% confidence interval (box) and range (whiskers).

Fig 5. In utero exposure to EMIP induces a cognitive phenotype in offspring that is rescued by genetic and pharmacological blockade of C5a-C5a receptor signaling.

(a) Maternal parasitaemia (day one to seven post infection) expressed as percent of infected red blood cells (iRBCs) per total red blood cells counted in wild-type (n = 5) and C5ar-/- dams (n = 5). (b) Weight from one to six weeks of age in wild-type malaria unexposed (n = 14) and wild-type malaria exposed offspring (n = 13), C5ar-/- malaria unexposed (n = 13) and C5ar-/- malaria exposed (n = 14) offspring. (c) Testing Performance (preference ratio) and (d) total exploration time of wild-type unexposed (WT UE, n = 14), wild-type malaria exposed (WT EX, n = 13), C5a receptor knockout unexposed (C5ar-/- UE, n = 13) and C5ar-/- malaria exposed (C5ar-/- EX, n = 14) offspring in the NOR test (P > 0.05). (e) Performance of WT UE (n = 15), WT EX (n = 15), C5ar-/- UE (n = 13) and C5ar-/- EX (n = 15) offspring in the TST test. (f) Testing Performance (preference ratio) and (g) total exploration time of wild-type unexposed (WT UE, n = 11), wild-type malaria exposed (WT EX, n = 12), C5a receptor knockout unexposed (C5ar-/- UE, n = 13) and C5ar-/- malaria exposed (C5ar-/- EX, n = 9) offspring tested at 20 weeks of age in the NOR test. (h) Performance of WT UE (n = 12), WT EX (n = 11), C5ar-/- UE (n = 12) and C5ar-/- EX (n = 10) offspring tested at 20 weeks of age in the TST test. (i) Testing Performance (preference ratio) and (j) total exploration time of UE offspring (n = 11), EX offspring (n = 11), EX offspring of control rabbit antisera treated dams (R-EX, n = 12) and EX offspring of C5a antisera treated dams (αC5a-EX, n = 12) offspring in the NOR test (P > 0.05 for total exploration time). (k) Performance of UE (n = 11), EX (n = 12), R-EX (n = 10) and αC5a EX (n = 12) offspring in the TST test. *P < 0.05, **P < 0.01, ***P < 0.005; one-way ANOVA and post-test. Data are presented as mean +/- SD.

Fig 6. Reduced tissue levels of biogenic amine transmitters were observed in wild-type malaria exposed, but not in C5a receptor knockout offspring, relative to unexposed controls.

Brain tissue level of (a) dopamine (DA) and (b) serotonin (5HT) in the frontal cortex, (c) norepinephrine (NE) in the temporoparietal cortex and (d) serotonin (5HT) in the striatum, (e) norepinephrine and (f) serotonin in the cerebellum of wild type malaria exposed offspring (WT EX, n = 15) expressed relative to malaria unexposed wild type offspring and C5a receptor knockout unexposed offspring expressed relative to malaria unexposed C5ar-/- offspring (C5ar-/-EX, n = 15). In utero exposure to EMIP induced dysregulated messenger ribonucleic acid (mRNA) transcription level of BDNF in the fetal brain at gestational day 19. Fetal brain mRNA transcript level expressed as normalized copy number of (g) BDNF. *P < 0.05, **P < 0.01, ***P < 0.005; T-test (a-f) and one-way ANOVA (g). Box plots depict median, 95% confidence interval (box) and range (whiskers).