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Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys
Experimental Neurology 184 (2003) 659 – 668 www.elsevier.com/locate/yexnr
Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys S.D. Hurley, a,* M.K. O’Banion, a,b D.D. Song, c F.S. Arana, d J.A. Olschowka, a and S.N. Haber a,d a
Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, NY 14642, USA b Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, USA c Department of Neurology, University of California, San Diego, La Jolla, CA 92093, USA d Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA Received 27 January 2003; revised 28 April 2003; accepted 19 May 2003
Abstract Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration—by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences. D 2003 Elsevier Inc. All rights reserved. Keywords: Substantia nigra; Caudate nucleus; Putamen; Striatum; Globus pallidus; Tyrosine hydroxylase; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Neuronophagia
Introduction The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is used to selectively lesion dopaminergic neurons in the substantia nigra pars compacta (SNc) which give rise to the mesostriatal dopamine (DA) system (Gerlach and Riederer, 1996; Langston et al., 1984; Schmidt and Ferger, 2001; Song and Haber, 2000). Lesioning the mesostriatal DA system with MPTP can produce behavioral and neuropathological changes that mimic the human neurodegenerative disorder, Parkinson’s disease (PD) (Langston et al., 1984). While MPTP-induced neurodegeneration does
* Corresponding author. Department of Neurobiology and Anatomy, University of Rochester Medical Center, 601 Elmwood Avenue, Box 603, Rochester, NY 14642, USA. E-mail address: [email protected] (S.D. Hurley). 0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00273-5
not faithfully mimic all of the features seen in advanced PD [i.e., Lewy bodies (Langston et al., 1983; Schmidt and Ferger, 2001)], animal studies using MPTP have helped to elucidate cellular mechanisms of human PD (Przedborski and Jackson-Lewis, 1998; Tipton and Singer, 1993). Microglia are the resident macrophages of the central nervous system (CNS). Although ubiquitous throughout the CNS, microglia are found at much higher density in the substantia nigra than in other brain regions (Kim et al., 2000; Lawson et al., 1990). Many investigators have reported extensive microglial activation in the mouse substantia nigra following a high-dose MPTP treatment (Dehmer et al., 2000; Francis et al., 1995; Kohutnicka et al., 1998; Kurkowska-Jastrzebska et al., 1999; Liberatore et al., 1999; Vila et al., 2001; Wu et al., 2002). In response to brain injury, microglia undergo transformation from a ‘‘resting’’ morphology into a hypertrophic ‘‘activated’’ morphology (Streit et al., 1988). Associated
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Table 1 Qualitative comparison of TH immunostaining, behavioral change, regional silver degeneration labeling, and regional microglial labeling in five MPTP-treated monkeys and two controls Animal
Control 1 Control 2 101 95 103 125 100
TH staining
Behavioral change
+++ +++ +++ + + + +/
n/a n/a mild moderate moderate moderate severe
Silver degeneration labeling
LN3 labeling of microglia
Substantia nigra
Globus pallidus
Striatum
Substantia nigra
Nigrostriatal tract
Globus pallidus
+++ +++ +++ +++ +++
++ ++ ++ ++ ++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
Putamen
Caudate nucleus
+ + +
TH immunostaining in the substantia nigra was assessed using the following scale: +++, normal levels of TH immunostaining; ++, some loss of labeling; +, significant and substantial loss of labeling; and +/ , nearly complete loss of labeling, particularly in the ventral tier. The behavioral assessment is detailed in Methods. Silver degeneration labeling was assessed using the following scale: , no silver degeneration labeling; +, some labeling, scattered throughout the region; ++, prominent labeling throughout the region; and +++, dense, intense labeling throughout the region, especially of diffuse fibers in neutropil. Microglial immunolabeling with LN3 was assessed using the following scale: , none or few activated microglia; +, some activated microglia, scattered throughout the region; ++, numerous activated microglia; and +++, numerous activated microglia with many multicellular microglia.
with this morphological change, activated microglia express immunogenic cell-surface proteins, proliferate, and actively secrete pro-inflammatory cytokines (Streit et al., 1988,
1999). Activated microglia have long been suspected of contributing to neuronal death, via production of neuronotoxins, nitric oxide, and free radicals (Giulian et al., 1993;
Fig. 1. Histochemical characterization of neurodegeneration in control and MPTP-treated monkeys. (A – D) TH immunostaining of the caudal striatum of control (A) and severely affected (B), moderately affected (C), and mildly affected (D) MPTP-treated monkeys. Note that the partial dopaminergic lesion diminishes TH immunolabeling in the globus pallidus as well as in the striatum. (E – L) TH immunostaining (E – H) and cresyl violet staining (I – L) of midbrain in control (E, I) and severely affected (F, J), moderately affected (G, K), and mildly affected (H, L) MPTP-treated monkeys. In MPTP-treated animals, there is a correlation between loss of TH immunolabeling and neuron loss in the SNc. Scale bar, 1 mm.
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Vila et al., 2001). Indeed, use of the activation inhibitor minocycline suggests that activated microglia significantly augment MPTP-induced neurodegeneration (Du et al., 2001; Wu et al., 2002). However, injury-associated brain macrophages, including activated microglia, are also associated with neuronal sprouting and regeneration (Rabchevsky and Streit, 1997; Streit et al., 1999). For example, microglia and macrophages associated with a striatal stab wound expressed both brain-derived neurotrophic factor and glial-derived neurotrophic factor, and were associated with DA neuron sprouting (Batchelor et al., 1999). Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum, with some behavioral recovery, in monkeys that had received a partial DA lesion with MPTP. Consistent with this plasticity, there was an increase in GAP-43 mRNA expression in the dorsal tier of the SNc (Song and Haber, 2000). A partial DA lesion was induced in these animals using a chronic low-dose MPTP administration paradigm (described in Methods). Animals were sacrificed 4 to 5 weeks following the last
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administration of MPTP when behavioral deficits were stable. Following this administration paradigm, some DA neurons remain in SNc. This model was designed to examine compensatory mechanisms following a partial lesion. In these animals, there was clear evidence of neurodegeneration, 5 weeks following cessation of MPTP administration, as noted by the loss of striatal TH immunoreactivity and the presence of silver degeneration in the striatum and SNc (Song and Haber, 2000). The aims of this study were to characterize the distribution of activated microglia in basal ganglia structures and to characterize the type of microglial response within each structure in these animals. To do this, we utilized the antibody LN3, or HLA-DR, which recognizes the g-invariant chain of the class II major histocompatibility complex (MHC II). MHC II is an activation-associated cell surface glycoprotein that is essential for antigen presentation. This antibody has been used to label microglia in aging monkeys (Sheffield and Berman, 1998; Sloane et al., 1999) and in affected regions of human PD (McGeer et al., 1988a,b,c).
