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PRIORITY COMMUNICATION Contrasting Effects of Haloperidol and Lithium on Rodent Brain Structure: A Magnetic Resonance Imaging Study with Postmortem Confirmation Anthony C. Vernon, Sridhar Natesan, William R. Crum, Jonathan D. Cooper, Michel Modo, Steven C.R. Williams, and Shitij Kapur Background: Magnetic resonance imaging (MRI) studies suggest that antipsychotic -treated patients with schizophrenia show a decrease in gray-matter volumes, whereas lithium-treated patients with bipolar disorder show marginal increases in gray-matter volumes. Although these clinical data are confounded by illness, chronicity, and other medications, they do suggest that typical antipsychotic drugs and lithium have contrasting effects on brain volume. Methods: Rodent models offer a tractable system to test this hypothesis, and we therefore examined the effect of chronic treatment (8 weeks) and subsequent withdrawal (8 weeks) with clinically relevant dosing of an antipsychotic (haloperidol, HAL) or lithium (Li) on brain volume using longitudinal in vivo structural MRI and confirmed the findings postmortem using unbiased stereology. Results: Chronic HAL treatment induced decreases in whole brain volume (⫺4%) and cortical gray matter (⫺6%), accompanied by hypertrophy of the corpus striatum (⫹14%). In contrast, chronic Li treatment induced increases in whole-brain volume (⫹5%) and cortical gray matter (⫹3%) without a significant effect on striatal volume. Following 8 weeks of drug withdrawal, HAL-induced changes in brain volumes normalized, whereas Li-treated animals retained significantly greater total brain volumes, as confirmed postmortem. However, the distribution of these contrasting changes was topographically distinct: with the haloperidol decreases more prominent rostral, the lithium increases were more prominent caudal. Conclusions: The implications of these findings for the clinic, potential mitigation strategies, and further drug development are discussed. Key Words: Antipsychotic, brain volume, haloperidol, lithium, magnetic resonance imaging, schizophrenia he effects of psychotropic medications have predominantly been presumed to be neurochemical. However, it is increasingly being realized that chronic psychotropic treatment may lead to structural remodeling of the brain, although it remains to be determined whether these changes are related to their positive effects or whether they have any negative implications (1– 4). However, recent data emerging from clinical studies suggest an intriguing picture: chronic treatment with antipsychotic drugs is associated with a decrease in brain cortical volume, whereas treatment with the mood stabilizer lithium (Li) is associated with an increase in cortical brain volume. Haloperidol (HAL), a prototypical typical antipsychotic drugs, has been widely cited to induce changes in brain volume, including enlargement of the caudate nucleus (1,5– 8) and reductions in cortical gray matter (1,2,6,9 –11). In contrast, increasing brain gray matter is well documented with Li treatment (3,4,12–15). Understandably, these data have limitations. Patients are not assigned to placebo for long periods of time, and the interpretations are often confounded by illness duration, chronicity, and severity thereafter. Furthermore, patients have concomitant illnesses and medications. Nonetheless, these data support a T From the Departments of Psychosis Studies (ACV, SN, SK), Institute of Psychiatry, King’s College London; Department of Neuroimaging (WRC, SCRW), Centre for Neuroimaging Sciences, Institute of Psychiatry; Department of Neuroscience (JDC, MM), Institute of Psychiatry, The James Black Centre, King’s College London, London, United Kingdom. Authors ACV and SN contributed equally to this work. Address correspondence to Shitij Kapur, M.D., Ph.D., FRCPC, King’s College London, Institute of Psychiatry, Box P0 01, De Crespigny Park, London, United Kingdom, SE5 8AF; E-mail: [email protected]. Received Sep 28, 2011; revised Nov 22, 2011; accepted Dec 1, 2011. 0006-3223/$36.00 doi:10.1016/j.biopsych.2011.12.004 testable hypothesis that HAL and Li have contrasting effects on brain volume. Rodent models offer an effective way to test this hypothesis, affording precise control over drug exposure, age, concomitant medications, and conditions. However, preclinical studies often lack clinically comparable dosing regimens, and longitudinal noninvasive imaging technology has rarely been used. The two most common approaches to bridge clinical and preclinical studies are to match brain receptor occupancy or therapeutically acceptable drug plasma levels (16,17). Because human dosing intervals are of the order of one pharmacokinetic half-life, animals would have to be injected several times a day to maintain a comparable human plasma concentration for HAL (rodent t1/2 1.5 hour vs. human t1/2 12–36 hours) and Li (rodent t1/2 3.5 hours versus human t1/2 20 –24 hours) (16,18). We have developed a rat model using clinically relevant drug exposure through use of continuous infusion pumps that provide constant drug delivery and matched clinical dosing in combination with longitudinal magnetic resonance imaging (MRI) (19). In the current study, we have used this powerful systems-level approach to address four new questions: 1) Does chronic treatment with HAL and Li result in contrasting effects on brain volume? 