Journal of NeuroVirology, 7: 501±510, 2001c° 2001 Taylor & Francis ISSN 1355±0284/01 $12.00+.00

Activation of microglia cells is dispensablefor the induction of rat retroviral spongiformencephalopathy

Regine Hansen,1 Christian Sauder,2 Stefanie Czub,3 Eva Bachmann,3 Simone Schimmer,1 Annette Hegyi,

1

and Markus Czub1

1Institut fur Virologie und Immunbiologie, Universitat Wurzburg, Germany; 2Abteilung Virologie, Institut furMedizinische Mikrobiologie und Hygiene der Albert-Ludwigs-Universitat Freiburg, Germany; and 3PathologischesInstitut, Universitat Wurzburg, Germany

In the course of retroviral CNS infections, microglia activation has beenobserved frequently, and it has been hypothesized that activated microgliaproduce and secrete neurotoxic products like proin�ammatory cytokines, bythis promoting brain damage. We challenged this hypothesis in a rat modelfor neurodegeneration. In a kinetic study, we found that microglia cells of ratsneonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40became infected in vivo to maximal levels within 9–13 days postinoculation(d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolizationof neurons and neuropil were found in cerebellar and other brain-stem nu-clei. Elevated numbers of activated microglia cells—as revealed by immuno-histochemical staining with monoclonal antibody ED1—were �rst detected at19 d.p.i. and were always locally associated with degenerated areas but notwith nonaltered, yet infected, brain regions. Both neuropathological changesand activated microglia cells increased in intensity and numbers, respectively,with ongoing infection but did not spread to other than initially affected brainregions. By ribonuclease protection assays, we were unable to detect differ-ences in the expression levels of tumor-necrosis-factor-® (TNF-®), interleukin-1¯ (IL-1¯), and interleukin-6 (IL-6) in microglia cells nor in total brains frominfected versus uninfected rats. Our results suggest that the activation of mi-croglia in the course of MuLV neurodegeneration is rather a reaction to, andnot the cause of, neuronal damage. Furthermore, overt expression of the proin-�ammatory cytokines TNF-®, IL-1¯, and IL-6 within the CNS is not requiredfor the induction of retroviral associated neurodegeneration in rats. Journal ofNeuroVirology (2001) 7, 501–510.

Keywords: spongiform encephalopathy; microglia; activation; retrovirus;neurodegeneration; cytokines

Introduction

Microglia cells are ubiquitous within the central ner-vous system (CNS) parenchyma and comprise 5 to20% of all glia cells (Lawson et al, 1991). Althoughthe function of resting microglia is still unknown,

Address correspondence to Markus Czub, Canadian ScienceCentre for Human and Animal Health, Pathogenesis of biosafetylevel 4 agents, 1015 Arlington Street, Winnipeg, MB, R3E 3M4,Canada. E-mail: markus czub@hc-sc.gc.ca

Received 27 January 2001; accepted 17 July 2001.

microglia cells transform rapidly from a resting toan activated state under patho-physiological con-ditions. This activation-associated transformationis characterized by overt morphological and im-munophenotypical changes (reviewed by Streit

et al,

1999). Activated microglia cells are the centralcellular element in the brain to initiate defensemechanisms against exogenous and endogenousnoxae and to facilitate regenerative processes (Lotanand Schwartz, 1994; Rabchevsky and Streit, 1997).However, activated microglia cells are also able todamage neighboring cells, especially neurons, bysecreting potentially cytotoxic molecules including

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free radicals, nitrogen oxides (Boje and Arora, 1992;Chao et al, 1992; Merrill et al, 1993), proteases,arachidonic acid, platelet-activating factor, quinoli-nate, cysteine, proin�ammatory cytokines such asinterleukin-1 (IL-1), interleukin-6 (IL-6), and tumor-necrosis-factor-® (TNF-®). Microglia-derived neuro-toxicity was demonstrated in a variety of experimen-tal and natural encephalopathies (Benveniste, 1997;Oleszak et al, 1997; Zujovic et al, 2000).