Fig. 2. Comparison of silver degeneration labeling with LN3 labeling of microglia in the level of the caudal striatum (A, B) and midbrain (C, D) in MPTPtreated monkeys. Bright-field photomontages of LN3-positive microglia (B, D) have been color-inverted to better compare microglial distribution with darkfield images of silver degeneration labeling (A, C). Silver degeneration labeling (A) and LN3 labeling (B) of microglia in the caudal striatum of animal 101. Silver degeneration labeling is prominent throughout the putamen. Significant labeling, presumably of fibers of passage, also exists in the globus pallidus. In contrast, LN3-positive microglia are nearly absent from the putamen; yet, a prominent microglial reaction is clearly visible in the globus pallidus. Animal 101 was the animal with the mildest MPTP-induced degeneration. Other animals in this study had similar silver degeneration labeling in the globus pallidus but more silver degeneration labeling in the striatum. Silver degeneration labeling (C) and LN3 labeling (D) of microglia in the midbrain of animal 125. Silver degeneration labeling is prominent throughout the substantia nigra but especially in the ventral tier. Microglial reactivity is prominent throughout the substantia nigra and VTA. Scale bar, 1 mm.
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Methods MPTP treatment and behavioral assessment Seven adult male monkeys (Macaca nemistrina) were used for this study. Food and water were available ad libitum. Animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Five monkeys were treated with 0.15 mg/kg of MPTP two or three times a week, and two served as normal controls. MPTP treatment was terminated within 48 h of the emergence of bradykinesia, action tremor, stooped posture, or change in gait. One case was mildly affected behaviorally with minimal bradykinesia and a small tremor of the left hand. Three cases were moderately affected with moderate bradykinesia, stooped postures, and some difficulties with balance and gait, but were able to independently feed and groom. One case was severely affected with frequent akinesia (freezing), very stooped posture, and severe difficulty in moving. This animal required assisted feeding and did not groom. Histology All cases were euthanized 4 to 5 weeks following the last MPTP treatment. Monkeys were deeply anesthetized with pentobarbital and killed by intracardiac perfusion with saline followed by a 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4. The brains were removed and cryoprotected in increasing gradients of sucrose (10%, 20%, and finally 30%). Fifty-micrometer serial sections
were cut on a freezing microtome. Sections were processed for cresyl violet staining or for TH and LN3 immunohistochemistry. In addition, selected sections from each animal were processed by Neuroscience Associates (Knoxville, TN) for degeneration using the aminocupric acid silver stain (de Olmos et al., 1994). Tissue processed for LN3 immunolabeling was incubated with the LN3 antibody (1:100, ICN) overnight at 4 jC in 0.1 M phosphate buffer with 0.4% Triton X-100 and 1% normal goat serum. For TH immunohistochemistry, sections were incubated with antisera to TH (1:20,000; Eugene Tech) for four nights at 4 jC in 0.1 M phosphate buffer with 0.3% Triton X-100 and 10% normal goat serum. Both immunostains were completed using the avidin – biotin method (Elite Vectastain ABC kit; Vector Laboratories) followed by incubation with filtered 0.05% 3,3V-diaminobenzidine tetrahydrochloride solution in Tris buffer, using H2O2 as a reaction catalyst. Charting of labeled cells LN3-labeled sections containing caudal striatum or midbrain were selected for charting. Using dark-field illumination with a camera lucida drawing tube, a map of basal ganglia structures with anatomical landmarks was prepared for each section. Following this, a digital photomontage of LN3 labeling within basal ganglia structures was constructed. This was performed using Photoshop software (Adobe) with images taken from a Spot RT color camera (Diagnostic Instruments) attached to an Axioplan II microscope (Zeiss). The map of basal ganglia structures was then matched with the photomontage using Illustrator software (Adobe). Using
Fig. 3. Charts of the distribution of activated and multicellular LN3-positive microglia at the level of the caudal striatum (A – D) and midbrain (E – H) in control (A, E) and severely affected (B, F), moderately affected (C, G), and mildly affected (D, H) MPTP-treated monkeys. Each dot represents one LN3-positive microglia that had either an activated or multicellular morphology. These morphologies were mostly absent from the controls (A, E) and were predominantly seen in the substantia nigra and globus pallidus of MPTP-treated animals (B – D, F – H). Relatively few activated morphologies and no multicellular forms were seen in the caudate nucleus or putamen of MPTP-treated animals. The distribution of activated and multicellular LN3-positive microglia was remarkably similar across all MPTP-treated animals, regardless of phenotypic severity.
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Illustrator, this composite image was magnified to facilitate morphological classification of microglia. Individual activated and multicellular microglia were charted. Activated microglia were identified as possessing a swollen perinuclear cytoplasm with swollen, shortened processes. Multicellular microglia were identified on the basis of size, being at least twice as large as activated microglia. Ramified, LN3-labeled microglia were not charted.
Results Characterization of lesion severity MPTP-treated monkeys were assessed behaviorally and histologically (Song and Haber, 2000). Animals were grouped into three categories according to the degree of Parkinson-like symptoms (Table 1): mildly affected, moderately affected, and severely affected. Nigral cell loss was substantial in moderate and severely affected animals (Fig. 1). This loss was primarily in the ventral tier. Loss of TH-positive immunoreactivity within
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the substantia nigra was also obvious in moderate and severely affected animals. In adjacent sections, the loss of TH immunoreactivity seemed to coincide with the presence of silver degeneration labeling (data not shown). In moderate and mildly affected animals, silver degeneration labeling was most prominent in the ventral tier. The loss of striatal TH-positive immunoreactivity was most severe in the severely affected animal (Fig. 1). In moderately affected animals, TH immunostaining was absent in the caudate nucleus and dorsal putamen and largely present in the ventral striatum. TH immunostaining in the mildly affected animal was similar to the pattern seen in naı¨ve control. Intense silver degeneration labeling was seen in the striatum of all MPTP-treated animals (Table 1; Fig. 2; see also: Song and Haber, 2000). However, the intensity of silver degeneration labeling seemed related to TH-positive fiber loss, with the most severely affected animal showing the most intense striatal silver degeneration labeling. Previously we have shown, using double labeling, that TH immunostaining and silver degeneration labeling do not label the same nerve fibers in the striatum (Song and Haber, 2000).
Fig. 4. Heterogeneous microglial involvement in the degenerating substantia nigra following MPTP treatment. (A) Microglia respond in a heterogeneous manner with numerous commingled ramified, activated, and multicellular LN3-positive morphologies scattered throughout the substantia nigra. (B) In nontreated, control monkeys, few, lightly stained, highly ramified microglia are present. (C) A representative multinuclear microglia found in MPTP-treated substantia nigra. The presence of multicellular microglia (C) in the substantia nigra of MPTP-treated monkeys provides indirect evidence of active phagocytosis as does the presence, in many MPTP-treated monkeys, of strongly labeled fat granules (D). (D) A fat granule (arrow), an end product of phagocytosis, is contiguous with a lightly labeled ramified microglia (arrowhead). (E) An example of marginating LN3-positive blood-borne cells, in VTA of animal 101, a mildly affected animal. Margination was also observed in the VTA of animal 100, the most severely affected animal. This phenomenon was present around a few scattered vessels in the VTA. Scale bar, 50 Am.
