archives of oral biology 59 (2014) 749–755
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Up-regulation of PKMz expression in the anterior cingulate cortex following experimental tooth movement in rats Yinzi Xin a,b, Xingyu Liu d, Yang Cao a,b,*, Yu Chen a,b, Chufeng Liu c a
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China c Department of Orthodontics, Guangdong Provincial Stomatological Hospital, Affiliated Stomatological Hospital of Southern Medical University, Guangzhou, China d Department of Orthodontics, Changsha Stomatological Hospital, Changsha, China b
article info
abstract
Article history:
Objective: To explore the involvement of synaptic plasticity in pain induced by experimental
Accepted 8 April 2014
tooth movement, we evaluated the expression of protein kinase M zeta (PKMz), an enzyme
Keywords:
(ACC).
PKMz
Methods: Male Sprague-Dawley rats weighing 250–300 g were used. The change of the
Pain
expression of PKMz in the ACC was measured by western blot, and the mRNA of PKMz
necessary for maintaining long-term potentiation (LTP) in the anterior cingulate cortex
Anterior cingulate cortex
was detected by quantitative real-time PCR 1, 3, 7 days after experimental tooth movement.
Experimental tooth movement
The average time spent on mouth-wiping behaviour of rats involved in pain perception was
ZIP
detected. After that a selective PKMz inhibitor, called myristoylated z-pseudosubstrate inhibitory peptide (ZIP) was injected into ACC, and the effects of ZIP were evaluated. Results: The mouth-wiping behaviour of rats was significantly increased 1, 3, and 7 days after experimental tooth movement. Changes in PKMz levels were not detected on day 1 but were found to be increased 3 days following the tooth movement, and then declined to the baseline 7 days after tooth movement in the ACC. PKMz mRNA levels were not significantly different between the experimental and sham-treated groups at the three time points. Time spent on mouth-wiping behaviour was reduced after ZIP was injected into ACC 3 days after tooth movement, and the analgesic effect last for at least 24 h. Conclusion: PKMz in the ACC acts to maintain the pain induced by experimental tooth movement. Increased expression of PKMz protein is attributed to persistent translation of PKMz mRNA. Synaptic plasticity may be involved in the development of tooth movement pain. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, Guangdong 510060, China. Tel.: +86 020 83866394; fax: +86 020 83822807. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.archoralbio.2014.04.002 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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1.
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Introduction
Pain is an unpleasant sensory and emotional experience associated with tissue damage. Pathological pain or chronic pain causes major suffering among patients, and there are two common associated pathological conditions: allodynia and hyperalgesia. Orthodontic pain, the most cited side effect arising from orthodontic force application, is a major concern for clinicians, patients, and parents. Ninety-five percent of patients treated with fixed appliances reported pain after 24 h. The pain disrupts patients’ daily lives including chewing and sleep, and 8% of a study population even discontinued treatment because of pain.1–3 The mechanism of the pain caused by orthodontic treatment is still not well known. Furstman and Bernick suggested that periodontal pain is caused by a process of pressure, ischaemia, inflammation, and oedema.4 Previous studies indicated that pain caused by orthodontic tooth movement involves changes in blood flow in the periodontium and dental pulp, as well as the activation of inflammatory reactions. Many cytokines such as IL-1, IL-6, TNF contribute to pain perception. It has also been found that neuropeptides such as calcitonin gene-related peptide (CGRP) and galanin (GAL) are increased in PDL during experiment tooth movement.5–9 Further studies suggest that changes occur in mediators and receptors in the trigeminal nerve such as C-fos and P2X3 receptor correlate with orthodontic pain.10,11 Most studies have mostly focused on peripheral nerves, but less is known about what type of cortical changes in pain after tooth movement. Therefore, we hypothesized that the central nervous system is involved in pain signal regulation during tooth movement. In recent years, theories have suggested that the mechanisms underlying synaptic plasticity used for learning and memory may also be responsible for chronic pain. Synaptic plasticity, including long-term potentiation (LTP) and longterm depression (LTD), plays a significant role within the brain.12–19 LTP is a widespread phenomenon exhibited by most excitatory synapses, and it can be divided into two phases: early induction, which triggers potentiation, and maintenance, which commences at least 3 h after the initiating event sustains it over time. Multiple protein kinases have been implicated in LTP induction, such as CaMK II, PI3K, MAPK, PKA, and so on.20,21 However, the mechanism of the maintenance stage is poorly understood. In the past few years, Todd and colleagues have proved that PKMz plays a critical role in late-LTP maintenance and memory consolidation.22–31 PKMz is a constitutively active, atypical isoform of protein kinase C (PKC), and is produced by a unique PKMz mRNA. It is expressed exclusively in neural tissue and is enriched in the forebrain.32 PKMz maintains the late phase of LTP by increasing the number of a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors at postsynaptic sites.33–35 Several cortical areas have been reported to contribute to chronic pain, including the anterior cingulate cortex (ACC), which is a key area for pain-related perception.12 Recent studies from Zhuo Min and colleagues have shown that the protein levels of PKMz in ACC were increased 3 days after nerve injury. Cumulative results suggest that PKMz is
necessary for maintaining LTP in the ACC and maybe responsible for pathological pain.12–15,18,19 Therefore, the present study aims to explore whether synaptic plasticity is involved in experimental tooth movement pain by recording changes in time spent on face-grooming behaviour of mouthwiping and in PKMz expression in the ACC of rats. Effects of a selective cell-permeable PKMz inhibitor ZIP were also investigated to further verify the role of PKMz.
2.
Materials and methods
2.1.
Animals
Male Sprague-Dawley rats (8–10 weeks old) weighing 250–300 g were obtained from the experimental animal centre of Sun Yat-sen University. The animals were housed individually with stable temperature (23–25 8C) and light/dark cycle (12 h/ 12 h). Soft food and water were provided ad libitum. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and steps were taken to minimize the number of animals and their discomfort.
2.2.
