Comparative anatomical studies on the cranial nerves of the fully formed embryos of the Nile tilapia Oreochromis niloticus (Ostiechthyes-Cichlidae). I. Nervus glossopharyngeus

The organization of the roots, ganglia and the peripheral distribution of the cranial nerves of the fully formed embryos of Oreochromis niloticus are examined in the transverse serial sections. These nerves carry fibers, which were also analyzed. The results of this study demonstrated that the glossopharyngeal nerve originates by means of only one root, which leaves the cranium through the glossopharyngeal foramen. This nerve gives fibers (visceromotor) to the first internal and external levator arcus branchialis muscles. There is a single epibranchial (petrosal) ganglion located extracranially. Nervus glossopharyngeus has three rami; pharyngeus, pretramticus and posttrematicus. The ramus pharyngeus carries only viscerosensory fibers; general for the pharyngeal epithelium and special ones for the pseudobranch. General viscerosensory fibers are also carried by rami pretrematicus and posttrematicus for the pharyngeal epithelial lining. The special sensory fibers are carried by the ramus pretrematicus for the taste buds and by ramus posttrematicus for the gill filaments. The ramus pretrematicus also carries visceromotor fibers for the first adductor arcus branchialis and to the first obliquus ventralis muscles.

Comparative characteristics of changes in subchondral bone of rats after antenatal glucocorticoid administration and modeling of osteoporosis

Background. Osteoporosis is a progressive systemic bone disease that results in decreased bone mineral density and, as a result, increases the risk of bone fractures. Changes that occur in the subchondral bone in osteoporosis or because of hormones can cause degenerative changes in the articular cartilage that underlie osteoarthritis. Objective. The aim of the study was to identify and compare morphological changes that occur in the subchondral bone in experimental simulations of osteoporosis and in adult rats that were born from females that were exposed to glucocorticoid solution administration in the 3rd trimester of pregnancy. Methods. Tibias of 26 white mature laboratory rats were studied. In serial sections, the relative areas occupied by bone trabeculae and lacunae were calculated. Results. The relative area involving the bone trabeculae (23,2 ± 3,70%) statistically significantly decreased at 21 day in the group of animals that undergone a simulation of osteoporosis in comparison with the control group. Similar changes are observed in the group of experimental animals that were born from females that were exposed to glucocorticoid solution administration in the 3rd trimester of pregnancy. Conclusion. Thus, the results of the study demonstrate the similarity of morphological changes occurring in the subchondral bone in rats that undergone a simulation of osteoporosis and rats that were born from females that were exposed to glucocorticoid solution administration in the 3rd trimester of pregnancy.

High-Throughput Strategy for Profiling Sequential Section With Multiplex Staining of Mouse Brain

The brain modulates specific functions in its various regions. Understanding the organization of different cells in the whole brain is crucial for investigating brain functions. Previous studies have focused on several regions and have had difficulty analyzing serial tissue samples. In this study, we introduced a pipeline to acquire anatomical and histological information quickly and efficiently from serial sections. First, we developed a serial brain-slice-staining method to stain serial sections and obtained more than 98.5% of slices with high integrity. Subsequently, using the self-developed analysis software, we registered and quantified the signals of imaged sections to the Allen Mouse Brain Common Coordinate Framework, which is compatible with multimodal images and slant section planes. Finally, we validated the pipeline with immunostaining by analyzing the activity variance in the whole brain during acute stress in aging and young mice. By removing the problems resulting from repeated manual operations, this pipeline is widely applicable to serial brain slices from multiple samples in a rapid and convenient manner, which benefits to facilitate research in life sciences.

Rehydration of freeze substituted brain tissue for preembedding immuno-electron microscopy

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

Image-based representation of massive spatial transcriptomics datasets

We present STIM, an imaging-based computational framework for exploring, visualizing, and processing high-throughput spatial sequencing datasets. STIM is built on the powerful ImgLib2, N5 and BigDataViewer (BDV) frameworks enabling transfer of computer vision techniques to datasets with irregular measurement-spacing and arbitrary spatial resolution, such as spatial transcriptomics data generated by multiplexed targeted hybridization or spatial sequencing technologies. We illustrate STIM's capabilities by representing, visualizing, and automatically registering publicly available spatial sequencing data from 14 serial sections of mouse brain tissue.

