Laboratory Investigation

Up-regulation of neuropoiesis generating glial progenitors that infiltrate rat

intracranial glioma

Christopher Duntsch1, Qihong Zhou1, James D. Weimar1, Bruce Frankel1, Jon H. Robertson1 and TayebehPourmotabbed1,21Department of Neurosurgery; 2Department of Molecular Sciences, The University of Tennessee Health ScienceCenter, Memphis, TN, USA

Key words: glioblastoma, glioma, neural stem cell, neuropoiesis, subventricular zone

Summary

To investigate adult neural stem cell (NSC) biology in relation to glioma, the C6 glioma cell line was tagged withgreen fluorescent protein (GFP) and inoculated into the brain of adult rats. The in vivo biological response of thebrain to glioma was studied using immunohistochemical analysis of the subventricular zone (SVZ), peritumoralareas, and glioma. Nestin immunoreactive cells were found infiltrating glioma, but the distribution of abnormalimmunoreactivity was restricted to the dorsal and medial border of the tumor relative to the ipsilateral ventricle.The SVZ was found to be hypertrophic, hypercellular, and up-regulated nestin expression. Furthermore, a densecontiguous population of nestin immunoreactive cells could be found streaming from ipsilateral dorsal tip of theSVZ, tracking along the ventral margin of the corpus callosum, and fanning out to encompass and infiltrate theproximal tumor border. Although most cells were either nestin or glial fibrillary acidic protein (GFAP) immuno-reactive in the SVZ and along the ventral margin of the corpus callosum, the number of cells co-expressing bothmarkers increased proportionally as the tumor was approached so that the predominant cell population along theproximal tumor border was GFAP immunoreactive. Finally, we demonstrated that a significant proportion of cellsfound in areas of abnormal immunoreactivity were proliferating, especially in peritumoral areas. In summary, thereis an induction of neuropoietic activity in a rat intracranial glioma model that results in an infiltration andaccumulation of abnormal nestin and GFAP expressing cells with proliferative potential along the dorsal andmedial border of intracranial C6 glioma.

Introduction

The most prevalent form of adult primary CNS tumorsis collectively referred to as glioma, and the most com-mon and devastating glioma is glioblastoma multiforme(GBM) [1,2]. The definitive cellular origin of GBMsremains to be elucidated. Classically, oligodendroglio-mas are thought to arise from oligodendrocytes, astro-cytomas from astrocytes, with both generated secondaryto neoplastic events resulting in cellular proliferationand uncontrolled growth [3]. Because oligodendrocytesand astrocytes are differentiated cells with relativelylittle capacity to divide under normal conditions, it isdifficult to explain the number of oncogenic eventsrequired to achieve transformation. Furthermore, thedifficulty identifying truly pre-malignant lesions thatlead to glioma has precluded studies aimed at under-standing initial tumorigenic events [3].Although controversial, there is growing evidence to

support the hypothesis that adult neural stem cells(NSCs) play some role in the genesis, development, andgeneral biology of gliomas [4,5]. The intermediate fila-ment nestin is an accepted marker for NSCs and neuralprogenitors in the developing and adult mammalianCNS [6–8]. Nestin is sequentially replaced by otherintermediate filaments such as vimentin, microtubule-associated protein 2 (MAP-2), and GFAP during mat-uration and/or differentiation of neural precursor cells

[6,9,10]. Studies of NSCs and neural progenitors in theadult brain have revealed a population of proliferatingcells in the SVZ that express nestin and have tremendousmigratory ability [11–17].Reports that some CNS tumors may arise from NSCs

are beginning to emerge. Glial tumors have been shownto be associated with markers specific for undifferenti-ated neural cells [9,18]. Zhu et al. [19] and others haverecently suggested that gangliogliomas may arise frommultipotential stem cells which have the ability to dif-ferentiate into glial and neuronal lineages [20,21]. Dug-gal et al. [22] have suggested that this same phenomenonmay be a contributing factor in the development ofcortical dysplasias. These data collectively imply thatsome adult CNS tumors may arise from or interact insome way with NSCs.We have used a rat intracranial glioma model with a

fluorescent tag to study the activity and fate of intrinsicNSCs and progenitors in the context of glioma in adultrats. We demonstrate a significant accumulation of nes-tin and GFAP immunoreactive cells that are distributedasymmetrically along the dorsal and medial border ofintracranial glioma relative to the ventricle. We furtherdemonstrate up-regulation of neuropoiesis in the SVZ asindicated by increased nestin expression and cell density.Finally, we demonstrate that all areas of increased orabnormal nestin expression are significantly immunore-active for the proliferation marker Ki67. Thus, our data