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The control globus pallidus showed less TH immunoreactivity than the striatum. Nonetheless, a noticeable loss of palladial TH-positive immunoreactivity was seen in moderate and severely affected animals. This was accompanied by some silver degeneration labeling (Table 1). The intensity of silver degeneration labeling was much less in the globus pallidus than in the striatum, especially in moderately and severely affected animals. The microglial reaction In control monkeys, LN3 labeling of microglia was nearly negligible in basal ganglia structures. In contrast, LN3 antibody labeling revealed a pattern of microglial activation that was common to all monkeys exposed to MPTP. In particular, microglial activation was prominent in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus (Table 1, Figs. 2 and 3). Relatively little microglial labeling was seen in either the caudate nucleus or putamen. This is in marked contrast to the pattern of degeneration, as noted by loss of TH immunostaining (Fig. 1) and the presence of silver degeneration labeling (Fig. 2). In control animals, few, lightly labeled LN3-positive microglia were observed scattered throughout the substantia nigra and ventral tegmental area (VTA) (Fig. 4B). In all MPTP-treated monkeys, microglia were strongly labeled with LN3 throughout the entire extent of the substantia nigra and VTA (Fig. 4A). These immunolabeled microglia were morphologically heterogeneous. Microglia in the substantia nigra of MPTP-treated monkeys had three distinct phenotypes. The most common phenotype was a ramified morphology, with scant perinuclear cytoplasm and many branching processes. This morphology is typical of microglia in the naı¨ve (control) CNS, though staining intensity was stronger. Also seen were many microglia with an activated morphology. These microglia had a swollen perinuclear cytoplasm and, compared to ramified microglia, fewer and enlarged processes (Fig. 4A). Also prevalent in the substantia nigra and VTA were multicellular microglia. These giant cells were at least two, often five, times as large as an activated microglia. These cells displayed relatively few, shortened processes. All three morphologies were distributed more or less evenly, in a commingled fashion, throughout the substantia nigra and VTA of MPTP-treated monkeys. Within the substantia nigra and VTA of MPTP-treated animals, there was ample indirect evidence of active phagocytosis. This included the presence of numerous multicellular microglia (Fig. 4C). In addition, most but not all MPTP-treated monkeys possessed strongly labeled fat granules, an end product of phagocytosis (Fig. 4D). These fat granules varied in size between 10 and 30 Am in diameter, and were not seen in the most severely affected monkey. Fat granules were most often associated with ramified microglia.
In addition to the microglial response, marginating LN3positive blood-borne cells were present in the VTA of two animals, the mildly affected and the severely affected animals (Fig. 4E). Margination was not seen in the substantia nigra proper nor in other basal ganglia structures. There was almost no LN3 labeling of microglia in the control nigrostriatal tract (Fig. 5B). In contrast, there was a prominent microglial reaction in MPTP-treated monkeys (Fig. 5A). This reaction, as in the substantia nigra, was marked by the presence of intensely labeled, commingled ramified, activated, and multicellular microglia. Numerous fat granules were seen in all animals except in the most severely affected case. LN3 labeling of the striatum in control animals showed few labeled microglia. Rather, the most common cell type labeled with this antibody in control animals were scattered perivascular macrophages—a non-microglial cell population. In MPTP-treated monkeys, there was little change in the striatal LN3 staining pattern (Figs. 6C, D). A few activated microglia were visible. These were scattered widely throughout the striatum. Most of the microglia that were labeled in the striatum possessed a ramified morphol-
Fig. 5. A prominent microglial response is present in the nigrostriatal tract of MPTP-treated monkeys. (A) A heterogeneous response, similar to that in the substantia nigra, is observed in the nigrostriatal tract with numerous multicellular and activated LN3-positive microglia commingled with LN3positive ramified microglia. (B) In contrast, almost no staining is present in the same brain region of control animals. Scale bar, 50 Am.
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Fig. 6. Both internal and external segments of the globus pallidus display a prominent, heterogeneous microglial response to MPTP treatment, whereas the caudate nucleus and putamen do not. (A) The microglial response to MPTP treatment in the globus pallidus is similar to that in the substantia nigra and nigrostriatal tract, with numerous activated and multicellular microglia commingled with morphologically ramified microglia. (B) The globus pallidus in control animals shows relatively few, lightly labeled, highly ramified LN3-labeled microglia. (C, D) The putamen (C) and caudate nucleus (D) possess some scattered LN3-positive ramified microglia, with few activated and no multicellular microglia. Scale bar, 50 Am.
ogy. There appeared to be a lack of active phagocytosis as no multicellular microglia and no fat granules were observed in the striatum of MPTP-treated animals. In control animals, lightly labeled LN3-positive ramified microglia were observed throughout the globus pallidus (Fig. 6B). In MPTP-treated animals, both the internal and external segments of the globus pallidus (Fig. 6A) displayed widespread microglial activation. As in the substantia nigra and nigrostriatal tract of MPTP-treated animals, the microglial reaction in the globus pallidus was heterogeneous with commingled ramified, activated, and multicellular morphologies. Also present were scattered fat granules, although less prevalent than in the substantia nigra. As within the substantia nigra, these fat granules were not seen in the most severely affected case.
Discussion The microglial reaction appears to be independent of degeneration Five weeks following chronic, low-dose MPTP treatment, we observed an intense microglial reaction in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically
heterogeneous and was notable for the presence of multicellular microglia, a hallmark of an active phagocytic response. Most animals also displayed the presence of numerous fat granules, an end product of phagocytosis. Surprisingly, there was little evidence of microglial reactivity in the striatum. This pattern of microglial reactivity was independent of the behavioral and morphological consequences of MPTP neurotoxicity. The observed pattern of microglial reactivity was the same in all MPTP-treated animals regardless of the degree of degeneration. It is clear in this and in our previous study (Song and Haber, 2000) that there is significant degeneration in both the striatum and SNc with this animal model. In contrast, the segments of the globus pallidus are thought to be spared from MPTP-mediated neurotoxicity (Langston et al., 1984): the globus pallidus is a relatively minor target for DA innervation (Jan et al., 2000) and there is no neuronal loss in the pallidum with PD (Hardman and Halliday, 1999a,b). However, it is worth noting that there is some degeneration in the pallidum, given both the presence of silver degeneration labeling and a decreased level of TH immunostaining. It is likely that much of the microglial reaction in the globus pallidus is stimulated by the degeneration of fibers of passage—nigral projections that pass through the globus pallidus to the striatum.
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Thus, the microglial response in this animal model is enigmatic. Microglia are reactive in the SNc, a region with significant continuing degeneration, as well as in the globus pallidus, a region with relatively minor degeneration. Yet, the microglial reaction appears to be entirely lacking in the striatum, where there is significant evidence of degeneration.
morphological heterogeneity seen in PD and in our animal model represents a common feature of chronic neurodegenerative processes. Indeed, similar patterns of microglial reactivity are seen in other neurodegenerative diseases, including Alzheimer’s disease, as well as during Wallerian degeneration (McGeer et al., 1988a,b; Perlmutter et al., 1992; Schmitt et al., 2000).