Microglia cells are the major target cells for human(Dickson et al, 1991; Brinkmann et al, 1992; Dicksonet al, 1994) and simian immunode�ciency virus(HIV, SIV) (Brinkmann et al, 1993; Czub et al,1996) in the brain. However, the mechanistic linkbetween retroviral CNS infection and neurologicaldeterioration, like the AIDS dementia complex,is unclear. A widely regarded hypothesis is thatmicroglial activation plays a signi�cant role in medi-ating retrovirus-induced disease. This hypothesis ismainly based on in vitro studies showing increasedlevels of proin�ammatory cytokines released frommicroglia cell cultures (Sopper et al, 1996), probablyinduced by retroviral gene products (Dawson et al,1993; Toggas et al, 1994; Kong et al, 1996). Recentinvestigations on brain material from HIV-infectedpatients demonstrated increased levels of TNF-®gene products in the CNS of patients with HIVdementia in comparison to HIV-infected patientswithout neurological impairment (Tyor et al, 1992;Seilhean et al, 1997; Wesselingh et al, 1997). Thissuggests that TNF-® may participate in the develop-ment of neurodegenerative disease. However, formalproof that activated microglia cells really cause thedevelopment of neurodegenerative lesions and/orclinical signs in patients with HIV dementia or otherneurodegenerative diseases such as Alzheimer’sDisease or multiple sclerosis, is lacking.

Microglia cells are the major target cells forneurovirulent murine leukemia viruses (MuLV) inthe brain (Baszler and Zachary, 1990; Lynch et al,1991; Czub et al, 1995). The infection of susceptiblemice and rats with neurovirulent MuLV serves as asimple and well-de�ned animal model for analyzingthe molecular causes leading to the developmentof retroviral induced neurodegeneration in vivo(reviewed in Portis and Lynch, 1998). MuLV-NT40belongs to a group of neurovirulent ecotropic C-typeleukemia viruses that cause a progressive, nonin�am-matory spongiform encephalopathy predominantlyof brain-stem areas when inoculated into susceptibleneonatal rats. The disease is not accompanied byimmunopathological phenomenons and proceedsregularly with regard to the incidence of neurologi-cal disease, the incubation period and neurologicalsigns. Neurological signs are manifested as ataxiaand paresis, which progress to hindlimb paralysesaccompanied by the atrophy of the skeleton muscles(Czub et al, 1995). The aim of our investigationon retroviral-induced neurodegeneration was toelucidate whether microglial activation and the

release of proin�ammatory cytokines (IL-1¯ , IL-6,and TNF-®) were involved in the induction ofretrovirus-associated neurodegeneration.

Results

The role of microglia activation in the pathogenesisof MuLV-NT40-induced neurodegeneration was in-vestigated by a kinetic analysis on the chronologicalcourse of retroviral CNS infection, microglia activa-tion, histopathological CNS lesions, and of clinicalsigns.

Dynamics of viral envelope protein expressionin the CNS By means of viral envelope-speci�cimmunohistochemistry (IHC), immunopositive cellswere �rst detected at 8 days postinfection (p.i.) in theCNS. Immunopositive cells had an elongated nucleusand were associated with blood vessels, indicatinginfection of endothelial cells. At later stages, ram-i�ed (beginning at 11 days p.i.) and activated (be-ginning at 19 days p.i.) microglia cells as de�nedby their morphology (Figure 2A–C, G–H) and bydouble-labeling (Figure 2M) were also found to beinfected. Immunopositive cells increased in numberbetween day 9 p.i. and day 19 p.i., reaching maxi-mum levels at 19 days p.i. (379 cells/0.8 mm2, nD 2).The number of immunopositive cells persisted atthis level until the last day of quanti�cation wasperformed (36 day p.i.: 356 cells/0.8 mm2, nD 2)(Figure 1).

