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Lithium reduces ischemia-induced hippocampal CA1 damage and behavioral deficits in gerbils
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Lithium reduces ischemia-induced hippocampal CA1 damage and behavioral deficits in gerbils Qingming Biana , Tao Shi a , De-Maw Chuang b , Yanning Qiana,⁎ a
Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People's Republic of China Molecular Neurobiology Section, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1363, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Lithium is a major drug used for the treatment of bipolar mood disorder and has recently
Accepted 15 September 2007
been shown to have neuroprotective properties. In this study we investigated the
Available online 29 September 2007
neuroprotective effects of lithium in gerbils subjected to global cerebral ischemia, an animal model of stroke. The ischemia-induced exploratory behavior changes, measured by
Keywords:
open field testing, were largely suppressed by lithium treatment for 7 days prior to ischemic
Lithium
onset. Similarly, memory impairments, measured by T-maze testing, were prevented by
Neuroprotection
lithium pretreatment. This is believed to be the first report of lithium-induced protection
Global ischemia
against hyperactivity in a novel open field and memory impairment in a gerbil model of
Habituation
global ischemia. These behavioral benefits were associated with an increase in viable cells
Working memory
as measured by hematoxylin and eosin staining and a decrease in apoptotic TUNEL-positive
Gerbil
cells in the CA1 hippocampal area of ischemic gerbils. Moreover, the lithium-induced neuroprotection was accompanied by down-regulation of pro-apoptotic p53 in the CA1 but up-regulation of anti-apoptotic Bcl-2 and heat shock protein 70 (HSP70) in the ischemic brain. These results underscore the ability of lithium to improve functional behavioral outcome in gerbil and rodent cerebral ischemic models and further indicate the potential therapeutic use of lithium in certain human stroke conditions. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Lithium is a major drug used for the treatment of bipolar mood disorder. Despite intensive research, the underlying therapeutic mechanisms remain elusive. Increasing evidence from both in vitro and in vivo studies supports that lithium has neuroprotective properties. In cultured cells, lithium protects against a variety of insults, notably glutamate-induced excitotoxicity which has been implicated in a variety of neurodegenerative diseases including stroke (reviewed in Chuang, 2004; Chuang
and Priller, 2006). The neuroprotective mechanisms are complex, likely involving multiple mechanisms such as inactivation of N-methyl-D-aspartate (NMDA) receptors, changes in the expression of pro-apoptotic and anti-apoptotic genes as well as activation of cell survival factors (Chuang, 2004; Chuang and Priller, 2006; Rowe and Chuang, 2004). Lithium also displays neuroprotection in a variety of animal models of neurodegenerative diseases including stroke, Huntington's disease, Parkinson's disease, Alzheimer's disease and HIV-I infection (Chuang and Priller, 2006). In the rat ischemia model of stroke
⁎ Corresponding author. Fax: +86 25 83724440. E-mail address: [email protected] (Y. Qian). Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DAB, diaminobenzidine; HE staining, hematoxylin–eosin staining; HSE, heat shock element; HSP70, heat shock protein 70; NBT, nitroblue tetrazolium; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.09.054
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using middle cerebral artery occlusion, it had been shown that pre- or post-insult treatment with lithium decreases brain infarct volume and suppresses neurological deficits (Nonaka and Chuang, 1998; Ren et al., 2003; Xu et al., 2003). The neuroprotective effects of lithium involve super-induction of heat shock protein 70 (HSP70) (Ren et al., 2003), but inhibition of ischemia-induced up-regulation of Bax and caspase-3 (Xu et al., 2003). Despite these positive results, little or no information is available on the effects of lithium in the gerbil cerebral ischemia model, which was established by the complete occlusion of the bilateral common carotid arteries (Hunter et al., 1995), resulting in global rather than focal ischemia. The present study was undertaken to investigate the effects of lithium pretreatment on cerebral ischemia-induced hippocampal dysfunction of habituation to novelty, memory impairment, as well as apoptosis in the CA1 area of the gerbil hippocampus, a brain region particularly vulnerable to global ischemia. In addition, we studied potential underlying neuroprotective mechanisms with a special emphasis on the changes in the expression levels of pro-apoptotic and antiapoptotic proteins.
2.
Results
2.1.
Behavioral testing
The open field test has been shown to be a sensitive indicator of an animals' ability to habituate to a new environment. An increase in activity in an open field correlates well with ischemic injury to the CA1 of the hippocampus, but not other brain regions in gerbils (Dowden et al., 1999; Colbourne and Corbett, 1995; Wang and Corbett, 1990; Gerhardt and Boast, 1988). We thus first performed open field tests to examine the effects of
Fig. 1 – Lithium pretreatment suppresses hyperactivity in an open field test in gerbils subjected to common carotid artery occlusion. Conditions of lithium treatment and common carotid artery occlusion in gerbils were described in the Experimental procedures section. On day 3 and day 7 after ischemic onset, open field tests were performed and the number of squares entered during a 10 min period was recorded. The data shown are means ± SEM from a group of 6 gerbils in each of the SH, LI-SH, IR and LI-IR groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group. +, p < 0.05 between the IR groups of day 3 and day 7.
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Fig. 2 – Lithium pretreatment prevents memory impairment, measured by T-maze test, in gerbils subjected to global ischemia. Experimental conditions are as described in the legend to Fig. 1 except that on day 4, day 5 and day 6, the T-maze tests were performed to assess memory function. The number of correct choices per 10 pairs in T-maze testing was recorded. The data are means ± SEM from a group of 6 gerbils in each of the SH, LI-SH, IR and LI-IR groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group. +, p < 0.05 between the IR groups of day 4 and day 5; ++, p < 0.01 between the IR groups of day 4 and day 6.
lithium pretreatment in gerbils subjected to occlusion of common carotid arteries for 5 min. The number of squares entered by gerbils subjected to ischemia–reperfusion (in the IR group) during a 10-min period was significantly increased on day 3 and day 7 post-ischemia, compared with the sham-operated (SH) group (Fig. 1). This increase of the number of squares entered at both time points was robustly suppressed by 1 week
Fig. 3 – Lithium pretreatment suppresses the loss of CA1 pyramidal cells following global ischemia in gerbils. Experimental conditions are as described in the legend to Fig. 1 except that on day 1, day 3 and day 7, HE stainings for viable cells in the CA1 were performed as described in the Experimental procedures section. The numbers of HE-positive cells in 0.04 mm2 in the corresponding areas was determined. Data are means ± SEM from six gerbils in each of the SH, LI-SH, IR and LI-IR groups. *, p < 0.05 and **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group.
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ingly, there was a slight improvement in working memory on day 5 and day 6 in the IR group, and lithium pretreatment also blocked ischemia-induced memory impairment at these two time points. There was no statistical difference between the SH group and LI-SH group in the number of correct choices in the T-maze test. This observation differs from that of rats subjected to 30 daily injections of lithium (2 mmol/kg) which showed enhanced memory (Tsaltas et al., 2007).
