Journal of Histochemistry and Cytochemistry, Vol. 50, 147-158, February 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Immunohistochemical Detection of Nestin in Pediatric Brain Tumors

Per M. Almqvista, Richard Mahd, Urban Lendahlb, Björn Jacobssonc, and Glenda Hendsond
a Department of Clinical Neuroscience, Section of Neurosurgery
b Department of Cell and Molecular Biology
c Department of Pathology and Cytology
d Karolinska Institute, Stockholm, Sweden, and Department of Pathology, British Columbia's Children's Hospital, Vancouver, Canada

Correspondence to: Per M. Almqvist, Dept. of Neurosurgery, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: per.almqvist@ks.se


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology. (J Histochem Cytochem 50:147–158, 2002)

Key Words: first trimester, telencephalon, nestin, vimentin, S-100, GFAP, immunohistochemistry, antigen retrieval, PNET, ependymoma, glioma


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

THE ADVENT of immunohistochemical (IHC) techniques to identify the expression of specific proteins has significantly advanced the typing of CNS tumors. These techniques have been of particular value in identification of intermediate filament proteins (IFPs) (Herrman and Aebi 2000 ) because these proteins are invaluable markers of cell origin within the CNS. Vimentin, glial fibrillary acidic protein (GFAP), and S-100ß are the markers most often used for identifying astrocytic cells in the intact CNS and in primary CNS neoplasms (Ridet et al. 1997 ). GFAP is the most specific marker for gliomas available (McKeever 1998 ), but more markers would be required to unambigously identify and grade these tumors (Kurpad et al. 1995 ).

Nestin is an interesting IFP in this regard (Lendahl et al. 1990 ; Dahlstrand et al. 1992b ). During development, nestin is expressed by primitive neuroepithelial cells in all regions of the central and peripheral nervous systems (Hockfield and McKay 1985 ), whereas in the adult CNS nestin is a marker for neural stem cells lining the ventricular wall and the central canal (Johansson et al. 1999 ). Here nestin is also expressed by endothelial and periendothelial cells (pericytes) (Alliot et al. 1999 ).

Nestin and vimentin are co-expressed in proliferating neuroepithelial cells in fetal rodent and human brain (Tohyama et al. 1992 ; Liem 1993 ; Dahlstrand et al. 1995 ; Takano et al. 1996 ), as well as in neuronal and glial progenitor cells (Fliegner et al. 1994 ). Developmental studies on postmortem human tissues have detected extensive nestin expression in neuroepithelial cells in the ventricular layer at 11 weeks post-conceptional age (PCA) in all parts of the CNS, whereas nestin immunoreactivity diminishes during the second and third trimesters (Takano et al. 1996 ; Takano and Becker 1997 ). During or after migration away from the proliferative ventricular layer, nestin expression is sharply downregulated in post-mitotic neurons and is replaced by {alpha}-internexin, first detected at 15 weeks PCA in the developing hippocampus (Arnold and Trojanowski 1996 ). This process is followed by expression of the low molecular weight (Mr) neurofilament (NF) protein (NF-L) and mid-Mr NF protein (Liem 1993 ). Whereas expression of both nestin and vimentin is sharply downregulated in post-mitotic neuroblasts (Ho and Liem 1996 ), vimentin is transiently expressed, along with GFAP, in radial glial cells in rat CNS (Dahl 1981 ; Dahl et al. 1981 ) and in late first trimester spinal cord and brainstem (Sarnat 1992 ).

Nestin can be re-expressed in astrocytes of the adult CNS in response to cellular stress, including blunt focal skull trauma (Holmin et al. 1997 ), penetrating injury to the brain (Lin et al. 1995 ) and spinal cord (Frisen et al. 1995 ), CNS ischemia (Lin et al. 1995 ; Li and Chopp 1999 ), kainic acid lesions (Clarke et al. 1994 ), and neoplastic transformation (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ). This complex pattern of nestin expression during development and adulthood is controlled by regulatory elements in the second intron of the nestin gene (Lothian and Lendahl 1997 ; Zimmerman et al. 1994 ). This regulatory element functions as an enhancer, directing nestin gene expression to precursor cells of the CNS (Zimmerman et al. 1994 ; Lothian and Lendahl 1997 ; Lothian et al. 1999 ). Consequently, nestin has been detected in malignancies derived from these tissues, including primitive neuroectoderml tumors (PNETs), ependymomas, malignant astrocytomas and oligodendrogliomas (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ), medulloepithelioma (Khoddami and Becker 1997 ), melanomas (Florenes et al. 1994 ), and pediatric rhabdomyosarcomas (Kobayashi et al. 1998 ). Despite the use of the #130 polyclonal antiserum for nestin detection, these studies report inconsistent results with respect to localization and expression levels of nestin in different types of primary CNS tumors.

This justifies a re-evaluation of nestin expression in tumor specimens processed by formalin fixation (Dahlstrand et al. 1992a ) and compared to cryostat-sectioned tumor tissue fixed with an organic solvent before immunostaining (Tohyama et al. 1992 ).