Journal of Neuro-Oncology (2005) 71: 245–255 � Springer 2005

shows the novel and potentially important observationthat there is enhanced neuropoiesis in the presence ofintracranial glioma that may contribute to the overallpathophysiology of gliomas by generating abnormalglial cells that infiltrate tumor and have proliferativepotential. Further studies to evolve this model will leadto a better understanding of glioma biology and poten-tial new therapeutic options for treating GBMs.

Materials and methods

Cells and cell culture

The rat C6 glioma cell line (CCL-107, American TypeCulture Collection, Manassas, VA) used in these studieswas first induced by exposing Wistar–Furth rats toN,N¢-nitroso-methylurea [23] and has been described in detailelsewhere [23–30]. This glioma cell line was maintained asa monolayer culture at 37 �C, 5% CO2 in Hams/F12supplemented with 15% heat-inactivated horse serum(American Type Culture Collection, Manassas, VA),2.5% heat-inactivated fetal bovine serum (Hyclone, Lo-gan, UT), 100 i.u./ml penicillin, and 100 lg/ml strepto-mycin (Hyclone, Logan, UT). The C6 glioma cell line wasstably transduced with the pFB retrovirus (Stratagene,La Jolla, CA) expressing GFP to allow for enhancedvisual analysis. Cells stably transduced with GFP weresorted using flow cytometry to generate a cell populationhomogeneously expressing high levels of GFP. For gen-erating intracranial glioma, cells in exponential growthwere harvested by EDTA/Trypsin (Hyclone, Logan, UT)for 5 min at 37 �C. Trypsinization was stopped with thecomplete media described above and the cells were cen-trifuged for 5 min at 1000 rpm. The pellets were resus-pended in sterile phosphate buffered saline (PBS) at aconcentration of 1 · 104 ll)1 and placed on ice.

Generation of rat intracranial glioma models

Adult Sprague–Dawley rats 250–350 g in weight wereused for all studies. The animals were handled inaccordance with protocols approved by the Animal Careand Use Committee at the University of TennesseeHealth Science Center. Rats were anesthetized withintraperitoneal injection of ketamine/xylazine at a dos-age of 8.7/1.3 mg/100 g body weight. Rats were placedon a stereotaxic frame. An incision approximately 5 mmin length was made in the midline, and the sagital andcoronal sutures were located. A small burr hole wasmade with a handheld drill 3 mm lateral from midlinealong the bregma suture over the right hemisphere. PBSalone (sham-operated) or 1 · 105 GFP positive C6 gli-oma cells resuspended in PBS in a volume of 10 ll wereinjected over a period of 5 min at a depth of 5 mm fromthe brain surface using a Hamilton syringe (Hamilton,Reno, NV). Cells were kept in suspension during theexperiment using gentle pippetting. Once the needle wasremoved, a plug of bone wax was inserted into the burrhole. The area was thoroughly irrigated with sterilesaline and the wound was closed with sutures. Rats wereobserved daily for signs of infection, alertness, feeding

habits, and neurological deficits. The animals were sac-rificed at 2 weeks after tumor inoculation, or earlier ifsignificant neurological deficit was noted, by transcar-dial perfusion with 4% paraformaldehyde. The brainswere sectioned and prepared for histology and immu-nohistochemistry.

Hematoxylin–eosin staining

Rat brains were cut on a cryostat at 20 lm and collectedon clean slides. The sections were stored at 4 �C beforeprocessing. Hematoxylin–eosin (H&E) staining wasperformed using the following procedures. The sectionswere immersed in 95% ethanol and rehydrated beforebeing stained with 2% hematoxylin (245–655, FisherDiagnostics, Middletown, VA) for 1 min. After threerinses in water, the sections were treated with Scott’s tapwater substitute blueing solution (CS410-4, Fisher Sci-entific, Pittsburgh, PA). After a brief rinse in water, thesections were dehydrated in 95% ethanol for 1 min andstained with eosin (LC14030-2, LabChem, Inc., Pitts-burgh, PA) for 3 min. The sections were coverslippedafter dehydration in graded ethanol and xylene.

