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Reduced corpus callosum white matter microstructural integrity revealed by diffusion tensor eigenvalues in abstinent methamphetamine addicts
NeuroToxicology 30 (2009) 209–213
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NeuroToxicology
Reduced corpus callosum white matter microstructural integrity revealed by diffusion tensor eigenvalues in abstinent methamphetamine addicts In-Sung Kim a, Yang-Tae Kim b, Hui-Jin Song a, Jae-Jun Lee a, Do-Hoon Kwon b, Hui Joong Lee c, Myoung-Nam Kim a, Done-Sik Yoo d, Yongmin Chang a,c,e,* a
Department of Medical & Biological Engineering, Kyungpook National University, Daegu, Republic of Korea Department of Psychiatry, Bugok National Hospital, Gyeongsang nam-do, Republic of Korea Department of Radiology, Kyungpook National University College of Medicine, Daegu, Republic of Korea d IT-BT Group, ETRI, Daejeon, Republic of Korea e Department of Molecular Medicine, Kyungpook National University College of Medicine, Daegu, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2008 Accepted 9 December 2008 Available online 24 December 2008
The purpose of the current study was to determine whether abstinent methamphetamine (MA) abusers demonstrate differences in white matter (WM) integrity of the corpus callosum (CC) due to possible neurotoxic effects of long-term MA abuse, compared with control subjects. In addition to fractional anisotropy (FA), the eigenvalues of the diffusion ellipsoid were used to evaluate the microstructural source of abnormal change in abstinent MA abusers if there occurred a difference in white matter integrity of the CC between healthy controls and abstinent MA abusers. Results showed significantly reduced FA in the genu of the corpus callosum in MA-dependent subjects compared with controls. Furthermore, the eigenvalues offered a unique opportunity to assess the microstructural source of abnormal changes in the genu of the CC. The relationships between Wisconsin Card Sorting Test (WCST) performance and the values of tensor measures also suggest that altered myelination is a possible source of FA reduction observed in the genu of the CC in MA abusers. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Corpus callosum Diffusion tensor imaging Methamphetamine White matter
1. Introduction Methamphetamine (MA), also known as ‘‘ice’’ or ‘‘crystal,’’ is a cationic lipophilic molecule with potent action on the central nervous system. It stimulates the mesolimbic reward pathway, causing euphoria and excitement, and is thus a highly addictive substance associated with a range of serious health problems that include cognitive impairment (Steketee, 2003; Nordahl et al., 2003). The neurotoxic effects of MA have been extensively reviewed (Axt et al., 1994; Kleven and Seiden, 1992; O’Callaghan and Miller, 2000; Davidson et al., 2001), and the various types of brain abnormalities present in long-term MA abusers have been reported in neuroimaging studies (Paulus et al., 2002; Sekine et al., 2003; Jernigan et al., 2005). Although most neuroimaging studies of MA abusers have focused on damage to the frontal gray matter, a magnetic resonance spectroscopy (MRS) study also reported neurochemical deficits of the frontal white matter in MA-abusing
* Corresponding author at: Department of Molecular Medicine & Radiology, Kyungpook National University and Hospital, 50, Samduk-Dong 2Ga, Chung-Gu, Daegu 700-721, Republic of Korea. Tel.: +82 53 420 5471; fax: +82 53 422 2677. E-mail address: [email protected] (Y. Chang). 0161-813X/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.12.002
subjects (Ernst et al., 2000). Several positron emission tomography (PET) studies demonstrated that MA abusers exhibit lower levels of dopamine transporters in the striatum (McCann et al., 1998; Sekine et al., 2001; Volkow et al., 2001) and prefrontal cortex (Sekine et al., 2003), and differences in regional cerebral glucose metabolism (Volkow et al., 2001; London et al., 2004) compared with corresponding measures in control subjects. A recent diffusion tensor imaging (DTI) study reported that abstinent MA abusers had disrupted integrities in the frontal white matter (WM) (Chung et al., 2007). The results of these neuroimaging studies suggest that damage to frontal brain regions after MA abuse may contribute to the wide range of cognitive deficits observed in MA abusers. The corpus callosum (CC) is the largest white matter structure in the brain, consisting mainly of interhemispheric fibers. Although structural MRI studies have confirmed alterations in brain structures, including the CC, in patients with drug addiction (Hommer et al., 1996; Thompson et al., 2004), diffusion tensor imaging has increasingly been employed in investigating the neuropathology of addiction. In patients with alcohol dependence, DTI showed white matter microstructural abnormalities in the CC consistent with reduced fractional anisotropy (FA) and intervoxel coherence of fibers (Pfefferbaum and Sullivan, 2005). In cocaine dependence, results showed significantly reduced FA in the genu
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and rostral body of the anterior CC compared with controls (Moeller et al., 2005). Most recently, Moeller et al. (2007) reported significantly smaller longitudinal (l1) eigenvalues along the principal diffusion axis in the rostral body of the corpus callosum in 3,4-methylenedioxymethamphetamine (MDMA, known as ‘‘ecstasy’’) users. DTI is a unique method for characterizing white matter microintegrity because it relies on the principle that water diffusion is highly anisotropic in brain white matter structures (Beaulieu, 2002). Many DTI studies reported that fractional anisotropy was decreased in lesions attributable to different neuropsychiatric conditions that may reflect edema, demyelination, and axonal loss (Kubicki et al., 2002; Ardekani et al., 2003); however, in many neuropsychiatric DTI studies, the possible physiological sources of FA alteration are not fully discussed. Recent evidence (Moeller et al., 2007) suggests that longitudinal (l1) and transverse (l2 and l3) eigenvalues provide more specific information about myelination and the axonal morphology of white matter than do FA and mean diffusivity (Dav). In addition, while previous studies have explored abnormalities of the frontal lobe of the brain related to cognitive impairment in MA use, none have investigated the white matter abnormalities of CC in MA abusers, despite its functional significance. Because frontal white matter tracts cross between hemispheres in the CC, it is possible that white matter integrity in the CC could also be altered in drug-dependent subjects. For example, Moeller et al. (2005) observed compromised white matter integrity in the genu and rostral body of the CC in cocaine users. In the present study, we used DTI to observe whether abstinent MA abusers demonstrate differences in the white matter integrity of the CC, compared with control subjects. Specifically, it was of interest whether FA is a good functional marker for MA-related change in the CC; if so, the individual eigenvalues might provide more specific information regarding damage to the white matter integrity of the CC. No previous DTI studies have clearly defined abnormalities of CC integrity in abstinent MA abusers. We hypothesized that MA abusers would show abnormalities of the corpus callosum not observed in those of non-drug users, reflecting alteration of white matter integrity caused by long-term MA use. Furthermore, we sought to interpret the microstructural source of abnormal changes in abstinent MA abusers in cases of a difference in white matter integrity in the CC of healthy controls and abstinent MA abusers by analyzing the eigenvalues of the diffusion ellipsoid. Finally, we assessed the relationship between white matter integrity and impaired cognitive function in abstinent MA abusers, using the Wisconsin Card Sorting Test (WCST).