The masking of antigens induced by fixation of tissues in formaldehyde-containing fixatives is a well-recognized problem. Several different methods have been used in an attempt to recover immunoreactivity, including trypsinization (Finley and Petrusz 1982 ), alkaline hydrolysis (Shi et al. 1992 ), treatment with detergents (Meehan et al. 1989 ), and the use of formic acid (Kitamoto et al. 1987 ). The most successful has been microwave (MW) oven heating of tissue sections (Shi et al. 1991 ), and the optimal result is correlated with the product of heating temperature and time of heating treatment (Shi et al. 1997 ). Since the introduction of epitope retrieval methods, the sensitivity of many antibodies has been enhanced and the ability to retrieve a wide range of antigens in formalin-fixed, paraffin-embedded CNS tissue has greatly increased the range of investigations that can be carried out on this type of stored material (McQuaid et al. 1995 ). Marked increase in sensitivity with no loss of specificity was reported for several IFPs, including vimentin (Shi et al. 1991 ), GFAP (McQuaid et al. 1995 ), (Evers and Uylings 1996 ) and, most recently, for nestin by the use of Rat-401 mouse monoclonal antibody on rodent tissues (Mokry and Nemecek 1998 ).

The present study confirms the value of heat-induced antigen retrieval for the localization of nestin in formalin-fixed tissue, and we emphasize the importance of proper tissue processing for optimal antigen detection using the #4350 nestin antiserum in IHC. We found that tumor cells of malignant gliomas expressed high levels of nestin, whereas pilocytic astrocytomas showed low but consistent nestin expression by tumor cells in contrast to normal brain tissue.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Tissue Processing
Tumor specimens derived from surgical biopsies and autopsies were selected from the files of British Columbia's Children's Hospital. Surgical biopsies were from 1995 to 1998. One glioblastoma specimen was used as a nestin-positive control sample and five brain metastases, including three adenocarcinomas and two low-differentiated carcinomas, were used as nestin-negative controls. All tumors were processed according to routine protocols for histopathological diagnosis and were graded according the WHO grading system (Kleihues et al. 1993 ). For eight patients, surgical tumor specimens were processed for both cryostat sectioning of fresh frozen tissue and formalin-fixed, paraffin-embedded for comparison of nestin immunoperoxidase staining. CNS tissue from a first-trimester fetus 9.6 weeks PCA was also used for the study. The fetus was derived from a spontaneous abortion after placental abruption and had been fixed in 10% buffered formalin, serially cut in the coronal plane, and embedded in paraffin for sectioning.

Nestin Antibodies
The anti-nestin antisera #130 and #4350 were produced in rabbits immunized with a bacterially produced fusion protein containing the 1300 carboxy-terminal amino acid residues of the rat nestin protein (#130) (Tohyama et al. 1992 ) and a region of the fourth exon, corresponding to amino acids 1454–1618 in the human nestin protein (#4350) (Grigelioniene et al. 1996 ). Specificities of the antibodies used have been documented previously (Tohyama et al. 1992 ; Grigelioniene et al. 1996 ).

Immunohistochemistry
The formalin-fixed, paraffin-embedded tissue specimens were sectioned at 5 µm. Sections were mounted on silanized slides, deparaffinized in xylene, blocked in 3% H2O2 in absolute methanol, and processed for antigen retrieval by MW heating according to Ashraf Imam and co-workers 1995 . Briefly, the slides containing the tissue sections were placed in a plastic jar containing 0.1 M sodium citrate buffer, pH 6.0, and heated twice for 5 min in an MW oven with the highest power setting of 600 W. After heating the slides were allowed to remain in the jar until they returned to ambient temperature. Before immunostaining the slides were washed in PBS.

For localization of IFPs and S-100 protein in tissue sections, an indirect IHC staining technique was used (Level 2 Ultra Streptavidin Detection System; Signet Laboratories, Dedham, MA). Both nestin rabbit antisera #130 and #4350 and rabbit antiserum against S-100 (Innovations Foundation; Toronto, ONT, Canada) were diluted 1:2000. Mouse MAbs to human GFAP and vimentin (Dako; Glostrup, Denmark) were used diluted 1:1000 and 1:100, respectively. Antibodies bound were visualized with 3-amino-9-ethyl-carbazole (AEC) (Sigma; St Louis, MO) as substrate chromogen. Slides were counterstained with Carazzi's hematoxylin and coverslipped using Entellan (Merck; Darmstadt, Germany). Brightfield microscopy with conventional photography was performed using a Leica DMRB microscope and a Nikon FII camera with Fuji T64 film.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cytoarchitecture of Fetal Forebrain
Brain tissue from an aborted first-trimester fetus was used for the study of nestin expression at an early developmental stage, for evaluation of staining protocols, and for titration of the nestin antisera. The tissue specimen had been paraformaldehyde-fixed, paraffin-embedded, and processed for IHC without previous storage. Hematoxylin (Carazzi) staining of tissue sections showed the characteristic cytoarchitecture of the outer telencephalic wall at the time of transition from the embryonic to the fetal stage, including the germinal matrix of the ventricular layer (VL) with the highest density of cells, the intermediate layer (IL), the cortical plate (CP) and subplate zone (SPZ), and the subpial layer (SPL) (Fig 1). The cortical plate was present on the surface of the entire neopallium at this stage (Fig 1, inset), with the subpial layer half the width of the cortical plate. External to the ventricular layer is the IL, followed by the SPZ. The subplate was at least twice the thickness of the CP, based on an increase of fibers travelling perpendicular to the developing cerebral cortex (O'Rahilly and Muller 1994 ).