A Morphological and Histological Investigation of Imperfect Lungfish Fin Regeneration

Regeneration, the replacement of body parts in a living animal, has excited scientists for centuries and our knowledge of vertebrate appendage regeneration has increased significantly over the past decades. While the ability of amniotes to regenerate body parts is very limited, members of other vertebrate clades have been shown to have rather high regenerative capacities. Among tetrapods (four-limbed vertebrates), only salamanders show unparalleled capacities of epimorphic tissue regeneration including replacement of organ and body parts in an apparently perfect fashion. The closest living relatives of Tetrapoda, the lungfish, show regenerative abilities that are comparable to those of salamanders and recent studies suggest that these high regenerative capacities may indeed be ancestral for bony fish (osteichthyans) including tetrapods. While great progress has been made in recent years in understanding the cellular and molecular mechanisms deployed during appendage regeneration, comparatively few studies have investigated gross morphological and histological features of regenerated fins and limbs. Likewise, rather little is known about how fin regeneration compares morphologically to salamander limb regeneration. In this study, we investigated the morphology and histology of regenerated fins in all three modern lungfish families. Data from histological serial sections, 3D reconstructions, and x-ray microtomography scans were analyzed to assess morphological features, quality and pathologies in lungfish fin regenerates. We found several anomalies resulting from imperfect regeneration in regenerated fins in all investigated lungfish species, including fusion of skeletal elements, additional or fewer elements, and distal branching. The similarity of patterns in regeneration abnormalities compared to salamander limb regeneration lends further support to the hypothesis that high regenerative capacities are plesiomorphic for sarcopterygians.

May the palps be with you – new insights into the evolutionary origin of anterior appendages in Terebelliformia (Annelida)

Background Head appendages in Annelida contribute significantly to the immense morphological diversity in this spiralian taxon. Nevertheless, the evolutionary origin of annelid antennae, palps, cirri and tentacles are part of vast theories and debates that took place over decades. One of these heavily discussed groups are the Terebelliformia, which bear numerous anterior tentacles originating from different regions of the head. The question, whether these tentacles are homologous to feeding palps in other annelids or if these structures evolved convergently in terebellids and the remaining taxa, has been highly debated in the past. Results By using morphological methods including immunohistochemistry, confocal microscopy, Azan-stained serial sections and 3D-visualisation, we are able to shed new light and a fresh look on the old question of the evolutionary origin of the buccal tentacles and their associated head structures in Terebelliformia. Our investigations show that the brains of the ampharetid Hypania invalida and the aulophora larvae of Lanice conchilega (Terebellidae) consist of a dorsal, more prominent and a more slender, ventral brain region. Neurite bundles innervating the buccal tentacles split off from the ventral and dorsal root within the ventral brain region and thus originate from the dorsal and ventral root of the circumoesophageal connectives. Hence, the observed neurite bundles fulfil the morphological criteria for the innervating neurite bundles of feeding palps known from Paleoannelida. Conclusions We disagree with former conclusions that buccal tentacles are part of the alimentary canal. Based on the presented data, the buccal tentacles of terebelliform taxa are innervated by neurite bundles and can be homologized with peristomial feeding palps of other Annelida. Our comparative investigations reveal important insights into morphological changes during the evolution of anterior head appendages in Terebelliformia and Annelida in general. Nevertheless, our analyses also illustrate the gaps in knowledge and that more investigations throughout the annelid tree are necessary to explain and understand the huge diversity of annelid anterior appendages.

51 A novel cross-site analysis of Vectra® Polaris™ multiplex fluorescence PD-1/PD-L1 immunohistochemistry on colorectal cancer with high and low microsatellite instability