2) Are drug effects on brain volume global or limited to specific regions? 3) Are drug effects on brain volume reversible after drug withdrawal? 4) Are changes seen on the MRI verifiable postmortem? Methods and Materials Animals Male Sprague-Dawley rats (Charles River UK, Kent, United Kingdom), initial body weight 240 to 250 g (9 weeks of age) were housed 4 per cage under a 12-hour light– dark cycle (7 AM lights on) with food and water available ad libitum. Room temperature was maintained at 21° ⫾ 2°C and relative humidity at 55% ⫾ 10%. Animals were habituated for 7 days before experimental procedures. Animal experiments were carried out with local ethical approval and in BIOL PSYCHIATRY 2012;xx:xxx © 2012 Society of Biological Psychiatry 2 BIOL PSYCHIATRY 2012;xx:xxx accordance with the Home Office Animals (Scientific Procedures) Act, United Kingdom. Experimental Design The experimental design was as described previously (19), and none of experiments in the present study overlapped with the previous one. Briefly, animals were randomly assigned to one of four treatment groups: 1) vehicle (-hydroxypropylcyclodextrin, 20% w/v, acidified by ascorbic acid, to pH 6); 2) .5 mg/kg/day HAL (low-HAL); 3) 2 mg/kg/day HAL (high-HAL); (Sigma-Aldrich, Dorset, United Kingdom); or 4) 2 mmol/L equiv/kg/day lithium chloride (Sigma-Aldrich). Each treatment group initially comprised 10 animals. Vehicle or drugs were administered subcutaneously from the 10th week of life, using MRI-safe osmotic minipumps (Alzet Model 2ML4, 28 days; Alzet, Cupertino, California) for 8 weeks, equivalent to approximately 5 human years assuming 11.8 rat days equals 1 human year (20). During drug treatment, Li-treated animals were given access to .9% saline instead of tap water to minimize diuretic properties of Li (17), and this was provided to all animals in all treatment groups. Subsequently, animals underwent drug withdrawal for 8 weeks. The doses of HAL were chosen on the basis of 65% to 90% striatal D2 receptor occupancy (16,21), whereas the Li dose was chosen on the basis of previous rodent in vivo data reporting serum plasma levels within the clinical therapeutic range (.4 – 1.2 mmol/mL/L) (17,22–24). Minipumps filled with drug or vehicle solutions were inserted subcutaneously on the back flank under isoflurane anaesthesia (5% induction, 1.5% maintenance) and replaced once after 28 days. In vivo MRI scans were acquired at baseline before surgery, 8 weeks after the start of drug treatment and another 8 weeks after the end of drug treatment. Animals were then sacrificed by cardiac perfusion (.9% saline followed by 4% paraformaldehyde) under terminal anaesthesia (sodium pentobarbital, 60 mg/kg intraperitoneal). Postmortem brain volumes were measured using the Cavalieri estimator probe from Nissl-stained serial coronal sections (40 m, interval 1/12) as previously described (19). Dyskinetic behavior, that is, vacuous chewing movements (VCM) and body weight were assessed at baseline then biweekly until termination as previously described (19). A blood sample was collected after 8 weeks of drug treatment for commercial estimation of plasma drug levels. MRI Acquisition and Analysis In vivo T2-weighted magnetic resonance (MR) images were acquired from each animal in a random order interspersed with phantoms to ensure consistent operation. Images were acquired using a 7.0-T horizontal small bore magnet (Varian, Palo Alto, California), as previously described (19,25) (see also Supplement 1). Before analysis, MR images were visually inspected for motion, intensity, or gross anatomical malformation, and scans displaying such artifacts were excluded from further analysis. One animal from the vehicle group did not survive the 8-week MRI scan because of respiratory failure. Therefore, final MRI scans analyzed per group at each time point were vehicle, n ⫽ 9; low-HAL, n ⫽ 7; high-HAL, n ⫽ 8; Li, n ⫽ 9. Whole-brain volume, total intracranial volume, cerebral cortex, and subcortical structures (lateral ventricles, hippocampal formation, and corpus striatum) volumes were delineated manually from in vivo MR images by two raters (ACV and SN) blinded to treatment group, using the region-of-interest tool in JIM software (v5.0; Xinapse Systems, Northants, United Kingdom) (19,25). Anatomic landmarks and reference to the rodent brain atlas (26) were used to define region-of-interest contours (Table S1 in