Immunohistochemistry

The primary antibodies used in the experiments wereobtained from the following sources: mouse anti-nestin(556309, BD Biosciences, San Diego, CA), rabbit anti-GFAP (G 9269, Sigma-Aldrich Corp., St. Louis, MO),and mouse anti-Ki67 (MAB4062, Chemicon Interna-tional, Temecula, CA). The immunohistochemistry wasperformed using the following procedures. The frozenbrain sections were (1) quenched with 3% hydrogenperoxide and 10% methanol for 20 min with subsequentrinses with PBS; (2) incubated in 2% non-fat milk and0.3% Triton-X in PBS for 1 h; (3) incubated in antibodyof interest at the following dilutions: nestin 1:100, GFAP1:400, or Ki67 1:100, with 3% donkey serum and 0.1%Triton-X overnight at room temperature (for negativecontrols, the sections were incubated with normal mouseor rabbit serum); (4) washed with PBS for three times; (5)incubated in appropriate Cye3 or 5-conjugated Affini-Pure donkey anti-mouse or rabbit IgG (1:250, JacksonImmunoResearch Laboratories, Inc., West Grove, PA)with 2% donkey serum and 0.1% Triton-X for 4 h indark at room temperature; (6) washed with PBS for threetimes and dehydrated through graded ethanol, clearedwith xylene, and coverslipped with DPX mountingmedium (44581, Fluka Biochemika). The immunoreac-tivity was visualized with a Bio-Rad confocal microscopeand images collected on a computer for later analysis.

Results

Nestin immunoreactive cells infiltrate the dorsal andmedial border of rat intracranial C6 glioma

In order to distinguish C6 glioma from normal CNS,GFP was stably introduced into the rat C6 glioma cell

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line using retroviral strategies. Figure 1A shows a rep-resentative H&E coronal section of a rat intracranial C6glioma. Figure 1B and C show low and high-powerfluorescent micrographs demonstrating the GFP fluo-rescence of tumor cells. Figure 2A shows a coronal mapof the brain with the structures studied below labeled inrelation to the ventricle and tumor body. We firstinvestigated whether nestin immunoreactive cells couldbe found surrounding or infiltrating tumor. As shown inFigure 2, a significant population of nestin immunore-active cells (red) could be seen accumulating at the tumorborder (Figure 2D, demarcated by green fluorescence)and infiltrating the tumor itself (Figure 2C). However,nestin expression was anatomically restricted to thedorsal and medial border of the tumor relative to theipsilateral ventricle (Figure 2D, area 5 from Figure 2A).

Indeed, nestin expression along the rest of the tumorborder was low to undetectable as shown in a represen-tative photomicrograph from area 6 of Figure 2A (Fig-ure 2E). Nestin immunoreactivity in similar brainparenchymal areas of rats inoculated with sterile PBS(sham-operated group) or healthy adult rats could not bedetected (Figure 2B and data not shown). Althoughweak and sporadic nestin expression was detected in theC6 glioma cells, the majority of nestin immunoreactivecells were non-GFP expressing cells indicating that theyoriginated from the animal and not from the tumor. Insummary, a significant population of nestin expressingcells infiltrate intracranial C6 glioma along the dorsaland medial tumor border.

The SVZ is hypertrophic, hypercellular and up-regulatesnestin in rats with intracranial C6 glioma

The degree of nestin immunoreactivity observed alongthe proximal glioma border relative to the ipsilateralventricle prompted us to look at nestin expression in theSVZ of rats with intracranial C6 glioma. As shown inFigure 3A and B (area 2 and 1, respectively, from thecoronal map in Figure 2), the SVZ of rats with intra-cranial C6 glioma demonstrated enhanced nestinimmunoreactivity which was especially striking at thedorsal tip of the ipsilateral ventricle when compared tosham-operated and healthy adult rats (Figure 3D and Eand data not shown). H&E staining of the areas ofincreased nestin immunoreactivity indicated that areasof increased nestin expression correlated with increasedcellular density as well when compared to sham-oper-ated and healthy adult rats (Figure 3C and F and datanot shown). Thus, the presence of intracranial C6 gli-oma causes increased expression of nestin and hyper-cellularity in the SVZ that is especially prominent alongthe dorsal portion of the lateral ventricle.

