Microdissection of the supraoptic nucleus and posterior pituitary gland from fresh unfixed tissue.

The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular characterization of these regions, such as transcriptome and methylome profiling, it is necessary to obtain large quantities of high-quality RNA and DNA. Prior methods to isolate molecular material from these small regions required fixing or freezing and laser microdissection of whole tissue, which can compromise recovery and integrity. We have established a straight-forward method of dissecting out the SON and posterior pituitary gland from fresh, unfixed tissue that allows for the isolation of RNA or DNA without compromising nucleic acid integrity. Furthermore, this method can be used as a framework for the microdissection of any region of the brain to isolate any sensitive material. In this manuscript, we describe step-by-step instructions from the macro scale dissection, to brain sectioning, and finally the microdissection of the appropriate tissue.•Transcardial perfusion without fixative prevents the shortcomings of nucleic acid cross-linking.•A fast method and the maintenance of tissue in ice-cold HBSS during dissection and sectioning prevents nucleic acid degradation.•A vibratome is used for the sectioning of fresh brain tissue without freezing or gelatin embedding (i.e. cryostat or microtome).

Transcriptome and methylome of the supraoptic nucleus provides insights into the age-dependent loss of neuronal plasticity.

The age-dependent loss of neuronal plasticity is a well-known phenomenon that is poorly understood. The loss of this capacity for axonal regeneration is emphasized following traumatic brain injury, which is a major cause of disability and death among adults in the US. We have previously shown the intrinsic capacity of magnocellular neurons within the supraoptic nucleus to undergo axonal regeneration following unilateral axotomization in an age-dependent manner. The aim of this research was to determine the age-dependent molecular mechanisms that may underlie this phenomenon. As such, we characterized the transcriptome and DNA methylome of the supraoptic nucleus in uninjured 35-day old rats and 125-day old rats. Our data indicates the downregulation of a large number of axonogenesis related transcripts in 125-day old rats compared to 35-day old rats. Specifically, several semaphorin and ephrin genes were downregulated, as well as growth factors including FGF's, insulin-like growth factors (IGFs), and brain-derived neurotrophic factor (BDNF). Differential methylation analysis indicates enrichment of biological processes involved in axonogenesis and axon guidance. Conversely, we observed a robust and specific upregulation of MHCI related transcripts. This may involve the activator protein 1 (AP-1) transcription factor complex as motif analysis of differentially methylated regions indicate enrichment of AP-1 binding sites in hypomethylated regions. Together, our data suggests a loss of pro-regenerative capabilities with age which would prevent axonal growth and appropriate innervation following injury.

The choroid plexus and its role in the pathogenesis of neurological infections.

The choroid plexus is situated at an anatomically and functionally important interface within the ventricles of the brain, forming the blood-cerebrospinal fluid barrier that separates the periphery from the central nervous system. In contrast to the blood-brain barrier, the choroid plexus and its epithelial barrier have received considerably less attention. As the main producer of cerebrospinal fluid, the secretory functions of the epithelial cells aid in the maintenance of CNS homeostasis and are capable of relaying inflammatory signals to the brain. The choroid plexus acts as an immunological niche where several types of peripheral immune cells can be found within the stroma including dendritic cells, macrophages, and T cells. Including the epithelia cells, these cells perform immunosurveillance, detecting pathogens and changes in the cytokine milieu. As such, their activation leads to the release of homing molecules to induce chemotaxis of circulating immune cells, driving an immune response at the choroid plexus. Research into the barrier properties have shown how inflammation can alter the structural junctions and promote increased bidirectional transmigration of cells and pathogens. The goal of this review is to highlight our foundational knowledge of the choroid plexus and discuss how recent research has shifted our understanding towards viewing the choroid plexus as a highly dynamic and important contributor to the pathogenesis of neurological infections. With the emergence of several high-profile diseases, including ZIKA and SARS-CoV-2, this review provides a pertinent update on the cellular response of the choroid plexus to these diseases. Historically, pharmacological interventions of CNS disorders have proven difficult to develop, however, a greater focus on the role of the choroid plexus in driving these disorders would provide for novel targets and routes for therapeutics.

Host transcriptome response to Borrelia burgdorferi sensu lato.

The host immune response to infection is a well-coordinated system of innate and adaptive immune cells working in concert to prevent the colonization and dissemination of a pathogen. While this typically leads to a beneficial outcome and the suppression of disease pathogenesis, the Lyme borreliosis bacterium, Borrelia burgdorferi sensu lato, can elicit an immune profile that leads to a deleterious state. As B. burgdorferi s.l. produces no known toxins, it is suggested that the immune and inflammatory response of the host are responsible for the manifestation of symptoms, including flu-like symptoms, musculoskeletal pain, and cognitive disorders. The past several years has seen a substantial increase in the use of microarray and sequencing technologies to investigate the transcriptome response induced by B. burgdorferi s.l., thus enabling researchers to identify key factors and pathways underlying the pathophysiology of Lyme borreliosis. In this review we present the major host transcriptional outcomes induced by the bacterium across several studies and discuss the overarching theme of the host inflammatory and immune response, and how it influences the pathology of Lyme borreliosis.

The Lyme disease bacterium, Borrelia burgdorferi, stimulates an inflammatory response in human choroid plexus epithelial cells.

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.