Address correspondence and reprint requests to Xin-Min Li, Department of Psychiatry, University of Manitoba, PZ432-771, Bannatyne Ave., Winnipeg, MB, Canada R3E 3N4. E-mail: firstname.lastname@example.org (or) Qingrong Tan, Department of Psychiatry, Xijing Hospital, Fourth Military Medical University, 15 West Changle Road, Xi’an, Shanxi Province, China 710032. E-mail: email@example.com
White matter impairment is a feature of vascular depression. The anti-psychotic quetiapine has been shown to enhance the therapeutic effects of anti-depressants on vascular depression, but the mechanism remains unknown. In this study, we found that 2 weeks of treatment with quetiapine prior to bilateral carotid artery occlusion and reperfusion, in an animal model of vascular depression, resulted in reduced myelin breakdown and oligodendrocyte loss compared to placebo-treated mice on post-operative day (POD) 7. For late stage of recovery (POD40), quetiapine treatment resulted in enhanced oligodendrocyte maturation relative to placebo. The results suggest that quetiapine is a potential intervention for oligodendrocyte damage and this may contribute to its anti-depressant effects through white matter protection in vascular depression.
Late-life depression (LLD) is the depression that occurs in elderly patients with evidence of cerebrovascular impairments and prominent executive dysfunction (Herrmann et al. 2007). The vascular depression hypothesis suggests that LLD results from disrupted fiber tracts connecting cortical and subcortical structures, mainly the subcortical white matter; cerebrovascular risk factors (strokes, arthrosclerosis, diabetes, hypertension, and smoking, etc.) are highly associated with white matter hypersensitivities in LLD (reviewed in (Herrmann et al. 2008). This evidence indicated that white matter/oligodendrocyte changes may contribute to vascular depression and may be a therapeutic target.
Compared to early-life major depressive disorder, vascular depression is more resistant to conventional anti-depressant treatment. Atypical anti-psychotics have been used as augmenting agents in vascular depression. Quetiapine, a widely used atypical anti-psychotic, has shown superior therapeutic effects in treating late-life depression with cerebrovascular damage (Carta et al. 2007). However, the underlying mechanism remains unknown.
As white matter hyperintensities may predict the treatment effects of selective serotonin reuptake inhibitors in late-life depression (Alexopoulos et al. 2008; Sheline et al. 2010), we hypothesized that atypical anti-psychotics (e.g., quetiapine) may augment the anti-depressant effects by altering white matter impairments.
Our previous studies have shown that quetiapine alleviated depression-like behaviors and neuronal damage in mice with bilateral common carotid arteries occlusion (BCCAO) (Yan et al. 2007a,b), an animal model of global cerebral ischemia (GCI) and vascular depression (Gaur and Kumar 2010). In this study, we tested the hypothesis that pre-treatment with quetiapine protects the brain from OLG loss and enhances OLG regeneration in GCI mice.
Materials and methods
Male CD1 mice (18–20 g, Charles River, St. Constant, QC, Canada) received either quetiapine (10 mg/kg in saline, AstraZeneca, Canada, Montreal, QC, Canada) or vehicle (saline) treatment (i.p.) for 14 days. On day 15, the mice were assigned to a sham or a BCCAO surgery to induce GCI as previously described (Yan et al. 2007b), which generated four treatment groups: Sham (sham + saline), QTP (sham + quetiapine), GCI (GCI + saline), and G + Q (GCI + quetiapine). Briefly, mice were anesthetized with ketamine (110 mg/kg) and xylazine (15 mg/kg). Both common carotid arteries were exposed and occluded with cotton thread for 60 min, after which the thread was removed to allow reperfusion. The sham group underwent the same procedure without arterial occlusion. Animals recovered for either 7 or 40 days post-operative (POD) and were then killed for immunohistochemical analysis. All experiments were approved by the University of Saskatchewan Animal Care Committee protocol review.
Bromodeoxyuridine (BrdU), immunohistochemistry, and Western blot
To identify proliferating cells, mice were administered (BrdU, 150 mg/kg, i.p, Sigma, Oakville, ON, Canada) daily for 3 days, starting on POD4. Mice were deeply anesthetized and euthanized and transcardially perfused on POD7 or POD40. Serial sections of the right hemisphere were cut into 30 μm sections through the entire hippocampus for immunohistochemistry. The hippocampus from the left hemisphere was dissected and processed for western blot assay.
The following primary antibodies were used: rabbit anti-Glutathione S Transferase pi (GST-pi) (1 : 500; Assay Designs, Inc. Ann Arbor, MI, USA) to detect mature OLGs, mouse anti-O4 (1 : 300; Chemicon, Billerica, MA, USA) to detect pre-OLGs, goat anti-myelin basic protein (MBP) (1 : 250, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for myelin sheaths, rabbit anti-NG2 proteoglycan (1 : 200, Chemicon) for oligodendrocyte progenitor cells (OPCs), anti-glial fibrillary acidic protein (GFAP) antibodies (1 : 2000, Dako, Carpinteria, CA, USA) for astrocytes and mouse anti-BrdU antibody (1 : 200; Sigma-Aldrich) to detect the cell proliferation. Double immunofluorescent staining of BrdU and NG2, or GST-pi was used to determine the newborn OPCs and their maturation, respectively. The sections treated without primary antibodies were used as negative controls.