2.2.
Hematoxylin–eosin (HE) staining and TUNEL staining
HE staining was employed to assess the number of viable cells in the CA1 of gerbils following ischemic insult. There was a slight but significant reduction (by about 15%) in the number of HE-positive cells 24 h (on day 1) after the onset of ischemia (Fig. 3). This was followed by a reduction by more than 50% on day 3 and almost 80% on day 7. At all three time points,
Fig. 4 – Lithium decreases the numbers of TUNEL-positive cells in the CA1 of ischemic gerbils. On day 3 and day 7 post-ischemia, gerbils from the IR and LI-IR groups were sacrificed and TUNEL stainings for cells undergoing DNA damage in the CA1 were performed. (A) The number of TUNEL-positive cells in 0.04 mm2 in corresponding areas was calculated. Data are means ± SEM from six gerbils in each of the IR and LI-IR groups. ##, p < 0.01 compared with the respective IR group; +, p < 0.05 between the IR groups of day 3 and day 7. Typical TUNEL staining in corresponding areas of the CA1 is also shown: (B) SH on day 3; (C) IR on day 3; (D) LI-IR on day 3; (E) IR on day 7; (F) LI-IR on day 7. Scale bar = 50 μm.
daily injections with 3 mEq/kg lithium chloride (in the LI-IR group). There was no statistical difference between the SH group and SH gerbils pretreated with lithium (LI-SH group) in the number of squares crossed by gerbils. Cerebral ischemia in gerbils and rats causes impairment in working memory which can be demonstrated with the T-maze test (Dowden et al., 1999; Colbourne and Corbett, 1995; Volpe et al., 1988). Our results showed that there was a marked reduction in the number of correct choices in the T-maze test on day 4 post-ischemia in the IR group, compared with those in the SH group, and this memory impairment was prevented by lithium pretreatment in the LI-IR group (Fig. 2). Interest-
Fig. 5 – Lithium decreases the numbers of p53-positive cells in the CA1 of ischemic gerbils. On day 1, day 3 and day 7 post-ischemia, gerbils from the SH, LI-SH, IR and LI-IR groups were sacrificed and p53 immunostaining in the CA1 was performed as described in the Experimental procedures section. (A) Quantified results of the number of p53-positive cells in 0.04 mm2 in corresponding areas of the CA1 were determined. Data are means ± SEM from six gerbils in each of the SH, LI-SH, IR and LI-IR LI groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group; ++, p < 0.01 between the IR groups of day 1 and day 3, day 3 and day 7. Typical p53 immunostaining in corresponding areas of the CA1 is also shown: (B) IR on day 1; (C) LI-IR on day 1; (D) IR on day 3; (E) LI-IR on day 3. Scale bar = 50 μm.
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lithium pretreatment largely suppressed the loss of CA1 HEstained cells. Conversely, TUNEL staining was used to detect DNA damage in cells of the CA1 following cerebral ischemia. TUNEL-positive CA1 cells in the SH group, and LI-SH group were exceedingly low in number and difficult to detect (data not shown). However, TUNEL-positive cells were abundant in the CA1 of the IR group, and the number was robustly decreased by lithium treatment on day 3 and day 7 after ischemia (Figs. 4A–F).
2.3. Immunohistochemical staining of p53, Bcl-2 and HSP70 In an attempt to explore potential underlying neuroprotective mechanisms of lithium, levels of pro-apoptotic and neuroprotective proteins were assessed by immunohistochemistry. In the IR group, the number of p53-immunopositive cells in the CA1 was time-dependently increased compared with the SH group, with a significant increase on day 1 and maximal increase (by more than 10-fold) on day 3 followed by a decline on day 7. On day 1, 3 and 7, the ischemia-induced increase in the number of p53-expressing cells was almost completely prevented by lithium pretreatment (Fig. 5A). Typical immunohistochemical stainings of p53 in the CA1 sections of the IR and LI-IR groups at day 1 and day 3 are shown in Figs. 5B–E. The levels of the neuroprotective proteins, Bcl-2 and HSP70 determined by immunohistochemical staining, were exceedingly low in the CA1 region in our experimental conditions (data not shown), which were in accordance with the previous results (Aoki et al., 1993; Kirino et al., 1991). Because the hippocampal CA1 region receives synaptic input from the entorhinal cortex (Witter and Amaral, 1991), we investigated the immunohistochemistry of both proteins in the entorhinal cortex. Our results showed that both the number and intensity of Bcl-2 (Figs. 6A–C) and HSP70 (Figs. 6D–F) immunostained cells were higher in the section from the LI-IR group than those from the IR group.
2.4.
Fig. 6 – Lithium up-regulates Bcl-2 and HSP70 immunostaining in the entorhinal cortex following global ischemia in gerbils. On day 1 post-ischemia, immunostaining for Bcl-2 and HSP70 was performed as described in the Experimental procedures section. (A) Quantified results of the number of Bcl-2-positive cells in 0.04 mm2 of the entorhinal cortex of IR and LI-IR groups. Data are means ± SEM from six gerbils in each group. #, p < 0.05 compared with the IR group. Bcl-2 immunostaining in the corresponding areas of the entorhinal cortex from gerbils of the IR (B) and LI-IR (C) groups is shown. (D) Quantified results of the number of HSP-70-positive cells in 0.04 mm2 of the entorhinal cortex of IR and LI-IR groups. Data are means ± SEM from six gerbils in each group. ##, p < 0.01 compared with the IR group. HSP-70 immunostaining in the corresponding areas of the entorhinal cortex from gerbils of the IR (E) and LI-IR (F) groups is also shown. Scale bar = 50 μm.
Serum lithium concentration
Serum level of lithium determined by flame atomic absorption spectrometry at 24 h after the last injection of 7 daily injections of LiCl (3 mEq/kg) was found to be 0.49 ± 0.09 mM (n = 6). This serum lithium level is within the therapeutic plasma level of lithium for treating bipolar disorder (0.4–1.2 mM) and comparable with the value in rats determined at the same time point following a single injection (s.c.) of 3 mEq/kg LiCl, namely, 0.79 ± 0.01 (Senatorov et al., 2004).
3.
