Microglia Density Decreases WithAge in a Mouse Model of

Huntington’s DiseaseLI MA,1 A. JENNIFER MORTON,2 AND LOUISE F.B. NICHOLSON1*

1Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand2Department of Pharmacology, University of Cambridge, Cambridge, U.K.

KEY WORDS transgenic; gliosis; aging; forebrain

ABSTRACT Huntington’s disease (HD) is characterized by selective neuronal lossand reactive gliosis. In the R6/2 transgenic HD mouse model, there is no selective cellloss, although astrocytosis has been reported. Since there have been no previous studieson microglia in this model, we have undertaken a detailed investigation of microglia insix different forebrain regions in the R6/2 mouse and their wild-type littermates at twotime points. Microglia were identified using the histochemical marker isolectin B4 andinteractions of genotype, region, and age were analyzed. Results showed that there wasa significant decrease in the number of microglia with age in both wild-type and R6/2brains, which was more pronounced in the transgenic mouse. There were also morpho-logical changes with age observed in both genotypes. As early as 7 weeks of age,structural microglial abnormalities could be seen in R6/2 brains, including bulbousswellings and long stringy processes; comparable changes were seen at 16 weeks inwild-type brains. At 14.5 weeks, microglia in R6/2 mouse brains were smaller in sizewith condensed nuclei and fragmentation of their processes. We suggest that the densityand morphology of microglia change with normal aging and that this process is accel-erated in R6/2 brains. Such changes in the dynamic status of microglia may lead to animpairment of their neurosupportive functions. Further studies are needed to under-stand better the role of microglia in aging and neurodegeneration. © 2003 Wiley-Liss, Inc.

INTRODUCTION

Huntington’s disease (HD) is an autosomal dominantneurodegenerative disease with a neuropathologycharacterized by a selective loss of the medium spinyprojection neurons in the striatum (Graveland et al.,1985) and a more generalized loss of the large pyrami-dal neurons in the cerebral cortex (Hedreen et al.,1991; Sotrel et al., 1991). In addition to the selectiveneuronal loss in the HD brain, there is a reactive gliosis(Vonsattel et al., 1985; Myers et al., 1991). Morphomet-ric studies indicate that this gliosis can be attributed toa marked increase in reactive astrocytes and oligoden-droglia in the striatum and to oligodendroglia in thecortex (Sotrel et al., 1991). Changes in microglia in theHD brain had not been reported until recently, whenSapp et al. (2001) showed an early and progressiveaccumulation of reactive microglia in the striatum andcortex of the HD brain. Microglia have also been found

to associate with neurons that contain neuronal in-tranuclear inclusions (NIIs) of the mutant protein hun-tingtin (Sapp et al., 2001).

Microglia comprise a significant proportion of cells inthe adult central nervous system (CNS) with estimatednumbers ranging from 5% to 20% of the cell population(Lawson et al., 1990). They have been broadly de-scribed as the macrophages of the CNS (Afifi and Berg-man, 1998). More recently, however, a role for acti-vated microglia in the neurodegenerative disease

Grant sponsor: the New Zealand Neurological Foundation.

*Correspondence to: Dr. Louise F.B. Nicholson, Department of Anatomy withRadiology, University of Auckland, Private Bag 92019, Auckland, New Zealand.E-mail: lfb.nicholson@auckland.ac.nz

Received 17 March 2003; Accepted 26 March 2003

DOI 10.1002/glia.10261

GLIA 43:274–280 (2003)

© 2003 Wiley-Liss, Inc.

process has been proposed. In Alzheimer’s disease (AD)brain, activated microglia surround senile amyloidplaques and it has been suggested that they contributeto the degeneration of adjacent neuronal processes.Indeed, the beneficial effects of anti-inflammatory med-ication also support the involvement of microglia in AD(Hull et al., 2002). Furthermore, both in vitro and invivo studies in human have shown that microglia, stim-ulated by �-amyloid or fragments of �-amyloid, expressinducible nitric oxide synthase (iNOS), an enzyme in-volved in the production of reactive oxygen species anda marker of activated microglia (Heneka et al., 2001;Haas et al., 2002). In the transgenic mouse model ofhuman amyotrophic lateral sclerosis (ALS), the mutantcopper/zinc superoxide dismutase (mSOD1) transgenicmouse, microglia are significantly activated in parallelwith motoneuron loss, providing further evidence forthe involvement of microglia in the disease process(Hall et al., 1998; Almer et al., 1999). Furthermore,when transgenic mice from a demyelinating neurolog-ical disorder (globoid cell dystrophy) are crossed withMHC class II knockout mice, macrophage/microglia inthe CNS are markedly reduced and there is significantclinical improvement in these mice (Matsushima et al.,1994). Thus, it appears that microglia contribute toprogressive neurodegeneration.