Korea are reported to be attributable to intravenous drug injections (Kim et al., 2003). After a detailed explanation of the study design and potential risks, all subjects gave written informed consent. All study protocols were approved by the local Internal Review Board. 2.2. DTI acquisition All subjects were imaged on a 3.0-T clinical whole body magnet (VHi; General Electric Medical, Milwaukee, WI, USA). Diffusion tensor imaging was performed using the single-shot spin-echo, echo-planar imaging technique with Stejskal–Tanner diffusionsensitizing pulses. The DTI imaging parameters were as follows: 220 mm 220 mm field of view, 128 128 matrix, 12–16 axial slices, 4 mm slice thickness, repetition time = 8000 ms, echo time = 71 ms. Diffusion was measured along 25 non-collinear directions. For each slice and gradient direction, two images were acquired: with no diffusion weighting (b = 0 s/mm2) and with diffusion weighting (b = 1000 s/mm2). Fast spin-echo (FSE) T2weighted images were acquired as anatomical images. 2.3. Diffusion tensor analysis for scalar measures The eigenvalues l1, l2, l3 and eigenvectors were calculated using the 25 diffusion-weighted images. Fractional anisotropy was calculated from the eigenvalues from the tensor, on a voxel-byvoxel basis (Pierpaoli and Basser, 1996). The tensor scalar measure (FA) was deduced using the eigenvalues vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u u3ððl1 Dav Þ2 þ ðl2 Dav Þ2 þ ðl3 Dav Þ2 Þ FA ¼ t ; 2ðl21 þ l22 þ l23 Þ where Dav ¼ ðl1 þ l2 þ l3 Þ=3: 2.4. ROI placement To enable ROI placement in the genu and splenium of the CC across subjects and minimize the possible inclusion of gray matter, FA and eigenvalue images were segmented using segmentation software developed in-house based on Matlab version 7.2 (The Mathworks Inc., Natick, MA, USA). After segmentation of the CC, multiple small circular ROIs (24 mm2) were placed on the genu and splenium of the CC in the segmented FA maps (Fig. 1). An average value was computed from multiple circular ROIs to reduce the number of statistical tests performed. The ROI location was fixed for each ROI of each individual to avoid user-dependent variation in ROI selection.
2. Methods
2.5. Wisconsin Card Sorting Test (WCST)
2.1. Subjects
A computerized version of the WCST (Heaton, 1993), a commonly used measure of concept formation and flexibility of abstract thought, was administered using the standardized guidelines provided in the test manual. WCST performance was scored in terms of the number of perseveration errors, nonperseveration errors, and total errors.
Thirteen healthy, right-handed male subjects and 11 abstinent male MA abusers participated in this study. All abstinent MA subjects recruited for this study were diagnosed solely with MA dependence and were without lifetime exposure to any other addictive substance except nicotine and caffeine. In addition, all MA subjects who had administered MA intravenously met the following inclusion criteria: (1) no previous history of comorbid psychiatric disorders, (2) no alcohol dependence, and (3) abuse period of more than 5 years. These MA subjects were selected among a number of potential MA subjects by screening process. Current abuse of MA, cocaine, and marijuana was examined by urine screening using the Redwood Biotech urine strip (Santa Rosa, CA, USA). Screening for HIV status was not conducted due to ethical and legal issues; only 1.1% of HIV transmissions in
2.6. Statistical analysis Student’s t-test was used to examine group differences for l1, l2, l3, and FA in the genu and splenium of the CC, using SPSS (SPSS Inc., USA). Pearson correlation analysis was performed to determine whether a relationship existed between the WCST scores and the tensor measures for the two regions of the CC that differed significantly between the two groups. Two-tailed tests were employed; statistical significance was defined at p < 0.05.
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in the genu of MA abusers was significantly lower than that in controls, whereas the eigenvalues l2 and l3 were significantly higher. The FA index decreased because of contrasting degrees of change in the eigenvalues in the three principal axes of the diffusion ellipsoid. In the genu of the CC of MA subjects, there was no statistically significant correlation between duration of MA abuse and FA (p = 0.06). For eigenvalues, the years of MA use did not correlate with l1 (p = 0.457) and l3 (p = 0.221) whereas l2 showed statistically significant correlation with years of MA abuse (p = 0.014). Total dose over years of MA use did not show any correlation with either FA (p = 0.066) or eigenvalues (p = 0.124 for l1; p = 0.141 for l2; p = 0.125 for l3). In comparing the splenium of the CC, MA abusers had slightly higher eigenvalues than those of control subjects; however, group difference in the splenium was not statistically significant. Although there was a limited age span in MA users, correlation analysis showed statistically significant correlation between age and l2 (p = 0.014). However, other tensor measures (FA, l1, l3) did not correlate with age. 3.3. Relationship between cognitive measures and DTI
Fig. 1. Example of ROI locations in the genu and splenium of the CC in the segmented FA map.