Time course of lesion development Neuropathol-ogy induced by MuLV-NT40 is characterized by ex-tensive vacuolar degeneration within the neuropiland in neurons, predominantly of brain-stem areas(Czub et al, 1995) including the deep cerebellar nu-clei, the vestibulares nuclei, the cochleares nuclei,the reticular formation of the mesencephalon andmetencephalon, and parts of the corpus callosum.There were some notable regions that were spared bydegenerative changes. These included the cerebellarcortex, the hippocampus, the olfactory bulb, and thecortex parietalis and perirhinalis of the cerebrum. In-�ammatory changes were never detected.

Clear evidence of spongiform degeneration was�rst noticed in one of �ve rats at 13 days p.i. Evenat this early timepoint, evidence of spongiform de-generation was observed in virtually all brain regionsthat were also affected in advanced stages of disease.In general, the vacuoles were heterogenous in dimen-sion and varied from round to ovoid. Often, vacuoleswere divided by thin membranes (Figure 2). In brainregions where spongiform lesions had occurred early,the intensity of spongiform lesions (see Materials andmethods) increased in parallel with time. Maximalintensity of brain lesions was reached at 25 days p.i.(Figure 1).

Microglia activation Activated microglia cells asassessed by ED1 immunostaining were rarely foundin noninfected control rats at any time point (data

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R Hansen et al 503

Figure 1 Kinetic analysis of MuLV-NT40 infection. RetroviralCNS infection (upper panel), induction of histopathological le-sions (second upper panel), activation of microglia cells (secondlower panel), and inauguration of neurologicalsigns (lower panel),were analyzed on individual rats in a time-course manner. Impor-tant time points in the pathogenesis of MuLV-induced neurode-generation are represented by vertical lines in red: at 13 days p.i.(left red line), we noticed a widespread expression of retroviralgene products in de�ned brain regions, such as deep nuclei cere-bellares, nuclei cochleares, nuclei vestibulares, nucleus facialis,and cortex cerebri (upper panel). At this time point, we observedhistopathologicallesions in only one of �ve animals (second upperpanel) and no increased numbers of activated microglia cells (sec-ond lower panel; horizontal dotted line indicates cutoff betweennormal and elevated numbers of acivated microglia cells). Also,clinical signs were not observed (lower panel). Histopathologicallesions reached maximum levels at 25 days p.i. (right red line),which was accompanied by few activated microglia cells. At thistime point, we recognizedneurological signs in some MuLV-NT40-infected rats. Until the end of the observation period (36 days p.i.),numbers of activatedmicroglia cells and the extent of neurologicalsigns increased continously. Each symbol represents data from anindividual animal.

not shown). In brains of MuLV-NT40-infected rats, wealso noticed a few ED-1-positive cells between day 8and 17 p.i., however, there was no difference in thenumbers of activated microglia cells in noninfectedversus infected rat brains up to 18 days p.i. Begin-ning at day 19 p.i., we found a progressive increaseof activated microglia cells until the last time pointanalysed (Figure 1, Figure 2D–F, I–K).

No activation of microglia cells in the cortexcerebri Microglia activation seemed to be an eventrather in response to the histopathological brain le-sions but not a direct consequence of retroviral in-fection. To further elucidate this point, we examinedthe parietal and perirhinal cortex cerebri, i.e., brainregions that are spared from spongiform lesions, butnot from retroviral infection of endothelial and mi-croglia cells (Figure 2H). Activation of microglia cellswas not observed in these areas (Figure 2K). Thus, toactivate microglia cells retroviral infection alone wasnot suf�cient but needed to be accompanied by obvi-ous brain damage.

Clinical signs and incidence of neurologicaldisease The incidence of neurological disease wasdetermined in 64 rats for a time period between22 and 45 days p.i. First clinical signs were re�exabnormalities of the hind- and forelimbs, followedby ataxia and undersized growth. Neurological signswere �rst observed day 24 p.i., at this time point theincidence was 2.3%, and continously increasing to83.9% until the last point of investigation (45 dayp.i.) (Figure 1).

