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1 Kobe University Repository : Kernel タイトル Title 著者 Author(s) 掲載誌 巻号 ページ Citation 刊行日 Issue date 資源タイプ Resource Type 版区分 Resource Version 権利 Rights DOI JaLCDOI URL Expression of S-100 Protein and Neuron-Specific Enolase in the Developing and Adult Human Retina : An Immunohistochemical Study on 24 Cases Miki, Akinori / Itoh, Hiroshi / Karim, Mohammed Mohibul Bulletin of allied medical sciences Kobe : BAMS (Kobe),12: Departmental Bulletin Paper / 紀要論文 publisher PDF issue:

2 Expression of S-100 Protein and Neuron-Specific Enolase in the Developing and Adult Human Retina: An Immunohistochemical Study on 24 Cases Mohammed Mohibul Karim \ Akinori Miki 2 and Hiroshi Itoh 3 Imm unohistochemically, we examined the developmental expression of S-lOO protein and neuron-specific enolase (NSE) in the human retina. The expression of S- 100 protein examined with polyclonal antiserum against S-lOO protein was almost the same as that examined with monoclonal antibodies in a previous study.l The result of the present and the previous studies confirmed that S-100 protein is expressed preferentially in the ganglion cells and astrocytes, but not in the Muller cells, and suggest that the expression of this protein might be closely related to the differentiation and/or maturation of these cells. On the other hand, NSE began to be expressed in the human retina slightly earlier than S-lOO protein, and not only in the ganglion cells but also in the cells of the outer and inner neuroblastic layers. At later developmental stages, NSE was strongly expressed in the inner plexiform layer. NSE was not expressed in astrocytes or Muller cells at any stages examined in the present study. These findings suggest that NSE could be used as a reliable maker for the neuronal elements in the human retina, and that the strong expression of NSE in the inner plexiform layer might be closely related to the synaptogenesis arid/or synaptic functions in the related regions. Key Words S-100 protein, Neuron-specific enolase, Developmental expression, Imm unohistochemistry, Human retina. INTRODUCTION S-IOO protein is one of the acidic proteins of the brain 1 and in a previous study, using monoclonal anti Green Road, Dhaka-1205, Bangladesh, Faculty of Health Science 2 and First Department of Pathology3, Kobe University School of Medicine, Kobe, Japan bodies, we examined immunohistochemically the developmental expression of this protein in the human retina, and reported that the expression of this protein might be closely related to the differentiation of ganglion cells and astrocytes. 2 We also reported that this protein was expressed preferentially in the ganglion cells and astrocytes, but the other kinds of cellular elements were not immunolablelled though several investigators observed the expression of S-IOO protein in the Muller cells of the mammal Ian retina.. Inc 1 u d' lng man I t IS. well known that monoclonal antibodies have higher specificity but. lower sensitivity than polyclonal anti-. bodies. It is also possible that the Vol. 12, 1996 Bulletin of Allied Medical Sciences, Kobe 115

