Recent Neuropathology Concepts

Abstract

The last session of the 2024 European Society of Toxicologic Pathology Congress was dedicated to recent neuro concepts, encompassing various topics including artificial intelligence (AI) applied to toxicologic pathology, new technologies for visualization and/or analysis of tissues, as well as specific case reports. Four presentations detailed the role of AI and computational pathology for toxicologic pathologists, usage of cryofluorescent tomography in neuropathology, blood-brain barrier organoids in early drug screening, and spatial transcriptomics in toxicologic pathology. In addition, there were two short presentations about a spontaneous brain lesion in Beagle dogs as well as central nervous system lesions related to adeno-associated virus vectors administration in various preclinical species. This session underscored the current and prospective impact of emerging technologies on drug screening and development.

Computational Pathology Applied to Toxicologic Pathology: Learning to Ride AI

Wave of Opportunity

Dr Julie Boisclair (Novartis) presented the current and future potential usage of artificial intelligence (AI) in the field of toxicologic pathology.

It highlighted the changes from a digital evolution to AI revolution in the field, emphasizing the benefits of digital primary read, peer review, and whole slide scanning as catalysts to the AI transformation of toxicologic pathology and covering forward-looking approaches in AI-assisted pathology evaluation. The presentation underlined the need for a multidisciplinary collaboration, emphasizing the integral role of the pathologist in partnership with computational scientists in AI model creation. Challenges of AI in toxicologic pathology include the anatomic complexity and 3-dimensional aspects of histology, along with confounding factors such as artifactual changes, study blocking variability, and the inherent diversity of pathological findings. The potential for use of AI in reducing animal use through the use of histologic virtual controls was discussed, along with the considerations for implementation of test facility–based digital pathology virtual controls. Dr Boisclair then explored the different kinds of AI models and methods that can serve for different goals and objectives in toxicologic pathology. The use of AI in extraction and comparison of histological features from whole slide images (WSI) was explained, concluding the presentation with the necessary pathways of consideration and action for establishing validation of AI workflows.

This was followed by open and active discussion with the audience, which covered multiple aspects regarding the use of digital pathology and AI, both from the discovery and the regulatory perspectives, with different perspectives on their impact on the future of the profession. In particular, the quantification of specific microscopic findings and the usage of virtual control groups, either for pathology (digital images) or for clinical pathology, were actively discussed, as was the validation of digital pathology systems in the context of Good Laboratory Practice regulations and incorporation of the Organisation for Economic Co-operation and Development guidelines for digital peer review, primary reads, and raw data considerations in these contexts.

Neuropathology Insights via Cryofluorescent Tomography

Dr Elizabeth Galbreath presented an overview of the use of cryofluorescent tomography (CFT) and its application in biodistribution studies. Her presentation emphasized that one area where pathologists can provide tremendous value is through augmenting the effectiveness of drug development teams by identifying tissue and cellular deposition and distribution of therapeutics in the context of histopathology. Frequently, Absorption, Distribution, Metabolism and Excretion/Drug Metabolism and Pharmacokinetics (ADME/DMPK) tissue distribution studies are conducted through either selective dissection of specific tissues followed by tissue homogenization (destroying cellular and sub-anatomic information) or via low-resolution quantitative whole-body autoradiography (QWBA) analyzed by phosphor imaging. Whole animal, or in the case of large animals, whole organ, assessment by QWBA lacks the resolution to provide information on the localization and identification of the therapeutic in specific cell types. Although thin sections of selected tissues can be evaluated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in conjunction with a pathologist to potentially identify cellular and sub-anatomic localization of a therapeutic, this is a time- and labor-intensive approach that is not feasible for whole animal assessment and does not allow additional assessment of the tissue section. Ideally, localization of the distribution of a therapeutic, or the cellular expression in response to nucleic acid or gene therapy modality, would be readily evaluated in the whole animal in a quantitative manner and at a cellular to subcellular level of resolution. CFT is a transformative 3-dimensional imaging technique that provides whole-body anatomical and fluorescence images at up to a 20-µm-level resolution. The CFT images can be overlaid with their corresponding bright light images for enhanced visualization of on- and off-targeted drug biodistribution and protein expression, with the added benefit of being able to collect these tissue sections for secondary molecular, immunohistochemistry (IHC), in situ hybridization, single-cell, or spatial imaging analysis in the context of the CFT-imaged step sections through the whole animal or organ. The presentation encompassed examples of whole-body antisense oligonucleotides and adeno-associated virus vector (AAV) biodistribution and expression, while reviewing current insights on cerebrospinal fluid drainage pathways relevant to intrathecal administration, and provided examples of where whole-body CFT provided previously unreported AAV7 and AAV9 biodistribution and AAV-directed gene expression in nasal olfactory epithelium (olfactory sensory receptor cells), within odontoblasts and odontoblast processes of the teeth, and several other unexpected cell types and tissues. ²