Expression of proin�ammatory cytokines inmicroglia cells One possible mechanism for the in-duction of retroviral-induced neurodegeneration isa release of potentially neurotoxic substances likeproin�ammatory cytokines IL-1¯ , IL-6, and TNF-®from activated microglia (Sopper et al, 1996). Thesecytokines may initiate and/or promote the develop-ment of neurodegenerative lesions. Therefore, we in-vestigated whether the proin�ammatory cytokinesIL-1¯ , IL-6, and TNF-® were expressed in the courseof MuLV-NT40-induced neurodegeneration.This wasaccomplished by examining the expression of mRNAfor these proin�ammatory cytokines in microgliacells isolated ex vivo (MGexvi) at three different timepoints. Day 14 p.i. was selected because retroviral in-fection had spread to de�ned brain regions, howeverwithout microglia activation. The second time point(day 25 p.i.—data not shown) was chosen becauseretroviral infection persisted on maximum levels andwas accompanied by some activated microglia cells.Finally, we analyzed the expression of proin�amma-tory cytokines at 74 days p.i.

Basal expression of the proin�ammatory cytokinemRNAs IL-1¯ , IL-6, and TNF-® was detected inmicroglia cells isolated from all animals examined(Figure 3). Expression levels of cytokine mRNAfrom MuLV-NT40-infected rats were similar in com-parison to those of noninfected control rats. Up-regulation of cytokine gene expression was never

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504 R Hansen et al

observed, at any time point. Quantitative compari-son of mRNA from LPS-stimulated microglia cells tomRNA from nonstimulated microglia cells showedan 150-fold increase of IL-1¯, IL-6, and TNF-® mRNA(Figure 3). This result indicates that microglia hadnot lost their functions with respect to their capabil-ity to secrete proin�ammatory cytokines. From ourresults, we conclude that activated microglia cells

Figure 2 Immunohistochemicalanalyses of rat brains. Rat brain tissues were analyzed by immunohistochemistry at various time pointsafter MuLV-NT40 inoculation. Antiviral staining was performed with a polyclonal goat antiserum, which recognizes the viral envelopeprotein (Figure 2: A–C, G, H, barD 17 ¹m),and with monoclonalantibodyED1 (Dijkstra etal, 1985) to visualizeactivatedmicroglia cells (D–F, I, bar D 17 ¹m; K, barD 26 ¹m). The photomicrographsof each timepoint represent brain sections of one respective rat. Immunopositivecells were identi�ed by their brown color. In the deep cerebellar nuclei, we found similar expression levels of retroviral gene products ina time interval ranging from 13 to 67 days p.i. (A–C, G). Microglia cells were the main target cells for MuLV infection (!), but infectionof endothelial cells was also observed ( ). Maximal levels of retroviral infection were detected beginning from 13 days p.i. (A). However,activated microglia cells could not be observed (D). Activated microglia cells ( ) were rarely found at 23 days p.i., a time point whenspongiform lesions were clearlyestablished (E). At later timepoints, numbers of activatedmicroglia cells had increased(F, I), thus followingthe extent of spongiform lesions (D–F, I). Retroviral infection alone was not suf�cient to induce activation of microglia cells as shown bycomparison of the deep cerebellar nuclei (G, I) and the cerebral cortex (H, K). In both the deep cerebellar nuclei (G) and in the cerebralcortex (H), similar numbers of microglia cells were infected. Whereas in the deep cerebellar nuclei extensive spongiform lesions wereaccompaniedby numerous activatedmicroglia cells (I), the cortex cerebri failed to exhibit spongiform lesions and activatedmicroglia cells(K). A negativecontrol for antiviral staining is shown in L (barD 26 ¹m). Double staining with antiviral antibody gp70 (light brown) and themonoclonal antibody ED-1 (dark brown) clearly showed that microglia cells are the main infected target cell in the brain (M). (Continued)

do not release enhanced levels of proin�ammatorycytokines, under the given circumstances. Moreover,MuLV-NT40 infection alone is not suf�cient to in-duce cytokine gene expression in microglia cells, atthe time points tested.

