Behavioural Brain Research 278 (2015) 411–416
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
Behavioral impairments and changes of nitric oxide and inducible nitric oxide synthase in the brains of molarless KM mice Qian Pang a , Xingxue Hu b , Xinya Li c , Jianjun Zhang c , Qingsong Jiang a,∗ a b c
Department of Prosthodontics, Beijing Stomatology Hospital and School of Stomatology, Capital Medical University, Beijing 100050, China Advanced Standing Program, Boston University Henry M. Goldman School of Dental Medicine, 100 East Newton Street, Boston, MA 02118, USA Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
h i g h l i g h t s • Molar extraction may result in the impairments of spatial learning and memory of the KM mouse. • The expression of NO and iNOS increases in the hippocampus of the KM mouse after molar extraction. • The changes of NO and iNOS in the hippocampus may be involved in the behavioral changes in the molarless condition.
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 14 October 2014 Accepted 17 October 2014 Available online 28 October 2014 Keywords: Mastication Hippocampus Nitric oxide Inducible nitric oxide synthase
a b s t r a c t More studies showed that as a common disorder in senior population, loss of teeth could adversely affect human cognitive function, and nitric oxide (NO) might play an important role in the cognitive function. However, the underlying mechanism has not yet been well-established. The objectives of this study are to evaluate behavior changes of KM mice after loss of molars, and levels of NO and inducible nitric oxide synthase (iNOS) in the brain in molarless condition. It is hypothesized that loss of molars of the mice tested results in the cognitive impairments and that the process is mediated by NO in the brain through the signaling pathways. Morris water maze is used to test the behavioral changes after 8 weeks of the surgery. The changes of NO and iNOS are evaluated by using Griess assay, western blot, and immunohistochemistry method. The results show that 8 weeks after loss of molars, the spatial learning and memory of KM mice impair and the levels of NO and iNOS in mice hippocampus increase. These findings suggest that molar extraction is associated with the behavioral impairment, and that the changes of NO and iNOS in the hippocampus may be involved in the behavioral changes in the molarless condition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Loss of teeth is one of common disorders in the geriatric population. The elders with long-term loss of teeth may suffer from reduced mastication, inarticulate pronunciation, temporomandibular joint dysfunction, and further appear a poor nutritional status and decreased daily living skills [1,2]. The association between mastication and brain function recently gains more attentions. Animal and human experimental studies confirmed the causal relationship between mastication and cognition function [3], and tooth loss was associated with an increased risk of both
∗ Corresponding author at: Beijing Stomatological Hospital & School of Stomatology, Capital Medical University, No. 4 Tiantan Xili, Beijing 100050, China. Tel.: +86 10 57099309; fax: +86 10 57099310. E-mail address: [email protected] (Q. Jiang). http://dx.doi.org/10.1016/j.bbr.2014.10.020 0166-4328/© 2014 Elsevier B.V. All rights reserved.
dementia and cognitive decline [4]. Alzheimer disease is considered the most common cause of cognitive dysfunction among the aged, and tooth loss might be a risk factor for Alzheimer-type dementia [5]. It was found that the construct of vascular contributions to cognitive impairment and dementia is sufficiently important [6], and that chewing lead to an increased blood flow [7], superior learning and word recalling [8]. It was beneficial for cognitive impairment during the early intervention in that vascular risk factors could be found and easily controlled [6]. Dental prosthesis could significantly stimulate masticatory muscle and dorsal prefrontal cortex activities [9], and increase cerebral regional blood volume [10]. It is known that nitric oxide (NO) involves the aspects of regulating blood flows, transferring neural signals and mediating stress responses. In addition to its vasoactive and immunological properties, NO has significant neurophysiological functions such as long-term potentiation (LTP), long-term depression (LTD), elucidation of calcium-dependent and NMDAR-mediated activation of
412
Q. Pang et al. / Behavioural Brain Research 278 (2015) 411–416
neuronal nitric oxide synthase (nNOS) [11]. Abnormal NO signaling was attributed to a variety of neurodegenerative pathologies such as stroke/excitotoxicity, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease [11,12]. Nitric oxide is an enzymatic product of nitric oxide synthase (NOS). One of the three isoforms of NOS, inducible nitric oxide synthase (iNOS) generates NO and NOderived reactive nitrogen species (e.g. peroxy nitrite). Inducible nitric oxide synthase was involved in the mechanisms of cerebral ischemic insult [13]. However, few studies have been reported regarding the association between the changes of NO and iNOS and the damage of learning and memory caused by the condition of loss of teeth. The objectives of this study were to investigate the behavior changes of KM mice after loss of molars, and levels of nitric oxide and nitric oxide synthase in the brains of the molarless mice. It was hypothesized that loss of molars results in the cognitive impairments of the KM mice tested and that the process is mediated by NO through the signaling pathways. 2. Materials and methods 2.1. Animals Sixty male KM mice (10–11 months old) (Vital River Laboratory Animal Technology Co. Ltd.) were employed. All mice were habituated at least 7 days before the start of experiments. Five mice were housed in every standard polycarbonate cage with free access to food and water and a reversed day/night cycle with lights on at 10:00 P.M. and off at 10:00 A.M. The experiments complied with the guidelines for care and use of laboratory animals from National Institutes of Health (NIH). 