Neurol Sci (2015) 36:315–321 DOI 10.1007/s10072-014-1943-x
ORIGINAL ARTICLE
Protective effects of lithium chloride treatment on repeated cerebral ischemia–reperfusion injury in mice Mingyue Fan • Chunfeng Song • Tianjun Wang Ling Li • Yanhong Dong • Wei Jin • Peiyuan LU
•
Received: 10 June 2014 / Accepted: 1 September 2014 / Published online: 7 September 2014 Ó Springer-Verlag Italia 2014
Abstract Lithium is a renowned pharmacological treatment for mood disorders. Recent studies suggest that lithium chloride (LiCl) performs neuroprotective effects on cerebrovascular diseases. The present study is to investigate the protective effects of LiCl treatment on the hippocampus of mice with repeated cerebral ischemia– reperfusion (IR). Mice were subjected to IR through repeated bilateral common carotid artery occlusion. LiCl (2 mmol/kg) was administered daily postoperative until the mice were sacrificed. Swimming time was prolonged and error count increased in the model group through learning and memory tests. Pathological changes such as reduction in cell count and obvious pyknosis were seen in haematoxylin–eosin staining, and apoptosis was detected by TUNEL staining in hippocampal CA1 regions in the model group. The model animals exhibited more phospho-Akt Ser473 and phospho-GSK3b Ser9 than the sham group when measured by Western blot. LiCl treatment mitigated the prolonged swimming time and the increased error count compared with NaCl-treated group and improved the pathological changes. Meanwhile, LiCl further up-regulated phospho-Akt Ser473 and phospho-GSK3b Ser9 expression. The highest level of diversity was at 4 weeks
M. Fan P. LU (&) Department of Neurology, Hebei Medical University, Shijiazhuang 050017, China e-mail: [email protected] C. Song Electron Microscopy Center, Hebei Medical University, Shijiazhuang 050017, China T. Wang L. Li Y. Dong W. Jin P. LU Department of Neurology, Hebei General Hospital, Shijiazhuang 050051, China
postoperative. Therefore, repeated IR can severely damage the hippocampus and decrease the learning and memory functions in mice. Changes in the Akt and GSK3b protein activity were involved in the IR process. LiCl treatment exerted a neuroprotective effect on learning and memory by potentiating the Akt/GSK3b cell-signaling pathway. Keywords Repeated cerebral ischemia–reperfusion Learning Memory Akt Glycogen synthase kinase-3b Lithium chloride
Introduction Repeated ischemia–reperfusion (IR) in rodent animal models can lead to cell apoptosis, which results in cognitive malfunction and vascular dementia (VD) [1–4]. Different signaling pathways, such as those mediated by tyrosine receptors kinase (Trk) can participate in the cell survival or death decision [5]. Serine–threonine protein kinases are shown to decrease the damaged ischemicanoxemic neurons. Protein kinase B (Akt) is a major serine–threonine kinase and a major upstream modulator of glycogen synthase kinase-3b (GSK3b) in neurotrophindependent signaling pathways. Akt/GSK3b signal pathway has a major function in learning and memory in the hippocampus improved by environmental enrichment [6]. Lithium is a renowned pharmacological treatment for mood disorders. Recently lithium has also been recognized as a neuroprotective agent for managing neurodegenerative diseases [7] and cerebrovascular damage [8, 9]. Post-insult treatment with lithium in rodent ischemic models decreased the infarct volume, the ameliorated neurological deficits and improved functional recovery [10]. Son H et al. [11] proved that lithium can enhance long-term potentiation in the
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hippocampus of rat dentate gyrus. The current study was to determine whether Akt and GSK3b activities are the essential components in the repeated IR injury, and if so, whether lithium enhances or counteracts this action.