Comparative AAV9-AMIR-SOD1–Related Findings in the Nervous System of Cynomolgus Monkeys, New Zealand White Rabbits, and C57BL/6 Mice

In this case presentation, Dr Ingrid Pardo (Biogen) outlined the comparative histopathology findings in the dorsal root ganglia (DRG), trigeminal ganglia (TG), spinal cord, and nerves associated with administration of AAVs in cynomolgus monkeys (NHP) and two other species: New Zealand White rabbits and C57BL/6 mice. The histopathology findings were compared in these 3 preclinical species at 4 or 6 weeks after a single lumbar intrathecal (L-IT) or intra-cisterna magna (ICM), injection of AAV-directed vector genome encoding an artificial miRNA targeting superoxide dismutase 1 (AAV-amiR-SOD1). The amiR sequence targeted SOD1 in NHPs and mice but not in rabbits. In one 6-week study, 2.5- to 3.5-year-old, male and female NHPs were dosed in the L-IT space L4/L5 with 1.5 × 10E14 or 3.0 × 10E14 genome copies (GC)/dose of AAV-amiR-SOD1. In a 6-week study, 6- to 7-week-old male and female mice were dosed in the L-IT space 6 with 8.0 × 10E11 or 1.6 × 10E12 GC/dose of AAV-amiR-SOD1. In a 4-week study, male rabbits were dosed with 8.31E12 to 1.14E13 GC/dose of AAV-amiR-SOD1 in the ICM. AAV-amiR-SOD1–related histopathology findings in NHP, rabbit, and mice were all characterized by degeneration/necrosis of neurons associated with mononuclear cell infiltrate (MCI) in the DRG and TG, and nerve fiber degeneration in the dorsal funiculi (of the spinal cord), dorsal nerve roots, sciatic nerve, saphenous nerve (mice only), and the trigeminal nerves (attached to TG). DRG findings were of higher incidence and greater severity in the lumbar regions than cervical and thoracic regions. The severity of the nerve fiber degeneration in nerves and spinal cord was not bilaterally symmetric in intensity or distribution. Dr Pardo reported 3 major histopathologic variations among these species. First, the MCI reaction associated with neuronal changes in the DRG was not as prominent in mice as it was in NHP and rabbit, although providing an increased dose in mice resulted in a dose-dependent severity of neuronal lesions. Second, the presence of test article–related vacuoles in neurons undergoing degeneration/necrosis in the DRG and TG, along with nerve fiber degeneration in the lateral funiculi, was a typical finding in rabbits. Third, rabbits tend to develop more severe lesions in the TG compared to NHPs, while mice have not to date been reported to develop TG lesions even when DRG neuronal changes are present. The ICM route of administration may be associated with the presence of TG changes in rabbits. In conclusion, NHPs, rabbits, and mice develop similar AAV-related lesions in target tissues with some variations in severity, neuroanatomic predilection, and pathology features that could provide insights into different mechanisms of toxicity and the translatability of these findings to humans.

