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calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy
Neuroscience 256 (2014) 302–308
THE EFFECTS OF INTRAGANGLIONIC INJECTION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II INHIBITORS ON PAIN-RELATED BEHAVIOR IN DIABETIC NEUROPATHY A. JELICIC KADIC, M. BORIC, S. KOSTIC, D. SAPUNAR AND L. PULJAK *
2005). Thus, more studies are necessary about potential treatments and treatment modalities for neuropathic pain. Many drugs used to treat neuropathic pain are delivered systematically or intrathecally, which results in a number of adverse events. An alternative approach involves regional delivery of therapies, such as delivery of drugs into the dorsal root ganglion (DRG) (Sapunar et al., 2012). The DRG contains bodies of primary sensory neurons, which may generate ectopic discharges and become an important source of hyperexcitability and increased nociceptive signaling (Sapunar et al., 2005; Xie et al., 2006). Therefore, targeted delivery of pharmacological agents into the DRG could prevent development of ectopic discharges (Sapunar et al., 2012) and reduce neuroinflammation (Sapunar et al., 2011; Kostic et al., 2013). Previous results indicate that intraganglionic (i.g.) injection per se is a secure procedure because injection of saline into the DRG did not result in a significant inflammatory response and it did not cause increase of pain-related behavior (Puljak et al., 2009a; Fischer et al., 2011). Calcium/calmodulin protein kinase II (CaMKII) is a serine/threonine protein kinase, which has four distinct isoforms: a, b, c and d (Tobimatsu and Fujisawa, 1989). It plays an important role in molecular memory and excitability of neurons (Eshete and Fields, 2001) by turning transient changes in calcium concentration into changes of cell activity (Morris and Torok, 2001). Autophosphorylation of CaMKII enables continued and efficient activation of the enzyme and of downstream pathways, even after the calcium influx, caused by injury for instance, subsides (Saitoh and Schwartz, 1985; Lucic et al., 2008). Previous findings have indicated that CaMKII is localized in specific subpopulations of sensory neurons, and that DRG neurons containing CaMKIIa may be involved in the processing of nociceptive information. As such, it has been suggested that CaMKII is critical for modulation of nociceptor activity and plasticity of primary sensory neurons (Carlton and Hargett, 2002). CaMKII has been implicated in the pathophysiology of many types of neuropathic pain, specifically in injury model (Kawano et al., 2009; Kojundzic et al., 2010) and in inflammation model (Carlton, 2002). Studies about the role of CaMKII in diabetic neuropathy are scarce. In our recent studies we have found that CaMKII may have a role in pathophysiology of neuropathic pain in diabetes mellitus. Changes in the expression of CaMKII in DRG neurons were accompanied with an increased
Laboratory for Pain Research, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia
Abstract—Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transmission of nociceptive input in diabetic neuropathy. The aim of this study was to test whether intraganglionic (i.g.) injection of CaMKII inhibitors may alleviate pain-related behavior in diabetic rats. Diabetes was induced in Sprague–Dawley rats using 55 mg/kg streptozotocin intraperitoneally. Two weeks after diabetes induction, CaMKII inhibitors myristoil-AIP and KN93 were injected directly into the right L5 dorsal root ganglion (DRG). Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2 and 24 h after the i.g. injection. The expression of total CaMKII and its alpha isoform in DRG neurons was analyzed using immunohistochemistry. CaMKII inhibitors attenuated pain-related behavior in a modality-specific fashion. Attenuation of nociceptive behavior was accompanied with a corresponding decrease of CaMKII alpha expression in DRG neurons on the side of injection. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons. In conclusion, our study provides evidence that CaMKII inhibitors are potential pharmacological agents that should be further explored for treatment of diabetic neuropathy symptoms. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: diabetes mellitus, dorsal root myristoil-AIP, KN-93, pain-related behavior.
