Task Progress:
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FINAL REPORTING DECEMBER 2021
Leveraging on an existing hindlimb suspension (HS) analog in rats, we studied the molecular aspects of CSF production and outflow modulation as a result of HS in the tissues involved in processes of cerebrospinal fluid (CSF) dynamics, namely choroid plexus (CP) and arachnoid villi (AV). On available tissue shared from the parent animal experiment, we performed differential gene expression profiling by RNA sequencing in the CP of rats subjected to HS and their normal posture controls, and in animals exposed to an enriched CO2 air composition versus those exposed to a normal atmosphere. The trasncriptomic profiles of each experimental group were clearly segregated, evidencing an effect of both the HS treatment and the 1% CO2 exposure.
In addition, we examined the ultrastructure of the CP and AV and the histologic localization and distribution of putative targets implicated in CSF dynamics (aquaporin 4) in normal posture and in HS rats within each cohort. This work has demonstrated that these minute structures in the brain can be accessed for their investigation, gaining a study tool to elucidate the molecular basis of the biological processes involved in CSF dynamics changes generated by HS and CO2 exposure.
ANNUAL REPORTING JULY 2020
Currently, it is hypothesized that weightlessness-induced cephalad fluid shift, possibly associated with a chronic elevation of intracranial pressure (ICP), may play a critical role in the pathophysiology of the Spaceflight Associated Neuro-Ocular Syndrome (SANS). Changes in cerebrospinal fluid (CSF) dynamics might also be involved in the ICP increase. It is not known whether CSF production and/or outflow are altered in microgravity, but changes at the molecular and cellular level in the structures that produce and regulate the transcellular and paracellular secretion and reabsorption of CSF may be relevant. These structures are the choroid plexus (CP) and the arachnoid granulations, which in rodents are more rudimentary and called arachnoid villi (AV). Their morphology, ultrastructure, and gene expression profiles might be subject to change by conditions of weightlessness or cephalad fluid shifts. This project examines the responses to extended durations of rodent hindlimb suspension (HS) at various levels: (a) changes within the ultrastructure of the CP and AV, (b) the localization and distribution of key proteins involved in the production and reabsorption of CSF, and (c) the transcriptomic differences in the CP of rats subjected to HS compared to their normal posture controls. Male 9-month-old Long Evans rats were subjected to HS for 14 and 90 days, with a subset of animals completing 90-day HS further studied for recovery periods at normal posture of 14 and 90 days. All HS rats had age-matched cage controls. The rats were sacrificed under anesthesia and the brain carefully removed, cut in 5 mm-thick coronal blocks, and preserved for various analyses. Transmission electron microscopy of glutaraldehyde-fixed sections were used to evaluate the ultrastructure of the CP from the lateral ventricles and AV from the cerebral convexities. Samples stored in Bouin’s fixative were used for immunohistochemical analysis of specific targets involved in CSF regulation, such as aquaporins. In addition, similar coronal blocks were frozen without fixation for processing by laser capture microdissection to isolate RNA specifically from the CP for gene expression profiling by RNA sequencing analysis.
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Abstracts for Journals and Proceedings
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Zanello SB, Theriot CA, Chevez-Barrios P, Rivera A. "A gene expression and histologic approach to study production and outflow of cerebrospinal fluid in hindlimb suspended rats." 2022 NASA Human Research Program Investigators' Workshop, Virtual, February 7-10, 2022. Abstracts. 2022 NASA Human Research Program Investigators' Workshop, Virtual, February 7-10, 2022. , Feb-2022
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