Microglial and astrocytic changes in the striatum of methamphetamine abusers

Legal Medicine 12 (2010) 57–62

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Microglial and astrocytic changes in the striatum of methamphetamine abusers Osamu Kitamura a,*, Toshiaki Takeichi a, Elaine Lu Wang a, Itsuo Tokunaga b, Akiko Ishigami b, Shin-ichi Kubo c a

Department of Legal Medicine, Kanazawa Medical University, Japan Department of Forensic Medicine, Institute of Health Bioscience, The University of Tokushima Graduate School, Japan c Department of Forensic Medicine, Fukuoka University School of Medicine, Japan b

a r t i c l e

i n f o

Article history: Received 9 June 2009 Received in revised form 19 October 2009 Accepted 4 November 2009 Available online 27 January 2010 Keywords: Methamphetamine Neuroroxicity Striatum Microglia Astrocytes Drug addiction

a b s t r a c t Little is known about the role of glial cells in the striatum of chronic methamphetamine (METH) users. In this study, we immunohistochemically examined glial reactions in the striatum of chronic METH users who did not abstain from METH use and died of drug intoxication. Human glucose transporter 5 (hGLUT), a useful marker of microglia, and CR3.43, a major histocompatibility complex class II antigen specific for reactive microglia, were immunostained. Glial fibrillary acidic protein (GFAP) and S100D were used for astrocyte immunohistochemistry. We analyzed 12 chronic METH users and 13 control subjects, and detected a 200–240% increase in the number of hGLUT5-positive cells in chronic METH users (p < 0.01). However, we did not detect any proliferation of CR3.43-positive cells. The number of GFAPpositive astrocytes increased, but this increase was not significant (p > 0.05). Moreover, S100B-positive cell density between the two groups was not significant (p > 0.05). This study demonstrates the absence of reactive gliosis in the striatum of chronic METH users who did not abstain for prolonged periods from METH use. The results suggest that chronic METH use by itself did not activate glial cells in humans and reactive gliosis may not be involved in the mechanism underlying the loss of control in drug intake, which is a characteristic feature of drug addiction. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Methamphetamine (METH) is a powerful stimulant drug of abuse with potent addictive and neurotoxic properties. Single or repeated administration of high METH doses induces neurotoxicity in the striatum of animals, which is characteristic of long-term depletion of dopamine and its metabolites, or dopaminergic terminal degeneration [1–8]. Although the mechanisms of METH neurotoxicity are not clearly understood, it has been proposed that the METH-induced release of dopamine generates reactive hydrogen species and dopamine quinones, which are suggested to play an important role in METH neurotoxicity [9–11]. Several human studies using positron emission tomography (PET) studies have demonstrated dopamine transporter reduction in abstinent METH users with psychomotor impairment [12,13]. Postmortem studies have revealed dopaminergic terminal marker deficits in the striatum of chronic METH users [14,15]. Several investigations have focused on the role of microglia in METH neurotoxicity. It is well known that microglia, which are immunocompetent cells, exhibit proliferation and activation

* Corresponding author. Address: Department of Legal Medicine, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. Tel.: +81 76 218 8099; fax: +81 76 286 5242. E-mail address: [email protected] (O. Kitamura). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.11.001

(microgliosis) in pathological states such as neurodegenerative disease, trauma, and ischemia [16–19]. Some studies have suggested that microglial activation mediates METH neurotoxicity, based on the findings that microgliosis accompanies METH neurotoxicity and that attenuation of microglial activation reduced dopamine depletion or dopamine transporter (DAT)-immunoreactivity (IR) deficit [20–24]. Like microglia, astrocytes contribute to many functions, including support during central nervous system development, ion homeostasis, neurotransmitter uptake, the maintenance of blood–brain barrier integrity, and contribution to the central nervous system, immune system, neuromodulation [25–27]. Astrogliosis is also induced in various kinds of pathological conditions such as neurodegenerative disease, ischemia or trauma, and is considered to be one of the markers for neuronal injury [28]. In addition, astrocytes are sensitive to toxicant-induced damage of the central nervous system [29]. Previous studies have demonstrated that increases in the glial fibrillary acidic protein (GFAP) level and proliferation of reactive astrocytes (astrogliosis) are induced by neurotoxic METH administration and are associated with METH neurotoxicity in animals [1,2,4,5,30]. Recently, a PET study using [11C](R)-(1-[2-chrlorophenyl]-Nmethyl-N-[1-methylpropyl]-3-isoquinoline carbamide) ([11C](R)PK11195), which has been proposed to be a useful radiotracer for activated microglia [31], has demonstrated increases in the radio-

