Neurol Sci DOI 10.1007/s10072-013-1605-4
ORIGINAL ARTICLE
Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation Di Liu • Hong Zhang • Wenjuan Gu Mengren Zhang
•
Received: 17 August 2013 / Accepted: 10 December 2013 Ó Springer-Verlag Italia 2013
Abstract Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFHDA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucosetreated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the
D. Liu W. Gu M. Zhang (&) Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, No. 1 Shuaifuyuan, Dongcheng District, Beijing 100730, China e-mail: [email protected] H. Zhang Department of Cell Resource Center, Institute of Basic Medical Science, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China
neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation. Keywords Hippocampal neurons High glucose Diabetes mellitus Cognitive impairment Oxidative stress
Introduction The incidence of diabetes mellitus is increasing worldwide, and studies have showed that both type 1 and type 2 diabetes mellitus have been associated with reduced performance on numerous domains of cognitive function [1, 2]. Furthermore, numerous epidemiological studies have linked T2DM with an increased risk of cognitive dysfunction [3]. Therefore, a great deal of researches has focused on the mechanism of diabetic cognitive impairment. Previous studies indicated that diabetic cognitive impairment has a close relation to hippocampus dysfunction. Diabetes mellitus could not only cause the structural changes of hippocampus, such as the degeneration in synaptic structures [4], reductions in hippocampal volume, but also induce the physiology abnormalities of hippocampal neurons, for example, the impairment of neural coordination [5], reductions in levels of brain-derived neurotrophic factor [6], deficits in neurogenesis [7]. Hyperglycemia plays a critical role in hippocampus dysfunction which has been considered to be related to abnormalities in cognitive function in patients with both type 1 and type 2 diabetes mellitus [2]. Consistent with this, it was recently reported that the hippocampal glucose concentration was significantly increased in the diabetic rats [8]. Accumulating evidence suggested that reactive
123
Neurol Sci
oxygen species (ROS) induced by hyperglycemia plays critical roles in the pathogenesis of diabetic neuropathy [9]. It was found that ROS concentrations in brain are significantly increased in diabetic rats [10], but there is little research about the effect of high glucose on oxidative stress in primary cultured hippocampal neurons. Therefore, it is essential to explore the effects of different concentrations of high glucose and different exposure time on hippocampal neurons in vitro, especially the effects on oxidative stress. In the present study, we used the primary cultured hippocampal neurons exposed to different concentrations of high glucose for different time to test the glucotoxicity.
Methods Primary cultures of hippocampal neurons All experimental procedures were performed in accordance with Chinese Academy of Medical Sciences experimental standards as well as international guidelines on the ethical treatment of experimental animals. Newborn Sprague– Dawley (SD) rats, \24-h old, were purchased from the Peking University Health Science Center. Primary cultures of hippocampal neurons were prepared as described previously [11, 12] with a few modifications. Hippocampi were dissected from the brain on ice and minced in sterile icecold D-Hanks’ (Peking union cell resource center, China) with the blood vessels and meninges thoroughly removed. The tissues were digested with 0.25 % trypsin (Peking union cell center, China) for 15 min at 37 °C and then the digestion procedure was terminated by adding 5 ml fetal bovine serum (FBS). Cell suspension was passed through a 200-mesh cell strainer and separated by density gradient centrifugation at for 20 min at 2,000 rpm. Then the cell suspension containing the desired cell fractions was carefully collected and centrifuged within D-Hanks’ for 5 min at 1,500 rpm and then resuspended within 15–20 ml DMEM medium (Peking union cell resource center, China). The cells were then seeded on poly-D-lysine (Sigma, USA) coated-glass coverslips, 96-well plates or 24-well plates at a density of 5 9 105 cell/ml after counting the cell density by using a hemacytometer and maintained at 37 °C in a humidified 5 % CO2 incubator (Forma, USA). Neurons were cultured in DMEM medium and supplemented with 10 % FBS, 20 mmol/L sodium pyruvate, 1 mmol/L glutamine, 100 U/ml Penicillin, 100 lg/ml streptomycin, 10 ng/ml nerve growth factor (NGF) (Millpore, USA).