View larger version (144K):
[in this window]
[in a new window]
 
Figure 1. Brightfield photomicrograph of a Carazzi hematoxylin-stained section of the telencephalic wall derived from a 9.5-week-old human fetus, with the characteristic cytoarchitecture including the ventricular layer (VL) with its germinal matrix, the intermediate layer (IL), the subplate zone (SPZ), the cortical plate (CP), and the subpial layer (SPL). Bar = 100 µm. (Inset) Tissue section of the intact cerebral hemisphere at low-power magnification. The boxed area is magnified in Fig 1. Bar = 1 mm.

Nestin Expression in Fetal Forebrain
To perform a comparative analysis of nestin antisera #130 and #4350, tissue sections of formalin-fixed human fetal forebrain were processed for IHC using a standard protocol. Nestin #130 antiserum specifically stained the ependymal cell layer, scattered cells in the germinal matrix of the ventricular layer, and radial fibers projecting to the SPZ (Fig 2A), whereas nestin #4350 antiserum gave a very faint immunostaining (Fig 2B). After antigen retrieval by MW heating of tissue slides, nestin immunostaining was enhanced for both nestin antisera, giving equally strong immunostaining at 1:2000 dilution and overnight incubation with primary antiserum (Fig 2C and Fig 2D). In addition, the staining patterns were identical for the two nestin antisera, with strong nestin immunoreactivity detected in ependymal cells lining the ventricle, in scattered cells of the germinal matrix of the ventricular layer, in radial glial cells extending fibers from the ventricular layer, traversing the intermediate layer towards the SPZ (Fig 2C and Fig 2D). There was a steep gradient of nestin immunoreactivity, with very intense staining of the columnar epithelium lining the ventricle and the nestin expression diminishing as the cells were located away from the germinal matrix. No nestin-positive cell bodies were seen outside the ventricular layer.



View larger version (136K):
[in this window]
[in a new window]
 
Figure 2. Nestin immunoperoxidase staining of the outer telecephalic wall with Carazzi counterstain. (A,C) Staining with nestin antiserum #130. (B,D) Staining with nestin antiserum #4350. Tissue sections in (C,D) were processed for antigen retrieval immunohistochemistry. (A,B) Slides processed according to a standard protocol. The four figures A–D show the immunostained tissue sections at two magnifications. Bars = 100 µm and 50 µm, respectively. Note the increased immunoreactivity after antigen retrieval without loss of specificity.

Similar to nestin, vimentin was expressed by ependymal cells, by cells in the germinal matrix, and by radial fibers as they traversed the ventricular and the intermediate layer. In addition, vascular endothelial cells were strongly positive for vimentin. No GFAP immunoreactivity could be detected before or after heat-induced antigen retrieval (data not shown).

Immunohistochemical Staining for IFPs in Formalin-fixed Tissue
Sectioned material from formalin-fixed, paraffin-embedded surgical specimens of 20 pediatric brain tumors was analyzed for nestin expression by IHC after heat-induced antigen retrieval. The specimens consisted of seven ependymomas, seven PNETs, five pilocytic astrocytomas, and one glioblastoma multiforme (GBM).

Ependymomas. All seven ependymomas examined arose within the posterior fossa of children 2–10 years old, with a median age at diagnosis of 4 years. Six of these tumors (numbers 1–6 in Table 1) were classic ependymomas characterized by moderate cellularity, formation of perivascular pseudorosettes, nuclear-free zones, and fibrillary cell processes extending to the endothelium of the vessels. One anaplastic ependymoma was analyzed and showed increased cellularity and mitoses compared to the classic ependymomas.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Immunostaining of formalin-fixed tumor tissuea

By standard IHC techniques, the classic ependymomas showed no immunoperoxidase staining with any of the two nestin antisera. However, after antigen retrieval there was cytoplasmic staining of the tumor cells and endothelial cells in all tumor specimens tested. The most prominent staining was noted in the cell processes of the perivascular spaces. In these areas the intermediate filaments stained as fine fibrillary processes that extended from the cytoplasm of the ependymal cells to the endothelium lining the vascular spaces. The intensity of staining and the staining pattern were very similar with the two nestin antisera. More than 50% of tumor cells were nestin-positive in all specimens, and staining intensity varied slightly. The vascular endothelium of ependymomas stained for nestin with high intensity in all samples tested (Table 1).

Staining of an anaplastic ependymoma (number 7, Table 1) with nestin antiserum #130 without antigen retrieval showed both tumor cell cytoplasmic staining and endothelial cell staining (Fig 3A), whereas nestin #4350 antiserum failed to give any specific immunostaining (Fig 3C). The staining intensity increased after antigen retrieval (Table 1). Fig 3B and Fig 3D show strong immunoperoxidase staining of tumor cells and vascular endothelial with either #130 or #4350 antiserum.



