title: Rescuing Perishable Neuroanatomical Information from a Threatened Biodiversity Hotspot: Remote Field Methods for Brain Tissue Preservation Validated by Cytoarchitectonic Analysis, Immunohistochemistry, and X-Ray Microcomputed Tomography.
abstract: Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue. Third, immersion-fixation of lizard brains, prepared under identical environmental conditions, was readily compatible with subsequent iodine-enhanced X-ray microcomputed tomography, which facilitated the non-destructive imaging of the intact brain within its skull. In summary, we have validated multiple approaches to preserving intact lizard brains in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Systems Neuroscience Laboratory, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Biodiversity Collections, University of Texas at El Paso, El Paso, Texas, United States of America.|Doctoral Program in Ecology & Evolutionary Biology, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Hughes
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Systems Neuroscience Laboratory, University of Texas at El Paso, El Paso, Texas, United States of America.|Doctoral Program in Environmental Pathobiology, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Walker
authors.affiliation: Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma, United States of America.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Gignac
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Systems Neuroscience Laboratory, University of Texas at El Paso, El Paso, Texas, United States of America.|Doctoral Program in Environmental Pathobiology, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Anais
authors.initials: A
authors.lastname: Martinez
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Systems Neuroscience Laboratory, University of Texas at El Paso, El Paso, Texas, United States of America.|Masters Program in Biology, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Negishi
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Biodiversity Collections, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Carl S
authors.initials: CS
authors.lastname: Lieb
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Biodiversity Collections, University of Texas at El Paso, El Paso, Texas, United States of America.|Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Eli
authors.initials: E
authors.lastname: Greenbaum
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas, United States of America.|UTEP Systems Neuroscience Laboratory, University of Texas at El Paso, El Paso, Texas, United States of America.|Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas, United States of America.
authors.forename: Arshad M
authors.identifier: 0000-0003-0254-3546
authors.initials: AM
authors.lastname: Khan
mesh_terms: D000818:Animals; D044822:Biodiversity; D001921:Brain / Q000473:pathology*; D003247:Conservation of Natural Resources / Q000379:methods*; D004463:Ecology; D017753:Ecosystem; D056727:Endangered Species; D005843:Geography; D006321:Heart / Q000502:physiology; D007150:Immunohistochemistry; D008116:Lizards; D009445:Neuroanatomy / Q000379:methods*; D010477:Perfusion; D014021:Tissue Preservation; D014454:Uganda; D055114:X-Ray Microtomography