2.2. Surgery Mice were randomly divided into 4 groups (15 mice each group): maxillary extraction group (E1), maxillary sham group (S1), mandibular extraction group (E2) and mandibular sham group (S2). The mice were anesthetized via intraperitoneal (i.p.) injection of 10% chloral hydrate (400 mg/kg). In the sham group, small amounts of bilateral maxillary or mandibular alveolar bone were removed with rongeur from the toothless gap region between molars and canines in the superior alveolar ridge. In the molarless group, all the bilateral maxillary or mandibular molars were removed. If there was root fracture, all the tooth structure visible on the gum was removed to eliminate occlusal contacts [14]. After the operation, the mice were allowed free access to routine pelleted diet and water. General conditions and body weights of each mouse were monitored during the entire progress. 2.3. Morris water maze (MWM) The Morris water maze test was performed according to the previous study [15]. The maze consisted of a circular plastic pool with 120 cm in diameter and 60 cm deep, the interior of which was painted black [16]. The plastic escape platform (10 cm in diameter and 28 cm high) was positioned in the pool. The pool was filled with water 1 cm above the platform at 22 ± 1 ◦ C. Behavior of the mice in the pool was recorded by a video camera positioned over the pool. During the whole test, the lighting of the testing room was placed indirectly to the pool and the environment (e.g. experimenter, work table, door, and pipes, etc.) was kept consistent. Eight weeks after operation, the acquisition training session was performed. On the first day, the mice were placed into the pool without the platform one by one, and each mouse swam separately and freely for 60 s. A similar procedure was repeated in a pool with the platform that was fixed with a red flag and placed in the center
of one of the four quadrants of the pool. In this procedure, each mouse was placed into the water at the opposite position to the platform, and they were observed to find the flag in 60 s so as to eliminate the influence of impaired vision on the measurement. In the place navigation test, mice were required to find the hidden platform to examine the spatial learning and memory. The platform was placed in the northwest quadrant of the pool (Quadrant II), which differed from the position set in the training session. Two points of the pool (south and east) were used as the starting positions. The animals were given two trials per day for 5 days. Trials began with the mice placed in the pool facing the side wall at a start position and ended once the animal found the platform; if it did not find the platform within 60 s, the mouse was taken out of the water and placed onto the platform for 10 s. Then the mouse was immediately replaced in the pool at another start position for the next trial. The swim paths, distances, speed and latencies taken to swim to the platform were monitored with a CCD video camera linked to a computer system. The probe test was carried out 24 h after the last trial on Day 5. The starting position was set opposite to the original platform position with the platform removed. Each mouse was placed to face the wall. The time of the mouse first passing the platform and the frequency of passing the platform were recorded in 60 s. 2.4. Griess assay Nitric oxide could be released and quantified using the Griess assay [17]. After finishing the MWM test, the productions of NO were determined by an assay for nitrite. Eight mice of each group were decapitated and the whole brain was removed. Then the cortex and hippocampus area were rapidly separated on the ice plate and weighed. The tissue was grinded, adding nine times with phosphate-buffered saline and 0.3% TritonX100, bounce shocking for 3 s and water bathing for 5 min, then centrifugation (12,000 × g, 5 min) at 4 ◦ C, keeping the culture of the supernatant. The procedure followed the instruction of the total nitric oxide assay kit (Biyotime institute of biotechnology, S0024). The optical density of the assay samples was measured spectrophotometrically at 540 nm. 2.5. Immunohistochemistry Four mice of each group were anesthetized with 10% chloral hydrate (400 mg/kg), and then perfused with phosphate-buffered saline (PBS, pH = 7.4) containing 4% parafomaldehyde in 0.1 M phosphate buffer, following postfixation for 24 h in the same buffer. The tissue was then imbedded into paraffin. Sections with 4 m thickness were prepared on a microslicer and then processed according to a standard immunohistochemical protocol. After rinsed with PBS, the sections were incubated with 30% H2 O2 for 10 min to quench endogenous peroxidase activity and then blocked with 5% BSA for 20 min at room temperature, followed by incubation for 2 days at 4 ◦ C with rabbit anti-iNOS antibody, diluted 1:100. The sections were rinsed in PBS for 2 min and 3 times, and incubated for 20 min at 37 ◦ C with anti-rabbit IgG, and then with ABC reagent in PBS, before being treated for about 10 min at room temperature with diaminobenzidine (DAB). After a final wash with ddH2 O2 , the sections were ready for light microscopy. 2.6. Western blot analysis Following decapitation, the cortex and hippocampus of 3 mice of each group were dissected, weighted, recorded and snap frozen in liquid nitrogen. Proteins were extracted with Applygen total protein extraction kit. Protein concentration was normalized using Coomassie brilliant blue G-250 staining. Equal amounts of proteins were separated by SDS-PAGE on 10% polyacrylamide gel, and the