View larger version (147K):
[in this window]
[in a new window]
 
Figure 3. Immunoperoxidase-stained tissue sections of an anaplastic ependymoma (#7, Table 1). (A,B) Tumor sections immunostained with nestin antiserum #130. (C,D) Sections stained with nestin antiserum #4350. Sections in A and C were processed according to a standard protocol. Sections in B and D were processed for antigen retrieval immunohistochemistry. Bar = 100 µm.

Figure 4. Consecutive tissue sections of an anaplastic ependymoma (#7 in Table 1; see also Fig 3), stained for hematoxylin and eosin (A), vimentin (B), GFAP (C), and S-100 (D). All immunoperoxidase stainings were done after antigen retrieval. Note that tumor cells stained for vimentin, GFAP, and S-100, and for nestin (see Fig 3), whereas endothelial cells stained only for nestin and vimentin. Bar = 100 µm.

Three ependymomas (numbers 5–7 in Table 1) were analyzed for vimentin, GFAP, and S-100 expression. Clearly, all three of these glial markers were co-expressed with nestin in tumor cells (Fig 4). Endothelial cells expressed nestin (Fig 3) and vimentin (Fig 4B), whereas they were devoid of GFAP and S-100 (Fig 4C and Fig 4D).

Primitive Neuroectodermal Tumors. All PNETs examined arose within the posterior fossa of children 2.5–16 years old. Median age at diagnosis was 7.5 years. These tumors were characterized by dense cellularity and were composed of sheets of cells with hyperchromatic, angulated nuclei and minimal amounts of cytoplasm. True rosettes were identified in some areas of the tumors. None of the tumor samples showed further differentiation along glial or neuronal cell lines. Mitoses and karryhorectic cells were present in the tumor.

Neither of the nestin antisera showed distinct immunostaining of tumor cells before antigen retrieval, although weak staining of endothelial cells by the #130 antiserum was observed in some cases. After antigen retrieval, 5/7 specimens showed coarse tumor cell cytoplasmic staining by the #130 nestin antiserum. More than 50% of cells were positive in all samples and intensity of staining was intermediate (Table 1). Antiserum #4350 stained tumor cells in all PNETs tested. Staining pattern and intensity varied extensively among tumor specimens, while these parameters correlated well in 5/7 tumors comparing #130 and #4350 antisera (Fig 5A and Fig 5B) Vascular endothelial cells showed very high staining intensity for both nestin antisera in all PNETs tested (Table 1).



View larger version (210K):
[in this window]
[in a new window]
 
Figure 5. Nestin-immunostained formalin-fixed tumor tissue. (A,B) PNET; (C,D) GBM; (E,F) pilocytic astrocytoma. Tissue sections were processed for antigen retrieval immunohistochemistry and stained with nestin antiserum #130 (A,C,E) and nestin antiserum #4350 (B,D,F). Bar = 100 µm.

GFAP and S-100 expression were detected in a minority of tumor cells after antigen retrieval. The tumor cells showed cytoplasmic staining with a very high intensity in two of the samples tested. Endothelial cells were devoid of GFAP and S-100 immunoreactivity but stained positive for vimentin. In contrast, vimentin could not be detected in tumor cells of the two PNETs tested.

Glioblastoma Multiforme. Tumor tissue of a GBM located in the suprasellar region of a 14-year-old boy was used as a positive tumor control sample for the analyses of IFP expression. The histological picture was characterized by increased cellularity, mitoses, and endothelial proliferation. The tumor cells were plump, with abundant eosinophilic cytoplasm, and were devoid of astrocytic morphology. Areas of necrosis were present.

Nestin antiserum #130 stained the majority of tumor cells, whereas no staining was obtained with the #4350 antiserum before antigen retrieval. After antigen retrieval, the cell cytoplasm of most tumor cells, if not all, stained intensely with both nestin antisera (Fig 5C and Fig 5D). In addition, these cells showed strong staining for vimentin, GFAP, and S-100. Endothelial cells within the tumor tissue were stained for nestin and vimentin but not for GFAP or S-100 (Table 1).

Pilocytic Astrocytomas. Pilocytic astrocytomas were from children 7–12 years of age, of whom none was diagnosed with neurofibromatosis 1 (NF1). Four of five tumors originated from the cerebellum and one was located in the thalamus. Histologically, these astrocytomas were characterized by a biphasic pattern with microcysts filled with mucin, bordered by round astrocytes, and alternating with dense fibrillary areas composed of astrocytes with hair-like cell processes. One of the astrocytomas showed many Rosenthal fibers.

Nestin immunoreactivity of tumor cells was negative before antigen retrieval, whereas nestin #130 antiserum showed occasional staining of endothelial cells only. After antigen retrieval, both nestin antisera stained the vast majority of tumor cells at a low but consistent intensity (Fig 5E and Fig 5F). Tumor cells showed a delicate nestin immunoperoxidase staining of the cytoplasm with its hair-like cell processes, and staining pattern and intensity correlated well between the two nestin antisera (Table 1; Fig 5E and Fig 5F). The glial markers vimentin, GFAP, and S-100 were all expressed by tumor cells, whereas only nestin and vimentin were found in endothelial cells (Table 1).