Materials and methods Chemicals LiCl was purchased from Amresco (Solon, OH, USA). Apoptosis Detection Kit was purchased from Chemicon (Temecula, CA, USA). Akt, phospho-Akt, GSK3b and phospho-GSK3b were purchased from cell-signaling technology (Boston, MA, USA). b-actin was purchased from Santa Cruz Biotechnology, Inc (Delaware Avenue, CA, USA). Animals and treatments A total of 144 C57Bl/6 male mice weighing 22–25 g were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). They were allowed free access to food and water and were housed at a constant humidity and temperature, with 12-h light–dark cycles. All animal studies have been approved by the China Ethics Committee and performed in accordance with the ethical standards. Experiment design The mice were randomly distributed into four groups: (1) NaCl-treated sham group (with sham operation, physiological saline administered, n = 36); (2) NaCl-treated model group (repeated IR, physiological saline administered, n = 36); (3) LiCl-treated sham group (with sham operation, LiCl administered, n = 36); and (4) LiCl-treated model group (repeated IR, LiCl administered, n = 36). Each group was investigated for 2, 4 and 6 weeks postoperatively (subgroup, n = 12). The LiCl-treated mice were intraperitoneally administered with 2 mmol/kg of LiCl (dissolved in physiological saline) daily postoperative until the mice were terminated. The NaCl-treated mice were administered with physiological saline. Repeated cerebral ischemia–reperfusion model Mice were anesthetised intraperitoneally with 10 % chloral hydrate (0.3 g/kg body weight). Repeated IR operation was conducted through bilateral common carotid artery (BCCA) occlusion according to the described method [12] with some modifications. BCCAs were separated from the surrounding tissue and occluded with 4–0 silk for 20 min repeated thrice with an interval of 10 min. Sham-group mice were only exposed to BCCA.
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Fig. 1 Mouse water maze apparatus. S starting point. E1-4 non-exit 1-4. L escape ladder. Mice were individually placed into the starting point (S) with the head toward the wall during training. There are four non-exits (E1-4), which are blind ends in the maze. Mice could get rest by reaching the escape ladder (L) at the finishing point
Cognitive function test by water maze Cognitive impairments are reflected in learning and memory in the water maze test as previously reported [13]. The maze had four non-exits (E1-4) to form irregular paths (Fig. 1). The finishing point of the maze had only one escape ladder (L) for rest. The time used to find the ladder (swimming time) and the frequency of entering non-exits (error count) were considered as index measures. The duration longer than 60 s was recorded as 60 s. The whole test included a 4-day training period. To gradually increase the degree of difficulty, there were two non-exits on the first day (the gates of E3 and E4 were closed), three on the second day (the gate of E4 was closed), and four on the later 2 days. After the second trail on the third day, a third trail was created to record the swimming time and error count as the learning score. No learning trail was conducted on the fourth day. The swimming time and error count were recorded directly as the memory score. Pathological examination by hematoxylin–eosin (HE) staining and TUNEL assay Shortly after behavioral evaluation, the mice were transcardially perfused with 0.01 M PBS followed by 4 % paraformaldehyde. Brain samples between the optic chiasma and mamillary body were removed to prepare 5 lm of continuous coronal sections for HE staining and TUNEL assay according to the manufacturer’s instructions. Two slides that were selected from the same site of each mouse were observed under light microscopy (Nikon 50i, Japan). Western blot assay for protein expression Shortly after behavioral evaluation, hippocampal tissue was removed from the brains and protein was extracted. A total of 50-lg sample was separated on 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
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PAGE), and electro-blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 0.5 h at room temperature in a fresh blocking buffer containing 5 % fat-free milk and then incubated with either anti-Akt (1:2,000), anti-phospho-Akt (1:2,000), antiGSK3b (1:1,000), anti-phospho-GSK3b (1:1,000), or antib-actin antibody (1:500) overnight with gentle agitation at 4 °C. Following three washes with TBS–T, membranes were incubated with fluorescent secondary antibody (1:3,000) in TBS for 1 h. Membranes were analyzed by Odyssey Infrared Imaging System (LI-COR, USA).
Results Mortality The mortality rates were 16.67 and 11.11 % in the NaCland LiCl-treated model groups. The numbers of deaths at 2, 4, and 6 weeks postoperative were listed as 2, 1, and 3 (NaCl-treated model group) and 1, 1, and 2 (LiCl-treated model group), respectively. No mortality was observed in the sham groups. Effect of lithium on behavior changes
Statistical analysis All data are expressed as mean ± SEM and analyzed by three-way ANOVA followed by Student–Newman–Keuls multiple range tests using SPSS 17.0 software. P \ 0.05 was considered significant. Results from behavioral test, HE staining, TUNEL staining and Western blot were analyzed by investigators who were blinded to the experimental treatment. NaCl treat sham group LiCl treat sham group
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Cognitive abnormalities were observed before and after the surgical operation. No difference was observed in learning and memory function among each group preoperative (0 week). Figure 2 shows that the swimming time and error count in learning and memory function both increased in model groups compared with those in sham groups. LiCl treatment decreased the prolonged swimming time and reduced the increased error count compared with NaCl-
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Fig. 2 Effect of treatment with LiCl on cognition. Swimming time and error count in learning and memory function both increased in model groups compared with those in sham groups. Moreover, LiCl treatment decreased the prolonged swimming time and reduced the increased error count compared with NaCl-treated groups.
