Systematic validation and assessment of immunohistochemical markers for central nervous system pathology in cetaceans, with emphasis on auditory pathways

Abstract

Cetacean neuropathology is a developing field that aims to assess structural and neurochemical changes involved in neurodegenerative, infectious and traumatic processes, however markers used previously in cetaceans have rarely undergone systematic validation. This is a prerequisite to investigating the potential damage inflicted on the cetacean auditory system by anthropogenic noise. In order to assess apoptotic, neuroinflammatory and structural aberrations on a protein level, the baseline expression of biomarker proteins has to be characterized, implementing a systematic approach to validate the use of anti-human and anti-laboratory animal antibodies in dolphin tissues. This approach was taken to study 12 different antibodies associated with hypoxic-ischemic, inflammatory, plastic and excitatory-inhibitory changes implicated in acoustic trauma within the ventral cochlear nuclei and inferior colliculi of 20 bottlenose dolphins (Tursiops truncatus). Out of the 12 tested antibodies, pro-apoptotic protease factor 1 (Apaf-1), diacylglycerolkinase-ζ (DGK-ζ), B-cell lymphoma related protein 2 (Bcl-2), amyloid-β peptide (Aβ) and neurofilament 200 (NF200) were validated employing Western blot analyses and immunohistochemistry (IHC). The results of the validation process indicate specific patterns of immunoreactivity that are comparable to those reported in other mammals, thus suggesting a key panel of IHC biomarkers of pathological processes in the cetacean brain. As a consequence, the antibodies tested in this study may constitute a valid tool for supporting existing diagnostic methods in neurological diseases. The approach of systematic validation of IHC markers in cetaceans is proposed as a standard practice, in order for results to be transparent, reliable and comparable.

LKB1 and CRMP1 cooperatively promote the repair of the sciatic nerve injury

“…In acute peripheral nerve injury, S100β released from activated SCs promotes SC migration, thereby promoting peripheral nerve regeneration. NF200 is a cytoskeletal protein of nerve cells that can fully display the morphology and distribution of the entire nerve cell (Orekhova et al., 2022; Trojanowski et al., 1986). Axonal regeneration is frequently detected by immunofluorescence staining of axons with NF200 labels.…”

Different responses of skeletal muscles to femoral artery ligation-induced ischemia identified in BABL/c and C57BL/6 mice

“…Then muscle segments were incubated overnight at 4 °C with Alexa Fluor ® 594 labeled donkey anti-mouse (A21203, Thermo Fisher Scientific, Waltham, MA) and anti-rabbit (A21207, Thermo Fisher Scientific, Waltham, MA) IgGs, and Alexa Fluor ® 488 labeled α-bungarotoxin (α-BTX, B13422, Thermo Fisher Scientific, Waltham, MA). As molecular markers of nerve fibers, nerve terminals, and nicotinic acetylcholine receptors (nAChRs), the specificity of these primary antibodies has been validated in previous published studies (Berg et al, 1972;Mu et al, 2017;Orekhova et al, 2022). Finally, images of muscle segments mounted on glass slides were captured using a laser scanning confocal microscope (Zeiss LSM 800) to analyze immunohistochemically labeled NMJs, including motor nerve terminals and nAChR clusters.…”