In addition to microglia cells, astrocytes, and otherbrain cells are also potential candidates for releas-ing cytokines in the brain (Schobitz et al, 1994).

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Figure 2 (Continued).

Astrocytes or other brain cells may release proin�am-matory cytokines, consequently leading to an activa-tion of microglia cells (Choe et al, 1998). Therefore,we performed RNase protection assays using wholebrain mRNA from MuLV-NT40-infected rats in com-parison to noninfected control rats. The same timepoints as before were investigated. Expression levelsof the proin�ammatory cytokine mRNAs IL-1¯, IL-6,and TNF-® in brain material was much lower than inMGexvi. Again, we found no differences in cytokineexpression between MuLV-NT40-infected rats andnoninfected controls, at days 14 and 25 p.i. (data notshown). From these results, we conclude that proin-�ammatory cytokines such as IL-1¯, IL-6, and TNF-®are not initially involved in MuLV-NT40-associatedneurodegeneration.

Discussion

In this study, we show that spongiform encephalopa-thy induced by MuLV-NT40 was followed by activa-

tion of microglia cells and not vice versa. Our resultsclearly demonstrate that microglia activation was notresponsible for the induction of brain lesions. Fur-thermore, microglia activation was not accompaniedby increased levels of the proin�ammatory cytokinesIL-1¯ , IL-6, and TNF-® indicating that these cytokinesare dispensable for the induction of MuLV-associatedneurodegeneration.

Activation of microglia cells is frequently ob-served in many neurodegenerative as well as in-�ammatory diseases such as Alzheimer’s disease(Eikelenboom and Veerhuis, 1996), multiple sclerosis(Gonzalez-Scarano and Baltuch, 1999), experimentalallergic encephalomyelitis (EAE) (Benveniste, 1997),prion diseases (Giese et al, 1998), and retroviral en-cephalopathies induced by HIV (Weis et al, 1994),SIV (Czub et al, 2000), and neurovirulent MuLV(Gravel et al, 1993; Robertson et al, 1997; Choe et al,1998). At present, it is not clear how microglia cellsare implicated in the pathogenic process. However, anumber of in vitro studies have shown that microgliacells activated by different stimuli are directly or

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Figure 3 Expression of proin�ammatory cytokines in microgliacells isolated ex vivo. We investigated the expression of the proin-�ammatorycytokines IL-1¯, IL-6, and TNF-® in microglia cells iso-lated ex vivo from rats neonatally infected with MuLV-NT40 dayspi (dpi) or non-infected rats days postpartum (dpp), by means ofRPA. Each lane represents data from an individual animal. Threedifferent time points were chosen for our analysis (days 14, 74pi/pp, and day 25 pi/pp—data not shown). In all samples frommicroglia cells isolated ex vivo (MGexvi) basal expression of mRNAfor the proin�ammatory cytokines IL-1¯, IL-6, and TNF-® was de-tected (lanes 5–9, 11–14). An upregulation of cytokine mRNA ex-pression in MuLV-NT40 infected rats (lanes 5–8, 11–13) versusnoninfected control rats (lanes 9, 14) was not observed. Quantita-tive evaluation (on a Molecular Dynamics Phosphorimager usingImageQuant software) of levels of LPS-stimulated MGexvi mRNAcompared to levels of non-stimulated MGexvi mRNA revealed an150-fold increase of IL-1¯, IL-6, and TNF-® mRNA (lane 4, 10).

indirectly neurotoxic to cultured neurons. Mechanis-tically, nitric oxide, glutamate, cytokines, and non-de�ned microglia-derived neurotoxins, respectively,might be responsible for neuronal cell death (Bojeand Arora, 1992; Chao et al, 1992; Piani et al, 1992).Such in vitro studies are helpful to analyze pheno-typical characteristics and possible functions of mi-croglia cells, but it is quite clear that these studiescannot really re�ect the in vivo situation.

