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Anatomy and physiology
Korean Journal of Audiology 2009;13(2):135-139.
Immunocytochemical Characterization of Schwann Cells Cultured from Guinea Pig Spiral Ganglions
Kyoung Ho Park1, Shi-Nae Park1, Jae-Hyun Seo1, Hyug-Gi Choi1, Helge Rask-Andersen2, Sang W. Yeo1
1Department of Otolaryngology Head & Neck Surgery, The Catholic University of Korea, School of Medicine, Seoul, Korea
2Department of Otolaryngology Otosurgery, Uppsala University Hospital Uppsala, Sweden
Immunocytochemical Characterization of Schwann Cells Cultured from Guinea Pig Spiral Ganglions
Kyoung Ho Park1, Shi-Nae Park1, Jae-Hyun Seo1, Hyug-Gi Choi1, Helge Rask-Andersen2, and Sang W. Yeo1
2Department of Otolaryngology Head & Neck Surgery, The Catholic University of Korea, School of Medicine, Seoul, Korea

Background and Objectives
The nervous system is built from two broad categories of cells: the neurons and glial cells. The main glial cell in the peripheral nervous system (PNS) is the Schwann cell. The best known function of glial cells in the adult PNS is the formation of myelin sheaths around axons allowing for the fast conduction of signaling essential for nervous system function. In addition, they may be essential regulators of the formation, maintenance and function of synapses, the key functional units of the nervous system. We hypothesized that different shapes of Schwann cells, named type 1 and type 2, may have different functions similar to the central nervous system (CNS) astrocyte and oligodendrocyte cells, which we attempt to prove here. 

Materials and Methods
Guinea pig spiral ganglion (SG) cells were harvested and cultured in vitro. The cells were grown and differentiated in culture medium together with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF). After 1 week of culturing, the cells were fixed and immunocytochemical stainings with β-III tubulin, S-100, polysialic acid (PSA) and neural cell adhesion molecules (NCAMs) were performed. 

Phenotypic characterization of guinea pig Schwann cells showed the presence of two types of cells, one spindle shaped and one more flattened, both showing immunopositivity for S-100. The flat shaped cell (type 2) was morphologically similar to astrocyte cells in the CNS. The spindle shaped cell (type 2) often showed physical interaction with adjacent neurites. Similar findings were made in human SG cultures. 

We conclude that there are two morphologically and immunocytochemically distinct Schwann cell types in guinea pig SG cultures. We speculate that these two types of cells may have different functional characteristics similar to astrocyte and oligodendrocyte cells in the CNS.

Keywords: Spiral ganglion;Schwann cells;Guinea pig.

Address for correspondence : Sang W. Yeo, MD, Department of Otolaryngology Head & Neck Surgery, The Catholic University of Korea, School of Medicine, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea
Tel : +82-2-2258-6210, Fax : +82-2-533-1349, E-mail : swyeo@catholic.ac.kr

서     론

The nervous system is built from two broad categories of cells, the neurons and glial cells. The main glial cell in the peripheral nervous system (PNS) is the Schwann cell. The best known function of glial cells in the adult PNS is the formation of myelin sheaths around axons allowing the fast conduction of signaling essential for nervous system function. In addition, they may be essential regulators of the formation, maintenance and function of synapse, the key functional unit of the nervous system.1,2)
Schwann cells ensheathing axons in peripheral nerves are found to be of two types; myelinating and non-myelinating. The myelinating Schwann cells form insulating sheaths around axons that are comparable in structure and function to those made by oligodendrocytes in the central nervous system (CNS). The non-myelinating cells are likely to have metabolic and mechanical support functions similar to astrocytes in CNS. There is evidence that Schwann cells are indispensable for neuronal survival during development as well as in damaged nerves. Thus, Schwann cells may control successful regeneration and restoration of function. 
In spiral ganglion (SG), there are specific characteristics in the human and other mammalian species, especially in the degree of myelination. In most forms, two main types of perikarya may be observed namely the type 1 and type 2 cells.3,4,5) In order to prove the morphological and functional different patterns of glial cells in the mammalian SG similar to CNS glia, SG cells were harvested and cultured in vitro. Cultures of Schwann cells facilitate analysis of both normal Schwann cell morphology and function. The cells were grown and differentiated in the culture medium together with brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and glial cell line derived neurotrophic factor (GDNF). 
In the present study, we demonstrated the presence of two types of glial cells in the cultured SG showing different morphological and immunocytochemical features. We hypothesized that each Schwann cell named type 1 and type 2 may have different function similar to the CNS astrocyte and oligodendrocyte cells.