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tracer binding in the brain of human METH abusers [32]. This suggests that reactive microgliosis can be induced by chronic METH use. In addition, a report has suggested the possibility that some subnormal glial changes are induced in METH users although glial cells were not quantitatively analyzed [33]. However, little is known about the extent of proliferation and morphology of microglia and astrocytes in the striatum of METH users. In this study, we applied immunohistochemical methods to examine the status of microglial and astrocytic reactions in the striatum of chronic METH users who died of METH intoxication following chronic drug use. Various kinds of markers have been used for the immunohistochemical detection of microglia. It has been suggested that human glucose transporter 5 (hGLUT5), a member of the glucose transporter family, is a novel marker for microglia on paraffin sections microglia [34–37]. Human complement receptor 3 (CR3.43), a major histocompatibility complex (MHC) class II antigen, stains only reactive microglia, and MHC class II-positive microglia are a sensitive index of neuropathological changes [35,37]. Therefore, we immunostained hGLUT5 for resting and reactive microglia, and CR3.43 was used for reactive microglia. In addition to GFAP, S100B, a member of the EF-hand Ca2+-binding protein family, was used for immunostaining astrocytes. S100Bpositive astrogliosis was found in Alzheimer’s disease, ischemia or head injury although this protein can be expressed in other glial cells or neurons [38–40].

2. Materials and methods 2.1. Cases Brain samples from 12 chronic METH users (10 males, 2 females, mean age; 31.6 ± 9.1 years old [mean ± SD]) and 13 drugfree control subjects (8 males, 5 females, mean age; 32.2 ± 7.3 years old) were examined. All cases were selected from those obtained at judicial autopsy performed in our facilities. The anonymity of the cases was strictly protected, though informed consent was not obtained because contact with the bereaved is prohibited by criminal autopsy regulations in Japan. Information on the cases in this study is summarized in Table 1. To select cases, toxicological screening was tested with Triage (Biotest, San Diego, USA). All cases were evaluated for common drugs of abuse and alcohol, and positive urine screens were confirmed by quantitative analysis of blood and urine. Screening test confirmed that urine samples of all METH users were negative for cocaine, opiate, marijuana, benzodiazepine and other drugs for medication for psychiatric illnesses. They seem to have had no history of other substance abuse including alcohol. METH quantification was performed by gas chromatography/mass spectrometry, as reported [15]. Toxicology diagnosed the cause of death as METH intoxication in 11 of 12 cases. Although the blood METH was negative in case 11, we determined that the case died of METH intoxication more than 24 h after lethal administration by detection of a high concentration of myoglobin, a skeletal muscle protein, was detected in the urine, which would be indicative of rhabdomyosis accompanying METH intoxication [41]. We diagnosed 5 cases as having suffered from hyperthermia based on a rectal temperature that was comparatively high for the postmortem interval, while a higher rectal temperature was not evident in other seven cases. We could not obtain detailed drug histories of the METH users because of the lack of a reliable source in most of cases. Hair samples of all METH cases were METH-positive, which indicated that all METH subjects in this study used chronically the drug until death. None of the methamphetamine users in this study seems to have received the medication for methamphetamine dependence. Our immunohistochemical study revealed that 11 of 12 cases (cases 1–11 in Ta-

Table 1 Information on METH users and control subjects in this study. Case No.