Supplement 1) exemplified in Figure S1 of Supplement 1. Volumes were calculated by multiplying the sum of the areas of a given structure on all slices www.sobp.org/journal A.C. Vernon et al. measured by the slice thickness (.6 mm). Intrarater and interrater reliability were assessed following repeated measurements using the intraclass correlation coefficient (27). Total cortex (CTX) volume for each individual slice from each animal acquired after 8 weeks of drug or vehicle treatment were also divided into rostral (slices 1– 6, equivalent to approximately ⫹3.00 to ⫺.12 mm from bregma), midlevel (slices 7–13; ⫺.72 to ⫺3.36 mm from bregma), or caudal (slices 14 –21; ⫺3.96 to ⫺7.56 mm from bregma) regions. Postmortem Tissue Handling and Regional Volume Measurements After termination, brains were removed and cryoprotected in buffered 30% sucrose for 48 hours before storage in tissue cryoprotection solution (25% glycerin [v/v] 30% ethylene glycol [v/v] in .2 mol phosphate buffer). Measurements of brain weight and volume were repeated at each step in the tissue processing, as described elsewhere (19). Serial coronal sections (40 m, interval 1/12) were prepared and Nissl stained using cresyl fast violet solution (10% w/v, 30 min, room temperature) as previously described (19). A single observer (ACV), blinded to experimental groups, measured the volume of the cerebral cortex and corpus striatum using the Cavalieri estimator probe as previously described (19,28). Gunderson coefficients of error (m1) were calculated and were always less than .05 (29,30). Data and Statistical Analysis Statistics were performed using SPSS 19.0 software (SPSS, Chicago, Illinois). Longitudinal assessment of variables was performed using two-way repeated-measures analysis of variance with one between-subject factor (treatment) and one within-subject factor (time) followed by post hoc Bonferroni test for multiple comparisons. Data for baseline versus Week 8 and baseline versus Week 16 were analyzed separately. Postmortem brain weight/volume and Cavalieri probe estimates of brain volume were analyzed using one-way analysis of variance followed by post hoc Bonferroni test for multiple comparisons. VCMs due to drug treatment were analyzed using the repeated-measures nonparametric Friedman test followed by pairwise nonparametric comparisons using a Wilcoxon t test. An ␣ level of .05 was selected. Results Plasma Levels and Behavior Administration of psychotropic drugs by osmotic pump achieved clinically relevant plasma levels (mean ⫾ SD) of 4.7 ⫾ .32 ng/mL (low HAL), 23.3 ⫾ 1.5 ng/mL (high HAL) and .49 ⫾ .04 mmol/L (Li). All animals increased in body weight over time irrespective of treatment group (Figure 1A; Table 1). HAL-treated animals gained less weight compared with controls, but this did not reach statistical significance. Chronic Li-treated animals displayed polyuria, but body weight was comparable to vehicle controls. No other adverse effects were observed, and animals appeared healthy throughout the treatment duration. Stereotypical VCM behavior developed only in HAL-treated animals 2 weeks after initiation of drug treatment, irrespective of dose, increasing to a maximum by 8 weeks of drug treatment (Figure 1B). Upon withdrawal of HAL, VCM behavior decreased differentially in a dose-dependent manner, returning to levels comparable to vehicle-treated animal levels, faster in low-HAL-treated animals than high-HAL-treated animals. Dose-Dependent Effects of Chronic Haloperidol Treatment on Whole-Brain and Regional Brain Volumes To establish the global effects of HAL, whole-brain (WBV) and total intracranial volumes (TIV) were measured from in vivo T2- BIOL PSYCHIATRY 2012;xx:xxx 3 A.C. Vernon et al. icantly increased with time (Table 2; Figure S3 in Supplement 1), it did so comparably across treatment groups, hence there was no effect of HAL on growth per se. Across all treatment groups, WBV significantly increased with time, and significant main effects of drug treatment and a time ⫻ treatment interaction were detected (Table 2; Figure 2A). HAL treatment significantly decreased WBV in a dose-related manner (low-HAL vs. control ⫺1.9%; p ⬍ .05; high-HAL vs. control ⫺3.7%; p ⬍ .01; Figure 2A). To pinpoint areas in the brain that account for smaller WBV observed after chronic HAL treatment, analysis of regional brain volumes was conducted. For CTX volume, significant main effects of drug treatment and a time ⫻ treatment interaction were detected (Table 2). HAL treatment resulted in a significant decrease in CTX volume compared with vehicle-treated controls in a dose-related manner (low-HAL vs. control ⫺1.0%; p ⬍ .05; high-HAL vs. control ⫺5.9%; p ⬍ .01; Figure 2B). Cortical slice profile analysis of Week 8 MR images revealed significant main effects of drug treatment and slice position, but no slice position ⫻ treatment interaction in rostral, midlevel, and caudal cortical regions (Table 3). Volume decreases in the CTX of HAL-treated animals were confined to the rostral and midlevel cortex, and more widespread cortical slices were affected in high- compared with low-HAL-treated animals (Figure 3A–3F). The volumes of the total corpus striatum (STR; left ⫹ right hemisphere) were significantly increased following chronic treatment with HAL (Table 2) compared with vehicle-treated animals (low-HAL ⫹8.6%; p ⬍ .05; high-HAL⫹13.6%; p ⬍ .01; Figure 2C). There were no statistically significant effects of chronic HAL treatment on total lateral ventricle or total hippocampal volume (Table 2; Figure S3 in Supplement 1). Figure 1. (A) Changes in body weight during chronic psychotropic drug treatment and withdrawal. Both low-haloperidol (HAL)-treated (low-HAL; n ⫽ 10) and high-HAL-treated (n ⫽ 10) animals gained less weight compared with vehicle-treated controls (n ⫽ 9) after 8 weeks of treatment, although this did not reach statistical significance. Chronic lithium chloride (LiCl) treatment had no effect on animal body weight (n ⫽ 10). Data shown are body weight (mean ⫾ SEM) for each treatment group at each time point. (B) Chronic HAL but not LiCl treatment induces stereotypical vacuous chewing movements (VCM) by 2 weeks, maintained until 8 weeks of drug treatment. Upon withdrawal of HAL, VCM behavior decreased differentially in a dosedependent manner, **p ⬍ .01 low-HAL and high-HAL versus vehicle, respectively. Data shown are mean VCM score ⫾ SEM across time for vehicle and psychotropic drug treated animals. BL, baseline. weighted MR images. At baseline, before drug administration, there were no significant differences in brain volumes across the treatment groups (Figure S2 in Supplement 1). Although TIV signif- Effects of Chronic Lithium Treatment on Whole-Brain and Regional Brain Volumes We used an identical approach to investigate chronic Li treatment. Although TIV significantly increased with time, it did so comparably across treatment groups, hence there was no effect of Li on growth per se (Table 2; Figure S3 in Supplement 1). Li treatment resulted in a significant increase in WBV compared with vehicle (⫹4.8%; p ⬍ .05; Figure 2A), low-HAL (⫹6.7%; p ⬍ .01), or high-HALtreated animals (⫹8.5%; p ⬍ .01). Further analysis of regional brain volumes was conducted to account for increased WBV. Li treatment resulted in a significant increase in CTX volume when compared with vehicle (⫹3.3%; p ⬍ .01; Table 2; Figure 2B) and either low-HAL (⫹4.0%; p ⬍ .01) or high-HAL (⫹9.2%; p ⬍ .01) treated animals. Slice profile analysis of Table 1. Change in Body Weight over Time in Vehicle- and Psychotropic Drug-Treated Animals Body Weight (g) Independent On Drug Off Drug Time Point Vehicle Low-HALa High-HALb Lithiumc Baseline Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 Week 14 Week 16 242.7 ⫾ 2.7 308.7 ⫾ 3.0 338.4 ⫾ 4.7 368.0 ⫾ 5.8 392.6 ⫾ 6.6 388.9 ⫾ 7.0 411.7 ⫾ 7.5 425.6 ⫾ 7.3 421.6 ⫾ 7.3 241.6 ⫾ 3.3 298.6 ⫾ 7.6 320.8 ⫾ 9.4 349.5 ⫾ 8.3 362.2 ⫾ 13.4 374.0 ⫾ 13.7 386.6 ⫾ 15.1 402.4 ⫾ 16.5 398.3 ⫾ 16.1 248.3 ⫾ 2.4 289.5 ⫾ 8.8 324.3 ⫾ 4.4 341.6 ⫾ 5.5 363.2 ⫾ 4.7 375.1 ⫾ 7.1 395.4 ⫾ 6.4 407.3 ⫾ 8.2 403.5 ⫾ 8.7 241.3 ⫾ 4.2 306.8 ⫾ 5.2 341.6 ⫾ 6.3 370.0 ⫾ 7.5 389.4 ⫾ 7.9 396.5 ⫾ 7.7 418.2 ⫾ 7.8 430.7 ⫾ 8.7 426.1 ⫾ 8.6 Data shown are body weight (mean ⫾ SEM) for each treatment group across time. HAL, haloperidol. a .5 mg/kg/day subcutaneous. b 2 mg/kg/day subcutaneous. c 2 mmol/L equiv/kg/day subcutaneous. www.sobp.org/journal 4 BIOL PSYCHIATRY 2012;xx:xxx A.C. Vernon et al. Table 2. Results of Two-Way Repeated-Measures ANOVA Statistics for Longitudinal In Vivo Magnetic Resonance Imaging Volume Measurements Effect of Treatment ANOVA Within Subjects Between Groups Time Time ⫻ Treatment Interaction F(1,29) ⫽ 4133; p ⬍ .05 F(1,29) ⫽ 2503; p ⬍ .05 F(3,29) ⫽ 3.98; nss F(3,29) ⫽ 12.69; p ⬍ .05 F(3,29) ⫽ 1.43; nss F(3,29) ⫽ 6.50; p ⬍ .05 Cerebral Cortex F(1,29) ⫽ 749.2; p ⬍ .05 F(3,29) ⫽ 9.35; p ⬍ .05 F(3,29) ⫽ 19.32; p ⬍ .05 Corpus Striatum F(1,29) ⫽ 708.6; p ⬍ .05 F(3,29) ⫽ 6.06; p ⬍ .05 F(3,29) ⫽ 4.23; p ⬍ .05 Hippocampal Formation Lateral Ventricles Baseline vs. 16 Weeks Total Intracranial Volume Whole Brain Volume F(1,29) ⫽ 281.8; p ⬍ .05 F(1,29) ⫽ 79.5; p ⬍ .05 F(3,29) ⫽ 1.02; nss F(3,29) ⫽ 1.46 nss F(3,29) ⫽ .21; nss F(3,29) ⫽ .41; nss F(1,29) ⫽ 2831; p ⬍ .05 F(1,29) ⫽ 3391; p ⬍ .05 F(3,29) ⫽ 1.42; nss F(3,29) ⫽ 6.97; p ⬍ .05 F(3,29) ⫽ .44; nss F(3,29) ⫽ 2.64; nss Cerebral Cortex Corpus Striatum Hippocampal Formation Lateral Ventricles F(1,29) ⫽ 983.5; p ⬍ .05 F(1,29) ⫽ 600.7; p ⬍ .05 F(1,29) ⫽ 312; p ⬍ .05 F(1,29) ⫽ 179.5; p ⬍ .05 F(3,29) ⫽ 0.42; nss F(3,29) ⫽ .92; nss F(3,29) ⫽ .63; nss F(3,29) ⫽ .54; nss F(3,29) ⫽ 2.79; nss F(3,29) ⫽ 1.87; nss F(3,29) ⫽ 1.48; nss F(3,29) ⫽ .50; nss Variable Baseline vs. 8 Weeks Total Intracranial Volume Whole Brain Volume Treatment Post Hoc Testa nd p ⬍ .05 vehicle vs. low-HAL p ⬍ .01 vehicle vs. high-HAL p ⬍ .05 