Appliance for experimental tooth movement
A fixed, Ni-Ti alloy closed-coil spring appliance was constructed for mesial movement of the maxillary first molar, as described by Ashizawa and Sahara.36 After an intraperitoneal injection of sodium pentobarbital at a dose of 40 mg/kg body wt., the closed-coil spring was hooked between the maxillary first molar and upper incisor through a stainless steel ligature and then cement-wrapped through the stainless steel ligature. A 0.5–1.0 mm groove was produced in the cervical region of the labial and proximal surfaces of the incisor to enhance retention. The appliance provided an initial force of 80 g. The sham-treated rats received the same procedure as the experimental rats, but the springs in their mouths were not activated. In all experimental groups, there was no reactivation of the appliance during the experimental periods. The animals were sacrificed for biochemical studies 1, 3, and 7 days after manipulation as noted.
2.3.
Brain cannulation surgery and microinjection of ZIP
Rats were anaesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg). Scalp was shaved and sterilized with iodophor. The head of rats was restrained in a stereotaxic apparatus (Benchmark, myNeurolab.com, USA) with the incisor bar set at approximately 3.0 mm below horizontal zero to make the skull position flat. An incision was made at midline to expose the skull. Two holes were drilled in the skull at stereotaxic coordinates: AP: 1.7 mm anterior to the bregma and L: 0.6 mm lateral to the midline. Then two guide cannulas (23 gauge, Plastics One Inc., Roanoke, VA, USA) were implanted bilaterally 1.0 mm above the ACC into the holes (V: 1.6 mm ventral to the surface of the skull).37 Dummy inner cannulas (Plastics One Inc., Roanoke, VA, USA) of the same extension were positioned inside the guide cannulas to close
archives of oral biology 59 (2014) 749–755
holes. Rats were allowed to recover for 7 days before undergoing experimental tooth movement manipulation. The PKMz inhibitor ZIP (myr-SIYRRGARRWRKL-OH, Tocris Bioscience) was prepared in saline to a concentration of 10 nmol/mL. Rats were confined in a plastic cone which has a hole above the injection site. After removing the dummy cannula from the guide cannula, a injector cannula (30 gauge, Plastics One Inc., Roanoke, VA, USA) connected to a tubing (PE 10, Plastics One Inc., Roanoke, VA, USA) was extended 1 mm further from the tip of the guide cannula. Microinjection was performed bilaterally using a 5.0 mL glass Hamilton syringe. The injection volume of ZIP or saline was 1.0 mL per side over I min.27 The injector cannula was left in position for additional 1 min to prevent any possible solution backflow through the guide cannula. After the microinjection, the exposed cranium was sealed with bone wax, and the scalp was sutured and treated with antibiotic ointment. Rats were kept in a sterilized transparent plastic cage for observation.
2.4.
Behaviour testing
The face-grooming activity of mouth-wiping has been proved to be a reliable nociceptive response for assessing pain perception of experimental tooth movement in freely moving rodents.38,39 The mouth-wiping behaviour was recorded by a video camera 1, 3, and 7 days after experimental tooth movement between 09:00 and 11:00 h. The behaviour was videotaped for 10 min, starting 20 min after placement in a transparent plastic cage. Each animal was videotaped three times at intervals of 20 min.11 For microinjection rats, mouth wiping behaviour was recorded 2 h prior to injection and 2 h, 24 h after injection respectively. Each rat was videotaped 3 times, each time for 10 min at 10 min intervals. The average time consumed in wiping the mouth was calculated off-line by two independent and blinded observers.
2.5.
Preparation of tissues for western blot
After being anaesthetized by intraperitoneal injection with sodium pentobarbital, the animals were perfused through the aorta for 10 min with 0.9% saline. The anterior fontanelle was marked with a stereotaxic instrument. The brain tissue containing ACC was cut into 1 mm slices by a slicing machine for 3 slices. The ACC region was acquired on coronal sections according to Stereotaxic Coordinates of George and Charles.37 Then, the anterior cingulate cortex was homogenized in RIPA buffer (50 mM pH 7.6 Tris–Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton-100, 0.1% SDS, 1 mM DTT, 0.5% sodium deoxycholate) containing protease inhibitor cocktail. After centrifugation, the supernatants were used for protein quantification by the Coomassie Plus (Bradford) Assay Kit (Pierce, Rockford, USA) and boiling in SDS/sample buffer for 10 min.
2.6.
Western blot analysis
Equal amounts of total protein (20 mg/cm-lane) were subjected to 10% SDS-polyacrylamide gels. The proteins were transferred to polyvinylidene fluoride (PVDF) transfer membrane
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(Pierce, Rockford, USA) and blocked for 2 h in 5% skim milk. The PVDF membranes were incubated overnight at 4 8C with rabbit anti-PKC zeta polyclonal antisera (1:1500, Abcam, Cambridge, UK). Immunostaining for glycera-ldehyde-3-phosphate dehydrogenase GAPDH (1:10,000, Sigma. St. Louis, USA) was performed to normalize protein levels of PKMz concerning protein loading. After washing with TBSN, the membranes were incubated in secondary antibody (1:15,000, Jackson ImmunoResearch, Lancaster, USA) with an HRP-conjugated for 1 h at room temperature. The immunoreactive proteins were visualized by SuperSignal West Dura Chemiluminescent Substrate (Pierce, Rockford, USA). The protein density on the immune-blots was analysed with Image J. Representative bands from these strips are showed in Fig. 2.
2.7.
Real-time PCR analysis
After dissection, total RNA was extracted with Trizol-Reagent (Ambion, Carlsbad, USA) according to the manufacturer’s instruction. Then, the RNA was diluted in sterilized nucleasefree water for quantification with the Nanodrop 2000 (ThermoFisher Scientific, Pittsburgh, USA). The quality of the total RNA was measured by formaldehyde gel electrophoresis and spectrophotometry. Then, total RNA was reverse transcribed at 37 8C for 15 min, heated to 85 8C for 5 s, and then chilled to 4 8C, using the PrimeScript RT Master Mix (TaKaRa, Dalian, China). Nucleotide numbers for PKMz mRNA were the same as in Ref. 32. For amplification of PKCz cDNA, the specific forward primer was F 50 -CCACCTTCGGTAGAGCATAA-30 , and the reverse primer was R 50 -GCGGTAGATGGACTTGTCTT-30 . The glycera-ldehyde-3-phosphate dehydrogenase (GAPDH) primers, F 50 -ACGGCAAGTTCAACGGCACAG-30 , and R 50 -CGCCAGTAGACTCCACGACAT30 were used as a control. Then, the cDNA was quantified with the Roche LightCycler 480 with SYBR Green as the fluorescent dye. The reaction pre-denatured cDNA at 95 8C for 30 s, then comprised a 40-cycles programme of a 5-s hold at 95 8C and a 20-s cool-down to 60 8C. After the reaction, the melting curve was analysed at 65 8C for 15 s.