ganglion,
INTRODUCTION Treatment of neuropathic pain is inadequate and a gap between understanding the mechanisms of pain and its treatment has been well recognized (Brennan et al., 2007). There are a very small number of pharmacological treatments that are effective for neuropathic pain in different disorders (Finnerup et al., *Corresponding author. Tel: +385-21-557-807; fax: +385-21-557811. E-mail addresses: [email protected] (A. Jelicic Kadic), [email protected] (M. Boric), [email protected] (S. Kostic), [email protected] (D. Sapunar), [email protected] (L. Puljak). Abbreviations: CaMKII, calcium/calmodulin-dependent protein kinase II; DRG, dorsal root ganglion; i.g., intraganglionic; mAIP, myristoylated autocamtide-2-related inhibitory peptide; PBS, phosphate-buffered saline; STZ, streptozotocin; tCaMKII, total CaMKII.
0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.10.032 302
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pain-related behavior (Ferhatovic et al., 2013a,b) and increased CaMKII expression was present in dorsal horn neurons in long-term diabetes (Boric et al., 2013). Activation of CaMKII is a key event in calcium signaling. Others have shown that the altered calcium homeostasis may be an early molecular marker linked to the onset of diabetic sensory neuropathy, characterized with enhanced excitability of sensory neurons (Voitenko et al., 1999; Hall et al., 2001; Jagodic et al., 2007). A definite prolongation of the decay phase of the calcium current transients was observed under diabetic conditions, providing further evidence that changes of calcium signaling in nociceptive neurons may contribute to the development of neuropathy and its symptoms in the early stages of diabetes mellitus (Kostyuk et al., 2001). A number of studies have shown that intrathecal therapy with CaMKII inhibitors can attenuate painrelated symptoms in different models of neuropathic pain (Suh et al., 1997; Choi et al., 2005a, 2006a; Chen et al., 2009; Hasegawa et al., 2009; Nakayama et al., 2010; Katano et al., 2011; Shirahama et al., 2012). Systemic administration of drugs is often accompanied with unacceptable adverse events, and therefore regional approach is preferred. In our preliminary results we found that intrathecal application of CaMKII inhibitors does not alleviate pain-related behavior in diabetic rats (Jelicic Kadic et al., 2013). Therefore, we hypothesized that more direct approach using i.g. injection of CaMKII inhibitors could reduce pain-related behavior and the expression of CaMKII in the DRG of diabetic rats.
EXPERIMENTAL PROCEDURES Experimental animals Ethics Committee of the University of Split, School of Medicine approved the study. Experimental procedures and protocols followed the International Association for the Study of Pain (IASP) Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals, as well as the European Communities Council Directive of 24 November 1968 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering. A total of 28 male Sprague–Dawley rats (160–200 g) were included in the study. All rats were raised under controlled conditions (temperature 22 ± 1 °C, light schedule: 12 h of light and 12 h of dark) at the University of Split Animal Facility. After beginning of the experiment, animals were reared for 2 weeks in pairs in plastic cages with sawdust flooring. Diabetes induction and validation Rats were injected with 55 mg/kg of streptozotocin (STZ) dissolved in freshly prepared citrate buffer (pH = 4.5) after overnight fasting. Rats were fed ad libitum with normal laboratory chow (4RF21 GLP, Mucedola srl, Settimo Milanese, Italy). Plasma glucose was measured with glucometer (OneTouchVITa, LifeScan, High Wycombe, UK). The rats which showed plasma glucose
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levels > 300 mg/dl were considered diabetic. One rat was considered non-diabetic because the glucose level was lower than 300 mg/dl. Two rats died before the end of experiment. The number of remaining rats was 25. Rats were assigned to the following experimental groups: rats treated with regular saline DRG injection (N = 8), rats treated with myristoylated autocamtide-2related inhibitory peptide (mAIP) DRG injection (N = 9) and rats treated with KN-93 DRG injection (N = 8). I.g. injection One of two different conventional blockers of CaMKII, including KN-93 and more specific mAIP, was delivered into the L5 DRG using our previously described method for DRG injection in rats (Puljak et al., 2009b; Fischer et al., 