Association between hyperglycemia and cognition instreptozotocin induced diabetic Kunming mice

Zhang Yue, Feng Si-miao, Hu Shu, Ma Yan-lin, Liu Song-ling, Zheng Yan-jiang△

(1.Class 2014, Chengdu No.7 High School, Sichuan Chengdu 610041; 2.West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan Chengdu 610041)

英文論著

Association between hyperglycemia and cognition instreptozotocin induced diabetic Kunming mice

Zhang Yue, Feng Si-miao1, Hu Shu2, Ma Yan-lin2, Liu Song-ling1, Zheng Yan-jiang2△

(1.Class 2014, Chengdu No.7 High School, Sichuan Chengdu 610041; 2.West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan Chengdu 610041)

Objective：To zdetermine the effects of hyperglycemia on learning and memory abilities in streptozotocin (STZ)-induced diabetic mice. Methods: Diabetes mellitus (DM) was induced in age-matched Kunming mice by STZ injection (50 mg·kg-1body weight, intraperitoneally, once a day for 5 days). Diabetic status was assessed by blood glucose monitor. A fasting blood glucose level of more than or equal to 11 mmol·L-1were considered as diabetic mice. The learning and memory abilities of the mice (12 for the diabetic mice and 10 for control) were evaluated by Morris water maze and the Shuttle box. Diabetic related pathological alternations were observed in kidney, pancreases, and aortaventralis. Results: Compared with sham group, STZ treatment significantly increased food consumption and water consumption as well as body weight loss (P<0.05). DM mice did not show difference on learning and memory abilities compared with the control mice at the end of 4 weeks (P>0.05), but they were mild sluggish and showed reduced navigation ability in the transfer test and the hidden platform test at end of 12 weeks (P<0.05). Conclusion: Hyperglycemia itself did not induce cognitive dysfunction in STZ-induced diabetic mice. Further study should be carried out to clear the relationship between diabetes and cognitive disorder.

Cognition; Diabetes mellitus; Maze learning, Kunming mouse

Author: Yue Zhang, Female, Class 2014, Chengdu No.7 High School, Email: yuezhang1217@gmail.com.

△Corresponding author: Yanjiang Zheng, Male, Research fellow of the experimental technology of medical function, Email: zhengyj@scu.edu.cn.

Diabetes mellitus (DM) is one of the most prevalent diseases in the world. There are about 422 million adults who are living with DM[1]. The mortality and morbidity of DM are determined by various complications, such as heart attack, stroke, amputation, and kidney failure[2]. Almost all complications are caused by reduced or blocked blood flow when hyperglycemia damaged the blood vessels in a particular organ. The DM patients may have brain ischemic changes which are basic pathological feature of both Alzheimer′s disease and vascular dementia.

Studies have indicated that people with diabetes are at higher risk of developing Alzheimer′s dementia or other dementias eventually[3, 4]. The learning and memory abilities of the diabetic rats decreased compared with those of the age-matched control rats[5, 6]. Hippocampal atrophy[7], Aβ aggregation[3], and synapse loss[8]have been observed in the diabetic rats compared with the control rats. Data from Cassandra C. Brady suggested that working memory and processing speed were negative correlation with the duration of diabetes[9]. But not all the data support the association between DM and cognitive dysfunction. A meta-analysis showed no association between blood glucose and cognitive function[10].

To confirm whether hyperglycemia affect cognition or not, we examined learning and memory function in streptozotocin (STZ) induced hyperglycemia Kunming mice to find the association between hyperglycemia and cognition in this study.

1 Material and Methods

1.1 Laboratory Animal

Twenty male adult (body weight 25.3±0.6 g) Kunming mice were obtained from the Laboratory Animal Centre of Sichuan University, Chengdu, China. Mice were maintained in a standard housing condition (room temperature: 25±1℃ and humidity: 55%-65%) on a 12 h light/12 h dark cycle. Four mice were kept in one cage. All experimental procedures were conducted according to the Provision and General Recommendation of the Chinese Laboratory Association.

