TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Dynamic intravital imaging reveals reactive vessel-associated microglia play a protective role in cerebral malaria coagulopathy.

Vascular congestion and coagulopathy have been shown to play a role in human and experimental cerebral malaria (eCM), but little is known about the role of microglia, or microglia-vascular interactions and hypercoagulation during disease progression in this fatal infection. Recent studies show microglia bind to fibrinogen, a glycoprotein involved in thrombosis. An eCM model of Plasmodium chabaudi infection in mice deficient in the regulatory cytokine IL-10 manifests neuropathology, including hypercoagulation with extensive fibrin(ogen) deposition and neuroinflammation. Intravital microscopy and immunofluorescence are applied to elucidate the role of microglia in eCM. Results show microgliosis and coagulopathy occur early in disease at 3 dpi (day post-infection), and both are exacerbated as disease progresses to 7dpi. Vessel associated microglia increase significantly at 7 dpi, and the expression of the microglial chemoattractant CCL5 (RANTES) is increased versus uninfected and localized with fibrin(ogen) in vessels. PLX3397 microglia depletion resulted in rapid behavioral decline, severe hypothermia, and greater increase in vascular coagulopathy. This study suggests that microglia play a prominent role in controlling infection-initiated coagulopathy and supports a model in which microglia play a protective role in cerebral malaria by migrating to and patrolling the cerebral vasculature, potentially regulating degree of coagulation during systemic inflammation.

Semi-automated micro-computed tomography lung segmentation and analysis in mouse models.

Computed Tomography (CT) is a standard clinical tool utilized to diagnose known lung pathologies based on established grading methods. However, for preclinical trials and toxicity investigations in animal models, more comprehensive datasets are typically needed to determine discriminative features between experimental treatments, which oftentimes require analysis of multiple images and their associated differential quantification using manual segmentation methods. Furthermore, for manual segmentation of image data, three or more readers is the gold standard of analysis, but this requirement can be time-consuming and inefficient, depending on variability due to reader bias. In previous papers, microCT image manual segmentation was a valuable tool for assessment of lung pathology in several animal models; however, the manual segmentation approach and the commercial software used was typically a major rate-limiting step. To improve the efficiency, the semi-manual segmentation method was streamlined, and a semi-automated segmentation process was developed to produce:•Quantifiable segmentations: using manual and semi-automated analysis methods for assessing experimental injury and toxicity models,•Deterministic results and efficiency through automation in an unbiased and parameter free process, thereby reducing reader variance, user time, and increases throughput in data analysis,•Cost-Effectiveness: portable with low computational resource demand, based on a cross-platform open-source ImageJ program.

Label-Free Imaging and Histo-Optical Evaluation of Head and Neck Cancers with Multiphoton Autofluorescence Microscopy.

Depth-resolved label-free optical imaging by the method of multiphoton autofluorescence microscopy (MPAM) may offer new ways to examine cellular and extracellular atypia associated with epithelial squamous cell carcinoma (SCC). MPAM was evaluated for its ability to identify cellular and microstructural atypia in head and neck tissues from resected discarded tumor tissue. Three-dimensional image volumes were obtained from tissues from the floor of the mouth, tongue, and larynx, and were then processed for histology. MPAM micrographs were evaluated for qualitative metrics of cell atypia and quantitative measures associated with nuclear pleomorphism. Statistical analyses correlated MPAM endpoints with histological grade from each imaged site. Cellular overcrowding, discohesion, anisonucleosis, and multinucleated cells, as observed through MPAM, were found to be statistically associated with dysplasia and SCC grading, but not in histologically benign regions. A quantitative measure of the coefficient of variance in nuclear size in SCC and dysplasia was statistically elevated above histologically benign regions. MPAM also allowed for the identification of cellular heterogeneity across transitional areas and other features, such as inflammatory infiltrates. In the future, MPAM could be evaluated for the non-invasive detection of neoplasia, possibly as an adjunct to traditional conventional examination and biopsy.