In HD, the development of a number of transgenicmouse models has led to an increased understanding ofthis disease. The R6/2 line, which is transgenic for exon1 of the human huntingtin gene under the control of itsnative promoter (Mangiarini et al., 1996), develops aprogressive behavioral phenotype with an age of onsetnormally between 8 and 11 weeks (Carter et al., 1999).Notably, while cognitive deficits have been observed asearly as 4 weeks (Lione et al., 1999), there is no signif-icant neuronal degeneration observed within the stri-atum of these mice before 14–14.5 weeks (Turmaine etal., 2000). After this time, however, condensing neu-rons can be seen in the anterior cingulate cortex andthe striatum and vermis of cerebellum (Turmaine etal., 2000). This change in cellular appearance is pre-ceded by a progressive increase in the size, number,and location of abnormal aggregates of protein in thebrain (Davies et al., 1997; Morton et al., 2000) and adecrease in neurotransmitter receptor binding (Cha etal., 1999). There is also evidence of a decrease in bothbrain and body weight (Mangiarini et al., 1996; Carteret al., 1999), with significant progressive atrophy ofstriatal neurons (Ferrante et al., 2000). Curiously,since gliosis is considered a hallmark of HD pathology(Vonsattel and DiFiglia, 1998), there is no indication ofan accompanying reactive gliosis in this animal model,although efforts have been made to detect this response(Mangiarini et al., 1996; Turmaine et al., 2000). Thus,in most studies using the R6/2 and the N171-82Qmouse, neither neuronal loss nor astrogliosis has beenreported (Schilling et al., 1999; Andreassen et al.,2001). In some other transgenic mouse models of HD,however, selective neuronal loss and reactive gliosis

have been reported (Reddy et al., 1998; Lin et al.,2001).

In view of the importance of selective neuronal lossand reactive gliosis in the human condition, the diver-sity of observations seen in the different animal modelsof HD, and the popular use of the R6/2 mouse as amodel of HD, we have undertaken a detailed investi-gation of microglia in the cortex, basal ganglia, thala-mus, and associated white matter of the corpus callo-sum in the R6/2 mouse using the histochemical markerfor microglia isolectin B4.

MATERIALS AND METHODSR6/2 Mice

The brains of transgenic R6/2 mice and their wild-type littermates were taken from a colony maintainedin the Department of Pharmacology, University ofCambridge (Cambridge, U.K.). Mice were killed withan overdose of sodium pentobarbital and transcardiallyperfused with chilled normal saline followed by 4%paraformaldehyde. Whole brains were removed fromthe skull and stored in the same fixative for 16 h andcryoprotected in 30% sucrose. The sucrose-embeddedmouse brains were then shipped to New Zealand.Floating 30 �m coronal sections were cut on a freezingmicrotome and stored in PBS with 0.1% sodium azideuntil required. A total of 18 mice were used in thisstudy: 3 transgenic/mice R6/2 and 7 wild-type micekilled at 7 weeks of age (early symptomatic group), and4 R6/2 mice killed at 14.5 weeks and 4 wild-type micekilled 16 weeks of age (postsymptomatic group).

Histochemical Staining With Isolectin B4

Floating sections were washed with PBS/Triton be-fore endogenous peroxidase was quenched with 1%H2O2/50% methanol. Sections were then washed withPBS/Triton and stained with horseradish peroxidase-conjugated Griffonia simplicifolia isolectin B4 (10 �g/ml, SIGMA) at room temperature overnight. Afterwashing with PBS, microglia labeling was visualizedusing a diaminobenzidine (DAB; Sigma) color reaction.The sections were then mounted onto poly-L-lysine-coated slides, air-dried overnight, rehydrated in waterand dehydrated through ethanol and xylene, andmounted in DPX mount medium. The staining wasobserved using a Lecia Diaplan light microscope.