3. Results 3.1. Demographics The mean age of the MA abusers was 34.4 2.9 years, ranging from 28 to 39 years. The mean age of the healthy controls was 35.5 2.1 years, ranging from 33 to 38 years. There was no statistically significant difference in age between the groups (t = 1.079, df = 22, p = 0.292); thus, DTI measures (FA and eigenvalues) were unaffected by age-related changes. The mean duration of MA abuse was 10.6 2.7 years, ranging from 6 to 15 years. The dose per year ranged from 6.7 to 85.0 g. The mean abstinence period was 18.0 7.0 days. All MA abusers reported intravenous MA use and had no history of abusing other classes of drugs beside MA. There were no significant differences in handedness or social alcohol drinking habits between MA abusers and healthy comparison subjects. 3.2. Quantitative regional DTI measures Table 1 summarizes the mean FA values for MA abusers and healthy controls, and the three eigenvalues (l1, l2, l3) in the genu and splenium of the CC. Compared with healthy control subjects, MA abusers had lower FA values in the genu and splenium regions of the CC; however, group difference in the splenium was not statistically significant (p = 0.592, two-tailed t-test); difference in the genu was statistically significant (p = 0.003). The eigenvalue l1
The mean WCST total error score of the healthy controls was 10.08 0.65 and the mean WCST total error of the MA subjects was 42.00 5.06. There was statistically significant difference in WCST score between two groups (t = 6.263, df = 10.326, p < 0.0005). Within abstinent MA subjects, correlation analysis demonstrated a significant negative correlation between WCST total error and FA in the genu region of the CC (r = 0.750, p = 0.008, Fig. 2a). The eigenvalue (l2) in the genu region showed a significant positive correlation with WCST total error (r = 0.810, p = 0.003, Fig. 2b). However, the eigenvalues (l1) and (l3) in the genu did not show statistically significant correlation with WCST total error (r = 0.100, p = 0.770 for (l1) and r = 0.364, p = 0.272 for (l3) respectively). There were no significant correlations between WCST total error and tensor measures, including FA in the splenium region of the CC. 4. Discussion In the present study, DTI-derived scalar measures (FA and eigenvalues of the diffusion ellipsoid) revealed change in white matter integrity in abstinent MA abusers compared with healthy comparison subjects. MA abusers had reduced FA values in the genu of the CC. Our finding is consistent with the results of previous studies that showed reduced FA in the anterior CC in cocaine-dependent subjects (Moeller et al., 2005). Because all MA subjects were without lifetime exposure to other drugs, drug use other than MA could not have accounted for our finding of reduced FA in the MA abusers. Thus, it is possible that the reduced FA seen in MA abusers was due to the effects of MA. Our finding of reduced FA values in the genu of the CC of MA abusers suggests potential damage to the anterior corpus callosum WM in the MA abusers. Group differences in tensor scalar measures (l1, l2, l3, FA) in the splenium of the CC between MA abusers and healthy controls were not statistically significant. Controversy exists regarding
Table 1 DTI scalar measures of corpus callosum in abstinent methamphetamine abusers and healthy comparison subjects. Genu of CC Control (N = 13) 3
3
l1 (10 mm /s) l2 (103 mm3/s) l3 (103 mm3/s) TRACE (103 mm3/s) FA *
1.9742 0.1243 0.3181 0.0574 0.1436 0.0471 2.4359 0.1361 0.8704 0.0295
MA (N = 11) 1.8706 0.0495 0.3761 0.0587 0.1863 0.0449 2.4329 0.1358 0.8326 0.0255
Statistically significant difference (two sample t-test, p < 0.05).
Statistics
Splenium of CC
p-Value
Control (N = 13)
MA (N = 11)
p-Value
1.7456 0.0661 0.3775 0.0915 0.1837 0.0332 2.3068 0.1327 0.8211 0.0311
1.7774 0.1053 0.3949 0.0839 0.1953 0.0419 2.3675 0.1790 0.8145 0.0278
0.393 0.635 0.458 0.351 0.592
*
0.014 0.023* 0.034* 0.958 0.003*
Statistics
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Fig. 3. A simplified schematic of a myelinated white matter fiber.
Fig. 2. (a) Negative correlation between Wisconsin Card Sorting Test (WCST) total error and FA in the genu of the corpus callosum (CC) and (b) positive correlation between Wisconsin Card Sorting Test (WCST) total error and l2 in the genu of the corpus callosum (CC) within MA-dependent subjects.
drug-related white matter change to the splenium of the CC. Moeller et al. (2005) found no white matter pathology in the splenium of the CC in cocaine-dependent subjects; however, DTI studies in chronic alcoholism confirmed a reduction in FA in the genu and the splenium of the CC in alcoholic men (Rosenbloom et al., 2003). In this context, pathologic change in the splenium is therefore more subtle than that in the genu of the CC; FA may fail to show subtle change to the splenium in MA abusers. Anisotropic water diffusion, which is often measured in terms of FA, is closely related to the well-oriented arrangement of the myelinated fibers in white matter. For simplicity, white matter is described as an ordered axonal system consisting of myelin sheath surrounding the axons, axonal membrane, and the neurofibrils (Fig. 3). All these microstructural components are longitudinally oriented structures that could hinder water diffusion across the length of the axon and cause the perpendicular diffusion coefficients (l2 and l3) to be smaller than the parallel diffusion coefficient (l1). Therefore, the difference between parallel and perpendicular diffusion coefficients determines diffusion anisotropy, and these eigenvalues along the three principal axes of diffusion offer a unique opportunity to assess the microstructural source of abnormal changes in white matter. Our eigenvalue analysis showed that FA reduction in the genu of CC in abstinent MA abusers results from both parallel diffusion (l1) decrease and perpendicular diffusion (l2