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a Swimming time in learning function. b Error count in learning function. c Swimming time in memory function. d Error count in memory function. Values are expressed as mean ± SEM. mp \ 0.05, mm p \ 0.01 vs sham groups; #p \ 0.05, ##p \ 0.01 vs NaCl-treated groups
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Fig. 3 Effect of treatment with LiCl on pathology. (HE 9 400) Numerous cells tightly arranged and ordered pyramidal in the sham groups; the nucleus was circinal and large with a clear nucleolus. NaCl-treated model groups showed less pyramidal cells, and loosely arranged. The nuclei also showed pyknosis, were deeply stained and with no clear nucleolus seen. LiCltreated model groups showed less cell reduction and pyknosis. a 2w NaCl-treated sham group. b 2w LiCl-treated sham group. c 2w NaCl-treated model group. d 2w LiCl-treated model group. e 4w NaCl-treated model group. f 4w LiCl-treated model group. g 6w NaCl-treated model group. h 6w LiCl-treated model group. Values are expressed as mean ± SEM (n = 4). m P \ 0.05, mmp \ 0.01 vs. sham groups; #p \ 0.05, ## p \ 0.01 vs. NaCl-treated groups
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treated groups. These lithium treatment effects were highly significant in both learning and memory functions. The peak value was observed at 4 weeks in 2, 4 and 6 weeks of observation period.
postoperative, which showed only several neurons alive in some areas. However, the animals treated with LiCl showed less cell reduction and pyknosis. The number of apoptotic cells decreased, especially at 4 and 6 weeks.
Effect of lithium on the degree of lesion on the hippocampal CA1 region neurons
Effect of lithium on Akt and GSK3b expression
HE staining demonstrated many tightly arranged and ordered pyramidal cells in the hippocampal areas of the NaCl- and LiCl-treated sham groups. The nucleus were circinal and large with a clear nucleolus (Fig. 3), and TUNEL-positive cells were difficult to detect (Fig. 4). By contrast, the 4-and 6-week NaCl-treated model groups showed fewer pyramidal cells and were loosely arranged in the hippocampal area. The nuclei also showed pyknosis, were deeply stained, and with no clear nucleolus seen. The TUNEL-positive cells were abundant as compared with sham group. Most damage was observed 4 weeks
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In the experiments for determining whether a lower dose of lithium influences the Akt and GSK3b expression, 2 mmol/ kg lithium increased both phospho-Akt Ser473 and phospho-GSK3b Ser9 protein expression. The protein levels were higher in the LiCl-treated group than in the NaCltreated group (Fig. 5). The model animals exhibit to some extent higher phospho-Akt Ser473 and phospho-GSK3b Ser9 than those of the sham group. However, the levels of these changes were much lower than those of the LiCltreated group. The expressions of the two proteins varied at different time points postoperative, and the highest amount was observed at 4 weeks postoperative.
Neurol Sci (2015) 36:315–321 Fig. 4 A Representative images of the hippocampal CA1 region, stained for TUNELpositive apoptotic nuclei. B Quantification of the percentage of TUNEL-positive cells over the total number of cells in each group. a 2w NaCltreated sham group. b 2w LiCltreated sham group. c 2w NaCltreated model group. d 2w LiCltreated model group. e 4w NaCl-treated model group. f 4w LiCl-treated model group. g 6w NaCl-treated model group. h 6w LiCl-treated model group. Values are expressed as mean ± SEM (n = 4). m p \ 0.05, mmp \ 0.01 versus sham groups; #P \ 0.05, ## p \ 0.01 versus NaCl-treated groups