Powassan Virus Induces Structural Changes in Human Neuronal Cells In Vitro and Murine Neurons In Vivo.

Powassan virus (POWV) is a tick-borne flavivirus (TBFV) that can cause severe encephalitis in humans with a case-fatality rate as high as 11%. Patients who survive severe encephalitic disease can develop long-term neurological sequelae that can be debilitating and life-long. In this study, we have sought to characterize a primary human fetal brain neural stem cell system (hNSC), which can be differentiated into neuron and astrocyte co-cultures, to serve as a translational in vitro system for infection with POWV and a comparative mosquito-borne flavivirus (MBFV), West Nile virus (WNV). We found that both viruses are able to infect both cell types in the co-culture and that WNV elicits a strong inflammatory response characterized by increased cytokines IL-4, IL-6, IL-8, TNF-α and IL-1ß and activation of apoptosis pathways. POWV infection resulted in fewer cytokine responses, as well as less detectable apoptosis, while neurons infected with POWV exhibited structural aberrations forming in the dendrites. These anomalies are consistent with previous findings in which tick-borne encephalitis virus (TBEV) infected murine primary neurons formed laminal membrane structures (LMS). Furthermore, these structural aberrations are also recapitulated in brain tissue from infected mice. Our findings indicate that POWV is capable of infecting human primary neurons and astrocytes without causing apparent widespread apoptosis, while forming punctate structures reminiscent with LMS in primary human neurons and in vivo.

Age dependence of retinal vascular plexus attenuation in the triple transgenic mouse model of Alzheimer's disease.

The influence of Alzheimer's disease (AD) progression and severity on the structural and functional integrity of the cerebral vasculature is well recognized. The retina is an extension of the brain; thus, changes in retinal vascular features may serve as markers of AD cerebrovascular pathologies. However, differentiating normal aging-versus AD-induced retinal vascular changes is unresolved. Therefore, we compared and quantified changes in superficial (SVP), intermediate (IVP), and deep (DVP) retinal vascular plexuses in young, middle-age, and old triple transgenic mouse model of AD (3xT-AD) to the changes that occur in age-matched controls (C57BL/6j). We used immunostaining combined with a novel tissue optical clearing approach along with a computational tool for quantitative analysis of vascular network alterations (vessel length and density) in SVP, IVP, and DVP. All three layers had comparable structural features and densities in young 3xTg-AD and control animals. In controls, IVP and DVP densities decreased with aging (-14% to -32% change from young to old, p < 0.05), while no changes were observed in SVP. In contrast, vascular parameters in the transgenic group decreased in all three layers with aging (-12% to -49% change from young to old, p < 0.05). Furthermore, in the old group, SVP and DVP vascular parameters were lower in the transgenics compared to age-matched controls (p < 0.05). Our analysis demonstrates that normal aging and progression of AD lead to various degrees of vascular alterations in the retina. Specifically, compared to normal aging, changes in vascular features of SVP and DVP regions of the retina are accelerated during AD progression. Considering recent advances in the field of depth-resolved imaging of retinal capillary network and microangiography, noninvasive quantitative monitoring of changes in retinal vascular network parameters of SVP and DVP may serve as markers for diagnosis and staging of Alzheimer's disease and discriminating AD-induced vascular attenuation from age-related vasculopathy.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception.

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Correction: Role of microglia in the dissemination of Zika virus from mother to fetal brain.

[This corrects the article DOI: 10.1371/journal.pntd.0008413.].

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Light-Sheet Microscopy of the Optic Nerve Reveals Axonal Degeneration and Microglial Activation in NMDA-Induced Retinal Injury.