and l3) increase; however, the percentage change in the perpendicular diffusion coefficients (l2 and l3) was greater than that in the parallel diffusion coefficient (l1). This finding is consistent with the previous observation (Song et al., 2002) that decreased FA in the dysmyelinated WM tracts of shiverer mice was due to an increase in the perpendicular diffusion coefficients (l2 and l3) alone. Because the perpendicular diffusion coefficients (l2 and l3) are closely related to altered structures in myelin and the axonal membrane, our tensor eigenvalue findings in the genu of the CC suggest that WM deficits in abstinent MA abusers may be related to either abnormal myelination or damage to the axonal membrane. In previous studies, abnormal myelination was reported in cocaine abusers (Alberson et al., 2004) and in an animal experiment using MA (Melo et al., 2006). MA exposure is also known to cause cell body injury by inducing Wallerian degeneration of axons (Deng et al., 2001). Although the decreased parallel diffusion coefficient (l1) suggests MA-induced cell body damage, this mechanism seems to be less important in the current case. In the case of Wallerian degeneration of axons, previous studies demonstrated a severe reduction in FA of the neural fiber tracts by significant alteration of the parallel diffusion coefficient (l1) in addition to the perpendicular diffusion coefficients (l2 and l3) (Beaulieu et al., 1996; Thomalla et al., 2004). However, because the detailed mechanisms that underlie differential changes in directional diffusion coefficients remain to be elucidated, it should be acknowledged that our interpretation is speculative rather than conclusive. The relationship between WCST performance and the values of tensor measures (l1, l2, l3, FA) in the genu of the CC in MA abusers also appear to support our interpretation that altered myelination is the possible source of FA reduction observed in the genu of the CC in MA abusers. Correlation analysis revealed a statistically significant correlation between total WCST error score in FA and the perpendicular diffusion coefficient (l2), but not with the parallel diffusion coefficient (l1) (r = 0.100, p = 0.770). It is however interesting that other perpendicular diffusion coefficient (l3) appears not to correlate with WCST score. When we keep in mind that diffusion is truly 3-dimensional process, our result seems to suggest that water diffusion is somewhat anisotropic even in perpendicular direction. These findings therefore demonstrate that the perpendicular diffusion coefficient (l2), which is closely related to myelin integrity, is the main source of FA reduction in the genu of the CC. Furthermore, in addition to the frontal WM deficits in MA abusers found by Chung et al. (2007), our findings suggest that corpus callosum damage secondary to frontal WM deficit is partly responsible for the impairment in frontal executive function observed in MA abusers, as the genu region of the CC is linked to the frontal WM. However, there is a possibility that alterations of the CC, both in size or thickness and density may also contribute to impairment in frontal gray matter (Downhill et al., 2000). Finally, the current study focused on the corpus callosum, neglecting examination of the frontal white matter tracts (which
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are also main targets of MA pathology) for the following reason. The large concentration of white matter in the corpus callosum enables clear separation of white matter from gray matter, which is a significant confounding factor in DTI analysis (Bhagat and Beaulieu, 2004). In the case of frontal or prefrontal WM tracts, ROI analysis is commonly hampered by gray matter contamination; thus, the corpus callosum is the most reliable white matter structure for evaluation of any drug-related alteration of WM integrity using ROI analysis. Future studies should focus on defining the mechanisms that mediate the anomalies reported here, while our data suggest neurotoxic effects of MA. MA is an addictive CNS stimulant, the repeated use of which causes long-term damage to the dopaminergic fiber pathways in the brain, while long-term use of MDMA causes damage to the serotonergic pathway (Ricaurte et al., 1980; Commins and Seiden, 1986). Kokoshka et al. (1998) found a loss of dopamine transporters in the striatal synaptosomes of MA-treated rats. Similarly, Pubill et al. (2003) found that MA induced a 35% loss of dopaminergic terminals in rats, generally without cell death. Therefore, further studies that combine different imaging modalities (e.g., DTI and PET dopamine receptor imaging) may implicate particular neural processes of the affected structures. In conclusion, this study reports change in the white matter integrity of the CC in terms of multiple tensor scalar measures in short-term abstinent MA users, and demonstrates that the eigenvalues along the three principal axes of diffusion offer a unique opportunity to assess the microstructural source of abnormal changes in WM. Because the corpus callosum consists mainly of interhemispheric white matter tracts, these white matter changes in the genu of the CC might be associated with impairment of frontal executive function. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by Advanced Research Center for Recovery of Human sensibility funded by Ministry of Health and Welfare (Grant #02-PJ3-PG6-EV10-0001). This work was also supported by the Brain Korea 21 Project in 2008. References Alberson DN, Pruetz B, Schmidt CJ, Khun DM, Kapatos G, Bannon MJ. Gene expression profile of the nucleus accumbens of human cocaine abusers: evidence for dysregulation of myelin. Journal of Neurochemistry 2004;88:1211–9. Ardekani BA, Nierenberg J, Hoptman MJ, Javitt DC, Lim KO. MRI study of white matter diffusion anisotropy in schizophrenia. Neuroreport 2003;14:2025–9. Axt KJ, Mamounas LA, Molliver ME. Structural features amphetamine neurotoxicity in the brain. In: Cho AK, Segal DS, editors. Amphetamine and its analogs: psychopharmacology, toxicology and abuse. San Diego: Academic; 1994315–67. Beaulieu C, Does MD, Snyder RE, Allen PS. Changes in water diffusion due to Wallerian degeneration in peripheral nerve. Magnetic Resonance in Medicine 1996;36:627– 31. Beaulieu C. The basis of anisotropic water diffusion in the nervous system—a technical review. NMR in Biomedicine 2002;15:435–55. Bhagat YA, Beaulieu C. Diffusion anisotropy in subcortical white matter and cortical gray matter: changes with aging and the role of CSF-suppression. Journal of Magnetic Resonance Imaging 2004;20:216–27. Chung A, Lyoo IK, Kim SJ, Hwang J, Bae SC, Sung YH, et al. Decreased frontal whitematter integrity in abstinent methamphetamine abusers. The International Journal of Neuropsychopharmacology 2007;10:765–75. Commins DL, Seiden LS. Alpha-methyltyrosine blocks methylamphetamine-induced degeneration in the rat somatosensory cortex. Brain Research 1986;365:15–20. Davidson C, Gow AJ, Lee TH, Ellinwood EH. Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Research Reviews 2001;36:1–22. Downhill JE, Buchsbaum J, Wei MST, Spiegel-Cohen J, Hazlett EA, Haznedar MM, et al. Shape and size of the corpus callosum in schizophrenia and schizotypal personality disorder. Schizophrenia Research 2000;42:193–208.
