Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies

Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure.

BackgroundIntra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine model of intra-amniotic C. albicans infection. In addition, we tested whether these potential adverse outcomes of the fetal brain were improved in utero by antifungal treatment with fluconazole.MethodsPregnant ewes received an intra-amniotic injection of 10(7) colony-forming units C. albicans or saline (controls) at 3 or 5 days before preterm delivery at 0.8 of gestation (term ~ 150 days). Fetal intra-amniotic/intra-peritoneal injections of fluconazole or saline (controls) were administered 2 days after C. albicans exposure. Post mortem analyses for fungal burden, peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed to determine the effects of intra-amniotic C. albicans and fluconazole treatment.ResultsIntra-amniotic exposure to C. albicans caused a severe systemic inflammatory response, illustrated by a robust increase of plasma interleukin-6 concentrations. Cerebrospinal fluid cultures were positive for C. albicans in the majority of the 3-day C. albicans-exposed animals whereas no positive cultures were present in the 5-day C. albicans-exposed and fluconazole-treated animals. Although C. albicans was not detected in the brain parenchyma, a neuroinflammatory response in the hippocampus and white matter was seen which was characterized by increased microglial and astrocyte activation. These neuroinflammatory changes were accompanied by structural white matter injury. Intra-amniotic fluconazole reduced fetal mortality but did not attenuate neuroinflammation and white matter injury.ConclusionsIntra-amniotic C. albicans exposure provoked acute systemic and neuroinflammatory responses with concomitant white matter injury. Fluconazole treatment prevented systemic inflammation without attenuating cerebral inflammation and injury

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed

Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems

Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals

Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Dynamic Gene Expression in the Human Cerebral Cortex Distinguishes Children from Adults

In comparison with other primate species, humans have an extended juvenile period during which the brain is more plastic. In the current study we sought to examine gene expression in the cerebral cortex during development in the context of this adaptive plasticity. We introduce an approach designed to discriminate genes with variable as opposed to uniform patterns of gene expression and found that greater inter-individual variance is observed among children than among adults. For the 337 transcripts that show this pattern, we found a significant overrepresentation of genes annotated to the immune system process (pFDR≅0). Moreover, genes known to be important in neuronal function, such as brain-derived neurotrophic factor (BDNF), are included among the genes more variably expressed in childhood. We propose that the developmental period of heightened childhood neuronal plasticity is characterized by more dynamic patterns of gene expression in the cerebral cortex compared to adulthood when the brain is less plastic. That an overabundance of these genes are annotated to the immune system suggests that the functions of these genes can be thought of not only in the context of antigen processing and presentation, but also in the context of nervous system development