2011; Sapunar et al., 2011; Kostic et al., 2013). Rats were injected with 10 lM mAIP (189482, Merck Millipore, Nottingham, United Kingdom) and 1 mM KN93 (422711, Merck Millipore, Nottingham, United Kingdom). Only one DRG was injected in each animal (right L5 DRG), and each animal was injected only with one inhibitor. I.g. injection of saline was used as a control. The injected volume was 3 lL. I.g. injection was performed as described previously (Kostic et al., 2013). An incision was made along the midline of the back and the right paravertebral region was exposed. Right paraspinal muscles were separated from the transverse process at the L5 spinal level, and connective tissue and muscles were removed by iris scissors, until the right L5 intervertebral foramen could be identified. The L5 nerve and DRG were exposed using a micro bone rongeur. A 29-gauge needle with a slightly bent beveled tip was then advanced 2–4 mm into the foramen. The DRG injection was performed with about a 60° angle relative to the spine. Our validation studies indicate a reliable and precise delivery of injectate and uniform distribution of injected volume within the DRG without leakage to adjacent tissues (Puljak et al., 2009b). Behavioral testing Behavioral testing was initially performed on the day preceding the STZ injection. On the 15th experimental day, behavioral tests were performed to confirm increased pain-related behavior following successful induction of diabetes. Rats were then injected i.g. with one of the CaMKII inhibitors or saline and behavioral testing was performed 2 and 24 h after i.g. injection. The experimenter who performed the behavioral tests was blinded to the treatment group of the animals. Tests used in this study were chosen based on their relevance to changes noted in clinical practice (Lindblom and Verrillo, 1979). For behavioral testing, rats were placed individually in clear plastic enclosures (10 25 30 cm) on a mesh-wire surface (3 3 mm). Tests included stimulation of the plantar skin of both hind paws of unrestrained rats. Cold sensitivity was assessed using acetone test. For heat sensitivity, analgesia meter for mice and rats model PE34 was used with maximal temperature of 50 °C (IITC Life Science, Woodland Hills, CA, USA). Mechanical
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hyperalgesia was tested by applying a noxious stimulus using a needle pin-prick test and noting simple withdrawal and a complex hyperalgesia-type response characterized by sustained paw lifting, shaking, and licking (Hogan et al., 2004). For assessment of mechanical allodynia, von Frey test was used. Furthermore, we observed animals for signs of autotomy, paw licking and chewing, and we did not observe any changes in spontaneous pain behavior between the experimental groups. Tissue collection and immunohistochemistry Rats were sacrificed 16 days after diabetes induction under general anesthetic exposure (Isoflurane; Forane, Abbott Laboratories Ltd., Queenborough, UK). The left and right L5 ganglia were removed and postfixed for 24 h in Zamboni’s fixative (4% paraformaldehyde and 0.19% picric acid in 0.1 M phosphate-buffered saline (PBS)) at pH 7.4. Tissues were left for 24 h in 0.01 M PBS. Following overnight cryoprotection in 30% sucrose, ganglia were embedded in Optimal Cutting Temperature freezing medium (Tissue Tek, Tokyo, Japan). Sections 7 lm thick were cut parallel along the long axis of the ganglia on a cryostat (Thermo Shandon Cryotome, Pittsburgh, PA, USA) and placed on glass slides. Immunohistochemical analysis was performed for detection of total CaMKII (tCaMKII) and its alpha isoform. Primary rabbit polyclonal antibodies were used in dilution of 1:100 for detection of tCaMKII (sc-9035, lot# F0304, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphorylated CaMKII alpha isoform (sc-12886-R, lot# K2305, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubation was done overnight at room temperature and PBS containing 0.3% Triton X-100 (648466, lot# B65241, Merck KGaA, Darmstadt, Germany) was used for rinsing. Secondary detection of tCaMKII and alpha isoform was performed using secondary antibody with Rhodamin red X-conjugated (Donkey Anti-rabbit IgG (H + L) Jackson ImmunoResearch, Lot No 106114, dilution 1:300). After final rinsing in PBS, all slides were mounted, air-dried, and cover slipped (Immu-Mount, Shandon, Pittsburgh, PA, USA). Staining controls included omission of primary antibody from the staining procedure, which resulted in no staining of DRG tissue.