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proteins were transferred to PVDF membrane. After blocking with 0.1% TBST containing 5% non-fat milk at room temperature for 2 h. Primary antibody (iNOS 2977s, 1:1000, from Cell Signaling, Cell Signaling Technology, CA) was added for overnight incubation. The membrane was rinsed with 0.1% TBST three times, 10 min each, and incubated with secondary antibody (ZB2301, 1:5000, from Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h. Color development was performed using the ECL kit. Images were acquired using the Fuji Digital Science Imager, and analyzed with Gelpro Analyzer (Version 4.0) to measure the integrated optimal density (IOD) values of specific bands. 2.7. Statistical analysis Statistical analysis was performed using SPSS Statistics V17.0 software (SPSS Inc.). Treatment differences in the escape latency in the water maze task were analyzed using a repeated measures ANOVA. One-way ANOVA was used for the probe trials. Other data were evaluated using student’s t tests. The statistical significance level is 0.05. 3. Results 3.1. No differences in body weight between extraction and sham groups After 1 week, there was no difference in body weight or food and water consumption between the extraction groups (E1, E2) and sham groups (S1, S2). Body weights of all the mice increased gradually in the following weeks, yet in the whole process the extraction groups and sham groups did not show a significant difference (p = 0.22). Either the maxillary group or mandibular group showed the similar results. Body weight of the molarless mice decreased after the procedure of extraction but recovered to the preoperative
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levels within 1 week, which is consistent to the previous studies [18,19]. 3.2. Molarless mice exhibit a decreased spatial learning ability in the Morris water maze Before the test, all the mice tested showed similar physical abilities and normal visions. There was a significant main effect of training day on measures of escape latency (F(1,28) = 12.87, p = 0.0001 of maxillary groups; F(1,28) = 19.03, p = 0.0001 of mandibular groups) and of cumulative distance to the platform center (F(1,28) = 9.19, p = 0.0001 of maxillary groups; F(1,28) = 43.45, p = 0.0001 of mandibular groups) (Fig. 1A and B). There was no significant difference of swimming speed in each group (p = 0.49) (Fig. 1C). After training, the hidden platform was removed and the probe test was carried out. The percentage of quadrant II compared to the other quadrants of the mice of extraction group showed lower value than the sham groups (F(1,28) = 10.04, p = 0.005 of maxillary groups; F(1,28) = 10.60, p = 0.004 of mandibular groups) (Fig. 1D). By tracking the trajectory of mice in each group swimming records showed quite different results. The mice of the sham groups found the platform directly. They showed a typical preference for searching in the quadrant II than all the other three quadrants. Yet the behaviors of the mice of extraction groups in the pool showed obviously unpurposed. These results confirmed that the mice of extraction groups had impaired memory for the location of the hidden platform in the water maze, which was similar to the previous studies [20]. 3.3. The release of NO in the hippocampus after molar extraction increased After 8 weeks of molarless condition, the NO concentration in the hippocampus of the extraction group increased (t(14) = 5.28, p = 0.0002 of maxillary groups; t(14) = 3.99, p = 0.0001 of mandibular
Fig. 1. Molarless condition reduced the spatial learning and memory (n = 15). (A). All mice showed improvement in escape latency with continuous days of training, yet the improvement of extraction groups were significantly lower than the corresponding sham groups. (B). The path length of the extraction groups was significantly shorter than the corresponding sham groups. (C). No significant differences were observed in the extraction groups and sham groups as reflected by the swimming speed. (D) Molar extraction treatment significantly decreased the time spent searching for the hidden platform in the target quadrant of the mice. Values represent group mean ± SEM. *p < 0.05.
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Fig. 2. Molarless condition promoted NO production in the hippocampus area of the mice (n = 8). In the hippocampus, NO production of extraction groups was significantly higher than sham groups. The hippocampus of the extraction groups released significantly more NO than in the cortex. Values represent group mean ± SEM. **p < 0.001.
Fig. 4. The ratio of expression of iNOS in the hippocampus and cortex showing that the extraction groups expressed more iNOS than sham groups (n = 3). Values represent group mean ± SEM. *p < 0.05, **p < 0.001.
4. Discussion groups) (Fig. 2). The concentration of NO of extraction group in the hippocampus was significantly higher than that of the cortex (t(14) = 9.66, p = 0.0007 of maxillary groups; t(14) = 9.62, p = 0.0004 of mandibular groups). The NO release in the cortex area had no difference between the groups (p = 0.32). 3.4. iNOS inhibition in the hippocampus increased by molarless condition After measuring the expression of iNOS (Fig. 3A and B) using Gelpro Analyzer (Version 4.0), the ratio of expression of iNOS of extraction group in the hippocampus increased (t(4) = 6.88, p = 0.002 of maxillary groups; t(4) = 6.97, p = 0.002 of mandibular groups). And the hippocampus of the extraction groups expressed significantly more iNOS than in the cortex (t(4) = 5.13, p = 0.007 of maxillary groups; t(4) = 4.35, p = 0.012 of mandibular groups) (Fig. 4). The iNOS expression in the cortex area has no difference between the groups (p = 0.72). These results are similar to the NO release. Western blot analysis indicated that molar extraction led to iNOS protein expression mainly in the hippocampus, which the immunohistochemistry results confirmed (Fig. 5). In the hippocampus, compared with the sham group, the number of iNOS positive cells significantly increased (t(6) = 15.45, p = 0.0001 of maxillary groups; t(6) = 9.15, p = 0.0001 of mandibular groups) (Fig. 6).
Fig. 3. Expression of iNOS after molar extraction in the hippocampus (A) and cortex (B) (n = 3). Western blot results showing that molarless condition induced iNOS protein expression in the hippocampus.