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Discussion Our findings show that a lower dose of lithium exerts a neuroprotective effect in improving the learning and memory functions in the mouse brain in vivo against repeated IR. The neuroprotective effect was induced by increasing phosphorylation of Akt and GSK3b. The VD model in previous studies was prepared by electro-cauterizing vertebral artery and occluding bilateral common arteries on rats [2–4]. The operation was complex and the animal mortality was high although the different repeated IR intervals can both induce injury and cognitive impairment [2–4]. Our previous research reports that mice undergoing 30-min ischemia repeated thrice with an interval of 10-min presented neuronal damages, as well as long-term and obvious lower learning and memory dysfunction in the water maze test [14], as reflected in other report [13]. Recent work selected C57Bl/6 mice as the experimental animal because they are most vulnerable to cerebral ischemia among mice strains. Their posterior communicating arteries are also poorly developed, and thus
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cannot compensate for frontal brain ischemia [15]. This condition aided the induction of the VD model. Lithium has been used as a mood stabilizer. But it has a comparatively low therapeutic index. Thus, lithium overdose and toxicity are relatively common. High dose treatment can cause renal toxicity, weight loss, and even death [16, 17]. Base on these, it may be more suitable to investigate the therapeutic effect of low dose. Recently, low lithium dose has been reported to improve the memory and mitigate the brain injury after stoke and hypoxia–ischemia [8, 18]. We observed that 2 mmol/kg LiCl treatment significantly improved the learning and memory changes, as well as the pathological lesions after repeated IR. Thus, lithium may be a potentially effective therapy for cognitive deficits. Considerable evidence has recently supported that repeated IR stimulates brain-derived neurotrophic factor expression (BDNF). The BDNF combines with the superficial membrane receptor TrkB to activate the downstream signaling pathway—PI3 K/Akt [19, 20], which then participates in the apoptosis after IR and helps the body resist
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Fig. 5 Effect of treatment with LiCl on protein expression. Model animals exhibited higher phospho-Akt Ser473 and phospho-GSK3b Ser9 than those of the sham group. LiCl-treated group express much higher levels than in the NaCl-treated group, and the highest amount was observed at 4 week postoperative. a, b phospho-Akt Ser473. c,
d phospho-GSK3b Ser9. 1 NaCl-treated sham group, 2 NaCl-treated model group, 3 LiCl-treated sham group, 4 LiCl-treated model group. Values are expressed as mean ± SEM (n = 5). mP \ 0.05, mm p \ 0.01 versus sham groups; #P \ 0.05, ##p \ 0.01 versus NaCl-treated groups
ischemic damage [21]. We detected in our study that repeated IR dramatically up-regulated the expression of phospho-Akt Ser473 and phospho-GSK3b Ser9 protein. However, we believe this process cannot survive from ischemic injury with this self-protection alone based on the findings that mice in the model groups exhibited learning and memory deficits and neuronal loss. Compared with stress-activated self-protection, LiCl further increased the phospho-Akt Ser473 and phospho-GSK3b Ser9 protein expression and significantly attenuated cognitive and morphologic damages. Recent studies demonstrate that Akt and GSK3b are associated with learning and memory. The PI3 K/Akt-signaling pathway is involved in exercise-induced neurogenesis and associated with synaptic plasticity [22]. Inactivated GSK3b can alleviate the suppression of cAMP response elements binding protein that is closely related to learning and memory functions [23]. GSK-3 also plays a negative feedback regulatory role in encoding memory and cognitive processing [24]. Lithium can activate the PI3K/ Akt pathway and inhibit GSK-3b activity through direct or indirect mechanisms [25, 26]. These reports are consistent with our results that LiCl treatment up-regulated phospho-
Akt Ser473 and phospho-GSK3b Ser9, as well as improved cognitive function. In conclusion, all of these outcomes suggest that changes in the Akt and GSK3b activities can contribute to the occurrence of learning and memory damage following repeated IR. Lithium treatment can improve the pathological changes and cognitive outcome. This treatment also upregulates phospho-Akt Ser473 and phospho-GSK3b Ser9 expressions after LiCl. Thus, the activation of the Akt/ GSK3b pathway is probably responsible for the protective effects of lithium against repeated IR brain injury.