Discussion
The present study demonstrated that 1 week pretreatment with LiCl attenuated global ischemia-induced open field hyperactivity and learning/memory impairment in gerbils. Thus, the increase in the activity in the open field test, which correlated well with ischemia-induced hippocampal CA1 damage in gerbils (Dowden et al., 1999; Colbourne and Corbett, 1995; Wang and Corbett, 1990; Gerhardt and Boast, 1988), was largely suppressed by lithium pretreatment (Fig. 1). Similarly,
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learning/memory impairment, measured by the T-maze test, resulting from ischemia-induced brain damage was blocked by lithium pretreatment (Fig. 2). To our knowledge, this is the first report of lithium-induced protection against dysfunction of habituation to novel environments and impairment of learning/memory in a gerbil model of global ischemia. It has been reported that, in a rat model of permanent or transient focal ischemia, lithium administered before or after the ischemic onset attenuates neurological deficits in sensory, motor, reflex and other functions (Nonaka and Chuang, 1998; Ren et al., 2003; Yan et al., 2007). Together, the results from the present study and previous reports underscore the ability of lithium to improve functional behavioral outcome in gerbil and rodent cerebral ischemic models. The robust increase in HE-positive cells in the hippocampal CA1 of the LI-IR group compared with the IR group suggests a protection by lithium against ischemia-induced cell death (Fig. 3). This notion is further supported by our observation of the suppression by lithium of TUNEL-positive cells in the hippocampal CA1 following global ischemia (Fig. 4). A previous study reported that lithium fails to prevent the decrease in neuronal density in the hippocampal CA1 of gerbils subjected to 5 min occlusion of both common carotid arteries, although this drug does induce hypothermia (Yoshida et al., 1991). The discrepancy between the present and previous reports could be related to the conditions of lithium treatment. In the previous study, a higher dose (5 mEq/kg) of LiCl was given and the pretreatment was extended only up to 2 days prior to ischemia. The results in this study are compatible with those in other reports using a rat focal ischemia model to show that lithium treatment decreases brain infarct volume, neurological deficit and the number of TUNEL-positive cells in the ischemic hemispheres (Nonaka and Chuang, 1998; Ren et al., 2003; Xu et al., 2003). The neuroprotective effects of lithium were associated with a dramatic suppression of the number of p53-expressing cells in the hippocampal CA1 region (Fig. 5). The role of p53 in cerebral ischemia is suggested by the observations that p53 is up-regulated in the brain of rats subjected to focal ischemia (Watanabe et al., 1999) and that pifithrin-α, a p53 inhibitor, decreases the expression of p53-targeted genes and decreases the number of apoptotic cells in the ischemic brain (Leker et al., 2004). Among many actions of p53 is the up-regulation of expression of another pro-apoptotic protein, Bax, but downregulation of expression of the cytoprotective protein Bcl-2 (Miyashita et al., 1994). Bcl-2 immunostaining was found to be up-regulated by lithium pretreatment in the ischemic gerbil brain cortex (Fig. 6A–C). Lithium-induced up-regulation of Bcl2 has been reported in cultured neurons and rat brain following protracted or short-term treatment (Chen and Chuang, 1999; Chen et al., 1999; Senatorov et al., 2004). Bcl-2 has multiple neuroprotective actions including its ability to decrease Bax-induced cytochrome c release from mitochondria and to inhibit subsequent caspase activation (Chuang, 2004; Chuang and Priller, 2006) and may have been a component of the lithium-induced protection in this study. Similar to Bcl-2, HSP70 immunostaining was up-regulated in the ischemic brain of gerbils pretreated with lithium (Figs. 6D–F). These results are similar to those using a rat focal ischemia model to show that post-insult treatment with
lithium enhances transcription factor binding to heat shock element (HSE) and super-induces HSP70 in the ischemic brain (Ren et al., 2003; Xu et al., 2006). Over-expression of HSP70 in mice is neuroprotective against apoptotic and necrotic cell death during cerebral ischemia (Rajdev et al., 2000; Hoehn et al., 2001). It should be noted that down-regulation of p53 and up-regulation of Bcl-2/HSP70 can all be mediated by inhibition of glycogen synthase kinase-3 (GSK-3), a direct and indirect target of lithium (reviewed in Grimes and Jope, 2001; Rowe and Chuang, 2004). Thus, it is conceivable that the neuroprotective effects of lithium in our gerbil model are triggered by lithiuminduced inhibition of GSK-3, resulting in the regulation of an array of transcription factors. Consistent with this review is the report that induction of Dickkopf-1, a negative modulator of the Wnt-GSK-3 signaling pathway, is involved in the ischemiainduced neuronal death in the brain of gerbils and rats (Cappuccio et al., 2005). However, the causal relationship in the regulation among GSK-3, HSP70, p53, and Bcl-2 in the brain of ischemic gerbils treated with lithium requires further investigation. It has also been suggested that lithium-induced neuroprotection against glutamate excitotoxicity in cultured brain neurons involves induction of brain-derived neurotrophic factor and activation of its receptor TrkB (Hashimoto et al., 2002b; Yasuda et al., in press), rapid activation of the cell survival factor Akt (Chalecka-Franaszek and Chuang, 1999) and inactivation of NMDA receptors through inhibition of tyrosine phosphorylation (Nonaka et al., 1998; Hashimoto et al., 2002a). Their roles in lithium neuroprotection in the gerbil ischemic model await to be defined. In summary, we provided evidence that lithium pretreatment protected gerbils from global ischemia-induced open field hyperactivity and learning/memory impairment. The behavioral functional improvement was accompanied with an increase in the number of HE-positive cells and a decrease in the number of TUNEL-positive cells in the CA1 of the hippocampus. Furthermore, lithium-induced neuroprotection was associated with down-regulation of pro-apoptotic p53 protein but up-regulation of cytoprotective Bcl-2 and HSP70 proteins in the ischemic brain.
4.
Experimental procedures
4.1.
Animal surgery and drug treatments
Seventy-two male Mongolian gerbils weighing 55–70 g were randomly divided into 4 groups, sham-operated (SH group, n = 18), sham control group subjected to lithium chloride pretreatment (LI-SH group, n = 18), ischemia–reperfusion (IR group, n = 18) and ischemia–reperfusion group pretreated with lithium chloride (LI-IR group, n = 18). The SH, LI-SH, IR and LI-IR groups were further divided into 3 subgroups, respectively, according to the time of reperfusion, with 6 gerbils in each group. Midline incision was made in the ventral neck, and both common carotid arteries were occluded for 5 min with microaneurysm clips. Complete reperfusion was visually verified after removal of clips and the incision was closed. Body temperature was closely monitored with a rectal probe and maintained in the range of 37.0 ± 0.5 °C during and after surgery with a heating blanket until gerbils recovered
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from anesthesia. Gerbils in the LI-SH, LI-IR group were injected intraperitoneally with lithium chloride (3 mEq/kg) once a day for 7 consecutive days before surgery. This dose of lithium is relevant to those used in rats and is neuroprotective (Nonaka and Chuang, 1998; Wei et al., 2001; Ren et al., 2003; Senatorov et al., 2004). Normal saline was used instead of lithium in the SH and IR groups as a vehicle control. Global ischemia was induced at 24 h after the last injection of lithium chloride. Blood samples were collected for measurement of serum lithium concentration using the method of flame atomic absorption spectrometry.
4.2.