3 M. M. Karim et al. differences in the tissue processing methods affect the sensitivity of the immunoreaction. Thus, in the present study, we applied polyclonal antiserum against S-100 protein, and immunohistochemically reexamined in the developing and mature human retina. It is generally known that neuronspecific enolase (NSE) exists III almost all types of neurons, and is well established that NSE could be used.- as a good index of neronal maturation. 6 It is also said that the expression of NSE is closely related to the syna~togenesis in the developing brain. 7, This suggest that NSE could also be used as a good marker for neuronal differentiation in the neural retina. In the present study, therefore, we examined the developmental expression of NSE in the human retina and simultaneously the results were compared with those of S- 100 protein. MATERIALS AND METHODS Specimens A total of 24 normal human eyes from the registry of First Department of Pathology, Kobe University School of Medicine were used in this study. Informed consent was obtained from all persons and authorities, and the principles of the Declaration of Helsinki were followed. Eyes were collected following abortions or autopsies. All specimens were fixed with 4% paraformaldehyde dissolved in phosphate buffered saline (PBS, ph 7.4). These eyes were cut anteroposteriorly parallel to the optic axis and a band-shaped portion of retina was taken from the temporal half excluding macula and fovea. These materials were dehydrated with alcohol and embedded in paraffin as usual. Sections of 6,um in thickness were used for immunohistochemical analysis. Immunohistochemistry The rabbit polyclonal antiserum against S-100 protein purified from the cow brain (Dako, Glostrup, Denmark), was used at a dilution of 1 : 200 in this study. This antiserum cross-reacts storongly with human S- 100 A and B. The rabbit antiserum against neuron-specific enolase (NSE) purified from human brain (Dako, Glostrup, Denmark) reacts with the y subunits of NSE and used at a dilution of 1:100. During the immunohistochemical procedure, the endogenous peroxidase activity was quenched by incubating the specimens in 3% hydrogen peroxide for 10 minutes. The sections were incubated with the primary antibodies and followed by a labelled streptavidin biotin staining procedure. The immunohistochemical reactions were visualized with freshly prepared diaminobenzidine tetrahydrochloride. As positive controls, surgical specimens of nomal human brain were stained in parallel. For negative controls serial sections were incubated with non-immune rabbit serum at a comparable dilution Instead of the primary antibodies. RESULTS protein immunohistochemistry (Figs. 1-6 and Table 1) Between 13th and 22nd gestational week (GW), the human neural retina consisted of thick inner and outer 116 Bulletin of Allied Medical Sciences, Kobe

4 Epression of S-l 00 protein and NSE in retina neuroblastic layers, and a thin nerve fiber layer was noted inside the inner neuroblastic layer. At these stages, distinct immunoreactivity for S-lOO protein was not detected anywhere in the neural retina. At 24th CW, a thin ganglion cell layer was discerned just inside the inner neuroblastic layer, and an inner plexiform lnyer became visible between the inner neuroblastic and ganglion cell layers. At this stage, large round cells exhibiting the distinct immunoreaction were first demonstrated and formed the ganglion cell layer. A few small spindle-shaped cells exhibiting the imm uno reaction were seen in the nerve fiber layer. These immunoreactive cells were observed only around the optic disc. Thereafter, the immunoreactive area gradually extended toward the ora serrata with the progress of development. At the same time, intensity of the immunoreaction in the individual cell gradually became' stronger. At 40th CW, expression of S-lOO protein in the retina was light microscopically almost the same as that in the adult human retina. The developmental pattern of the immunoreactivity examined with the polyclonal antiserum was principally similar to that examined with the monoclonal antibodies in a previous study. A t any stage examined in the present study, S-lOO protein was not detected in the Muller cells. Fgiure 1-6. These figures show immrnoreactivity for S-100 protein stained with the labelled. streptavibin biotin method_ Sections were lightly counterstained with hematoxylin. Scale bar: 10J1m. In all pictures, single arrow indicates astrocytes and double arrows in dicate ganglion cells. At all stages the immunoreaction was detected only in the ganglion cell and nerve fiber layers_ The outer and inner neuroblastic (granular) layers were negative in the immunoreaction. NFL: nerve fiber layer, GeL: ganglion cell layer, IPL:inner plexiform layer, IGL: inner granular (neuroblastic) layer, OPL: outer plexiform layer, OGL: outer neuroblastic (granular) layer. Fig_ 1: At 24th CW, weakly labelled immunoreactive cells scatteredly visible in the ganglion cell layer. These cells were noted only in the vicinity of the optic disc Fig. 2 and 3 also show immunoreactive cells in the region near to the optic disc at 26th CW and 37th CW respectively. Fig. 4: In the adult human retina, ganglion cells and astrocytes exhibit strong immunoreactivity for S-100 protein near the optic disc. Fig. 5: At 40th CW, immunoreactive cells are observed near the ora serrata. Fig. 6: Immunoreaction is evident in the adult human retina near the ora serrata. Vol. 12,