1.2 Induction and assessment ofdiabetes mellitus in mice

Streptozotocin (STZ), was obtained from Sigma (St Louis, MO, USA), and dissolved in 0.01 M citrate buffer, pH 4.5 on ice. After fasting overnight, the concentration of blood glucose was measure by Accu-Chek Aviva Connect Blood Glucose Monitoring System (German). Mice with fasting blood glucose level lower than 11 mmol·L-1were administrated intraperitoneally with 50 mg·kg-1STZ, once a day for 5 days[11]. Age-matched mice received an equal volume of citrate buffer and were designated as the control group.

The diabetes model was verified after 5 days’ STZ injection. Blood samples were collected through the tail vein and glucose levels were measured. Animals with a fasting blood glucose level of more than or equal to 11 mmol·L-1were considered as diabetic mice. Totally 12 out of 22 STZ treatment mice were diagnosed as diabetic mice and used in subsequent experiments.

1.3 Evaluation of general physical condition

The metabolic parameters were monitored. In brief, the mice were monitored for 24 h after 8 hours’ fasting. Water and food consumption in 24 hours were measured in the interim (1, 2, 3, 4 and 8 weeks after the beginning of treatment).

1.4 Histopathology

Four mice from each group were sacrificed by neck dislocation at the end of 8 weeks. Organs were removed under aseptic conditions and kept in 10% buffered formalin for 24 hours. Then, routinehistopathological process was performed to obtain slides stained with hematoxylin and eosin (H&E) for histological evaluation.

1.5 Morriswater maze test

Twelve diabetes mice and 10 control mice performed a learning task in the Morris water maze with the blind method[12]. The maze consisted of a white circular plastic pool (diameter=120 cm, height=50 cm) filled with opaque whitened water using nontoxic white gouache (26±2℃). The behavior of the mice in the pool was traced with a digital camera connected to a WMT-100 analysis system (Taimeng Bioinstrumentation Ltd, Chengdu, China). The pool was divided into four quadrants: Position A was assigned to the northwest quadrant, B was the northeast quadrant, C was the southeast quadrant, and D was the southwest quadrant (Fig.1).

Figure 1 Schematic diagram of Mazing tank and site of the platform.

1.5.1 The hidden platform test

Place navigation requires the mice to learn to swim from any starting position to an escape platform with fixed-position, thereby acquiring a long-term memory of the platform’s spatial location. A circular platform (diameter=65 mm) was submerged 2 cm below the water surface in the middle of the target quadrant. Each day involved training the mice in the four quadrants with 30 min intervals. Each trial was started by placing a mouse with its back facing toward the platform at the starting points. The trial was terminated when the mouse stood on the platform. However, when the mouse did not find the platform within 120 s, it was guided on the platform for 15 s. On each day after training, the mouse was removed from the pool, dried, and then returned to its cage.

1.5.2 Transfer test

The transfer test provides an index of the mice’s tendency to persist around the previous location of platform and is generally considered to be a measure of retention (http://www.panlab.com/en/tests-solutions/morris-water-maze-test). After completion of the Hidden Platform test, the trials were given in which the platform was removed from the pool to measure spatial bias. This is accomplished by measuring the time and distance traveled in the zone of the previous target platform.

1.6 Shutter box escape/avoidance task

An automatic reflex conditioner (shuttle box) for escape/active avoidance was used[13]. The experiment was carried out in shutter boxes consisting of two equal-sized compartments (25×25×28 cm) connected by an opening (8×10 cm). Before the first trial of learning session, mice were acclimatized inside the shutter box shock area for 5 min in order to be familiarized with the learning environment. A memory retention session was performed after 4 consecutive days of learning by given the conditioned stimulus (light) for 5 s, followed by an unconditioned stimulus (a 24 V, 50 Hz electric shock though the grid floor) for 5 s. One round of task was complete when the animal crossed to the other compartment, with crossing during the conditioned or unconditioned stimulation being considered as avoidance and escape responses, respectively. A camera was placed in the center of the ceiling in each box to record and analyze the mice’s movement.