Nestin immunoreactive cells track along the ventralcorpus callosum border and merge with the proximalglioma border

Having demonstrated increased nestin expression in theSVZ and along the proximal tumor border relative to theipsilateral ventricle, we investigated nestin immunore-activity in the areas between the ventricle and tumor. Asshown in Figure 4A (area 2 from the coronal map inFigure 2), a significant amount of nestin immunoreac-tivity could be found streaming from the SVZ at thedorsal tip of the lateral ventricle. Although nestinexpressing cells were found in a similar distribution insham-operated rats and healthy adult rats, the densityand degree of nestin immunoreactivity was much greaterin the presence of tumor (compare Figure 4A with G).Furthermore, a contiguous pattern of nestin immuno-reactivity could be detected along a path from the dorsalportion of the lateral ventricle SVZ to the tumor runningroughly along the ventral border of the corpus callosum(Figure 4B, area 3 from the coronal map in Figure 2).Nestin immunoreactive cells were detected in this area insham-operated rats as well, but again the density ofimmunoreactivity was much less impressive (compare

Figure 1. The rat C6 intracranial glioma model. (A, B), Photomicro-

graphs of coronal sections of the rat C6-GFP intracranial glioma

model. (A) H&E staining (1.25·) demonstrating tumor growth in the

right hemisphere that has compressed the ventricles and pushed the

midline toward the contralateral hemisphere. (B) Fluorescent micros-

copy (1.25·) of the same animal demonstrating strong fluorescence of

the tumor body. (C) Fluorescent confocal microscopy (60·) demon-

strating fluorescence of individual cells in the C6 tumor body.

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Figure 4B with H). Interestingly, as the nestin immu-noreactive cells approached the tumor, the distributionof nestin-expressing cells intensified, spread out, andmerged with the C6 glioma along its dorsal and medialborder (Figure 4C, area 4 from the coronal map inFigure 2, also see Figure 2D). Very little nestin expres-sion was detected in these areas in sham-operated andhealthy adult rats (Figure 4I and data not shown). Asdemonstrated previously in Figure 3, the distribution ofnestin immunoreactivity correlated well with increased

cell density as demonstrated by H&E analysis (Fig-ure 4D–F versus J–L).

A significant population of GFAP immunoreactive cellsinfiltrate the dorsal and medial border of rat intracranialC6 glioma and co-express nestin

To better define the fate of nestin immunoreactive cellsaccumulating on the proximal border of intracranial C6glioma, immunohistochemical markers of various glial

Figure 2. Nestin immunoreactivity in intracranial glioma and peritumoral areas. (A) A coronal drawing of the rat brain is shown for anatomical

reference: V: ventricle; Tu: tumor; (1) lateral border of the ventricle; (2) dorsal tip of the lateral ventricle; (3) corpus callosum; (4) transition area

between the glioma and the ventricle or corpus callosum; (5) proximal tumor border relative to the ventricle; and (6) distal tumor border relative

to the ventricle. (B) There is little nestin immunoreactivity in a sham-operated rat in the same area of tumor inoculation used for experimental

rats. Photomicrographs of nestin immunoreactivity taken from the center of the tumor (C), proximal tumor edge (D, area 5 in the coronal map),

and distal tumor edge (E, area 6 in the coronal map) relative to the ipsilateral ventricle demonstrating that there is striking accumulation of nestin

immunoreactive cells along the proximal tumor border, but little abnormal cellular activity along the distal tumor border. Green: C6-GFP tumor

cells. Red: nestin immunoreactivity.