buffered saline (PBS) before being fixed in 4 % paraformaldehyde for 1 h at room temperature. The coverslips were then washed three times in cold PBS (5 min each). Prior to staining, cells were permeabilised in PBS containing 0.01 % Triton for 10 min at room temperature and then washed three times in cold PBS (3 min each). Nonspecific antibody binding was blocked by incubation at room temperature for 20 min in normal goat serum. Each coverslip was incubated with 20 ll rabbit anti-NSE primary antibody 1:10 (Bioworld, USA) in a petri dish which was placed at 4 °C overnight. Negative controls were incubated identically but without primary antibody. Coverslips were then washed three times in PBS, then incubated with a goat anti-rabbit secondary antibody conjugated to FITC 1:50 Sigma for 30 min at room temperature, washed six times in cold PBS 5 min each. Then coverslips were incubated with 20 ll DAPI for 5 min at room temperature in the dark. Cells were observed and photographed with a volocity demo imaging system (PerkinElmer, USA). High glucose exposures Primary hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h, respectively. The control group was exposed to normal medium which contains 25 mM glucose. The osmolality of all glucose exposure was between 260 and 320 mOsm/kg which had no effect on cell normal metabolism. Cell viability assays After exposure to various concentrations of high glucose for different time, the viability of cells was determined using the MTT assay system (Peking union cell resource center, China). Briefly, the cells were seeded on 96-well culture plates at a density of 5 9 105 cell/ml. After the indicated timeframe, 100 ll 5 mg/ml MTT solution 1:10 was added to each well and then incubated for 4 h at 37 °C. After 4 h incubation, the medium was removed and then the precipitated dye was dissolved within 100 ll DMSO solution for another 30 min incubation at 37 °C. Eventually, the OD value of each well was detected at 570 nm with a MultiSkan3ELISA Reader (Thermo fisher scientific, USA). Cell viability was calculated as follows: treated group OD/24 h control group OD 9100 %. Assessment of apoptosis: nuclear morphology assay
NSE immunostaining Coverslips with their attached cells were removed from the culture medium and washed three times in phosphate
123
Nuclear morphology was used to assess cells undergoing apoptosis (cells with condensed of fragmented nuclei). Cells were gently washed with warm PBS and then
Neurol Sci
Fig. 1 NSE immunostaining results. Immunocytochemical staining with NSE for neurons, while DAPI for all cells. Magnification 9200; scale bar 50 lm (A and B ); magnification 9600; scale bar 23 lm (C )
incubated with 2 lg/mL fluorescent dye Hoechst 33342 (Sigma, USA) in PBS for 10 min at room temperature. Fluorescence of stained chromatin was examined using an inverted fluorescence microscope (LEICA DMI4000B, Japan) and cells with condensed/fragmented chromatin were scored. For each preparation six random fields were counted. Apoptosis ratio treatment group/control group was expressed as percent, with 24 h control group being 100 %.
Neuronal morphological changes Cell morphological changes were assessed by a live cell imaging system. As shown in Fig. 2, hippocampal neurons treated with normal medium (control) exhibited large, vacuole-free cell bodies, with elaborate networks of neurites. However, exposure to different concentrations of high glucose for 72 h all resulted in obvious cell loss, with the disappearance of neurites, appearance of disrupted membranes and shrinkage of cell bodies.
Measurement of intracellular reactive oxygen species Intracellular reactive oxygen species were measured using the fluorescent probe DCFH-DA, which can be oxidized to the highly fluorescent compound dichlorofluorescin (DCF). Cells were incubated with 10 lM DCFH-DA at 37 °C for 30 min and then were washed twice for 5 min each with ice-cold PBS. Cellular fluorescence intensity was detected using a live cell imaging system (Olympus LCS SYSTEM, Japan) with excitation at 485 nm and emission at 530 nm. Statistical analysis All data are expressed as mean ± SD. Differences between groups were examined for statistical significance using a one-way ANOVA with Dunnett’s test as a post hoc analysis. P \ 0.05 was considered significantly different.
Results NSE immunostaining As shown in Fig. 1, nuclei of all cells were stained with DAPI, while cells stained positive for NSE were identified as neurons. Statistically, the ratio of neurons was approximately 90 % (for each preparation six random fields were counted).
Effect of exposure to high glucose on viability of hippocampal neurons The cell viability of all high glucose-treated groups started to decrease dramatically at 72 h and the cell viability of 50, 125, 150 mM high glucose groups reduced significantly as compared to 48 h (P \ 0.05) (Fig. 3). In neurons exposed to high glucose, the cell viability all decreased remarkably after 72 h compared with timed controls (P \ 0.05). Interestingly, there was no significant difference among the high glucose-treated groups from 72 to 96 h (P [ 0.05). Effect of exposure to high glucose on apoptosis of hippocampal neurons Consistent with the MTT results, there was a significant increase of apoptotic nuclei in all high glucose-treated groups after 72 h compared with control group (P \ 0.05) (Fig. 4). The apoptotic nuclei of high glucose-treated groups at 72 h all increased dramatically compared with that of 48 h. In comparison, there was no significant change in controls from 24 to 96 h. We also observed that the percentage of apoptotic neurons showed no statistical difference among high glucose-treated groups from 24 to 96 h (P [ 0.05). Therefore, we select treatment with 50 mM high glucose for further experiment according to the results of MTT assay and Hoechst staining.
123
Neurol Sci
Fig. 2 Neuronal morphological changes, A hippocampal neurons treated with normal medium (control), B hippocampal neurons exposed to 50 mM high glucose, C hippocampal neurons exposed to 75 mM high glucose, D hippocampal neurons exposed to 100 mM
high glucose, E hippocampal neurons exposed to 125 mM high glucose, F hippocampal neurons exposed to 150 mM high glucose. Magnification 9600; scale bar 17 lm
Effect of exposure to high glucose on intracellular ROS accumulation in hippocampal neurons Cells were exposed to 50 mM glucose for the indicated times (3–72 h), and the cellular ROS levels were measured by DCF-DA staining. The cellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h (P \ 0.01) (Fig. 5A). It was observed that ROS accumulation levels of glucose-treated group peaked at 24 h with sixfold higher than the control (P \ 0.01) and began to decrease beyond 24 h (Fig. 5B). Differently, the cellular ROS levels of control group peaked at 3 h and decreased dramatically from 6 h (P \ 0.01).
