Neurofilament Accumulation in Rabbit Retinas

J. Comp. Path. 2015, Vol. -, 1e4

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SPONTANEOUSLY ARISING DISEASE

Neurofilament Accumulation in Rabbit Retinas K. Kawasako, T. Oshikata, T. Kanno and M. Hamamura Pathology Department, Nonclinical Research Center, Drug Development Service Segment, LSI Medicine Corporation, 1285 Kurisaki-machi, Uto, Kumamoto, Japan

Summary An eosinophilic body (EB) was observed in the inner nuclear layer and the outer plexiform layer of the anterior dorsal region of the retina in New Zealand White, Japanese White and Dutch rabbits. Immunohistochemistry confirmed that the EB was an accumulation of neurofilaments (NFs). Ultrastructurally, intermediate filaments of approximately 10 nm in diameter were observed in the EB, but there were no intracellular organelles. These results suggested that the NFs had accumulated in the neurites of the horizontal cells in the retina. This is the first description of a new pattern of NF accumulation in the mammalian retina. The prevalence of the EBs increased significantly in 44e56-week-old male Dutch rabbits (38.9 %) compared with 18e23-week-old (12.9 %) rabbits, suggesting that the formation of EBs in the rabbit retina could be an age-related change. Ó 2015 Elsevier Ltd. All rights reserved. Keywords: eosinophilic body; neurofilaments; rabbit; retina

Neurofilaments (NFs) play a major role in the development, maintenance, regeneration and plasticity of the neuronal cytoskeleton in the central and peripheral nervous system (Zhang et al., 1998; Zhu et al., 1998; Ruiz-Ederra et al., 2004; Liu et al., 2011). NF accumulation is caused by gene mutation, dysregulated synthesis, defective axonal transport and abnormal phosphorylation/glycosylation/ oxidation, and induces selective degeneration and death of the neuron (Zhu et al., 1998; Ruiz-Ederra et al., 2004; Liu et al., 2011). In the human retina, one of the eosinophilic oval or circular structures has been previously described as a ‘cytoid body’ (Eagle, 1999). A cytoid body is a spheroid, which is a focal axonal swelling of the nerve fibre layer (NFL) resulting from a disturbance in axonal transport, and is localized in the NFL. In the cytoid body, NFs accumulate in the axon accompanied by cytoplasmic organelles such as microtubules, endoplasmic reticulum and mitochondria. However, to our knowledge, an accumulation of NFs in the retina, Correspondence to: K. Kawasako (e-mail: kawasako.kazufumi@mx. medience.co.jp). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2015.07.002

except for the cytoid body, has not yet been reported in any animals or in man. New Zealand White (NZW) rabbits (23-week-old males and 19-week-old females) were purchased from Kitayama Laboratories, Nagano, Japan. The animals were part of a group being kept to obtain background data for toxicity studies. They were housed in aluminum cages, maintained in an airconditioned animal room with a 12 h light/dark cycle, and cared for according to the principles outlined in the guidelines for the care and use of laboratory animals prepared by the Japanese Association for Laboratory Animal Science and our laboratory. They were allowed free access to feed and water. The rabbits showed no clinical abnormalities and received no prior treatments. Necropsy examination was performed after humane destruction. Eyes from eight males aged between 24 and 26 weeks and 12 females aged between 23 and 24 weeks were used for histological, immunohistochemical and ultrastructural examinations. The eyes were fixed in Davidson’s solution, trimmed along the dorsoventral axis and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE) and periodic Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kawasako K, et al., Neurofilament Accumulation in Rabbit Retinas, Journal of Comparative Pathology (2015), http://dx.doi.org/10.1016/j.jcpa.2015.07.002