Similarities and differences with rodent MPTP models
Caveats with using an antibody to MHC II
In rodent models, there is extensive microglial reactivity in both the substantia nigra and striatum following highdose, acute MPTP treatment regimens (Dehmer et al., 2000; Francis et al., 1995; Kohutnicka et al., 1998; Kurkowska-Jastrzebska et al., 1999; Liberatore et al., 1999; Vila et al., 2001; Wu et al., 2002). This activation is accompanied by MHC II expression (Kurkowska-Jastrzebska et al., 1999). While not specifically addressed in their results, many of the figures from these papers document a heterogeneous microglial reaction—with commingling of multicellular, activated, and some ramified microglia (see especially: Kurkowska-Jastrzebska et al., 1999). In these rodent models, the process of neuron loss occurs in a matter of days, while the microglial reaction begins to diminish within 1 week (Kurkowska-Jastrzebska et al., 1999). Thus, despite morphological similarities, there are key differences in the time course and location of the microglial reaction to MPTP treatment in mice and in our monkeys. These differences are likely to be due to differences in MPTP administration paradigms (the time course and dosage of administration), although species differences in the microglial responsiveness cannot be discounted.
One concern in doing monkey or human work is the unavailability of ‘‘good’’ markers for microglia—markers that label all microglia and allow one to clearly distinguish morphological phenotypes. Antibodies to antigens that are routinely stained in mouse or rat often fail to work in human or monkey fixed tissue [i.e., the type 3 complement receptor (also known as OX-42 or Mac-1)]. Many immunoantigens that work in human often fail to label the full microglial morphology (Knott et al., 1999; Mirza et al., 2000). This is also the case with other microglial markers, like cyclooxygenase-1 (Yermakova et al., 1999). In staining for human microglia, LN3, an antibody to MHC II, represents a useful compromise. MHC II expression by microglia is widely variable in postmortem samples, but tends to be more sensitive than the lectin Ricinus communis (Engel et al., 1996). In addition, labeling of MHC II with LN3 tends to increase with increasing age. Thus, in samples from middle-aged or elderly individuals, one can be confident that nearly all microglia will be labeled and that microglial morphologies can be clearly discerned (Streit and Sparks, 1997). However, this is not the case in our monkey tissue. In control brains, relatively few microglia were labeled, and those that were labeled were labeled lightly. This was also the case in cortical and thalamic regions of MPTP-treated monkeys—brain regions thought to be unaffected by MPTP-induced neurodegeneration. Therefore, one must be careful in interpreting an apparent lack of a microglial staining. However, MHC II expression by microglia has been consistently linked with phagocytosis in Wallerian degeneration (Konno et al., 1989; Schmitt et al., 2000; Watanabe et al., 1999), in response to ricin-induced neurodegeneration (Streit et al., 1988), and, in vitro, in response to apoptosis by commingled T cells (Magnus et al., 2001). Given this, we interpret the lack of MHC II expression by microglia in the striatum as indicating that there is at most a negligible microglial reaction to degeneration in the striatum.
Similarities to human PD Our findings are similar to what has been described in postmortem samples of PD patients. McGeer et al. (1988a,b,c) described numerous activated and phagocytic microglia in the substantia nigra and nigrostriatal tract of PD patients. These microglia were morphologically heterogeneous, and there was ample evidence of active phagocytosis. These findings have been confirmed by two other groups (Knott et al., 1999; Mirza et al., 2000). Interestingly, these investigators failed to find any microglial reactivity in the striatum (Knott et al., 1999; Mirza et al., 2000). Microglia in PD and in our MPTP administration paradigm respond in a morphologically heterogeneous fashion. Classically, microglial morphology is the best indicator of microglial phenotype (Rio-Hortega, 1932; Streit et al., 1988). In response to acute brain injury, like middle cerebral artery occlusion (Morioka et al., 1993), spreading depression (Gehrmann et al., 1993), or traumatic brain injury (Carbonell and Grady, 1999), microglia within the zone of injury will all show an activated or phagocytic morphology (Streit et al., 1988). We suggest that the
A disconnect between microglial reactivity and neuropathology? Our results appear to illustrate a disconnect between microglial reactivity and neuropathology—a concept previously suggested by McGeer et al. (1988a). First, we saw no microglial reactivity in the striatum, a brain region with
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substantial MPTP-induced neurodegeneration. At the same time, we saw an unexpectedly substantial microglial reaction in an area thought to be unaffected by MPTP-induced neurodegeneration, the globus pallidus. Second, the pattern of microglial reactivity was similar across all MPTPtreated animals regardless of the behavioral or morphological severity of neurodegeneration. Third, this disconnection between microglial reactivity and neuropathology does not appear related to the temporal aspects of our animal model (i.e., to the fact that the animals were euthanized 4 to 5 weeks following their last dose of MPTP). In one animal that was sacrificed at the cessation of MPTP administration, the pattern of microglial labeling was similar to that described here—with a substantial microglial reaction visible in the substantia nigra and globus pallidus, but no apparent striatal microglial reactivity (data not shown). Finally, it is likely that the silver degeneration labeling that we see in the striatum represents DA processes that have never been phagocytosed. Silver degeneration labeling binds to exposed neurofilaments from neurons that have lost cellular integrity (Scallet, 1995). Yet, despite the large quantity of striatal cellular debris, there is no appreciable microglial reaction. Thus, in this model, it appears that microglial reactivity colocalizes with neurodegeneration in some brain regions—the substantia nigra, the nigrostriatal tract, and the globus pallidus—but not in striatum. Nonetheless, a great body of literature supports the notion that microglia are ‘‘neuropathological sensors’’ within the CNS (Kreutzberg, 1996)—the idea that microglia undergo rapid activation in response to even minor pathological changes. Perhaps the clue to resolve this discrepancy lies in the morphological heterogeneity itself. In response to many injuries, microglia, which are exquisitely plastic cells, alter their morphology (Rio-Hortega, 1932; Streit et al., 1988). This alteration can occur within minutes of an acute traumatic injury. The signals that trigger microglial activation are only beginning to become known. These include pro-inflammatory factors like cytokines and chemokines, extracellular ATP (Inoue, 2002), neuropeptides (Priller et al., 1995), changes in extracellular potassium (Abraham et al., 2001), and serum proteins (Perry et al., 1992). It is also appreciated that the CNS itself is an ‘‘immunosuppressed’’ environment (Perry and Gordon, 1991). One contributing factor to the endogenous immunosuppression of the CNS is the expression of OX-2 (CD200) by neurons (Hoek et al., 2000). The fact that ‘‘activated’’ morphologies are commingled with ‘‘resting’’ morphologies suggests a microenvironment where pro-inflammatory and anti-inflammatory stimuli compete for microglial attention. This line of reasoning could potentially explain the failure to observe a striatal microglial reaction in our model and in PD: there is the possibility that anti-inflammatory signals may predominate in the striatum relative to other basal ganglia structures.
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Acknowledgments The authors thank Andrew Corey and April Whitbeck for their technical expertise. This work was supported by NIH NS38577 (S.D.H., J.A.O.) and NIH MH63324 (S.N.H.).