2.8.
Statistical analysis
SPSS 13.0 was applied for data analysis. Independent-samples t-test and paired-samples t-test were used for comparison. Data were displayed by as Mean values SEM and P < 0.05 was considered statistically significant.
3.
Results
3.1. Directed mouth-wiping behaviour following experimental tooth movement The mouth-wiping behaviour of rats were significantly increased 1 day (n = 10 for each group, independent-samples t test, t18 = 18.418, P < 0.001), 3 days (n = 10 for each group, independent-samples t test, t18 = 10.987, P < 0.001), and 7 days (n = 11 for each group, independent-samples t test, t20 = 8.693, P < 0.001) after experimental tooth movement compared to that of the sham-treated groups (see Fig. 1).
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Fig. 1 – Time spent on mouth-wiping. The mouth-wiping behaviour of rats was significantly increased 1, 3, and 7 days following experimental tooth movement, compared with the sham-treated groups. *P < 0.05. Error bars represent SEMs.
Fig. 4 – The mRNA level of PKMz showed no statistically significant difference between the sham and experimental groups on day 1, day 2, and day 7. Error bars represent SEMs.
3.3. The expression of PKMz mRNA levels following experimental tooth movement
Fig. 2 – Western blots for PKMz in the ACC of rats obtained on day 1, day 3 and day 7 from the sham and experimental groups.
Fig. 3 – Protein levels of PKMz in the ACC of rats from the sham and experimental groups. PKMz expression increased significantly 3 days after experimental tooth movement. *P < 0.05. Error bars represent SEMs.
Data from real-time PCR showed no statistically significant difference in PKMz mRNA levels between the experimental groups and sham-treated groups 1 day (n1 = 10, n2 = 8, independent-samples t test, t16 = 0.930, P > 0.05), 3 days (n = 10 rats for each group, independent-samples t test, t18 = 0.729, P > 0.05) and 7 days (n = 8 rats for each group, independent-samples t test, t14 = 0.873, P > 0.05) after experimental tooth movement (see Fig. 4).
3.4. Effects on behavioural responses after microinjecting ZIP into ACC following experimental tooth movement To further understand the role of increased PKMz in pain, a selective PKMz inhibitor, z-pseudosubstrate inhibitory peptide (ZIP) (10 nmol/mL, 1.0 mL per side) was bilaterally microinjected into the ACC. Bilateral microinjection of ZIP into the ACC produced a significant reduction in mouth-wiping behaviour 2 h after injection on day 3 (n = 7, paired-samples t test, t6 = 8.372, P < 0.001; Fig. 5). On the other hand, saline injection into the control group did not show a reduction in mouthwiping (n = 6, paired-samples t test, t5 = 1.190, P > 0.05; Fig. 5). A significant difference was observed between ZIP group and control group (independent-samples t test, t11 = 9.404,
3.2. Expression changes on protein levels of PKMz following experimental tooth movement PKMz protein levels were unchanged on day 1 (n = 10 for each group, independent-samples t test, t18 = 0.254, P > 0.05) after initiation of experimental tooth movement, but were increased 3 days after tooth movement (n = 10 for each group, independent-samples t test, t18 = 2.636, P < 0.05) compared to sham-treated groups. The protein levels of PKMz declined to baseline 7 days (n = 11 for each group, independent-samples t test, t20 = 0.939, P > 0.05) after the activation of appliance (see Figs. 2 and 3).
Fig. 5 – Time spent on wiping the mouth of rats was significantly reduced by microinjecting ZIP (black) into the ACC 3 days after experimental tooth movement compared to baseline. Microinjection of saline (grey) had no analgesic effect compared to baseline. *P < 0.05. Error bars represent SEMs.
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Fig. 6 – The time difference of mouth-wiping before and after the injection of ZIP and saline. Mouth-wiping behaviour was significantly attenuated by injecting ZIP into ACC on day 3 compared to saline group, whereas no significant reduction was observed on day 1 and 7. *P < 0.05. Error bars represent SEMs.
Fig. 7 – The analgesic effect sustained 2 and 24 h after microinjection of ZIP on day 3. The grey arrow indicated the injection time of ZIP. *P < 0.05. Error bars represent SEMs.
P < 0.001; Fig. 6). However, ZIP did not produce a significant reduction in mouth-wiping on day 1 (n = 6 for each group, independent-samples t test, t10 = 0.741, P > 0.05; Fig. 6), day 7 (n = 7 for each group, independent-samples t test, t12 = 1.164, P > 0.05; Fig. 6) after tooth movement, compared to saline group. To investigate how long the analgesic effect can persist, we also recorded behaviour responses 24 h after microinjection of ZIP on day 3. Time spent on mouth wiping behaviour still remained below the basal level (n = 5, paired-samples t test, t4 = 3.091, P < 0.05; Fig. 7).
4.
