In this study, we have evaluated two standard laboratory brain fixation methods for use in remote field locations where resources are limited and environmental conditions for tissue preservation are suboptimal. First, we found that transcardial perfusion-based methods to preserve lizard brain tissue, performed in parts of a remote biodiversity hotspot, are comparable to laboratory-based use of these methods in maintaining tissue quality at the cellular level. This conclusion is based on careful validation of the field fixation methods against laboratory methods by semi-quantitative cytoarchitectonic analysis of the processed brain tissue and by indirect immunohistofluorescence cytochemistry. Second, we found that immersion fixation in the field preserves gross neuroanatomical features of the brain very well, as evaluated using diceCT imaging. Moreover, the visualization of myelinated and unmyelinated components of the brain using diceCT imaging supports the efficacy of our field immersion fixation approach. To our knowledge, this paper is the first published account of a successful attempt to perform and rigorously validate protocols for transcardial perfusion and immersion fixation of brain tissue in a completely mobile field setting.

4.1 Methodological considerations

We found that field-perfused lizard brains are similar in condition to lizard brains perfused under standard laboratory conditions. In particular, cytoarchitectural features normally found in Nissl-stained brain sections were evident within the field specimens we processed, including discrete nuclear boundaries and structurally intact patterns of lamination. Moreover, the general appearance of the tissue, cleared completely of any blood, indicated complete perfusion. Whereas traditional methods to preserve brain tissue involve perfusing animals with ice-cold fixative solutions [51, 52], our collected animals were perfused with fixative solutions that were exposed to environmental conditions produced in the rainy season of a humid tropical climate, where we had no access to ice or cold storage. Specifically, our field-collected samples were subjected to high ambient temperatures (33–37°C) and a wide range of climates during their shipment from Central Africa to the southwestern United States. Considering the potential damage to delicate brain tissues from exposure to environmental variables and warm fixative solutions, our observations of no demonstrable differences between field- and lab-processed tissues for the first two criteria we evaluated (presence of blood and evenness of staining) is somewhat surprising. However, it has been shown that varying formaldehyde concentration within fixative solutions has little effect on the size of nuclei over a 10-fold range (1–20%), and extreme changes have only been observed in tissues fixed in at least 40% formaldehyde [53]. Furthermore, tissue shrinkage was not evident with the naked eye, which is known to occur in tissues incompletely fixed in formaldehyde or those subjected to varying temperatures [53].

The greatest negative effect on tissue quality for Nissl staining is arguably the interval between the time post mortem and the time of fixation [54]. It has been observed that fixation within 10 h post mortem has no effect on the intensity of stains, yet the ability for the tissue to be stained gradually deteriorates as the time interval increases, until staining capacity is entirely lost if fixation occurs 60–72 h post mortem [55]. On average, our field-collected animals were fixed in under an hour. Therefore, the mean duration between the time post mortem and the time of fixation for our field procedure does not compromise tissue quality. However, to minimize operational time, adequate training and multiple practice runs in a controlled environment are warranted before trying this procedure in the field.

4.1.1 Cytoarchitectonics. We used a semi-quantitative approach to evaluate the cytoarchitecture of the tissue sets processed under laboratory and field conditions of perfusion fixation. A recent survey of semi-quantitative methods used to evaluate histology has found that there is no accepted standard for the types of criteria used for such evaluations [56]. Given the many diverse approaches used to rate the quality of brain tissue, the study recommended that at the very least, investigators should provide a rationale for the specific criteria they use [56]. In line with this recommendation, we note here that the criteria we used were selected on the basis of the goals of our larger experimental research program involving these species, which are to examine the gross neuroanatomical relationships among their gray and white matter structures and the general cytoarchitectonic features of their brain tissue such as aggregations of neurons forming nuclei and laminae. These scales of comparative analysis are informed, in part, by seminal comparative neuroanatomical studies published at the turn of the twentieth century by Ramón [50], Edinger [57], and Brodmann [58]. Our approach necessarily constrains the criteria we use to those listed in Section 2.4.7, guided as we are by a rationale of general histological evaluation at the tissue level rather than its examination at the single-cell or subcellular levels. If the focus were on more fine-grained studies of the morphology or intracellular structure of the cells (e.g., the appearance of neurites, the condition of the organelles), then the criteria chosen using Nissl-based methods would likely be different (e.g., see Chapters X–XIV of Barker [59]), and alternatives to the Nissl method would also have been considered (e.g., see Chapter II in Vol. I of Cajal [60]). Our statistical analyses show that, on the basis of the two major criteria we used to evaluate fixation efficacy (presence of blood and evenness of stain), no differences in tissue quality were observed between field- and laboratory-perfused specimens. This result supports our qualitative observations of the tissue samples. Further, on the basis of the remaining four criteria (integrity of tissue in center, integrity of tissue at edges, visualization of lamination patterns, visualization of cell clustering and nuclei), the field-perfused tissue apparently displayed significantly better tissue quality than lab-perfused tissue. While these results may be somewhat surprising given the more controllable fixation conditions generally available in a laboratory setting, we must interpret these findings with caution for a few reasons. First, from a qualitative standpoint, it is difficult to separate these four criteria from underlying effects that could be due to other confounding factors, such as tissue damage that was incurred during the mounting of the tissue sections onto glass slides, tissue adherence to the slides during their mechanical transfer through separate reagent reservoirs during the thionin staining procedure, and any differences in tissue stability resulting from variations in section thickness or in the pH and salt composition of the fixative solutions we used. Second, our analysis is limited by the variability we observed among the three independent raters, despite the fact that the mixed model we used to analyze our results mitigates this issue to some extent. Finally, these latter four criteria are interdependent to a large degree on the first two criteria; this interdependence of predictors makes it possible that the statistically significant effects we obtained may be more apparent than real, given that the first two major criteria (which are not dependent, or as dependent, on issues such as mounting and mechanical transfer) show no differences between the two groups.