KM mouse was used in this study. KM mouse was derived in 1944 from a pair of Swiss mice that were introduced from Hoffline Institution of Hindustan into the providence of Kunming, China, and this type of mice shows strong disease resistance and adaptability, high reproduction rate and survival rate [21]. Also KM mice are less expensive than SAMP. There are several possible underlying biological mechanisms regarding the relationship between loss of molars and cognitive impairments. Diminished sensory input possibly leads to reduced cell growth and development, as seen in animal studies [22,23]. It was reported that loss of teeth could cause a long-term decrease of the neuron activity of the brain and cerebral blood flow [24]. The cholinergic neurotransmitter system appeared to be functionally impaired during loss of teeth [25]. Some stress responses caused by loss of teeth might be associated with increased corticosteroid levels due to disruption of the hypothalamic–pituitary–adrenal axis (HPA-axis), and the concentration of NO and expression of iNOS in the hippocampus significantly increases in the molarless condition of the rat dentate gyrus [26]. It is known that the molecule NO could freely cross cell membranes, and plays a role as a neurotransmitter in the brain. iNOS is not commonly found in healthy tissues in the central nervous system, but it could be expressed after brain insult in astrocytes, neurons, and endothelial cells, where the enzyme triggers the production of high amounts of NO [27]. Overproduction of NO may lead to neuronal damage and death, and the toxic NO produced by iNOS may damage the cells in the hippocampus. Since the function of hippocampus is associated with learning and memory, it might be an explanation why the changes of NO and iNOS were found in the hippocampus instead of in the cortex in this study. The spatial learning and memory impairments reflect the dysfunction of the hippocampus that might attribute to the changes of the contents of NO and iNOS in the hippocampus. It could be through the nerve or blood ways that the tooth extraction involves changes in the brain. Teeth extraction might result in a lower cerebral blood flow, a decrease in sensatory input, or an increase in stress, which could be pathological factors activating the expression of iNOS. After iNOS was activated, the iNOS protein transcription induced and the iNOS expression increased, which might be due to a large number of DNA regulation; within a few weeks, excessive NO was produced while iNOS still remained active [28]. The cytotoxicity of the excessive NO could cause the hippocampus-related nerve neurotransmitter and neuronal
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Fig. 5. The expression of iNOS in the hippocampus CA1 area (100×) (n = 4).
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81371165 and 30801311), and Beijing NOVA Program (No. 2008B60). References
Fig. 6. The positive cells of iNOS in the hippocampus CA1 area of extraction were significantly more than the sham groups (n = 4). Values represent group mean ± SEM. *p < 0.05, **p < 0.001.
damage, and eventually leads to a reduced ability of learning and memory in mice. More studies regarding the mechanisms of the changes of NO and iNOS and their subsequent influences on the cognitive function after the molar extraction are needed. Within the limitations of the study, it is concluded that molar extraction is associated with the behavioral impairment, and that the changes of NO and iNOS in the hippocampus may be involved in the behavioral change in the molarless condition. Conflict of interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
[1] Nordenram G, Ljunggren G, Cederholm T. Nutritional status and chewing capacity in nursing home residents. Aging 2001;13:370–7. [2] Takata Y, Ansai T, Awano S, Sonoki K, Fukuhara M, Wakisaka M, et al. Activities of daily living and chewing ability in an 80-year-old population. Oral Dis 2004;10:365–8. [3] Weijenberg RAF, Scherder EJA, Lobbezoo F. Mastication for the mind: the relationship between mastication and cognition in ageing and dementia. Neurosci Biobehav Rev 2011;35:483–97. [4] Batty GD, Li Q, Huxley R, Zoungas S, Taylor BA, Neal B, et al. Oral disease in relation to future risk of dementia and cognitive decline: prospective cohort study based on the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified-Release Controlled Evaluation (ADVANCE) trial. Eur Psychiatry 2011;28:49–52. [5] Kondo K, Niino M, Shido K. A case control study of Alzheimer’s disease in Japan: significance of life styles. Dement Geriatr Cogn Disord 1994;5:314–26. [6] Gorelick PB, Scuteri A, Black SE, Decarli C, Greenberg SM, Iadecola C, et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the American heart association/American stroke association. Stroke 2011;42:2672–713. [7] Miyake S, Wada-Takahashi S, Honda H, Takahashi SS, Sasaguri K, Sato S, et al. Stress and chewing affect blood flow and oxygen levels in the rat brain. Arch Oral Biol 2012;57:1491–7. [8] Rickman S, Johnson A, Miles C. The impact of chewing gum resistance on immediate free recall. Br J Psychol 2012;104:339–46. [9] Narita N, Kamiya K, Yamamura K, Kawasaki S, Matsumoto T, Tanaka N. Chewing-related prefrontal cortex activation while wearing partial denture prosthesis: pilot study. J Prosthodont Res 2009;53:126–35. [10] Miyamoto I, Yoshida K, Tsuboi Y, Iizuka T. Rehabilitation with dental prosthesis can increase cerebral regional blood volume. Clin Oral Implants Res 2005;16:723–7. [11] Steinert JR, Chernova T, Forsythe ID. Nitric oxide signaling in brain function, dysfunction, and dementia. Neuroscientist 2010;16:435–52. [12] Law A, Gauthier S, Quiriona R. Say NO to Alzheimer’s disease: the putative links between nitric oxide and dementia of the Alzheimer’s type. Brain Res Rev 2001;35:73–96. [13] O’Mahony D, Kendall MJ. Nitric oxide in acute ischaemic stroke: a target for neuroprotection. J Neurol Neurosurg Psychiatry 1999;67:1–3.