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321 16. Linakis JG, Eisenberg MS, Lacouture PG, Maher TJ, Lewander WJ, Driscoll JL, Woolf A (1992) Multiple-dose sodium polystyrene sulfonate in lithium intoxication: an animal model. Pharmacol Toxicol 70:38–40 17. Klemfuss H (1995) Dietary potassium effects on lithium concentration and toxicity in hamsters. Biol Psychiatry 37:42–47 18. Li Q, Li H, Roughton K, Wang X, Kroemer G, Blomgren K, Zhu C (2010) Lithium reduces apoptosis and autophagy after neonatal hypoxia-ischemia. Cell Death Dis 1:e56 19. Barnabe´-Heider F, Miller FD (2003) Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 23:5149–5160 20. Callaghan CK, Kelly AM (2012) Differential BDNF signaling in dentate gyrus and perirhinal cortex during consolidation of recognition memory in the rat. Hippocampus 22:2127–2135 21. Miyawaki T, Ofengeim D, Noh KM, Latuszek-Barrantes A, Hemmings BA, Follenzi A, Zukin RS (2009) The endogenous inhibitor of Akt, CTMP, is critical to ischemia-induced neuronal death. Nat Neurosci 12:618–626 22. Bruel-Jungerman E, Veyrac A, Dufour F, Horwood J, Laroche S, Davis S (2009) Inhibition of PI3K-Akt signaling blocks exercisemediated enhancement of adult neurogenesis and synaptic plasticity in the dentate gyrus. PLoS One 4:e7901 23. Tong L, Balazs R, Soiampornkul R, Thangnipon W, Cotman CW (2008) Interleukin-1 beta impairs brain derived neurotrophic factor-induced signal transduction. Neurobiol Aging 29:1380–1393 24. Xu X, Zhan M, Duan W, Prabhu V, Brenneman R, Wood W, Firman J, Li H, Zhang P, Ibe C, Zonderman AB, Longo DL, Poosala S, Becker KG, Mattson MP (2007) Gene expression atlas of the mouse central nervous system: impact and interactions of age, energy intake and gender. Genome Biol 8:R234 25. Chalecka-Franaszek E, Chuang DM (1999) Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc Natl Acad Sci USA 96:8745–8750 26. Kirshenboim N, Plotkin B, Shlomo SB, Kaidanovich-Beilin O, Eldar-Finkelman H (2004) Lithium-mediated phosphorylation of glycogen synthase kinase-3beta involves PI3 kinase-dependent activation of protein kinase C-alpha. J Mol Neurosci 24:237–245
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ORIGINAL ARTICLE
Protective effects of lithium chloride treatment on repeated cerebral ischemia–reperfusion injury in mice Mingyue Fan • Chunfeng Song • Tianjun Wang Ling Li • Yanhong Dong • Wei Jin • Peiyuan LU
•
Received: 10 June 2014 / Accepted: 1 September 2014 / Published online: 7 September 2014 Ó Springer-Verlag Italia 2014
Abstract Lithium is a renowned pharmacological treatment for mood disorders. Recent studies suggest that lithium chloride (LiCl) performs neuroprotective effects on cerebrovascular diseases. The present study is to investigate the protective effects of LiCl treatment on the hippocampus of mice with repeated cerebral ischemia– reperfusion (IR). Mice were subjected to IR through repeated bilateral common carotid artery occlusion. LiCl (2 mmol/kg) was administered daily postoperative until the mice were sacrificed. Swimming time was prolonged and error count increased in the model group through learning and memory tests. Pathological changes such as reduction in cell count and obvious pyknosis were seen in haematoxylin–eosin staining, and apoptosis was detected by TUNEL staining in hippocampal CA1 regions in the model group. The model animals exhibited more phospho-Akt Ser473 and phospho-GSK3b Ser9 than the sham group when measured by Western blot. LiCl treatment mitigated the prolonged swimming time and the increased error count compared with NaCl-treated group and improved the pathological changes. Meanwhile, LiCl further up-regulated phospho-Akt Ser473 and phospho-GSK3b Ser9 expression. The highest level of diversity was at 4 weeks
M. Fan P. LU (&) Department of Neurology, Hebei Medical University, Shijiazhuang 050017, China e-mail: [email protected] C. Song Electron Microscopy Center, Hebei Medical University, Shijiazhuang 050017, China T. Wang L. Li Y. Dong W. Jin P. LU Department of Neurology, Hebei General Hospital, Shijiazhuang 050051, China
postoperative. Therefore, repeated IR can severely damage the hippocampus and decrease the learning and memory functions in mice. Changes in the Akt and GSK3b protein activity were involved in the IR process. LiCl treatment exerted a neuroprotective effect on learning and memory by potentiating the Akt/GSK3b cell-signaling pathway. Keywords Repeated cerebral ischemia–reperfusion Learning Memory Akt Glycogen synthase kinase-3b Lithium chloride
Introduction Repeated ischemia–reperfusion (IR) in rodent animal models can lead to cell apoptosis, which results in cognitive malfunction and vascular dementia (VD) [1–4]. Different signaling pathways, such as those mediated by tyrosine receptors kinase (Trk) can participate in the cell survival or death decision [5]. Serine–threonine protein kinases are shown to decrease the damaged ischemicanoxemic neurons. Protein kinase B (Akt) is a major serine–threonine kinase and a major upstream modulator of glycogen synthase kinase-3b (GSK3b) in neurotrophindependent signaling pathways. Akt/GSK3b signal pathway has a major function in learning and memory in the hippocampus improved by environmental enrichment [6]. Lithium is a renowned pharmacological treatment for mood disorders. Recently lithium has also been recognized as a neuroprotective agent for managing neurodegenerative diseases [7] and cerebrovascular damage [8, 9]. Post-insult treatment with lithium in rodent ischemic models decreased the infarct volume, the ameliorated neurological deficits and improved functional recovery [10]. Son H et al. [11] proved that lithium can enhance long-term potentiation in the
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hippocampus of rat dentate gyrus. The current study was to determine whether Akt and GSK3b activities are the essential components in the repeated IR injury, and if so, whether lithium enhances or counteracts this action.