Negative Tumor Controls. Five brain metastases of non-neuroepithelial origin were used as tumor control samples. All five were negative for nestin (Table 1). However, some reactive astrocytes surrounding two of these metastases were weakly nestin-immunoreactive, as were some endothelial cells in the metastatic tumors (not shown).

Nestin Immunostaining of Fresh Frozen Tissue
Freshly frozen tumor specimens were available for eight of the 20 tumors studied (Table 2). These samples were cryostat-sectioned, acetone-fixed, and processed for nestin IHC as described in the Materials and Methods. In all tissue sections, nestin antiserum #4350 stained tumor cells and endothelial cells without preparatory MW heating. Antigen retrieval further increased the intensity of nestin staining in most tissue sections studied, whereas MW heating had the opposite effect on antiserum #130 immunostaining. This antiserum gave weak immunoperoxidase staining of cryostat sections and failed to detect nestin in more than half of the tissue sections tested (Table 2).


 
View this table:
[in this window]
[in a new window]
 
Table 2. Nestin immunostaining of fresh frozen tumor tissuea


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Nestin is a commonly used marker for undifferentiated cells in the developing CNS and for CNS tumors. In this report we establish an improved protocol for immunohistochemical detection of nestin, based on antigen retrieval. Using this protocol, we show that nestin is abundantly expressed by tumor cells and endothelial cells of ependymomas and PNETs, whereas pilocytic astrocytomas showed low but consistent nestin expression by tumor cells in contrast to normal brain tissue.

Effects of Microwave Heating
Encouraged by recent reports on improved immunoreactivity of IFPs without loss of specificity by heat-induced antigen retrieval, we have evaluated the use of antiserum #4350 to identify nestin in formalin-fixed CNS tissue and tumors. Nestin antiserum #4350 (Grigelioniene et al. 1996 ) was used in parallel with a control nestin antiserum #130, previously used to localize nestin in tissue sections (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ). To provide a proper substrate for this test, formalin-fixed, paraffin-embedded human fetal forebrain was sectioned and processed for IHC with and without MW heating of tissue sections at 100C twice for 5 min in a citrate buffer of pH 6. This tissue has the advantage of a well-defined histology and an expected high level of nestin expression by neuroepithelial precursor cells in the germinal matrix.

Microwave heating greatly increased the sensitivity of both nestin antisera. Although being weakly reactive before heating, antiserum #4350 gave strong immunostaining of formalin-fixed embryonic human telencephalon, identical to that of nestin antiserum #130, after microwave heating (Fig 2). Therefore, antigen retrieval did not affect the staining specificity but gave increased sensitivity with improved signal-tonoise ratio optimal at a 1:2000 dilution of either nestin antiserum.

Although the exact mechanism of antigen retrieval is not yet understood, it is believed that the crosslinks between proteins are broken, which makes the antigen accessible to the antibody. Improved detection of several intermediate filament proteins in formalin-fixed brain tissue has been reported, including phosphorylated and non-phosphorylated NF (Evers and Uylings 1996 ), vimentin (Ashraf Imam et al. 1995 ; Shi et al. 1991 ; Williams et al. 1997 ) and GFAP (McQuaid et al. 1995 ) and, when compared to protease digestion, MW pretreatment was clearly superior with antigens such as vimentin (Shi et al. 1991 ). Antigen retrieval IHC was used to study nestin expression in developing and adult rodent tissue by use of the mouse MAb Rat-401 (Mokry and Nemecek 1998 ). Following a protocol similar to ours, these authors reported an enhanced signal for nestin in formalin-fixed tissues, with the highest nestin expression in developing CNS and skeletal muscle.

Nestin and Vimentin Expression During CNS Development
Previous studies have shown that vimentin and nestin are concurrently expressed during rodent CNS development, from neural tube closure to the end of gliogenesis (Hockfield and McKay 1985 ; Frederiksen and McKay 1988 ). Vimentin and nestin are expressed by proliferating cells of the ventricular layer during embryogenesis (Lendahl et al. 1990 ) and are most highly expressed by ependymal cells and, at lower levels, in the rapidly proliferating SVZ progenitor cells in adult mammals (Doetsch et al. 1997 ). Recently, ependymal cells lining the ventricles were identified as neural stem cells with the ability to generate new neurons and to proliferate and migrate in response to injury, and to differentiate to astrocytes at the site of the lesion (Johansson et al. 1999 ). A recent molecular study showed that vimentin expression is a prerequisite for nestin to assemble into an IF-like network in cultured cells (Marvin et al. 1998 ). Nestin's dependence on vimentin for assembly may explain why the two IFPs are always co-expressed during CNS development. In the human embryo, vimentin is present in all neuroepithelial cells at 4 weeks of GA (Stagaard and Mollgard 1989 ) and by 9–10 weeks of GA vimentin is expressed by cells in close proximity to the ventricular wall and by radial glial processes traversing the wall of the neural tube (Stagaard and Mollgard 1989 ; Sarnat 1992 ). Similar to vimentin, nestin was identified in primitive neuroepithelial cells and radial glial fibers of the spinal cord at 6 and 11 weeks of gestation, whereas only endothelial cells were nestin-positive from 20 weeks of age (Tohyama et al. 1992 ). These authors also report nestin and vimentin immunoreactive cells in the germinal matrix of the telencephalic wall at 17 and 20 weeks of gestation. As expected, we found nestin expressed by cells in the germinal matrix of the 9.5-week outer telencephalic wall (Fig 2). The immunostained tissue sections showed a sharp gradient of immunoreactivity, with high expression levels by ependymal cells and gradually decreasing immunoreactivity with increasing distance from the ventricular wall. Nestin-expressing neural precursor cells were limited to the ventricular layer, whereas immunoreactive radial glial fibers extended into the intermediate layer (Fig 2). Parallel immunostainings for vimentin showed a virtual co-expression with nestin. Although similar to previous reports (Sarnat 1992 ), we failed to detect any GFAP in the telencephalic wall at this developmental stage.