Discussion
Face-grooming activity directed towards the irritated area, which in our study is mouth wiping, has proved to be a reliable indication of orofacial pain in rats.39 Previous studies have demonstrated that behavioural responses to pain induced by experimental tooth movement in rats were significantly increased on day 1, 3, 5 and 7, with a peak on day 1.38 Our findings that mouth-wiping behaviour of rats was significantly
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increased 1, 3, and 7 days after experimental tooth movement coincides with former research. Clinically, pain and discomfort were perceived by patient from day 1 to day 7 or even longer, and with a peak on first two days, which correlated with our findings.1–3,40 Zhuo Min and colleagues showed that protein levels of PKMz in the ACC are markedly increased 3 days and declined to baseline 14 days following peripheral nerve injury.19 The present study indicates that protein levels of PKMz in the ACC were increased 3 days after initiation of experimental tooth movement, but the protein levels of PKMz in the experimental groups were unchanged 1 day or 7 days after orthodontic force was applied, which is similar to the studies by Zhuo Min et al. Our results suggest that PKMz play a important role in pain maintenance induced by tooth movement. PKMz is an atypical isoform of PKC and has been identified as an LTP-specific plasticity-related protein. It is necessary and sufficient for LTP maintenance.22,31 PKMz enhances synaptic transmission by persistently increasing the number of AMPA receptors at active postsynaptic sites.33–35 Thus, up-regulation of the amount of PKMz also reveals that long-term synaptic plasticity in the ACC may be involved in chronic pain induced by experimental tooth movement. In our study, face-grooming activities persisted on day 1 while no marked change of PKMz was observed. We presume that there is a relationship between the emergence of pain-related substances and objectives’ perception of orthodontic pain. Previously studied substances like c-fos, NMDAR1 may predominate in the early stage of pain, that is, they may work on an acute state of orthodontic pain. NMDA receptor antagonists have been found to block the initial encoding but not the maintenance of memory.41 Compared with the control group, the application of an NMDA receptor antagonist, MK-801, 1 day after the orthodontic force applied dramatically reduced the pain behaviour caused by experimental tooth movement.38 PKMz, on the other hand, probably comes into effect in a later stage as a mechanism of LTP maintenance and transition to a chronic pain state. During the induction of long-term potentiation, the activation of N-methyl-D-aspartate (NMDA) receptors and the subsequent influx of calcium, trigger a cascade of second messengers. Those second messengers activate calcium-dependent protein kinases which releases inhibition for PKMz translation, then PKMz is formed in large quantities and maintains the late phase of LTP.21,31,32 Another possible interpretation of the discrepancy between behavioural responses and PKMz changes may be attributed to the different properties of pain. In human imaging studies, ACC is closely related to the affective features of pain, such as subjective feelings of unpleasantness, which is distinct from the simple sensory dimensions of pain.42 Thus, it is reasonable that PKMz in the ACC was not increased immediately following experimental tooth movement. This assumption requires future research. The role of PKMz in pain maintenance is further verified by microinjection of the myristoylated z-pseudosubstrate inhibitory peptide (ZIP), which potently and selectively inhibits PKMz by reconstituting auto inhibition of the absent PKMz regulatory domain and reverses the late phase of LTP.22,25–27,29 In our study, bilateral microinjection of ZIP exerted an
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analgesic effect on day 3, not on day 1, which correlates with development of PKMz expression. However, the effect of ZIP persisted 24 h after experimental tooth movement, which is different from Min Zhuo’s results.19 The inconsistency may be due to differences of animal species and doses, as the effect of ZIP has been reported to be dose dependent.29 The fact that protein levels of PKMz returned to baseline 7 days after experimental tooth movement whereas the perception of pain persisted, may be due to the increase of phosphorylated PKMz. Immediately after translation, PKMz has low levels of activity until it binds to phosphoinositidedependent protein kinase 1 (PDK1). Subsequently, PKMz is constitutively phosphorylated and converted into its maximally active state for the maintenance of LTP.24 Zhuo Min and colleagues demonstrated that the level of p-PKMz in the ACC remained elevated 7 and 14 days after peripheral nerve injury.19 Upon further investigation, we will determine whether levels of p-PKMz change after experimental tooth movement. PKMz is produced by a unique PKMz mRNA, which is generated by an internal promoter within the PKCz gene and then transported to dendrites of neuronal dendrites. However, under basal conditions, translation is inhibited by PIN (protein interacting with NIMA1). During LTP induction, CaMK II, PI3K, MAPK, PKA, mTOR and actin filament are formed, after which the translational block on PKMz is released and the efficiency of PKMz translation may be persistently increased to maintain the late stage of LTP.43–45 Todd and colleagues demonstrated that PKMz initiated a positive feedback loop through the inhibition of PIN1 to continuously increase translation, which subsequently increases PKMz levels.46 In our study, although the protein level of PKMz was increased 3 days after orthodontic force applied, the level of PKMz mRNA did not changed 1 day, 3 and 7 days after experimental tooth movement. Therefore, this suggests that the amount of PKMz may be increased through persistent translation of preexisting PKMz mRNA not through the transcription of new PKMz mRNA. This is the only way rapid de novo protein synthesis can support long-lasting forms of LTP and synaptic efficacy. This is similar to the results from Kelly and colleagues.23,47,48 In conclusion, synaptic plasticity may be the main mechanism behind chronic pain and PKMz in the ACC may play a role in maintain the pain attributed to experimental tooth movement. Up-regulation of PKMz can be attributed to the continuous translation of pre-existing mRNA. Therefore, PKMz in the ACC has been revealed as a new target in understanding the central mechanisms behind orthodontic pain.
Funding The research was supported by the NSFC (National Natural Science Foundation of China) (www.nsfc.gov.cn) (No. 81170990) and Guangdong Provincial Science & Technology Projects (http://www.gdstc.gov.cn/) (No. 2011B090400097). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests None declared.
Ethical approval All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Sun Yatsen University (IACUC-2011-0906).
Acknowledgements We sincerely thank Dr. Todd Sacktor for the antisera and advice.
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mRNA encoding an independent protein kinase C zeta catalytic domain. Implications for the molecular mechanism of memory. J Biol Chem 2003;278(41):40305–16. Ling DS, Benardo LS, Sacktor TC. Protein kinase Mzeta enhances excitatory synaptic transmission by increasing the number of active postsynaptic AMPA receptors. Hippocampus 2006;16(5):443–52. Yao Y, Kelly MT, Sajikumar S, Serrano P, Tian D, Bergold PJ, et al. PKM zeta maintains late long-term potentiation by Nethylmaleimide-sensitive factor/GluR2-dependent trafficking of postsynaptic AMPA receptors. J Neurosci 2008;28(31):7820–7. Migues PV, Hardt O, Wu DC, Gamache K, Sacktor TC, Wang YT, et al. PKMzeta maintains memories by regulating GluR2dependent AMPA receptor trafficking. Nat Neurosci 2010;13(5):630–4. Ashizawa Y, Sahara N. Quantitative evaluation of newly formed bone in the alveolar wall surrounding the root during the initial stage of experimental tooth movement in the rat. Arch Oral Biol 1998;43(6):473–84. George P, Charles W. The rat brain in stereotaxic coordinates. 6th ed. London: Academic Press; 2007. Yang Z, Luo W, Hou J, Zhao Z, Jian F, Wamalwa P, et al. Development of a behavior model of pain induced by experimental tooth movement in rats. Eur J Oral Sci 2009;117(4):380–4. Vos BP, Hans G, Adriaensen H. Behavioral assessment of facial pain in rats: face grooming patterns after painful and non-painful sensory disturbances in the territory of the rat’s infraorbital nerve. Pain 1998;76:173–8. Erdinc AM, Dincer B. Perception of pain during orthodontic treatment with fixed appliances. Eur J Orthod 2004;26(1): 79–85. Day M, Langston R, Morris RG. Glutamate-receptormediated encoding and retrieval of paired-associate learning. Nature 2003;424(6945):205–9. Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC. Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science 1997;277(5328):968–71. Muslimov IA, Nimmrich V, Hernandez AI, Tcherepanov A, Sacktor TC, Tiedge H. Dendritic transport and localization of protein kinase Mzeta mRNA: implications for molecular memory consolidation. J Biol Chem 2004;279(50):52613–22. Kelly MT, Yao Y, Sondhi R, Sacktor TC. Actin polymerization regulates the synthesis of PKMzeta in LTP. Neuropharmacology 2007;52(1):41–5. Sacktor TC. PINing for things past. Sci Signal 2010;3 (114):er1. Westmark PR, Westmark CJ, Wang S, Levenson J, O’Riordan KJ, Burger C, et al. Pin1 and PKMzeta sequentially control dendritic protein synthesis. Sci Signal 2010;3(112):a18. Kelleher RR, Govindarajan A, Tonegawa S. Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron 2004;44(1):59–73. Lin YT, Huang CC, Hsu KS. Oxytocin promotes long-term potentiation by enhancing epidermal growth factor receptor-mediated local translation of protein kinase Mzeta. J Neurosci 2012;32(44):15476–88.