vehicle vs. Li p ⬍ .05 vehicle vs. low-HAL p ⬍ .01 vehicle vs. high-HAL p ⬍ .01 vehicle vs. Li p ⬍ .01 vehicle vs. low-HAL p ⬍ .01 vehicle vs. high-HALM p ⬎ .05 vehicle vs. Li nd nd nd p ⬎ .05 vehicle vs. low-HAL p ⬎ .05 vehicle vs. high-HAL p ⬍ .05 vehicle vs. Li nd nd nd nd Drug treatment served as the between-subject factor and time as the within-subject factor. Data shown are comparing baseline to the Week 8 and Week 16 time points. ANOVA, analysis of variance; HAL, haloperidol; Li, Lithium chloride; nd, no difference; nss, no statistical significance. a Bonferroni’s test for multiple comparisons among treatment groups. Week 8 MR images revealed that CTX volume increases in Li-treated animals were predominantly in the midlevel and caudal cortical areas (Table 3; Figure 3G–3I). However, closer examination of the cortical slice profiles in Li-treated animals suggests a small persisting increase across the whole cortical profile, although not statistically significant (Figure 3G–3I). Li treatment had no significant effect on STR volume compared with vehicle-treated controls (Table 2; Figure 2C). There were no statistically significant effects of chronic Li treatment on total lateral ventricle and total hippocampal volume (Table 2; Figure S3 in Supplement 1). Effects of Drug Withdrawal on Whole-Brain and Regional Brain Volumes Following drug withdrawal (8 weeks), there were no longer any significant differences in WBV between vehicle-treated and either low-HAL- or high-HAL-treated animals (Table 2; Figure 2A). In contrast, to the effects of HAL, WBV remained significantly elevated in Li-treated animals (Table 2) compared with vehicle (⫹6.3%; p ⬍ .05; Figure 2A), low-HAL-treated (⫹6.2%; p ⬍ .01) and high-HAL-treated animals (⫹4.9%; p ⬍ .01). To determine which brain regions were altered following drug withdrawal, analysis of regional brain volumes was repeated. After drug withdrawal, no significant differences in total CTX volume were observed between vehicle-treated and either low-HAL or high-HAL-treated animals, or between Li-treated animals and either vehicle, low-, or high-HAL-treated animals (no difference; Table 2; Figure 2B). No significant differences were observed in CTX volume. Slice profile analysis of Week 16 MR images confirmed cortical volume differences normalized across the three cortical areas following drug withdrawal comparing HAL and vehicle-treated animals (Figure S4 in Supplement 1). Striatal hypertrophy induced by www.sobp.org/journal HAL was reversed after drug withdrawal, with no significant differences in total STR volume between low- and high-HAL-treated animals and vehicle- or Li-treated animals (ns; Table 2; Figure 2C). Trends toward ventricular hypertrophy were noted in Li-treated animals, but no statistically significant effects of drug withdrawal were found for total lateral ventricle and total hippocampal volume (Figure S3 in Supplement 1). Postmortem Confirmation of In Vivo MRI To confirm in vivo MRI measurements made after drug withdrawal, postmortem measurements of brain weight and volume were obtained and the volume of the CTX and STR assessed histologically using unbiased stereology. The mean fresh weights and volumes of brains extracted from Li-treated animals were significantly heavier compared with vehicle, low-, or high-HAL-treated animals (p ⬍ .05; Figure 4A and 4B). Importantly, during the three phases of tissue processing, brain weight and volume, respectively, changed in a similar manner across the four exposure groups (Figure S5 in Supplement 1). Histological measurement of the total volume (left ⫹ right hemisphere) of the CTX and STR using unbiased stereology (Cavalieri estimator probe) revealed no significant differences in either total CTX volume [F (3,35) ⫽ 2.697; p ⬎ .05; Figure 4C] or total STR volume [F (3,35) ⫽ .967; p ⬎ .05; Figure 4D] between treatment groups, thus confirming the in vivo MRI data. Discussion This is the first longitudinal in vivo MRI study in rodents to compare the effects of two commonly prescribed psychotropic medications on different brain regions. We observed contrasting effects of treatment (8 weeks) with therapeutically relevant concen- BIOL PSYCHIATRY 2012;xx:xxx 5 A.C. Vernon et al. Figure 2. Chronic treatment with haloperidol (HAL) or lithium chloride (LiCl) results in contrasting effects on whole brain and regional brain volumes. Chronic HAL treatment (8 weeks) dose-dependently decreased (A) whole brain volume and (B) total cortical volume, but increased striatal volume (C), compared with vehicle-treated animals. Following drug withdrawal (8 weeks), HAL-induced volumetric changes were reversed (A–C). In contrast, chronic LiCl treatment increased (A) whole brain volume and (B) total cortical volume but had no effect on striatal volume (C). Effects of LiCl on whole brain volume were maintained even after drug withdrawal (A), whereas the cortical increase normalized (B). Data shown are mean volume ⫾ SEM; *p ⬍ .05; **p ⬍ .01 HAL versus vehicle and LiCl versus vehicle, respectively. Total magnetic resonance imaging scans analyzed per group at each time point: vehicle, n ⫽ 9; low-HAL, n ⫽ 7; high-HAL, n ⫽ 8; LiCl, n ⫽ 9 (see Methods and Materials). trations of HAL and Li. Specifically, HAL-treatment resulted in doserelated reductions in WBV (up to ⫺3.7%) and CTX (up to ⫺5.9%) accompanied by hypertrophy of the STR (up to ⫹14%) compared with vehicle controls. In contrast, Li-treatment resulted in significant increases in WBV (up to ⫹4.8%) and CTX volume (up to ⫹3.3%), with no effect on the STR, when compared with controls. Following drug withdrawal (8 weeks, 16 weeks from baseline), the effects of either dose of HAL on WBV, CTX, and STR volume were reversed. In contrast, the WBV of Li-treated animals remained elevated, although the effects of Li on CTX volume alone were no longer significant. These MRI data were confirmed histologically using an unbiased stereological method, lending certainty to our in vivo findings. Comparison of the Effects of Chronic Treatment with HAL or Li on Brain Morphology The volume, location and profile of decreases in WBV and CTX in HAL-treated animals are similar to our previous in vivo study, con- firming that initial finding (19). These data are consistent with clinical neuroimaging studies of the trajectory of HAL-induced volume changes in schizophrenia patients (1,2,31). In the current study, as a result of a larger sample size, we were able to detect striatal hypertrophy due to HAL treatment, which was only observed at trend level in our previous study (19). These data are consistent with previous studies in rodents (32,33) and clinical studies (1,2,5–7). Studies reporting on the loss of gray matter in schizophrenic patients treated with antipsychotics include a 2.4% decrease in frontal lobe gray matter volume (2), which is related to dose and duration of antipsychotic treatment (1). The dosing regimen used in this study was tailored to capture clinical practice. The lower dose of HAL chosen for the current study (.5 mg/kg/day) achieves a drug plasma level within the recommended clinical range of 2 to 5 ng/mL (16, 34) and results in ⬃1.0% decrease in total cortex volume. The higher dose of HAL (2 mg/kg/day) achieves plasma levels higher than the recommended range but still commonly within the range Table 3. Results of Two-Way ANOVA Statistics for In Vivo Magnetic Resonance Imaging Volume Measurements of Cortical Slice Profiles (Week 8) ANOVA Within Subjects Variable Frontal Cortexa Medial Cortexb Caudal Cortexc Between Groups Slice Position Slice ⫻ Treatment Interaction Treatment F(6,203) ⫽ 96.78; p ⬍ .05 F(6,203) ⫽ 129.5; p ⬍ .05 F(6,203) ⫽ 180.9; p ⬍ .05 F(18,203) ⫽ 1.540; p ⬎ .05 F(18,203) ⫽ .876; p ⬎ .05 F(18,203) ⫽ .936; p ⬎ .05 F(3,203) ⫽ 47.20; p ⬍ .05 F(3,203) ⫽ 65.30; p ⬍ .05 F(3,203) ⫽ 41.67; p ⬍ .05 Drug treatment served as between-subject factor and slice position as within-subject factor. ANOVA, analysis of variance. a Rostral cortex is defined as ⫹3.00 to ⫺.12 mm from bregma. b Midlevel cortex is defined as ⫺.72 to ⫺3.36 mm from bregma. c Caudal cortex is defined as ⫺3.96 to ⫺7.56 mm from bregma (see Methods and Materials). www.sobp.org/journal 6 BIOL PSYCHIATRY 2012;xx:xxx A.C. Vernon et al. Figure 3. Slice profile analysis of cortical volume changes following 8 weeks of drug or vehicle treatment suggests significant volume decreases in haloperidol (HAL)-treated animals (A–F) are confined predominantly to rostral and midlevel cortical regions, with more widespread cortical areas affected in high-HAL (D-F) treated animals than low-HAL (A-C) treated animals, when compared to vehicle-treated controls. In contrast, lithium chloride (LiCl)-treated animals displayed significant cortical volume increases predominantly in the midlevel and caudal cortical areas (G–I). However, closer examination of the cortical slice profile in LiCl-treated animals suggests a small, persisting increase across the whole cortical profile, although this does not reach statistical significance at every slice (G–I). Data shown are mean slice volume at each position ⫾ SEM; *p ⬍ .01 HAL versus vehicle and Li versus vehicle, respectively. Total MRI scans analyzed per group at each time point: vehicle, n ⫽ 9; low-HAL, n ⫽ 7; high-HAL, n ⫽ 8; LiCl, n ⫽ 9 (see Methods and Materials). encountered in clinical practice (10 –20 ng/mL) leading to a 6% decrease in CTX volume. Therefore, the animal data show roughly comparable findings, which are dose dependent, in the ranges that are encountered in clinical practice. Yet no effects of HAL treatment on the volume of the lateral ventricles (LV) or hippocampus were observed during drug treatment or after withdrawal. LV hypertrophy and decreased hippocampus volume are two of the most replicated imaging findings in schizophrenia (35–37), indicating that not all