Behavioral testing
Open field testing was assessed on the 3rd and 7th day postischemia as described (Colbourne and Corbett, 1995; Dowden et al., 1999). Briefly, the floor of the open field apparatus (72 × 76 × 57 cm) was electronically divided into 25 equal squares. The number of squares which gerbils entered per min over a 10 min test period was counted by two investigators blinded to the animals' treatments. The open field was performed in a sound-proof environment with fixed lighting conditions. The environmental cues were held constant throughout testing. T-maze testing was performed as described (Colbourne and Corbett, 1995). On the 3rd day after ischemia, all gerbils were trained for three 5-min sessions to become familiar with the T-maze (60 cm in stem length, 30 cm in arm length, 10 cm wide, and 12 cm high). During these training sessions, gerbils had free access to both arms where they found and ate a shelled sunflower seed located in the food cup at the end of each arm. On the 4th, 5th and 6th days post-ischemia, gerbils were given 10 pairs of trials per day. Each pair consisted of a forced and choice trial. On the forced trial, gerbils were allowed entry into only one arm (door blocked opposite arm) where they ate the reward (1/2 sunflower seed) and then returned to the starting area (door closed). On the choice trail, the starting area door was opened to allow gerbil entry to enter either choice arm, but gerbils were only rewarded (1/2 sunflower seed) if they chose the opposite arm to the forced trial. The number of correct choices per 10 pairs was then used for analysis.
4.3.
Hematoxylin–eosin (HE) staining
The gerbils were deeply re-anesthetized with pentobarbital and perfused intracardially with 0.9% saline until the effluent was cleared of blood followed by perfusion of 500 ml 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were removed quickly and kept in 4% paraformaldehyde in 0.1 M PBS for 48 h. Two-mm-thick coronal brain sections were cut 2 mm posterior to the optic chiasm and embedded in paraffin. Five-micron-thick sections were stained with HE. Under light microscopy, the CA1 pyramidal cell density (cells/mm) was counted at a magnification of 400×. Neurons that had shrunken cell bodies with ambient empty spaces were excluded.
4.4.
TUNEL staining
The tissue sections were deparaffinated and rehydrated. After incubation with proteinase K (20 μg/ml) for 20 min, the tissue
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sections were incubated in the TUNEL reaction mixture (Roche, Mannheim, Germany) for 60 min at 37 °C in a humidified chamber according to the manufacturer's instructions. Antifluorescein antibody, Fab fragment from sheep, conjugated with alkaline phosphatase (Converter-AP) was then incubated with the tissue sections at 37 °C for 30 min followed by addition of the NBT/BCIP solution. The sections were analyzed under a light microscope.
4.5.
Immunohistochemistry
The tissue sections adjacent to those used for TUNEL staining were deparaffinated and rehydrated. Endogenous peroxidases were blocked by incubating the sections in 3% hydrogen peroxide for 20 min followed by boiling for 20 min in 10 mM citrate buffer, pH 6.0. For immunostains, the sections were then incubated at 4 °C overnight with rabbit anti-p53, rabbit anti-Bcl-2 or mouse anti-HSP70 antibody (Santa Cruz, CA) used at a dilution of 1:80, 1:50 and 1:50, respectively. The sections were further treated with biotinylated goat anti-rabbit IgG or goat antimouse IgG working solution for 45 min and horseradish peroxide conjugated streptavidin working solution for 30 min. Between each procedure, the tissue sections were rinsed 3 times in PBS for 3 min and then incubated with 3,3′-diaminobenzidine (DAB) solution. As a negative control, PBS solution was used instead of the first antibody.
4.6.
Quantitative analysis
In each section, six sampled images (in an area of 0.04 mm2) in the CA1 region of the bilateral hippocampus were chosen randomly at 400× magnification using an Olympus camera linked to a microscope. The total number of viable neurons, TUNEL-positive cells and p53-positive cells per image (in an area of 0.04 mm2) were calculated to obtain an average value for each animal. For HSP70 and Bcl-2 immunoreactive-positive cells quantitative analysis, sixteen sampled images (in an area of 0.04 mm2) in the bilateral cortex were chosen randomly at 400× magnification using an Olympus camera linked to a microscope. Total number of HSP70 and Bcl-2 immunoreactive-positive cells per image (in an area of 0.04 mm2) was calculated to obtain an average value for each animal.
4.7.
Statistical methods
All data were expressed as means ± SEM. Statistical analysis was performed using SPSS11.0 software in all experiments. The statistical significance between two groups was analyzed by Student's t-test. The statistical significance among multiple (three or more than three groups) groups was analyzed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD-t) or Dunnett's test for post hoc tests. A p value of ≤0.05 was considered to be statistically significant.
Acknowledgments We thank Peter Leeds in the Molecular Neurobiology Section, National Institute of Mental Health, National Institutes of
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Health, USA for excellent editorial assistance. This work was supported in part by a grant from the Personnel Ministry of the People's Republic of China. De-Maw Chuang was supported by the intramural program of NIMH, NIH, USA.
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ischemia in gerbil hippocampal neurons. J. Cereb. Blood Flow Metab. 11, 299–307. Leker, R.R., Aharonowiz, M., Greig, N.H., Ovadia, H., 2004. The role of p53-induced apoptosis in cerebral ischemia: effects of the p53 inhibitor pifithrin α. Exp. Neurol. 187, 478–486. Miyashita, T., Krajewski, S., Krajewska, M., Wang, H.G., Lin, H.K., Liebermann, D.A., et al., 1994. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9, 1799–1805. Nonaka, S., Chuang, D.M., 1998. Neuroprotective effects of chronic lithium on focal cerebral ischemia in rats. Neuroreport 99, 2081–2084. Nonaka, S., Hough, C., Chuang, D.M., 1998. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx Proc. Natl. Acad. Sci. U. S. A. 95, 2642–2647. Rajdev, S., Hara, K., Kokubo, Y., Mestril, R., Dillmann, W., Weinstein, P.R., Sharp, F.R., 2000. Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction Ann. Neurol. 47, 782–791. Ren, M., Senatorov, V.V., Chen, R.W., Chuang, D.M., 2003. Postinsult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat ischemia/reperfusion model. Proc. Natl. Acad. Sci. U. S. A. 100, 6210–6215. Rowe, M.K., Chuang, D.M., 2004. Lithium neuroprotection: molecular mechanisms and clinical implications. Expert Rev. Mol. Med. 6, 1–18. Senatorov, V.V., Ren, M., Kanai, H., Wei, H., Chuang, D.M., 2004. Short-term lithium treatment promotes neuronal survival and proliferation in rat striatum infused with quinolinic acid, an excitotoxic model of Huntington's disease. Mol. Psychiatry 9, 371–385. Tsaltas, E., Kontis, D., Boulougouris, V., Papakosta, V.M., Giannou, H., Poulopoulou, C., Soldatos, C., 2007. Enhancing effects of chronic lithium on memory in the rat. Behav. Brain Res. 177, 51–60. Volpe, B.T., Waczek, B., Davis, H.P., 1988. Modified T-maze training demonstrates dissociated memory loss in rats with ischemic hippocampal injury. Behav. Brain Res. 27, 259–268. Wang, D., Corbett, D., 1990. Cerebral ischemia, locomotor activity and spatial mapping. Brain Res. 533, 78–82. Watanabe, H., Ohta, S., Kumon, Y., Sakaki, S., Sakanaka, M., 1999. Increase in p53 protein expression following cortical infarction in the spontaneously hypertensive rat. Brain Res. 837, 38–45. Wei, H., Qin, Z.H., Senatorov, V.V., Wei, W., Wang, Y., Qian, Y., Chuang, D.M., 2001. Lithium suppresses excitotoxicity-induced striatal lesions in a rat model of Huntington's disease. Neuroscience 106, 603–612. Witter, M.P., Amaral, D.G., 1991. Entorhinal cortex of the monkey: projections to the dentate gyrus, hippocampus, and subicular complex. J. Comp. Neurol. 