BackgroundColorectal cancer (CRC) is the third most diagnosed cancer in the United States with a projected 52,980 deaths in 2021.1 Microsatellite instability-high (MSI-H) CRCs with deficiencies in mismatch repair (MMR) are significantly associated with positive response to immunotherapy and improved outcomes when treated with immune checkpoint inhibitors. Programmed cell death ligand-1 (PD-L1) is an effective biomarker of MSI-H status to identify CRC patients who will respond to treatment, however, reproducible quantification of programmed cell death receptor-1 (PD-1)/PD-L1 in the tumor microenvironment (TME) across laboratory sites has been under-reported.2–3 In this study, our group directly addressed this issue by interrogating PD-1/PD-L1 cross-site at Akoya Biosciences and NeoGenomics Laboratories by employing the MOTiF™ PD-1/PD-L1 Panel kit along with the Vectra Polaris imaging system.MethodsSerial sections from 40 CRC samples with known MSI status were stained at Akoya and NeoGenomics Laboratories using a modified MOTiF PD-1/PD-L1 Lung Panel Kit on the Leica BOND RX. Sections were scanned using the Vectra Polaris imaging system at both sites. Inter-site staining reproducibility was assessed using image analysis algorithms developed with inForm tissue analysis software. Cell counts and densities were calculated using the R-script package PhenoptrReports and correlations were plotted per marker.ResultsThe average signal intensity for all markers/Opal fluorophores was within the recommended ranges of 10–30 normalized counts, with the exception of Polaris 780, which has an advised range of 1–10. This indicates the protocol stained successfully and reproducibly across all serial sections at both sites. Inter-site concordance analysis of cell densities for each marker yielded an average R2 value of ≥0.70. H-Score of PD-L1 quantified at the cell membrane trended with MSI status (stable/high).ConclusionsThis study demonstrated that the MOTiF PD-1/PD-L1 Panel kit imaged in conjunction with the Vectra Polaris is not only a reliable assay that can be run across different sites, based on the concordant cross-site data, but that re-optimization of the kit allows for the assay panel to be successfully adapted to other cancers, such as CRC, which can then capture biological differences across a multitude of samples.ReferencesAmerican Cancer Society https://www.cancer.org/cancer/colon-rectal-cancer/about/key-statistics.htmlYi M, Jiao D, Xu H, Liu Q, Zhao W, Han X, et al. Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors. Mol Cancer 2018;17(1):129Lemery S, Keegan P, Pazdur R. First FDA approval agnostic of cancer site - when a biomarker defines the indication. N Engl J Med 2017;377(15):1409–12.

Reproducible, high-dimensional imaging in archival human tissue by Multiplexed Ion Beam Imaging by Time-of-Flight (MIBI-TOF)

Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute's (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 +/- 0.04) and frequency (R2 = 0.95 +/- 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 +/- 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between replicates yielded an average correlation of R2 = 0.92 +/- 0.06. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.

Study of triclabendazole effects on Fasciola hepatica’s life-supporting organs, spines and suckers responsible for stable position of the parasite in the host

The purpose of the research is to study triclabendazole effects on the Fasciola’s life-supporting organs, spines and suckers which are responsible for stable position of the parasite in the host.Materials and methods. The study material was trematodes Fasciola hepatica (Linneus 1758, family Fasciolidae Railliet 1895), which were collected after the action of triclabendazole (fasinex) (chemically 5-chloro-6-(2,3-dichlorophenoxy)-2-methylthiobenzimidazole)on the 7th day after the drug administered at a single dose of 10 mg/kg for the Active Substance in the treatment of ovine fasciolosis. F. hepatica from untreated animals served as control. Mature F. hepatica collected after treatment with triclabendazole, and marita from the control groups were dehydrated in ascending alcohol series for 1–2 days after fixation; then passed through a mixture of chloroform and absolute alcohol (in a ratio of 1:1), and through pure chloroform in two portions for 10–15 minutes. The material was then soaked in a mushy mixture of chloroform and paraffin in a thermostat at 37 °C for 12–18 hours, and in paraffin in a thermostat at 56 °C for 30–45 minutes; and then embedded in paraffin with added wax. The resulting paraffin blocks were broken down into serial sections of 5–7 μm thick, then stained and examined under a light microscope.Results and discussion. Pathomicromorphological analysis of F. hepatica’s spines and suckers, organs that come into adhesive contact with the host organism revealed destructive changes in them after the action of triclabendazole. After the action of triclabendazole on fascioles, the spines look enlarged and swollen, and have a more rounded shape and some changes in color, absorbing eosin in greater concentration. The muscle fibers of the fascioles’ oral and abdominal suckers also look swollen after the action of triclabendazole. Although the musculature of the F. hepatica’s pharynx retained its structure, it has changes. It thickened sharply, which is clearly visible on the transverse and longitudinal sections of the helminths; neurosecretory cells are destroyed, and voids are observed in their place.