Fluorescence intensity of neuronal cytoplasm and surface area of each neuron was measured. Further analysis was done on three separate groups of DRG neurons based on their soma diameter: small (d 6 30 lm), medium (30 lm 6 d 6 40 lm) and large (d P 40 lm). The diameter of DRG neurons was calculated using formula: diameter = (length + width)/2. Statistical analysis Comparisons between expression in ipsilateral (injected) and contralateral (non-injected) DRG neurons were analyzed using Student’s t-test. The same test was used for behavioral analyses (Statistica 7.0; StatSoft, Tulsa, OK, USA). The data were presented as mean and standard deviation (M ± SD). Any difference with p < 0.05 was considered statistically significant.
RESULTS CaMKII expression in DRG after CaMKII inhibition The expression of tCaMKII did not change significantly in ipsilateral and contralateral DRG neurons of diabetic rats after injecting saline, mAIP or KN-93 into the right L5 DRG (Fig. 1A). The expression of CaMKIIa was significantly reduced after i.g. KN-93 injection (t(7) = 2.68; p = 0.0317), but not after injection of saline and mAIP (both: p > 0.05) (Fig. 1B).
Quantitative analysis for immunohistochemistry Every fourth section of each DRG was examined with a microscope (BX61, Olympus, Tokyo, Japan). Microphotographs were taken with a cooled digital camera (DP71, Olympus, Tokyo, Japan) under the same magnification (40), exposition, binning and gain for each image. Measurements were performed using Image J (National Institutes of Health, Bethesda, MD, USA) where they were examined as monochromic microphotographs (2040 1536 pixels, 12 bits, 0–4096 gray scale), following background subtraction. Only the neurons with clearly visible nuclei were analyzed.
Fig. 1. The effect of intraganglionic injection of saline, mAIP and KN93 on the expression of total CaMKII (A) and CaMKIIa (B) in diabetic rats. White columns denote the left DRG (not injected) and black columns indicate the right DRG (injected). Data are presented as M ± SD. Asterisk denotes significant difference from the contralateral DRG (t-test; p < 0.05).
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Analysis of the tCaMKII and CaMKIIa expression after i.g. injection of saline, mAIP and KN-93 was also analyzed in neurons based on their size. The CaMKII expression analysis based on the size of neurons was conducted because of the previous findings that CaMKII is preferentially expressed in small-diameter neurons (Carlton, 2002), and that small-diameter neurons expressing CaMKII are affected after injury (Kojundzic et al., 2010), strongly suggesting that they are nociceptive neurons. The expression of tCaMKII after injection of mAIP and KN-93 was the same in all neurons, regardless of their diameter. There was no difference in the expression of CaMKIIa after injection of mAIP in neuronal groups based on their diameter. The expression of CaMKIIa was significantly reduced after injection of KN-93 in small- (t(7) = 2.65; p = 0.033) and medium-sized neurons (t(7) = 5.14; p = 0.001), but not in large neurons (data not shown). Representative tCaMKII and CaMKIIa staining of ipsilateral and contralateral DRG neurons after injection of saline, mAIP and KN-93 is shown in Figs. 2 and 3.
CaMKII inhibition and pain-related behavior Compared to baseline, diabetic rats developed increased sensitivity to cold and mechanical stimuli 2 weeks after induction of diabetes (Figs. 4A, B) but not in heat sensitivity and mechanical allodynia tests (data not shown). Sensitivity to cold was significantly increased after 2 weeks of diabetes. Sensitivity to cold was reduced after treatment with mAIP but not with KN-93 (Fig. 4A). A significant reduction in hypersensitivity to cold was observed 2 h after i.g. injection of mAIP (t(40) = 2.73; p = 0.030).