Therefore, we performed a kinetic study on Fisherrats neonatally infected with neurovirulent MuLV-NT40. We simultaneously analyzed four parametersper individuum, i.e., retroviral CNS infection, induc-tion of histopathological lesions, activation of mi-croglia cells, and inauguration of neurological signs,in a time-course manner. Activation of microgliacells was determined by staining with antibody ED1,which is a highly sensitive marker for microglia acti-vation (Dijkstra et al, 1985). It is possible though, thatdifferent forms of activated microglia exist and thatnot all of these different forms would be equally welldetected by ED1 immunostaining. Increased num-bers of activated microglia cells in rats neonatallyinfected with MuLV were only found in brain re-gions where neurodegenerative lesions had already

occurred, but increased numbers of activated mi-croglia cells were never detected before morpholog-ical damage had been established. Furthermore, wedid neither observe increased numbers of activatedmicroglia cells in cortical areas where microglia in-fection but no histopathological alterations occur.These results clearly indicate that microglia activa-tion was rather a consequence than the cause of retro-viral-induced neurodegeneration.

Brain damage and neurological impairment inAIDS encephalopathy induced by the retrovirusesHIV or SIV is thought to result at least partially fromproin�ammatory cytokines released by infected, ac-tivated microglia cells (Dickson et al, 1993). This hy-pothesis is supported by studies showing increasedexpression of proin�ammatory cytokines such asIL-1, IL-6, and TNF-® in the cerebrospinal �uid(Grimaldi et al, 1991; Perrella et al, 1992; Laverdaet al, 1994) as well as in brain autopsy material ofpatients infected with HIV (Tyor et al, 1993; An et al,1996; Seilhean et al, 1997; Wesselingh et al, 1997).In contrast to these studies, we were unable to de-tect enhanced levels of IL-1¯, IL-6, and TNF-® inMuLV-NT40-infected rats, neither in microglia cellsisolated ex vivo nor in total brain material. Thus, wedemonstrate both that the proin�ammatory cytokinesIL-1¯, IL-6, and TNF-® were dispensable for the in-duction and sustenance of MuLV-associated neurode-generation and that activated microglia cells do notnecessarily express the proin�ammatory cytokinesIL-1¯, IL-6, and TNF-®. The reason for the differencebetween cytokine involvement of MuLV and lentivi-ral induced encephalopathy might originate fromthe fact that lentiviral CNS infections are often ac-companied by in�ammatory changes. CNS-invadingleukocytes might be a direct or an indirect source ofproin�ammatory cytokines.

Our results clearly contrast observations obtainedon brains from mice infected with other neurovir-ulent MuLV (Nagra et al, 1994; Choe et al, 1998).Both TNF-® and Fas were detected in astrocytesand in some microglial cells (Choe et al, 1998).The study suggested that MuLV-induced neural celldeath was likely due to Fas- and TNF-®-mediatedcell death mechanisms (Choe et al, 1998). Simi-larly, Nagra et al (1994) found enhanced levels ofTNF-®, IL-6, and quinolic acid in MuLV-degeneratedmouse brains. Although virological, immunological,as well as histopathological data point to a uni-form pathomechanism of MuLV neurodegenerationin both mice (Czub et al, 1991; Lynch et al, 1991)and rats (Kai and Furuta, 1984; Czub et al, 1995), dif-ferences in viral and/or host genetics could have ac-counted for the contrasting cytokine expression pat-terns observed in Choe’s and Nagra’s as comparedto our study. Alternatively, technical aspects couldbe considered to explain the con�icting results: ourstudy demonstrates basal expression of proin�am-matory cytokines in microglia cells and brains ofboth infected and noninfected rats. However, there

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might have been regional, probably minor, differ-ences in the expression levels of proin�ammatory cy-tokines as suggested by the other studies (Choe et al,1998).