307, 437–459. Xu, J., Culman, J., Blume, A., Brecht, S., Gohlke, P., 2003. Chronic treatment with a low dose of lithium protects the brain against ischemic injury by reducing apoptotic death. Stroke 34, 1287–1292. Xu, X.H., Zhang, H.L., Han, R., Gu, Z.L., Qin, Z.H., 2006. Enhancement of neuroprotection and heat shock protein induction by combined prostaglandin A1 and lithium in rodent models of focal ischemia. Brain Res. 1102, 154–162. Yan, X.B., Wang, S.S., Hou, H.L., Ji, R., Zhou, J.N., 2007. Lithium improves the behavioral disorder in rats subjected to transient global cerebral ischemia. Behav. Brain Res. 177, 282–289. Yasuda, S., Liang, M.H., Marinova, Z., Yahyavi, A., Chuang, D.M., in press. The mood stabilizers lithium and valproate selectively activate the promoter IV of brain-derived neurotrophic factor. Mol Psychiatry. Yoshida, S., Kirino, T., Tamura, A., Basugi, N., Sano, K., 1991. Lithium ion does not protect brain against transient ischemia in gerbils. Stroke 22, 84–89.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Lithium reduces ischemia-induced hippocampal CA1 damage and behavioral deficits in gerbils Qingming Biana , Tao Shi a , De-Maw Chuang b , Yanning Qiana,⁎ a
Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People's Republic of China Molecular Neurobiology Section, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1363, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Lithium is a major drug used for the treatment of bipolar mood disorder and has recently
Accepted 15 September 2007
been shown to have neuroprotective properties. In this study we investigated the
Available online 29 September 2007
neuroprotective effects of lithium in gerbils subjected to global cerebral ischemia, an animal model of stroke. The ischemia-induced exploratory behavior changes, measured by
Keywords:
open field testing, were largely suppressed by lithium treatment for 7 days prior to ischemic
Lithium
onset. Similarly, memory impairments, measured by T-maze testing, were prevented by
Neuroprotection
lithium pretreatment. This is believed to be the first report of lithium-induced protection
Global ischemia
against hyperactivity in a novel open field and memory impairment in a gerbil model of
Habituation
global ischemia. These behavioral benefits were associated with an increase in viable cells
Working memory
as measured by hematoxylin and eosin staining and a decrease in apoptotic TUNEL-positive
Gerbil
cells in the CA1 hippocampal area of ischemic gerbils. Moreover, the lithium-induced neuroprotection was accompanied by down-regulation of pro-apoptotic p53 in the CA1 but up-regulation of anti-apoptotic Bcl-2 and heat shock protein 70 (HSP70) in the ischemic brain. These results underscore the ability of lithium to improve functional behavioral outcome in gerbil and rodent cerebral ischemic models and further indicate the potential therapeutic use of lithium in certain human stroke conditions. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Lithium is a major drug used for the treatment of bipolar mood disorder. Despite intensive research, the underlying therapeutic mechanisms remain elusive. Increasing evidence from both in vitro and in vivo studies supports that lithium has neuroprotective properties. In cultured cells, lithium protects against a variety of insults, notably glutamate-induced excitotoxicity which has been implicated in a variety of neurodegenerative diseases including stroke (reviewed in Chuang, 2004; Chuang
and Priller, 2006). The neuroprotective mechanisms are complex, likely involving multiple mechanisms such as inactivation of N-methyl-D-aspartate (NMDA) receptors, changes in the expression of pro-apoptotic and anti-apoptotic genes as well as activation of cell survival factors (Chuang, 2004; Chuang and Priller, 2006; Rowe and Chuang, 2004). Lithium also displays neuroprotection in a variety of animal models of neurodegenerative diseases including stroke, Huntington's disease, Parkinson's disease, Alzheimer's disease and HIV-I infection (Chuang and Priller, 2006). In the rat ischemia model of stroke
⁎ Corresponding author. Fax: +86 25 83724440. E-mail address: [email protected] (Y. Qian). Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DAB, diaminobenzidine; HE staining, hematoxylin–eosin staining; HSE, heat shock element; HSP70, heat shock protein 70; NBT, nitroblue tetrazolium; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.09.054
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using middle cerebral artery occlusion, it had been shown that pre- or post-insult treatment with lithium decreases brain infarct volume and suppresses neurological deficits (Nonaka and Chuang, 1998; Ren et al., 2003; Xu et al., 2003). The neuroprotective effects of lithium involve super-induction of heat shock protein 70 (HSP70) (Ren et al., 2003), but inhibition of ischemia-induced up-regulation of Bax and caspase-3 (Xu et al., 2003). Despite these positive results, little or no information is available on the effects of lithium in the gerbil cerebral ischemia model, which was established by the complete occlusion of the bilateral common carotid arteries (Hunter et al., 1995), resulting in global rather than focal ischemia. The present study was undertaken to investigate the effects of lithium pretreatment on cerebral ischemia-induced hippocampal dysfunction of habituation to novelty, memory impairment, as well as apoptosis in the CA1 area of the gerbil hippocampus, a brain region particularly vulnerable to global ischemia. In addition, we studied potential underlying neuroprotective mechanisms with a special emphasis on the changes in the expression levels of pro-apoptotic and antiapoptotic proteins.
2.
Results
2.1.
Behavioral testing
The open field test has been shown to be a sensitive indicator of an animals' ability to habituate to a new environment. An increase in activity in an open field correlates well with ischemic injury to the CA1 of the hippocampus, but not other brain regions in gerbils (Dowden et al., 1999; Colbourne and Corbett, 1995; Wang and Corbett, 1990; Gerhardt and Boast, 1988). We thus first performed open field tests to examine the effects of
Fig. 1 – Lithium pretreatment suppresses hyperactivity in an open field test in gerbils subjected to common carotid artery occlusion. Conditions of lithium treatment and common carotid artery occlusion in gerbils were described in the Experimental procedures section. On day 3 and day 7 after ischemic onset, open field tests were performed and the number of squares entered during a 10 min period was recorded. The data shown are means ± SEM from a group of 6 gerbils in each of the SH, LI-SH, IR and LI-IR groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group. +, p < 0.05 between the IR groups of day 3 and day 7.
