Quantifying Drug‐Induced Neurotoxicity in Brain, Using Image Analysis Algorithms to Evaluate Digitalized Histopathology Sections
Authors: Negi Geeta , Liachenko Serguei , Paule Merle G , Sarkar Sumit , Hanig Joseph P 
Published: November 2023

Histopathology provides valuable tools for the assessment of the effects of various pathological conditions, drugs or neurotoxicants in brain. Sequential brain sections have long been employed in the objective measurement of cellular, molecular and often biochemical events that occur in brain. To date, studies to correlate toxicity and neurodegeneration have been predominantly qualitative or semi‐quantitative. In this study we aim to quantitatively describe the extent of neurodegeneration in rats using argyrophilic staining after neurotoxicant administration. Rats were administered the known neurotoxic compounds, kainic acid and pyrithiamine orappropriate vehicle for controls (5–12 animals per treatment group) using published dosing regimens known to produce different types of brain lesions. At the end of the treatment, animals were perfusion‐fixed and brains were dissected out from the calvarium and embedded in a gelatin based matrix. Serial coronal sections (40 μm) were collected on glass slides and stained using amino‐cupricsilver stain. These slides were digitized by scanning at 20X magnification using a whole slide imaging instrument (Aperio ScanScope XT, Leica Biosystems). For quantifying histopathological changes associated with neurotoxicant administration, we applied tissue classification and area quantification algorithms available in HALO™ image analysis software. Analysis of 81 serial coronalbrain sections using amino‐cupric silver stain showed different toxicity profiles for kainic acid and pyrithiamine. While kainic acid affected a wide range of brain structures and nuclei including olfactory bulb, insular cortex, thalamic nuclei, hippocampus, prerhinal cortex, entorhinal cortex and retrosplenial cortex, toxicity of pyrithiamine was rather confined to thalamus and globus pallidus. We quantified the areas of degeneration after kainic acid and pyrithiamine administration. Kainic acid treatment resulted in degeneration of 9.38%, 7.53%,7.52% and 6.61% of total area in olfactory lobe, hippocampus, cortex and thalamus respectively which was found to be <0.2% in saline treated animals. Effect of pyrithiamine however, was limited to thalamus where 32.5% of to talarea was found to be affected as compared to 0.4 % in saline rats. Results of histopathology image analysis showed concordance with neuropathology scores as the affected area were given a score of 3/4 (moderated/marked neurodegeneration). The results obtained in this study demonstrate that automated methods of histological quantification, when applied carefully, can be very effective and unbiased approaches for executing high‐throughput histological analysis and handling the accompanying data. They can be successfully employed for locating pathological features including neurodegeneration and quantitatively measuring toxicevents.Support or Funding InformationFDA‐Center for Drug Evaluation & Research (CDER) Critical Path Program, CDER/OPQ Office of Testing & Research and the National Center for Toxicological Research (NCTR)/FDA (E0741801) provided financial support for performing this study