anatomic changes are drug-related. In contrast to HAL, Li-treatment induces an increase in both WBV and CTX volume, consistent with human studies of Li treatment in healthy volunteers (38). These data strongly suggest usage of Li potentially contributes to the heterogeneity in neuroimaging studies of bipolar disorder (39,40). Indeed, Li treatment of patients with bipolar disorder induces increases in total gray matter (3,4,12,13), cortical gray matter (12,41), hippocampal (14,15,42) volumes, and www.sobp.org/journal LV volumes (40) relative to unmedicated patients and healthy controls after as little as 4 weeks treatment. Our findings in rats are consistent with these human data, although some notable differences were apparent. In particular, chronic Li treatment did not significantly alter hippocampal volume in rats. This may be explained by the lack of disease-related neuroanatomic changes in the young healthy animals used for our model. Comparison of the Effects of Drug Withdrawal of HAL or Li on Brain Morphology Having demonstrated the contrasting effects of HAL and Li on brain volume, we next investigated how brain volume responds to withdrawal of drug treatment. Our in vivo MRI data suggest that HAL-induced morphologic changes are reversible upon drug withdrawal. These data were confirmed histologically and are supported by other studies in animals (32,43,44). Interestingly, a de- A.C. Vernon et al. BIOL PSYCHIATRY 2012;xx:xxx 7 Figure 4. Postmortem analysis of brain volume changes following drug withdrawal. Measurement of (A) fresh brain weight and (B) volume at dissection confirms a lack of significant differences between haloperidol (HAL)- and vehicle-treated animals following 8 wks of drug withdrawal. In contrast, lithium chloride (LiCl)-treated animals retained significantly enlarged brains compared to vehicle-treated controls, reinforcing the in vivo magnetic resonance imaging findings. Data shown are individual brain weight (A) or volume (B) (scatter plot with mean), *p ⬍ 0.05 Li vs. vehicle; nss, no statistical significance. Reversal of HAL and lithium chloride (L)-induced changes in cortical (C) and striatal (D) volume was confirmed histologically using the Cavalieri probe method. Data shown are mean estimated cortical or striatal volume ⫾ SEM. V, vehicle (n ⫽ 9); H(.5), HAL .5 mg/kg/day (n ⫽ 10); H(2), HAL 2 mg/kg/day (n ⫽ 10); L (n ⫽ 10). crease in caudate nucleus volume was found in a small number of schizophrenia patients in remission who discontinued atypical antipsychotic medication, whereas caudate nucleus volume increases were observed in those who continued, consistent with our findings (45). However, gray matter volume continued to decline in the same patients following discontinuation of antipsychotics (45). This reinforces the notion that schizophrenia is associated with progressive brain morphologic changes, at least in a subset of patients (46,47) to which antipsychotic drugs may contribute but are not the sole cause (1,2). Additional work is required to investigate this phenomenon fully. To our knowledge, no studies have examined the effect of Li discontinuation on brain volumes. This is relevant, because the osmotic effects of Li may lead to neuronal swelling, which might provide an alternative explanation of increased brain volumes as opposed to putative neurotrophic/neuroprotective mechanisms (17,38). The few animal studies on this matter are not consistent, probably reflecting methodologic differences (48,49). However, chronic (5-week) Li treatment induced a 3.1% increase in brain tissue water content in the rostral cortex and hippocampus of rats (49). In our study, slice profile analyses suggested no significant increases in the volume of the rostral cortex, nor were there any significant increases in hippocampal volume following chronic Li treatment. Furthermore, the observation that WBV remained elevated after drug withdrawal suggests at least a subset of brain volume changes induced by chronic Li treatment are maintained. Taken together, these data argue against our data being explained by purely osmotic effects of Li. However, we cannot ignore the finding that cortical volume alterations induced by Li were reversed following drug withdrawal, suggesting that other brain regions are contributing to the observed WBV increase. Further image analysis and corroborative histological work is underway to evaluate this. Neurobiology Underlying Volume Changes due to Chronic Treatment with Psychotropics The antipsychotic actions of HAL are attributed to blockade of D2/3 receptors. However, volume changes observed in our study include regions that express D2/3 receptors in high-density (striatum) and regions with sparse distribution (prefrontal cortex) (50). Sustained blockade of D2/3 receptors results in increased dopamine turnover (51), which may result in oxidative damage through production of cytotoxic free radicals