Age/sex

PMI (h)

METH concentration (lg/mL)

Cause of death

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

44/M 29/M 42/M 24/M 34/M 31/F 22/M 23/M 45/M 18/F 29/M 38/M 37/M 32/F 34/M 27/M 27/F 44/M 32/M 47/M 29/F 34/M 31/M 32/F 22/F

24 8 19 23 13 21 24 12 24 19 10 24 12 22 16 16 12 16 14 12 12 12 9 10 15

3.17 2.74 8.46 5.37 3.87 9.45 6.81 0.87 1.20 16.3 Negative 8.50 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication METH intoxication Bleeding due to stabbing Asphyxia Bleeding due to stabbing Bleeding due to stabbing Bleeding due to stabbing Ischemic heart disease Bleeding due to stabbing Bleeding due to stabbing Asphyxia Bleeding due to stabbing Bleeding due to stabbing Bleeding due to stabbing Bleeding due to stabbing

METH group: cases 1–12; Control group: cases 13–25. PMI: postmortem intervals. M = male; F = female.

ble 1) showed dopaminergic terminal marker deficits in the striatum, and a similar finding was observed in case 12 (data not shown). Normal controls had no history of drug abuse, brain ischemia, or psychiatric and neurological disorders. We selected the cases that were taking no medication, and in which the period from the onset of lethal events to death seemed to be less than half an hour to minimize glial reactions to short-term of agonal status would be minimal in such cases. The causes of death of the controls were bleeding due to stabbing (n = 10), asphyxia (n = 2), and ischemic heart disease (n = 1). Although a serological human immunodeficiency virus antigen test was not performed, histopathological examination of systemic organs demonstrated no AIDS relatedfindings, including HIV-related alterations, in all cases.

2.2. Immunohistochemistry Brains were collected at forensic autopsy and fixed in 10% buffered formalin. The nucleus accumbens, caudate and putamen were embedded in paraffin and cut into at 6 lm. After deparaffinization, the sections were incubated with anti-hGLUT5 (monoclonal, mouse, 1:1000, IBL, Fujioka, Japan), anti-CR3.43 (polyclonal, rabbit, 1:100, DakoCytomation, Kyoto, Japan) and anti-S100B (polyclonal, rabbit, 1:400, abcam, Tokyo, Japan) antibodies. Immunolabeling was detected and visualized using an ENVISION kit (DakoCytomation) according to the manufacturer’s instructions. Before immunostaining these sections were pretreated in 0.01 M citrate buffer, pH 6.0 with hydrated autoclaving for 10 min. For GFAP immunohistochemistry, the sections were incubated with an anti-human GFAP antibody (monoclonal, mouse, EPOS, DakoCytomation), and then visualized with 3–30 -diaminobenzidine tetrahydrochloride. After visualization, the sections were counterstained with Mayer’s hematoxyline. As a positive control for activated glial cells, the sections of the hippocampus of a 41-year old woman with ischemic brain damage were used.

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2.3. Quantification of immunopositive cells and statistical analysis Images were captured by a digital camera (DXM1200F, Nikon, Tokyo, Japan) connected to a microscope (Eclipse 80i, Nikon) with an objective 20. Immunopositive cells including the nucleus were counted over ten fields (415  320 lm/field) selected randomly from the sections. Counting was performed blindly. All analysis was performed using Statview 5.0 (SAS Inc., Cary, NC). Results are expressed as mean (±SEM) per field. To compare the number of immunopositive cells between METH and control groups, statistical analysis was performed using the Mann–Whitney U test. Differences were considered statistically significant at p < 0.05. Correlations between hGLUT5-positive cells or GFAP-positive cells and age were determined using the Pearson correlation test. 3. Results 3.1. Microglia and astrocyte density in METH and control groups Immunostaining of hGLUT5 revealed that most microglia were in the resting state in all cases examined (Fig. 1A). Statistical analysis

