Chronic mild stress exacerbates the effects of permanent bilateral common carotid artery occlusion on CA1 neurons

Brain Research 1014 (2004) 228 – 235 www.elsevier.com/locate/brainres

Research report

Chronic mild stress exacerbates the effects of permanent bilateral common carotid artery occlusion on CA1 neurons Lesley J. Ritchie, Maxine De Butte, Bruce A. Pappas * Institute of Neuroscience, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 Accepted 13 April 2004 Available online 1 June 2004

Abstract The effect of chronic mild stress (CMStress) was examined in an animal model of chronic cerebral hypoperfusion. Eight-month-old male Sprague – Dawley rats underwent permanent bilateral occlusion of the carotid arteries (2VO) or sham surgery. At 7 days postsurgery, animals from these groups were randomly assigned to undergo CMStress consisting of relatively mild stressor exposure 6 days a week for 6 weeks or a no-stress regimen. They were perfused 24 h thereafter and stereology was used to estimate the total number of hippocampal CA1 and CA3 pyramidal cells. Glial fibrillary acid protein (GFAP) immunoreactivity in the hippocampus was also measured. Degenerating neurons were quantified with the Fluoro-Jade B staining technique. CMStress significantly potentiated CA1 cell loss in 2VO rats (17% loss), compared to a 7% loss of CA1 cells in nonstressed 2VO rats. CMStress had no effect on CA3 cell number. CMStress also caused a significant reduction in GFAP-immunoreactive astrocyte density in CA1, CA3, and the hilus of both sham and 2VO rats. Fluoro-Jade staining was absent, indicating that cell loss probably occurred in the early stage of combined 2VO and CMStress. It was concluded that CMStress exacerbates the consequences of chronic cerebral hypoperfusion on CA1 probably by reducing astrocytes, thereby increasing extracellular glutamate and/or diminishing free radical defense systems. These findings have particular relevance to understanding the contribution of chronic stress to Alzheimer’s disease, which, in its premorbid stage, is characterized by cerebral hypoperfusion, and, in its clinical stage, is characterized by CA1 cell loss. D 2004 Elsevier B.V. All rights reserved. Keywords: Chronic cerebral hypoperfusion; Chronic stress; CA1; Neurodegeneration; Glia; Alzheimer’s disease

1. Introduction During normal human aging, there is a decline of brain blood flow and metabolism [19,36]. This decline is not due to neuron loss since the loss is slight and regionally localized during normal aging [22,50]. Alzheimer’s disease (AD) is characterized by even more extreme hypoperfusion of the brain, particularly in the temporoparietal association cortex, posterior cingulate and hippocampus, and entorhinal cortex [54]. A similar pattern of hypoperfusion also occurs in mild cognitive impairment (MCI). MCI is characterized by modest cognitive deficits, most notably episodic memory, but not clinical dementia [1], and it is thought to be a prodromal phase to AD [33]. Individuals with MCI who progressed to AD 2 years later also exhibit decreased * Corresponding author. Tel.: +1-613-520-7494; fax: +1-613-5204052. E-mail address: [email protected] (B.A. Pappas). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.04.036

cerebral perfusion, in a pattern similar to that observed for AD [14]. Permanent ligation of the common carotid arteries (2VO) in the rat mimics the chronic cerebral hypoperfusion that characterizes aging and MCI. 2VO causes a moderate and most likely permanent reduction of cortical and cerebral blood flow to about 70– 80% of normal level [47,48]. 2VO rats also show subtle spatial memory impairment [27,32]. As well, there is an unusually delayed 9 –20% loss in CA1 and a 12% loss in CA3 after f 180 days of 2VO [32]. No cell loss is observed at 14 days. Using unbiased stereology, which provides a more accurate count that is free of the confounds inherent in the counting techniques used in these earlier studies [51], we have recently shown a 10% reduction of CA1 and CA4 cells after 6 months of 2VO [8]. At this time, there is also an increase of hippocampal neurons that are labeled by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, indicative of DNA strand breakage [3].