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Fig. 2 – Lithium pretreatment prevents memory impairment, measured by T-maze test, in gerbils subjected to global ischemia. Experimental conditions are as described in the legend to Fig. 1 except that on day 4, day 5 and day 6, the T-maze tests were performed to assess memory function. The number of correct choices per 10 pairs in T-maze testing was recorded. The data are means ± SEM from a group of 6 gerbils in each of the SH, LI-SH, IR and LI-IR groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group. +, p < 0.05 between the IR groups of day 4 and day 5; ++, p < 0.01 between the IR groups of day 4 and day 6.
lithium pretreatment in gerbils subjected to occlusion of common carotid arteries for 5 min. The number of squares entered by gerbils subjected to ischemia–reperfusion (in the IR group) during a 10-min period was significantly increased on day 3 and day 7 post-ischemia, compared with the sham-operated (SH) group (Fig. 1). This increase of the number of squares entered at both time points was robustly suppressed by 1 week
Fig. 3 – Lithium pretreatment suppresses the loss of CA1 pyramidal cells following global ischemia in gerbils. Experimental conditions are as described in the legend to Fig. 1 except that on day 1, day 3 and day 7, HE stainings for viable cells in the CA1 were performed as described in the Experimental procedures section. The numbers of HE-positive cells in 0.04 mm2 in the corresponding areas was determined. Data are means ± SEM from six gerbils in each of the SH, LI-SH, IR and LI-IR groups. *, p < 0.05 and **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group.
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ingly, there was a slight improvement in working memory on day 5 and day 6 in the IR group, and lithium pretreatment also blocked ischemia-induced memory impairment at these two time points. There was no statistical difference between the SH group and LI-SH group in the number of correct choices in the T-maze test. This observation differs from that of rats subjected to 30 daily injections of lithium (2 mmol/kg) which showed enhanced memory (Tsaltas et al., 2007).
2.2.
Hematoxylin–eosin (HE) staining and TUNEL staining
HE staining was employed to assess the number of viable cells in the CA1 of gerbils following ischemic insult. There was a slight but significant reduction (by about 15%) in the number of HE-positive cells 24 h (on day 1) after the onset of ischemia (Fig. 3). This was followed by a reduction by more than 50% on day 3 and almost 80% on day 7. At all three time points,
Fig. 4 – Lithium decreases the numbers of TUNEL-positive cells in the CA1 of ischemic gerbils. On day 3 and day 7 post-ischemia, gerbils from the IR and LI-IR groups were sacrificed and TUNEL stainings for cells undergoing DNA damage in the CA1 were performed. (A) The number of TUNEL-positive cells in 0.04 mm2 in corresponding areas was calculated. Data are means ± SEM from six gerbils in each of the IR and LI-IR groups. ##, p < 0.01 compared with the respective IR group; +, p < 0.05 between the IR groups of day 3 and day 7. Typical TUNEL staining in corresponding areas of the CA1 is also shown: (B) SH on day 3; (C) IR on day 3; (D) LI-IR on day 3; (E) IR on day 7; (F) LI-IR on day 7. Scale bar = 50 μm.
daily injections with 3 mEq/kg lithium chloride (in the LI-IR group). There was no statistical difference between the SH group and SH gerbils pretreated with lithium (LI-SH group) in the number of squares crossed by gerbils. Cerebral ischemia in gerbils and rats causes impairment in working memory which can be demonstrated with the T-maze test (Dowden et al., 1999; Colbourne and Corbett, 1995; Volpe et al., 1988). Our results showed that there was a marked reduction in the number of correct choices in the T-maze test on day 4 post-ischemia in the IR group, compared with those in the SH group, and this memory impairment was prevented by lithium pretreatment in the LI-IR group (Fig. 2). Interest-
Fig. 5 – Lithium decreases the numbers of p53-positive cells in the CA1 of ischemic gerbils. On day 1, day 3 and day 7 post-ischemia, gerbils from the SH, LI-SH, IR and LI-IR groups were sacrificed and p53 immunostaining in the CA1 was performed as described in the Experimental procedures section. (A) Quantified results of the number of p53-positive cells in 0.04 mm2 in corresponding areas of the CA1 were determined. Data are means ± SEM from six gerbils in each of the SH, LI-SH, IR and LI-IR LI groups. **, p < 0.01 compared with the respective SH group; ##, p < 0.01 compared with the respective IR group; ++, p < 0.01 between the IR groups of day 1 and day 3, day 3 and day 7. Typical p53 immunostaining in corresponding areas of the CA1 is also shown: (B) IR on day 1; (C) LI-IR on day 1; (D) IR on day 3; (E) LI-IR on day 3. Scale bar = 50 μm.
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lithium pretreatment largely suppressed the loss of CA1 HEstained cells. Conversely, TUNEL staining was used to detect DNA damage in cells of the CA1 following cerebral ischemia. TUNEL-positive CA1 cells in the SH group, and LI-SH group were exceedingly low in number and difficult to detect (data not shown). However, TUNEL-positive cells were abundant in the CA1 of the IR group, and the number was robustly decreased by lithium treatment on day 3 and day 7 after ischemia (Figs. 4A–F).
2.3. Immunohistochemical staining of p53, Bcl-2 and HSP70 In an attempt to explore potential underlying neuroprotective mechanisms of lithium, levels of pro-apoptotic and neuroprotective proteins were assessed by immunohistochemistry. In the IR group, the number of p53-immunopositive cells in the CA1 was time-dependently increased compared with the SH group, with a significant increase on day 1 and maximal increase (by more than 10-fold) on day 3 followed by a decline on day 7. On day 1, 3 and 7, the ischemia-induced increase in the number of p53-expressing cells was almost completely prevented by lithium pretreatment (Fig. 5A). Typical immunohistochemical stainings of p53 in the CA1 sections of the IR and LI-IR groups at day 1 and day 3 are shown in Figs. 5B–E. The levels of the neuroprotective proteins, Bcl-2 and HSP70 determined by immunohistochemical staining, were exceedingly low in the CA1 region in our experimental conditions (data not shown), which were in accordance with the previous results (Aoki et al., 1993; Kirino et al., 1991). Because the hippocampal CA1 region receives synaptic input from the entorhinal cortex (Witter and Amaral, 1991), we investigated the immunohistochemistry of both proteins in the entorhinal cortex. Our results showed that both the number and intensity of Bcl-2 (Figs. 6A–C) and HSP70 (Figs. 6D–F) immunostained cells were higher in the section from the LI-IR group than those from the IR group.
2.4.