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Contents lists available at ScienceDirect
NeuroToxicology
Reduced corpus callosum white matter microstructural integrity revealed by diffusion tensor eigenvalues in abstinent methamphetamine addicts In-Sung Kim a, Yang-Tae Kim b, Hui-Jin Song a, Jae-Jun Lee a, Do-Hoon Kwon b, Hui Joong Lee c, Myoung-Nam Kim a, Done-Sik Yoo d, Yongmin Chang a,c,e,* a
Department of Medical & Biological Engineering, Kyungpook National University, Daegu, Republic of Korea Department of Psychiatry, Bugok National Hospital, Gyeongsang nam-do, Republic of Korea Department of Radiology, Kyungpook National University College of Medicine, Daegu, Republic of Korea d IT-BT Group, ETRI, Daejeon, Republic of Korea e Department of Molecular Medicine, Kyungpook National University College of Medicine, Daegu, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2008 Accepted 9 December 2008 Available online 24 December 2008
The purpose of the current study was to determine whether abstinent methamphetamine (MA) abusers demonstrate differences in white matter (WM) integrity of the corpus callosum (CC) due to possible neurotoxic effects of long-term MA abuse, compared with control subjects. In addition to fractional anisotropy (FA), the eigenvalues of the diffusion ellipsoid were used to evaluate the microstructural source of abnormal change in abstinent MA abusers if there occurred a difference in white matter integrity of the CC between healthy controls and abstinent MA abusers. Results showed significantly reduced FA in the genu of the corpus callosum in MA-dependent subjects compared with controls. Furthermore, the eigenvalues offered a unique opportunity to assess the microstructural source of abnormal changes in the genu of the CC. The relationships between Wisconsin Card Sorting Test (WCST) performance and the values of tensor measures also suggest that altered myelination is a possible source of FA reduction observed in the genu of the CC in MA abusers. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Corpus callosum Diffusion tensor imaging Methamphetamine White matter
1. Introduction Methamphetamine (MA), also known as ‘‘ice’’ or ‘‘crystal,’’ is a cationic lipophilic molecule with potent action on the central nervous system. It stimulates the mesolimbic reward pathway, causing euphoria and excitement, and is thus a highly addictive substance associated with a range of serious health problems that include cognitive impairment (Steketee, 2003; Nordahl et al., 2003). The neurotoxic effects of MA have been extensively reviewed (Axt et al., 1994; Kleven and Seiden, 1992; O’Callaghan and Miller, 2000; Davidson et al., 2001), and the various types of brain abnormalities present in long-term MA abusers have been reported in neuroimaging studies (Paulus et al., 2002; Sekine et al., 2003; Jernigan et al., 2005). Although most neuroimaging studies of MA abusers have focused on damage to the frontal gray matter, a magnetic resonance spectroscopy (MRS) study also reported neurochemical deficits of the frontal white matter in MA-abusing
* Corresponding author at: Department of Molecular Medicine & Radiology, Kyungpook National University and Hospital, 50, Samduk-Dong 2Ga, Chung-Gu, Daegu 700-721, Republic of Korea. Tel.: +82 53 420 5471; fax: +82 53 422 2677. E-mail address: [email protected] (Y. Chang). 0161-813X/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.12.002
subjects (Ernst et al., 2000). Several positron emission tomography (PET) studies demonstrated that MA abusers exhibit lower levels of dopamine transporters in the striatum (McCann et al., 1998; Sekine et al., 2001; Volkow et al., 2001) and prefrontal cortex (Sekine et al., 2003), and differences in regional cerebral glucose metabolism (Volkow et al., 2001; London et al., 2004) compared with corresponding measures in control subjects. A recent diffusion tensor imaging (DTI) study reported that abstinent MA abusers had disrupted integrities in the frontal white matter (WM) (Chung et al., 2007). The results of these neuroimaging studies suggest that damage to frontal brain regions after MA abuse may contribute to the wide range of cognitive deficits observed in MA abusers. The corpus callosum (CC) is the largest white matter structure in the brain, consisting mainly of interhemispheric fibers. Although structural MRI studies have confirmed alterations in brain structures, including the CC, in patients with drug addiction (Hommer et al., 1996; Thompson et al., 2004), diffusion tensor imaging has increasingly been employed in investigating the neuropathology of addiction. In patients with alcohol dependence, DTI showed white matter microstructural abnormalities in the CC consistent with reduced fractional anisotropy (FA) and intervoxel coherence of fibers (Pfefferbaum and Sullivan, 2005). In cocaine dependence, results showed significantly reduced FA in the genu
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and rostral body of the anterior CC compared with controls (Moeller et al., 2005). Most recently, Moeller et al. (2007) reported significantly smaller longitudinal (l1) eigenvalues along the principal diffusion axis in the rostral body of the corpus callosum in 3,4-methylenedioxymethamphetamine (MDMA, known as ‘‘ecstasy’’) users. DTI is a unique method for characterizing white matter microintegrity because it relies on the principle that water diffusion is highly anisotropic in brain white matter structures (Beaulieu, 2002). Many DTI studies reported that fractional anisotropy was decreased in lesions attributable to different neuropsychiatric conditions that may reflect edema, demyelination, and axonal loss (Kubicki et al., 2002; Ardekani et al., 2003); however, in many neuropsychiatric DTI studies, the possible physiological sources of FA alteration are not fully discussed. Recent evidence (Moeller et al., 2007) suggests that longitudinal (l1) and transverse (l2 and l3) eigenvalues provide more specific information about myelination and the axonal morphology of white matter than do FA and mean diffusivity (Dav). In addition, while previous studies have explored abnormalities of the frontal lobe of the brain related to cognitive impairment in MA use, none have investigated the white matter abnormalities of CC in MA abusers, despite its functional significance. Because frontal white matter tracts cross between hemispheres in the CC, it is possible that white matter integrity in the CC could also be altered in drug-dependent subjects. For example, Moeller et al. (2005) observed compromised white matter integrity in the genu and rostral body of the CC in cocaine users. In the present study, we used DTI to observe whether abstinent MA abusers demonstrate differences in the white matter integrity of the CC, compared with control subjects. Specifically, it was of interest whether FA is a good functional marker for MA-related change in the CC; if so, the individual eigenvalues might provide more specific information regarding damage to the white matter integrity of the CC. No previous DTI studies have clearly defined abnormalities of CC integrity in abstinent MA abusers. We hypothesized that MA abusers would show abnormalities of the corpus callosum not observed in those of non-drug users, reflecting alteration of white matter integrity caused by long-term MA use. Furthermore, we sought to interpret the microstructural source of abnormal changes in abstinent MA abusers in cases of a difference in white matter integrity in the CC of healthy controls and abstinent MA abusers by analyzing the eigenvalues of the diffusion ellipsoid. Finally, we assessed the relationship between white matter integrity and impaired cognitive function in abstinent MA abusers, using the Wisconsin Card Sorting Test (WCST).