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and neuronal lineages (O1, oligodendrocytes; MAP-2,neurons; and GFAP, astrocytes) were used to determinewhat cell types were present around the tumor. Al-though MAP-2 and O1 immunoreactivity were detectedin a normal distribution in healthy areas of brain, nei-ther marker could be detected in the peritumoral areaswhere we found nestin immunoreactivity to be signifi-cantly up-regulated (data not shown). However, GFAPwas detected and cells expressing GFAP appeared to bethe predominant cell population surrounding and infil-trating tumor in these areas, aggregating in a dense,scar-like distribution. Furthermore, the distribution ofGFAP immunoreactivity around the tumor paralleledthat of nestin, being found mainly along the dorsal andmedial border of the tumor relative to the ipsilateralventricle (Figure 5A and B, area 6 and 5, respectively,from the coronal map in Figure 2). Although someGFAP immunoreactivity could be detected along theventral corpus callosum border as previously demon-strated in Figure 4B for nestin (Figure 5C, area 3 fromthe coronal map in Figure 2), the areas closer to thetumor where the nestin staining was more diffuse dem-onstrated intense GFAP immunoreactivity (Figure 5D,area 4 from the coronal map in Figure 2). GFAPimmunoreactivity in sham-operated rats from a similararea appeared normal and is shown in Figure 5E and Ffor comparison.Having observed a similar abnormal presence and

asymmetric distribution of nestin and GFAP expressingcells in the brain of rats with intracranial C6 glioma, weinvestigated whether any of these cell populations co-expressed both markers. Interestingly, the predominantpopulation of cells in the SVZ at the dorsal ventricular

tip (Figure 6A, area 2 from the coronal map in Figure 2)and in the nestin immunoreactive stream along theventral margin of the corpus callosum first described inFigure 4B (Figure 6B, area 3 from the coronal map inFigure 2) were found to be either nestin or GFAPimmunoreactive, but not both. However, as the streamapproached and spread out around the proximal tumorborder, the proportion of cells that co-expressed nestinand GFAP increased significantly (Figure 6C and D,area 4 and 5, respectively, from the coronal map inFigure 2). Isotype matched serum controls using eithermouse (Figure 6E) or rabbit (Figure 6F) were takenfrom the proximal tumor border where nestin andGFAP immunoreactivity was the strongest and clearlyindicate a lack of non-specific binding.

Demonstration of significant proliferative activity in areasof abnormal nestin and GFAP immunoreactivity

The density of abnormal cellular activity focused alongthe proximal tumor border relative to the ipsilateralventricle led us to speculate that there may be abnormalproliferative activity by non-glioma cells in these areas.We used the proliferation marker Ki67 to delineatepossible areas of abnormal proliferative activity in thebrain of rats with intracranial C6 glioma. As shown inFigure 7, the dorsal tip of the SVZ of the lateral ven-tricle (area 2 from the coronal map in Figure 2) hadsignificant Ki67 immunoreactivity in rats with intra-cranial C6 glioma (Figure 7A), sham-operated rats(Figure 7D), and healthy adult rats (data not shown).However, a significant population of Ki67 staining cellscould only be demonstrated in rats with intracranial C6

Figure 3. Up-regulation of nestin in the SVZ of the lateral wall of the ventricle in rats with intracranial glioma. Confocal photomicrographs of

nestin immunoreactivity are shown for the SVZ of the dorsal tip (A, area 2 in the coronal map from Figure 2) and lateral wall (B, area 1 from the

coronal map in Figure 2) of the lateral ventricle in rats with intracranial glioma. Nestin immunoreactivity in similar areas (D, dorsal tip of lateral

ventricle; and E, lateral wall of the lateral ventricle) in sham-operated rats is shown for comparison. A robust increase in nestin immunoreactivity

was observed in the SVZ along the lateral wall of the ventricle relative to the sham-operated controls that was especially striking in the dorsal

portion of the lateral ventricle. C and F show the H&E staining of the same areas shown in A and D, respectively, and demonstrate that areas of

increased nestin immunoreactivity correlated with increased cell density. V: ventricle.

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glioma along the ventral border of the corpus callosum(Figure 7B, area 3 from the coronal map in Figure 2), inthe area between the ventricle and tumor (Figure 7C,area 4 from the coronal map in Figure 2), and along theproximal tumor border (Figure 7G, area 5 from thecoronal map in Figure 2). The degree of Ki67 immu-noreactivity was low to undetectable in these areas insham-operated rats and healthy adult rats (Figure 7Fand data not shown). In summary, there is a direct

correlation of significant proliferative activity with areasof abnormal nestin and GFAP immunoreactivity in ratswith intracranial C6 glioma.