The number of cells positive for O4, NG2, BrdU, GST-pi, GFAP, and double-stained cells were counted in six fields of view in the dentate gyrus (DG) and cornu ammonis 1(CA1) of the hippocampus from three sections between Bregma −1.28 mm to −2.12 mm (Franklin and Paxinos 1997). The immunopositive cells were counted and presented as numbers per mm2. Myelin basic protein (MBP) was measured in six fields of the hippocampus and presented as integrated optical density (IOD) by multiplying average density and MBP-positive area in the total scanned area.
The colocalization of BrdU and NG2, or GST-pi in representative sections from each animal was determined using an Olympus confocal microscope. Each double-staining cell was manually examined in its full ‘z’ dimension with use of split panel analysis to ensure the BrdU + nucleus was unambiguously associated with the lineage-specific marker.
A two-way anova (analysis of variance) followed by the Newman–Keul’s test was used to assess group differences. A p-value < 0.05 was considered statistically significant. Comparisons included GCI versus sham and G + Q versus GCI.
The IOD score of MBP immunostaining was significantly lower in the hippocampus of the mice in the GCI group relative to the sham group on POD7 or POD40. The mice in the G + Q group had significantly more IOD of MBP staining in the hippocampus relative to GCI mice on both POD7 and POD40 (p < 0.05). This suggests that GCI reduced myelin whereas quetiapine prevented myelin loss in the hippocampus of GCI animals (Fig. 1a–c).
The number of O4-positive cells in the CA1 was significantly lower in the GCI group relative to the sham group on both POD7 (p < 0.01) and POD40 (p < 0.05). The number of O4-positive cells in the CA1 region of the G + Q group was significantly greater compared to the GCI group on POD7 (p < 0.01) and POD40 (p < 0.05) (Fig. 2a–d). There were no significant group differences in the DG region. This suggests that GCI caused a decrease in pre-myelinating OLGs (Cai et al. 2006), particularly at POD7, whereas quetiapine promoted pre-myelinating OLGs in the CA1 region of the hippocampus, but not in the DG.
In the CA1 region, GCI mice had a significantly lower number of GST-pi-positive cells (p < 0.05) on POD7 and POD40 relative to sham mice (Fig. 3a and b). The G + Q group had a significantly higher number of GST-pi positive cells relative to the GCI group on POD7 (p < 0.05) and POD40 (p < 0.05). In the DG region, there were no significant differences (Fig. 3c). Pre-treatment with quetiapine resulted in a higher number of mature OLGs after GCI, particularly with a longer recovery time.
In the CA and DG regions of the hippocampus, the GCI and the G + Q groups had significantly (p < 0.01) higher number of NG2-positive cells (OPCs) relative to the sham group on POD7 (Fig. 3d–f). There was no significant difference between the G + Q and GCI groups. There were no significant differences among the groups in the CA and DG regions of the hippocampus on POD40.
In the CA1 region, GCI mice had a significantly higher number of GFAP-positive cells on POD7 (p < 0.01) and POD40 (p < 0.05) relative to sham mice, respectively (Fig. 3g and h). The G+Q group had a significantly lower number of GFAP-positive cells relative to the GCI group on POD7 (p < 0.01) and POD40 (p < 0.05), respectively. In the DG region, GCI mice had a significantly higher number of GFAP-positive cells on POD7 (p < 0.05). The G + Q group had a significantly lower number of GFAP-positive cells relative to the GCI group on POD7 (p < 0.05). There were no significant differences on POD40 (Fig. 3i).
In the CA1 region, the BrdU-positive cells were significantly higher in the GCI group relative to the sham group on POD7 (p < 0.05) and POD40 (p < 0.01, Fig. 4a and b). The G + Q group had BrdU cell counts that were significantly lower than the GCI group on POD7 (p < 0.01) and POD40 (p < 0.05). The GCI group had a significantly higher number of BrdU cells in the DG region relative to the sham group on POD7 and POD40 (p < 0.01) (Fig. 4c). The G + Q group had BrdU cell counts that were significantly lower than the GCI group on POD7 (p < 0.01) and POD40 (p < 0.05). There were fewer cell proliferations in the hippocampus of the quetiapine-treated GCI group relative to the untreated group on POD7.
The numbers of NG2 and BrdU double-labeled cells in the CA1 and DG regions were significantly higher in the GCI and G + Q groups than in the sham group (p < 0.01) (Fig. 4d and e). There was no difference between the GCI and Q + C groups. There were few double-labeled cells on POD40. The results suggest that there were more newborn OPCs in the early period after GCI, and quetiapine had no effect on suppressing the proliferation of OPCs. No double-labeled GST-pi+ BrdU+ cells were observed on POD7 for all groups. The number of GST-pi+ BrdU+ cells was significantly lower in the GCI mice (p < 0.05) on POD40 relative to the sham group. The Q + C group showed a significantly higher number of GST-pi+ BrdU+ double-stained cells compared with the GCI group (p < 0.01). There were no significant group differences in the number of GST-pi+ BrdU+ cells in DG area (Fig. 4d and f). The results suggest there were more newly matured OLGs in the quetiapine-treated GCI group than in the untreated on POD40.