Materials and methods Chemicals LiCl was purchased from Amresco (Solon, OH, USA). Apoptosis Detection Kit was purchased from Chemicon (Temecula, CA, USA). Akt, phospho-Akt, GSK3b and phospho-GSK3b were purchased from cell-signaling technology (Boston, MA, USA). b-actin was purchased from Santa Cruz Biotechnology, Inc (Delaware Avenue, CA, USA). Animals and treatments A total of 144 C57Bl/6 male mice weighing 22–25 g were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). They were allowed free access to food and water and were housed at a constant humidity and temperature, with 12-h light–dark cycles. All animal studies have been approved by the China Ethics Committee and performed in accordance with the ethical standards. Experiment design The mice were randomly distributed into four groups: (1) NaCl-treated sham group (with sham operation, physiological saline administered, n = 36); (2) NaCl-treated model group (repeated IR, physiological saline administered, n = 36); (3) LiCl-treated sham group (with sham operation, LiCl administered, n = 36); and (4) LiCl-treated model group (repeated IR, LiCl administered, n = 36). Each group was investigated for 2, 4 and 6 weeks postoperatively (subgroup, n = 12). The LiCl-treated mice were intraperitoneally administered with 2 mmol/kg of LiCl (dissolved in physiological saline) daily postoperative until the mice were terminated. The NaCl-treated mice were administered with physiological saline. Repeated cerebral ischemia–reperfusion model Mice were anesthetised intraperitoneally with 10 % chloral hydrate (0.3 g/kg body weight). Repeated IR operation was conducted through bilateral common carotid artery (BCCA) occlusion according to the described method [12] with some modifications. BCCAs were separated from the surrounding tissue and occluded with 4–0 silk for 20 min repeated thrice with an interval of 10 min. Sham-group mice were only exposed to BCCA.
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Fig. 1 Mouse water maze apparatus. S starting point. E1-4 non-exit 1-4. L escape ladder. Mice were individually placed into the starting point (S) with the head toward the wall during training. There are four non-exits (E1-4), which are blind ends in the maze. Mice could get rest by reaching the escape ladder (L) at the finishing point
Cognitive function test by water maze Cognitive impairments are reflected in learning and memory in the water maze test as previously reported [13]. The maze had four non-exits (E1-4) to form irregular paths (Fig. 1). The finishing point of the maze had only one escape ladder (L) for rest. The time used to find the ladder (swimming time) and the frequency of entering non-exits (error count) were considered as index measures. The duration longer than 60 s was recorded as 60 s. The whole test included a 4-day training period. To gradually increase the degree of difficulty, there were two non-exits on the first day (the gates of E3 and E4 were closed), three on the second day (the gate of E4 was closed), and four on the later 2 days. After the second trail on the third day, a third trail was created to record the swimming time and error count as the learning score. No learning trail was conducted on the fourth day. The swimming time and error count were recorded directly as the memory score. Pathological examination by hematoxylin–eosin (HE) staining and TUNEL assay Shortly after behavioral evaluation, the mice were transcardially perfused with 0.01 M PBS followed by 4 % paraformaldehyde. Brain samples between the optic chiasma and mamillary body were removed to prepare 5 lm of continuous coronal sections for HE staining and TUNEL assay according to the manufacturer’s instructions. Two slides that were selected from the same site of each mouse were observed under light microscopy (Nikon 50i, Japan). Western blot assay for protein expression Shortly after behavioral evaluation, hippocampal tissue was removed from the brains and protein was extracted. A total of 50-lg sample was separated on 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
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PAGE), and electro-blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 0.5 h at room temperature in a fresh blocking buffer containing 5 % fat-free milk and then incubated with either anti-Akt (1:2,000), anti-phospho-Akt (1:2,000), antiGSK3b (1:1,000), anti-phospho-GSK3b (1:1,000), or antib-actin antibody (1:500) overnight with gentle agitation at 4 °C. Following three washes with TBS–T, membranes were incubated with fluorescent secondary antibody (1:3,000) in TBS for 1 h. Membranes were analyzed by Odyssey Infrared Imaging System (LI-COR, USA).