1.7 Statistical analysis

Statistical analyses were performed with Prism5. Data were presented as mean±standard error of the mean and were analyzed with the unpaired student’sttest.TheresultsfromtheMorriswatermazeandavoidancetaskwereanalyzedbytwo-wayanalysisofvariance(ANOVA),P<0.05wasconsideredstatisticallysignificant.

2 Results

2.1 Establishment of diabetic mouse model

Plasma glucose levels were highly increased in mice after STZ treatment, as compared to control mice with citric buffer treatment. After 5 days, there are 80% mice with a fasting blood glucose level over 11 mmol·L-1, which were considered as DM and used for the further study. We monitored blood glucose levels every week as shown as Fig.2A.

The classic clinic symptoms of diabetes are polydipsia, polyphagia, polyuria, and emaciation. So we measured mice metabolic parameters, including body weight, water and food consumption for 24 h at the end of 1, 2, 3, 4 and 8 weeks. The results showed that the body weight (Fig.2B) of the diabetic mice was reduced compared with that of the control group. The water consumption (Fig.2C) and food consumption (Fig.2D) of the diabetic mice increased compared with non-diabetes mice (P<0.05).

Figure 2 Metabolic parameters of mice at 4 consecutive weeks and at the end of 8 weeks after STZ injection.A. Plasma glucose (mM), B. Body weight (gram), C. Water consumption (ml water per day), D. Food consumption (gram food per day).Compared with control group,*P<0.05. n=10 for control group and n=12 for diabetic mice.

Moreover, we detected the urine sugar by urine test strip and results were negative till 8 weeks. It suggested that the blood glucose level did not exceed renal threshold of glucose, the proximal tubule reabsorbed all glucose back to the blood.

2.2 Pathological changes of major organ in DM mice

The primary complications of diabetes, which is due to the damages in small blood vessels, usually affect the kidneys and the nerves. Herein, pathological examinations of DM mice were performed. We euthanized mice at the end of experiment,and tissues of spinal nerve, kidney, pancreas, hippocampus and aorta were collected. Histopathological examination was performed by H&E staining (Fig.3). (The neuronal integrity and orderliness in the hippocampus is on working.)

Figure 3 Histopathologic changes of the represented tissues from STZ induced diabetes in Kunming mice at 12 weeks. (H&E stain; magnification: 200×).Spinal nervous tissue did not show obvious pathological changes. In kidney, modest glomerular lesions were noted in diabetic group, including glomerulosclerosis, hyaline degeneration of glomerular capillaries, atrophy in renal tubule, and chronic inflammatory cell infiltration. The parenchyma of pancreatic islets showed atrophy and fibroplastic proliferation. Arteriosclerosis and hyaline degeneration can be observed in aorta.

In the diabetic group, a decrease in both the amount or size of pancreatic islet was observed. The parenchyma of pancreatic islets showed atrophy and fibroplastic proliferation. In kidney, modest glomerular lesions were noted in diabetic group, including glomerulosclerosis, hyaline degeneration of glomerular capillaries, atrophy in renal tubule, and chronic inflammatory cell infiltration. Arteriosclerosis and hyaline degeneration can be observed in aorta. All above histologic changes further confirmed the diagnosis of DM.

2.3 Cognitive function analyzed by the Morris water maze test

Morris water maze test took place over 5 days as described in Method. Swimming time before the mice got the platform was recorded and demonstrated as an escape latency. The latency time indicated that mice were learning to navigate the maze (Fig.4A). On the contrary, mice will be guided to platform if they cannot find it within 120 seconds (Fig.4B).