Quantification

Six brain regions were studied: the cingulate andfrontal cortex (Cx), striatum (ST), globus pallidus (GP),thalamus, corpus callosum medial (cc/m), and corpuscallosum lateral (cc/l). Representative sections of thespecific area of interest were selected from a rostralcaudal series. For Cx, ST, thalamus, and cc/l, a total of

275MICROGLIA IN R6/2 MICE

10 counting squares, 5 on each side of these brainregions, each measuring 170 � 240 �m, were sampledon the section and the number of microglia was countedat 500� magnification. For GP and cc/m, respectively,six and eight such counting squares were sampled persection and the number of microglia was counted at thesame magnification. Only microglia with a clearly vis-ible cell body were counted.

Statistical analysis of counts was carried out usingan SAS (SAS Institute, Cary, NC) mixed linear modelfitted to the square root of the numbers of microglia,including hemisphere and area within region as ran-dom effects and region as a repeated effect for eachbrain. Age and genotype of mouse were also included inthe model. The interaction of genotype of mouse, re-gion, and age was investigated to see whether mousegenotype had a different effect at different ages and indifferent regions. As an interaction was found, the re-gions were subsequently analyzed separately with theinteraction testing for a difference in the effect of agefor different genotypes. Where an interaction was de-tected, the analysis was split on age.

RESULTS

Isolectin B4 labeling was detected in cells withsmall- to medium-sized cell bodies and fine branchingprocesses. These cells were morphologically similar tomicroglia identified by others using OX-42 and F4/80and described in early studies (Lawson et al., 1990;Eriksson et al., 1993). In 7-week wild-type mice, micro-glia had fine branching processes characteristic of qui-escent microglia (Fig. 1A and E). In the brains of 16-week wild-type mice, most microglia had fewerprocesses with a less elaborate arborization than thoseat 7 weeks, although some had unusually long stringyprocess (Fig. 1B). In 7-week-old R6/2 brain, microgliaexhibited structural abnormalities, including longstringy processes or bulbous swellings on cytoplasmicprocesses as shown in Figure 1C, F, and G. In 14.5-week R6/2 mouse brain, few microglia could be seen. Ofthose microglial cells present, some were smaller insize than the microglia seen in 7-week-old R6/2 brainand had a condensed nucleus and fragmentation of thecytoplasm within processes (Fig. 1D and H).

Cell counts for the isolectin B4-positively stainedmicroglia in the six different brain regions in wild-typeand R6/2 mice have been presented graphically in Fig-ure 2. The median values for the number of microgliafor both wild-type and R6/2 mice have been plotted foreach of the two time points.

The effect of both genotype (wild-type and R6/2) andage (7 and 14.5–16 weeks) on the total number ofmicroglia in each of the six brain regions was analyzed.There was evidence of an overall difference in the effectof genotype at the two ages differing in the regions ofthe brain (F5,158 � 2.5; P � 0.03). Further analysis wastherefore undertaken on each of the brain regions. Inthe cortex, a difference in the effect of genotype at the

two ages was demonstrated (genotype-age interactionF1,33 � 6.6; P � 0.01). When the two ages were consid-ered separately, no effect of genotype could be demon-strated at 7 weeks (F1,18 � 1.2; P � 0.29), but at14.5–16 weeks there was evidence of an effect of geno-type (F1,15 � 7.5; P � 0.02). This is shown in Figure 2Aand B, where the number of microglia in the wild-typeand R6/2 mice has been plotted for each of the six brainregions at 7 and 14.5–16 weeks of age. In the otherbrain regions, there was no evidence of a difference inthe effect of age for different genotypes. The age-geno-type interaction was therefore dropped from the anal-ysis for all other brain regions and the main effects ofgenotype and age were investigated. In the striatum,while no effect of genotype was seen (Fig. 2A and B),there was a strong effect of age (F1,33 � 21.6; P �0.0001) within genotype as seen in Figure 2C and D. Inthe thalamus, there was a trend toward an effect ofgenotype and age (F1,33 � 3.3, P � 0.08 and F1,33 � 5.5,P � 0.03, respectively; Fig. 2C and D). In the globuspallidus, there was no evidence of an effect of genotypeor age (P � 0.79 and 0.6), while in the corpus callosum(both lateral and medial), there was evidence of aneffect of age only (F1,42 � 4.9, P � 0.03 and F1,42 � 5.4,P � 0.03, respectively). Clearly, age is a very importantdeterminant of microglia numbers in most regions ofthe brain studied; this can be seen in Figure 2.