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[14] Jiang QS, Liang ZL, Wu MJ, Feng L, Liu LL, Zhang JJ. Reduced brain-derived neurotrophic factor expression in cortex and hippocampus involved in the learning and memory deficit in molarless SAMP8 mice. Chin Med J 2011;124:1540–4. [15] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47–60. [16] Ikonen S, Schmidt BH, Riekkinen Jr P. Characterization of learning and memory behaviors and the effects of metrifonate in the C57BL strain of mice. Eur J Pharmacol 1999;372:117–26. [17] Griess P. Bemerkungen zu der abhandlung der H. H. Weselsky und Benedikt Ueber einige azoverbindungen. Chem Ber 1879;12:426–8. [18] Andoh T, Sakuma Y, Yamamoto S, Matsuno A, Maeda T, Kotani J. Influences of molar loss of rat on learning and memory. J Prosthodont Res 2009;53:155–60. [19] Kubo KY, Iwaku F, Watanabe K, Fujita M, Onozuka M. Molarless-induced changes of spines in hippocampal region of SAMP8 mice. Brain Res 2005;1057:191–5. [20] Watanabe K, Ozono S, Nishiyama K, Saito S, Tonosaki K, Fujita M, et al. The molarless condition in aged SAMP8 mice attenuates hippocampal Fos induction linked to water maze performance. Behav Brain Res 2002;128:19–25. [21] Zhang GM, Yao GH. A survey on the genetic background document of Chinese Kunming mouse (KM mouse). China J Lab Zool 1997;7(4):246–51 [in Chinese].
[22] Tsutsui K, Kaku M, Motokawa M, Tohma Y, Kawata T, Fujita T, et al. Influences of reduced masticatory sensory input from soft-diet feeding upon spatial memory/learning ability in mice. Biomed Res 2007;28:1–7. [23] Yamamoto T, Hirayama A. Effects of soft-diet feeding on synaptic density in the hippocampus and parietal cortex of senescence-accelerated mice. Brain Res 2001;902:255–63. [24] Kato T, Usami T, Noda Y, Hasegawa M, Ueda M, Nabeshima T. The effect of the loss of molar teeth on spatial memory and acetylcholine release from the parietal cortex in aged rats. Behav Brain Res 1997;83: 239–42. [25] Onozuka M, Watanabe K, Fujita M, Tomida M, Ozono S. Changes in the septohippocampal cholinergic system following removal of molar teeth in the aged SAMP8 mouse. Behav Brain Res 2002;133:197–204. [26] Aoki H, Kimoto K, Hori N, Hoshi N, Yamamoto T, Onozuka M. Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus. Neurosci Lett 2010;469:44–8. [27] Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat Rev Drug Discov 2002;1:939–50. [28] Aktan F. iNOS mediated nitric oxide production and its regulation. Life Sci 2004;6:639–53.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
Behavioral impairments and changes of nitric oxide and inducible nitric oxide synthase in the brains of molarless KM mice Qian Pang a , Xingxue Hu b , Xinya Li c , Jianjun Zhang c , Qingsong Jiang a,∗ a b c
Department of Prosthodontics, Beijing Stomatology Hospital and School of Stomatology, Capital Medical University, Beijing 100050, China Advanced Standing Program, Boston University Henry M. Goldman School of Dental Medicine, 100 East Newton Street, Boston, MA 02118, USA Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
h i g h l i g h t s • Molar extraction may result in the impairments of spatial learning and memory of the KM mouse. • The expression of NO and iNOS increases in the hippocampus of the KM mouse after molar extraction. • The changes of NO and iNOS in the hippocampus may be involved in the behavioral changes in the molarless condition.
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 14 October 2014 Accepted 17 October 2014 Available online 28 October 2014 Keywords: Mastication Hippocampus Nitric oxide Inducible nitric oxide synthase
a b s t r a c t More studies showed that as a common disorder in senior population, loss of teeth could adversely affect human cognitive function, and nitric oxide (NO) might play an important role in the cognitive function. However, the underlying mechanism has not yet been well-established. The objectives of this study are to evaluate behavior changes of KM mice after loss of molars, and levels of NO and inducible nitric oxide synthase (iNOS) in the brain in molarless condition. It is hypothesized that loss of molars of the mice tested results in the cognitive impairments and that the process is mediated by NO in the brain through the signaling pathways. Morris water maze is used to test the behavioral changes after 8 weeks of the surgery. The changes of NO and iNOS are evaluated by using Griess assay, western blot, and immunohistochemistry method. The results show that 8 weeks after loss of molars, the spatial learning and memory of KM mice impair and the levels of NO and iNOS in mice hippocampus increase. These findings suggest that molar extraction is associated with the behavioral impairment, and that the changes of NO and iNOS in the hippocampus may be involved in the behavioral changes in the molarless condition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Loss of teeth is one of common disorders in the geriatric population. The elders with long-term loss of teeth may suffer from reduced mastication, inarticulate pronunciation, temporomandibular joint dysfunction, and further appear a poor nutritional status and decreased daily living skills [1,2]. The association between mastication and brain function recently gains more attentions. Animal and human experimental studies confirmed the causal relationship between mastication and cognition function [3], and tooth loss was associated with an increased risk of both
∗ Corresponding author at: Beijing Stomatological Hospital & School of Stomatology, Capital Medical University, No. 4 Tiantan Xili, Beijing 100050, China. Tel.: +86 10 57099309; fax: +86 10 57099310. E-mail address: [email protected] (Q. Jiang). http://dx.doi.org/10.1016/j.bbr.2014.10.020 0166-4328/© 2014 Elsevier B.V. All rights reserved.