5 M. M. Karim et al. 118 Bulletin of Allied Medical Sciences, Kobe

6 Epression of S-l 00 protein and NSE in retina Neuron-specific enolase immunohistochemistry (Figs. 7, 8) At 22nd CW faint immunoreactivity for neuron-specific enolase (NSE) was first detected in the outer and inner neuroblastic layers, and at 24th, CW, when the ganglion cell layer began to form, the faint lmm unoreacti v ity was also detected in this layer. At these stages, the immunoreactive area was limited only near the optic disc. At 28th CW, the cells in the outer and inner neuroblastic layers exhibited distinct immunoreaction in the cytoplasm, and a slightly weaker reaction was diffusely demonstrated In the outer and inner plexiform layers. Thereafter, the immunoreactive area gradually extended toward the ora serrata, with the advancement of development. At 40th CW, th~ immunoreactivity in the outer neuroblastic and outer plexiform layers declined, but the cells in the inner neuroblastic layer (inner granular layer) and in the ganglion cell layer still exhibited distinct immunoreactivity. The inner plexiform layer exhi-' bited extremely strong immunoreactivity, while the neve fiber layer, very Fgiure 7 and 8. These pictures show immunoreactivity for neuron-specific enolase (NSE) stained with the labelled streptavidin biotin method_ Scale bar: lo,um. NFL:nerve fiber layer, GeL: ganglion cell layer, fpl: inner plexiform layer, fpl: inner plexiform layer, fgl: inner granular (neuroblastic) layer, OPL: outer plexiform layer, OGL: outer granular (neuroblastic) layer. Fig. 7: At 28th CW, moderate immunoreaction is seen in the outer and inner granular (neuroblastic) layers and the outer plexiform layer. A few cells (arrow) in the ganglion cell laver show weak immunoreaction. Nerve fiber layer lacks apparent immunoreaction. Fig. 8: At 40th CW immunoreaction in the outer granular (neuroblastic) layer becomes weak, but the reaction in the inner granular (neuroblastic) layer and ganglion cells (arrows) show distinct immunoreaction. The inner plexiform layer displays extremely strong immunoreaction, but the nerve fiber layer, very weak or no reaction. Vol. 12,

7 M. M. Karim et al. Table 1. S-100 protein immunohistochemical reaction patterns of the retina Age NFL GCL GW No No 24 GW Weak Weak GW Larger area Larger area GW Strong Strong 40 GW - Adult Larger area Larger area NFL- Nerve fiber layer, GCL- Ganglion cell layer, GW- Gestational weeks weak or no immunoreactivity. Light microscopically, the expression of NSE in the retina at this stage was essentially similar to that in the adult human retina. DISCUSSION Developmental expression of S-IOO protein in the human retina examined with polyclonal antiserum in the present study was almost the same as that examined with monoclonal antibodies in a previous study. 2 In these expriments, S-IOO protein was expressed in the ganglion cell and nerve fiber layers. On the basis of the morphological characteristics and localization, the immunoreactive cells were considered to be ganglion cells and astrocytes. Taken together these findings suggest that in the human retina, S-IOO protein is specifically expressed in these cells, but not in the Muller cells although several authors reported the expression of S-IOO protein in the Muller cells. 3.4,5 This discrepancy might be due the differences in antibody specificity or due to the tissue preparation processes. It was reported that in the mammalian central nervous system, S-IOO protein exists not only in the glial cells,l,9,10 but also in neurons,l1,12 and in the peripheral nervous system, in Schwann cells and satellite cells. 13 As to the functions of S-IOO protein, it was reported that this protein has a calcium-binding site, and takes part in the transport of G ABA across the axolemma, and increases the activity of RNA-polymerase in the nucleus. 13 Although at present the roles of S- 100 protein in the human retina is still obscure, it is suggested that this protein might be involved in some neuronal and glial functions specific for the ganglion cells and astrocytes. We showed in the present study that neuron-specific enolase (NSE) began to be expressed in the developing human retina slightly earlier than S-IOO protein. Furthermore, localization of NSE was quite different from that of S-IOO protein, i.e., NSE was demonstrated in the neuronal 120 Bulletin of Allied Medical Sciences, Kobe