Purpose: Optic nerve degeneration is a feature of neurodegenerative eye diseases and causes irreversible vision loss. Therefore, understanding the degenerating patterns of the optic nerve is critical to find the potential therapeutic target for optic neuropathy. However, the traditional method of optic nerve degeneration has the limitations of losing spatiotemporal tissue information. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique that allows capturing 3D images rapidly with a high spatial optical resolution. In this study, we evaluated the availability of LSFM on the optic nerve with NMDA injected Thy1-CFP mice. Methods: NMDA injected to both eyes of Thy1-CFP mice. After 7 days from the injection, the retina and optic nerve were collected and immunostained with anti-Iba1 antibody. NMDA excitotoxicity induced RGC, and its axon loss and microglial activation in the retina were observed using confocal microscopy. The immunostained optic nerve was completed the optical clearing process with TDE and mounted for LSFM imaging. Results: We found that retinal flatmounts confirmed significant loss of CFP-expressing RGC and axon degradation and loss in Thy1-CFP mice at 7 days after NMDA injection. Together with these data verifying that NMDA induces RGC and its axon loss, we confirmed that NMDA excitotoxicity induced microglia activation and leukostasis, such as increased microglia number, transform its morphology to ameboid or round, and increase in attached leukocytes in vessels. Using LSFM, we observed that CFP expressing nerve fiber was well organized and arranged parallel in vehicle treated optic nerve, whileas NMDA injected optic nerve showed axon swelling and fragmentation and loss of axon density from the anterior to the posterior regions. Furthermore, LSFM enabled the observation of microglia phenotype transformation in the entire optic nerve. Unlike microglia in vehicle injected optic nerve, microglia in NMDA injected optic nerve displayed larger soma and short process with high Iba1 expression through the entire optic nerve from the anterior to posterior. Conclusions: In summary, we examined the applicability of the modified optic clearing protocol for the optic nerve and verified it enabled to acquiring of the 3D images of the optic nerve successfully revealing the complex spatial relationships between the axons, microglia and vasculature throughout the entire organ with single acquisitions. With these optimized techniques, we successfully obtained the high-resolution 3D images of NMDA-induced optic neuropathy, including the clues for optic nerve degeneration such as axon swelling, axonal fragmentation, and microglia activation. Overall, we believe that our current study could help understand the pathology of the optic nerve in neurodegenerative diseases, and it will be the basis for translational research.

Characterization of the Ovine Vaginal Microbiome and Inflammation Patterns as an Improved Testing Model of Human Vaginal Irritation.

The development of therapies targeted to improve the health of women has utilized direct vaginal delivery as a more effective and less toxic method of protection from HIV and other pathogens. Vaginal applicants and delivery devices that provide sustained effects have been met with increasing acceptability, but the efficacy and toxicity outcomes have not been successfully predicted by preclinical in vitro studies and animal modeling. We have explored the utilization of sheep as a model for testing the safety of vaginal applicants and devices based on spatial and structural similarities to the human vagina. As recently noted by the FDA, an additional safety measure is an impact on the vaginal microbiome (VMB) that is known to contribute to vaginal health and influence pathogen susceptibility and drug metabolism. To advance the utility of the sheep vaginal model, we completed a thorough molecular characterization of the ovine VMB utilizing both next-generation sequencing (NGS) and PCR methods. The process also created a custom PCR array to quantify ovine VMB community profiles in an affordable, higher throughput fashion. The results from vaginal swabs (>475 samples) collected from non-pregnant crossbred Dorset and Merino ewes treated with selected vaginal applicants or collected as sham samples established 16 VMB community types (VMB CTs). To associate VMB CTs with eubiosis or dysbiosis, we also completed custom ELISAs for six cytokines identifying IL1B, IL8, TNFa, and CXCL10 as useful markers to support the characterization of ovine vaginal inflammation. The results indicated that Pasteurella, Actinobacillus, Pseudomonas, Bacteroides, Leptotrichia, and E. coli were common markers of eubiosis (low inflammatory marker expression), and that Haemophilus, Ureaplasma, and Corynebacterium were associated with dysbiosis (high cytokine levels). Utilizing the optimized workflow, we also confirmed the utility of three commonly used vaginal applicants for impact on the VMB and inflammatory state, producing a dataset that supports the recommendation for the use of sheep for testing of vaginal applicants and devices as part of preclinical pipelines.

Surrogate post-coital testing for contraceptive efficacy against human sperm activity in the ovine vaginal model†.