Available online at www.sciencedirect.com
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Up-regulation of PKMz expression in the anterior cingulate cortex following experimental tooth movement in rats Yinzi Xin a,b, Xingyu Liu d, Yang Cao a,b,*, Yu Chen a,b, Chufeng Liu c a
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China c Department of Orthodontics, Guangdong Provincial Stomatological Hospital, Affiliated Stomatological Hospital of Southern Medical University, Guangzhou, China d Department of Orthodontics, Changsha Stomatological Hospital, Changsha, China b
article info
abstract
Article history:
Objective: To explore the involvement of synaptic plasticity in pain induced by experimental
Accepted 8 April 2014
tooth movement, we evaluated the expression of protein kinase M zeta (PKMz), an enzyme
Keywords:
(ACC).
PKMz
Methods: Male Sprague-Dawley rats weighing 250–300 g were used. The change of the
Pain
expression of PKMz in the ACC was measured by western blot, and the mRNA of PKMz
necessary for maintaining long-term potentiation (LTP) in the anterior cingulate cortex
Anterior cingulate cortex
was detected by quantitative real-time PCR 1, 3, 7 days after experimental tooth movement.
Experimental tooth movement
The average time spent on mouth-wiping behaviour of rats involved in pain perception was
ZIP
detected. After that a selective PKMz inhibitor, called myristoylated z-pseudosubstrate inhibitory peptide (ZIP) was injected into ACC, and the effects of ZIP were evaluated. Results: The mouth-wiping behaviour of rats was significantly increased 1, 3, and 7 days after experimental tooth movement. Changes in PKMz levels were not detected on day 1 but were found to be increased 3 days following the tooth movement, and then declined to the baseline 7 days after tooth movement in the ACC. PKMz mRNA levels were not significantly different between the experimental and sham-treated groups at the three time points. Time spent on mouth-wiping behaviour was reduced after ZIP was injected into ACC 3 days after tooth movement, and the analgesic effect last for at least 24 h. Conclusion: PKMz in the ACC acts to maintain the pain induced by experimental tooth movement. Increased expression of PKMz protein is attributed to persistent translation of PKMz mRNA. Synaptic plasticity may be involved in the development of tooth movement pain. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Orthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, Guangdong 510060, China. Tel.: +86 020 83866394; fax: +86 020 83822807. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.archoralbio.2014.04.002 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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1.
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Introduction
Pain is an unpleasant sensory and emotional experience associated with tissue damage. Pathological pain or chronic pain causes major suffering among patients, and there are two common associated pathological conditions: allodynia and hyperalgesia. Orthodontic pain, the most cited side effect arising from orthodontic force application, is a major concern for clinicians, patients, and parents. Ninety-five percent of patients treated with fixed appliances reported pain after 24 h. The pain disrupts patients’ daily lives including chewing and sleep, and 8% of a study population even discontinued treatment because of pain.1–3 The mechanism of the pain caused by orthodontic treatment is still not well known. Furstman and Bernick suggested that periodontal pain is caused by a process of pressure, ischaemia, inflammation, and oedema.4 Previous studies indicated that pain caused by orthodontic tooth movement involves changes in blood flow in the periodontium and dental pulp, as well as the activation of inflammatory reactions. Many cytokines such as IL-1, IL-6, TNF contribute to pain perception. It has also been found that neuropeptides such as calcitonin gene-related peptide (CGRP) and galanin (GAL) are increased in PDL during experiment tooth movement.5–9 Further studies suggest that changes occur in mediators and receptors in the trigeminal nerve such as C-fos and P2X3 receptor correlate with orthodontic pain.10,11 Most studies have mostly focused on peripheral nerves, but less is known about what type of cortical changes in pain after tooth movement. Therefore, we hypothesized that the central nervous system is involved in pain signal regulation during tooth movement. In recent years, theories have suggested that the mechanisms underlying synaptic plasticity used for learning and memory may also be responsible for chronic pain. Synaptic plasticity, including long-term potentiation (LTP) and longterm depression (LTD), plays a significant role within the brain.12–19 LTP is a widespread phenomenon exhibited by most excitatory synapses, and it can be divided into two phases: early induction, which triggers potentiation, and maintenance, which commences at least 3 h after the initiating event sustains it over time. Multiple protein kinases have been implicated in LTP induction, such as CaMK II, PI3K, MAPK, PKA, and so on.20,21 However, the mechanism of the maintenance stage is poorly understood. In the past few years, Todd and colleagues have proved that PKMz plays a critical role in late-LTP maintenance and memory consolidation.22–31 PKMz is a constitutively active, atypical isoform of protein kinase C (PKC), and is produced by a unique PKMz mRNA. It is expressed exclusively in neural tissue and is enriched in the forebrain.32 PKMz maintains the late phase of LTP by increasing the number of a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors at postsynaptic sites.33–35 Several cortical areas have been reported to contribute to chronic pain, including the anterior cingulate cortex (ACC), which is a key area for pain-related perception.12 Recent studies from Zhuo Min and colleagues have shown that the protein levels of PKMz in ACC were increased 3 days after nerve injury. Cumulative results suggest that PKMz is
necessary for maintaining LTP in the ACC and maybe responsible for pathological pain.12–15,18,19 Therefore, the present study aims to explore whether synaptic plasticity is involved in experimental tooth movement pain by recording changes in time spent on face-grooming behaviour of mouthwiping and in PKMz expression in the ACC of rats. Effects of a selective cell-permeable PKMz inhibitor ZIP were also investigated to further verify the role of PKMz.