Tissue Technology in Blood-Brain Barrier Organoids as Combined Efficacy/Toxicity High-Throughput Spatial Readouts in Early Drug Screening

Dr Luisa Bell, PhD (Roche) gave a presentation about the use of complex in vitro models (CIVMs) in toxicologic pathology, specifically focusing on the characterization and validation of blood-brain barrier (BBB) models. She addressed various technical challenges, such as adapting standard histopathology workflows to CIVM due to their small size, differences in culturing devices, and cell composition/origin compared to animal tissue evaluation. Dr Bell presented different histotechniques using BBB organoids to support model engineering, model characterization, and compound distribution, as well as efficacy/toxicity evaluation in early drug screening. Their histotechniques allowed IHC and multiplex immunofluorescence–guided characterization with spatial evaluation of cell types and organoid architecture. Additionally, histo-embedding of these organoids enabled spatial evaluation of compounds via MALDI MS and IHC for both small and large molecules, respectively. Additionally, to complement the standard in vitro cell death assessment (5× standard Caspase assay), histotechniques, and digital workflows using hematoxylin and eosin (H&E) WSI scans allowed for light microscopic evaluation of single-cell toxicity. To provide quantitative assessment of the WSI at high resolution (40×), a single-cell morphologic AI algorithm was developed to detect necrotic/apoptotic nuclei on these H&E scans. Overall, these histotechniques developed in vitro assays and interpretations provided highly informative readouts for assessing efficacy and toxicity in a reproducible, robust, sensitive, and high-throughput manner in preclinical drug development. ¹

Unveiling CNS Complexity and Drug Responses Through Spatial Transcriptomics

Dr Kerstin Hahn and Benedek Pesti from Roche gave a presentation on spatial transcriptomics (ST) technologies and their applications in uncovering the pathogenesis of CNS diseases, as well as their use in toxicologic pathology. ST represents a revolutionary technology that allows for the visualization and quantification of transcriptomic profiles in the context of microscopic tissue morphology. Most platforms entail the possibility to map the transcriptome signatures to the standard H&E slide through image registration that aligns the H&E with the ST readout with immunofluorescent labeling of protein and/or RNA detection. This provides pathologists the unique opportunity to correlate histomorphological changes and/or therapeutic distribution or biodistribution patterns with transcriptomic signatures of the tissue condition or response. The presentation further highlighted the importance and the value-added benefits of integrating the role of toxicologic and discovery pathologists with molecular biology and drug distribution, metabolism, and pharmacokinetic scientists in the overall study design, experimental setup, data analysis, and interpretation. The presenters demonstrated, across therapeutic modalities and preclinical animal models, how ST can support the development of CNS-targeting compounds (see the corresponding manuscript in this special proceedings issue).

Bilateral Basal Nuclei Vacuolar Lesions—A Novel and Emerging Potential

Background Finding in Beagle Dogs

In this case presentation, Dr Stefanie Arms (Boehringer Ingelheim) outlined the characterization, terminology, and interpretation of a condition that has recently emerged as a background lesion in Beagle dogs. The lesion affects the caudate nucleus bilaterally and is characterized by vacuolation of the neuropil and white matter tracts, variable (astro)gliosis with the presence of gemistocytic astrocytes, and demyelination. To date, this lesion has been observed in a total of 26 male and female young adult dogs used in nonclinical safety studies by various institutions worldwide, affecting animals across all dose groups, including controls, in approximately similar proportions. Given the presence of this basal nucleus lesion in several control animals, the evidence suggests that this change should be regarded as a novel background finding in the Beagle brain with the possibility to be exacerbated by a given test item. Given the likelihood of this lesion appearing in a test item–treated animal by chance, misinterpreting it as a test item–related change could significantly impact the risk-benefit assessment and decision-making regarding the further development of a compound. The etiology remains unknown, and investigations, including genealogy and whole genome sequencing with substantial support from the breeder, are currently underway. Please refer to the more detailed manuscript that is included in this special proceedings publication.

Conclusion

The presentations highlighted that advanced tools like AI, CFT, and ST could significantly transform toxicologic pathology by enabling more precise and efficient assessments of drug efficacy and toxicity, potentially reducing reliance on animal models. These technologies offer promising future applications in improving the accuracy of pathological evaluations, facilitating high-throughput screening, and uncovering detailed molecular mechanisms, thus enhancing the overall drug development and safety processes. Furthermore, integrating these tools with traditional histopathological methods can help address challenges such as anatomical complexity, compound/therapeutic distribution, and drug-related effects by introducing new visualization and analysis quantification techniques. In conclusion, the adoption of these advanced technologies in toxicologic pathology holds significant promise for the future.