Methodology and Neuromarkers for Cetaceans’ Brains
Abstract
:1. Introduction
2. Materials and Methods
2.1. The Workflow for Cetaceans’ Brain Examination: From Fresh to Fixed Samples
2.2. Opening of the Skull
- one dorsal parallel to the nuchal ridge (crista occipitalis externa) at 1 cm from it (slightly more caudally in some species such as beaked whales or BW and pilot whales),
- another ventral and parallel to (a), bypassing the occipital condyles; and
- two lateral and perpendicular to (a) and (b), passing through the parietal and the squamosal bones, in the temporal fossa.
2.3. Cautious Sampling of Fresh Brain at Necropsy: A Key Step
2.4. Fixation of Cetaceans’ Brains: A Challenge
2.5. Sectioning of the Whole Brain and Postfixation
2.6. Routinary Neuropathologic Investigations (FFPE)
- -
- telencephalon: cortex (2 to 4 samples, at least frontopolar and occipital cortex) (a and f, stars), corpus striatum (b, arrowheads), amygdala (c, blue circle; h, Am), and hippocampus (h, hip);
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- diencephalon: thalamus and hypothalamus (c, star and rectangle);
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- mesencephalon: tectum (colliculus rostralis and caudalis) (d, star) and tegmentum (with the substantia nigra) (d and i, arrowheads);
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- rhombencephalon: at least pons (with the locus ceruleus) (e, rectangle), trapezoid body (with the cochlear nuclei) (f and k, rectangle), medulla oblongata (g, rectangle) and at least two samples of the cerebellum (included a sagittal section of the vermis) (e and g, arrowhead and star);
- -
- choroid plexus (d, g, i, j, and k, circle);
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- spinal cord (Figure 2, left): at least pars cervicalis and pars thoracica.
2.7. Immunoperoxidase Staining: Paraffin Embedded Tissues (p-IHC)
2.8. Cryoprotection and Preparation of the Sample (FFCS)
2.9. Immunoperoxidase Staining: Free-Floating Immunohistochemistry (ff-IHC)
2.10. Nissl Staining
2.11. TUNEL Staining
3. Results
3.1. Evaluation of Brain Quality
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- Provide longitudinal cuts to expose the lateral ventricles and allow the entry of the fixative;
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- Make cross-sections of the brain after at least 72 h of immersion in the fixative. Once a great percentage of the fixation process was achieved, serial cuts of the brain allowed a greater fixation;
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- Finally, the post-fixation of the selected samples permitted to complete the fixation of the tissues and provide the necessary firmness for their next processing;
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- Reposition of new fixative was made after cross sectioning the brain (immersion in a smaller container during 48 h) and then after sampling (postfixed during 24 h).
- -
- Tissue rupture and wearing, dark, shrunken and pycnotic neurons of that brains incorrectly handled during necropsy;
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- Poor fixation of the deepest subcortical structures of those brains, which did not receive cross-sections. This resulted in poor tissue quality, predisposing to easy rupture of the sections, especially during the continuous manipulation in ff-IHC, poor immunoreactivity to the neuromarkers, and a very low affinity to thionine;
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- The prolonged permanence in the fixative resulted in the loss of tissue quality, which predisposed it to an easy rupture of the sections, loss of antigenicity, and a very low affinity to thionine. In addition, formalin pigment accumulation was observed, as a background deposition and occasionally within the neurons mimicking the neuromelanin pigment;
3.2. Neuromarkers for Neuroanatomical Studies
3.3. Neuromarkers for Neuropathological Studies
3.4. Neuromarkers for Acoustic Trauma Research
4. Discussion
- -
- Several infectious pathogens including virus, bacteria, fungi, and parasites might cross the blood-cerebrospinal fluid barrier, entering the central nervous system and leading to inflammatory infectious diseases like meningitis and meningoencephalitis [42], very common in these animals;
- -
- Opening the ventricular system is a crucial step which allows its checking for exudates, space-occupying lesions, asymmetries, abnormal cerebrospinal fluid, and/or any alterations affecting the choroid plexus (i.e., cystic lesions as in Figure 3j or swelling). It is highly important to sample the choroid plexus, as a fundamental site of invasion of bacteria, virus (distemper), and protozoa. In fact, lesions may only be confined to the periventricular areas [43];
- -
- Thus, the opening of the ventricular system ensures a rapid uniform penetration of the fixative and the best possible preservation of the tissues. It is important to respect the proportions of fixative because of the large size and rounded shape of cetaceans’ brains. As fixative molecules bind to the tissue, they are depleted. Inadequate fixative volume will result in inadequate tissue fixation [44];
- -
- No macroscopic changes may be detected during necropsy and sampling but severe histopathological hallmarks may be present;
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- Random slicing and sampling of the brain may result in confused neuropathological interpretations. In addition, a strong knowledge of neuroanatomical structures is a critical advantage in order to boost the interpretation of neuropathological changes and their etiopathogenesis. Hence, another important aspect is respecting the international anatomical terminology (Nomina Anatomica) of the International Committee on Veterinary Gross Anatomical Nomenclature [45], which evolves over time;
- -
- A strong sampling protocol should not forbear to preserve the bilaterality of the brain, which permits to draw the specific pattern of distributions of the lesions, the first important step in neuropathological diagnosis [41];
- -
- Even if we usually lack clinical data on stranded animals, when brain lesions are the cause of the stranding, they are usually severe enough, large, and/or diffused.