Korea are reported to be attributable to intravenous drug injections (Kim et al., 2003). After a detailed explanation of the study design and potential risks, all subjects gave written informed consent. All study protocols were approved by the local Internal Review Board. 2.2. DTI acquisition All subjects were imaged on a 3.0-T clinical whole body magnet (VHi; General Electric Medical, Milwaukee, WI, USA). Diffusion tensor imaging was performed using the single-shot spin-echo, echo-planar imaging technique with Stejskal–Tanner diffusionsensitizing pulses. The DTI imaging parameters were as follows: 220 mm 220 mm field of view, 128 128 matrix, 12–16 axial slices, 4 mm slice thickness, repetition time = 8000 ms, echo time = 71 ms. Diffusion was measured along 25 non-collinear directions. For each slice and gradient direction, two images were acquired: with no diffusion weighting (b = 0 s/mm2) and with diffusion weighting (b = 1000 s/mm2). Fast spin-echo (FSE) T2weighted images were acquired as anatomical images. 2.3. Diffusion tensor analysis for scalar measures The eigenvalues l1, l2, l3 and eigenvectors were calculated using the 25 diffusion-weighted images. Fractional anisotropy was calculated from the eigenvalues from the tensor, on a voxel-byvoxel basis (Pierpaoli and Basser, 1996). The tensor scalar measure (FA) was deduced using the eigenvalues vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u u3ððl1 Dav Þ2 þ ðl2 Dav Þ2 þ ðl3 Dav Þ2 Þ FA ¼ t ; 2ðl21 þ l22 þ l23 Þ where Dav ¼ ðl1 þ l2 þ l3 Þ=3: 2.4. ROI placement To enable ROI placement in the genu and splenium of the CC across subjects and minimize the possible inclusion of gray matter, FA and eigenvalue images were segmented using segmentation software developed in-house based on Matlab version 7.2 (The Mathworks Inc., Natick, MA, USA). After segmentation of the CC, multiple small circular ROIs (24 mm2) were placed on the genu and splenium of the CC in the segmented FA maps (Fig. 1). An average value was computed from multiple circular ROIs to reduce the number of statistical tests performed. The ROI location was fixed for each ROI of each individual to avoid user-dependent variation in ROI selection.
2. Methods
2.5. Wisconsin Card Sorting Test (WCST)
2.1. Subjects
A computerized version of the WCST (Heaton, 1993), a commonly used measure of concept formation and flexibility of abstract thought, was administered using the standardized guidelines provided in the test manual. WCST performance was scored in terms of the number of perseveration errors, nonperseveration errors, and total errors.
Thirteen healthy, right-handed male subjects and 11 abstinent male MA abusers participated in this study. All abstinent MA subjects recruited for this study were diagnosed solely with MA dependence and were without lifetime exposure to any other addictive substance except nicotine and caffeine. In addition, all MA subjects who had administered MA intravenously met the following inclusion criteria: (1) no previous history of comorbid psychiatric disorders, (2) no alcohol dependence, and (3) abuse period of more than 5 years. These MA subjects were selected among a number of potential MA subjects by screening process. Current abuse of MA, cocaine, and marijuana was examined by urine screening using the Redwood Biotech urine strip (Santa Rosa, CA, USA). Screening for HIV status was not conducted due to ethical and legal issues; only 1.1% of HIV transmissions in
2.6. Statistical analysis Student’s t-test was used to examine group differences for l1, l2, l3, and FA in the genu and splenium of the CC, using SPSS (SPSS Inc., USA). Pearson correlation analysis was performed to determine whether a relationship existed between the WCST scores and the tensor measures for the two regions of the CC that differed significantly between the two groups. Two-tailed tests were employed; statistical significance was defined at p < 0.05.
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in the genu of MA abusers was significantly lower than that in controls, whereas the eigenvalues l2 and l3 were significantly higher. The FA index decreased because of contrasting degrees of change in the eigenvalues in the three principal axes of the diffusion ellipsoid. In the genu of the CC of MA subjects, there was no statistically significant correlation between duration of MA abuse and FA (p = 0.06). For eigenvalues, the years of MA use did not correlate with l1 (p = 0.457) and l3 (p = 0.221) whereas l2 showed statistically significant correlation with years of MA abuse (p = 0.014). Total dose over years of MA use did not show any correlation with either FA (p = 0.066) or eigenvalues (p = 0.124 for l1; p = 0.141 for l2; p = 0.125 for l3). In comparing the splenium of the CC, MA abusers had slightly higher eigenvalues than those of control subjects; however, group difference in the splenium was not statistically significant. Although there was a limited age span in MA users, correlation analysis showed statistically significant correlation between age and l2 (p = 0.014). However, other tensor measures (FA, l1, l3) did not correlate with age. 3.3. Relationship between cognitive measures and DTI
Fig. 1. Example of ROI locations in the genu and splenium of the CC in the segmented FA map.