DISCUSSION

Resting microglia are activated by a variety of cues,ranging from significant changes in the brain’s struc-tural integrity to very subtle alterations in their micro-environment (Kreutzberg, 1996). Whether activatedmicroglia are beneficial or harmful to neurons has beenthe focus of a long-standing debate. Numerous studieshave shown that microglia can be activated under cul-ture conditions to release several potentially cytotoxicsubstances (Paris et al., 1999; Badie et al., 2000; Liu etal., 2001; He et al., 2002; Ryu et al., 2002), which leadto a negative downstream effect. Several in vivo stud-ies, however, using peripheral and central nerve injurymodels showed clear evidence that activated microgliaare involved in nerve regeneration (Barron et al., 1990;Kreutzberg, 1996; Tseng et al., 1996; Streit et al.,2000). There are also in vitro studies that have shownthat microglia can express neurotrophic factors, pro-viding evidence for a supportive role of microglia forneurons (Elkabes et al., 1996; Polazzi et al., 2001).

Pathological activation of microglia has also beenreported in a wide range of disease conditions, includ-ing cerebral ischemia, Alzheimer’s disease, prion dis-eases, multiple sclerosis, and acquired immune defi-ciency syndrome (AIDS) dementia (Stoll and Jander,1999; Minagar et al., 2002). The role of microglia inAlzheimer’s disease has been extensively investigated.It is now generally accepted that �-amyloid and itsassociated molecules deposited in the senile plaquestrigger the activation of microglia leading to an inflam-

276 MA ET AL.

matory reaction, which can induce the apoptotic celldeath of neurons by bystander damage. Very recently,a new concept of “senescent and/or dysfunction of mi-croglia with normal aging” has been proposed (Streit,

2002), offering a new explanation for the pathogenesisof neurodegenerative diseases. The author proposedthat if microglia become disabled gradually, throughsome genetic and epigenetic risk, their functional ca-

Fig. 1. Isolectin B4 staining in the cortex of wild-type (A and B) andR6/2 (C and D) mouse brains of two different ages. A: In 7-week-oldwild-type brains, most of the stained microglia had fine branchingprocesses characteristic of resting microglia (arrows). B: At 16 weeksof age, microglia in wild-type brains had fewer processes with lessarborization (arrows); some cells had unusually long stringy processes(arrowhead). C: In 7-week-old R6/2 brains, obvious structural abnor-malities in microglia could be seen, including long stringy processes(arrows) and bulbous swellings on cytoplasmic processes as shown athigher magnification in G (arrow head). D: In the 14.5-week-old R6/2

mouse brains, very few microglia could be seen. Among the microgliapresent, some were smaller in size, with a condensed nucleus andfragmentation of the cytoplasm within processes (arrow). E–H arehigh-power photomicrograph of single cells exhibiting the differentmorphologies described above. E: Resting microglia with fine branch-ing processes. F: Microglia with long stringy processes (arrowhead).G: Microglia with a bulbous swelling on a cytoplasmic process (arrow-head). H: Microglia with a condensed cell nucleus and fragmentationof the cytoplasm within processes (arrowhead). Scale bar for A–D, 60�m; for E–H, 20 �m.

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pacity to support neurons also becomes diminished,and hence neurons slowly degenerate (Streit, 2002).

In this study, we have used the R6/2 transgenicmouse model of HD to investigate the status of micro-glia at two different time points. We have observedmorphological changes in microglia in wild-type andR6/2 mice at both time points. In 7-week-old wild-typemice, most microglia had the morphological appear-ance of resting microglia. By comparison, in 16-week-old wild-type mice, some of the microglia had the mor-phological appearance of activated-like microglia.These microglia had fewer processes with less arboriza-tion. Some microglia had long and stringy processes,which is thought to be a morphological sign of aging ofmicroglia. Although the morphology of these microgliawas different between the two age groups in wild-typemice, we cannot draw inference with respect to anyfunctional changes of microglia with age. A similarmorphological change in microglia has also been ob-served in normal human brain (Streit and Sparks,

1997). Using immunohistochemical staining for MHCII antigen, Streit and Sparks (1997) showed that mi-croglia became progressively more activated in appear-ance with normal aging. In R6/2 mice, we have ob-served structural abnormalities at the age of 7 weekswhen the R6/2 mouse has not started to show obviousovert motor symptoms. Some microglia had unusuallylong stringy processes similar to those seen in 16-week-old wild-type mice; this may well be indicative of apremature aging process in the 7-week-old R6/2 mousebrain. Furthermore, other microglia had bulbous swell-ings on their cytoplasmic processes. In 14.5-week-oldR6/2 mice, microglia with long stringy processed couldstill be seen, but bulbous swellings were absent. Frag-mentation of cytoplasm within processes (stained withisolectin B4) could be easily identified. Similar struc-tural abnormalities have been reported previously inAlzheimer’s disease (Streit, 2002) and HD brains (Sappet al., 2001) and are thought to be indicative of senes-cent microglia (Streit, 2002). Clearly, morphological