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These data suggest that after 2VO, CA1 neurons may be unhealthy and, after considerable delay, they die. Thus, 2VO provides an attractive model for assessing factors that may speed up the death of unhealthy neurons and, in doing so, may provide insight into variables that affect the progression toward dementia in aged humans. In this regard, it has been speculated that psychological stress may promote neurodegeneration and dementia [2,11]. However, there has been very little research that directly addresses this possibility, although it has been established that elevated plasma levels of the stress hormone cortisol are associated both with hippocampal atrophy and memory decline in aged humans and the severity of cognitive decline in AD [16,35]. Research with animal models supports the debilitating effect of stress on vulnerable neurons. For example, relatively brief social stress or immobilization significantly exacerbates cortical infarct volume after occlusion of the middle cerebral artery [9,17,46]. Hippocampal neurons may be especially vulnerable, as suggested by the findings that chronic administration of corticosterone to rats causes dendritic regression of CA3 neurons [18] and exacerbates CA1 pyramidal cell loss after acute ischemia [23,41]. The following experiment examined the effect of chronic mild stress (CMStress) on CA1 and CA3 neuron survival in 2VO rats. The CMStress procedure was initially developed to serve as an animal model of depression and has since been used to test the effectiveness of new and traditional forms of antidepressants [25,26] and to facilitate further investigation of the pathogenesis of depression [52]. The procedure reproduces in rats many features of human depression such as anhedonia [21] and disturbance of circadian rhythm [13], and it has been extended to mice [12]. The key elements are that the stressors are chronic, relatively mild, and are scheduled quasi-randomly to minimize habituation [52]. We employed primarily processive stressors (e.g., predator odour, novel environment), rather than physically aversive, or systemic stressors. CMStress was applied for 6 weeks after 2VO, following which we undertook stereological counts of CA1 and CA3. Fluoro-Jade B staining, a sensitive marker for degenerating neurons [43], was also carried out to determine if neurons were still actively dying at the termination of the experimental treatments. As well, we measured immunoreactivity for glial fibrillary acidic protein, a marker for astrocytes [10] that is known to be downregulated by corticosterone [28,29].

They were individually housed in a reversed 12-h light/dark cycle with ad libitum food and water. The care and handling of the animals followed the guidelines for humane treatment as established by the Canadian Council of Animal Care. The project was approved by the Carleton University Animal Care Committee.

2. Materials and methods

2.3.4. Fox odour The rats were exposed to fox urine (Ottawa Archery, Ottawa, ON) in their home cages. Fox urine was placed on a Q-tip and administered by air puff once every minute for 5 min in a testing room. They were exposed to this stressor in groups of five, in the morning and afternoon.

2.1. Animals Forty-eight male 8-month-old Sprague – Dawley rats from Harlan Sprague – Dawley (Indianapolis, IN) were used.

2.2. 2VO surgery The animals were randomly assigned to the sham (n = 24) or 2VO (n = 24) groups. The rats were anaesthesized with 2% halothane. 2VO surgery followed previously described procedures [8,32]. Briefly, a ventral midline incision was made to bilaterally expose the common carotid arteries and the arteries were dissected from the vagus nerve and surrounding tissues. The carotids of the 2VO animals were permanently doubly ligated with 5-0 silk. Animals in the sham treatment group underwent the same surgical procedure with the exception of ligation. All animals were given 7 days of recovery. The numbers decreased to 38 due to surgical complications. The sham and 2VO animals were then randomly assigned to one of four treatment groups: CONT (n = 9), Stress (n = 10), 2VO (n = 9), and 2VO Stress (n = 10). Animal in the stress groups were housed separately from nonstressed animals. 2.3. CMStress procedure CMStress rats underwent stressor exposure 6 days a week for 6 weeks. They were exposed to one of eight possible stressors (plus one exposure to a startle procedure), which were randomly assigned such that each stressor was experienced once within an 8-day span. All rats experienced the same sequence of stressors. The stressors consisted of the following. 2.3.1. Physical restraint Rats were placed for 15 min in a plastic decapitation bag containing a nose hole in the small end. 2.3.2. Saline injection The rats were injected with 0.2 ml of 9% saline solution (i.p.) in the morning and afternoon. 2.3.3. Open field Animals were placed in an 0.8  0.8  0.6-m large gray Plexiglas box for 10 min.