dementia and cognitive decline [4]. Alzheimer disease is considered the most common cause of cognitive dysfunction among the aged, and tooth loss might be a risk factor for Alzheimer-type dementia [5]. It was found that the construct of vascular contributions to cognitive impairment and dementia is sufficiently important [6], and that chewing lead to an increased blood flow [7], superior learning and word recalling [8]. It was beneficial for cognitive impairment during the early intervention in that vascular risk factors could be found and easily controlled [6]. Dental prosthesis could significantly stimulate masticatory muscle and dorsal prefrontal cortex activities [9], and increase cerebral regional blood volume [10]. It is known that nitric oxide (NO) involves the aspects of regulating blood flows, transferring neural signals and mediating stress responses. In addition to its vasoactive and immunological properties, NO has significant neurophysiological functions such as long-term potentiation (LTP), long-term depression (LTD), elucidation of calcium-dependent and NMDAR-mediated activation of
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neuronal nitric oxide synthase (nNOS) [11]. Abnormal NO signaling was attributed to a variety of neurodegenerative pathologies such as stroke/excitotoxicity, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease [11,12]. Nitric oxide is an enzymatic product of nitric oxide synthase (NOS). One of the three isoforms of NOS, inducible nitric oxide synthase (iNOS) generates NO and NOderived reactive nitrogen species (e.g. peroxy nitrite). Inducible nitric oxide synthase was involved in the mechanisms of cerebral ischemic insult [13]. However, few studies have been reported regarding the association between the changes of NO and iNOS and the damage of learning and memory caused by the condition of loss of teeth. The objectives of this study were to investigate the behavior changes of KM mice after loss of molars, and levels of nitric oxide and nitric oxide synthase in the brains of the molarless mice. It was hypothesized that loss of molars results in the cognitive impairments of the KM mice tested and that the process is mediated by NO through the signaling pathways. 2. Materials and methods 2.1. Animals Sixty male KM mice (10–11 months old) (Vital River Laboratory Animal Technology Co. Ltd.) were employed. All mice were habituated at least 7 days before the start of experiments. Five mice were housed in every standard polycarbonate cage with free access to food and water and a reversed day/night cycle with lights on at 10:00 P.M. and off at 10:00 A.M. The experiments complied with the guidelines for care and use of laboratory animals from National Institutes of Health (NIH). 2.2. Surgery Mice were randomly divided into 4 groups (15 mice each group): maxillary extraction group (E1), maxillary sham group (S1), mandibular extraction group (E2) and mandibular sham group (S2). The mice were anesthetized via intraperitoneal (i.p.) injection of 10% chloral hydrate (400 mg/kg). In the sham group, small amounts of bilateral maxillary or mandibular alveolar bone were removed with rongeur from the toothless gap region between molars and canines in the superior alveolar ridge. In the molarless group, all the bilateral maxillary or mandibular molars were removed. If there was root fracture, all the tooth structure visible on the gum was removed to eliminate occlusal contacts [14]. After the operation, the mice were allowed free access to routine pelleted diet and water. General conditions and body weights of each mouse were monitored during the entire progress. 2.3. Morris water maze (MWM) The Morris water maze test was performed according to the previous study [15]. The maze consisted of a circular plastic pool with 120 cm in diameter and 60 cm deep, the interior of which was painted black [16]. The plastic escape platform (10 cm in diameter and 28 cm high) was positioned in the pool. The pool was filled with water 1 cm above the platform at 22 ± 1 ◦ C. Behavior of the mice in the pool was recorded by a video camera positioned over the pool. During the whole test, the lighting of the testing room was placed indirectly to the pool and the environment (e.g. experimenter, work table, door, and pipes, etc.) was kept consistent. Eight weeks after operation, the acquisition training session was performed. On the first day, the mice were placed into the pool without the platform one by one, and each mouse swam separately and freely for 60 s. A similar procedure was repeated in a pool with the platform that was fixed with a red flag and placed in the center
of one of the four quadrants of the pool. In this procedure, each mouse was placed into the water at the opposite position to the platform, and they were observed to find the flag in 60 s so as to eliminate the influence of impaired vision on the measurement. In the place navigation test, mice were required to find the hidden platform to examine the spatial learning and memory. The platform was placed in the northwest quadrant of the pool (Quadrant II), which differed from the position set in the training session. Two points of the pool (south and east) were used as the starting positions. The animals were given two trials per day for 5 days. Trials began with the mice placed in the pool facing the side wall at a start position and ended once the animal found the platform; if it did not find the platform within 60 s, the mouse was taken out of the water and placed onto the platform for 10 s. Then the mouse was immediately replaced in the pool at another start position for the next trial. The swim paths, distances, speed and latencies taken to swim to the platform were monitored with a CCD video camera linked to a computer system. The probe test was carried out 24 h after the last trial on Day 5. The starting position was set opposite to the original platform position with the platform removed. Each mouse was placed to face the wall. The time of the mouse first passing the platform and the frequency of passing the platform were recorded in 60 s. 2.4. Griess assay Nitric oxide could be released and quantified using the Griess assay [17]. After finishing the MWM test, the productions of NO were determined by an assay for