Nestin Expression in Brain Tumors
Two previous studies report on nestin expression in CNS tumors. Interestingly, their results differ despite the use of nestin antiserum #129 in both studies (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ). In the first study, tumor tissue analyzed was derived from surgical biopsies and autopsies. Tissue specimens were fresh-frozen, cryostat-sectioned, and fixed in acetone or methanol. These sections were subsequently treated with detergent (0.1% SDS) and MW-heated briefly for 3 min before immunoperoxidase staining (Tohyama et al. 1992 ). These authors report high levels of nestin expression in malignant primary CNS tumors, including PNETs, anaplastic astrocytomas, and ependymomas, whereas 2/4 low-grade astrocytomas were weakly positive and two were negative. Nestin was located in the cytoplasm of tumor cells and in endothelial cells of the tumor bed.

The study by Dahlstrand and co-workers 1992a made use of two nestin antisera, #129 and #130, produced in separate rabbits immunized with the same antigen (Redies et al. 1991 ). These two antisera gave very similar staining patterns when applied to formalin-fixed tumor samples derived from neurosurgical biopsies. However, the frequency of nestin-positive primary CNS tumors and the expression levels were both significantly lower compared to the report by Tohyama and co-workers 1992 . Dahlstrand and co-workers 1992a report that the majority of gliomas tested (34/50) were negative for nestin, including 4/6 anaplastic astrocytomas, 6/7 astrocytomas grade II, 6/6 pilocytic astrocytomas, 2/5 anaplastic oligodendrogliomas grade III, 2/5 oligodendrogliomas grade I-II, 3/6 ependymomas, and 11/15 PNETs. In 14/16 nestin-positive tumors, less than 5% of tumor cells were immunoreactive. GBMs were the only primary CNS tumors with high nestin expression levels.

In view of these differences, the aim of this study was to establish an optimal protocol for nestin IHC and to analyze expression in primary CNS tumors. To this end, we used the #130 antiserum previously used and the recently developed #4350 antiserum against human nestin. With the #130 antiserum, we could confirm the previous findings of apparently low nestin immunoreactivity in formalin-fixed tumor tissue samples using a standard staining protocol. After MW heating for 10 min in acetate buffer, nestin immunoreactivity increased significantly without loss of specificity. The morphological resolution and low background staining were maintained. These findings are in agreement with the work by Tohyama and co-workers 1992 using antiserum #129 for detection of nestin by antigen retrieval IHC with similar detection levels.

Although no immunoreactivity could be detected by #4350 antiserum before MW heating of tumor sections, #4350 antiserum gave a weak signal, as analyzed by standard IHC of human fetal forebrain. This is probably a result of different processing of fetal and tumor tissue specimens. Whereas the fetal tissue specimens were processed immediately after fixation, tumor samples had been stored, paraffin-embedded, for 1 to several years. Extensive paraformaldehyde fixation and/or prolonged storage was reported to have a negative effect on antigenic sites and required extended heating for antigen retrieval (Shi et al. 1997 ).

A GBM tissue sample was included as a control sample in the tumor group because it had been reported previously to be high in nestin (Dahlstrand et al. 1992a , Dahlstrand et al. 1992b ; Tohyama et al. 1992 ). Both nestin antisera gave cytoplasmic staining of tumor cells of all four types of tumors tested, with high nestin levels in GBM, PNETs, and ependymomas, and low nestin levels in benign pilocytic astrocytomas. It has previously been reported that nestin expression is augmented with increasing tumor grade in astrocytic tumors, and our results are in agreement with these reports (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ). In addition, we found some variability in nestin expression within each type of tumor, but further studies are needed to determine whether the levels of nestin expression are correlated to the clinical outcome.

Nestin expression in astrocytic tumors has previously been considered to be restricted to astrocytomas of WHO grade II-IV (Dahlstrand et al. 1992a ; Tohyama et al. 1992 ). In the present study, we found consistently low-level nestin expression in all pilocytic astrocytomas of WHO grade I tested, with the piloid cell processes staining positive for nestin and for the glial markers GFAP, vimentin, and S-100. We also found low but variable nestin expression in reactive astrocytes surrounding brain metastases in two cases. The latter must be taken into account when immunodetection is used as a diagnostic tool in CNS tumors.