Discussion

High-grade gliomas are the most common and devas-tating of all primary brain tumors. The prognosis for

Figure 4. Nestin immunoreactive cells are distributed along the ventral border of the corpus callosum and in areas between the corpus callosum

and tumor in rats with intracranial C6 glioma. Fluorescent photomicrographs of nestin immunoreactivity streaming from the SVZ of the dorsal

tip of the lateral ventricle (A, area 2 in the coronal map from Figure 2) and demonstrating these cells track in a uniform manner along the ventral

border of the corpus callosum (B, stream is indicated by arrow, area 3 in the coronal map from Figure 2). Nestin expression in the area between

the tumor and the corpus callosum (C, area 4 in the coronal map from Figure 2). Nestin expression spreads out from the stream identified in B as

the tumor is approached and eventually merges with the proximal tumor border (as demonstrated in Figure 2D). (D–F) H&E staining of the same

areas shown in A–C, respectively, demonstrating that areas of increased nestin immunoreactivity correlate with increased cell density. G–I and J–

L demonstrating nestin immunoreactivity and H&E staining, respectively, in sham-operated controls in the areas corresponding to A–C. These

photomicrographs demonstrate that although nestin expression is present in the sham-operated controls, it is much less, and associate with a

lower cell density in the absence of tumor. V: ventricle.

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patients diagnosed with high-grade gliomas has notimproved significantly over the last 40 years [1,2].Clearly a better understanding of the biological mech-anisms critical to glioma biology is a prerequisite todeveloping more effective means of diagnosis andtreatment. We have used a rat intracranial glioma modelto investigate cellular activity in the SVZ and paren-chymal brain tissue in response to developing glioma.We observed that nestin immunoreactive cells infiltratedintracranial C6 glioma (Figure 2). In a striking displayof polarized and specific abnormal cellular distribution,the majority of nestin immunoreactivity was localizedanatomically along the dorsal and medial border of thetumor relative to the ipsilateral ventricle (Figure 2).The dorsal portion of the lateral ventricular SVZ

ipsilateral to the tumor was found to be hypertrophic,hypercellular, and up-regulated the expression of nestin(Figure 3). Furthermore, we observed a contiguous andanatomically specific pattern of nestin immunoreactiv-

ity that streamed from the SVZ at the dorsal tip of theventricle and tracked along the ventral margin of thecorpus callosum towards the tumor, fanning out toeventually merge with the dorsal and medial tumormargins (Figure 4). One possible explanation is thatnestin immunoreactive cells originating in the SVZ aremigrating towards the tumor along predefined micro-anatomical intracranial pathways of cell transit. Theseobservations implicate a potential in vivo inherent tro-pism of these cells for glial tumors. Such a finding isconsistent with Aboody et al. [11] who previouslydemonstrated that NSCs implanted heterotopically intorats with intracranial glioma migrated towards andinfiltrate throughout the tumor bed.Assuming that nestin-immunoreactive cells found

infiltrating the tumor are part of a biological response ofthe brain to the presence of tumor, we investigatedvarious neural lineage markers and found that the pre-dominant cell population infiltrating the tumor was glial

Figure 5. GFAP immunoreactivity in intracranial glioma and peritumoral areas. Fluorescent photomicrographs demonstrate that there is an

uneven distribution of GFAP immunoreactivity with much greater cell density and expression found along the proximal tumor border (B) than

the distal tumor border (A) relative to the ipsilateral ventricle (area 6 and 5, respectively, from the coronal map in Figure 2). GFAP immu-

noreactivity along the ventral border of the corpus callosum (C, indicated by arrow, area 3 from the coronal map in Figure 2) and near the

proximal tumor border (D, area 5 from the coronal map in Figure 2) in similar areas where nestin immunoreactivity was previously demonstrated

(Figures 2 and 4). E and F correspond to C and D, respectively, and show GFAP immunoreactivity in sham-operated controls for comparison.

Green: C6 tumor cells; Red: GFAP immunoreactivity.