Discussion Fig. 3 Effect of high glucose on the viability of hippocampal neurons. Cell viability was assessed by the MTT reduction assay. The results are representative of three independent experiments, and are expressed as percentage of control at 24 h
123
Diabetes is associated with multiple adverse effects on the brain, some of which may result primarily from direct consequences of chronic hyperglycemia. Clinical study
Neurol Sci Fig. 4 Effect of high glucose on apoptosis of hippocampal neurons. Total cells and cells with condensed/fragmented nuclei (cells undergoing apoptosis) were counted in six random fields in each coverslip using Hoechst staining. The results represent the mean ± SD of at least three independent experiments, and are expressed as percentage of control; *P \ 0.05, significantly different from control; # P \ 0.05, compared with 48 h, the apoptotic nuclei of high glucose groups increased significantly at 72 h
has demonstrated that the blood control level is closely related to the cognitive function [13]. Furthermore, experiment on diabetic rats also has proved that blood glucose level could affect synaptic plasticity and the ability to learn [6]. The importance of the hippocampal formation for memory is well established on the basis of neuroanatomical and electro-physiological studies [14]. Chronic hyperglycemia can induce the hippocampal neurons dysfunction and apoptosis through a variety of mechanisms, including oxidative stress [9], increased formation of advanced glycation end products [15], mitochondrial dysfunction [16] and so on. Moreover, long-term exposure to high glucose could induce changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons and a significant increase of apoptotic nuclei [17]. Since hyperglycemia is considered the primary pathogenic factor for the development of diabetic cognitive impairments and hippocampus function is closely related to cognition, we explore the effects of different concentrations of high glucose and different exposure time on primary cultured hippocampal neurons. Long-term exposure to high glucose could impair the mitochondrial function [18] and cause the decrease of cell viability. In present study, we observed that there was a significant decrease of the cell viability and a significant increase of apoptotic nuclei in high glucose-treated groups at 72 and 96 h which mimic chronic hyperglycemic conditions. Consistent with previous researches where apoptosis was found in neural cells exposed to high glucose [17–19] and in the hippocampus of diabetic animals [20, 21], the results give support to that high glucose exposure for long time could induce the hippocampal neurons dysfunction and apoptosis.
From the results of MTT assay and Hoechst staining, we can see that the cell viability and percentage of apoptotic neurons had no significant difference among different concentration of high glucose groups from 72 to 96 h (P [ 0.05). Hence, exposure to 50 mM high glucose for 72 h was enough to cause obvious hippocampal neurons damage and could maintain the effects with the extension to 96 h. This can be considered as a basis of establishment of the high-glucose-insulted primary culture rat hippocampal neuronal cell model. Increasing evidences in both vivo and vitro experiments suggest that there is a close link between hyperglycemia, oxidative stress, and diabetic neuropathy. Hyperglycemia has been shown to induce oxidative stress through multiple pathways such as redox imbalances secondary to enhanced aldose reductase (AR) activity, increased advanced glycation end products (AGE), altered protein kinase C (PKC) activity, prostanoid imbalances, and mitochondrial overproduction of superoxide [22]. ROS can lead to tissue injury and cell death through damaging DNA and inactivating antioxidant defense enzymes [23, 24]. Increased brain mitochondrial ROS production may cause the opening of mitochondrial permeability transition, leading to brain mitochondrial swelling and the depolarization of mitochondrial membrane potential [25]. Moreover, it has been reported that mitochondrial ROS production of diabetic rat hippocampus was significantly increased [10]. Consistent with this study, our result showed that cellular ROS generation can be markedly induced by high glucose. In 50-mM glucose group, cell viability reduced and apoptotic nuclei increased significantly at 72 h while ROS generation declined, which may be partly caused by increased neuronal death on one hand. On the other hand, it has been suggested that ROS generation precede disruption
123
Neurol Sci
Fig. 5 Effect of high glucose on intracellular ROS accumulation in hippocampal neurons. Hippocampal neurons were exposed to 50 mM high glucose for 3–72 h. A Representative photographs of DCF staining in different groups, a hippocampal neurons exposed to 50 mM high glucose for 3 h, b hippocampal neurons exposed to 50 mM high glucose for 6 h, c hippocampal neurons exposed to 50 mM high glucose for 12 h, d hippocampal neurons exposed to
50 mM high glucose for 24 h, e hippocampal neurons exposed to 50 mM high glucose for 48 h, f hippocampal neurons exposed to 50 mM high glucose for 72 h. Scale bar 17 lm. B Quantitative analysis of the DCF fluorescence intensity among groups. The results represent the mean ± SD of at least three independent experiments, and are expressed as percentage of control at 3 h
of membrane potential of mitochondria and the reduction of Dwm is an early step of apoptosis, followed by cytochrome c release in many cell types [26]. As is observed in
many studies [19, 27, 28], ROS generation peaked in early phase but did not sustain at the latter stage when cell apoptosis increased obviously. Therefore, ROS generation
123
Neurol Sci
might be an initial signal in high glucose-induced apoptosis. And the precise mechanism of ROS triggered apoptosis in high glucose-treated hippocampal neurons requires further investigation. In summary, we showed that exposures to different concentrations of high glucose for 72 and 96 h could cause cell viability reduction and apoptosis in hippocampal neurons, supporting previous findings which have proved hyperglycemia to be a critical contributor to hippocampus changes. Our study also suggested that high glucose can also cause hippocampal neurons damage by mediating intracellular ROS production and sheds lights on the pathogenesis of diabetic neuropathy. Furthermore, the mechanisms that cause hippocampal neurons injury in the exposure to high glucose still need to be well explored and understood. Acknowledgments We are grateful to Professor Yuqin Liu and Bei Gu for their helpful technical assistance and scientific suggestions. Conflicts of interest
None declared.