Fig. 2. Comparison of the number of microglia in different brainregions of wild-type and transgenic R6/2 mice at two different ages (Aand B) and between the two different ages within each genotype (Cand D). At 7 weeks of age, R6/2 mouse brains did not show anysignificant difference in the number of microglia in all six brainregions examined when compared to wild-type littermates (A). At thelater age (14.5–16 weeks), the number of microglia in the R6/2 mousebrain remained at approximately the same level as that seen inwild-type mouse in all the brain regions except cortex (B), where theR6/2 mouse had significantly less microglia than their wild-type lit-

termates (P � 0.02). The number of microglia decreased with age inboth the wild-type (C) and R6/2 mouse (D) in all brain regions [Cx (P �0.01), Str (P � 0.0001), thalamus (P � 0.03), cc/m (P � 0.03), and cc/l(P � 0.03)] except in the GP. The decrease in the number of microgliawith time in R6/2 brains was more dramatic than that seen in wild-type mouse. Bars are the median of the number of microglia percounting area. Asterisk indicates P � 0.05; WT, wild-type. Abbrevia-tions below bars indicate different regions of the brain that werestudied. Cx, cingulate and frontal cortex; ST, striatum; GP, globuspallidus; cc/m, corpus callosum medial; cc/l, corpus callosum lateral.

278 MA ET AL.

changes with age are dramatic and must be contribut-ing to their functional status.

What is most interesting, however, are the changesobserved in the number of microglia in different brainregions of the wild-type and R6/2 mice at various timepoints. Wild-type mice showed a significant decrease inthe number of microglia with age in almost all thebrain regions studied except the globus pallidus, sup-porting the idea that a loss of microglial cells in thebrain may well be part of the aging process. In R6/2mice, a decrease in the number of microglia with agewas also observed in the same brain regions; however,the decrease was more significant. The cause of thisloss of microglia is unknown, but age is clearly a veryimportant determinant of microglia numbers. Based onthese data, we propose that the status of microglia isnot constant through life, but is a dynamic process,with the morphology, number of microglia and possiblytheir function changing with normal aging. The accel-erated change in the status of microglia with aging, asobserved in the R6/2 mice in our study, may lead to animpairment of the neuron-supporting functions of mi-croglia and thus contribute to neuronal cell death.

The question then arises as to the cause of the de-crease of microglia density in the brain with aging.Previous studies have shown that apoptotic cell deathof microglia occurs both during development (Streit,2001; de Louw et al., 2002) and in some neurodegen-erative diseases. In Alzheimer’s disease, while apopto-tic neuronal death is extremely rare, DNA fragmenta-tion was seen in glial cells (Jellinger and Stadelmann,2000). In Parkinson’s disease, Lewy body dementia,multisystem atrophy, and corticobasal degeneration, insitu terminal dUTP nick-end labeling (TUNEL)-posi-tive staining and increased expression of apoptosis-related proteins (ARPs) or activated caspase-3 haveonly been seen in microglia and oligodendrocytes withcytoplasmic inclusions, but not in neurons (Jellingerand Stadelmann, 2000). In HD, DNA fragmentationhas been reported and is positively correlated withCAG repeat length and pathological grade (Dragunowet al., 1995; Butterworth et al., 1998). In these twostudies, the authors were unable to identify the cellsexhibiting DNA fragmentation. However, in the high-grade HD cases where most DNA fragmentation wasfound, there were not enough remaining neurons toaccount for the observed TUNEL staining. Some of theTUNEL-positive cells were therefore likely to be glialcells. Further studies are needed to address the ques-tion of whether the quantitative decrease and morpho-logical changes in microglia in the R6/2 mouse is aconsequence of the cell death via an apoptotic pathway.