The hidden platform test of the Morris water maze study was performed at the end of 4 weeks after DM model was setup. Our data revealed that elevated blood glucose in diabetic mice did not affected the escape latency compared to control group on day 2, 3, 4 and 5 (P>0.05), as shown in Fig.4F, 4G, 4H and 4I. But mice with 12 weeks’ hyperglycemia had significant extended escape latency compared to non-diabetic rats (Fig.4J and 4L) (P<0.05).

The transfer test in the Morris water maze was a method to analyze expressing memory retention in terms of selective search behavior, which specifically correlates with learning impairment. Representative swimming tracks of the hidden platform test and the transfer test were shown as Fig.4C to 4D.Fig.4E showed that the results of transfer test did not have significant difference between DM mice and age-matched control mice, either at 4 weeks or at 12 weeks of diabetes (P>0.05).

Figure 4 Results of the Morris water maze.A-D show the representative swimming tracks of the hidden platform tests and the transfer test. In A and C, the mice successfully reached the platform hid under water about 2 cm. In B and D, the mice failed to reach the platform in 120 s. The green arrows showed the position of the hidden platform. Figure E showed the transfer test results. The platform was removed, then the number of the mice passed through the position of removed platform were counted. Figure F to I showed the results of the hidden platform in 4 quatrants of the mice with 4 weeks hyperglycemia. Figure F to I showed the results of the hidden platform in 4 quatrants of the mice with 12 weeks hyperglycemia. Data are expressed as mean±SEM.*P<0.05. n=10 for control group and n=12 for diabetic mice.

2.4 Cognitive function analyzed by escape/avoidance test employing the shuttle box

The shuttle boxescape/avoidance tests were applied to analyze the cognitive function of mice. The cognitive function was evaluated after DM model was setup for 4 weeks. The frequency of active avoidance (conditioned reflex, Figure 5A), passive avoidances (unconditioned reflex, Figure 5B), escaping failure (false reflex, Figure 5C), and the incubation time (freezing time, Figure 5D) in 20-trial sessions were recorded. All results of above 4 parameters did not show significant difference between DM and control mice.

Figure 5 Learning performance in Shutter box escape/avoidance task at 4 weeks after STZ administration.A. Frequency of active avoidance happened after a light stimulation. B. Frequency of passive avoidance happened after an electronic stimulation. C. Occurrence frequency of aimless action. D. Freezing time. Data are expressed as mean±SEM.

3 Discussion

DM is a disease not only related with increased blood glucose, but also involving multiple metabolic related dysfunctions such as hyperlipidemia, hypertension, and aging, et al[14]. Alterations of central nervous system (CNS) functions, including cognitive decline, are often detected in DM patients. It is shown that diabetes can be a risk factor for dementia, including Alzheimer’s disease, vascular dementia and other types of dementia, since cardiovascular problems associated with diabetes are also associated with dementia[15]. It is hard to distinguish pathological differences between the brains of diabetes patients and the brains of patients with Alzheimer’s disease. Impaired blood vessels, high cholesterol and high blood pressure accompanied with the process of DM can cause the same pathological changes in brain of Alzheimer’s disease[16, 17].

In an epidemiological study of over 6000 person for up to 6 years,Ott, et al found that older adults apparently face a greater risk of vascular dementia, but this effect cannot be accounted only through vascular factors[18]. Recent reports also suggested that cognitive alterations observed in T2D appear to require the interaction of ageing factors with diabetes[19-21]. A study conducted in a large Hispanic urban population found no association between mild cognitive dysfunction and glycemic control, though it was noted by the authors that assistance from informal care givers could not be ruled out[22-23]. Diabetes-induced changes of brain properties might in fact share many properties with brain ageing, obesity, circulation problems, high cholesterol and high blood pressure.