References 1. Cukierman T, Gerstein HC, Williamson JD (2005) Cognitive decline and dementia in diabetes–systematic overview of prospective observational studies. Diabetologia 48:2460–2469. doi:10.1007/s00125-005-0023-4 2. McCrimmon RJ, Ryan CM, Frier BM (2012) Diabetes and cognitive dysfunction. Lancet 379:2291–2299. doi:10.1016/S01406736(12)60360-2 3. Saczynski JS, Jonsdottir MK, Garcia ME et al (2008) Cognitive impairment: an increasingly important complication of type 2 diabetes: the age, gene/environment susceptibility–Reykjavik study. Am J Epidemiol 168:1132–1139. doi:10.1093/aje/kwn228 4. Hou Y, Zhou L, Yang QD et al (2012) Changes in hippocampal synapses and learning-memory abilities in a streptozotocin-treated rat model and intervention by using fasudil hydrochloride. Neuroscience 200:120–129. doi:10.1016/j.neuroscience.2011.10. 030 5. Liu K, Xie KN, Qiao ZM et al (2009) Impaired neural coordination in hippocampus of diabetic rat. Sheng Li Xue Bao 61:417–423 6. Stranahan AM, Norman ED, Lee K et al (2008) Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats. Hippocampus 18:1085–1088. doi:10.1002/hipo.20470 7. Stranahan AM, Arumugam TV, Cutler RG et al (2008) Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons. Nat Neurosci 11:309–317. doi:10.1038/nn2055 8. Duarte JM, Carvalho RA, Cunha RA et al (2009) Caffeine consumption attenuates neurochemical modifications in the hippocampus of streptozotocin-induced diabetic rats. J Neurochem 111:368–379. doi:10.1111/j.1471-4159.2009.06349.x 9. Yi SS, Hwang IK, Kim DW et al (2011) The chronological characteristics of SOD1 activity and inflammatory response in the hippocampi of STZ-induced type 1 diabetic rats. Neurochem Res 36:117–128. doi:10.1007/s11064-010-0280-6
10. Pipatpiboon N, Pratchayasakul W, Chattipakorn N et al (2012) PPARgamma agonist improves neuronal insulin receptor function in hippocampus and brain mitochondria function in rats with insulin resistance induced by long term high-fat diets. Endocrinology 153:329–338. doi:10.1210/en.2011-1502 11. Kaech S, Banker G (2007) Culturing hippocampal neurons. Nat Protoc 1:2406–2415 12. Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490–1498. doi:10.1038/ nprot.2007.207 13. Yaffe K, Blackwell T, Kanaya AM et al (2004) Diabetes, impaired fasting glucose, and development of cognitive impairment in older women. Neurology 63:658–663 14. Eichenbaum H (1999) The hippocampus and mechanisms of declarative memory. Behav Brain Res 103:123–133 15. Wang SH, Sun ZL, Guo YJ et al (2009) Diabetes impairs hippocampal function via advanced glycation end product mediated new neuron generation in animals with diabetes-related depression. Toxicol Sci 111:72–79. doi:10.1093/toxsci/kfp126 16. Ye L, Wang F, Yang RH (2011) Diabetes impairs learning performance and affects the mitochondrial function of hippocampal pyramidal neurons. Brain Res 1411:57–64. doi:10.1016/j. brainres.2011.07.011 17. Gaspar JM, Castilho A, Baptista FI et al (2010) Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons. Neuroscience 171:981–992. doi:10.1016/j.neuroscience. 2010.10.019 18. Santiago AR, Cristovao AJ, Santos PF et al (2007) High glucose induces caspase-independent cell death in retinal neural cells. Neurobiol Dis 25:464–472 19. Russell JW, Golovoy D, Vincent AM et al (2002) High glucoseinduced oxidative stress and mitochondrial dysfunction in neurons. FASEB J 16:1738–1748 20. Zhao CH, Liu HQ, Cao R et al (2012) Effects of dietary fish oil on learning function and apoptosis of hippocampal pyramidal neurons in streptozotocin-diabetic rats. Brain Res 1457:33–43. doi:10.1016/j.brainres.2012.03.067 21. Li ZG, Zhang W, Grunberger G et al (2002) Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 946:221–231 22. Pop-Busui R, Sima A, Stevens M (2006) Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 22:257–273 23. Rosen P, Nawroth PP, King G et al (2001) The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCOMCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17:189–212 24. Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820. doi:10.1038/ 414813a 25. Sedlic F, Sepac A, Pravdic D et al (2010) Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2?. Am J Physiol Cell Physiol 299:C506–C515. doi:10.1152/ajpcell.0 0006.2010 26. Orrenius S, Gogvadze V, Zhivotovsky B (2007) Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47:143–183. doi:10.1146/annurev.pharmtox.47.120505. 105122 27. Ni Y, Gong XG, Lu M et al (2008) Mitochondrial ROS burst as an early sign in sarsasapogenin-induced apoptosis in HepG2 cells. Cell Biol Int 32:337–343. doi:10.1016/j.cellbi.2007.12.004 28. Yokoyama Y, Nohara K, Okubo T et al (2007) Generation of reactive oxygen species is an early event in dolichyl phosphateinduced apoptosis. J Cell Biochem 100:349–361. doi:10.1002/ jcb.21024