Fig. 6 – Lithium up-regulates Bcl-2 and HSP70 immunostaining in the entorhinal cortex following global ischemia in gerbils. On day 1 post-ischemia, immunostaining for Bcl-2 and HSP70 was performed as described in the Experimental procedures section. (A) Quantified results of the number of Bcl-2-positive cells in 0.04 mm2 of the entorhinal cortex of IR and LI-IR groups. Data are means ± SEM from six gerbils in each group. #, p < 0.05 compared with the IR group. Bcl-2 immunostaining in the corresponding areas of the entorhinal cortex from gerbils of the IR (B) and LI-IR (C) groups is shown. (D) Quantified results of the number of HSP-70-positive cells in 0.04 mm2 of the entorhinal cortex of IR and LI-IR groups. Data are means ± SEM from six gerbils in each group. ##, p < 0.01 compared with the IR group. HSP-70 immunostaining in the corresponding areas of the entorhinal cortex from gerbils of the IR (E) and LI-IR (F) groups is also shown. Scale bar = 50 μm.
Serum lithium concentration
Serum level of lithium determined by flame atomic absorption spectrometry at 24 h after the last injection of 7 daily injections of LiCl (3 mEq/kg) was found to be 0.49 ± 0.09 mM (n = 6). This serum lithium level is within the therapeutic plasma level of lithium for treating bipolar disorder (0.4–1.2 mM) and comparable with the value in rats determined at the same time point following a single injection (s.c.) of 3 mEq/kg LiCl, namely, 0.79 ± 0.01 (Senatorov et al., 2004).
3.
Discussion
The present study demonstrated that 1 week pretreatment with LiCl attenuated global ischemia-induced open field hyperactivity and learning/memory impairment in gerbils. Thus, the increase in the activity in the open field test, which correlated well with ischemia-induced hippocampal CA1 damage in gerbils (Dowden et al., 1999; Colbourne and Corbett, 1995; Wang and Corbett, 1990; Gerhardt and Boast, 1988), was largely suppressed by lithium pretreatment (Fig. 1). Similarly,
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learning/memory impairment, measured by the T-maze test, resulting from ischemia-induced brain damage was blocked by lithium pretreatment (Fig. 2). To our knowledge, this is the first report of lithium-induced protection against dysfunction of habituation to novel environments and impairment of learning/memory in a gerbil model of global ischemia. It has been reported that, in a rat model of permanent or transient focal ischemia, lithium administered before or after the ischemic onset attenuates neurological deficits in sensory, motor, reflex and other functions (Nonaka and Chuang, 1998; Ren et al., 2003; Yan et al., 2007). Together, the results from the present study and previous reports underscore the ability of lithium to improve functional behavioral outcome in gerbil and rodent cerebral ischemic models. The robust increase in HE-positive cells in the hippocampal CA1 of the LI-IR group compared with the IR group suggests a protection by lithium against ischemia-induced cell death (Fig. 3). This notion is further supported by our observation of the suppression by lithium of TUNEL-positive cells in the hippocampal CA1 following global ischemia (Fig. 4). A previous study reported that lithium fails to prevent the decrease in neuronal density in the hippocampal CA1 of gerbils subjected to 5 min occlusion of both common carotid arteries, although this drug does induce hypothermia (Yoshida et al., 1991). The discrepancy between the present and previous reports could be related to the conditions of lithium treatment. In the previous study, a higher dose (5 mEq/kg) of LiCl was given and the pretreatment was extended only up to 2 days prior to ischemia. The results in this study are compatible with those in other reports using a rat focal ischemia model to show that lithium treatment decreases brain infarct volume, neurological deficit and the number of TUNEL-positive cells in the ischemic hemispheres (Nonaka and Chuang, 1998; Ren et al., 2003; Xu et al., 2003). The neuroprotective effects of lithium were associated with a dramatic suppression of the number of p53-expressing cells in the hippocampal CA1 region (Fig. 5). The role of p53 in cerebral ischemia is suggested by the observations that p53 is up-regulated in the brain of rats subjected to focal ischemia (Watanabe et al., 1999) and that pifithrin-α, a p53 inhibitor, decreases the expression of p53-targeted genes and decreases the number of apoptotic cells in the ischemic brain (Leker et al., 2004). Among many actions of p53 is the up-regulation of expression of another pro-apoptotic protein, Bax, but downregulation of expression of the cytoprotective protein Bcl-2 (Miyashita et al., 1994). Bcl-2 immunostaining was found to be up-regulated by lithium pretreatment in the ischemic gerbil brain cortex (Fig. 6A–C). Lithium-induced up-regulation of Bcl2 has been reported in cultured neurons and rat brain following protracted or short-term treatment (Chen and Chuang, 1999; Chen et al., 1999; Senatorov et al., 2004). Bcl-2 has multiple neuroprotective actions including its ability to decrease Bax-induced cytochrome c release from mitochondria and to inhibit subsequent caspase activation (Chuang, 2004; Chuang and Priller, 2006) and may have been a component of the lithium-induced protection in this study. Similar to Bcl-2, HSP70 immunostaining was up-regulated in the ischemic brain of gerbils pretreated with lithium (Figs. 6D–F). These results are similar to those using a rat focal ischemia model to show that post-insult treatment with
lithium enhances transcription factor binding to heat shock element (HSE) and super-induces HSP70 in the ischemic brain (Ren et al., 2003; Xu et al., 2006). Over-expression of HSP70 in mice is neuroprotective against apoptotic and necrotic cell death during cerebral ischemia (Rajdev et al., 2000; Hoehn et al., 2001). It should be noted that down-regulation of p53 and up-regulation of Bcl-2/HSP70 can all be mediated by inhibition of glycogen synthase kinase-3 (GSK-3), a direct and indirect target of lithium (reviewed in Grimes and Jope, 2001; Rowe and Chuang, 2004). Thus, it is conceivable that the neuroprotective effects of lithium in our gerbil model are triggered by lithiuminduced inhibition of GSK-3, resulting in the regulation of an array of transcription factors. Consistent with this review is the report that induction of Dickkopf-1, a negative modulator of the Wnt-GSK-3 signaling pathway, is involved in the ischemiainduced neuronal death in the brain of gerbils and rats (Cappuccio et al., 2005). However, the causal relationship in the regulation among GSK-3, HSP70, p53, and Bcl-2 in the brain of ischemic gerbils treated with lithium requires further investigation. It has also been suggested that lithium-induced neuroprotection against glutamate excitotoxicity in cultured brain neurons involves induction of brain-derived neurotrophic factor and activation of its receptor TrkB (Hashimoto et al., 2002b; Yasuda et al., in press), rapid activation of the cell survival factor Akt (Chalecka-Franaszek and Chuang, 1999) and inactivation of NMDA receptors through inhibition of tyrosine phosphorylation (Nonaka et al., 1998; Hashimoto et al., 2002a). Their roles in lithium neuroprotection in the gerbil ischemic model await to be defined. In summary, we provided evidence that lithium pretreatment protected gerbils from global ischemia-induced open field hyperactivity and learning/memory impairment. The behavioral functional improvement was accompanied with an increase in the number of HE-positive cells and a decrease in the number of TUNEL-positive cells in the CA1 of the hippocampus. Furthermore, lithium-induced neuroprotection was associated with down-regulation of pro-apoptotic p53 protein but up-regulation of cytoprotective Bcl-2 and HSP70 proteins in the ischemic brain.