High unintended pregnancy rates are partially due to lack of effective nonhormonal contraceptives; development of safe, effective topical vaginal methods will address this need. Preclinical product safety and efficacy assessment requires in vivo testing in appropriate models. The sheep is a good model for the evaluation of vaginally delivered products due to its close similarities to humans. The study objective was to develop an ovine model for efficacy testing of female nonhormonal contraceptives that target human sperm. Fresh human semen was pooled from male volunteers. Nonpregnant female Merino sheep were treated with control or vaginal contraceptive product (IgG antibody with action against sperm or nonoxynol-9 [N9]). Pooled semen was added to the sheep vagina and mixed with product and vaginal secretions. Microscopic assessment of samples was performed immediately and progressive motility (PM) of sperm was compared between treatments. Cytokines CXCL8 and IL1B were assessed in vaginal fluid after instillation of human semen. No adverse reactions or elevations in proinflammatory cytokines occurred in response to human semen. N9 produced signs of acute cellular toxicity while there were no cellular changes after IgG treatment. N9 and IgG had dose-related effects with the highest dose achieving complete sperm immobilization (no sperm with PM). Surrogate post-coital testing of vaginally administered contraceptives that target human semen was developed in an ovine model established for vaginal product preclinical testing. This expanded model can aid the development of much needed nonhormonal topical vaginal contraceptives, providing opportunities for rapid iterative drug development prior to costly, time-intensive human testing.

Multimodal widefield fluorescence imaging with nonlinear optical microscopy workflow for noninvasive oral epithelial neoplasia detection: a preclinical study.

SIGNIFICANCE: Early detection of epithelial cancers and precancers/neoplasia in the presence of benign lesions is challenging due to the lack of robust in vivo imaging and biopsy guidance techniques. Label-free nonlinear optical microscopy (NLOM) has shown promise for optical biopsy through the detection of cellular and extracellular signatures of neoplasia. Although in vivo microscopy techniques continue to be developed, the surface area imaged in microscopy is limited by the field of view. FDA-approved widefield fluorescence (WF) imaging systems that capture autofluorescence signatures of neoplasia provide molecular information at large fields of view, which may complement the cytologic and architectural information provided by NLOM. AIM: A multimodal imaging approach with high-sensitivity WF and high-resolution NLOM was investigated to identify and distinguish image-based features of neoplasia from normal and benign lesions. APPROACH: In vivo label-free WF imaging and NLOM was performed in preclinical hamster models of oral neoplasia and inflammation. Analyses of WF imaging, NLOM imaging, and dual modality (WF combined with NLOM) were performed. RESULTS: WF imaging showed increased red-to-green autofluorescence ratio in neoplasia compared to inflammation and normal oral mucosa (p < 0.01). In vivo assessment of the mucosal tissue with NLOM revealed subsurface cytologic (nuclear pleomorphism) and architectural (remodeling of extracellular matrix) atypia in histologically confirmed neoplastic tissue, which were not observed in inflammation or normal mucosa. Univariate and multivariate statistical analysis of macroscopic and microscopic image-based features indicated improved performance (94% sensitivity and 97% specificity) of a multiscale approach over WF alone, even in the presence of benign lesions (inflammation), a common confounding factor in diagnostics. CONCLUSIONS: A multimodal imaging approach integrating strengths from WF and NLOM may be beneficial in identifying oral neoplasia. Our study could guide future studies on human oral neoplasia to further evaluate merits and limitations of multimodal workflows and inform the development of multiscale clinical imaging systems.

Scattering Angle Resolved Optical Coherence Tomography Detects Early Changes in 3xTg Alzheimer's Disease Mouse Model.

