Histological Evaluation of Brain Tissue

Histological evaluation of the brain in rodent models and human postmortem tissue primarily addresses biological alterations identified by other methods, for example, hyperintense regions in MRI⁶⁷ or Gd presence in specific brain regions.²³ To obtain tissue sections with high morphological preservation, rat brains should be carefully collected to minimize structural artifacts after exsanguination, buffer-perfusion, and fixation or postfixation by immersion.⁶⁹ Postfixation appears adequate during general toxicity studies, but perfusion fixation provides an optimal tissue preservation for neurotoxicity studies.⁷⁰ Neutral buffered 10% formalin followed by paraffin embedding is most efficient for routine analysis. However, cryosectioned material is preferable for some stains (eg, immunochemistry, silver degeneration). Guidelines for organ sampling and trimming in rodents toxicity studies have been published.⁷¹

General staining methods such as hematoxylin and eosin stain (HES) can serve as a first overall evaluation of the tissue to detect gross alterations. However, HES is known to have limited sensitivity in detecting possibly subtle changes associated with potential Gd/GBCA-induced neurotoxicity.⁷² Therefore, specific stains or immunohistochemistry are helpful to detect specific markers. Inflammation is associated with microglia (ionized calcium binding adaptor molecule 1, Iba1), activated microglia (CD68/ED-1), and astrocytes (glial fibrillary acidic protein, GFAP, a sensitive biomarker that labels most reactive astrocytes that respond to CNS injuries)⁷³ (Fig. ​(Fig.4).4). Selection of the stains is best left at the discretion of the pathologist. A well-described qualitative histological evaluation is usually sufficient but becomes most effective when combined with digital image analysis and quantification,⁷⁴ for example, quantification of the astrocyte marker GFAP to evaluate reactive astrocytosis.^72,75

Histological examination of parallel slices (4–6 μm, formalin-fixed paraffin-embedded, FFPE) of a rat brain in the region of the deep cerebellar nuclei (DCN, in red) using different staining procedures: HES (B and C), Nissl Stain with cresyl violet (D), immunohistochemistry for astrocytes with glial fibrillary acidic protein, GFAP (E), and microglia cells with ionized calcium-binding adaptor molecule 1, Iba1 (F) (From Lohrke et al²³). The full slice (B) is compared with the correct position in a rat atlas (A, Paxinos and Watson,⁵⁰ Copyrights Elsevier Inc, with permission).

It is very important that the analyzed brain regions, independent of the method used (histological staining, laser ablation or MRI), are aligned and compared with standard anatomical data sets or literature to precisely confirm their localization^50,76 (Figs. ​(Figs.22 and ​and44).

Pathologist blinding should be avoided^23,72 when there is no a priori defined spectrum of lesions, a situation that applies to administration of GBCAs. In blinded studies, only changes considered to be clearly outside of a reference range can be recorded, an approach that would reduce the sensitivity of the pathological study.⁷⁷