123
ORIGINAL ARTICLE
Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation Di Liu • Hong Zhang • Wenjuan Gu Mengren Zhang
•
Received: 17 August 2013 / Accepted: 10 December 2013 Ó Springer-Verlag Italia 2013
Abstract Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFHDA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucosetreated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the
D. Liu W. Gu M. Zhang (&) Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, No. 1 Shuaifuyuan, Dongcheng District, Beijing 100730, China e-mail: [email protected] H. Zhang Department of Cell Resource Center, Institute of Basic Medical Science, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China
neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation. Keywords Hippocampal neurons High glucose Diabetes mellitus Cognitive impairment Oxidative stress
Introduction The incidence of diabetes mellitus is increasing worldwide, and studies have showed that both type 1 and type 2 diabetes mellitus have been associated with reduced performance on numerous domains of cognitive function [1, 2]. Furthermore, numerous epidemiological studies have linked T2DM with an increased risk of cognitive dysfunction [3]. Therefore, a great deal of researches has focused on the mechanism of diabetic cognitive impairment. Previous studies indicated that diabetic cognitive impairment has a close relation to hippocampus dysfunction. Diabetes mellitus could not only cause the structural changes of hippocampus, such as the degeneration in synaptic structures [4], reductions in hippocampal volume, but also induce the physiology abnormalities of hippocampal neurons, for example, the impairment of neural coordination [5], reductions in levels of brain-derived neurotrophic factor [6], deficits in neurogenesis [7]. Hyperglycemia plays a critical role in hippocampus dysfunction which has been considered to be related to abnormalities in cognitive function in patients with both type 1 and type 2 diabetes mellitus [2]. Consistent with this, it was recently reported that the hippocampal glucose concentration was significantly increased in the diabetic rats [8]. Accumulating evidence suggested that reactive
123
Neurol Sci
oxygen species (ROS) induced by hyperglycemia plays critical roles in the pathogenesis of diabetic neuropathy [9]. It was found that ROS concentrations in brain are significantly increased in diabetic rats [10], but there is little research about the effect of high glucose on oxidative stress in primary cultured hippocampal neurons. Therefore, it is essential to explore the effects of different concentrations of high glucose and different exposure time on hippocampal neurons in vitro, especially the effects on oxidative stress. In the present study, we used the primary cultured hippocampal neurons exposed to different concentrations of high glucose for different time to test the glucotoxicity.
Methods Primary cultures of hippocampal neurons All experimental procedures were performed in accordance with Chinese Academy of Medical Sciences experimental standards as well as international guidelines on the ethical treatment of experimental animals. Newborn Sprague– Dawley (SD) rats, \24-h old, were purchased from the Peking University Health Science Center. Primary cultures of hippocampal neurons were prepared as described previously [11, 12] with a few modifications. Hippocampi were dissected from the brain on ice and minced in sterile icecold D-Hanks’ (Peking union cell resource center, China) with the blood vessels and meninges thoroughly removed. The tissues were digested with 0.25 % trypsin (Peking union cell center, China) for 15 min at 37 °C and then the digestion procedure was terminated by adding 5 ml fetal bovine serum (FBS). Cell suspension was passed through a 200-mesh cell strainer and separated by density gradient centrifugation at for 20 min at 2,000 rpm. Then the cell suspension containing the desired cell fractions was carefully collected and centrifuged within D-Hanks’ for 5 min at 1,500 rpm and then resuspended within 15–20 ml DMEM medium (Peking union cell resource center, China). The cells were then seeded on poly-D-lysine (Sigma, USA) coated-glass coverslips, 96-well plates or 24-well plates at a density of 5 9 105 cell/ml after counting the cell density by using a hemacytometer and maintained at 37 °C in a humidified 5 % CO2 incubator (Forma, USA). Neurons were cultured in DMEM medium and supplemented with 10 % FBS, 20 mmol/L sodium pyruvate, 1 mmol/L glutamine, 100 U/ml Penicillin, 100 lg/ml streptomycin, 10 ng/ml nerve growth factor (NGF) (Millpore, USA).