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2.3.5. Cat odour Animals were individually placed in empty cages containing cat feces for 1 h in the morning and the afternoon. 2.3.6. Plus maze Rats were placed in the centre of a raised apparatus (0.5 m high) consisting of four arms (0.52  0.12 m). Opaque white polyvinyl chloride walls 0.4 m high enclosed two of the arms and the other two arms were open. Entry into the enclosed arms was prevented. The animals were free to roam for 10 min. 2.3.7. Food deprivation (water ad libitum) Animals were food-deprived for 16 h (6 a.m. – 10 p.m.). 2.3.8. Forced swim The rats were placed in a large white polypropylene pool (1.6 m in diameter) filled with tepid water for 3 min. This was repeated three times at 1-min intervals.

microscope fitted with a 450-nm FITC filter at 40  magnification. Quantitation of hippocampal CA1 pyramidal cells was accomplished by stereological techniques [51] of hematoxylin-stained sections. Every sixth section containing the hippocampus was selected from a random starting point. Stereological determination of CA1 estimates of the total cell number was carried out using the StereoInvestigator Software package (MicroBrightfield, Colchester, VT) and a light microscope (Olympus BH-2) at 100  magnification. All stereological estimates of total cell number and all densitometry measures were carried out by an investigator who was blind to group membership. 2.5. Data analysis The data were first analysed by 2  2 analysis of variance (ANOVA) with Stress (Nonstress vs. Stress) and 2VO (Sham vs. 2VO) as between-group variables. Post-hoc

2.3.9. Startle The rat was placed in a plastic (0.1  0.19 m) cylinder attached to a startle platform and exposed to random bursts of 85– 115 dB tones for 20 min. Startle was administered only once, on the second to the last day of CMStress. 2.4. Histology Twenty-four hours after the last stress procedure, the animals were deeply anaesthesized with pentobarbitol and transcardially perfused with 75 ml of heparinized saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were cut down the midline and the halves were postfixed as described elsewhere [4]. The right hemisphere was sucrose-cryoprotected and stored at 4 jC until processed. The left hemisphere was dehydrated and paraffin-embedded. For cell counting, 40-Am sagittal sections containing the hippocampus were cut and then stained with hematoxylin. To determine astrocyte areal density, three 40-Am paraffin sections from each rat were immunohistochemically reacted with a glial fibrillary acid protein (GFAP) monoclonal antibody (1:600; Sigma) as described elsewhere [4,13]. A DAB (3,3-diaminobenzidine; Sigma) reaction was performed to visualize the antibody complex. GFAPimmunoreactive (IR) cell density was quantified by optical densitometry for CA1 stratum oriens, CA3 (oriens), and the hilus of the dentate gyrus, using MCID imaging analysis software (Imaging Research, St. Catherine’s, ON) to determine the proportion of area displaying GFAP-IR. To quantify degenerating cells, two representative paraffin sections (medial and medial – lateral) were selected for Fluoro-Jade B staining [43]. Positive Fluoro-Jade fluorescent cells were counted using an Olympus BX51 light

Fig. 1. The upper panel shows the mean number of CA1 pyramidal cells in the four groups. The bottom panel shows the means for CA3. The error bars represent S.E.M. Groups that have the same character above their bar significantly differed from one another.

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comparisons of groups consisted of one-way ANOVA followed by Tukey’s test.

3. Results 3.1. CA1 and CA3 cell number The results for CA1 and CA3 cell number are shown in Fig. 1. Fig. 2 (upper two panels) is a photomicrograph showing CA1 from an unstressed sham rat and a 2VO Stress rat. For CA1, ANOVA revealed a significant effect of 2VO [ F(1,31) = 27.212; p>0.01], as well as a Stress  2VO interaction [ F(1,31) = 5.367; p < 0.05]. It can be seen from Fig. 1 that the interaction occurred because stress had no effect on CA1 cell number in control rats, but it added to the decrease in cell number in 2VO rats. The 2VO Stress rats had the lowest total CA1 cell numbers (83.3% of controls), followed by 2VO (92.8% of controls). Tukey’s test indicated that the 2VO Stress rats had significantly fewer CA1 cells than both Control rats ( p < 0.01) and Stress rats ( p < 0.01). The 2VO group did not significantly differ from these groups. As shown in the bottom half of Fig. 1, there were no significant effects for CA3 cell number (all F < 1.0). The 2VO rats had 6.7% fewer CA3 cells than the controls, while the 2VO Stress had 5.8% fewer. Neither reduction was statistically significant.