Although activated rat microglia cells were notinvolved in the initial formation of neurodegener-ative MuLV alterations, it is striking that clinical,i.e., neurologic disease emerged simultanously withthe activation of microglia cells. This observationraises the possibility that the clinical outcome of neu-ropathological diseases might be strongly in�uencedby the number of activated microglia cells. In consis-tency with this hypothesis are studies on the AIDS-encephalopathy, demonstrating a positive correlationbetween the number of activated microglia cells andthe severity of neurological disease (Glass et al, 1995;Berman et al, 1999). Indirect support for the signif-icance of activated microglia cells for the severityof neurological disease comes from observations onother murine MuLV models in which spongiosis wascertainly induced but neither activation of microgliacells nor clinical disease (Askovic et al, 2000). How-ever, in the fastest model for MuLV-induced neurode-generation, i.e., infection of mice with FrCasE (Portiset al, 1990), there is neither microglia activation, inspite of severe neurological signs (Lynch et al, 1995).From this, it appears unlikely that microglia acti-vation contributes to formation of clinical signs inMuLV encephalopathy.

Thus, in the course of MuLV-induced neurodegen-eration, different stages of activation of microgliacells appear to be associated with various biolog-ical effects: during the early phase, morphologicalCNS damage is initiated. For the induction of thisCNS alteration, retroviral infection of microglia cellswas previously shown to be essential and suf�cient(Lynch et al, 1995). Whether gain or rather loss ofmicroglia functions brings on morphological dam-age remains to be elucidated. However, our resultsstand in favor of deprived microglial competence,at the beginning of MuLV-induced neurodegenera-tion, but not of microglial hyperfunction. In the sec-ond phase of MuLV neurodegeneration, microgliosisturns up, probably as a reaction to severe morpho-logical damage or to other signals. Although beingactivated, microglia cells did not secrete proin�am-matory cytokines. This makes us believe that ratherregenerative than detrimental functions were exe-cuted at this time, e.g., phagocytosis of dead cells. Fi-nally, neurological disease turns up, in parallel withstill activated microglia cells. However, additionalsigns of microglial hyperfunctions are still missingat this time point, as we did not see elevated lev-els of proin�ammatory cytokines, although release ofother microglia-derived toxins cannot be excluded.It is also possible, at this �nal point of the disease,that the microglia cells are deadly exhausted, withthe consequence of lacking protective and regenera-tive support to sustain normal brain functions. Ac-cording to this concept, neurological disease arises

upon a variety of microglial reactions, which are inno way stereotypic but vain to combat the effects ofexogenous insult.

Material and methods

Virus MuLV-NT40 was isolated after serial pas-sage of a molecular clone of Friend-MuLV, FB29through Fisher rats (Czub et al, 1995). Viral stockswere prepared from con�uently infected Fisher ratembryo cells. The titer of viral stock used varied be-tween 1.0 £ 105 and 5 £ 105 FFU/ml. Viral titrationswere performed using a focal immunoassay (Czubet al, 1995).

Experimental design Inbred F344-Rats were in-fected intraperitoneally at 24–48 h postnatally withMuLV-NT40 and were observed for signs of clinicalneurologic disease (Czub et al, 1995) and/or were sac-ri�ced at various times after inoculation for furtheranalyses. A kinetic analysis (time interval rangingfrom 8 to 36 days after inoculation) was performed bymeans of histopathological and immunohistochemi-cal examination on 41 brains of rats neonatally inoc-ulated with MuLV-NT40.