nitrite. Eight mice of each group were decapitated and the whole brain was removed. Then the cortex and hippocampus area were rapidly separated on the ice plate and weighed. The tissue was grinded, adding nine times with phosphate-buffered saline and 0.3% TritonX100, bounce shocking for 3 s and water bathing for 5 min, then centrifugation (12,000 × g, 5 min) at 4 ◦ C, keeping the culture of the supernatant. The procedure followed the instruction of the total nitric oxide assay kit (Biyotime institute of biotechnology, S0024). The optical density of the assay samples was measured spectrophotometrically at 540 nm. 2.5. Immunohistochemistry Four mice of each group were anesthetized with 10% chloral hydrate (400 mg/kg), and then perfused with phosphate-buffered saline (PBS, pH = 7.4) containing 4% parafomaldehyde in 0.1 M phosphate buffer, following postfixation for 24 h in the same buffer. The tissue was then imbedded into paraffin. Sections with 4 m thickness were prepared on a microslicer and then processed according to a standard immunohistochemical protocol. After rinsed with PBS, the sections were incubated with 30% H2 O2 for 10 min to quench endogenous peroxidase activity and then blocked with 5% BSA for 20 min at room temperature, followed by incubation for 2 days at 4 ◦ C with rabbit anti-iNOS antibody, diluted 1:100. The sections were rinsed in PBS for 2 min and 3 times, and incubated for 20 min at 37 ◦ C with anti-rabbit IgG, and then with ABC reagent in PBS, before being treated for about 10 min at room temperature with diaminobenzidine (DAB). After a final wash with ddH2 O2 , the sections were ready for light microscopy. 2.6. Western blot analysis Following decapitation, the cortex and hippocampus of 3 mice of each group were dissected, weighted, recorded and snap frozen in liquid nitrogen. Proteins were extracted with Applygen total protein extraction kit. Protein concentration was normalized using Coomassie brilliant blue G-250 staining. Equal amounts of proteins were separated by SDS-PAGE on 10% polyacrylamide gel, and the
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proteins were transferred to PVDF membrane. After blocking with 0.1% TBST containing 5% non-fat milk at room temperature for 2 h. Primary antibody (iNOS 2977s, 1:1000, from Cell Signaling, Cell Signaling Technology, CA) was added for overnight incubation. The membrane was rinsed with 0.1% TBST three times, 10 min each, and incubated with secondary antibody (ZB2301, 1:5000, from Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h. Color development was performed using the ECL kit. Images were acquired using the Fuji Digital Science Imager, and analyzed with Gelpro Analyzer (Version 4.0) to measure the integrated optimal density (IOD) values of specific bands. 2.7. Statistical analysis Statistical analysis was performed using SPSS Statistics V17.0 software (SPSS Inc.). Treatment differences in the escape latency in the water maze task were analyzed using a repeated measures ANOVA. One-way ANOVA was used for the probe trials. Other data were evaluated using student’s t tests. The statistical significance level is 0.05. 3. Results 3.1. No differences in body weight between extraction and sham groups After 1 week, there was no difference in body weight or food and water consumption between the extraction groups (E1, E2) and sham groups (S1, S2). Body weights of all the mice increased gradually in the following weeks, yet in the whole process the extraction groups and sham groups did not show a significant difference (p = 0.22). Either the maxillary group or mandibular group showed the similar results. Body weight of the molarless mice decreased after the procedure of extraction but recovered to the preoperative
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levels within 1 week, which is consistent to the previous studies [18,19]. 3.2. Molarless mice exhibit a decreased spatial learning ability in the Morris water maze Before the test, all the mice tested showed similar physical abilities and normal visions. There was a significant main effect of training day on measures of escape latency (F(1,28) = 12.87, p = 0.0001 of maxillary groups; F(1,28) = 19.03, p = 0.0001 of mandibular groups) and of cumulative distance to the platform center (F(1,28) = 9.19, p = 0.0001 of maxillary groups; F(1,28) = 43.45, p = 0.0001 of mandibular groups) (Fig. 1A and B). There was no significant difference of swimming speed in each group (p = 0.49) (Fig. 1C). After training, the hidden platform was removed and the probe test was carried out. The percentage of quadrant II compared to the other quadrants of the mice of extraction group showed lower value than the sham groups (F(1,28) = 10.04, p = 0.005 of maxillary groups; F(1,28) = 10.60, p = 0.004 of mandibular groups) (Fig. 1D). By tracking the trajectory of mice in each group swimming records showed quite different results. The mice of the sham groups found the platform directly. They showed a typical preference for searching in the quadrant II than all the other three quadrants. Yet the behaviors of the mice of extraction groups in the pool showed obviously unpurposed. These results confirmed that the mice of extraction groups had impaired memory for the location of the hidden platform in the water maze, which was similar to the previous studies [20]. 3.3. The release of NO in the hippocampus after molar extraction increased After 8 weeks of molarless condition, the NO concentration in the hippocampus of the extraction group increased (t(14) = 5.28, p = 0.0002 of maxillary groups; t(14) = 3.99, p = 0.0001 of mandibular
Fig. 1. Molarless condition reduced the spatial learning and memory (n = 15). (A). All mice showed improvement in escape latency with continuous days of training, yet the improvement of extraction groups were significantly lower than the corresponding sham groups. (B). The path length of the extraction groups was significantly shorter than the corresponding sham groups. (C). No significant differences were observed in the extraction groups and sham groups as reflected by the swimming speed. (D) Molar extraction treatment significantly decreased the time spent searching for the hidden platform in the target quadrant of the mice. Values represent group mean ± SEM. *p < 0.05.
