Optimization and validation of two multiplex qPCR assays for the rapid detection of microorganisms commonly invading the amniotic cavity.

Microbial invasion of the amniotic cavity (MIAC) leading to infection is strongly associated with adverse pregnancy and neonatal outcomes. Limitations of current diagnostic assays to detect MIAC rapidly and accurately have hindered the ability of obstetricians to identify and treat intra-amniotic infections. We developed, optimized, and validated two multiplex quantitative polymerase chain reaction (qPCR) assays for the simultaneous detection and quantification of microbial taxa commonly associated with MIAC. The first assay allows for the quantification of general bacterial and fungal loads in amniotic fluid and includes a human reference gene to allow for assessing the integrity of clinical samples and the DNA extraction process. The second assay allows for the detection and quantification of four specific bacterial taxa commonly associated with MIAC: Ureaplasma spp., Mycoplasma hominis, Streptococcus agalactiae, and Fusobacterium nucleatum. The qPCR assays were validated by using both microbial isolates and clinical amniotic fluid samples. The assays were further validated by comparing qPCR amplification results to those from the microbial culture and bacterial 16S rRNA gene sequencing of amniotic fluid. Both assays demonstrated high reproducibility and are sensitive and specific to their intended targets. Therefore, these assays represent promising molecular diagnostic tools for the detection of MIAC. Most importantly, these assays may allow for administration of timely and targeted antibiotic interventions to reduce adverse perinatal outcomes attributed to intra-amniotic infections.

Correcting QUEST Magnetic Resonance Imaging-Sensitive Free Radical Production in the Outer Retina In Vivo Does Not Correct Reduced Visual Performance in 24-Month-Old C57BL/6J Mice.

Purpose: To test the hypothesis that acutely correcting a sustained presence of outer retina free radicals measured in vivo in 24-month-old mice corrects their reduced visual performance. Methods: Male C57BL/6J mice two and 24 months old were noninvasively evaluated for unremitted production of paramagnetic free radicals based on whether 1/T1 in retinal laminae are reduced after acute antioxidant administration (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]). Superoxide production was measured in freshly excised retina (lucigenin assay). Combining acute antioxidant administration with optical coherence tomography (i.e., QUEST OCT) tested for excessive free radical-induced shrinkage of the subretinal space volume. Combining antioxidant administration with optokinetic tracking tested for a contribution of uncontrolled free radical production to cone-based visual performance declines. Results: At two months, antioxidants had no effect on 1/T1 in vivo in any retinal layer. At 24 months, antioxidants reduced 1/T1 only in superior outer retina. No age-related change in retinal superoxide production was measured ex vivo, suggesting that free radical species other than superoxide contributed to the positive QUEST MRI signal at 24 months. Also, subretinal space volume did not show evidence for age-related shrinkage and was unresponsive to antioxidants. Finally, visual performance declined with age and was not restored by antioxidants that were effective per QUEST MRI. Conclusions: An ongoing uncontrolled production of outer retina free radicals as measured in vivo in 24 mo C57BL/6J mice appears to be insufficient to explain reductions in visual performance.

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

PURPOSE: The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision. METHODS: In dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking. RESULTS: In light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil. CONCLUSIONS: The sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.

Rod Photoreceptor Neuroprotection in Dark-Reared Pde6brd10 Mice.

Purpose: The purpose of this study was to test the hypothesis that anti-oxidant and / or anti-inflammation drugs that suppress rod death in cyclic light-reared Pde6brd10 mice are also effective in dark-reared Pde6brd10 mice. Methods: In untreated dark-reared Pde6brd10 mice at post-natal (P) days 23 to 24, we measured the outer nuclear layer (ONL) thickness (histology) and dark-light thickness difference in external limiting membrane-retinal pigment epithelium (ELM-RPE) (optical coherence tomography [OCT]), retina layer oxidative stress (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]); and microglia/macrophage-driven inflammation (immunohistology). In dark-reared P50 Pde6brd10 mice, ONL thickness was measured (OCT) in groups given normal chow or chow admixed with methylene blue (MB) + Norgestrel (anti-oxidant, anti-inflammatory), or MB or Norgestrel separately. Results: P24 Pde6brd10 mice showed no significant dark-light ELM-RPE response in superior and inferior retina consistent with high cGMP levels. Norgestrel did not significantly suppress the oxidative stress of Pde6brd10 mice that is only found in superior central outer retina of males at P23. Overt rod degeneration with microglia/macrophage activation was observed but only in the far peripheral superior retina in male and female P23 Pde6brd10 mice. Significant rod protection was measured in female P50 Pde6brd10 mice given 5 mg/kg/day MB + Norgestrel diet; no significant benefit was seen with MB chow or Norgestrel chow alone, nor in similarly treated male mice. Conclusions: In early rod degeneration in dark-reared Pde6brd10 mice, little evidence is found in central retina for spatial associations among biomarkers of the PDE6B mutation, oxidative stress, and rod death; neuroprotection at P50 was limited to a combination of anti-oxidant/anti-inflammation treatment in a sex-specific manner.

Age-related murine hippocampal CA1 laminae oxidative stress measured in vivo by QUEnch-assiSTed (QUEST) MRI: impact of isoflurane anesthesia.

