Kevin K. Noguchi

Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears.

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 by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might 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.

Dimethyl Sulfoxide (DMSO) Produces Widespread Apoptosis in the Developing Central Nervous System

Dimethyl sulfoxide (DMSO) is a solvent that is routinely used as a cryopreservative in allogous bone marrow and organ transplantation. 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.

Acute neonatal glucocorticoid exposure produces selective and rapid cerebellar neural progenitor cell apoptotic death

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.

Glucocorticoid receptor stimulation and the regulation of neonatal cerebellar neural progenitor cell apoptosis.

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 (11β-Hydroxysteroid-Dehydrogenase-Type 2; HSD2) naturally disappears. Here we show that HSD2 inhibition and raising corticosterone to adult physiological levels both can independently increase neural progenitor cell apoptosis in the neonatal mouse. Conversely, glucocorticoid receptor antagonism decreases natural physiological apoptosis in this same progenitor cell population suggesting that endogenous glucocorticoid stimulation may regulate apoptosis in the external granule layer. We also found that glucocorticoids which HSD2 can effectively metabolize generate less external granule layer apoptosis than glucocorticoids this enzyme is ineffective at breaking down. This finding may explain why glucocorticoids that 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 exposures produce external granule layer apoptosis but without appropriate control groups this effect becomes masked. These results are discussed in terms of their implications for glucocorticoid therapy and neurodevelopment during the perinatal period.

Long-term Effects of Multiple Glucocorticoid Exposures in Neonatal Mice.

Glucocorticoids (GCs) such as dexamethasone (DEX) or betamethasone are repeatedly administered for up to a month to prematurely born infants as a treatment for chronic lung dysfunction. Results of clinical trials have shown that the use of GCs in these infants induces long-term deficits in neuromotor function and cognition. We have previously shown that a single exposure to clinically relevant doses of DEX or other GCs in the mouse during a period corresponding to the human perinatal period produces a dramatic increase in apoptotic cell death of neural progenitor cells in the developing cerebellum. To provide a model approximating more chronic clinical dosing regimens, we evaluated possible behavioral effects resulting from repeated exposures to DEX and subsequent GC-induced neuronal loss where neonatal mouse pups were injected with 3.0 mg/kg DEX or saline on postnatal days 7, 9, and 11 (DEX3 treatment). Adult, DEX3-treated mice exhibited long-term, possibly permanent, neuromotor deficits on a complex activity wheel task, which requires higher-order motor co-ordination skills. DEX3 mice exhibited impaired performance on this task relative to saline controls in each of two independent studies involving separate cohorts of mice. Histopathology studies utilizing stereological neuronal counts conducted in behaviorally-tested mice showed that the DEX3 treatment resulted in a significant decrease in the number of neurons in the internal granule layer (IGL) of the cerebellum, although the number of neurons in the Purkinje cell layer were unchanged. The results suggest that multiple neonatal DEX exposures can produce chronic deficits in fine motor co-ordination that are associated with cerebellar IGL neuronal loss.

Isoflurane exposure leads to apoptosis of neurons and oligodendrocytes in 20- and 40-day old rhesus macaques

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 rhesus macaques to 5h of isoflurane anesthesia or no exposure (control animals). Brains were collected 3h 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 rhesus macaques, 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.

Propylene Glycol produces excessive apoptosis in the developing mouse brain, alone and in combination with Phenobarbital

Introduction:Propylene glycol (PG) is a common solvent used in medical preparations. It is generally recognized as safe at regulated concentrations; however, its apoptotic potential is unknown.Results:PG triggered widespread apoptotic neurodegeneration with the greatest damage at postnatal day 7 (P7). Significant apoptosis was observed at doses as low as 2 ml/kg. These findings have implications for the safety of drug preparations used in pediatric medicine. The anticonvulsant phenobarbital (PB), which alone produces apoptosis in the immature central nervous system (CNS) is prepared in 68% PG and 10% ethanol (EtOH). We assessed whether PG contributes to the neurotoxic potential of PB. The agents (both at subtoxic doses) produce significantly more apoptosis when used in combination.Discussion:In conclusion, finding an alternative non-apoptotic solvent that can be used as a substitute for PG may be beneficial to patients.Methods:C57BL/6 mice (P4–30) were exposed to PG to examine whether PG could produce apoptosis in the developing CNS.