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Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys S.D. Hurley, a,* M.K. O’Banion, a,b D.D. Song, c F.S. Arana, d J.A. Olschowka, a and S.N. Haber a,d a
Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, NY 14642, USA b Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, USA c Department of Neurology, University of California, San Diego, La Jolla, CA 92093, USA d Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA Received 27 January 2003; revised 28 April 2003; accepted 19 May 2003
Abstract Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration—by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences. D 2003 Elsevier Inc. All rights reserved. Keywords: Substantia nigra; Caudate nucleus; Putamen; Striatum; Globus pallidus; Tyrosine hydroxylase; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Neuronophagia
Introduction The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is used to selectively lesion dopaminergic neurons in the substantia nigra pars compacta (SNc) which give rise to the mesostriatal dopamine (DA) system (Gerlach and Riederer, 1996; Langston et al., 1984; Schmidt and Ferger, 2001; Song and Haber, 2000). Lesioning the mesostriatal DA system with MPTP can produce behavioral and neuropathological changes that mimic the human neurodegenerative disorder, Parkinson’s disease (PD) (Langston et al., 1984). While MPTP-induced neurodegeneration does
* Corresponding author. Department of Neurobiology and Anatomy, University of Rochester Medical Center, 601 Elmwood Avenue, Box 603, Rochester, NY 14642, USA. E-mail address: [email protected] (S.D. Hurley). 0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00273-5
not faithfully mimic all of the features seen in advanced PD [i.e., Lewy bodies (Langston et al., 1983; Schmidt and Ferger, 2001)], animal studies using MPTP have helped to elucidate cellular mechanisms of human PD (Przedborski and Jackson-Lewis, 1998; Tipton and Singer, 1993). Microglia are the resident macrophages of the central nervous system (CNS). Although ubiquitous throughout the CNS, microglia are found at much higher density in the substantia nigra than in other brain regions (Kim et al., 2000; Lawson et al., 1990). Many investigators have reported extensive microglial activation in the mouse substantia nigra following a high-dose MPTP treatment (Dehmer et al., 2000; Francis et al., 1995; Kohutnicka et al., 1998; Kurkowska-Jastrzebska et al., 1999; Liberatore et al., 1999; Vila et al., 2001; Wu et al., 2002). In response to brain injury, microglia undergo transformation from a ‘‘resting’’ morphology into a hypertrophic ‘‘activated’’ morphology (Streit et al., 1988). Associated
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Table 1 Qualitative comparison of TH immunostaining, behavioral change, regional silver degeneration labeling, and regional microglial labeling in five MPTP-treated monkeys and two controls Animal
Control 1 Control 2 101 95 103 125 100
TH staining
Behavioral change
+++ +++ +++ + + + +/
n/a n/a mild moderate moderate moderate severe
Silver degeneration labeling
LN3 labeling of microglia
Substantia nigra
Globus pallidus
Striatum
Substantia nigra
Nigrostriatal tract
Globus pallidus
+++ +++ +++ +++ +++
++ ++ ++ ++ ++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
+++ +++ +++ +++ +++
Putamen
Caudate nucleus
+ + +
TH immunostaining in the substantia nigra was assessed using the following scale: +++, normal levels of TH immunostaining; ++, some loss of labeling; +, significant and substantial loss of labeling; and +/ , nearly complete loss of labeling, particularly in the ventral tier. The behavioral assessment is detailed in Methods. Silver degeneration labeling was assessed using the following scale: , no silver degeneration labeling; +, some labeling, scattered throughout the region; ++, prominent labeling throughout the region; and +++, dense, intense labeling throughout the region, especially of diffuse fibers in neutropil. Microglial immunolabeling with LN3 was assessed using the following scale: , none or few activated microglia; +, some activated microglia, scattered throughout the region; ++, numerous activated microglia; and +++, numerous activated microglia with many multicellular microglia.
with this morphological change, activated microglia express immunogenic cell-surface proteins, proliferate, and actively secrete pro-inflammatory cytokines (Streit et al., 1988,
1999). Activated microglia have long been suspected of contributing to neuronal death, via production of neuronotoxins, nitric oxide, and free radicals (Giulian et al., 1993;
Fig. 1. Histochemical characterization of neurodegeneration in control and MPTP-treated monkeys. (A – D) TH immunostaining of the caudal striatum of control (A) and severely affected (B), moderately affected (C), and mildly affected (D) MPTP-treated monkeys. Note that the partial dopaminergic lesion diminishes TH immunolabeling in the globus pallidus as well as in the striatum. (E – L) TH immunostaining (E – H) and cresyl violet staining (I – L) of midbrain in control (E, I) and severely affected (F, J), moderately affected (G, K), and mildly affected (H, L) MPTP-treated monkeys. In MPTP-treated animals, there is a correlation between loss of TH immunolabeling and neuron loss in the SNc. Scale bar, 1 mm.
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Vila et al., 2001). Indeed, use of the activation inhibitor minocycline suggests that activated microglia significantly augment MPTP-induced neurodegeneration (Du et al., 2001; Wu et al., 2002). However, injury-associated brain macrophages, including activated microglia, are also associated with neuronal sprouting and regeneration (Rabchevsky and Streit, 1997; Streit et al., 1999). For example, microglia and macrophages associated with a striatal stab wound expressed both brain-derived neurotrophic factor and glial-derived neurotrophic factor, and were associated with DA neuron sprouting (Batchelor et al., 1999). Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum, with some behavioral recovery, in monkeys that had received a partial DA lesion with MPTP. Consistent with this plasticity, there was an increase in GAP-43 mRNA expression in the dorsal tier of the SNc (Song and Haber, 2000). A partial DA lesion was induced in these animals using a chronic low-dose MPTP administration paradigm (described in Methods). Animals were sacrificed 4 to 5 weeks following the last
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administration of MPTP when behavioral deficits were stable. Following this administration paradigm, some DA neurons remain in SNc. This model was designed to examine compensatory mechanisms following a partial lesion. In these animals, there was clear evidence of neurodegeneration, 5 weeks following cessation of MPTP administration, as noted by the loss of striatal TH immunoreactivity and the presence of silver degeneration in the striatum and SNc (Song and Haber, 2000). The aims of this study were to characterize the distribution of activated microglia in basal ganglia structures and to characterize the type of microglial response within each structure in these animals. To do this, we utilized the antibody LN3, or HLA-DR, which recognizes the g-invariant chain of the class II major histocompatibility complex (MHC II). MHC II is an activation-associated cell surface glycoprotein that is essential for antigen presentation. This antibody has been used to label microglia in aging monkeys (Sheffield and Berman, 1998; Sloane et al., 1999) and in affected regions of human PD (McGeer et al., 1988a,b,c).