Materials and Methods

Culture of Schwann cells from the human and guinea pig spiral ganglion
Human inner ear tissue was obtained during intracranial surgery for life threatening petroclival meningioma. In addition, fresh guinea pig cochleas were taken out from the temporal bone after anesthesia. Tissues were placed in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Paisley, UK) and immediately transported to the research department for further processing. The study conforms with the Declaration of Helsinki and was approved by the medical ethics committee at Uppsala University Hospital (No. 99398, 22/9 1999, 29/12 2003). Animals were treated according to the rules set up by the Ethical Committee of Animal Care and used in Uppsala, Sweden. For human tissue, patient consent was also obtained.
The SG in Rosenthal's canal was excised after midmodiolar division of the hard bone using a micro-saw under constant saline irrigation. This is the same as earlier written. The tissue was dissected out with a micro-elevator and rinsed 3 times in DMEM, and transferred to a 15 mL tube with 0.25% trypsin, and incubated at 37℃ for 20 minutes. DNase (10 mg/mL, Sigma, St Louis, MO, USA) was added and cells were carefully triturated and larger pieces allowed to settle for 2 minutes. The supernatant was transferred to a new tube and stopped digestion by 10% fetal calf serum (Gibco), and the cell suspension was centrifuged at 1,000 rpm for 5 minutes. The pellet was resuspended in neurobasal medium containing B27 supplements (Invitrogen, Carlsbad, CA, USA), L-Glutamin and gentamycin. 10 ng/mL of GDNF (Invitrogen), BDNF (Invitrogen), and NT-3 (Sigma) were added at the beginning of the incubation. Cells were then plated on culture plates coated with poly-DL-ornithine (Sigma). On every third day, half of the medium was renewed and fresh neurotrophic factors added. Individual neurons and Schwann cells were identified, labelled and photographed digitally each day using an Olympus camera with an imaging device (Olympus digital 4.1 megapixel C-404OZ00M).

Cells were immunostained using antibodies for S-100 (diluted 1 : 200, Sigma), Thy1.1 (CD90, diluted 1 : 200, Chemicon), β III tubulin (β-tubulin, diluted 1 : 300, BioSite Taby, Sweden), neural cell adhesion molecule (NCAM, diluted 1 : 100, Sigma) and polyclonal antipolysialic acid antibody (diluted 1 : 100, given by FA Troy). After 1 week culturing, cells were fixed for 10 minutes by 4% paraformaldehyde in phosphate-buffered saline (PBS), washed with PBS. To stain neurons and glial cells from cultured SG, primary antibodies were diluted in 1% polysialic acid (PSA) in PBS and then applied for 1 hour at room temprature. Cells were rinsed 3 times for 5 minutes with 0.1% Saponin in PBS. Blockage of staining was done in 3% H2O2 in methanol for 15 minutes. Secondary antibodies of biotinylated anti-mouse in horse 1 : 200 or anti-rabbit in goat 1 : 200 in 1% BSA in PBS were made and these antibodies were applied for 30 minutes at room temperature followed by the avidin-horseradish peroxidase (Vectastatin ABC
® kit, Burlingame, CA, USA) for 30 minutes. These were developed and counterstained with hematoxylin and mounted.


Morphological characterization of spiral ganglion Schwann cells 
From the guinea pig SG cell culture, two types of Schwann cells were found.
The first one was designated it as a type I Schwann cell in SG. It was found to be spindle shaped having bipolar processes sometimes showing 3 or 4 dendritic cytoplasmic projections. The nucleus was ovoid to spindle shape and had a smaller size than type 2 Schwann cells. The cells sometimes had neurite or neuron like appearance but was found to be negatively stained for β-tubulin but positive for S-100. This was marked contrast to the adjacent neurons and neurites which showed strong immunopositive reaction for β-tubulin and other neuron markers. The second one type II Schwann cells from cultured SGs had a flat shaped fibroblast like appearance that was differentiated from fibroblast cells by Thy 1.1 immunostaining. Fibroblasts which seldom occur in our serum free cultures show positive reactions for Thy1.1. Flat elongated and sometimes star shaped appearance of the Schwann cells were similar with cultured astrocytes from the cultured CNS tissue. Its nucleus was round, large and with prominent nucleoli. This type of Schwann cell was more prevalent than the type I cell and it was also similar in humans (Fig. 1, 2).

Immunocytochemical characteristics of type I and II Schwann cells
In order to differentiate the Schwann cells from contaminated fibroblast, we performed the experiments using Thy1.1 staining. No cells were stained positive with this antibody (Fig. 2). In cultured SG, neurons were positive for β-tubulin. In PSA and NCAM immunostaining, both type I and type II cells showed no staining for PSA while the SG neuronal cells were moderately stained with PSA. SG neuronal cells as well as type II Schwann cells were found positive for NCAM while the type I Schwann cells showed no expression of NCAM. In neurons, however, cell body and axon displayed slight to moderate staining. There was a various expression and immunostaining with NCAM of type II Schwann cells. Approximately 2/3 of the type II Schwann cells were found to show positive immunostaining with NCAM (Fig. 3).

Comparison of human and guinea pig spiral ganglion Schwann cells
For comparison between human and guinea pig cultured Schwann cells, we immunostained with β-tubulin and S-100 in cultured SGs. The shape of the Schwann cells and neurons of humans were found similar to guinea pigs. In S-100 staining of human and guinea pig Schwann cells, a similar discrete cell types with type I and type II Schwann cells were found. Type I Schwann cell was spindle shaped cells with ovoid nucleus and type II Schwann cell was fibroblast like and similar to the astrocyte in CNS and more prevalent than type I cell (Fig. 4). 