Our previous studies have shown that mice having undergone the BCCAO procedure present depression-like behaviors, and that quetiapine improves the behavioral deficits (Yan et al. 2007b). In this study, we further tested if GCI induced by BCCAO may result in white matter and OLG damage and if quetiapine can alleviate them. The results show that both mature (GST-pi-labeled) and pre-myelinating OLGs (O4-labled) were depleted in the CA1 region of the hippocampus, but not in the DG in early-period post-GCI (POD7). This difference may reflect the distinct distribution of OLG subtypes in the CA1 and DG regions of the hippocampus (Vinet et al. 2010), with the CA1 being more vulnerable to ischemia and the DG more resistant. Interestingly, a similar pattern of vulnerability was observed in neurons (Bernaudin et al. 1998), which suggests that neuronal survival may be associated with OLG viability. Excitotoxicity, oxidative stress, and inflammatory insults are the major contributors to OLG and neuronal death caused by ischemia (McTigue and Tripathi 2008). Our previous studies found that quetiapine can reduce cuprizone-induced brain demyelination and OLG damage in the mouse brains (Zhang et al. 2008) and promoted OLG development in vitro (Xiao et al. 2007). These protective effects may be because quetiapine regulates superoxide dismutase (SOD) protein expression (Zhang et al. 2008), scavenges the hydroxyl radicals produced in the Fenton system (Xu et al. 2008), and reverses excitotoxin-induced hippocampal neuropathy (Martin et al. 2005). In fact, quetiapine may exert a prominent neuroprotective effect on multiple cell lineages, as we also observed that quetiapine protected neuronal cells from GCI-induced cell death (Yan et al. 2007a). Considering its effects on white matter, and the tight relationship between vascular depression and white matter, quetiapine may be a potent augmenting agent in vascular depression.
In this study, there was remarkably enhanced cell proliferation and OLG genesis in both CA1 and DG areas on POD7 in response to ischemia. OPC proliferation has been regarded as a compensatory reaction to OLG loss (Tanaka et al. 2001). This study did not find a correlation between OPC accumulation and the amount of OLG loss in the DG. It appears that OPC proliferation was not activated by OLG loss in the DG. Other early signals may stimulate OPCs prior to OLG loss. Quetiapine significantly suppressed the overall cell proliferation, but not OLG genesis. This result suggests that quetiapine may inhibit the proliferation of non-OPCs and increase the OLG lineage portion relative to the total proliferating cells. In addition, despite the higher OLG genesis after GCI in the early stage of recovery (POD7), fewer OPCs developed into mature OLGs in the CA1 on POD40. The failure of OPCs to differentiate into functional OLGs may result in insufficient remyelination and OLG restoration after ischemic injuries (Miyamoto et al. 2010). Quetiapine treatment lead to the highest number of mature OLGs developed from newly generated OPCs, indicating that quetiapine facilitated the differentiation of OPCs or enhanced the survival of new OLGs. This is in accordance with an in vitro study that reported that quetiapine promoted OLG differentiation and maturation via the Erk1/2 pathway (Xiao et al. 2007), indicating that the effects of OLGs may play a critical role in the therapeutic potency of quetiapine in both schizophrenia and vascular depression. A recent clinical study (Zhang et al. 2009) showed that electroconvulsive therapy increased glial cell line-derived neurotrophic factor serum levels in patients with drug-resistant depression, and these researchers suggested that OLG/white matter protection is an emerging target in drug-resistant depression, including vascular depression. In addition to OLG lines, we also noted that astrocyte activation after BCCAO was inhibited by quetiapine treatment.
Astrocyte activation has been widely found in ischemia and brain injury with involvement in neuronal and OLG damage (Ouyang et al. 2007; Hazelton et al. 2010). Inhibition of astrocyte activation may also contribute to the neuroprotection and oligodendrocyte protection in the current research.
In summary, together with the neuroprotective effects reported in our previous studies, we demonstrated a new mechanism of action of quetiapine treatment and indicated a new therapeutic target in vascular depression. Further basic and clinical studies are needed for understanding more fully the therapeutic potency and elaborating the detailed mechanism.
This work was supported by the Canadian Institutes of Health Research (CIHR) China-Canada Collaborative Teams in Health Research, CIHR Canada-China Joint Health Research Initiative program grant, and Shanghai Municipal Health Bureau general program 2008087 and National Natural Science Foundation of China 81101010. The authors are grateful to Gabriel Stegeman and Dr. Monica Novotny for their excellent assistance. Quetiapine was provided by AstraZeneca Canada. Xin-Min Li has received funding from AstraZeneca and Pfizer. The other authors have no conflicts of interest to declare.