4.
Experimental procedures
4.1.
Animal surgery and drug treatments
Seventy-two male Mongolian gerbils weighing 55–70 g were randomly divided into 4 groups, sham-operated (SH group, n = 18), sham control group subjected to lithium chloride pretreatment (LI-SH group, n = 18), ischemia–reperfusion (IR group, n = 18) and ischemia–reperfusion group pretreated with lithium chloride (LI-IR group, n = 18). The SH, LI-SH, IR and LI-IR groups were further divided into 3 subgroups, respectively, according to the time of reperfusion, with 6 gerbils in each group. Midline incision was made in the ventral neck, and both common carotid arteries were occluded for 5 min with microaneurysm clips. Complete reperfusion was visually verified after removal of clips and the incision was closed. Body temperature was closely monitored with a rectal probe and maintained in the range of 37.0 ± 0.5 °C during and after surgery with a heating blanket until gerbils recovered
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from anesthesia. Gerbils in the LI-SH, LI-IR group were injected intraperitoneally with lithium chloride (3 mEq/kg) once a day for 7 consecutive days before surgery. This dose of lithium is relevant to those used in rats and is neuroprotective (Nonaka and Chuang, 1998; Wei et al., 2001; Ren et al., 2003; Senatorov et al., 2004). Normal saline was used instead of lithium in the SH and IR groups as a vehicle control. Global ischemia was induced at 24 h after the last injection of lithium chloride. Blood samples were collected for measurement of serum lithium concentration using the method of flame atomic absorption spectrometry.
4.2.
Behavioral testing
Open field testing was assessed on the 3rd and 7th day postischemia as described (Colbourne and Corbett, 1995; Dowden et al., 1999). Briefly, the floor of the open field apparatus (72 × 76 × 57 cm) was electronically divided into 25 equal squares. The number of squares which gerbils entered per min over a 10 min test period was counted by two investigators blinded to the animals' treatments. The open field was performed in a sound-proof environment with fixed lighting conditions. The environmental cues were held constant throughout testing. T-maze testing was performed as described (Colbourne and Corbett, 1995). On the 3rd day after ischemia, all gerbils were trained for three 5-min sessions to become familiar with the T-maze (60 cm in stem length, 30 cm in arm length, 10 cm wide, and 12 cm high). During these training sessions, gerbils had free access to both arms where they found and ate a shelled sunflower seed located in the food cup at the end of each arm. On the 4th, 5th and 6th days post-ischemia, gerbils were given 10 pairs of trials per day. Each pair consisted of a forced and choice trial. On the forced trial, gerbils were allowed entry into only one arm (door blocked opposite arm) where they ate the reward (1/2 sunflower seed) and then returned to the starting area (door closed). On the choice trail, the starting area door was opened to allow gerbil entry to enter either choice arm, but gerbils were only rewarded (1/2 sunflower seed) if they chose the opposite arm to the forced trial. The number of correct choices per 10 pairs was then used for analysis.
4.3.
Hematoxylin–eosin (HE) staining
The gerbils were deeply re-anesthetized with pentobarbital and perfused intracardially with 0.9% saline until the effluent was cleared of blood followed by perfusion of 500 ml 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were removed quickly and kept in 4% paraformaldehyde in 0.1 M PBS for 48 h. Two-mm-thick coronal brain sections were cut 2 mm posterior to the optic chiasm and embedded in paraffin. Five-micron-thick sections were stained with HE. Under light microscopy, the CA1 pyramidal cell density (cells/mm) was counted at a magnification of 400×. Neurons that had shrunken cell bodies with ambient empty spaces were excluded.
4.4.
TUNEL staining
The tissue sections were deparaffinated and rehydrated. After incubation with proteinase K (20 μg/ml) for 20 min, the tissue
275
sections were incubated in the TUNEL reaction mixture (Roche, Mannheim, Germany) for 60 min at 37 °C in a humidified chamber according to the manufacturer's instructions. Antifluorescein antibody, Fab fragment from sheep, conjugated with alkaline phosphatase (Converter-AP) was then incubated with the tissue sections at 37 °C for 30 min followed by addition of the NBT/BCIP solution. The sections were analyzed under a light microscope.
4.5.
Immunohistochemistry
The tissue sections adjacent to those used for TUNEL staining were deparaffinated and rehydrated. Endogenous peroxidases were blocked by incubating the sections in 3% hydrogen peroxide for 20 min followed by boiling for 20 min in 10 mM citrate buffer, pH 6.0. For immunostains, the sections were then incubated at 4 °C overnight with rabbit anti-p53, rabbit anti-Bcl-2 or mouse anti-HSP70 antibody (Santa Cruz, CA) used at a dilution of 1:80, 1:50 and 1:50, respectively. The sections were further treated with biotinylated goat anti-rabbit IgG or goat antimouse IgG working solution for 45 min and horseradish peroxide conjugated streptavidin working solution for 30 min. Between each procedure, the tissue sections were rinsed 3 times in PBS for 3 min and then incubated with 3,3′-diaminobenzidine (DAB) solution. As a negative control, PBS solution was used instead of the first antibody.
4.6.
Quantitative analysis
In each section, six sampled images (in an area of 0.04 mm2) in the CA1 region of the bilateral hippocampus were chosen randomly at 400× magnification using an Olympus camera linked to a microscope. The total number of viable neurons, TUNEL-positive cells and p53-positive cells per image (in an area of 0.04 mm2) were calculated to obtain an average value for each animal. For HSP70 and Bcl-2 immunoreactive-positive cells quantitative analysis, sixteen sampled images (in an area of 0.04 mm2) in the bilateral cortex were chosen randomly at 400× magnification using an Olympus camera linked to a microscope. Total number of HSP70 and Bcl-2 immunoreactive-positive cells per image (in an area of 0.04 mm2) was calculated to obtain an average value for each animal.
4.7.
Statistical methods
All data were expressed as means ± SEM. Statistical analysis was performed using SPSS11.0 software in all experiments. The statistical significance between two groups was analyzed by Student's t-test. The statistical significance among multiple (three or more than three groups) groups was analyzed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD-t) or Dunnett's test for post hoc tests. A p value of ≤0.05 was considered to be statistically significant.
Acknowledgments We thank Peter Leeds in the Molecular Neurobiology Section, National Institute of Mental Health, National Institutes of
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BR A I N R ES E A RC H 1 1 8 4 ( 2 00 7 ) 2 7 0 –27 6
Health, USA for excellent editorial assistance. This work was supported in part by a grant from the Personnel Ministry of the People's Republic of China. De-Maw Chuang was supported by the intramural program of NIMH, NIH, USA.
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