Fig. 2. Comparison of silver degeneration labeling with LN3 labeling of microglia in the level of the caudal striatum (A, B) and midbrain (C, D) in MPTPtreated monkeys. Bright-field photomontages of LN3-positive microglia (B, D) have been color-inverted to better compare microglial distribution with darkfield images of silver degeneration labeling (A, C). Silver degeneration labeling (A) and LN3 labeling (B) of microglia in the caudal striatum of animal 101. Silver degeneration labeling is prominent throughout the putamen. Significant labeling, presumably of fibers of passage, also exists in the globus pallidus. In contrast, LN3-positive microglia are nearly absent from the putamen; yet, a prominent microglial reaction is clearly visible in the globus pallidus. Animal 101 was the animal with the mildest MPTP-induced degeneration. Other animals in this study had similar silver degeneration labeling in the globus pallidus but more silver degeneration labeling in the striatum. Silver degeneration labeling (C) and LN3 labeling (D) of microglia in the midbrain of animal 125. Silver degeneration labeling is prominent throughout the substantia nigra but especially in the ventral tier. Microglial reactivity is prominent throughout the substantia nigra and VTA. Scale bar, 1 mm.
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Methods MPTP treatment and behavioral assessment Seven adult male monkeys (Macaca nemistrina) were used for this study. Food and water were available ad libitum. Animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Five monkeys were treated with 0.15 mg/kg of MPTP two or three times a week, and two served as normal controls. MPTP treatment was terminated within 48 h of the emergence of bradykinesia, action tremor, stooped posture, or change in gait. One case was mildly affected behaviorally with minimal bradykinesia and a small tremor of the left hand. Three cases were moderately affected with moderate bradykinesia, stooped postures, and some difficulties with balance and gait, but were able to independently feed and groom. One case was severely affected with frequent akinesia (freezing), very stooped posture, and severe difficulty in moving. This animal required assisted feeding and did not groom. Histology All cases were euthanized 4 to 5 weeks following the last MPTP treatment. Monkeys were deeply anesthetized with pentobarbital and killed by intracardiac perfusion with saline followed by a 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4. The brains were removed and cryoprotected in increasing gradients of sucrose (10%, 20%, and finally 30%). Fifty-micrometer serial sections
were cut on a freezing microtome. Sections were processed for cresyl violet staining or for TH and LN3 immunohistochemistry. In addition, selected sections from each animal were processed by Neuroscience Associates (Knoxville, TN) for degeneration using the aminocupric acid silver stain (de Olmos et al., 1994). Tissue processed for LN3 immunolabeling was incubated with the LN3 antibody (1:100, ICN) overnight at 4 jC in 0.1 M phosphate buffer with 0.4% Triton X-100 and 1% normal goat serum. For TH immunohistochemistry, sections were incubated with antisera to TH (1:20,000; Eugene Tech) for four nights at 4 jC in 0.1 M phosphate buffer with 0.3% Triton X-100 and 10% normal goat serum. Both immunostains were completed using the avidin – biotin method (Elite Vectastain ABC kit; Vector Laboratories) followed by incubation with filtered 0.05% 3,3V-diaminobenzidine tetrahydrochloride solution in Tris buffer, using H2O2 as a reaction catalyst. Charting of labeled cells LN3-labeled sections containing caudal striatum or midbrain were selected for charting. Using dark-field illumination with a camera lucida drawing tube, a map of basal ganglia structures with anatomical landmarks was prepared for each section. Following this, a digital photomontage of LN3 labeling within basal ganglia structures was constructed. This was performed using Photoshop software (Adobe) with images taken from a Spot RT color camera (Diagnostic Instruments) attached to an Axioplan II microscope (Zeiss). The map of basal ganglia structures was then matched with the photomontage using Illustrator software (Adobe). Using
Fig. 3. Charts of the distribution of activated and multicellular LN3-positive microglia at the level of the caudal striatum (A – D) and midbrain (E – H) in control (A, E) and severely affected (B, F), moderately affected (C, G), and mildly affected (D, H) MPTP-treated monkeys. Each dot represents one LN3-positive microglia that had either an activated or multicellular morphology. These morphologies were mostly absent from the controls (A, E) and were predominantly seen in the substantia nigra and globus pallidus of MPTP-treated animals (B – D, F – H). Relatively few activated morphologies and no multicellular forms were seen in the caudate nucleus or putamen of MPTP-treated animals. The distribution of activated and multicellular LN3-positive microglia was remarkably similar across all MPTP-treated animals, regardless of phenotypic severity.
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Illustrator, this composite image was magnified to facilitate morphological classification of microglia. Individual activated and multicellular microglia were charted. Activated microglia were identified as possessing a swollen perinuclear cytoplasm with swollen, shortened processes. Multicellular microglia were identified on the basis of size, being at least twice as large as activated microglia. Ramified, LN3-labeled microglia were not charted.
Results Characterization of lesion severity MPTP-treated monkeys were assessed behaviorally and histologically (Song and Haber, 2000). Animals were grouped into three categories according to the degree of Parkinson-like symptoms (Table 1): mildly affected, moderately affected, and severely affected. Nigral cell loss was substantial in moderate and severely affected animals (Fig. 1). This loss was primarily in the ventral tier. Loss of TH-positive immunoreactivity within
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the substantia nigra was also obvious in moderate and severely affected animals. In adjacent sections, the loss of TH immunoreactivity seemed to coincide with the presence of silver degeneration labeling (data not shown). In moderate and mildly affected animals, silver degeneration labeling was most prominent in the ventral tier. The loss of striatal TH-positive immunoreactivity was most severe in the severely affected animal (Fig. 1). In moderately affected animals, TH immunostaining was absent in the caudate nucleus and dorsal putamen and largely present in the ventral striatum. TH immunostaining in the mildly affected animal was similar to the pattern seen in naı¨ve control. Intense silver degeneration labeling was seen in the striatum of all MPTP-treated animals (Table 1; Fig. 2; see also: Song and Haber, 2000). However, the intensity of silver degeneration labeling seemed related to TH-positive fiber loss, with the most severely affected animal showing the most intense striatal silver degeneration labeling. Previously we have shown, using double labeling, that TH immunostaining and silver degeneration labeling do not label the same nerve fibers in the striatum (Song and Haber, 2000).
Fig. 4. Heterogeneous microglial involvement in the degenerating substantia nigra following MPTP treatment. (A) Microglia respond in a heterogeneous manner with numerous commingled ramified, activated, and multicellular LN3-positive morphologies scattered throughout the substantia nigra. (B) In nontreated, control monkeys, few, lightly stained, highly ramified microglia are present. (C) A representative multinuclear microglia found in MPTP-treated substantia nigra. The presence of multicellular microglia (C) in the substantia nigra of MPTP-treated monkeys provides indirect evidence of active phagocytosis as does the presence, in many MPTP-treated monkeys, of strongly labeled fat granules (D). (D) A fat granule (arrow), an end product of phagocytosis, is contiguous with a lightly labeled ramified microglia (arrowhead). (E) An example of marginating LN3-positive blood-borne cells, in VTA of animal 101, a mildly affected animal. Margination was also observed in the VTA of animal 100, the most severely affected animal. This phenomenon was present around a few scattered vessels in the VTA. Scale bar, 50 Am.