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acideSchiff (PAS). Microscopical examination revealed eosinophilic bodies (EBs) in the inner nuclear layer (INL) and the outer plexiform layer (OPL) of the retina (Fig. 1). The EB was oval or circular and stained strongly eosinophilic centrally and weakly eosinophilic in the periphery. There was an indistinct border to this structure. The EB was located in the anterior dorsal region of the eye in the sagittal sections. Microscopical examination using serial sections revealed that the EB continued with the neurite running horizontally in the OPL of the retina. The EB was not stained by PAS and there were no histological abnormalities in other levels of the retina including the ganglion cells or photoreceptor cells. Immunohistochemistry (IHC) was performed using a commercial kit (Dako LSAB K680; Dako, Burlingame, California, USA) with peroxidaseconjugated streptavidin and a mixture of biotinylated goat anti-rabbit and anti-mouse immunoglobulins as the link antibody. Mouse monoclonal anti-human NF protein (Dako) and anti-bovine synaptophysin (PROGEN Biotechnik GmbH., Heidelberg, Germany) were used as the primary antibodies. Negative controls were performed by omitting the primary antibody. The EB showed intense immunoreactivity for NF (Fig. 2), but was negative for synaptophysin. For electron microscopical examination, eyes were fixed in 2.5 % glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) and post-fixed in 1% phosphatebuffered osmium tetroxide. The samples were processed and embedded in resin. Ultrathin sections were stained with uranyl acetateelead citrate and examined with a transmission electron microscope (Hitachi, Ltd., Tokyo, Japan). The EBs were seen to comprise of fila-

Fig. 1. Anterior dorsal retina. An eosinophilic body is located in the inner nuclear layer (INL) to the outer plexiform layer (OPL) of the retina. It continues with the neurite running horizontally in the OPL of the retina. NFL, nerve fibre layer; GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments. HE. Bar, 20 mm.

Fig. 2. Anterior dorsal retina. An eosinophilic body is labelled for neurofilaments. IHC. Bar, 20 mm.

mentous structures that were more dense towards the centre than at the periphery (Fig. 3). Intermediate filaments of approximately 10 nm in diameter, dense bodies, vesicles and membranous structures were observed in the EB (Figs. 3 and 4). Intermediate filaments observed in the EB were identical to those in the neurite. There were no intracellular organelles such as mitochondria in the EB. These results indicated that the EB in the rabbit retina was an accumulation of NFs in the INL to

Fig. 3. Anterior dorsal retina. Filamentous structures are observed filling the eosinophilic body (EB) and are more dense towards the centre than the periphery. Dense bodies (asterisks), vesicles (arrows) and membranous structures (arrowheads) are also observed in the EB. N, neurite. TEM. Bar, 2 mm.

Please cite this article in press as: Kawasako K, et al., Neurofilament Accumulation in Rabbit Retinas, Journal of Comparative Pathology (2015), http://dx.doi.org/10.1016/j.jcpa.2015.07.002

Neurofilament Accumulation in Rabbit Retinas

Fig. 4. Anterior dorsal retina. Intermediate filaments and vesicles (arrow) are observed in the eosinophilic body. There are no mitochondria in the eosinophilic body. TEM. Bar, 500 nm.

the OPL. In addition to the cytoid body, another eosinophilic oval or circular structure has been described in man as a retinal corapora amylacea (Avendano et al., 1980). This is a polyglucosan body, staining red with PAS, and localized mainly to the inner plexiform layer (IPL) of the retina. The retinal corapora amylacea is composed of fibrils and glycogen granules as shown by ultrastructural examination. The morphological and immmunohistochemical characteristics of the EB in the rabbit retina were distinct from those of the eosinophilic oval or circular structures in the human retina as previously described. Immunohistochemically, the cells expressing NF in the mammalian retina vary between animal species (Lyser et al., 1994; Lohrke et al., 1995; Ruiz-Ederra et al., 2004). In the rabbit retina, NFs are expressed in the horizontal cells, the bipolar cells and the amacrine cells, other than the ganglion cells (Lohrke et al., 1995; Ruiz-Ederra et al., 2004). The EB in the INL to the OPL continued with the neurite running horizontally in the OPL, suggesting that the accumulation of NFs occurred in the neurites of the horizontal cells in the rabbit retina. Horizontal cells in the rabbit retina are of two types: A-type (axonless; HA) and B-type (short axon; HB) (Lyser et al., 1994; Lohrke et al., 1995; Ruiz-Ederra et al., 2004). The HA cells are labelled