Age-related impairments in spatial learning and memory often precede non-familial neurodegenerative disease. Ex vivo studies suggest that physiologic age-related oxidative stress in hippocampus area CA1 may contribute to prodromal spatial disorientation and to morbidity. Yet, conventional blood or cerebrospinal fluid assays appear insufficient for early detection or management of oxidative stress within CA1 sub-regions in vivo. Here, we address this biomarker problem using a non-invasive MRI index of CA1 laminae oxidative stress based on reduction in R1 (= 1/T1) after anti-oxidant administration. An R1 reduction reflects quenching of continuous and excessive production of endogenous paramagnetic free radicals. Careful motion-correction image acquisition, and avoiding repeated exposure to isoflurane, facilitates detection of hippocampus CA1 laminae oxidative stress with QUEnch-assiSTed (QUEST) MRI. Intriguingly, age- and isoflurane-related oxidative stress is localized to the stratum lacunosum of the CA1 region. Our data raise the possibility of using QUEST MRI and FDA-approved anti-oxidants to remediate spatial disorientation and later neurodegeneration with age in animals and humans.

D-cis-Diltiazem Can Produce Oxidative Stress in Healthy Depolarized Rods In Vivo.

Purpose: New perspectives are needed to understand decades of contradictory reports on the neuroprotective effects of the Cav1.2 L-type calcium channel blocker d-cis-diltiazem in retinitis pigmentosa (RP) models. Here, we address, in vivo, the following two knowledge gaps regarding d-cis-diltiazem's actions in the murine outer retina: (1) do normal mouse rods contain d-cis-diltiazem-insensitive Cav1.2 L-type calcium channels? (2) Can d-cis-diltiazem modify the normal rod redox environment? Methods: First, transretinal Cav1.2 L-type calcium channels were noninvasively mapped with manganese-enhanced magnetic resonance imaging (MRI) following agonist Bay K 8644 in C57BL/6 (B6) and in Cav1.2 L-type calcium channel BAY K 8644-insensitive mutant B6 mice. Second, d-cis-diltiazem-treated oxidative stress-vulnerable (B6) or -resistant [129S6 (S6)] mice were examined in vivo (QUEnch-assiSTed [QUEST] MRI) and in whole retina ex vivo (lucigenin). Retinal thickness was measured using MRI. Results: The following results were observed: (1) manganese uptake patterns in BAY K 8644-treated controls and mutant mice identified in vivo Cav1.2 L-type calcium channels in inner and outer retina; and (2) d-cis-diltiazem induced rod oxidative stress in dark-adapted B6 mice but not in light-adapted B6 mice or dark-adapted S6 mice (QUEST MRI). Oxidative stress in vivo was limited to inferior outer retina in dark-adapted B6 mice approximately 1-hour post d-cis-diltiazem. By approximately 4 hours post, only superior outer retina oxidative stress was observed and whole retinal superoxide production was supernormal. All groups had unremarkable retinal thicknesses. Conclusions: D-cis-diltiazem's unexpectedly complex spatiotemporal outer retinal oxidative stress pattern in vivo was dependent on genetic background and rod membrane depolarization, but not apparently dependent on Cav1.2 L-type calcium channels, providing a potential rationale for contradictory results in different RP models.

Dark Rearing Does Not Prevent Rod Oxidative Stress In Vivo in Pde6brd10 Mice.

Purpose: In cyclic light-reared Pde6brd10 mice, rod cell oxidative stress contributes to the degenerative phenotype. Dark rearing Pde6brd10 mice slows but does not prevent atrophy. This suggests that outer retinal oxidative stress occurs in Pde6brd10 mice independent of light exposure, a hypothesis tested in this study. Methods: Mouse strains Pde6brd10 and C57Bl/6 (wild type) were dark reared until postnatal day (P) 23 (P23) or P30. In subgroups of dark-reared mice, (1) layer-specific excessive free radical production (i.e., an oxidative stress biomarker) in vivo via QUEnch-assiSTed magnetic resonance imaging (QUEST MRI) was indicated by a significant reduction in the greater-than-normal spin-lattice relaxation rate R1 (1/T1) with methylene blue, (2) superoxide production was measured ex vivo in whole retina (lucigenin), and (3) retinal layer spacing and thickness were assessed in vivo (optical coherence tomography, MRI). Results: In P23 male Pde6brd10 mice, only the outer superior retina showed oxidative stress in vivo, as measured by QUEST MRI; a lucigenin assay confirmed supernormal superoxide production. In contrast, at P30, no evidence for retinal oxidative stress was observed. In P23 female Pde6brd10 mice, no retinal oxidative stress was apparent; however, at P30, oxidative stress was observed in superior inner and outer nuclear layers. Male and female Pde6brd10 mice at P23 had normal retinal thicknesses, whereas at P30, modest thinning was noted in inferior and superior retina. Conclusions: We confirmed that outer retinal oxidative stress occurs in male and female dark-reared Pde6brd10 mice. Male and female Pde6brd10 mice demonstrated similar degrees of retinal thinning, but with unexpectedly distinct spatial and temporal retinal oxidative stress patterns.