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The control globus pallidus showed less TH immunoreactivity than the striatum. Nonetheless, a noticeable loss of palladial TH-positive immunoreactivity was seen in moderate and severely affected animals. This was accompanied by some silver degeneration labeling (Table 1). The intensity of silver degeneration labeling was much less in the globus pallidus than in the striatum, especially in moderately and severely affected animals. The microglial reaction In control monkeys, LN3 labeling of microglia was nearly negligible in basal ganglia structures. In contrast, LN3 antibody labeling revealed a pattern of microglial activation that was common to all monkeys exposed to MPTP. In particular, microglial activation was prominent in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus (Table 1, Figs. 2 and 3). Relatively little microglial labeling was seen in either the caudate nucleus or putamen. This is in marked contrast to the pattern of degeneration, as noted by loss of TH immunostaining (Fig. 1) and the presence of silver degeneration labeling (Fig. 2). In control animals, few, lightly labeled LN3-positive microglia were observed scattered throughout the substantia nigra and ventral tegmental area (VTA) (Fig. 4B). In all MPTP-treated monkeys, microglia were strongly labeled with LN3 throughout the entire extent of the substantia nigra and VTA (Fig. 4A). These immunolabeled microglia were morphologically heterogeneous. Microglia in the substantia nigra of MPTP-treated monkeys had three distinct phenotypes. The most common phenotype was a ramified morphology, with scant perinuclear cytoplasm and many branching processes. This morphology is typical of microglia in the naı¨ve (control) CNS, though staining intensity was stronger. Also seen were many microglia with an activated morphology. These microglia had a swollen perinuclear cytoplasm and, compared to ramified microglia, fewer and enlarged processes (Fig. 4A). Also prevalent in the substantia nigra and VTA were multicellular microglia. These giant cells were at least two, often five, times as large as an activated microglia. These cells displayed relatively few, shortened processes. All three morphologies were distributed more or less evenly, in a commingled fashion, throughout the substantia nigra and VTA of MPTP-treated monkeys. Within the substantia nigra and VTA of MPTP-treated animals, there was ample indirect evidence of active phagocytosis. This included the presence of numerous multicellular microglia (Fig. 4C). In addition, most but not all MPTP-treated monkeys possessed strongly labeled fat granules, an end product of phagocytosis (Fig. 4D). These fat granules varied in size between 10 and 30 Am in diameter, and were not seen in the most severely affected monkey. Fat granules were most often associated with ramified microglia.
In addition to the microglial response, marginating LN3positive blood-borne cells were present in the VTA of two animals, the mildly affected and the severely affected animals (Fig. 4E). Margination was not seen in the substantia nigra proper nor in other basal ganglia structures. There was almost no LN3 labeling of microglia in the control nigrostriatal tract (Fig. 5B). In contrast, there was a prominent microglial reaction in MPTP-treated monkeys (Fig. 5A). This reaction, as in the substantia nigra, was marked by the presence of intensely labeled, commingled ramified, activated, and multicellular microglia. Numerous fat granules were seen in all animals except in the most severely affected case. LN3 labeling of the striatum in control animals showed few labeled microglia. Rather, the most common cell type labeled with this antibody in control animals were scattered perivascular macrophages—a non-microglial cell population. In MPTP-treated monkeys, there was little change in the striatal LN3 staining pattern (Figs. 6C, D). A few activated microglia were visible. These were scattered widely throughout the striatum. Most of the microglia that were labeled in the striatum possessed a ramified morphol-
Fig. 5. A prominent microglial response is present in the nigrostriatal tract of MPTP-treated monkeys. (A) A heterogeneous response, similar to that in the substantia nigra, is observed in the nigrostriatal tract with numerous multicellular and activated LN3-positive microglia commingled with LN3positive ramified microglia. (B) In contrast, almost no staining is present in the same brain region of control animals. Scale bar, 50 Am.
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Fig. 6. Both internal and external segments of the globus pallidus display a prominent, heterogeneous microglial response to MPTP treatment, whereas the caudate nucleus and putamen do not. (A) The microglial response to MPTP treatment in the globus pallidus is similar to that in the substantia nigra and nigrostriatal tract, with numerous activated and multicellular microglia commingled with morphologically ramified microglia. (B) The globus pallidus in control animals shows relatively few, lightly labeled, highly ramified LN3-labeled microglia. (C, D) The putamen (C) and caudate nucleus (D) possess some scattered LN3-positive ramified microglia, with few activated and no multicellular microglia. Scale bar, 50 Am.
ogy. There appeared to be a lack of active phagocytosis as no multicellular microglia and no fat granules were observed in the striatum of MPTP-treated animals. In control animals, lightly labeled LN3-positive ramified microglia were observed throughout the globus pallidus (Fig. 6B). In MPTP-treated animals, both the internal and external segments of the globus pallidus (Fig. 6A) displayed widespread microglial activation. As in the substantia nigra and nigrostriatal tract of MPTP-treated animals, the microglial reaction in the globus pallidus was heterogeneous with commingled ramified, activated, and multicellular morphologies. Also present were scattered fat granules, although less prevalent than in the substantia nigra. As within the substantia nigra, these fat granules were not seen in the most severely affected case.
Discussion The microglial reaction appears to be independent of degeneration Five weeks following chronic, low-dose MPTP treatment, we observed an intense microglial reaction in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically
heterogeneous and was notable for the presence of multicellular microglia, a hallmark of an active phagocytic response. Most animals also displayed the presence of numerous fat granules, an end product of phagocytosis. Surprisingly, there was little evidence of microglial reactivity in the striatum. This pattern of microglial reactivity was independent of the behavioral and morphological consequences of MPTP neurotoxicity. The observed pattern of microglial reactivity was the same in all MPTP-treated animals regardless of the degree of degeneration. It is clear in this and in our previous study (Song and Haber, 2000) that there is significant degeneration in both the striatum and SNc with this animal model. In contrast, the segments of the globus pallidus are thought to be spared from MPTP-mediated neurotoxicity (Langston et al., 1984): the globus pallidus is a relatively minor target for DA innervation (Jan et al., 2000) and there is no neuronal loss in the pallidum with PD (Hardman and Halliday, 1999a,b). However, it is worth noting that there is some degeneration in the pallidum, given both the presence of silver degeneration labeling and a decreased level of TH immunostaining. It is likely that much of the microglial reaction in the globus pallidus is stimulated by the degeneration of fibers of passage—nigral projections that pass through the globus pallidus to the striatum.
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Thus, the microglial response in this animal model is enigmatic. Microglia are reactive in the SNc, a region with significant continuing degeneration, as well as in the globus pallidus, a region with relatively minor degeneration. Yet, the microglial reaction appears to be entirely lacking in the striatum, where there is significant evidence of degeneration.
morphological heterogeneity seen in PD and in our animal model represents a common feature of chronic neurodegenerative processes. Indeed, similar patterns of microglial reactivity are seen in other neurodegenerative diseases, including Alzheimer’s disease, as well as during Wallerian degeneration (McGeer et al., 1988a,b; Perlmutter et al., 1992; Schmitt et al., 2000).