In this study, we noted that while a quantitativedecrease in the number of microglia was observed inalmost all brain regions of the R6/2 mouse, the anteriorcingulate cortex was the only brain region to show astatistically significant difference in the number of mi-croglia between genotypes at the postsymptomaticstage. This is perhaps not surprising since studies haveshown evidence for cortical abnormalities in R6/2 mice

(Cha et al., 1999) and NIIs formation in cortex as earlyas 3.5 weeks of age (Davies et al., 1997; Meade et al.,2002). Another study also showed that the specific typeof nonapoptotic cell death observed at 14 weeks of ageoccurred first and with more prominence in the ante-rior cingulate cortex (Turmaine et al., 2000). Neuronalpathology in the cortex of the R6/2 mouse may there-fore precede that occurring in the striatum. If observa-tions of microglia were to be made at even later timepoint, we may see similar significant changes in theR6/2 striatum.

Our results in the mouse model do not support thepublished findings of Sapp et al. (2001), where a semi-quantitative study of microglia in human HD diseasebrain was undertaken. In this study, an early andprogressive increase of reactive microglia was observedusing immunohistochemistry with thymosin �-4 as amarker of activated microglia. The difference in ourfindings may well be due to the fact that there is no cellloss prior to the premature death of the R6/2 mouse.However, a previous report of histopathological studiesin the HD brain also did not identify an inflammatoryreaction, although a fibrillary reactive astrocytosis andan increased density of oligodendrocytes have beenseen with progression of the disease (Vonsattel andDiFiglia, 1998). Notwithstanding this difference, earlymorphological changes in microglia (including unusuallong stringy processes and bleeding similar to thosedescribed here in 7-week-old R6/2 mice) have been ob-served in low-grade HD brain (Sapp et al., 2001).

In conclusion, we observed an age-related decreasein the number of microglia in normal mice. In R6/2mice, the decease in microglia number in the anteriorcingulate cortex, striatum, thalamus, and corpus callo-sum is more pronounced than in their wild-type litter-mates. The cause and the consequences of both themorphological and quantitative changes observed arenot known, although morphological changes in micro-glia appear in advance of any motor symptoms in thesemice. Further studies are needed to explore the role ofmicroglia in the R6/2 mouse model to understand bet-ter the role of microglia in the disease process of HD.

ACKNOWLEDGMENTS

The authors thank Ms. Joanna Stewart for excellentstatistic analysis of the data.

REFERENCES

Afifi AK, Bergman RA. 1998. Functional neuroanatomy: text andatlas. New York: McGraw-Hill.

Almer G, Vukosavic S, Romero N, Przedborski S. 1999. Induciblenitric oxide synthase up-regulation in a transgenic mouse model offamilial amyotrophic lateral sclerosis. J Neurochem 72:2415–2425.

Andreassen OA, Dedeoglu A, Ferrante RJ, Jenkins BG, Ferrante KL,Thomas M, Friedlich A, Browne SE, Schilling G, Borchelt DR,Hersch SM, Ross CA, Beal MF. 2001. Creatine increases survivaland delays motor symptoms in a transgenic animal model of Hun-tington’s disease. Neurobiol Dis 8:479–491.

279MICROGLIA IN R6/2 MICE

Badie B, Schartner J, Vorpahl J, Preston K. 2000. Interferon-gammainduces apoptosis and augments the expression of Fas and Fasligand by microglia in vitro. Exp Neurol 162:290–296.

Barron KD, Marciano FF, Amundson R, Mankes R. 1990. Perineuro-nal glial responses after axotomy of central and peripheral axons: acomparison. Brain Res 523:219–229.

Butterworth NJ, Williams L, Bullock JY, Love DR, Faull RL, Dra-gunow M. 1998. Trinucleotide (CAG) repeat length is positivelycorrelated with the degree of DNA fragmentation in Huntington’sdisease striatum. Neuroscience 87:49–53.

Carter RJ, Lione LA, Humby T, Mangiarini L, Mahal A, Bates GP,Dunnett SB, Morton AJ. 1999. Characterization of progressive mo-tor deficits in mice transgenic for the human Huntington’s diseasemutation. J Neurosci 19:3248–3257.

Cha JJ, Frey AS, Alsdorf SA, Kerner JA, Kosinski CM, Mangiarini L,Penney JB Jr, Davies SW, Bates GP, Young AB. 1999. Alteredneurotransmitter receptor expression in transgenic mouse modelsof Huntington’s disease. Philosoph Transact Royal Soc London SerB Biol Sci 354:981–989.

Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA,Scherzinger E, Wanker EE, Mangiarini L, Bates GP. 1997. Forma-tion of neuronal intranuclear inclusions underlies the neurologicaldysfunction in mice transgenic for the HD mutation. Cell 90:537–548.

de Louw AJ, de Vente J, Steinbusch HP, Gavilanes AW, SteinbuschHW, Blanco CE, Troost J, Vles JS. 2002. Apoptosis in the rat spinalcord during postnatal development: the effect of perinatal asphyxiaon programmed cell death. Neuroscience 112:751–758.