Histopathological and immunohistochemical(IHC) analyses We investigated brains of ratsneonatally inoculated with MuLV-NT40 at a timeinterval ranging from 8 to 36 days p.i., with a seriesof four coronal sections per animal. Rats were killedby CO2 inhalation and perfused through the leftventricle with PBS until the ef�uent ran clear.Subsequently, a perfusion with 4% formaldehyde inPBS was performed. Brains were removed and thetissues were �xed for 48 h in 4% formaldehyde inPBS and embedded in paraf�n. Sections (1 ¹m) werestained with hematoxylin and eosin. Vacuolizationin speci�ed areas (see Results) were evaluated at amagni�cation 200£ using an ocular grid (per area:�ve �elds/individuum). Results were recorded aspercent vacuolated of total area and categorizedinto four groups: none (0), mild (1), moderate (2),and severe (3). Sections without vacuolizationconstituted the �rst group. Mild lesions consisted offew and small vacuoles. Moderate lesions consistedof occasional vacuoles, occupying 20% to 40%of an 200£ visual �eld. Severe CNS lesions werecharacterized by extensive vacuolization, occupyingmore than 40% of the visual �eld.

For IHC, 1 ¹m thick sections from paraf�n-embedded tissues were deparaf�nated, rehydrated,and permeabilized in 0.5% Triton-X-100/phosphate-buffered-saline (PBS) with 1 mg/ml Proteinase Kat 37±C for 5 min. Endogenous peroxidase activ-ity was suppressed by incubation with 0.3% H2O2in methanol for 2 min. After washing, slides wereblocked in PBS with 10% normal serum (MerckTissue Gnost Uni-Pak Kit) for 15 min. A goat poly-clonal anti-MuLV-gp70 serum provided by Prof. R.Friedrich, Institute of Virology, Gießen, was used

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in a dilution 1:2000. Activated microglia cells werestained by monoclonal antibody ED1 (Dijkstra et al,1985) in a dilution of 1:100 (Serotec). Slides werewashed twice and then incubated with a biotiny-lated secondary antibody. Bound antibodies wererevealed with an avidin-biotin-peroxidase kit (Bio-Genex-Super Sensitive Immunodetection) at 37±C for30 min and diaminobenzidine as a substrate. Sectionswere counterstained with hematoxylin.

Infected CNS cells and activated microglia cells, re-spectively, were quanti�ed by counting immunopos-itive cells in �ve visual �elds of investigated brainregions (deep cerebellares nuclei, nuclei cochleares,nuclei vestibulares, nucleus facialis, cortex cere-brum) using an ocular grid with an total area of0.2 mm2.

Isolation of microglia cells Microglia cell isola-tion was performed as described in detail before(Hansen et al, 2000). Brie�y, brains were removedfrom perfused animals, stripped of meninges, enzy-matically digested, and subjected to a density gradi-ent. Microglia cells were collected from the 1.077/1.066 g/cm3 interface and analyzed further.

Detection and quantitation of cytokine mRNAsby ribonuclease protection assay (RPA) mRNA wasisolated from total brains and from microglia iso-

lated ex vivo, respectively employing Oligo(dT)25Dynabeads from Dynal following the recommenda-tions of the manufacturer. Cytokine mRNAs for IL-1¯,IL-6, and TNF-® were measured by RPA as describedin detail before (Sauder and de la Torre, 1999). Bandscorresponding to protected cytokine mRNAs werequantitated on a Molecular Dynamics Phosphorim-ager using ImageQuant software (Becton Dickinson)and were normalized to the amounts of L32, a ri-bosomal house keeping gene, in each lane. At eachtime point, mRNAs from microglia cells from in-dividual infected and noninfected rats were anal-ysed. Microglia cells isolated ex vivo from rats neona-tally infected with MuLV-NT40 were also stimulatedin vitro with LPS (100 ng/105 cells, 5 h), and iso-lated mRNA was used subsequently as a positivecontrol.

Acknowledgements

The authors wish to thank W. P. Lynch for critical re-view of the manuscript and helpful discussions. Thisstudy was supported by grants from the DFG (Cz 56/3-4; Sa 682/1-1) and the BMBF (01K/97 63/8).

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