buffered saline (PBS) before being fixed in 4 % paraformaldehyde for 1 h at room temperature. The coverslips were then washed three times in cold PBS (5 min each). Prior to staining, cells were permeabilised in PBS containing 0.01 % Triton for 10 min at room temperature and then washed three times in cold PBS (3 min each). Nonspecific antibody binding was blocked by incubation at room temperature for 20 min in normal goat serum. Each coverslip was incubated with 20 ll rabbit anti-NSE primary antibody 1:10 (Bioworld, USA) in a petri dish which was placed at 4 °C overnight. Negative controls were incubated identically but without primary antibody. Coverslips were then washed three times in PBS, then incubated with a goat anti-rabbit secondary antibody conjugated to FITC 1:50 Sigma for 30 min at room temperature, washed six times in cold PBS 5 min each. Then coverslips were incubated with 20 ll DAPI for 5 min at room temperature in the dark. Cells were observed and photographed with a volocity demo imaging system (PerkinElmer, USA). High glucose exposures Primary hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h, respectively. The control group was exposed to normal medium which contains 25 mM glucose. The osmolality of all glucose exposure was between 260 and 320 mOsm/kg which had no effect on cell normal metabolism. Cell viability assays After exposure to various concentrations of high glucose for different time, the viability of cells was determined using the MTT assay system (Peking union cell resource center, China). Briefly, the cells were seeded on 96-well culture plates at a density of 5 9 105 cell/ml. After the indicated timeframe, 100 ll 5 mg/ml MTT solution 1:10 was added to each well and then incubated for 4 h at 37 °C. After 4 h incubation, the medium was removed and then the precipitated dye was dissolved within 100 ll DMSO solution for another 30 min incubation at 37 °C. Eventually, the OD value of each well was detected at 570 nm with a MultiSkan3ELISA Reader (Thermo fisher scientific, USA). Cell viability was calculated as follows: treated group OD/24 h control group OD 9100 %. Assessment of apoptosis: nuclear morphology assay
NSE immunostaining Coverslips with their attached cells were removed from the culture medium and washed three times in phosphate
123
Nuclear morphology was used to assess cells undergoing apoptosis (cells with condensed of fragmented nuclei). Cells were gently washed with warm PBS and then
Neurol Sci
Fig. 1 NSE immunostaining results. Immunocytochemical staining with NSE for neurons, while DAPI for all cells. Magnification 9200; scale bar 50 lm (A and B ); magnification 9600; scale bar 23 lm (C )
incubated with 2 lg/mL fluorescent dye Hoechst 33342 (Sigma, USA) in PBS for 10 min at room temperature. Fluorescence of stained chromatin was examined using an inverted fluorescence microscope (LEICA DMI4000B, Japan) and cells with condensed/fragmented chromatin were scored. For each preparation six random fields were counted. Apoptosis ratio treatment group/control group was expressed as percent, with 24 h control group being 100 %.
Neuronal morphological changes Cell morphological changes were assessed by a live cell imaging system. As shown in Fig. 2, hippocampal neurons treated with normal medium (control) exhibited large, vacuole-free cell bodies, with elaborate networks of neurites. However, exposure to different concentrations of high glucose for 72 h all resulted in obvious cell loss, with the disappearance of neurites, appearance of disrupted membranes and shrinkage of cell bodies.
Measurement of intracellular reactive oxygen species Intracellular reactive oxygen species were measured using the fluorescent probe DCFH-DA, which can be oxidized to the highly fluorescent compound dichlorofluorescin (DCF). Cells were incubated with 10 lM DCFH-DA at 37 °C for 30 min and then were washed twice for 5 min each with ice-cold PBS. Cellular fluorescence intensity was detected using a live cell imaging system (Olympus LCS SYSTEM, Japan) with excitation at 485 nm and emission at 530 nm. Statistical analysis All data are expressed as mean ± SD. Differences between groups were examined for statistical significance using a one-way ANOVA with Dunnett’s test as a post hoc analysis. P \ 0.05 was considered significantly different.
Results NSE immunostaining As shown in Fig. 1, nuclei of all cells were stained with DAPI, while cells stained positive for NSE were identified as neurons. Statistically, the ratio of neurons was approximately 90 % (for each preparation six random fields were counted).
Effect of exposure to high glucose on viability of hippocampal neurons The cell viability of all high glucose-treated groups started to decrease dramatically at 72 h and the cell viability of 50, 125, 150 mM high glucose groups reduced significantly as compared to 48 h (P \ 0.05) (Fig. 3). In neurons exposed to high glucose, the cell viability all decreased remarkably after 72 h compared with timed controls (P \ 0.05). Interestingly, there was no significant difference among the high glucose-treated groups from 72 to 96 h (P [ 0.05). Effect of exposure to high glucose on apoptosis of hippocampal neurons Consistent with the MTT results, there was a significant increase of apoptotic nuclei in all high glucose-treated groups after 72 h compared with control group (P \ 0.05) (Fig. 4). The apoptotic nuclei of high glucose-treated groups at 72 h all increased dramatically compared with that of 48 h. In comparison, there was no significant change in controls from 24 to 96 h. We also observed that the percentage of apoptotic neurons showed no statistical difference among high glucose-treated groups from 24 to 96 h (P [ 0.05). Therefore, we select treatment with 50 mM high glucose for further experiment according to the results of MTT assay and Hoechst staining.
123
Neurol Sci
Fig. 2 Neuronal morphological changes, A hippocampal neurons treated with normal medium (control), B hippocampal neurons exposed to 50 mM high glucose, C hippocampal neurons exposed to 75 mM high glucose, D hippocampal neurons exposed to 100 mM
high glucose, E hippocampal neurons exposed to 125 mM high glucose, F hippocampal neurons exposed to 150 mM high glucose. Magnification 9600; scale bar 17 lm
Effect of exposure to high glucose on intracellular ROS accumulation in hippocampal neurons Cells were exposed to 50 mM glucose for the indicated times (3–72 h), and the cellular ROS levels were measured by DCF-DA staining. The cellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h (P \ 0.01) (Fig. 5A). It was observed that ROS accumulation levels of glucose-treated group peaked at 24 h with sixfold higher than the control (P \ 0.01) and began to decrease beyond 24 h (Fig. 5B). Differently, the cellular ROS levels of control group peaked at 3 h and decreased dramatically from 6 h (P \ 0.01).