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with anti-NF antibody, but the HB cells are not labelled, indicating that HA cells contain more NF than HB cells (Lohrke et al., 1995). NF expression is dependent on the location along the dorsoventral axis of the retina, such that dorsal HA cells contain more NF than ventral HA cells (Lohrke et al., 1995). In the present study, IHC for NF revealed that more positive signals were observed in the dorsal retina than in the ventral retina. In addition, more EBs labelled as NFs were observed in the dorsal retina than in the ventral retina. Therefore, it is suggested that the EBs represent NF accumulation in the dendrites of the dorsal HA cells of the rabbit retina. To clarify the prevalence of the EBs in rabbit retina, a histological examination was performed retrospectively using HE-stained sections of eyes prepared in previous toxicology studies. Eyes from 79 NZW rabbits (40 males and 39 females aged between 20 and 27 weeks), 89 Japanese White (JW) rabbits (49 males and 40 females aged between 20 and 27 weeks) and 103 Dutch rabbits (31 males aged between 18 and 23 weeks and 72 males aged between 44 and 56 weeks) were examined. Fisher’s exact test was used to compare the prevalence of the EBs between the strains, sexes and ages. P <0.05 was considered significant. EBs were observed in approximately 30e50% of the examined animals (48.1% in NZW, 31.5% in JW and 31.1% in Dutch rabbits). The prevalence of the EBs in NZW rabbits aged between 20 and 27 weeks was significantly higher compared with that in the age-matched JW rabbits (P ¼ 0.0393). There was no significant difference between the sexes of the NZW (47.5% in male and 48.7% in female) and JW (26.5% in male and 37.5% in female) strains. There are no descriptions concerning this change in other animal species. Therefore, it was suggested that these EBs were a rabbit-specific change and that their appearance could be associated with a species-specific pattern of NF expression in the retina. The prevalence of the EBs was increased significantly in male Dutch rabbits aged between 44 and 56 weeks (38.9%) compared with those between 18 and 23 weeks (12.9%) (P ¼ 0.0104), suggesting that EBs in the rabbit retina could be an age-related change. NF accumulation is also observed as an age-related change, except for that of many human neurodegenerative disorders and toxic neuropathy (Zhang et al., 1998; Zhu et al., 1998; Ruiz-Ederra et al., 2004; Liu et al., 2011). In old cats, age-related NF accumulation is observed in the perikarya in the nucleus lateralis reticularis in the brainstem and in the axon (intra-axonal accumulation of neurofilaments; IAAN) in the dorsal column nuclei of the brainstem (Zhang et al., 1997, 1998). IAAN

Please cite this article in press as: Kawasako K, et al., Neurofilament Accumulation in Rabbit Retinas, Journal of Comparative Pathology (2015), http://dx.doi.org/10.1016/j.jcpa.2015.07.002

K. Kawasako et al.

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contains abundant NFs, dense granules, vacuoles and mitochondria (Zhang et al., 1998). In rabbit retina, NF accumulation was observed in the dendrites and did not include mitochondria. Therefore, this was a distinct NF accumulation pattern compared with the age-related IAAN. In the present study, we have described a new pattern of NF accumulation in the dendrites of horizontal cells in the rabbit retina. There were no histological alterations other than the formation of EBs in the retina in rabbits up to 56 weeks of age. The pathological significance of the EBs could not be determined. To understand further the nature of the EBs it will be necessary to investigate the dysregulated synthesis or assembly, or abnormal posttranslational modification, of the accumulated NFs, using rabbits older than those in the present study. NF accumulation represents an important factor leading to neuronal cellular disruption in many neurodegenerative diseases (Zhu et al., 1998; RuizEderra et al., 2004; Liu et al., 2011). These diseases are investigated using transgenic animal models with changes made to the genes associated with NF abnormalities. Methods that could remove the abnormal accumulations of NFs would have potential clinical benefit (Liu et al., 2011), so further studies of the characteristics and mechanisms of accumulation of NFs in the rabbit retina may contribute to the establishment and development of effective therapeutics for neurodegenerative diseases.

Acknowledgments We thank Mr. R. Yoshida, Ms. H. Yamaguchi and Mr. Y. Ikeda for technical support and Mr. S. K. Filiatrault and Ms. K. Tamatsukuri for language editing. The authors declare that they received no financial support for their research and/or authorship of this article.

Conflict of Interest Statement The authors declare that they have no conflict of interest with respect to their authorship or the publication of this article.

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April 13th, 2015 ½ Received, Accepted, July 6th, 2015 

Please cite this article in press as: Kawasako K, et al., Neurofilament Accumulation in Rabbit Retinas, Journal of Comparative Pathology (2015), http://dx.doi.org/10.1016/j.jcpa.2015.07.002