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revealed a significant increase in hGLUT5-positive cells in the nucleus accumbens (METH: 10.783 ± 1.120, control: 5.446 ± 0.634, Mann–Whitney U = 23.0, p = 0.0028), putamen (METH: 5.192 ± 0.733, control: 2.415 ± 0.355, Mann–Whitney U = 26.0, p = 0.0047) and caudate (METH 3.450 ± 0.700, control: 1.439 ± 0.320, Mann– Whitney U = 29.5, p = 0.0083) of the METH group (Fig. 2A). CR3.43 immunostaining rarely detected activated microglia (Fig. 1B), and proliferation of the cells was not evident even in the striatum of the two groups (Fig. 1C and D). There were no differences between the number of CR3.43-positive cells in the METH and control groups (nucleus accumbens: METH 0.075 ± 0.035, control 0.015 ± 0.015, Mann–Whitney U = 57.5, p = 0.1103; putamen: METH 0.100 ± 0.051, control 0.039 ± 0.026, Mann–Whitney U = 63.5, p = 0.2924; caudate: METH 0.025 ± 0.018, control 0.015 ± 0.010, Mann–Whitney U = 76.0, p = 0.8645). GFAP immunostaining revealed that most of the astrocytes in both groups had normal morphology (Fig. 1E). As shown in Fig. 2B, the METH group exhibited a higher, although not significantly different GFAP-positive cell density in each subdivision of the striatum than the control group (nucleus accumbens: METH: 3.258 ± 0.657, control: 2.392 ± 0.478, Mann–Whitney U = 57.5,

Fig. 1. Resting microglia immunostained by hGLUT5 in the nucleus accumbens of a control subject (case 18) (A). A CR3.43-positive cell with a plump cytoplasm and shortened processes in the nucleus accunbens of a 29-year-old male METH abuser (case 11) (B). In the nucleus accumbens of a 42-year-old male METH abuser (case 2), resting microglia were observed (C), whereas few CR3.43-positive cells were detected (D). GFAP immunostained astrocytes showing no reactive form in the nucleus accumbens of case 2 (E). S100B-positive cells having no hypertrophic cell body in the caudate of a 38-year-old male METH abuser (case 12) (F). Scale bar = 10 lm (A, B, and F), 20 lm. (C–E).

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Number of labelled cells/field

A

hGLUT-positive cells p < 0.01

20

15

p = .0047 0.01 pp=<.0047

10

5

0

Control

METH

Control

NAc

B Number of labelled cells/field

p < 0.01

METH

PT

Control

METH

CN

GFAP-positive cells 10

5

0 Control

METH

NAc

Control

METH

PT

Control

METH

CN

Fig. 2. The density of hGLUT5-positive cells and GFAP-positive cells per field in each subdivision of the striatum in control or METH group. A: hGlut5, B: GFAP. NAc, nucleus accumbems; PT, putamen; CN, caudate nucleus.

p = 0.2646; putamen: METH: 2.133 ± 0.355, control: 1.353 ± 0.233, Mann–Whitney U = 49.5, p = 0.2532; caudate: METH 3.358 ± 0.643, control 2.439 ± 0.559, Mann–Whitney U = 57.0, p = 0.2532). S100Bpositive cells were found in each region of the striatum in METH and control groups. However, reactive astrocytes exhibiting hypertrophic cell bodies, shortening of cytoplasmic processes and nuclear enlargement were rarely detected (Fig. 1F). Furthermore, a significant difference in S100B-positive cell density was not found between the two groups (nucleus accumbens: METH: 1.142 ± 0.525, control: 0.585 ± 0.183, Mann–Whitney U = 72.5, p = 0.7648; putamen: METH: 2.950 ± 1.915, control: 2.725 ± 1.874, Mann–Whitney U = 55.0, p = 0.3408; caudate: METH 1.725 ± 1.124, control 1.875 ± 1.152, Mann–Whitney U = 67.0, p = 0.7728). 3.2. Correlation of glial density with age The nucleus accumbens and putamen in older METH abusers tended to have a tendency toward a higher density of hGLUT5-positive cells without conventional statistical significance (nucleus accumbens: r = 0.438, p = 0.1590; putamen: r = 0.525, p = 0.0804) although there was no correlation between hGLUT cell density in the caudate and age (r = –0.073, p = 0.8623). On the other hand, in METH and control subjects, there was no correlation between GFAP-positive cells in the striatum and age (data not shown). 4. Discussion In this study, we found increases in hGLUT5-positive microglia in the striatum of chronic METH users. However, there was no proliferation of the activated microglia characteristic of CR3.43 expression and the reactive form. In addition, GFAP and S100B