Purpose: Clinical intensity-based optical coherence tomographic retinal imaging is unable to resolve some of the earliest changes to Alzheimer's disease (AD) neurons. The aim of this pilot study was to demonstrate that scattering-angle-resolved optical coherence tomography (SAR-OCT), which is sensitive to changes in light scattering angle, is a candidate retinal imaging modality for early AD detection. SAR-OCT signal data may be sensitive to changes in intracellular constituent morphology that are not detectable with conventional OCT. Methods: In this cross-sectional study, retinas of a triple transgenic mouse model of AD (3xTg-AD) were imaged alongside age-matched control mice (C57BL/6J) using SAR-OCT. A total of 32 mice (12 control, 20 3xTg-Ad) at four ages (10, 20, 30, and 45 weeks) were included in this cross-sectional study, and three retinal feature sets (scattering, thickness, and angiography) were examined between the disease and control groups. Results: AD mice had significantly increased scattering diversity (lower SAR-OCT C parameter) at the earliest imaging time (10 weeks). Differences in the C parameter between AD and control mice were diminished at later times when both groups showed increased scattering diversity. AD mice have reduced retinal thickness compared to controls, particularly in central regions and superficial layers. No differences in vascular density or fractional blood volume between groups were detected. Conclusions: SAR-OCT is sensitive to scattering angle changes in a 3xTg-AD mouse model and could provide early-stage biomarkers for neurodegenerative diseases such as AD. Translational Relevance: Clinical OCT systems may be modified to record SAR-OCT images for non-invasive retinal diagnostic imaging of patients with neurodegenerative diseases such as AD.

Role of microglia in the dissemination of Zika virus from mother to fetal brain.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.

In Vivo Epithelial Metabolic Imaging Using a Topical Fluorescent Glucose Analog.

The demanding metabolic needs of cancer cells are met by aerobic glycolysis. While whole-body PET imaging methods exist for evaluating this metabolic response, these are not ideal for local, more detailed regions such as mucosal surfaces. Fluorescence imaging of glucose analogs with similarities to radiolabeled deoxyglucose used in PET, namely, fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), offers such an alternative, particularly as this glucose analog may be delivered by local topical delivery. In this chapter, methods for in vivo epithelial imaging in a preclinical hamster model for oral cancer and oral epithelial dysplasia are described. Outlined are methods for preparation and in vivo delivery of 2-NBDG by topical application to the oral mucosa followed by fluorescence imaging to compare fluorescence responses between neoplasia and control mucosa or to monitor changes in fluorescence signal with time in both groups.

Efficacy of Novel Highly Specific Bromodomain-Containing Protein 4 Inhibitors in Innate Inflammation-Driven Airway Remodeling.

NF-κB/RelA triggers innate inflammation by binding to bromodomain-containing protein 4 (BRD4), an atypical histone acetyltransferase (HAT). Although RelA·BRD4 HAT mediates acute neutrophilic inflammation, its role in chronic and functional airway remodeling is not known. We observed that BRD4 is required for Toll-like receptor 3 (TLR3)-mediated mesenchymal transition, a cell-state change that is characteristic of remodeling. We therefore tested two novel highly selective BRD4 inhibitors, ZL0420 and ZL0454, for their effects on chronic airway remodeling produced by repetitive TLR3 agonist challenges, and compared their efficacy with that of two nonselective bromodomain and extraterminal (BET) protein inhibitors, JQ1 and RVX208. We observed that ZL0420 and ZL0454 more potently reduced polyinosinic:polycytidylic acid-induced weight loss and fibrosis as assessed by microcomputed tomography and second harmonic generation microscopy. These measures correlated with the collagen deposition observed in histopathology. Importantly, the ZL inhibitors were more effective than the nonselective BET inhibitors at equivalent doses. The ZL inhibitors had significant effects on lung physiology, reversing TLR3-associated airway hyperresponsiveness and increasing lung compliance in vivo. At the molecular level, ZL inhibitors reduced elaboration of the transforming growth factor-ß-induced growth program, thereby preventing mucosal mesenchymal transition and disrupting BRD4 HAT activity and complex formation with RelA. We also observed that ZL0454 treatment blocked polyinosinic:polycytidylic acid-associated expansion of the α-SMA1+/COL1A+ myofibroblast population and prevented myofibroblast transition in a coculture system. We conclude that 1) BRD4 is a central effector of the mesenchymal transition that results in paracrine activation of myofibroblasts, mechanistically linking innate inflammation to airway hyperresponsiveness and fibrosis, and 2) highly selective BRD4 inhibitors may be effective in reversing the effects of repetitive airway viral infections on innate inflammation-mediated remodeling.