Similarities and differences with rodent MPTP models
Caveats with using an antibody to MHC II
In rodent models, there is extensive microglial reactivity in both the substantia nigra and striatum following highdose, acute MPTP treatment regimens (Dehmer et al., 2000; Francis et al., 1995; Kohutnicka et al., 1998; Kurkowska-Jastrzebska et al., 1999; Liberatore et al., 1999; Vila et al., 2001; Wu et al., 2002). This activation is accompanied by MHC II expression (Kurkowska-Jastrzebska et al., 1999). While not specifically addressed in their results, many of the figures from these papers document a heterogeneous microglial reaction—with commingling of multicellular, activated, and some ramified microglia (see especially: Kurkowska-Jastrzebska et al., 1999). In these rodent models, the process of neuron loss occurs in a matter of days, while the microglial reaction begins to diminish within 1 week (Kurkowska-Jastrzebska et al., 1999). Thus, despite morphological similarities, there are key differences in the time course and location of the microglial reaction to MPTP treatment in mice and in our monkeys. These differences are likely to be due to differences in MPTP administration paradigms (the time course and dosage of administration), although species differences in the microglial responsiveness cannot be discounted.
One concern in doing monkey or human work is the unavailability of ‘‘good’’ markers for microglia—markers that label all microglia and allow one to clearly distinguish morphological phenotypes. Antibodies to antigens that are routinely stained in mouse or rat often fail to work in human or monkey fixed tissue [i.e., the type 3 complement receptor (also known as OX-42 or Mac-1)]. Many immunoantigens that work in human often fail to label the full microglial morphology (Knott et al., 1999; Mirza et al., 2000). This is also the case with other microglial markers, like cyclooxygenase-1 (Yermakova et al., 1999). In staining for human microglia, LN3, an antibody to MHC II, represents a useful compromise. MHC II expression by microglia is widely variable in postmortem samples, but tends to be more sensitive than the lectin Ricinus communis (Engel et al., 1996). In addition, labeling of MHC II with LN3 tends to increase with increasing age. Thus, in samples from middle-aged or elderly individuals, one can be confident that nearly all microglia will be labeled and that microglial morphologies can be clearly discerned (Streit and Sparks, 1997). However, this is not the case in our monkey tissue. In control brains, relatively few microglia were labeled, and those that were labeled were labeled lightly. This was also the case in cortical and thalamic regions of MPTP-treated monkeys—brain regions thought to be unaffected by MPTP-induced neurodegeneration. Therefore, one must be careful in interpreting an apparent lack of a microglial staining. However, MHC II expression by microglia has been consistently linked with phagocytosis in Wallerian degeneration (Konno et al., 1989; Schmitt et al., 2000; Watanabe et al., 1999), in response to ricin-induced neurodegeneration (Streit et al., 1988), and, in vitro, in response to apoptosis by commingled T cells (Magnus et al., 2001). Given this, we interpret the lack of MHC II expression by microglia in the striatum as indicating that there is at most a negligible microglial reaction to degeneration in the striatum.
Similarities to human PD Our findings are similar to what has been described in postmortem samples of PD patients. McGeer et al. (1988a,b,c) described numerous activated and phagocytic microglia in the substantia nigra and nigrostriatal tract of PD patients. These microglia were morphologically heterogeneous, and there was ample evidence of active phagocytosis. These findings have been confirmed by two other groups (Knott et al., 1999; Mirza et al., 2000). Interestingly, these investigators failed to find any microglial reactivity in the striatum (Knott et al., 1999; Mirza et al., 2000). Microglia in PD and in our MPTP administration paradigm respond in a morphologically heterogeneous fashion. Classically, microglial morphology is the best indicator of microglial phenotype (Rio-Hortega, 1932; Streit et al., 1988). In response to acute brain injury, like middle cerebral artery occlusion (Morioka et al., 1993), spreading depression (Gehrmann et al., 1993), or traumatic brain injury (Carbonell and Grady, 1999), microglia within the zone of injury will all show an activated or phagocytic morphology (Streit et al., 1988). We suggest that the
A disconnect between microglial reactivity and neuropathology? Our results appear to illustrate a disconnect between microglial reactivity and neuropathology—a concept previously suggested by McGeer et al. (1988a). First, we saw no microglial reactivity in the striatum, a brain region with
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substantial MPTP-induced neurodegeneration. At the same time, we saw an unexpectedly substantial microglial reaction in an area thought to be unaffected by MPTP-induced neurodegeneration, the globus pallidus. Second, the pattern of microglial reactivity was similar across all MPTPtreated animals regardless of the behavioral or morphological severity of neurodegeneration. Third, this disconnection between microglial reactivity and neuropathology does not appear related to the temporal aspects of our animal model (i.e., to the fact that the animals were euthanized 4 to 5 weeks following their last dose of MPTP). In one animal that was sacrificed at the cessation of MPTP administration, the pattern of microglial labeling was similar to that described here—with a substantial microglial reaction visible in the substantia nigra and globus pallidus, but no apparent striatal microglial reactivity (data not shown). Finally, it is likely that the silver degeneration labeling that we see in the striatum represents DA processes that have never been phagocytosed. Silver degeneration labeling binds to exposed neurofilaments from neurons that have lost cellular integrity (Scallet, 1995). Yet, despite the large quantity of striatal cellular debris, there is no appreciable microglial reaction. Thus, in this model, it appears that microglial reactivity colocalizes with neurodegeneration in some brain regions—the substantia nigra, the nigrostriatal tract, and the globus pallidus—but not in striatum. Nonetheless, a great body of literature supports the notion that microglia are ‘‘neuropathological sensors’’ within the CNS (Kreutzberg, 1996)—the idea that microglia undergo rapid activation in response to even minor pathological changes. Perhaps the clue to resolve this discrepancy lies in the morphological heterogeneity itself. In response to many injuries, microglia, which are exquisitely plastic cells, alter their morphology (Rio-Hortega, 1932; Streit et al., 1988). This alteration can occur within minutes of an acute traumatic injury. The signals that trigger microglial activation are only beginning to become known. These include pro-inflammatory factors like cytokines and chemokines, extracellular ATP (Inoue, 2002), neuropeptides (Priller et al., 1995), changes in extracellular potassium (Abraham et al., 2001), and serum proteins (Perry et al., 1992). It is also appreciated that the CNS itself is an ‘‘immunosuppressed’’ environment (Perry and Gordon, 1991). One contributing factor to the endogenous immunosuppression of the CNS is the expression of OX-2 (CD200) by neurons (Hoek et al., 2000). The fact that ‘‘activated’’ morphologies are commingled with ‘‘resting’’ morphologies suggests a microenvironment where pro-inflammatory and anti-inflammatory stimuli compete for microglial attention. This line of reasoning could potentially explain the failure to observe a striatal microglial reaction in our model and in PD: there is the possibility that anti-inflammatory signals may predominate in the striatum relative to other basal ganglia structures.
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Acknowledgments The authors thank Andrew Corey and April Whitbeck for their technical expertise. This work was supported by NIH NS38577 (S.D.H., J.A.O.) and NIH MH63324 (S.N.H.).
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