Dragunow M, Faull RL, Lawlor P, Beilharz EJ, Singleton K, WalkerEB, Mee E. 1995. In situ evidence for DNA fragmentation in Hun-tington’s disease striatum and Alzheimer’s disease temporal lobes.Neuroreport 6:1053–1057.

Elkabes S, DiCicco-Bloom EM, Black IB. 1996. Brain microglia/mac-rophages express neurotrophins that selectively regulate microglialproliferation and function. J Neurosci 16:2508–2521.

Eriksson NP, Persson JK, Svensson M, Arvidsson J, Molander C,Aldskogius H. 1993. A quantitative analysis of the microglial cellreaction in central primary sensory projection territories followingperipheral nerve injury in the adult rat. Exp Brain Res 96:19–27.

Ferrante RJ, Andreassen OA, Jenkins BG, Dedeoglu A, Kuemmerle S,Kubilus JK, Kaddurah-Daouk R, Hersch SM, Beal MF. 2000. Neu-roprotective effects of creatine in a transgenic mouse model ofHuntington’s disease. J Neurosci 20:4389–4397.

Graveland GA, Williams RS, DiFiglia M. 1985. Evidence for degener-ative and regenerative changes in neostriatal spiny neurons inHuntington’s disease. Science 227:770–773.

Haas J, Storch-Hagenlocher B, Biessmann A, Wildemann B. 2002.Inducible nitric oxide synthase and argininosuccinate synthetase:co-induction in brain tissue of patients with Alzheimer’s dementiaand following stimulation with beta-amyloid 1-42 in vitro. NeurosciLett 322:121–125.

Hall ED, Oostveen JA, Gurney ME. 1998. Relationship of microglialand astrocytic activation to disease onset and progression in atransgenic model of familial ALS. Glia 23:249–256.

He BP, Wen W, Strong MJ. 2002. Activated microglia (BV-2) facilita-tion of TNF-alpha-mediated motor neuron death in vitro. J Neuro-immunol 128:31–38.

Hedreen JC, Peyser CE, Folstein SE, Ross CA. 1991. Neuronal loss inlayers V and VI of cerebral cortex in Huntington’s disease. NeurosciLett 133:257–261.

Heneka M, Wiesinger H, Dumitrescu-Ozimek L, Riederer P, FeinsteinD, Lolchgether T. 2001. Neuronal and glial coexpression of argini-nosuccinate synthetase and inducible nitric oxide synthase in Alz-heimer disease. J Neuropathol Exp Neurol 60:906–916.

Hull M, Lieb K, Fiebich B. 2002. Pathways of inflammatory activationin Alzheimer’s disease: potential targets for disease modifyingdrugs. Curr Med Chem 9:83–88.

Jellinger KA, Stadelmann CH. 2000. The enigma of cell death in neuro-degenerative disorders. J Neural Transmission 60 (Suppl):21–36.

Kreutzberg GW. 1996. Microglia: a sensor for pathological events inthe CNS. Trends Neurosci 19:312–318.

Lawson LJ, Perry VH, Dri P, Gordon S. 1990. Heterogeneity in thedistribution and morphology of microglia in the normal adult mousebrain. Neuroscience 39:151–170.

Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS,Crouse AB, Ren S, Li XJ, Albin RL, Detloff PJ. 2001. Neurologicalabnormalities in a knock-in mouse model of Huntington’s disease.Hum Mol Genet 10:137–144.

Lione LA, Carter RJ, Hunt MJ, Bates GP, Morton AJ, Dunnett SB.1999. Selective discrimination learning impairments in mice ex-

pressing the human Huntington’s disease mutation. J Neurosci19:10428–10437.

Liu B, Wang K, Gao HM, Mandavilli B, Wang JY, Hong JS. 2001.Molecular consequences of activated microglia in the brain: overac-tivation induces apoptosis. J Neurochem 77:182–189.

Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hether-ington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP.1996. Exon 1 of the HD gene with an expanded CAG repeat issufficient to cause a progressive neurological phenotype in trans-genic mice. Cell 87:493–506.

Matsushima GK, Taniike M, Glimcher LH, Grusby MJ, Frelinger JA,Suzuki K, Ting JP. 1994. Absence of MHC class II molecules re-duces CNS demyelination, microglial/macrophage infiltration, andtwitching in murine globoid cell leukodystrophy. Cell 78:645–656.

