SUMMARY Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1−/−) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.

SUMMARY Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, pan-uveitis and infection of the cornea, iris, optic nerve, and the ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, as Axl−/−, Mertk−/− or Axl−/− Mertk−/− DKO mice sustained similar levels of infection as control animals. We also detected abundant viral RNA in tears, suggesting that virus may be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye.

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Dimethyl sulfoxide (DMSO) is a solvent that is routinely used as a cryopreservative in allogous bone marrow and organ transplantion. We exposed C57Bl/6 mice of varying postnatal ages (P0–P30) to DMSO in order to study whether DMSO could produce apoptotic degeneration in the developing CNS. DMSO produced widespread apoptosis in the developing mouse brain at all ages tested. Damage was greatest at P7. Significant elevations above the background rate of apoptosis occurred at the lowest dose tested, 0.3 ml/kg. In an in vitro rat hippocampal culture preparation, DMSO produced neuronal loss at concentrations of 0.5% and 1.0%. The ability of DMSO to damage neurons in dissociated cultures indicates that the toxicity likely results from a direct cellular effect. Because children, who undergo bone marrow transplantation, are routinely exposed to DMSO at doses higher than 0.3 ml/kg, there is concern that DMSO might be producing similar damage in human children.

There has been a growing controversy regarding the continued use of glucocorticoid therapy to treat respiratory dysfunction associated with prematurity, as mounting clinical evidence has shown neonatal exposure produces permanent neuromotor and cognitive deficits. Here we report that, during a selective neonatal window of vulnerability, a single glucocorticoid injection in the mouse produces rapid and selective apoptotic cell death of the proliferating neural progenitor cells in the cerebellar external granule layer and permanent reductions in neuronal cell counts of their progeny, the cerebellar internal granule layer neurons. Our estimates suggest that this mouse window of vulnerability would correspond in the human to a period extending from approximately 20 weeks gestation to 6.5 weeks after birth. This death pathway is critically regulated by the proapoptotic Bcl-2 family member Puma and is independent of p53 expression. These rodent data indicate that there exists a previously unknown window of vulnerability during which a single glucocorticoid exposure at clinically relevant doses can produce neural progenitor cell apoptosis and permanent cerebellar pathology that may be responsible for some of the iatrogenically induced neurodevelopmental abnormalities seen in children exposed to this drug. This vulnerability may be related to the physiological role of glucocorticoids in regulating programmed cell death in the mammalian cerebellum.

Previously we reported that a 5-hour exposure of 6-day-old (P6) rhesus macaques to isoflurane triggers robust neuron and oligodendrocyte apoptosis. In an attempt to further describe the window of vulnerability to anesthetic neurotoxicity, we exposed P20 and P40 rhesusmacaques to 5 h of isoflurane anesthesia or no exposure (control animals). Brains were collected 3 h later and examined immunohistochemically to analyze neuronal and glial apoptosis. Brains exposed to isoflurane displayed neuron and oligodendrocyte apoptosis distributed throughout cortex and white matter, respectively. When combining the two age groups (P20 + P40), the animals exposed to isoflurane had 3.6 times as many apoptotic cells as the control animals. In the isoflurane group, approximately 66% of the apoptotic cells were oligodendrocytes and 34% were neurons. In comparison, in our previous studies on P6 rhesusmacaques, approximately 52% of the dying cells were glia and 48% were neurons. In conclusion, the present data suggest that the window of vulnerability for neurons is beginning to close in the P20 and P40 rhesus macaques, but continuing for oligodendrocytes.

A 3 h exposure to ISO is sufficient to induce widespread neurotoxicity in the developing primate brain. These results are relevant for clinical medicine, as many surgical and diagnostic procedures in children require anaesthesia durations similar to those modelled here. Further research is necessary to identify long-term neurobehavioural consequences of 3 h ISO exposure.

Glucocorticoids are used to treat respiratory dysfunction associated with premature birth but have been shown to cause neurodevelopmental deficits when used therapeutically. Recently, we established that acute glucocorticoid exposure at clinically relevant doses produces neural progenitor cell apoptosis in the external granule layer of the developing mouse cerebellum and permanent decreases in the number of cerebellar neurons. As the cerebellum naturally matures and neurogenesis is no longer needed, the external granule layer decreases proliferation and permanently disappears during the second week of life. At this same time, corticosterone (the endogenous rodent glucocorticoid) release increases and a glucocorticoid-metabolizing enzyme that protects the external granule layer against glucocorticoid receptor stimulation (11B-Hydroxysteroid-Dehydrogenase-Type 2; HSD2) naturally disappears. Here we show that HSD2 inhibition or raising corticosterone to adult physiological levels can both independently increase neural progenitor cell apoptosis in the neonatal mouse. Conversely, glucocorticoid receptor antagonism decreased natural physiological apoptosis in this same progenitor cell population suggesting endogenous glucocorticoid stimulation may regulate apoptosis in the external granule layer. We also found that glucocorticoids HSD2 can effectively metabolize generate less external granule layer apoptosis then glucocorticoids this enzyme is ineffective at breaking down. This finding may explain why glucocorticoids this enzyme can metabolize are clinically effective at treating respiratory dysfunction yet seem to produce no neurodevelopmental deficits. Finally, we demonstrate that both acute and chronic glucocorticoid exposure produces external granule layer apoptosis but without appropriate control groups this effect becomes masked. These results are discussed in terms of their implications for glucocortiocid therapy and neurodevelopment during the perinatal period.