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3.2. GFAP immunohistochemistry The results for GFAP are shown in Fig. 3. The lower panels of the photomicrograph (Fig. 2) show CA1 GFAP-IR in a nonstressed 2VO and a 2VO Stress rat. ANOVA revealed a Stress main effect in the CA1, CA3, and hilus regions of hippocampus. In the CA1 oriens region, significantly lower densities of GFAP immunoreactivity were observed in stressed animals [ F(1,37) = 10.647; p < 0.01]. Similar results were observed in the CA3 oriens layer [ F(1,37) = 5.464; p < 0.05] and the hilar region [ F(1,37) = 7.824; p < 0.01]. No effect of 2VO surgery (i.e., Sham vs. 2VO) was observed in any of these regions. Furthermore, no Stress  2VO interaction effect was observed in any of the three hippocampal regions, indicating that the effect of stress on GFAP was the same for control and 2VO groups. The decrease in GFAP-IR in stressed rats was obviously due to a decrease in cell number. This was confirmed by the counting of GFAP-IR cells in the CA1 (stratum oriens layer), indicating an average of 481 F 56 GFAP-IR cells per section for a nonstressed sham rat and 203 F 65 for a stressed sham ( p < 0.01; t test). 3.3. Fluoro-Jade Table 1 shows the results of the Fluoro-Jade-stained cell counts. Positive staining of approximately 10 –20 cells was

Fig. 2. Top panel: Photomicrograph of CA1 hippocampal pyramidal cell layer at 20 magnification from a control and a 2VO Stress rat. Bottom panel: Photomicrograph of GFAP immunoreactivity in the CA1 hippocampal pyramidal cell layer at 20 magnification in a 2VO and a 2VO Stress rat. The reduction of GFAP-IR cells in the stressed rats is clearly evident.

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4. Discussion

Fig. 3. Mean percentage of GFAP staining for the four groups in the CA1 stratum oriens layer (top panel), CA3 oriens (middle panel), and hilus of the dentate gyrus (bottom panel). In each area, the stressed rats had lower GFAP-IR than the nonstressed rats as indicated by a significant main effect of stress in the ANOVA. The error bars represent S.E.M.

observed in all three areas of the hippocampus for all groups and there was considerable variability within groups. The ANOVAs did not indicate significant group differences in any area.

Following 6 weeks of CMStress, stereological cell counting identified a significant 17% CA1 pyramidal cell loss in 2VO Stress rats. Nonstressed 2VO rats showed a nonsignificant 7.2% loss of cells. CMStress did not affect CA1 cell number in control rats. Thus, while not affecting control rats, CMStress exacerbated the CA1 pyramidal cell loss resulting from 2VO. This was not observed in CA3. In this sector, the 2VO rats showed a nonsignificant 6.7% loss of pyramidal neurons while the 2VO Stress group showed 5.8% fewer cells. The observed reductions in pyramidal cell number after 2VO are similar to what we have reported in the only other 2VO study to employ stereological counting; there, we found a 10% reduction of CA1 and a 7.2% reduction of CA4 cells 120 days after 2VO [8]. Thus, CA1 is more sensitive to 2VO than other pyramidal cell sectors. This is consistent with the selective vulnerability of CA1 neurons that has been observed with various acute ischemia models [6,30,31]. The experiment here suggests that not only are CA pyramidal cells selectively vulnerable to ischemia, but they are also more vulnerable to chronic stress when this is coincident with chronic hypoperfusion. Densitometric analysis indicated significant differences in GFAP expression between stressed and nonstressed rats. The stressed animals had significantly lower GFAP densities in CA1 (25% reduction), CA3 (17.4% reduction), and hilar (27% reduction) regions of the hippocampus. The counting of GFAP-IR cells confirmed what was obvious to the naked eye, namely, that there were reduced numbers of GFAP-IR astrocytes in the stressed rats. GFAP is a marker of differentiated astrocytes and neural injury invariably upregulates the proliferation of GFAP-IR astrocytes [10,45]. Unlike the effects of overt neural trauma, however, CMStress was found here to downregulate astrocyte differentiation. Hippocampal GFAP expression is upregulated by adrenalectomy and downregulated by high levels of the stress hormone corticosterone [28,29]. Surprisingly, to our knowledge, only a single study [15] has examined the effects of psychological stress on GFAP. In that experiment, food restriction for 6 days plus access to an activity wheel were found to increase GFAP cell number by 30% in CA3. In the present study, 6 weeks of brief, daily stress exposure decreased GFAP-IR astrocytes throughout the hippocampus. Hence, CMStress seems to have a very different effect on GFAP-IR astrocytes than does more acute stress. This needs Table 1 Fluoro-Jade B cell counts Fluoro-Jade B cell count

CA1

CA3

Dentate gyrus

Control Stress 2VO 2VO and Stress

10.8 F 1.6 16.1 F 4.4 22.6 F 10.7 11.0 F 1.5

18.6 F 7.0 13.4 F 4.1 19.2 F 9.5 8.8 F 2.0

19.4 F 4.1 16.7 F 4.4 19.8 F 5.0 11.9 F 1.3

The numbers shown are group mean F S.E.M.