Discussion Fig. 3 Effect of high glucose on the viability of hippocampal neurons. Cell viability was assessed by the MTT reduction assay. The results are representative of three independent experiments, and are expressed as percentage of control at 24 h
123
Diabetes is associated with multiple adverse effects on the brain, some of which may result primarily from direct consequences of chronic hyperglycemia. Clinical study
Neurol Sci Fig. 4 Effect of high glucose on apoptosis of hippocampal neurons. Total cells and cells with condensed/fragmented nuclei (cells undergoing apoptosis) were counted in six random fields in each coverslip using Hoechst staining. The results represent the mean ± SD of at least three independent experiments, and are expressed as percentage of control; *P \ 0.05, significantly different from control; # P \ 0.05, compared with 48 h, the apoptotic nuclei of high glucose groups increased significantly at 72 h
has demonstrated that the blood control level is closely related to the cognitive function [13]. Furthermore, experiment on diabetic rats also has proved that blood glucose level could affect synaptic plasticity and the ability to learn [6]. The importance of the hippocampal formation for memory is well established on the basis of neuroanatomical and electro-physiological studies [14]. Chronic hyperglycemia can induce the hippocampal neurons dysfunction and apoptosis through a variety of mechanisms, including oxidative stress [9], increased formation of advanced glycation end products [15], mitochondrial dysfunction [16] and so on. Moreover, long-term exposure to high glucose could induce changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons and a significant increase of apoptotic nuclei [17]. Since hyperglycemia is considered the primary pathogenic factor for the development of diabetic cognitive impairments and hippocampus function is closely related to cognition, we explore the effects of different concentrations of high glucose and different exposure time on primary cultured hippocampal neurons. Long-term exposure to high glucose could impair the mitochondrial function [18] and cause the decrease of cell viability. In present study, we observed that there was a significant decrease of the cell viability and a significant increase of apoptotic nuclei in high glucose-treated groups at 72 and 96 h which mimic chronic hyperglycemic conditions. Consistent with previous researches where apoptosis was found in neural cells exposed to high glucose [17–19] and in the hippocampus of diabetic animals [20, 21], the results give support to that high glucose exposure for long time could induce the hippocampal neurons dysfunction and apoptosis.
From the results of MTT assay and Hoechst staining, we can see that the cell viability and percentage of apoptotic neurons had no significant difference among different concentration of high glucose groups from 72 to 96 h (P [ 0.05). Hence, exposure to 50 mM high glucose for 72 h was enough to cause obvious hippocampal neurons damage and could maintain the effects with the extension to 96 h. This can be considered as a basis of establishment of the high-glucose-insulted primary culture rat hippocampal neuronal cell model. Increasing evidences in both vivo and vitro experiments suggest that there is a close link between hyperglycemia, oxidative stress, and diabetic neuropathy. Hyperglycemia has been shown to induce oxidative stress through multiple pathways such as redox imbalances secondary to enhanced aldose reductase (AR) activity, increased advanced glycation end products (AGE), altered protein kinase C (PKC) activity, prostanoid imbalances, and mitochondrial overproduction of superoxide [22]. ROS can lead to tissue injury and cell death through damaging DNA and inactivating antioxidant defense enzymes [23, 24]. Increased brain mitochondrial ROS production may cause the opening of mitochondrial permeability transition, leading to brain mitochondrial swelling and the depolarization of mitochondrial membrane potential [25]. Moreover, it has been reported that mitochondrial ROS production of diabetic rat hippocampus was significantly increased [10]. Consistent with this study, our result showed that cellular ROS generation can be markedly induced by high glucose. In 50-mM glucose group, cell viability reduced and apoptotic nuclei increased significantly at 72 h while ROS generation declined, which may be partly caused by increased neuronal death on one hand. On the other hand, it has been suggested that ROS generation precede disruption
123
Neurol Sci
Fig. 5 Effect of high glucose on intracellular ROS accumulation in hippocampal neurons. Hippocampal neurons were exposed to 50 mM high glucose for 3–72 h. A Representative photographs of DCF staining in different groups, a hippocampal neurons exposed to 50 mM high glucose for 3 h, b hippocampal neurons exposed to 50 mM high glucose for 6 h, c hippocampal neurons exposed to 50 mM high glucose for 12 h, d hippocampal neurons exposed to
50 mM high glucose for 24 h, e hippocampal neurons exposed to 50 mM high glucose for 48 h, f hippocampal neurons exposed to 50 mM high glucose for 72 h. Scale bar 17 lm. B Quantitative analysis of the DCF fluorescence intensity among groups. The results represent the mean ± SD of at least three independent experiments, and are expressed as percentage of control at 3 h
of membrane potential of mitochondria and the reduction of Dwm is an early step of apoptosis, followed by cytochrome c release in many cell types [26]. As is observed in
many studies [19, 27, 28], ROS generation peaked in early phase but did not sustain at the latter stage when cell apoptosis increased obviously. Therefore, ROS generation
123
Neurol Sci
might be an initial signal in high glucose-induced apoptosis. And the precise mechanism of ROS triggered apoptosis in high glucose-treated hippocampal neurons requires further investigation. In summary, we showed that exposures to different concentrations of high glucose for 72 and 96 h could cause cell viability reduction and apoptosis in hippocampal neurons, supporting previous findings which have proved hyperglycemia to be a critical contributor to hippocampus changes. Our study also suggested that high glucose can also cause hippocampal neurons damage by mediating intracellular ROS production and sheds lights on the pathogenesis of diabetic neuropathy. Furthermore, the mechanisms that cause hippocampal neurons injury in the exposure to high glucose still need to be well explored and understood. Acknowledgments We are grateful to Professor Yuqin Liu and Bei Gu for their helpful technical assistance and scientific suggestions. Conflicts of interest
None declared.
