Calbindin D-28 and microtubule-associated protein-2: Their use as sensitive immunohistochemical markers of cerebellar neurotoxicity in a regulatory toxicity study

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Abstract

The aim of this study was to develop an immunohistochemical (IHC) method for calbindin D-28 (CB-28) and microtubule-associated protein-2 (MAP-2) and evaluate their expression as markers in the detection, characterisation and grading of unexpected cerebellar toxicity in the rat. High power examination of H&E-stained brain sections of treated rats 2 days following a single oral dose of a novel compound revealed irregular vacuolation of the molecular layer and Purkinje cell degeneration. Animals killed after 14 days recovery showed Purkinje cell degeneration but vacuolation of the molecular layer was absent. In control animals, CB-28 and MAP-2 expression was high in Purkinje cell dendrites and cell bodies in the molecular layer. In treated animals, low power examination revealed loss of CB-28 and MAP-2 expression in degenerating neurons arranged in parasagittal stripes within the vermis. This is the first description of successful use of these two markers in a regulatory toxicity study using FFPE brain. In particular, CB-28 provides a sensitive method for characterising CNS toxicity which can be detected at low power enabling easier detection, screening and grading of neurotoxicity.

Introduction

Neurotoxicity can produce subtle morphological changes, which are usually first detected using conventiional stains such as haematoxylin and eosin (H&E) or cresyl violet. Immunohistochemistry (IHC) in the CNS can provide real benefits in terms of sensitivity and specificity and in helping characterise a morphological change. However, one of the problems with its routine use in detecting toxicity in regulatory studies for new pharmaceuticals and industrial chemicals is the successful application to routinely prepared material, e.g. formalin-fixed paraffin embedded (FFPE) sections. A wide range of neuronal and glial markers have been examined as part of research studies but these studies have used either frozen sections, perfused brains or free floating staining techniques (Fix et al., 1996). There is a need to be able to apply these techniques in a more regulated environment using material where prospective tissue collection of frozen material is not always possible. This paper describes the application of two immunohistochemical (IHC) markers, calbindin D-28K (CB-28) and microtubule-associated protein-2 (MAP-2), in the detection and evalution of an unexpected toxicity as part of a preclinical safety assessment of a novel compound using immersion-fixed, FFPE brain. These methods allow discrimination of minor differences between control and treated animals using low power examination and permit relatively rapid screening of large number of tissue sections. A further benefit is that this method enables examination of thinner sections of tissue (5 μm) with enhanced neuronal detail, rather than thicker vibratome sections (40–50 μm) (Garcia et al., 1996a).

The vitamin D3-dependent calcium-binding protein (CaBP) CB-28 is a member of a superfamily of CaBP involved in the regulation of intracellular calcium, located in neurons and found widely in the nervous system (Baimbridge et al., 1992). In some cases, CaBP-containing neurons are more able to survive ischaemic and excitotoxic insults than those which lack CaBP (Mattson et al., 1991). Further supporting evidence of a protective role of CaBP is provided by the observation that overexpression of CB-28 protects striatal neurons from transient focal cerebral ischemia (Yenari et al., 2001) and hippocampal neurons from kainic acid (a glutamate agonist) toxicity (Phillips et al., 1999). Although initially considered a neuron-specific marker within the central nervous system (CNS), astrocytes have also been shown to express CB-28 in response to brain injury and TNF-α (Mattson et al., 1995).

One of the major mechanisms controlling neuronal morphology is the phosphorylation of cytoskeletal proteins via changes in the relative activities in protein kinases and phosphatases (Sanchez et al., 2000). The MAP-2 family of proteins are an abundant group of cytoskeletal proteins which are predominantly expressed in neurons. MAP-2 phophorylation controls its association with the cytoskeleton and these proteins are involved in the maintenance of neuronal morphology and neurite outgrowth (Tucker, 1990).

The work reported here used rat brain sections obtained from regulatory toxicity studies. The test compound administered was in early development as a potential CNS-active therapeutic. The identity of this compound is not important to the conclusions of this report. However, the IHC methods reported here are of widespread practical use to those pathologists working with FFPE brain material.

Section snippets

Animals and experimental protocols

Eight to nine-week-old male and female Han Wistar rats (Charles River, UK) were housed in conventional solid bottom cages. Rats had free access to food and water. All animal experiments were approved by GlaxoSmithKline management and complied with the Animal (Scientific Procedures) Act (1986). Data from two separate toxicology studies is reported here; a single dose and a repeat dose toxicity study. In the single dose study, three rats per sex were dosed with a novel compound in early

Single dose study

Following single dosing of rats with the test compound, clinical signs of trembling were observed. Histopathological examination of animals killed 2 days after dosing revealed the following changes on routine examination of H&E-stained brain sections: irregular vacuolation of the molecular layer and Purkinje cell degeneration (both graded as very slight to slight) (Table 1). Degenerating Purkinje cells were identified by cellular eosinophilia and shrinkage, which was similar to that observed in

Discussion

This report documents two toxicology studies with a test compound in early development. Conventional microscopy of regulatory toxicity studies is usually limited to examination of H&E-stained sections of multiple tissues. In the studies reported here, initial examination of H&E-stained sections detected subtle morphological changes in the cerebellum which in some cases were only observed using high power objectives. There was clearly a need to develop methods to detect the toxicity more

Acknowledgements

We thank Stewart Jones for histology assistance and Sue Sparrow for critical review. We thank Jo Hunter and Gerard Pratt for helpful discussion.

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