2.
Materials and methods
2.1.
Animals
Male Sprague-Dawley rats (8–10 weeks old) weighing 250–300 g were obtained from the experimental animal centre of Sun Yat-sen University. The animals were housed individually with stable temperature (23–25 8C) and light/dark cycle (12 h/ 12 h). Soft food and water were provided ad libitum. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and steps were taken to minimize the number of animals and their discomfort.
2.2.
Appliance for experimental tooth movement
A fixed, Ni-Ti alloy closed-coil spring appliance was constructed for mesial movement of the maxillary first molar, as described by Ashizawa and Sahara.36 After an intraperitoneal injection of sodium pentobarbital at a dose of 40 mg/kg body wt., the closed-coil spring was hooked between the maxillary first molar and upper incisor through a stainless steel ligature and then cement-wrapped through the stainless steel ligature. A 0.5–1.0 mm groove was produced in the cervical region of the labial and proximal surfaces of the incisor to enhance retention. The appliance provided an initial force of 80 g. The sham-treated rats received the same procedure as the experimental rats, but the springs in their mouths were not activated. In all experimental groups, there was no reactivation of the appliance during the experimental periods. The animals were sacrificed for biochemical studies 1, 3, and 7 days after manipulation as noted.
2.3.
Brain cannulation surgery and microinjection of ZIP
Rats were anaesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg). Scalp was shaved and sterilized with iodophor. The head of rats was restrained in a stereotaxic apparatus (Benchmark, myNeurolab.com, USA) with the incisor bar set at approximately 3.0 mm below horizontal zero to make the skull position flat. An incision was made at midline to expose the skull. Two holes were drilled in the skull at stereotaxic coordinates: AP: 1.7 mm anterior to the bregma and L: 0.6 mm lateral to the midline. Then two guide cannulas (23 gauge, Plastics One Inc., Roanoke, VA, USA) were implanted bilaterally 1.0 mm above the ACC into the holes (V: 1.6 mm ventral to the surface of the skull).37 Dummy inner cannulas (Plastics One Inc., Roanoke, VA, USA) of the same extension were positioned inside the guide cannulas to close
archives of oral biology 59 (2014) 749–755
holes. Rats were allowed to recover for 7 days before undergoing experimental tooth movement manipulation. The PKMz inhibitor ZIP (myr-SIYRRGARRWRKL-OH, Tocris Bioscience) was prepared in saline to a concentration of 10 nmol/mL. Rats were confined in a plastic cone which has a hole above the injection site. After removing the dummy cannula from the guide cannula, a injector cannula (30 gauge, Plastics One Inc., Roanoke, VA, USA) connected to a tubing (PE 10, Plastics One Inc., Roanoke, VA, USA) was extended 1 mm further from the tip of the guide cannula. Microinjection was performed bilaterally using a 5.0 mL glass Hamilton syringe. The injection volume of ZIP or saline was 1.0 mL per side over I min.27 The injector cannula was left in position for additional 1 min to prevent any possible solution backflow through the guide cannula. After the microinjection, the exposed cranium was sealed with bone wax, and the scalp was sutured and treated with antibiotic ointment. Rats were kept in a sterilized transparent plastic cage for observation.
2.4.
Behaviour testing
The face-grooming activity of mouth-wiping has been proved to be a reliable nociceptive response for assessing pain perception of experimental tooth movement in freely moving rodents.38,39 The mouth-wiping behaviour was recorded by a video camera 1, 3, and 7 days after experimental tooth movement between 09:00 and 11:00 h. The behaviour was videotaped for 10 min, starting 20 min after placement in a transparent plastic cage. Each animal was videotaped three times at intervals of 20 min.11 For microinjection rats, mouth wiping behaviour was recorded 2 h prior to injection and 2 h, 24 h after injection respectively. Each rat was videotaped 3 times, each time for 10 min at 10 min intervals. The average time consumed in wiping the mouth was calculated off-line by two independent and blinded observers.
2.5.
Preparation of tissues for western blot
After being anaesthetized by intraperitoneal injection with sodium pentobarbital, the animals were perfused through the aorta for 10 min with 0.9% saline. The anterior fontanelle was marked with a stereotaxic instrument. The brain tissue containing ACC was cut into 1 mm slices by a slicing machine for 3 slices. The ACC region was acquired on coronal sections according to Stereotaxic Coordinates of George and Charles.37 Then, the anterior cingulate cortex was homogenized in RIPA buffer (50 mM pH 7.6 Tris–Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton-100, 0.1% SDS, 1 mM DTT, 0.5% sodium deoxycholate) containing protease inhibitor cocktail. After centrifugation, the supernatants were used for protein quantification by the Coomassie Plus (Bradford) Assay Kit (Pierce, Rockford, USA) and boiling in SDS/sample buffer for 10 min.
2.6.
Western blot analysis
Equal amounts of total protein (20 mg/cm-lane) were subjected to 10% SDS-polyacrylamide gels. The proteins were transferred to polyvinylidene fluoride (PVDF) transfer membrane
751
(Pierce, Rockford, USA) and blocked for 2 h in 5% skim milk. The PVDF membranes were incubated overnight at 4 8C with rabbit anti-PKC zeta polyclonal antisera (1:1500, Abcam, Cambridge, UK). Immunostaining for glycera-ldehyde-3-phosphate dehydrogenase GAPDH (1:10,000, Sigma. St. Louis, USA) was performed to normalize protein levels of PKMz concerning protein loading. After washing with TBSN, the membranes were incubated in secondary antibody (1:15,000, Jackson ImmunoResearch, Lancaster, USA) with an HRP-conjugated for 1 h at room temperature. The immunoreactive proteins were visualized by SuperSignal West Dura Chemiluminescent Substrate (Pierce, Rockford, USA). The protein density on the immune-blots was analysed with Image J. Representative bands from these strips are showed in Fig. 2.