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and more specifically, GFAP positive cells (Figure 5).These cells were arranged densely in a scar-like distri-bution that correlated well with that of nestin immu-noreactivity, being asymmetrically distributed along theproximal border of the tumor. Despite the overlap ofnestin and GFAP expression in peritumoral areas andbrain parenchyma situated between the ventricle, corpuscallosum, and tumor, the only cell populations thatsignificantly co-expressed nestin and GFAP were foundin closer proximity to the tumor’s dorsal and medialborder (Figure 6). However, we did observe that the

proportion of cells co-expressing each marker increasedinversely with the distance from the tumor (Figure 6).Finally, virtually all areas of abnormal nestin andGFAP expression exhibited significant Ki67 immuno-reactivity indicating that continuous proliferative activ-ity is occurring in these areas. One explanation for ourimmunoreactive findings might be explained by theinfiltration of the tumor by macrophages as recentlydescribed by Dutta et al. [31] with up-regulation of cellsurface PC receptor resulting in indiscriminate binding.However, arguments against that explanation include:

Figure 6. Immunohistochemical analysis of co-expression of nestin and GFAP in cell populations in the SVZ and peritumoral area. Fluorescent

photomicrographs showing nestin (red) and GFAP (blue) immunoreactivities in the SVZ of the dorsal tip of the lateral ventricle (A, area 2 from

the coronal map in Figure 2), ventral margin of the corpus callosum (B, stream is indicated by arrow, area 3 from the coronal map in Figure 2),

between the corpus callosum and tumor (C, area 4 from the coronal map in Figure 2), and at the proximal tumor border (D, area 5 from the

coronal map in Figure 2). Each figure is followed by an overlay for either nestin or GFAP to allow for differentiation of single expression of a

given marker from co-expression of both markers. Most cells streaming from the SVZ area of the dorsal tip of the lateral ventricle and tracking

along the ventral border of the corpus callosum appear to express either nestin or GFAP. However, the proportion of cells co-expressing each

marker increases inversely with the distance from the tumor. Isotype matched serum controls with either mouse (E) or rabbit (F) are shown at the

proximal tumor border where both nestin and GFAP staining were the strongest.

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(1) the lack of non-specific binding of MAP-2, O1, andcontrols (isotype and secondary antibody controls); and(2) the neuroanatomically restricted and specific patternof immunoreactivity demonstrated by nestin, GFAP,and Ki67 in both unique and overlapping patterns.These results are summarized in Table 1.Repair processes following brain injury are aimed at

recovery of tissue integrity and isolation of intact areasfrom surrounding damaged or necrotic tissue [32]. Thenestin immunoreactivity and dense, almost scar-liketissue architecture formed along the proximal tumorborder by GFAP immunoreactive cells in our ratintracranial C6 glioma model is reminiscent of the gli-osis occurring in ischemic or traumatic damage to braintissue [33–37]. With CNS tissue damage, astrocytesaccumulate at the site of injury, form a scar with char-acteristic reactive morphology, and undergo a variety offunctional changes including up-regulation of nestin sothat it is co-expressed with GFAP [32–40]. However, it isnot clear at this time whether in situ differentiated as-trocytes or migratory progenitor glial populations giverise to the scar-forming cells [41].Despite some similarities of our observations to

those previously described in the literature regardingnestin and GFAP staining patterns in ischemic andmechanical neurotrauma [33–37,40], there are alsoimportant differences. First, the collective biologicalresponse in our rat model of glioma is striking when

compared to that seen in ischemic and mechanicalneurotrauma models. Second and as described above,the polarized and anatomically restricted nature of theresponse, as well as its contiguous pattern between thetumor and the SVZ is not supportive of in situ pro-cesses alone. Our observations indicate that along anexpanding circular tumor border that is juxtaposed tobrain tissue (and thus theoretically inducing tissuedamage), only about a third of that tumor border (thedorsal and medial tumor border relative to the ipsi-lateral ventricle) demonstrates abnormal cellularactivity and immunoreactivities. Indeed, cell popula-tions in brain tissue interfacing with the tumor alongthe rest of its border are relatively quiescent and im-munostain for nestin, GFAP, MAP-2, and O1 in amanner similar to that seen in healthy adult rats. Insummary, the presence of nestin and/or GFAPexpressing cells with proliferative activity surroundingand infiltrating the tumor along its dorsal and medialborder are more consistent with nestin progenitors thatare migrating towards tumor from the SVZ and com-mitting to a glial fate, rather than in situ mature glialcells de-differentiating and up-regulating nestin so thatit is co-expressed with GFAP.Although it is possible that the collective cellular

activity observed in these studies are neurobiologicalprocesses occurring in response to brain tissue damagefrom developing glioma, there are still important con-