Imaging of Murine Whole Lung Fibrosis by Large Scale 3D Microscopy aided by Tissue Optical Clearing.

Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.

In-vivo topical mucosal delivery of a fluorescent deoxy-glucose delineates neoplasia from normal in a preclinical model of oral epithelial neoplasia.

Metabolic imaging of oral cavity mucosal surfaces could benefit early detection of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED). Fluorescent deoxy-glucose agents provide contrast for glucose metabolism similar to 18FDG-PET imaging and allow use of optical imaging, which provides high resolution and lower potential cost. However, in-vivo topical mucosal delivery of fluorescent deoxy-glucose agents without injection or tissue resection has not been shown. We introduce in-vivo optical imaging of neoplasia following mucosal delivery of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) in an OSCC/OED hamster model and demonstrate uptake into epithelium across the mucosal surface without injection or disrupting the epithelium. 2-NBDG fluorescence intensity following 30-minutes topical application was 6-fold and 4-fold higher in OSCC and OED, respectively, compared to normal mucosa. Receiver operator characteristic analysis show 83% sensitivity and 73% specificity for detection of neoplasia vs benign (normal and inflammation). Faster 2-NBDG fluorescence temporal decay in neoplasia indicated higher uptake and glucose metabolic rate than normal mucosa. Mucosal delivery of 2-NBDG by topical application to the in-vivo oral surface is feasible and delineates neoplasia from normal mucosa, providing in-vivo noninvasive molecular imaging of dysregulated glucose metabolism, which could benefit preclinical studies of carcinogenesis or be developed for use in early detection.

Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal outcome of Plasmodium infection. There are clear correlations between expression of inflammatory cytokines, severe coagulopathies, and mortality in human CM. However, the mechanisms intertwining the coagulation and inflammation pathways, and their roles in CM, are only beginning to be understood. In mice with T cells deficient in the regulatory cytokine IL-10 (IL-10 KO), infection with Plasmodium chabaudi leads to a hyper-inflammatory response and lethal outcome that can be prevented by anti-TNF treatment. However, inflammatory T cells are adherent within the vasculature and not present in the brain parenchyma, suggesting a novel form of cerebral inflammation. We have previously documented behavioral dysfunction and microglial activation in infected IL-10 KO animals suggestive of neurological involvement driven by inflammation. In order to understand the relationship of intravascular inflammation to parenchymal dysfunction, we studied the congestion of vessels with leukocytes and fibrin(ogen) and the relationship of glial cell activation to congested vessels in the brains of P. chabaudi-infected IL-10 KO mice. METHODS: Using immunofluorescence microscopy, we describe severe thrombotic congestion in these animals. We stained for immune cell surface markers (CD45, CD11b, CD4), fibrin(ogen), microglia (Iba-1), and astrocytes (GFAP) in the brain at the peak of behavioral symptoms. Finally, we investigated the roles of inflammatory cytokine tumor necrosis factor (TNF) and coagulation on the pathology observed using neutralizing antibodies and low-molecular weight heparin to inhibit both inflammation and coagulation, respectively. RESULTS: Many blood vessels in the brain were congested with thrombi containing adherent leukocytes, including CD4 T cells and monocytes. Despite containment of the pathogen and leukocytes within the vasculature, activated microglia and astrocytes were prevalent in the parenchyma, particularly clustered near vessels with thrombi. Neutralization of TNF, or the coagulation cascade, significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. CONCLUSIONS: These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature to neuropathology in malaria infection. Strikingly, localization of inflammatory leukocytes within intravascular clots suggests a mechanism for interaction between the two cascades by which cytokines drive local inflammation without considerable cellular infiltration into the brain parenchyma.