Lithium Protects Against Anaesthesia Neurotoxicity In The Infant Primate Brain.

Exposure of infant animals, including non-human primates (NHPs), to anaesthetic drugs causes apoptotic death of neurons and oligodendrocytes (oligos) and results in long-term neurodevelopmental impairment (NDI). Moreover, retrospective clinical studies document an association between anaesthesia exposure of human infants and significant increase in NDI. These findings pose a potentially serious dilemma because millions of human infants are exposed to anaesthetic drugs every year as part of routine medical care. Lithium (Li) at clinically established doses is neuroprotective in various cerebral injury models. We therefore investigated whether Li also protects against anaesthesia neurotoxicity in infant NHPs. On postnatal day 6 NHPs were anaesthetized with the widely used anaesthetic isoflurane (ISO) for 5 h employing the same standards as in a human pediatric surgery setting. Co-administration of Li completely prevented the acute ISO-induced neuroapoptosis and significantly reduced ISO-induced apoptosis of oligodendroglia. Our findings are highly encouraging as they suggest that a relatively simple pharmacological manipulation might protect the developing primate brain against the neurotoxic action of anaesthetic drugs while not interfering with the beneficial actions of these drugs. Further research is needed to determine Li’s potential to prevent long-term NDI resulting from ISO anaesthesia, and to establish its safety in human infants.

Lithium protects against glucocorticoid induced neural progenitor cell apoptosis in the developing cerebellum.

Respiratory dysfunction is one of the most common causes of death associated with premature birth (Barton et al., 1999). In the United States, 7-10% of pregnant women receive antenatal glucocorticoid (GC) therapy (Matthews et al., 2004), while approximately 19% of very low birth weight infants receive postnatal GC therapy (Jobe, 2009). Clinical research suggests that GC treatment causes permanent neuromotor and cognitive deficits (Yeh et al., 2004) and stunts cerebellar growth (Parikh et al., 2007; Tam et al., 2011). We previously reported that GC-mediated neural progenitor cell (NPC) apoptosis may be responsible for cerebellar neuropathology (Maloney et al., 2011; Noguchi et al., 2008, 2011). The goal of the current study was to determine whether lithium protects NPCs from GC neuroapoptosis in vivo and in vitro. Given that it protects against a range of brain insults, we hypothesized that lithium would significantly attenuate GC induced NPC toxicity. We report that acute lithium pretreatment provides potent, cell-intrinsic neuroprotection against GC induced NPC toxicity in vivo and in vitro.

Isoflurane exposure for three hours triggers apoptotic cell death in neonatal macaque brain

Background Retrospective clinical studies suggest there is a risk for neurodevelopmental impairment following early childhood exposure to anaesthesia. In the developing animal brain, including those of non-human primates (NHPs), anaesthetics induce apoptotic cell death. We previously reported that a 5 h isoflurane (ISO) exposure in infant NHPs increases apoptosis 13-fold compared with control animals. However, the majority of paediatric surgeries requiring anaesthesia are of shorter durations. We examined whether 3 h ISO exposure similarly increases neuroapoptosis in the NHP developing brain. Methods Six-day-old NHP infants ( Macaca mulatta ) were exposed to 3 h of a surgical plane of ISO ( n =6) or to room air ( n =5). Following exposure, NHP brains were screened for neuronal and oligodendrocyte apoptosis using activated caspase-3 immunolabelling and unbiased stereology. Results ISO treatment increased apoptosis (neurones + oligodendrocyte) to greater than four times that in the control group [mean density of apoptotic profiles: 57 (SD 22) mm -3 vs 14 (SD 5.2) mm -3 , respectively]. Oligodendrocyte apoptosis was evenly distributed throughout the white matter whereas neuroapoptosis occurred primarily in the cortex (all regions), caudate, putamen and thalamus. Conclusions 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.