References 1. Cukierman T, Gerstein HC, Williamson JD (2005) Cognitive decline and dementia in diabetes–systematic overview of prospective observational studies. Diabetologia 48:2460–2469. doi:10.1007/s00125-005-0023-4 2. McCrimmon RJ, Ryan CM, Frier BM (2012) Diabetes and cognitive dysfunction. Lancet 379:2291–2299. doi:10.1016/S01406736(12)60360-2 3. Saczynski JS, Jonsdottir MK, Garcia ME et al (2008) Cognitive impairment: an increasingly important complication of type 2 diabetes: the age, gene/environment susceptibility–Reykjavik study. Am J Epidemiol 168:1132–1139. doi:10.1093/aje/kwn228 4. Hou Y, Zhou L, Yang QD et al (2012) Changes in hippocampal synapses and learning-memory abilities in a streptozotocin-treated rat model and intervention by using fasudil hydrochloride. Neuroscience 200:120–129. doi:10.1016/j.neuroscience.2011.10. 030 5. Liu K, Xie KN, Qiao ZM et al (2009) Impaired neural coordination in hippocampus of diabetic rat. Sheng Li Xue Bao 61:417–423 6. Stranahan AM, Norman ED, Lee K et al (2008) Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats. Hippocampus 18:1085–1088. doi:10.1002/hipo.20470 7. Stranahan AM, Arumugam TV, Cutler RG et al (2008) Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons. Nat Neurosci 11:309–317. doi:10.1038/nn2055 8. Duarte JM, Carvalho RA, Cunha RA et al (2009) Caffeine consumption attenuates neurochemical modifications in the hippocampus of streptozotocin-induced diabetic rats. J Neurochem 111:368–379. doi:10.1111/j.1471-4159.2009.06349.x 9. Yi SS, Hwang IK, Kim DW et al (2011) The chronological characteristics of SOD1 activity and inflammatory response in the hippocampi of STZ-induced type 1 diabetic rats. Neurochem Res 36:117–128. doi:10.1007/s11064-010-0280-6
10. Pipatpiboon N, Pratchayasakul W, Chattipakorn N et al (2012) PPARgamma agonist improves neuronal insulin receptor function in hippocampus and brain mitochondria function in rats with insulin resistance induced by long term high-fat diets. Endocrinology 153:329–338. doi:10.1210/en.2011-1502 11. Kaech S, Banker G (2007) Culturing hippocampal neurons. Nat Protoc 1:2406–2415 12. Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490–1498. doi:10.1038/ nprot.2007.207 13. Yaffe K, Blackwell T, Kanaya AM et al (2004) Diabetes, impaired fasting glucose, and development of cognitive impairment in older women. Neurology 63:658–663 14. Eichenbaum H (1999) The hippocampus and mechanisms of declarative memory. Behav Brain Res 103:123–133 15. Wang SH, Sun ZL, Guo YJ et al (2009) Diabetes impairs hippocampal function via advanced glycation end product mediated new neuron generation in animals with diabetes-related depression. Toxicol Sci 111:72–79. doi:10.1093/toxsci/kfp126 16. Ye L, Wang F, Yang RH (2011) Diabetes impairs learning performance and affects the mitochondrial function of hippocampal pyramidal neurons. Brain Res 1411:57–64. doi:10.1016/j. brainres.2011.07.011 17. Gaspar JM, Castilho A, Baptista FI et al (2010) Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons. Neuroscience 171:981–992. doi:10.1016/j.neuroscience. 2010.10.019 18. Santiago AR, Cristovao AJ, Santos PF et al (2007) High glucose induces caspase-independent cell death in retinal neural cells. Neurobiol Dis 25:464–472 19. Russell JW, Golovoy D, Vincent AM et al (2002) High glucoseinduced oxidative stress and mitochondrial dysfunction in neurons. FASEB J 16:1738–1748 20. Zhao CH, Liu HQ, Cao R et al (2012) Effects of dietary fish oil on learning function and apoptosis of hippocampal pyramidal neurons in streptozotocin-diabetic rats. Brain Res 1457:33–43. doi:10.1016/j.brainres.2012.03.067 21. Li ZG, Zhang W, Grunberger G et al (2002) Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 946:221–231 22. Pop-Busui R, Sima A, Stevens M (2006) Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 22:257–273 23. Rosen P, Nawroth PP, King G et al (2001) The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCOMCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17:189–212 24. Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820. doi:10.1038/ 414813a 25. Sedlic F, Sepac A, Pravdic D et al (2010) Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2?. Am J Physiol Cell Physiol 299:C506–C515. doi:10.1152/ajpcell.0 0006.2010 26. Orrenius S, Gogvadze V, Zhivotovsky B (2007) Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47:143–183. doi:10.1146/annurev.pharmtox.47.120505. 105122 27. Ni Y, Gong XG, Lu M et al (2008) Mitochondrial ROS burst as an early sign in sarsasapogenin-induced apoptosis in HepG2 cells. Cell Biol Int 32:337–343. doi:10.1016/j.cellbi.2007.12.004 28. Yokoyama Y, Nohara K, Okubo T et al (2007) Generation of reactive oxygen species is an early event in dolichyl phosphateinduced apoptosis. J Cell Biochem 100:349–361. doi:10.1002/ jcb.21024
123