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The withdrawal response rate to mechanical stimuli was significantly increased after 2 weeks of diabetes (t(46) = 12.85; p < 0.001). I.g. injection of mAIP leads to a significant reduction of withdrawal responses 2 h (t(40); p = 0.036) but not 24 h following injection (Fig. 4B). KN-93 i.g. injection also leads to a decrease of withdrawal responses, but this result did not reach statistical significance. Diabetes induction did not change sensitivity to heat or decrease in von Frey withdrawal threshold.
DISCUSSION The aim of the present study was to explore whether DRG-targeted delivery of CaMKII inhibitors may attenuate pain-related behavior in rats with experimentally induced diabetes mellitus. Attenuation of nociceptive behavior was accompanied in a modalityspecific fashion with a corresponding decrease of CaMKII alpha expression. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons after treatment with KN-93. We found that two CaMKII inhibitors, with different mechanism of action, may attenuate pain-related behavior in a modality-specific fashion. Organic inhibitor KN-93 competitively inhibits calmodulin binding at the regulatory domain for CaMKII (Sumi et al., 1991). AIP is a highly specific membrane-permeable peptide inhibitor of CaMKII, which inhibits CaMKII by binding to its substrate-binding site for autophosphorylation, and its inhibition on CaMKII is not affected by calcium/ calmodulin (Ishida et al., 1995). Studies using i.g. delivery of pharmacological agents have been seldom used in the basic studies of nociception. It has been suggested that direct and selective DRG-targeted delivery of drugs directly may
Fig. 2. Representative images of CaMKIIa staining in the contralateral DRG of diabetic rats injected with saline (A), mAIP (C) and KN-93 (E) and in the ipsilateral DRG injected with saline (B), mAIP (D) and KN-93 (F).
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Fig. 3. Representative images of tCaMKII staining in the contralateral DRG of diabetic rats injected with saline (A), mAIP (C) and KN-93 (E) and in the ipsilateral DRG injected with saline (B), mAIP (D) and KN-93 (F).
Fig. 4. Behavioral changes in diabetic rats after direct injection of saline and CaMKII inhibitors mAIP and KN-93 into the dorsal root ganglion. Responses to cold (A), and needle (B) stimuli were recorded. One asterisk denotes a significant difference from baseline values, while two asterisks show a difference from values in diabetic rats. Legend is identical for A and B plates.
limit influences of drugs on other neuronal populations and provide basis for development of novel therapies (Kostic et al., 2013). Unlike i.g. route of delivery, intrathecal application of CaMKII inhibition has been used in various pain models. These studies indicate that CaMKII has a role in abnormal nociception in neuropathic conditions. However, one can observe a significant methodological diversity in previous studies on i.t. delivery of CaMKII inhibitors in rodent models. Different pain models, doses of CaMKII inhibitors and limited behavioral tests that were used leave plenty of room for further studies on the effects of CaMKII inhibition on nociception. Most of the studies are using only one CaMKII inhibitor (KN-93) and one-time assessment. Pretreatment, posttreatment, as well as combination of both were studied (Choi et al., 2005b, 2006b; Dai et al., 2005; Luo et al., 2008; Chen et al., 2009; Hasegawa et al., 2009; Katano et al., 2011;
Shirahama et al., 2012). In this study, only posttreatment approach was used, since diabetic neuropathy occurs after the onset of the disease (Veves et al., 2008). AIP binds to the autophosphorylation site of the enzyme (Ishida et al., 1995) and blocks both Ca2+dependent and Ca2+-independent activities (Lisman et al., 2002). KN-93 only blocks CaMKII activation but it does not inhibit CaMKII autonomous activity (Sumi et al., 1991). Our results suggest that inhibitors of activated/phosphorylated CaMKII are more effective in the condition following the changes that occur in diabetic neuropathy, and this may be the reason why KN-93 was not as successful as mAIP in alleviating pain behavior. We have also observed modality-specific behavioral response after CaMKII inhibition. It is not uncommon to encounter the results of behavioral analysis which show modality-specific changes. The