4.1.2 Immunohistochemistry. In addition to providing validation of our fixation procedures in the field by evaluating cytoarchitectonic criteria within Nissl-stained tissue sections, we sought indications that our fixation was compatible with standard chemoarchitectural localization methods. In particular, given that the field conditions required prolonged post-fixation in formalin-sucrose, we were concerned about the possibilities of over-fixing the tissue. The duration of formaldehyde fixation may lead to absent or weak binding for some epitopes, preventing effective chemoarchitectural studies with immunohistochemistry [61]. At times, poor penetration of the fixative can occur with too short of an exposure time and excessive cross-linkage can occur from prolonged exposure [62]. In addition, prolonged formalin fixation can cause irreversible damage to some epitopes, but this is largely dependent on the antibody used [63]. For our immunohistochemical procedures, we first aimed to identify dopamine-containing neurons in the periventricular hypothalamus, a well-studied neuronal subpopulation that has been documented previously to be present within the lizard brain [64–66]. Our results demonstrating robust TH-immunoreactivity (-ir) in neurons of field-fixed tissues that is comparable to that observed in laboratory-fixed tissues, confirms the findings of others [64–66] that have characterized this cell population as dopaminergic and extends them by demonstrating that the field conditions of prolonged post-fixation did not prevent chemical identification of these neurons. Relative to the field-fixed sample, the lab-fixed sample displayed apparently elevated levels of TH expression in cell bodies and low levels in fibers. Whether this staining difference indicates a difference in species, fixation efficacy, or peptide transport from the cell bodies to distal neurites between animals, is unclear. We also found comparable labeling of NPY-ir, reported to be present in chameleon brain [67], in cell bodies and/or axonal fibers within field- and lab-fixed tissues. These findings demonstrate the extensibility of our field-based fixation methods to antigens of different types (i.e., those that mark the presence of small neurotransmitters or those that mark neuropeptides). Although immunohistochemical labeling and visualization was feasible using our approach, further research is required to understand the degree of immunohistochemical reactivity across a broader range of antigens within field-perfused brains under our protocol conditions.

4.1.3 DiceCT imaging. In addition to cytoarchitectonic and immunohistochemical validation of our procedures in the field, diceCT scans from immersion-fixed field-collected samples show that our field protocol is compatible with the non-destructive visualization of gross neuroanatomical features in relation to other soft-tissue structures of the head as well as the skull. Our approach to prepare specimens for diceCT scans, immersion fixation and prolonged storage in fixative followed by iodine-enhancement, was demonstrated to have no negative effects on the contrast and visual quality of the scans. Gray and white matter regions were clearly distinguishable in the soft tissue, further demonstrating that our specimen preparation was successful. Importantly, diceCT can be achieved without encephalectomy, allowing for the interrelationships between central and peripheral components of the nervous system to be preserved. Another advantage of diceCT is that 3-D rendering software can be used to rapidly visualize the high-resolution scans as complex, 3-D soft-tissue anatomy [25, 26]. In turn, these datasets can be analyzed to quantify and compare neuroanatomical structures among different body regions and species [68]. Indeed, the possibility with field-fixed samples to visualize neural circuitry from the cellular level to that of entire brain regions sets up the potential for a comprehensive mapping of brain interconnectedness in three dimensions across multiple scales.