immunostaining revealed that most of the astrocytes did not exhibit reactive changes in METH users, although there was a trend toward higher density of astrocytes. Our findings on glial reactions in human METH users were inconsistent with those in experimental animals treated repeatedly with high doses of METH. Neurotoxic METH regimes have been demonstrated to induce prominent proliferation of activated microglia in areas with dopaminergic terminal degeneration [20–24]. In addition, toxic METH doses induce astrogliosis in animals [1,2,4,5,30], while METH has been suggested to cause directly on astrocytes in vitro to cause astrogliosis [42]. Therefore, microgliosis and astrogliosis have been suggested as markers for METH neurotoxicity [2,24,29]. Therefore, the extent of glial reactions would correlate with the severity of neurotoxicity in animals treated with high doses of METH. However, previous studies have shown differences in the extent of METH neurotoxicity between animals and human users. Neurotoxic METH regimes induced dopamine depletions as well as tyrosine hydroxylase (TH), DAT, and vesicular monoamine transporter 2 (VMAT2) deficits in the striatum of animals [1,3–8]. On the other hand, the ligandbinding activity of VMAT2, a stable marker for striatal dopaminergic terminal integrity [3], did not significantly decrease in the striatum of chronic METH users though TH and DAT level deficits were evident [14]. METH cases (cases 1–11 in Table 1) in this study showed significant decreases in TH in the nucleus accumbens and DAT in the nucleus accumbens and putamen, while statistical analysis revealed no significant decreases in VMAT2-IR [15]. These findings were consistent with those in a previous study using postmortem brains of chronic METH users [14]. In the present study, the numbers of hGLUT5-positive microglia with the resting form significantly increased in all areas of the striatum of METH users and there was no correlation between microglial density and dopaminergic terminal marker deficits (data not shown). From these findings, microglial changes as found in this study may not be affected by the severity of METH neurotoxicity in chronic METH users. A PET study demonstrated higher binding of [11C] (R)-PK11195, a radiotracer for activated microglia, in brain regions including the striatum, suggesting that chronic METH use induces microglial activation in the brain of human abusers [32]. These findings seem to be inconsistent with those obtained in this study. However, in this study, hair sample analysis demonstrated that chronic METH users did not abstain for prolonged periods from METH. Moreover, all cases of METH abusers died within 2 days, typically within 24 h after lethal administration. On the other hand, subjects in the PET study were recreational users of the drug, and had abstained from 6 months to 6 years. Further, in most of animal studies, reactive gliosis was observed 2 days or more after the last METH administration [1,2,4,5,20–24]. Therefore, one of the important factors affecting the extent of glial reactions might be the interval time following chronic METH administration. Therefore, long-term survival after METH binges might induce reactive gliosis, as well as the development of neurotoxicity, in chronic METH abusers. Previous studies demonstrated glial changes in chronic drug abusers. The hippocampus of victims of acute heroine intoxication showed enhanced expression of GFAP and/or a proliferation of microglia accompanied by primary ischemic neuronal loss [43]. Furthermore, it has been suggested that polydrug abuse could damage GFAP-positive astrocytes, and that microglia activation would be caused by drug-induced axonal damage [44]. However, the significance of glial reactions to METH abuse as observed in this study is unknown. In an animal study, METH administered for 14 days induced an increase in the percentage of cells expressing CD4 antigen, which is a microglial marker for rats in the thymus [45]. A previous study found that microglia significantly increased in the frontal lobe of human immunodeficiency virus (HIV)-positive METH users compared to those in HIV-positive non-METH