Although diabetes is one of risk factors for AD, it is still a question whether only increasing blood glucose can affect cognitive or not. This study focuses on evaluating the effect of hyperglycemia on memory and learning processes. We increased blood glucose concentration by administration of STZ in Kunming mice. The control group strictly matched DM group with age, gender, and body weight, and fed in the same condition as DM mice. Our data showed that hyperglycemia caused histologic pathological changes in kidney, pancreas, blood vessel, as well as the changes in general body conditions and metabolism level. But there was no significant influence on the mice navigation behaviors. Diabetic mice and control mice presented similar learning curves in the hidden platform test, the transfer test and the shutter box escape/avoidance task at the end of 4 weeks after STZ administration. These negative data could be explained as the insufficient brain vessel injury and brain dysfunction at the early stage of DM. As we prolong the training time to 12 weeks, the DM mice took more time to find platform. But this difference may not cause by hyperglycemia or dementia. The aged diabetes mice were seriously sick. The bad body condition made them swimming slower than the control mice, and this situation was significant enough to cause a false-positive result of navigation behavior analysis. Alternatively, if the cognition dysfunction happened in the late stage of DM, other methods should be chosen to analyze learning and memory function instead of Water morris maze.

The data in this study suggests that there is no direct cause-effect relationship between hyperglycemia and cognition in our model. According to preliminary data, comprehensive prevention and treatment are better than only controlling blood sugar for improving cognition. We are going to do further studyin vivo and by clinical questionnaire on diabetes and cognitive disorder.

Acknowledgement

We are immensely grateful to Dr.Yufang Wang and Dr. Ji Zhang who provided insight and expertise that greatly assisted the research. We thank pathologist Dr. Xueqin Chen to do pathological diagnosis.

Author Contributions

Conceived and designed the experiments: YZ, YJZ,SLL. Performed the experiments: YZ, SMF, SH, YLM. Analyzed the data: YZ, YJZ. Contributed reagents/materials/analysis tools: YJZ. Wrote the paper: YZ, SMF,YJZ

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2016-12-16)

鏈佐霉素誘導(dǎo)糖尿病昆明鼠認(rèn)知功能與高血糖的關(guān)系

張玥1馮思淼1胡舒2馬彥林2劉松齡1鄭焱江2△

(1.成都市第七中學(xué)2014級(jí)，四川 成都 610041； 2.四川大學(xué)華西基礎(chǔ)醫(yī)學(xué)與法醫(yī)學(xué)院，四川 成都 610041)

目的：研究昆明鼠經(jīng)鏈佐霉素誘導(dǎo)后，血糖升高與學(xué)習(xí)和記憶能力之間的相關(guān)性。方法：選取年齡、體重以及血糖接近的昆明小鼠，連續(xù)5 d腹腔注射50 mg·kg-1鏈佐霉素建立糖尿病小鼠模型。經(jīng)血糖儀測(cè)定尾靜脈血糖含量，其中空腹血糖大于或等于11 mmol·L-1作為糖尿病模型合格小鼠。糖尿病小鼠(n=12)和正常小鼠(n=10)通過水迷宮實(shí)驗(yàn)和穿梭實(shí)驗(yàn)檢測(cè)小鼠學(xué)習(xí)和記憶能力。病理組織學(xué)檢測(cè)糖尿病小鼠腎臟、胰腺和主動(dòng)脈等器官病理改變。結(jié)果：與對(duì)照組相比，鏈佐霉素處理組小鼠飲食、飲水量顯著提高并伴隨體重下降(P<0.05)。4周后，糖尿病小鼠和正常小鼠的學(xué)習(xí)和記憶能力并沒有顯著差別(P>0.05)。但是12周后，糖尿病小鼠行動(dòng)遲緩，比正常組小鼠定位巡航和空間探索能力均下降(P<0.05)。討論：持續(xù)12周的高血糖狀態(tài)并未導(dǎo)致STZ誘導(dǎo)的糖尿病小鼠出現(xiàn)認(rèn)知功能障礙，有關(guān)糖尿病和認(rèn)知功能之間的關(guān)系需要進(jìn)一步進(jìn)行研究。

認(rèn)知；糖尿病；水迷宮；昆明小鼠