3. Results 3.1. Demographics The mean age of the MA abusers was 34.4 2.9 years, ranging from 28 to 39 years. The mean age of the healthy controls was 35.5 2.1 years, ranging from 33 to 38 years. There was no statistically significant difference in age between the groups (t = 1.079, df = 22, p = 0.292); thus, DTI measures (FA and eigenvalues) were unaffected by age-related changes. The mean duration of MA abuse was 10.6 2.7 years, ranging from 6 to 15 years. The dose per year ranged from 6.7 to 85.0 g. The mean abstinence period was 18.0 7.0 days. All MA abusers reported intravenous MA use and had no history of abusing other classes of drugs beside MA. There were no significant differences in handedness or social alcohol drinking habits between MA abusers and healthy comparison subjects. 3.2. Quantitative regional DTI measures Table 1 summarizes the mean FA values for MA abusers and healthy controls, and the three eigenvalues (l1, l2, l3) in the genu and splenium of the CC. Compared with healthy control subjects, MA abusers had lower FA values in the genu and splenium regions of the CC; however, group difference in the splenium was not statistically significant (p = 0.592, two-tailed t-test); difference in the genu was statistically significant (p = 0.003). The eigenvalue l1
The mean WCST total error score of the healthy controls was 10.08 0.65 and the mean WCST total error of the MA subjects was 42.00 5.06. There was statistically significant difference in WCST score between two groups (t = 6.263, df = 10.326, p < 0.0005). Within abstinent MA subjects, correlation analysis demonstrated a significant negative correlation between WCST total error and FA in the genu region of the CC (r = 0.750, p = 0.008, Fig. 2a). The eigenvalue (l2) in the genu region showed a significant positive correlation with WCST total error (r = 0.810, p = 0.003, Fig. 2b). However, the eigenvalues (l1) and (l3) in the genu did not show statistically significant correlation with WCST total error (r = 0.100, p = 0.770 for (l1) and r = 0.364, p = 0.272 for (l3) respectively). There were no significant correlations between WCST total error and tensor measures, including FA in the splenium region of the CC. 4. Discussion In the present study, DTI-derived scalar measures (FA and eigenvalues of the diffusion ellipsoid) revealed change in white matter integrity in abstinent MA abusers compared with healthy comparison subjects. MA abusers had reduced FA values in the genu of the CC. Our finding is consistent with the results of previous studies that showed reduced FA in the anterior CC in cocaine-dependent subjects (Moeller et al., 2005). Because all MA subjects were without lifetime exposure to other drugs, drug use other than MA could not have accounted for our finding of reduced FA in the MA abusers. Thus, it is possible that the reduced FA seen in MA abusers was due to the effects of MA. Our finding of reduced FA values in the genu of the CC of MA abusers suggests potential damage to the anterior corpus callosum WM in the MA abusers. Group differences in tensor scalar measures (l1, l2, l3, FA) in the splenium of the CC between MA abusers and healthy controls were not statistically significant. Controversy exists regarding
Table 1 DTI scalar measures of corpus callosum in abstinent methamphetamine abusers and healthy comparison subjects. Genu of CC Control (N = 13) 3
3
l1 (10 mm /s) l2 (103 mm3/s) l3 (103 mm3/s) TRACE (103 mm3/s) FA *
1.9742 0.1243 0.3181 0.0574 0.1436 0.0471 2.4359 0.1361 0.8704 0.0295
MA (N = 11) 1.8706 0.0495 0.3761 0.0587 0.1863 0.0449 2.4329 0.1358 0.8326 0.0255
Statistically significant difference (two sample t-test, p < 0.05).
Statistics
Splenium of CC
p-Value
Control (N = 13)
MA (N = 11)
p-Value
1.7456 0.0661 0.3775 0.0915 0.1837 0.0332 2.3068 0.1327 0.8211 0.0311
1.7774 0.1053 0.3949 0.0839 0.1953 0.0419 2.3675 0.1790 0.8145 0.0278
0.393 0.635 0.458 0.351 0.592
*
0.014 0.023* 0.034* 0.958 0.003*
Statistics
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Fig. 3. A simplified schematic of a myelinated white matter fiber.
Fig. 2. (a) Negative correlation between Wisconsin Card Sorting Test (WCST) total error and FA in the genu of the corpus callosum (CC) and (b) positive correlation between Wisconsin Card Sorting Test (WCST) total error and l2 in the genu of the corpus callosum (CC) within MA-dependent subjects.
drug-related white matter change to the splenium of the CC. Moeller et al. (2005) found no white matter pathology in the splenium of the CC in cocaine-dependent subjects; however, DTI studies in chronic alcoholism confirmed a reduction in FA in the genu and the splenium of the CC in alcoholic men (Rosenbloom et al., 2003). In this context, pathologic change in the splenium is therefore more subtle than that in the genu of the CC; FA may fail to show subtle change to the splenium in MA abusers. Anisotropic water diffusion, which is often measured in terms of FA, is closely related to the well-oriented arrangement of the myelinated fibers in white matter. For simplicity, white matter is described as an ordered axonal system consisting of myelin sheath surrounding the axons, axonal membrane, and the neurofibrils (Fig. 3). All these microstructural components are longitudinally oriented structures that could hinder water diffusion across the length of the axon and cause the perpendicular diffusion coefficients (l2 and l3) to be smaller than the parallel diffusion coefficient (l1). Therefore, the difference between parallel and perpendicular diffusion coefficients determines diffusion anisotropy, and these eigenvalues along the three principal axes of diffusion offer a unique opportunity to assess the microstructural source of abnormal changes in white matter. Our eigenvalue analysis showed that FA reduction in the genu of CC in abstinent MA abusers results from both parallel diffusion (l1) decrease and perpendicular diffusion (l2