Results Mortality The mortality rates were 16.67 and 11.11 % in the NaCland LiCl-treated model groups. The numbers of deaths at 2, 4, and 6 weeks postoperative were listed as 2, 1, and 3 (NaCl-treated model group) and 1, 1, and 2 (LiCl-treated model group), respectively. No mortality was observed in the sham groups. Effect of lithium on behavior changes
Statistical analysis All data are expressed as mean ± SEM and analyzed by three-way ANOVA followed by Student–Newman–Keuls multiple range tests using SPSS 17.0 software. P \ 0.05 was considered significant. Results from behavioral test, HE staining, TUNEL staining and Western blot were analyzed by investigators who were blinded to the experimental treatment. NaCl treat sham group LiCl treat sham group
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Fig. 2 Effect of treatment with LiCl on cognition. Swimming time and error count in learning and memory function both increased in model groups compared with those in sham groups. Moreover, LiCl treatment decreased the prolonged swimming time and reduced the increased error count compared with NaCl-treated groups.
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a Swimming time in learning function. b Error count in learning function. c Swimming time in memory function. d Error count in memory function. Values are expressed as mean ± SEM. mp \ 0.05, mm p \ 0.01 vs sham groups; #p \ 0.05, ##p \ 0.01 vs NaCl-treated groups
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Fig. 3 Effect of treatment with LiCl on pathology. (HE 9 400) Numerous cells tightly arranged and ordered pyramidal in the sham groups; the nucleus was circinal and large with a clear nucleolus. NaCl-treated model groups showed less pyramidal cells, and loosely arranged. The nuclei also showed pyknosis, were deeply stained and with no clear nucleolus seen. LiCltreated model groups showed less cell reduction and pyknosis. a 2w NaCl-treated sham group. b 2w LiCl-treated sham group. c 2w NaCl-treated model group. d 2w LiCl-treated model group. e 4w NaCl-treated model group. f 4w LiCl-treated model group. g 6w NaCl-treated model group. h 6w LiCl-treated model group. Values are expressed as mean ± SEM (n = 4). m P \ 0.05, mmp \ 0.01 vs. sham groups; #p \ 0.05, ## p \ 0.01 vs. NaCl-treated groups
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treated groups. These lithium treatment effects were highly significant in both learning and memory functions. The peak value was observed at 4 weeks in 2, 4 and 6 weeks of observation period.
postoperative, which showed only several neurons alive in some areas. However, the animals treated with LiCl showed less cell reduction and pyknosis. The number of apoptotic cells decreased, especially at 4 and 6 weeks.
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HE staining demonstrated many tightly arranged and ordered pyramidal cells in the hippocampal areas of the NaCl- and LiCl-treated sham groups. The nucleus were circinal and large with a clear nucleolus (Fig. 3), and TUNEL-positive cells were difficult to detect (Fig. 4). By contrast, the 4-and 6-week NaCl-treated model groups showed fewer pyramidal cells and were loosely arranged in the hippocampal area. The nuclei also showed pyknosis, were deeply stained, and with no clear nucleolus seen. The TUNEL-positive cells were abundant as compared with sham group. Most damage was observed 4 weeks
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In the experiments for determining whether a lower dose of lithium influences the Akt and GSK3b expression, 2 mmol/ kg lithium increased both phospho-Akt Ser473 and phospho-GSK3b Ser9 protein expression. The protein levels were higher in the LiCl-treated group than in the NaCltreated group (Fig. 5). The model animals exhibit to some extent higher phospho-Akt Ser473 and phospho-GSK3b Ser9 than those of the sham group. However, the levels of these changes were much lower than those of the LiCltreated group. The expressions of the two proteins varied at different time points postoperative, and the highest amount was observed at 4 weeks postoperative.