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Fig. 2. Molarless condition promoted NO production in the hippocampus area of the mice (n = 8). In the hippocampus, NO production of extraction groups was significantly higher than sham groups. The hippocampus of the extraction groups released significantly more NO than in the cortex. Values represent group mean ± SEM. **p < 0.001.
Fig. 4. The ratio of expression of iNOS in the hippocampus and cortex showing that the extraction groups expressed more iNOS than sham groups (n = 3). Values represent group mean ± SEM. *p < 0.05, **p < 0.001.
4. Discussion groups) (Fig. 2). The concentration of NO of extraction group in the hippocampus was significantly higher than that of the cortex (t(14) = 9.66, p = 0.0007 of maxillary groups; t(14) = 9.62, p = 0.0004 of mandibular groups). The NO release in the cortex area had no difference between the groups (p = 0.32). 3.4. iNOS inhibition in the hippocampus increased by molarless condition After measuring the expression of iNOS (Fig. 3A and B) using Gelpro Analyzer (Version 4.0), the ratio of expression of iNOS of extraction group in the hippocampus increased (t(4) = 6.88, p = 0.002 of maxillary groups; t(4) = 6.97, p = 0.002 of mandibular groups). And the hippocampus of the extraction groups expressed significantly more iNOS than in the cortex (t(4) = 5.13, p = 0.007 of maxillary groups; t(4) = 4.35, p = 0.012 of mandibular groups) (Fig. 4). The iNOS expression in the cortex area has no difference between the groups (p = 0.72). These results are similar to the NO release. Western blot analysis indicated that molar extraction led to iNOS protein expression mainly in the hippocampus, which the immunohistochemistry results confirmed (Fig. 5). In the hippocampus, compared with the sham group, the number of iNOS positive cells significantly increased (t(6) = 15.45, p = 0.0001 of maxillary groups; t(6) = 9.15, p = 0.0001 of mandibular groups) (Fig. 6).
Fig. 3. Expression of iNOS after molar extraction in the hippocampus (A) and cortex (B) (n = 3). Western blot results showing that molarless condition induced iNOS protein expression in the hippocampus.
KM mouse was used in this study. KM mouse was derived in 1944 from a pair of Swiss mice that were introduced from Hoffline Institution of Hindustan into the providence of Kunming, China, and this type of mice shows strong disease resistance and adaptability, high reproduction rate and survival rate [21]. Also KM mice are less expensive than SAMP. There are several possible underlying biological mechanisms regarding the relationship between loss of molars and cognitive impairments. Diminished sensory input possibly leads to reduced cell growth and development, as seen in animal studies [22,23]. It was reported that loss of teeth could cause a long-term decrease of the neuron activity of the brain and cerebral blood flow [24]. The cholinergic neurotransmitter system appeared to be functionally impaired during loss of teeth [25]. Some stress responses caused by loss of teeth might be associated with increased corticosteroid levels due to disruption of the hypothalamic–pituitary–adrenal axis (HPA-axis), and the concentration of NO and expression of iNOS in the hippocampus significantly increases in the molarless condition of the rat dentate gyrus [26]. It is known that the molecule NO could freely cross cell membranes, and plays a role as a neurotransmitter in the brain. iNOS is not commonly found in healthy tissues in the central nervous system, but it could be expressed after brain insult in astrocytes, neurons, and endothelial cells, where the enzyme triggers the production of high amounts of NO [27]. Overproduction of NO may lead to neuronal damage and death, and the toxic NO produced by iNOS may damage the cells in the hippocampus. Since the function of hippocampus is associated with learning and memory, it might be an explanation why the changes of NO and iNOS were found in the hippocampus instead of in the cortex in this study. The spatial learning and memory impairments reflect the dysfunction of the hippocampus that might attribute to the changes of the contents of NO and iNOS in the hippocampus. It could be through the nerve or blood ways that the tooth extraction involves changes in the brain. Teeth extraction might result in a lower cerebral blood flow, a decrease in sensatory input, or an increase in stress, which could be pathological factors activating the expression of iNOS. After iNOS was activated, the iNOS protein transcription induced and the iNOS expression increased, which might be due to a large number of DNA regulation; within a few weeks, excessive NO was produced while iNOS still remained active [28]. The cytotoxicity of the excessive NO could cause the hippocampus-related nerve neurotransmitter and neuronal
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Fig. 5. The expression of iNOS in the hippocampus CA1 area (100×) (n = 4).
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81371165 and 30801311), and Beijing NOVA Program (No. 2008B60). References
Fig. 6. The positive cells of iNOS in the hippocampus CA1 area of extraction were significantly more than the sham groups (n = 4). Values represent group mean ± SEM. *p < 0.05, **p < 0.001.
damage, and eventually leads to a reduced ability of learning and memory in mice. More studies regarding the mechanisms of the changes of NO and iNOS and their subsequent influences on the cognitive function after the molar extraction are needed. Within the limitations of the study, it is concluded that molar extraction is associated with the behavioral impairment, and that the changes of NO and iNOS in the hippocampus may be involved in the behavioral change in the molarless condition. Conflict of interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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