Table 1.
Summary of the ID, age group, predominant pathological process detected histologically the CNS, and pooled histoscore averages of the protein marker immunoreactivity for the VCN and IC of the dolphins of this study (0—No immunoreactivity; 3—Very intense immunoreactivity).
Fig 1.
Results of WB analyses for antibodies against DGK-ζ, Bcl-2, Aβ, and NF200.
B.d.: Bottlenose dolphin; F.w.: Fin whale; B.w.: Beaked whale; S.d.: Striped dolphin; S.w.: Sperm whale; Lad: Ladder. Labels on the right of each panel signify molecular weight in kDa.
Table 2.
Summary of IHC parameters of the herein utilized antibodies, and their roles in the body depending on subcellular location.
Fig 2.
Haematoxylin-eosin/HE (left column), blank control/CTRL (middle column) and IHC patterns (right columns) for the investigated markers—magnification: 400x.
Panels marked a-f represent characteristic IHC patterns for each marker: (a) Apaf-1-IR in both neuronal cytoplasm and, unlike in most other examined animals, in the nucleus of ID319. (b) Nucleolar DGK-ζ-IR (arrowhead) in the VCN of a younger adult dolphin without brain lesions (ID146) as compared to (c) its cytoplasmic appearance in the VCN of a bycaught, older adult (ID203). In this animal, multifocal cytoplasmic IR in glial cells (arrowheads), as well as streaks of IR in the neuropil (asterisk) are apparent. (d) Lack of Bcl-2-IR in the VCN of a younger adult dolphin (ID89), representative of most adults. (e) Perineuronal enhancement of glial Aβ-IR in the VCN of a bycaught, hypoxic dolphin (ID203). (f) Aβ-IR marking a coalescing extracellular plaque between the VCN and superior olivary complex of older adult (ID319). An adjacent neuron shows light disseminated granular IR in its cytoplasm, but not in the nucleus, as in the VCN of most investigated adult dolphins <30 y.o. (inset in e). (g) Intense NF200-IR of the dendritosomatic and axonal cytoskeleton of VCN neurons (ID89, representative of all investigated animals).
Fig 3.
Comparison between histochemical and IHC staining results for a glial node surrounding T. gondii cysts (arrowheads) in the IC of ID142—Magnification: 200x.
Insets display detail of cysts at a magnification of 400x. (a) HE-stain. (b) NF200-IR within the glial node tissue (c) Aβ-IR which also localizes to the T. gondii cysts (inset); (d) T. gondii cysts and zoites recognized by a specific anti-T. gondii antibody; (e) Apaf-1-IR appears attenuated within the cell-rich microglial nodule in comparison to the granular IR and high background of the surrounding neuropil; (f) DGK-ζ-IR is mildly stronger within the glial cells of the parasitic nodule compared to the surrounding neuropil.
Fig 4.
Histochemical (a) and IHC (b-f) findings in vascular and perivascular tissues in the investigated dolphins—Magnification: 100x.
Panels a-c represent the same vessel in consecutive sections. (a) Microscopic appearance of a mid-caliber vessel in the IC of ID344 using a haematoxylin-eosin staining, with a mild perivascular infiltrate characterized by mononuclear inflammatory cells. (b) very mild cytoplasmic DGK-ζ-IR in few foci of perivascular glia. Inset: Moderate, multifocal IR in pericapillary neuropil of ID133 (arrow)—magnification: 200x. (c) The same vessel of ID344 displays intense Apaf-1-IR, mostly in the cytoplasm of pericytes, showing perivascular distribution and radiating cellular processes, visible also in transverse sections of the IC vessels of ID344 (d). (e) Clearly demarcated endothelial Apaf-1-IR in a VCN vessel of ID142. (f) Aβ-IR deposit in neuropil adjacent to a mid-caliber VCN vessel—the IR appears to extend into the vessel wall and endothelial lining (arrow).
Fig 5.
Relevant biological roles of the implemented biomarkers protein in programmed cell death PCD (PCD; green highlight) and Aβ-associated toxicity (orange highlight) as referred to in the main text.
Light blue circles—nuclei. Dashed line circles—nucleoli. Orange arrows—translocation and transformation of molecules/processes. Green arrows—activation or induction of molecules. Red arrows—inhibition of molecules. Blue highlight—interacting molecules. Blue dashed arrow: Association between focal ischemia and proteolysis of cytoskeletal neurofilaments (NF), leading to the development of spheroids. Abbreviations: APPsα—soluble APP α; BACE-1—Beta-site APP Cleaving Enzyme 1; BBB—blood brain barrier; Cas—caspase; iNOS—inducible nitric oxide synthase/NOS II; NO—nitric oxide; p53—(tumor) protein 53; PrpC—cellular prion protein; RAGE—Receptor for Advanced Glycation End Products; U—ubiquitin (degrades cytoplasmic DGK-ζ). Astrocytes are omitted from this figure for simplification, though they closely interact with both neurons and microglia under both physiological and pathological conditions.