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to be confirmed by a systematic examination of the effects of stressor chronicity (and other parameters) on GFAP-IR. The possibility of differential effects of stressor chronicity on microglia activation should also be explored. It is noteworthy that reduced GFAP and glial cell loss (shown by Nissl stain) are features of depression in humans, a psychiatric condition involving chronic stress [7]. Our finding here that CMStress reduces hippocampal GFAP-IR not only further validates CMStress as a model for human depression, but it should also serve as a model to unravel the functional implications of GFAP reduction in human depressive disorder. No significant group differences in Fluoro-Jade B staining were observed for any of the regions. In fact, for all groups, only minimal amounts of positive staining were observed. Thus, while a decrease in CA1 pyramidal cell number was observed in 2VO Stress rats, very little hippocampal neurodegeneration was actually occurring at the time of sacrifice (8 weeks postsurgery). Perhaps there is a limit to the extent to which daily stress can modulate the effects of 2VO, and this was reached sometime prior to 6 weeks. Alternatively, maybe only the initial stress exposures potentiated the effects of 2VO on cell death, although it should be noted that it has recently been reported that even after several weeks of CMStress, a stressor still elicits a corticosterone response [4,5]. The most obvious explanation as to how CMStress potentiated the effects of 2VO is that it caused the reduction of GFAP-IR astrocytes and this then potentiated the effects of 2VO. CMStress was shown here to downregulate GFAPIR in the sham 2VO rats, but by itself, CMStress was insufficient to induce CA1 pyramidal cell degeneration. We suggest that the decreased GFAP observed in the CMStress rats in this experiment resulted, at least in part, from repeated stressor-induced daily elevations of corticosterone, a stress hormone that is not only potentially toxic to hippocampal neurons [20,24,38 –42,44] but also decreases GFAP [28,29]. While we did not measure plasma corticosterone after the application of CMStress because we were concerned that the blood sampling procedure is itself a stressor and thus the unstressed control rats would also experience this stressor, based on previous research [4,5,23,37], we are confident that all of the stressors used here elicited a transient increase in plasma corticosterone. Astrocytes modulate neuron viability. They buffer extracellular glutamate, provide neurotrophins, and contribute to the brain’s antioxidant defense systems [7,45,49,53]. CMStress could impair these processes by way of repeated elevations of corticosterone. We suggest that the CMStressinduced reduction of GFAP is an indicator of either the loss of astrocytes or at least their dysfunction, and the subsequent compromising of the astrocytic uptake of glutamate. Acute ischemia increases extraneuronal glutamate [34]. The effects of chronic 2VO on extraneuronal glutamate are unknown. Nevertheless, it is certainly reasonable that the interaction between CMStress and 2VO, which resulted in

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CA1 cell loss, was a result of potentiated glutamate excitotoxicity. However, there are other possibilities. For example, dysfunction of astrocytes may have potentiated oxidative stress in 2VO rats, promoting free radical damage to neurons. Indeed, it is even possible that the reduced GFAP was simply coincidental to neuron loss and that some other effects of CMStress caused the latter. For example, chronic, repeated elevations of corticosterone by CMStress could precipitate the death of neurons that are already vulnerable as a result of hypoperfusion. In conclusion, exposure to CMStress exacerbated CA1 but not CA3 pyramidal cell death during chronic cerebral hypoperfusion. Thus, CA1 neurons seem to not only be selectively sensitive to ischemia, but also to psychological stress when they are coincidentally subjected to mild ischemia. CA1 neuron loss is a relatively early feature of AD. Hypoperfusion is also an early feature of MCI and AD. Our results suggest that chronic stress may speed the onset of dementia by promoting CA1 cell loss, perhaps by downregulating neuroprotective astrocytes.

Acknowledgements This research was supported by grants from the Ontario Heart and Stroke Foundation (B4138) and the NSERC to B.A.P. We thank Teresa Fortin for her technical assistance and Hymie Anisman for his advice regarding the stress procedure.

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