2.7.
Real-time PCR analysis
After dissection, total RNA was extracted with Trizol-Reagent (Ambion, Carlsbad, USA) according to the manufacturer’s instruction. Then, the RNA was diluted in sterilized nucleasefree water for quantification with the Nanodrop 2000 (ThermoFisher Scientific, Pittsburgh, USA). The quality of the total RNA was measured by formaldehyde gel electrophoresis and spectrophotometry. Then, total RNA was reverse transcribed at 37 8C for 15 min, heated to 85 8C for 5 s, and then chilled to 4 8C, using the PrimeScript RT Master Mix (TaKaRa, Dalian, China). Nucleotide numbers for PKMz mRNA were the same as in Ref. 32. For amplification of PKCz cDNA, the specific forward primer was F 50 -CCACCTTCGGTAGAGCATAA-30 , and the reverse primer was R 50 -GCGGTAGATGGACTTGTCTT-30 . The glycera-ldehyde-3-phosphate dehydrogenase (GAPDH) primers, F 50 -ACGGCAAGTTCAACGGCACAG-30 , and R 50 -CGCCAGTAGACTCCACGACAT30 were used as a control. Then, the cDNA was quantified with the Roche LightCycler 480 with SYBR Green as the fluorescent dye. The reaction pre-denatured cDNA at 95 8C for 30 s, then comprised a 40-cycles programme of a 5-s hold at 95 8C and a 20-s cool-down to 60 8C. After the reaction, the melting curve was analysed at 65 8C for 15 s.
2.8.
Statistical analysis
SPSS 13.0 was applied for data analysis. Independent-samples t-test and paired-samples t-test were used for comparison. Data were displayed by as Mean values SEM and P < 0.05 was considered statistically significant.
3.
Results
3.1. Directed mouth-wiping behaviour following experimental tooth movement The mouth-wiping behaviour of rats were significantly increased 1 day (n = 10 for each group, independent-samples t test, t18 = 18.418, P < 0.001), 3 days (n = 10 for each group, independent-samples t test, t18 = 10.987, P < 0.001), and 7 days (n = 11 for each group, independent-samples t test, t20 = 8.693, P < 0.001) after experimental tooth movement compared to that of the sham-treated groups (see Fig. 1).
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Fig. 1 – Time spent on mouth-wiping. The mouth-wiping behaviour of rats was significantly increased 1, 3, and 7 days following experimental tooth movement, compared with the sham-treated groups. *P < 0.05. Error bars represent SEMs.
Fig. 4 – The mRNA level of PKMz showed no statistically significant difference between the sham and experimental groups on day 1, day 2, and day 7. Error bars represent SEMs.
3.3. The expression of PKMz mRNA levels following experimental tooth movement
Fig. 2 – Western blots for PKMz in the ACC of rats obtained on day 1, day 3 and day 7 from the sham and experimental groups.
Fig. 3 – Protein levels of PKMz in the ACC of rats from the sham and experimental groups. PKMz expression increased significantly 3 days after experimental tooth movement. *P < 0.05. Error bars represent SEMs.
Data from real-time PCR showed no statistically significant difference in PKMz mRNA levels between the experimental groups and sham-treated groups 1 day (n1 = 10, n2 = 8, independent-samples t test, t16 = 0.930, P > 0.05), 3 days (n = 10 rats for each group, independent-samples t test, t18 = 0.729, P > 0.05) and 7 days (n = 8 rats for each group, independent-samples t test, t14 = 0.873, P > 0.05) after experimental tooth movement (see Fig. 4).
3.4. Effects on behavioural responses after microinjecting ZIP into ACC following experimental tooth movement To further understand the role of increased PKMz in pain, a selective PKMz inhibitor, z-pseudosubstrate inhibitory peptide (ZIP) (10 nmol/mL, 1.0 mL per side) was bilaterally microinjected into the ACC. Bilateral microinjection of ZIP into the ACC produced a significant reduction in mouth-wiping behaviour 2 h after injection on day 3 (n = 7, paired-samples t test, t6 = 8.372, P < 0.001; Fig. 5). On the other hand, saline injection into the control group did not show a reduction in mouthwiping (n = 6, paired-samples t test, t5 = 1.190, P > 0.05; Fig. 5). A significant difference was observed between ZIP group and control group (independent-samples t test, t11 = 9.404,
3.2. Expression changes on protein levels of PKMz following experimental tooth movement PKMz protein levels were unchanged on day 1 (n = 10 for each group, independent-samples t test, t18 = 0.254, P > 0.05) after initiation of experimental tooth movement, but were increased 3 days after tooth movement (n = 10 for each group, independent-samples t test, t18 = 2.636, P < 0.05) compared to sham-treated groups. The protein levels of PKMz declined to baseline 7 days (n = 11 for each group, independent-samples t test, t20 = 0.939, P > 0.05) after the activation of appliance (see Figs. 2 and 3).
Fig. 5 – Time spent on wiping the mouth of rats was significantly reduced by microinjecting ZIP (black) into the ACC 3 days after experimental tooth movement compared to baseline. Microinjection of saline (grey) had no analgesic effect compared to baseline. *P < 0.05. Error bars represent SEMs.
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Fig. 6 – The time difference of mouth-wiping before and after the injection of ZIP and saline. Mouth-wiping behaviour was significantly attenuated by injecting ZIP into ACC on day 3 compared to saline group, whereas no significant reduction was observed on day 1 and 7. *P < 0.05. Error bars represent SEMs.
Fig. 7 – The analgesic effect sustained 2 and 24 h after microinjection of ZIP on day 3. The grey arrow indicated the injection time of ZIP. *P < 0.05. Error bars represent SEMs.
P < 0.001; Fig. 6). However, ZIP did not produce a significant reduction in mouth-wiping on day 1 (n = 6 for each group, independent-samples t test, t10 = 0.741, P > 0.05; Fig. 6), day 7 (n = 7 for each group, independent-samples t test, t12 = 1.164, P > 0.05; Fig. 6) after tooth movement, compared to saline group. To investigate how long the analgesic effect can persist, we also recorded behaviour responses 24 h after microinjection of ZIP on day 3. Time spent on mouth wiping behaviour still remained below the basal level (n = 5, paired-samples t test, t4 = 3.091, P < 0.05; Fig. 7).