8 Epression of S-l 00 protein and NSE in retina cells of the outer and inner neuroblastic (granular) layers and gangliou cell layer, but not in the astrocytes. It is said that in the developing central nervous cells, NSE is not expressed in the ependymal layer which consists of proliferative neuroepithelial cells, and begins to be expressed in the cells which are in the stage of differentia bon." Th us, It.. IS propose d t h at NSE can be used as a reliable marker of neuronal maturation. 16 In the present study, NSE was detected in the outer and inner neuroblastic layers and in the ganglion cell layer at the early developmental stages, while at later stages, the expression of NSE in the outer neuroblastic layer (outer granular layer) declined. This developmental pattern was very similar to that in the developing rat retina. 16 Taken together these findings suggest that the expression of NSE in the neu-. ral retina might be closely related to the neuronal differentiation. and maturation. In the literature it has been described that NSE may be involved in the synaptogenesis and synaptic functions of the developing brain. 7,8 The findings of our study suggest that at early stages, NSE may be involved in the synaptogenesis, but at later stages strong NSE expression in the inner plexiform layer (IPL) may take part in the synaptic functions specific for IPL. REFERENCES 1. Moore BW: A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 19: , Karim MM, Miki A, Itoh H: Monoclonal antibodies analysis of S l 00 protein expression in the developing human retina..(in press Bull Allied Med Sci Kobe) 3. Das A, Pansky B, Budd GC, et al.: Immunohistochemistry of mouse and human retina with antisera to insulin and S-100 protein. Curr Eye Res 3: , Iwanaga T, Takahashi Y, Fujita T: Immunohistochemical localization of S-100 protein in the retina, ciliary body and iris of human fetuses. Cell Tissue Res 239: , Molnar ML, Stefansson K, Molnar GK, et al.: Species variations in distribution of S-l 00 in retina. Invest Opthalmol Vis Sci 26: , Schmechel DE, Brightman MW, Marangos PJ: Neuron switch from non-neuronal enolase to neuron-specific enolase during differentiation. Brain Res 190: :..214, Maxwell GD, Whitehead MC, Connolly SM, et al.: Development of neuron-specific enolase immunoreactivity in avian nervous tissue in vivo and in vitro. Dev Brain Res 3: , Whitehead MC, Marangos pj, Conolly SM, et al.: Synapse formation is related to the onset of neuron-specific enolase immunoreactisity in the avian auditory and vestibular systems. Dev Neurosci 5: , Moore BW, Perez VJ: Specific acidic proteins of the nervous system. Carlson FD (ed), In Physiological and Biochemical Aspects of Nervous Intergration, Englewood Cliffs, NJ, Prentice Hall, pp , Zomzely-Neurath CE, Waler Wa: Nervous system-specific proteins: , neuron-specific enolase and S-100 protein. In Proteins of the nevous system (2nd edition), Bradshaw RA, Schneider DM (ed), Raven Press, New York, ppl-57, Sviridov SM, Korochkin LI, Ivanov VN, et al.: Immunohistochemical studies of S-100 protein during postnatal ontogenesis of brain of two strains of rats. J Neurochem 19: , Hansson HA, Persson L, Ronnback L, et al.: Immuno-electron microscopic study of the distribution of the S-100 protein in brain glial cells. Cytobios 15: 45-48, 1976 Vol. 12,

9 M. M. Karim et al. l3. Stefansson K. Wollmann RL, Moore BW: Distribution of S 100 protein outside the central nevous system. Brain Res 234: , Cicero TJ, Cowan WM, Moore BW: Changes in the concentration of two brain proteins S-100 and proteins during the development of the avian optic tectum. Brain Res 24: 1-10, Cicero TJ, Ferrendelli JA, Suntzeff V, et al.: Regional changes of the S-100 and proteins during development and aging of the mouse. J Neurochem, 19: , Fujieda H, Sato T, Wake K: Expression of neuron-specific enolase in the developing rat retina as revealed by immunocytochemistry. Dev Brain Res 82: 69-80, Bulletin of Allied Medical Sciences, Kobe