Neurol Sci (2015) 36:315–321 Fig. 4 A Representative images of the hippocampal CA1 region, stained for TUNELpositive apoptotic nuclei. B Quantification of the percentage of TUNEL-positive cells over the total number of cells in each group. a 2w NaCltreated sham group. b 2w LiCltreated sham group. c 2w NaCltreated model group. d 2w LiCltreated model group. e 4w NaCl-treated model group. f 4w LiCl-treated model group. g 6w NaCl-treated model group. h 6w LiCl-treated model group. Values are expressed as mean ± SEM (n = 4). m p \ 0.05, mmp \ 0.01 versus sham groups; #P \ 0.05, ## p \ 0.01 versus NaCl-treated groups
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Discussion Our findings show that a lower dose of lithium exerts a neuroprotective effect in improving the learning and memory functions in the mouse brain in vivo against repeated IR. The neuroprotective effect was induced by increasing phosphorylation of Akt and GSK3b. The VD model in previous studies was prepared by electro-cauterizing vertebral artery and occluding bilateral common arteries on rats [2–4]. The operation was complex and the animal mortality was high although the different repeated IR intervals can both induce injury and cognitive impairment [2–4]. Our previous research reports that mice undergoing 30-min ischemia repeated thrice with an interval of 10-min presented neuronal damages, as well as long-term and obvious lower learning and memory dysfunction in the water maze test [14], as reflected in other report [13]. Recent work selected C57Bl/6 mice as the experimental animal because they are most vulnerable to cerebral ischemia among mice strains. Their posterior communicating arteries are also poorly developed, and thus
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cannot compensate for frontal brain ischemia [15]. This condition aided the induction of the VD model. Lithium has been used as a mood stabilizer. But it has a comparatively low therapeutic index. Thus, lithium overdose and toxicity are relatively common. High dose treatment can cause renal toxicity, weight loss, and even death [16, 17]. Base on these, it may be more suitable to investigate the therapeutic effect of low dose. Recently, low lithium dose has been reported to improve the memory and mitigate the brain injury after stoke and hypoxia–ischemia [8, 18]. We observed that 2 mmol/kg LiCl treatment significantly improved the learning and memory changes, as well as the pathological lesions after repeated IR. Thus, lithium may be a potentially effective therapy for cognitive deficits. Considerable evidence has recently supported that repeated IR stimulates brain-derived neurotrophic factor expression (BDNF). The BDNF combines with the superficial membrane receptor TrkB to activate the downstream signaling pathway—PI3 K/Akt [19, 20], which then participates in the apoptosis after IR and helps the body resist
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Fig. 5 Effect of treatment with LiCl on protein expression. Model animals exhibited higher phospho-Akt Ser473 and phospho-GSK3b Ser9 than those of the sham group. LiCl-treated group express much higher levels than in the NaCl-treated group, and the highest amount was observed at 4 week postoperative. a, b phospho-Akt Ser473. c,
d phospho-GSK3b Ser9. 1 NaCl-treated sham group, 2 NaCl-treated model group, 3 LiCl-treated sham group, 4 LiCl-treated model group. Values are expressed as mean ± SEM (n = 5). mP \ 0.05, mm p \ 0.01 versus sham groups; #P \ 0.05, ##p \ 0.01 versus NaCl-treated groups
ischemic damage [21]. We detected in our study that repeated IR dramatically up-regulated the expression of phospho-Akt Ser473 and phospho-GSK3b Ser9 protein. However, we believe this process cannot survive from ischemic injury with this self-protection alone based on the findings that mice in the model groups exhibited learning and memory deficits and neuronal loss. Compared with stress-activated self-protection, LiCl further increased the phospho-Akt Ser473 and phospho-GSK3b Ser9 protein expression and significantly attenuated cognitive and morphologic damages. Recent studies demonstrate that Akt and GSK3b are associated with learning and memory. The PI3 K/Akt-signaling pathway is involved in exercise-induced neurogenesis and associated with synaptic plasticity [22]. Inactivated GSK3b can alleviate the suppression of cAMP response elements binding protein that is closely related to learning and memory functions [23]. GSK-3 also plays a negative feedback regulatory role in encoding memory and cognitive processing [24]. Lithium can activate the PI3K/ Akt pathway and inhibit GSK-3b activity through direct or indirect mechanisms [25, 26]. These reports are consistent with our results that LiCl treatment up-regulated phospho-
Akt Ser473 and phospho-GSK3b Ser9, as well as improved cognitive function. In conclusion, all of these outcomes suggest that changes in the Akt and GSK3b activities can contribute to the occurrence of learning and memory damage following repeated IR. Lithium treatment can improve the pathological changes and cognitive outcome. This treatment also upregulates phospho-Akt Ser473 and phospho-GSK3b Ser9 expressions after LiCl. Thus, the activation of the Akt/ GSK3b pathway is probably responsible for the protective effects of lithium against repeated IR brain injury.
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