The PNS, the major glial cells are characterized as Schwann cells. It is known that there are two types of Schwann cells in the PNS, myelinating and non-myelinating. The Schwann cells perform similar functions to CNS astrocytes and oligodendrocytes: forming myelin, ensheathing synaptic junction and compartment glial small-diameter axons. Schwann cells are also likely to regulate the ionic environment of axons, provide neurotrophic support, and regulate periaxonal space. During development and nerve injury, it provide increased neurotrophic support for neurons and guidance both as a source of soluble neurotrophic factors and by providing a growth promoting substrate for axonal regrowth by synthesis of permissive molecules both on the cell surface and secreted into the extracellular matrix.1,2,6)
So far there is no morphologic characterization study for cultured Schwann cells in the SG. We tried to find indications of the existence of 2 morphologically and functionally different Schwann cells in the SG similar to CNS glia, such as astrocyte and oligodendrocyte. The results suggest that there are in fact two types of morphologically different Schwann cells of type I, spindle shaped type with bipolar processes sometimes showing dentritic processes. The cell was originally leaved to represent type II SG neurons but immunocytochemical staining using β-tubulin and S-100 showed that this was non-neural cell showing Schwann cell characteristics. The type II Schwann cells were found to be flattened fibroblast like but easily differentiated from fibroblast by absence of Thy1.1 immunostaining. Its shape was similar to cultured astrocytes from the CNS and was found to be more prevalent than the type I Schwann cells. The flattened Schwann cells often showed signs of physical interaction with adjacent neurites sometimes the cells could be seen to migrate along the neurite levels. Similar separation between type I and type II Schwann cells often occurred in human material.
To prove immunocytochemical characteristics of type I and II Schwann cells in cultured guinea pig SGs, we applied PSA and NCAM antibodies. PSA is sugar chains that are linear homopolymers consisting of N-acetylneuraminic acid (Neu5Ac) joined internally by α2, 8-ketosidic linkage. It is usually attached on cell adhesion molecules (CAMs) such as neural CAMs (NCAMs) are a class of high molecular weight cell surface sialoglycoproteins that function in cell adhesion and cell migration. PSA covalently modifies the embryonic form of neural cell adhesion molecules (PSA-NCAM), NCAM with PSA. PSA decreases NCAM dependent cell adhesion thereby negative regulator of cell-cell interaction. Nonpolysialated NCAM, so called adult form NCAM, aggregate much more readily than PSA-NCAM. In neural development, the presence of PSA on NCAM is closely correlated with axon pathfinding, synaptogenesis and neural cell migration and differentiation. But adult form NCAM is postulated to have a role in facilitating two adjacent cells to form regions of contact and stabilize mature tissue.7,8) In this study, type I and II Schwann cells were negative for PSA. But it is expressed in neurons. PSA-NCAM expression by the neuron is related to their sprouting capabilities. In this study, type I and type II Schwann cells were negative for PSA but positive neurons. PSA and NCAM expression by the neurons is related to their sprouting capability. So far there is no information about the role of PSA on Schwann cells in the PNS. However, some roles are known in the CNS. PSA on NCAM is an important cell-cell interaction regulator during development and in regeneration of the injured axon and remyelination. Its expression on axons acts as a negative regulator of myelination and loss of axonal PSA accelerates myelination by oligodendrocytes. PSA reappears on naked axons in pathologic conditions, such as multiple sclerosis.9,10,11) In the development of CNS glial cells, PSA-NCAM of astrocyte is switched to NCAM after onset of gliafibrillaryacidic protein (GFAP) expression.12) GFAP and S-100 are expressed same stage, so it may be the reason that the all Schwann cells which are positive for S-100 shows no immunoreactivity for PSA. But on the other hand, only the type II Schwann cell shows positive reactivity for NCAM, adult form NCAM without PSA. It is known that NCAM was expressed by non-myelinating Schwann cells.13,14) So, we hypothesize that type II Schwann cells are associated with mechanical and metabolic support function rather than myelination.
From the results of the experiment, the spindle shaped type I Schwann cells had definitely different morphologic appearance from the type II Schwann cells and with common characteristic immunostaining for S-100 and PSA. Only type II Schwann cells stained with NCAM. It is known that several cell adhesion molecules of immunoglobulin superfamily are expressed during the development and myelination of the PNS. NCAM does not express simultaneously with myelinspecific protein protein P0, so we considered that NCAM may be expressed in non-myelinating Schwann cells in the SG as Ran-2, a cell surface protein in astrocyte.15,16,17,18) 


In conclusion, type I and type II Schwann cell had different morphological and immunocytochemical features in cultured SG cells. It is believed that the type I Schwann cell may function similar to oligodendrocytes in the CNS for myelination and type II Schwann cells may be similar to the astrocyte cells in the CNS as mechanical and metabolic support functions.


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