Methodology and Neuromarkers for Cetaceans' Brains

Abstract

Cetacean brain sampling may be an arduous task due to the difficulty of collecting and histologically preparing such rare and large specimens. Thus, one of the main challenges of working with cetaceans' brains is to establish a valid methodology for an optimal manipulation and fixation of the brain tissue, which allows the samples to be viable for neuroanatomical and neuropathological studies. With this in view, we validated a methodology in order to preserve the quality of such large brains (neuroanatomy/neuropathology) and at the same time to obtain fresh brain samples for toxicological, virological, and microbiological analysis (neuropathology). A fixation protocol adapted to brains, of equal or even three times the size of human brains, was studied and tested. Finally, we investigated the usefulness of a panel of 20 antibodies (neuromarkers) associated with the normal structure and function of the brain, pathogens, age-related, and/or functional variations. The sampling protocol and some of the 20 neuromarkers have been thought to explore neurodegenerative diseases in these long-lived animals. To conclude, many of the typical measures used to evaluate neuropathological changes do not tell us if meaningful cellular changes have occurred. Having a wide panel of antibodies and histochemical techniques available allows for delving into the specific behavior of the neuronal population of the brain nuclei and to get a "fingerprint" of their real status.

Keywords: beaked whales; cetaceans; dolphins; immunohistochemistry; methodology; neuroanatomy; neuromarkers; neuropathology.

Figure 1

Sagittal section of a fixed dolphin head, showing the lengthening of the sphlancnocranium (S), the shortening of the neurocranium (N), and the position of the brain (B). bw, blowhole; mel, melon; nar, nares; t, tongue. Atlantic spotted dolphin. Stenella frontalis.

Figure 2

Fresh spinal cord (left) and fresh brain (right). In the inset, the lateral ventricle is exposed (arrow), in the proximity of the sagittal cleft. Striped dolphin, Stenella coeruleoalba.

Figure 3

Representative brain sampling in cross sections. Details are given in the main text (Section 2.6). Striations are due to the dentated blade of the slicing machine (i.e., in b). Blainville’s beaked whale, Mesoplodon densirostris (a–f). Sagittal section of the brainstem. P, pons; TB, trapezoid body. Atlantic spotted dolphin, Stenella frontalis (g). Dorsal section of the brain, obtained at the level of the ventral limit of the telencephalon. The amygdaloid complex (Am), is located rostral to the tiny hippocampus (hip) and lateral to the optic tract (ot). Pu, putamen; sf, sylvian fissure; T, thalamus; TL, temporal lobe; I-II-III, 1st-2nd-3rd ventricle. Atlantic spotted dolphin, Stenella frontalis (h). Cross section made as in (d) intended to show the substantia nigra (arrowheads). Gervais’ beaked whale, Mesoplodon europaeus (i). Cross section of the brain exposing the lateral ventricle. The choroid plexus presents cystic lesions (arrowheads). The animal was positive to herpesvirus and presented lesions in the brain [29]. Striped dolphin, Stenella coeruleoalba (j). Sample of the brainstem including the trapezoid body (TB), exposing de ventral cochlear nucleus (VCN), the facial nerve (7), and the vestibulocochlear nerve (8). Risso’s dolphin, Grampus griseus (k).

Figure 4

VCN and Dorsal Cochlear Nucleus (DCN). 7, facial nerve; 8 vestibulocochlear nerve; C, cerebellum. Thionine. Striped dolphin, Stenella coeruleoalba. Inset: intranuclear strong c-fos positivity in a giant neuron of the VCN. Paraffin embedded brain sample. DAB counterstained with thionine. Atlantic spotted dolphin, Stenella frontalis (a) Left Hippocampus. Archicortex: DG, Gyrus dentatus and CA, cornu Ammonis. Cplex, choroid plexus, lv, lateral ventricle. Thionine. Atlantic spotted dolphin, Stenella frontalis (b); Hypothalamic tuberal area: supraoptic nucleus (SON) and suprachiasmatic (SC) nucleus. ot, optic tract. Inset: neurons of the SON. Free-floating, not embedded brain sample. DAB. Vasopressin. Blainville’s beaked whale, Mesoplodon densirostris (c); Substantia nigra and crus cerebri. Distribution of the Tyrosine Hydroxylase immunoreactive neurons (arrowheads), which make up the mesencephalic substantia nigra. crce, crus cerebri; cC, caudal colliculus. Free-floating, not embedded brain sample. DAB. Atlantic spotted dolphin, Stenella frontalis (d); nNOS immunoreaction in the cortical neurons. Paraffin embedded brain sample. DAB not counterstained. Striped dolphin, Stenella coeruleoalba (e) Parvalbumin immunoreaction in the cortical neurons. Paraffin embedded brain sample. DAB not counterstained. Striped dolphin, Stenella coeruleoalba (f) Alpha-synuclein immunopositive round body in the neuropil of the mesencephalon. Inset: Ubiquitin immunopositivity in the same round body. Paraffin embedded brain sample. AEC counterstained with Mayer’s hematoxylin. Short-finned pilot whale, Globicephala macrorhynchus (g) Purkinje cells show diffuse granular and vacuolar (arrowhead) cytoplasmic NFT-positive labeling. Free-floating immunolabeling. DAB counterstained with thionine. Blainville’s beaked whale, Mesoplodon densirostris (h) Vascular amyloid deposition in the amygdaloid complex. Free-floating immunolabeling. DAB counterstained with thionine. Atlantic spotted dolphin, Stenella frontalis (i); Mild intranuclear TUNEL positivity in some multipolar small spherical and multipolar globular neurons (arrowheads) of the VCN. Counterstained with thionine. Cuvier’s beaked whale, Ziphius cavirostris. Inset: Positive control, VCN incubated with TACS-Nuclease. Neuronal and glial nuclear positivity. Counterstained with thionine. Newborn bottlenose dolphin, Tursiops truncatus. (j); Ubiquitin nuclear staining (arrowheads) in the anterior part of the VCN. Paraffin embedded brain sample. DAB counterstained with thionine. Atlantic spotted dolphin, Stenella frontalis (k).

Figure 5

Schematic representation of fingerprint-like cellular identikit, of “healthy” vs. pathological neurons. Adapted from [69]. HSPs, Heat Shock Proteins.