Figure 7. Expression of the proliferation antigen Ki67 in areas of abnormal nestin and GFAP immunoreactivity. Ki67 immunoreactivity is found

streaming from the dorsal SVZ tip (A, area 2 from the coronal map in Figure 2) and is also distributed along the ventral border of the corpus

callosum (B, stream is indicated by arrow, area 3 from the coronal map in Figure 2) in rats with intracranial C6 glioma. (C) Ki67 immunore-

activity in areas between the tumor and corpus callosum where nestin and GFAP immunoreactivity was shown to be more intense and diffusely

distributed (area 4 from the coronal map in Figure 2). D–F are taken from sham-operated controls and correspond to the same areas shown in

A–C, respectively. (G) Ki67 immunoreactivity along the proximal tumor border (arrows indicate the tumor border). Although Ki67 was also

found in the SVZ of the dorsal tip of the lateral ventricle in sham-operated controls, the immunoreactivity was much greater along the ventral

margin of the corpus callosum (E, compare to B) and in peritumoral areas (F, compare to C and G) in rats with intracranial glioma.

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siderations to be addressed. It appears that as the tumordevelops and infiltrates normal tissue, a reactive re-sponse may be generated enhancing activity in the SVZand giving rise to nestin-immunoreactive progenitorsthat invade the tumor and replace damaged tissue withglial scar. The degree of active proliferation in areas ofabnormal nestin and GFAP immunoreactivity occurringalong the proximal border relative to the ipsilateralventricle is an important observation. It begs the ques-tion of whether these cells may contribute somehow tooverall mass effect of the developing glioma either bytheir physical presence or in more indirect ways such asthe release of pro-inflammatory factors that are growth-inducing and angiogenic. Thus, it is possible that somespecific aspects of glioma biology may be driving theseprocesses or be driven by them. This is especially truewhen one considers that the most obvious differencebetween the biological processes of mechanical orischemic neurotrauma and those of neurotrauma in-duced by glioma is that with respect to the latter thereare malignant cells that can be impacted by the brain’sbiological response to the tissue damage. Thus, therealization that these biological phenomena are occur-ring is an important contribution of this work not pre-viously described and emphasizes the need for furtherresearch into the impact of these phenomena on gliomadevelopment.Despite the lack of information about early processes

in glioma development, this study does significantlycontribute to a better understanding of the relationshipbetween glial tumors and adult NSC biology after thetumor is established and actively developing. This studyargues that glioma and/or the response of brain tissue todeveloping glioma communicates in some manner withthe SVZ and surrounding tissue, possibly via solublefactors and adhesion proteins that enhance NSC activ-ity, chemo-attract NSCs, and commit these cells towardsa glial fate. Identification of these soluble factors andadhesion proteins will be a key component to under-standing these processes and the focus of future studies.Regardless of whether the activity we have observed isfrom glioma specific biological processes (i.e., secretionof growth factors, etc.), or a generic response to gliomaas a continuously developing trauma, the inflammatory/reactive response of the brain to the glioma may notonly promote glioma growth and development, but alsomake therapeutic attempts at eradication more difficult.Understanding the nature of the response of the brain toglioma and the impact of that response on glioma

development and treatment will be paramount and thefocus of future studies.

Acknowledgements

This work was supported by a research grant from theMethodist Foundation, Memphis, Tennessee. We thankDr. Xiaofei Wang, Mariya Nazarova, and DwightBordelon for their technical assistance. We also thankMrs. Tricia Weimar for her creative support and medi-cal illustrations.

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Transition areab 4 ++ ++ ++

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Address for offprints: Christopher Duntsch, Department of Neuro-

surgery, The University of Tennessee Health Science Center, 847

Monroe Avenue, Suite 427, Memphis, TN 38163, USA; Tel.: +1-901-

448-4493; Fax: +1-901-448-8468; E-mail: duntsch@bellsouth.net

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