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users [46]. These findings suggest that METH administration induces immunomodulation that may be closely linked to microglial reactions. Therefore, chronic drug use might induce higher density of microglia and produce immunomodulation in METH abusers. However, the mechanisms underlying drug addiction have not yet been fully understood. Further studies are required to elucidate the role of glial cells in the mechanisms of drug addiction in humans. There are some limitations to the current study. Because of the lack of detailed clinical information about the METH subjects, we were not able to clarify the correlations between glial responses and histories of METH use such as doses, duration or daily pattern of drug use. However, this study revealed that glial activation is not necessarily common in the striatum of METH users with dopaminergic terminal marker deficits. Moreover, this study focused on glial reactions in only active users who died of METH intoxication; further immunohistochemical studies in abstinent METH users may clarify the extent of glial reactions after the cessation of drug use. Lastly, there might have been some conditions such as other drug use that influence, at least to some extent, glial reactions in METH users although it seems that they had no history of other substance abuse that could induce or suppress glial reactions. In summary, this study found a significant increase in microglia without the reactive form and trends toward the elevation of resting astrocyte density in the striatum of chronic METH abusers who did not abstain for a long period although there was no evidence of reactive gliosis. Our observations suggest that chronic METH use by itself would not induce reactive gliosis in humans. Furthermore, reactive gliosis as shown in animals treated with a high dose of METH may not be involved in the mechanisms of escalating drug use. Further studies on glial cells in METH users could contribute to the understanding of the pathophysiology of METH addiction. Conflict of interest None of conflict of interest. Acknowledgment We would like to thank Kanako Ishikawa for her technical assistance. This work was supported by grants-in-aid for General Scientific Research (17590579 and 19590674) from the Ministry of Education, Science, Sports and Culture of Japan. References [1] Bowyer JF, Davies DL, Schmued L, Broening HW, Newport GD, Slikker Jr W, et al. Further studies of the role of hyperthermia in methamphetamine neurotoxicity. J Pharmacol Exp Ther 1994;268:1571–80. [2] O’Callaghan JP, Miller DB. Neurotoxicity profiles of substituted amphetamine in the C57BL/6J mouse. J Pharmacol Exp Ther 1994;270:741–51. [3] Frey K, Kilbourn M, Robinson T. Reduced striatal vesicular monoamine transporters after neurotoxic but not after behaviorally-sensitizing doses of methamphetamine. Eur J Pharmacol 1997;334:273–9. [4] Fukumura M, Cappon GD, Pu C, Broening HW, Vorhees CV. A single dose of methamphetamine-induced neurotoxicity in rats: effects on neostriatal monoamines and glial fibrillary acidic protein. Brain Res 1998;806:1–7. [5] Cappon GD, Pu C, Vorhees CV. Time-course of methamphetamine-induced neurotoxicity in rat caudate-putamen after single dose treatment. Brain Res 2000;863:106–11. [6] Johnson-Davis KL, Fleckenstein AE, Wilkins DG. The role of hyperthermia and metabolism as mechanisms of tolerance to methamphetamine neurotoxicity. Eur J Pharmacol 2003;482:151–4. [7] Segal DS, Kuczenski R, O’Neil ML, Melega WP, Cho AK. Escalating dose methamphetamine pretreatment alters the behavioral and neurochemical profiles associated with exposure to a high-dose methamphetamine binge. Neuropsychopharmacology 2003;28:1730–40. [8] Nakajima A, Yamada K, Nagai T, Uchiyama T, Miyamoto Y, Mamiya T, et al. Role of tumor–necrosis factor-a in methamphetamine-induced drug dependence and neurotoxicity. J Neurosci 2004;24:2212–25. [9] De Vito MJ, Wagner GC. Methamphetamine-induced neuronal damage: a possible role for free radicals. Neuropharmacology 1989;28:1145–50.

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