Consistent nestin expression was also found in vascular endothelial cells of normal CNS tissue and in tumor samples (Table 1). Immunostained consecutive tissue sections of an anaplastic ependymoma, as shown in Fig 3, clearly show that endothelial cells of tumor vessels co-express nestin and vimentin, whereas they are negative for GFAP and S-100. Previously, endothelial cells positive for nestin were reported in primary CNS tumors (Dahlstrand et al. 1992a ), and a detailed morphological study by Alliot and co-workers 1999 described nestin expression in endothelial and periendothelial cells of CNS vasculature in mice. Although the biological significance of these findings remains to be unraveled, nestin may become a useful marker for endothelial and periendothelial cells in the CNS, with special relevance for gene therapeutic efforts of targeting tumor vessels in the treatment of malignant brain tumors.

With special reference to surgical pathology, we wanted to study the immunoreactivity of nestin in fresh frozen tumor specimens that had been cryostat sectioned and simply fixed with cold acetone. On this substrate, both nestin antisera could detect nestin without prior antigen retrieval. However, antiserum #4350 was superior to antiserum #130 with respect to identification and intensity of immunostaining (Table 2). Antigen retrieval by MW heating, as described for formalin-fixed tissue sections, could further improve the immunoreactivity of nestin as detected by the #4350 antiserum, whereas it had no or a negative effect on immunostaining by the #130 antiserum. This finding indicates the potential use of nestin antiserum #4350 for immunostaining of tissue smears and cryostat-sectioned tissue samples from stereotactic biopsies and open surgery to identify neoplastic CNS cells.


  Acknowledgments

Supported by the Swedish Medical Research Council, grants B96-13X-11570, K96-17R-12045 and K97-17F-12023, the Foundations of the Karolinska Institute, the Magnus Bergvall Fund for Medical Research and the Foundations of Sven Jerring, of Tore Nilson, and of Barncancerfonden.

Received for publication March 12, 2001; accepted October 3, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Alliot F, Rutin J, Leenen PJM, Pessac B (1999) Pericytes and periendothelial cells of the brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A and nestin. J Neurosci Res 58:367-378[Medline]

Arnold SE, Trojanowski JQ (1996) Human fetal hippocampal development: II. The neuronal cytoskleton. J Comp Neurol 367:293-307[Medline]

Ashraf Imam S, Young L, Chaiwun B, Taylor CR (1995) Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. Anticancer Res 15:1153-1158[Medline]

Clarke SR, Shetty AK, Bradley JL, Turner DA (1994) Reactive astrocytes express the embryonic intermediate neuofilament nestin. NeuroReport 5:1885-1888[Medline]

Dahl D (1981) The vimentin-GFA protein transition in rat neuroglia cytoskeleton occurs at the time of myelination. J Neurosci Res 6(6):741-748[Medline]

Dahl D, Reuger DC, Bignami A, Weber K, Osborn M (1981) Vimentin, the 57 000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia. Eur J Cell Biol 24(2):191-196[Medline]

Dahlstrand J, Collins P, Lendahl U (1992a) Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 52:5334-5341[Abstract]

Dahlstrand J, Lardelli M, Lendahl U (1995) Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of the developing central nervous system. Dev Brain Res 84:109-129[Medline]

Dahlstrand J, Zimmerman LB, McKay RDG, Lendahl U (1992b) Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments. J Cell Sci 103:589-597[Abstract/Free Full Text]

Doetsch F, Garcia–Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046-5061[Abstract/Free Full Text]

Evers P, Uylings HBM (1996) An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods 72:197-207

Finley JCW, Petrusz P (1982) The use of proteolytic enzymes for improved localization of tissue antigens with immunocytochemistry. In Bullock GR, Petrusz P, eds. Techniques in Immunohistochemistry. Vol 1. London, Academic Press, 239-249

Fliegner KH, Kaplan MP, Wood TL, Pintar JE, Liem RK (1994) Expression of the gene for the neuronal intermediate filament protein alpha-internexin coincides with the onset of neuronal differentiation in the developing rat nervous system. J Comp Neurol 342:161-173[Medline]

Flørenes VA, Holm R, Myklebost O, Lendahl U, Fodstad Ø (1994) Expression of the neuroectodermal intermediate filament nestin in human melanomas. Cancer Res 54:354-356[Abstract]

Frederiksen K, McKay RDG (1988) Proliferation and differentiation of rat neuroepithelial precursor cells in vivo. J Neurosci 8:1144-1151[Abstract]

Frisen J, Johansson CB, Török C, Riesling M, Lendahl U (1995) Rapid, widespread and long-lasting induction of nestin contributes to the generation of glial scar tissue after CNS injury. J Cell Biol 131:453-464[Abstract]

Grigelioniené G, Blennow M, Törrök C, Fried G, Dahlin I, Lendahl U, Lagercrantz H (1996) Cerebrospinal fluid of newborn infants contains a deglycosylated form of the intermediate filament nestin. Pediatr Res 40:809-881[Abstract]

Herrman H, Aebi U (2000) Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol 12:79-90[Medline]

Ho C-L, Liem RKH (1996) Intermediate filaments in the nervous system: implications in cancer. Cancer Metastas Rev 15:483-497[Medline]

Hockfield S, McKay RDG (1985) Identification of major cell classes in the developing mammalian nervous system. J Neurosci 5:3310-3328[Abstract]