4.
Discussion
Face-grooming activity directed towards the irritated area, which in our study is mouth wiping, has proved to be a reliable indication of orofacial pain in rats.39 Previous studies have demonstrated that behavioural responses to pain induced by experimental tooth movement in rats were significantly increased on day 1, 3, 5 and 7, with a peak on day 1.38 Our findings that mouth-wiping behaviour of rats was significantly
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increased 1, 3, and 7 days after experimental tooth movement coincides with former research. Clinically, pain and discomfort were perceived by patient from day 1 to day 7 or even longer, and with a peak on first two days, which correlated with our findings.1–3,40 Zhuo Min and colleagues showed that protein levels of PKMz in the ACC are markedly increased 3 days and declined to baseline 14 days following peripheral nerve injury.19 The present study indicates that protein levels of PKMz in the ACC were increased 3 days after initiation of experimental tooth movement, but the protein levels of PKMz in the experimental groups were unchanged 1 day or 7 days after orthodontic force was applied, which is similar to the studies by Zhuo Min et al. Our results suggest that PKMz play a important role in pain maintenance induced by tooth movement. PKMz is an atypical isoform of PKC and has been identified as an LTP-specific plasticity-related protein. It is necessary and sufficient for LTP maintenance.22,31 PKMz enhances synaptic transmission by persistently increasing the number of AMPA receptors at active postsynaptic sites.33–35 Thus, up-regulation of the amount of PKMz also reveals that long-term synaptic plasticity in the ACC may be involved in chronic pain induced by experimental tooth movement. In our study, face-grooming activities persisted on day 1 while no marked change of PKMz was observed. We presume that there is a relationship between the emergence of pain-related substances and objectives’ perception of orthodontic pain. Previously studied substances like c-fos, NMDAR1 may predominate in the early stage of pain, that is, they may work on an acute state of orthodontic pain. NMDA receptor antagonists have been found to block the initial encoding but not the maintenance of memory.41 Compared with the control group, the application of an NMDA receptor antagonist, MK-801, 1 day after the orthodontic force applied dramatically reduced the pain behaviour caused by experimental tooth movement.38 PKMz, on the other hand, probably comes into effect in a later stage as a mechanism of LTP maintenance and transition to a chronic pain state. During the induction of long-term potentiation, the activation of N-methyl-D-aspartate (NMDA) receptors and the subsequent influx of calcium, trigger a cascade of second messengers. Those second messengers activate calcium-dependent protein kinases which releases inhibition for PKMz translation, then PKMz is formed in large quantities and maintains the late phase of LTP.21,31,32 Another possible interpretation of the discrepancy between behavioural responses and PKMz changes may be attributed to the different properties of pain. In human imaging studies, ACC is closely related to the affective features of pain, such as subjective feelings of unpleasantness, which is distinct from the simple sensory dimensions of pain.42 Thus, it is reasonable that PKMz in the ACC was not increased immediately following experimental tooth movement. This assumption requires future research. The role of PKMz in pain maintenance is further verified by microinjection of the myristoylated z-pseudosubstrate inhibitory peptide (ZIP), which potently and selectively inhibits PKMz by reconstituting auto inhibition of the absent PKMz regulatory domain and reverses the late phase of LTP.22,25–27,29 In our study, bilateral microinjection of ZIP exerted an
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analgesic effect on day 3, not on day 1, which correlates with development of PKMz expression. However, the effect of ZIP persisted 24 h after experimental tooth movement, which is different from Min Zhuo’s results.19 The inconsistency may be due to differences of animal species and doses, as the effect of ZIP has been reported to be dose dependent.29 The fact that protein levels of PKMz returned to baseline 7 days after experimental tooth movement whereas the perception of pain persisted, may be due to the increase of phosphorylated PKMz. Immediately after translation, PKMz has low levels of activity until it binds to phosphoinositidedependent protein kinase 1 (PDK1). Subsequently, PKMz is constitutively phosphorylated and converted into its maximally active state for the maintenance of LTP.24 Zhuo Min and colleagues demonstrated that the level of p-PKMz in the ACC remained elevated 7 and 14 days after peripheral nerve injury.19 Upon further investigation, we will determine whether levels of p-PKMz change after experimental tooth movement. PKMz is produced by a unique PKMz mRNA, which is generated by an internal promoter within the PKCz gene and then transported to dendrites of neuronal dendrites. However, under basal conditions, translation is inhibited by PIN (protein interacting with NIMA1). During LTP induction, CaMK II, PI3K, MAPK, PKA, mTOR and actin filament are formed, after which the translational block on PKMz is released and the efficiency of PKMz translation may be persistently increased to maintain the late stage of LTP.43–45 Todd and colleagues demonstrated that PKMz initiated a positive feedback loop through the inhibition of PIN1 to continuously increase translation, which subsequently increases PKMz levels.46 In our study, although the protein level of PKMz was increased 3 days after orthodontic force applied, the level of PKMz mRNA did not changed 1 day, 3 and 7 days after experimental tooth movement. Therefore, this suggests that the amount of PKMz may be increased through persistent translation of preexisting PKMz mRNA not through the transcription of new PKMz mRNA. This is the only way rapid de novo protein synthesis can support long-lasting forms of LTP and synaptic efficacy. This is similar to the results from Kelly and colleagues.23,47,48 In conclusion, synaptic plasticity may be the main mechanism behind chronic pain and PKMz in the ACC may play a role in maintain the pain attributed to experimental tooth movement. Up-regulation of PKMz can be attributed to the continuous translation of pre-existing mRNA. Therefore, PKMz in the ACC has been revealed as a new target in understanding the central mechanisms behind orthodontic pain.
Funding The research was supported by the NSFC (National Natural Science Foundation of China) (www.nsfc.gov.cn) (No. 81170990) and Guangdong Provincial Science & Technology Projects (http://www.gdstc.gov.cn/) (No. 2011B090400097). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests None declared.
Ethical approval All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Sun Yatsen University (IACUC-2011-0906).
Acknowledgements We sincerely thank Dr. Todd Sacktor for the antisera and advice.
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