Meade CA, Deng YP, Fusco FR, Del Mar N, Hersch S, Goldowitz D,Reiner A. 2002. Cellular localization and development of neuronalintranuclear inclusions in striatal and cortical neurons in R6/2transgenic mice. J Comp Neurol 449:241–269.

Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C.2002. The role of macrophage/microglia and astrocytes in the patho-genesis of three neurologic disorders: HIV-associated dementia,Alzheimer disease, and multiple sclerosis. J Neurol Sci 202:13–23.

Morton AJ, Lagan MA, Skepper JN, Dunnett SB. 2000. Progressiveformation of inclusions in the striatum and hippocampus of micetransgenic for the human Huntington’s disease mutation. J Neuro-cytol 29:679–702.

Myers RH, Vonsattel JP, Paskevich PA, Kiely DK, Stevens TJ,Cupples LA, Richardson EP, Bird ED. 1991. Decreased neuronaland increased oligodendroglial densities in Huntington’s diseasecaudate nucleus. J Neuropathol Exp Neurol 50:729–742.

Paris D, Town T, Parker TA, Tan J, Humphrey J, Crawford F, MullanM. 1999. Inhibition of Alzheimer’s beta-amyloid induced vasoactiv-ity and proinflammatory response in microglia by a cGMP-depen-dent mechanism. Exp Neurol 157:211–221.

Polazzi E, Gianni T, Contestabile A. 2001. Microglial cells protectcerebellar granule neurons from apoptosis: evidence for reciprocalsignaling. Glia 36:271–280.

Reddy PH, Williams M, Charles V, Garrett L, Pike-Buchanan L,Whetsell WO Jr, Miller G, Tagle DA. 1998. Behavioural abnormal-ities and selective neuronal loss in HD transgenic mice expressingmutated full-length HD cDNA. Nat Genet 20:198–202.

Ryu JK, Shin WH, Kim J, Joe EH, Lee YB, Cho KG, Oh YJ, Kim SU,Jin BK. 2002. Trisialoganglioside GT1b induces in vivo degenera-tion of nigral dopaminergic neurons: role of microglia. Glia 38:15–23.

Sapp E, Kegel KB, Aronin N, Hashikawa T, Uchiyama Y, Tohyama K,Bhide PG, Vonsattel JP, DiFiglia M. 2001. Early and progressiveaccumulation of reactive microglia in the Huntington disease brain.J Neuropathol Exp Neurol 60:161–172.

Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA,Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG,Price DL, Ross CA, Borchelt DR. 1999. Intranuclear inclusions andneuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 8:397–407.

Sotrel A, Paskevich PA, Kiely DK, Bird ED, Williams RS, Myers RH.1991. Morphometric analysis of the prefrontal cortex in Hunting-ton’s disease. Neurology 41:1117–1123.

Stoll G, Jander S. 1999. The role of microglia and macrophages in thepathophysiology of the CNS. Prog Neurobiol 58:233–247.

Streit WJ, Sparks DL. 1997. Activation of microglia in the brains ofhumans with heart disease and hypercholesterolemic rabbits. J MolMed 75:130–138.

Streit WJ, Hurley SD, McGraw TS, Semple-Rowland SL. 2000. Com-parative evaluation of cytokine profiles and reactive gliosis supportsa critical role for interleukin-6 in neuron-glia signaling duringregeneration. J Neurosci Res 61:10–20.

Streit WJ. 2001. Microglia and macrophages in the developing CNS.Neurotoxicology 22:619–624.

Streit WJ. 2002. Microglia as neuroprotective, immunocompetentcells of the CNS. Glia 40:133–139.

Tseng GF, Wang YJ, Lai QC. 1996. Perineuronal microglial reactivityfollowing proximal and distal axotomy of rat rubrospinal neurons.Brain Res 715:32–43.

Turmaine M, Raza A, Mahal A, Mangiarini L, Bates GP, Davies SW.2000. Nonapoptotic neurodegeneration in a transgenic mouse modelof Huntington’s disease. Proc Natl Acad Sci USA 97:8093–8097.

Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richard-son EP. 1985. Neuropathological classification of Huntington’s dis-ease. J Neuropathol Exp Neurol 44:559–577.

Vonsattel JP, DiFiglia M. 1998. Huntington disease. J NeuropatholExp Neurol 57:369–384.

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