and l3) increase; however, the percentage change in the perpendicular diffusion coefficients (l2 and l3) was greater than that in the parallel diffusion coefficient (l1). This finding is consistent with the previous observation (Song et al., 2002) that decreased FA in the dysmyelinated WM tracts of shiverer mice was due to an increase in the perpendicular diffusion coefficients (l2 and l3) alone. Because the perpendicular diffusion coefficients (l2 and l3) are closely related to altered structures in myelin and the axonal membrane, our tensor eigenvalue findings in the genu of the CC suggest that WM deficits in abstinent MA abusers may be related to either abnormal myelination or damage to the axonal membrane. In previous studies, abnormal myelination was reported in cocaine abusers (Alberson et al., 2004) and in an animal experiment using MA (Melo et al., 2006). MA exposure is also known to cause cell body injury by inducing Wallerian degeneration of axons (Deng et al., 2001). Although the decreased parallel diffusion coefficient (l1) suggests MA-induced cell body damage, this mechanism seems to be less important in the current case. In the case of Wallerian degeneration of axons, previous studies demonstrated a severe reduction in FA of the neural fiber tracts by significant alteration of the parallel diffusion coefficient (l1) in addition to the perpendicular diffusion coefficients (l2 and l3) (Beaulieu et al., 1996; Thomalla et al., 2004). However, because the detailed mechanisms that underlie differential changes in directional diffusion coefficients remain to be elucidated, it should be acknowledged that our interpretation is speculative rather than conclusive. The relationship between WCST performance and the values of tensor measures (l1, l2, l3, FA) in the genu of the CC in MA abusers also appear to support our interpretation that altered myelination is the possible source of FA reduction observed in the genu of the CC in MA abusers. Correlation analysis revealed a statistically significant correlation between total WCST error score in FA and the perpendicular diffusion coefficient (l2), but not with the parallel diffusion coefficient (l1) (r = 0.100, p = 0.770). It is however interesting that other perpendicular diffusion coefficient (l3) appears not to correlate with WCST score. When we keep in mind that diffusion is truly 3-dimensional process, our result seems to suggest that water diffusion is somewhat anisotropic even in perpendicular direction. These findings therefore demonstrate that the perpendicular diffusion coefficient (l2), which is closely related to myelin integrity, is the main source of FA reduction in the genu of the CC. Furthermore, in addition to the frontal WM deficits in MA abusers found by Chung et al. (2007), our findings suggest that corpus callosum damage secondary to frontal WM deficit is partly responsible for the impairment in frontal executive function observed in MA abusers, as the genu region of the CC is linked to the frontal WM. However, there is a possibility that alterations of the CC, both in size or thickness and density may also contribute to impairment in frontal gray matter (Downhill et al., 2000). Finally, the current study focused on the corpus callosum, neglecting examination of the frontal white matter tracts (which
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are also main targets of MA pathology) for the following reason. The large concentration of white matter in the corpus callosum enables clear separation of white matter from gray matter, which is a significant confounding factor in DTI analysis (Bhagat and Beaulieu, 2004). In the case of frontal or prefrontal WM tracts, ROI analysis is commonly hampered by gray matter contamination; thus, the corpus callosum is the most reliable white matter structure for evaluation of any drug-related alteration of WM integrity using ROI analysis. Future studies should focus on defining the mechanisms that mediate the anomalies reported here, while our data suggest neurotoxic effects of MA. MA is an addictive CNS stimulant, the repeated use of which causes long-term damage to the dopaminergic fiber pathways in the brain, while long-term use of MDMA causes damage to the serotonergic pathway (Ricaurte et al., 1980; Commins and Seiden, 1986). Kokoshka et al. (1998) found a loss of dopamine transporters in the striatal synaptosomes of MA-treated rats. Similarly, Pubill et al. (2003) found that MA induced a 35% loss of dopaminergic terminals in rats, generally without cell death. Therefore, further studies that combine different imaging modalities (e.g., DTI and PET dopamine receptor imaging) may implicate particular neural processes of the affected structures. In conclusion, this study reports change in the white matter integrity of the CC in terms of multiple tensor scalar measures in short-term abstinent MA users, and demonstrates that the eigenvalues along the three principal axes of diffusion offer a unique opportunity to assess the microstructural source of abnormal changes in WM. Because the corpus callosum consists mainly of interhemispheric white matter tracts, these white matter changes in the genu of the CC might be associated with impairment of frontal executive function. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by Advanced Research Center for Recovery of Human sensibility funded by Ministry of Health and Welfare (Grant #02-PJ3-PG6-EV10-0001). This work was also supported by the Brain Korea 21 Project in 2008. References Alberson DN, Pruetz B, Schmidt CJ, Khun DM, Kapatos G, Bannon MJ. Gene expression profile of the nucleus accumbens of human cocaine abusers: evidence for dysregulation of myelin. Journal of Neurochemistry 2004;88:1211–9. Ardekani BA, Nierenberg J, Hoptman MJ, Javitt DC, Lim KO. MRI study of white matter diffusion anisotropy in schizophrenia. Neuroreport 2003;14:2025–9. Axt KJ, Mamounas LA, Molliver ME. Structural features amphetamine neurotoxicity in the brain. In: Cho AK, Segal DS, editors. Amphetamine and its analogs: psychopharmacology, toxicology and abuse. San Diego: Academic; 1994315–67. Beaulieu C, Does MD, Snyder RE, Allen PS. Changes in water diffusion due to Wallerian degeneration in peripheral nerve. Magnetic Resonance in Medicine 1996;36:627– 31. Beaulieu C. The basis of anisotropic water diffusion in the nervous system—a technical review. NMR in Biomedicine 2002;15:435–55. Bhagat YA, Beaulieu C. Diffusion anisotropy in subcortical white matter and cortical gray matter: changes with aging and the role of CSF-suppression. Journal of Magnetic Resonance Imaging 2004;20:216–27. Chung A, Lyoo IK, Kim SJ, Hwang J, Bae SC, Sung YH, et al. Decreased frontal whitematter integrity in abstinent methamphetamine abusers. The International Journal of Neuropsychopharmacology 2007;10:765–75. Commins DL, Seiden LS. Alpha-methyltyrosine blocks methylamphetamine-induced degeneration in the rat somatosensory cortex. Brain Research 1986;365:15–20. Davidson C, Gow AJ, Lee TH, Ellinwood EH. Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Research Reviews 2001;36:1–22. Downhill JE, Buchsbaum J, Wei MST, Spiegel-Cohen J, Hazlett EA, Haznedar MM, et al. Shape and size of the corpus callosum in schizophrenia and schizotypal personality disorder. Schizophrenia Research 2000;42:193–208.
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