Holmin S, Almqvist P, Lendahl U, Mathiesen T (1997) Adult nestin-expressing subependymal cells differentiate to astrocytes in response to brain injury. Eur J Neurosci 9(1):65-75[Medline]

Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J (1999) Identification of a neural stem cell in the adult mammalian central nervous sytem. Cell 96:25-34[Medline]

Khoddami M, Becker LE (1997) Immunohistochemistry of medulloepithelioma and neural tube. Pediatr Pathol Lab Med 17:913-925[Medline]

Kitamoto T, Ogomori K, Tateishi J, Prusnier SB (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 58(2):230-236

Kleihues P, Burger PC, Schleithauer BW (1993) Histological typing of tumours of the central nervous system. International Histological Classification of Tumours. Berlin, World Health O Springer-Verlag

Kobayashi M, Sjöberg G, Söderhäll S, Lendahl U, Sandstedt B, Sejersen T (1998) Pediatric rhabdomyosarcomas express the intermediate filament nestin. Pediatr Res 43:386-392[Abstract]

Kurpad SN, Zhao X-G, Wikstrand CJ, Batra SK, McLendon RE, Bigner DD (1995) Tumor antigens in astrocytic gliomas. Glia 15:244-256[Medline]

Lendahl U, Zimmerman LB, McKay RDG (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595[Medline]

Li Y, Chopp M (1999) Temporal profile of nestin expression after focal cerebral ischemia in adult rat. Brain Res 838:1-10[Medline]

Liem RKH (1993) Molecular biology of neuronal intermediate filaments. Curr Opin Cell Biol 5:12-16[Medline]

Lin RCS, Matesic DF, Marvin M, McKay RDG, Brustle O (1995) Re-expression of the intermediate filament nestin in reactive astrocytes. Neurobiol Dis 2:79-85[Medline]

Lothian C, Lendahl U (1997) An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to the CNS progenitor cells and to early neural crest cells. Eur J Neurosci 9:452-462[Medline]

Lothian C, Prakash N, Lendahl U, Wahlström GM (1999) Identification of both general and region-specific embryonic CNS enhancer elements in the nestin promoter. Exp Cell Res 248:509-519[Medline]

Marvin MJ, Dahlstrand J, Lendahl U, McKay RDG (1998) A rod end deletion in the intermediate filament protein nestin alters its subcellular localization in neuroepithelial cells of transgenic mice. J Cell Sci 111:1951-1961[Abstract/Free Full Text]

McKeever PE (1998) Insights about brain tumors gained through immunohistochemistry and in situ hybridization of nuclear and phenotypic markers. J Histochem Cytochem 46:585-594[Abstract/Free Full Text]

McQuaid S, McConnell R, McMahon J, Herron B (1995) Microwave antigen retrieval for immunohistochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue. J Pathol 176:207-216[Medline]

Meehan JT, Cutlip RC, Lehmkuhl HD (1989) Evaluation of ethylenediaminotetra-acetic acid, tetrasodium salt dihydrate (EDTA)-Tween 20 treatment versus protease digestion of formalin-fixed tissue sections for detection of bovine respiratory syncytial virus antigen in infected bovine lung. Vet Pathol 26:322-325[Abstract]

Mokry J, Nemecek S (1998) Immunochemical detection of intermediate filament nestin. Acta Med 41:73-80

O'Rahilly R, Müller F (1994) The Embryonic Human Brain. New York, Wiley–Liss

Redies C, Lendahl U, McKay RDG (1991) Differentiation and heterogeneity in T-antigen immortalized precursor cell lines from mouse cerebellum. J Neurosci Res 30:601-615[Medline]

Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci 20:570-577[Medline]

Sarnat HB (1992) Regional differentiation of the human fetal ependyma: immunocytochemical markers. J Neuropathol Exp Neurol 51:58-75[Medline]

Shi S-R, Cote C, Kalra KL, Taylor CR, Tandon AK (1992) A technique for retrieving antigens in formalin-fixed, routinely acid decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry. J Histochem Cytochem 40:787-792[Abstract/Free Full Text]

Shi S-R, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem 45:327-343[Abstract/Free Full Text]

Shi S-R, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]

Stagaard M, Mollgård K (1989) The developing neuroepithelium in human embryonic and fetal brain studied with vimentin-immunohistochemistry. Anat Embryon 180:17-28

Takano T, Becker LE (1997) Developmental change of the nestin-immunoreactive midline raphe glial structure in human brainstem and spinal cord. Dev Neurosci 19:202-209[Medline]

Takano T, Rutka JT, Becker LE (1996) Overexpression of nestin and vimentin in ependymal cells in hydrocephalus. Acta Neuropathol (Berl) 92:90-97[Medline]

Tohyama T, Lee VM-Y, Rorke LB, Marvin M, McKay RDG, Trojanowski JQ (1992) Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells. Lab Invest 66:303-313[Medline]

Williams JH, Mepham BL, Wright DH (1997) Tissue preparation for immunocytochemistry. J Clin Pathol 50:422-428[Abstract]

Zimmerman LZ, Lendahl U, Cunningham M, McKay RDG, Parr B, Gavin B, Mann J, Vassileva G, McMahon A (1994) Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron 12:11-24[Medline]