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primary Antibody | Type | Specificity [Published Manuscript] | Diluition ff-IHC p-IHC | Antigen Retrieval (Only for FFPE) | NS | SA |
---|---|---|---|---|---|---|
c-Fos (4) Santa Cruz Biotechnology sc-52 | P/R | c-Fos Protein | 1/100 (both) | Pronase, 7 min | G | BaR |
c-Jun (D) Santa Cruz Biotechnology sc-44 | P/R | c-Jun Protein | 1/100 (both) | Citrate Buffer, 90–95 °C, 10 min (pH 6) | G | BaR |
HSP70 Abcam Ab6535 | M/Mo | Heat Shock Protein 70 kD [35] | 1/100 (both) | None | H | BaMo |
Ubiquitin Dako Z045801 | P/R | Human Ubiquitin | 1/100 (p-IHC) | None | G | BaR |
Neuroglobin Abcam Ab37258 | M/Mo | Neuroglobin | 1/100 (p-IHC) | None | H | BaMo |
Calretinin Swant 6B3 | M/Mo | Calretinin calcium-binding protein [35] | 1/500 (p-IHC) | Wet autoclave method of Shin 118° C, 5 min | H | BaMo |
Calbindin D-28k Swant 300 | M/Mo | Calbindin calcium-binding protein | 1/500 (both) | Wet autoclave method of Shin, 118° C, 5 min | H | BaMo |
Parvalbumin Swant 235 | M/Mo | Parvalbumin calcium-binding protein | 1/500 (p-IHC) | Wet autoclave method of Shin, 118° C, 5 min | H | BaMo |
GFAP DakoCytomation | P/R | Glial Fibrillary Acidic Protein | 1/120 (p-IHC) | None | G | BaR |
nNOS Millipore Ab5380 | P/R | Nitric Oxide Synthase | 1/300 (p-IHC) | Wet autoclave method of Shin, 118° C, 5 min | G | BaR |
TH Monosan MONX10786 | M/Mo | Tyrosine Hydroxylase [14] | 1/200 (ff-IHC) 1/50 (p-IHQ) | Wet autoclave method of Shin, 118° C, 5 min | H | BaMo |
CRF Abcam Ab59023 | P/G | Corticotropin Releasing Factor | 1/100 (ff-IHC) | N/A | R | BaG |
Vasopressin Abcam Ab39363 | P/R | Vasopressin | 1/500 (ff-IHC) | N/A | G | BaR |
HSV1 Abcam Ab9533 | P/R | Herpesvirus type I [29] | 1/50 (p-IHC) | Pronase 10 min | G | BaR |
CDV VMRD CDV-NP | M/Mo | Nucleoprotein of Canine Distemper Virus [36] | 1/100 (p-IHC) | Wet autoclave method of Shin, 118° C, 5 min | R | BaMo (1/20) |
Laforin Novus Biologicals NBP2-24474 | P/R | Human Laforin (EPM2A) | 1/100 (p-IHC) | Wet autoclave method of Shin, 118° C, 5 min | G | BaR |
B-Amyloid Invitrogen 700254 | M/R | Beta Amyloid (H31L21) [15] | 1/100 (ff-IHC) | N/A | G | BaR |
NFT AHB0161 | P/R | Neurofibrillary Tangles [15] | 1/100 (ff-IHC) | N/A | G | BaR |
α-Synuclein Abcam Ab27766 | M/Mo | Alpha-synuclein (LB 509) | 1/100 (ff-IHC) | N/A | H | BaMo |
α-Synuclein Invitrogen 35-8300 | M/Mo | Alpha-synuclein (Syn 505) | 1/100 (p-IHC) | Wet autoclave method of Shin, 118° C, 5 min | H | BaMo |
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Sacchini, S.; Herráez, P.; Arbelo, M.; Espinosa de los Monteros, A.; Sierra, E.; Rivero, M.; Bombardi, C.; Fernández, A. Methodology and Neuromarkers for Cetaceans’ Brains. Vet. Sci. 2022, 9, 38. https://doi.org/10.3390/vetsci9020038
Sacchini S, Herráez P, Arbelo M, Espinosa de los Monteros A, Sierra E, Rivero M, Bombardi C, Fernández A. Methodology and Neuromarkers for Cetaceans’ Brains. Veterinary Sciences. 2022; 9(2):38. https://doi.org/10.3390/vetsci9020038
Chicago/Turabian StyleSacchini, Simona, Pedro Herráez, Manuel Arbelo, Antonio Espinosa de los Monteros, Eva Sierra, Miguel Rivero, Cristiano Bombardi, and Antonio Fernández. 2022. "Methodology and Neuromarkers for Cetaceans’ Brains" Veterinary Sciences 9, no. 2: 38. https://doi.org/10.3390/vetsci9020038