due to auto-oxidation (52). However, the striatum that has the highest concentration of D2/3 receptors, paradoxically, shows hypertrophy in contrast to a volume decrease in the cortex. Therefore, other mechanisms (Akt and glycogen synthase kinase 3 signaling pathways) (53) or idiosyncratic mechanisms could be responsible. Of note, primate postmortem studies suggest chronic antipsychotic drug treatment induces a reduction in cortical astrocyte number (54,55). These data are consistent with postmortem studies in human schizophrenia brain tissue, suggesting loss of neuropil rather than overt neurodegeneration (56). In contrast, Li has pleiotropic biological actions, and both human and animal studies suggest potential neurotrophic effects of Li (57). In several brain regions including the cortex, Li affects multiple cellular pathways including oxidative stress, apoptosis, inflammation, glial dysfunction, excitotoxicity, and mitochondrial stability (58). Furthermore, Li increases expression of brain-derived neurotrophic factor and B-cell lymphoma-2, while inhibiting protein kinase C and glycogen synthase kinase-3. It is currently unclear which of these cellular actions may underlie the effects of Li on brain volume. Further image analysis and corroborative histology are currently underway to identify the cellular components and mechanisms underlying both antipsychotic drugs and Li-induced changes in brain volume. Limitations of the current study should be noted. First, young rats (spanning 10 –16 weeks of age), corresponding to late adolescence in humans, were used, not reflecting other age groups. Consequently, additional experimentation in younger and older animals is required. Second, there is a fundamental gap in our knowledge of how chronic psychotropic drug treatment affects cognition. Whereas chronic antipsychotic drug treatment induces cognitive deficits, Li has been shown to improve cognition in rodents (54 –59). Third, we focused our studies on gray matter, because we did not detect any gross effect of antipsychotics on white matter volume in our prior study (19) and human data suggest Li does not affect white matter volume (3,4). Nevertheless, our data do not preclude an effect of psychotropic drug treatment on white matter microstructure, which may be observed using more sensitive imaging techniques, such as diffusion tensor imaging. Fourth, www.sobp.org/journal 8 BIOL PSYCHIATRY 2012;xx:xxx we used manual segmentation to measure brain volumes, a robust, widely used method (19,25,59). However, it is prone to bias (although minimized by repeated measurements and blinding to subject treatment status) requires a priori hypotheses, and is relatively insensitive to subtle morphologic change (60). Fourth, because of the longitudinal nature of this in vivo study, postmortem analysis of brain volume changes at Week 8 is lacking. Nevertheless, we have previously confirmed HAL induced cortical volume change postmortem after 8 weeks of treatment (19). In conclusion, our study offers a powerful model system for further investigation of the effects of psychotropic drug treatment on brain morphology. Our data are consistent with clinical studies suggesting that antipsychotics may contribute to region-specific brain volume decreases in schizophrenia patients, whereas Li induces brain volume increases in bipolar disorder patients. Notably, our data suggest that the effects of HAL are dose-related and reversible, whereas some effects of Li are maintained following drug withdrawal. Nonetheless, our studies were done in normal rats, which do not capture the innate pathology of either schizophrenia or bipolar disorder. Moreover, because the mechanism(s) of these drug effects remain unknown, further studies are required, and one should be cautious in drawing clinical inferences. Strategic funding from the Medical Research Council (Grant Nos. G0701748 [85253] and G1002198), which we thank for its generous financial assistance, supported this study. We also thank the British Heart Foundation for supporting the 7T magnetic resonance imaging scanner at the King’s College London Preclinical imaging unit (KCLPIU). JC acknowledges support from the Batten Disease Support and Research Association, Batten Disease Family Association, and The Natalie Fund, which provided the microscope setup. SCRW and WRC acknowledge support from the National Institute for Health Research Biomedical Research Centre for Mental Health at the South London and Maudsley National Health Service Foundation Trust and Institute of Psychiatry, Kings College London, and the King’s College London Centre of Excellence in Medical Engineering funded by the Wellcome Trust and Engineering and Physical Sciences Research Council (Grant No. WT 088641/Z/09/Z). We acknowledge the technical support of Miss Lauren Heathcote M.Sc. and Dr. Po-Wah So (KCLPIU). 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