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possible explanation is that experimental manipulation affects specific cell phenotype responsible for affected sensory modality. An abnormality in CaMKII expression in DRG neurons of STZ-induced diabetic male rats indicates a possible important role of this enzyme in the pathophysiology of abnormal pain perception in diabetes (Ferhatovic et al., 2013a,b). Changes in the expression of CaMKII in DRG neurons corresponded to the changes in pain-related behavior and development of mechanical and thermal hyperalgesia in rat models of diabetes type 1. The animal model of diabetes type 2, induced with a combination of low-dose STZ and high-fat diet, did not exhibit changes in CaMKII expression or pain-related behavior. The two animal models of diabetes differed significantly in the level of hyperglycemia (Ferhatovic et al., 2013a). In studies of pain-related behavior, authors frequently use limited battery of behavioral tests, analyzing reactions either mechanical or thermal stimuli. In our study, we have used a full range of behavioral analyses, with hot and cold thermal stimuli and two types of mechanical stimuli, therefore providing data about the effect of CaMKII inhibition on several modalities of nociceptive behavior. Cellular mechanisms responsible for the development of hyperalgesia and allodynia in neuropathic pain are still poorly understood. It has been suggested that remodeling of ion channels can increase excitability of sensory neurons and thus contribute to development of neuropathic symptoms (Coderre et al., 1993; Woolf, 2004). Disturbed calcium homeostasis has been implicated in the pathogenesis of diabetic neuropathy (Biessels and Gispen, 1996). Pathophysiologically important changes in calcium currents, with increased cellular excitability and resultant axonal alterations, have been described in DRG neurons of diabetic rats (Hall et al., 2001; Kishi et al., 2002; Jagodic et al., 2007). As the end result, intracellular calcium overload may lead to CaMKII overexpression in primary afferent neurons and cause abnormal firing of DRG neurons (Coultrap et al., 2011). CaMKIIa is the most abundant isoform of CaMKII in neurons (Lucchesi et al., 2011). The present study shows that CaMKII inhibition via i.g. injection causes ipsilateral reduction in CaMKIIa fluorescence, providing further evidence that CaMKIIa is involved in nociception across different pain models. Furthermore, a significant reduction of CaMKIIa was found only in small- and medium-sized neurons. These small to medium DRG neurons are known to respectively give rise to C and Ad fibers, which are responsible for transmission of noxious stimuli (Julius and Basbaum, 2001).
CONCLUSION I.g. administration of CaMKII inhibitors in STZ-diabetic rats attenuated pain-related behavior in a modalityspecific fashion. Our study provides further evidence that CaMKII is a potential pharmacological agent that can be tested further for attenuation of pain-related behavior and nociception.
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AUTHOR CONTRIBUTIONS The experiments were performed in the Laboratory for Pain Research at the Department of Anatomy, Histology and Embryology of the University of Split School of Medicine. Conception and design of the experiments were performed by L.P. and D.S. Collection, analysis and interpretation of data were performed by A.J., M.B., S.K., L.P. and D.S. Drafting the article was performed by L.P. All authors discussed the results, commented on the manuscript and approved the final version to be submitted. Acknowledgements—The study was funded by the Croatian Foundation for Science (HRZZ) grant no. 02.05./28 awarded to Livia Puljak.
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(Accepted 14 October 2013) (Available online 23 October 2013)
THE EFFECTS OF INTRAGANGLIONIC INJECTION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II INHIBITORS ON PAIN-RELATED BEHAVIOR IN DIABETIC NEUROPATHY A. JELICIC KADIC, M. BORIC, S. KOSTIC, D. SAPUNAR AND L. PULJAK *
2005). Thus, more studies are necessary about potential treatments and treatment modalities for neuropathic pain. Many drugs used to treat neuropathic pain are delivered systematically or intrathecally, which results in a number of adverse events. An alternative approach involves regional delivery of therapies, such as delivery of drugs into the dorsal root ganglion (DRG) (Sapunar et al., 2012). The DRG contains bodies of primary sensory neurons, which may generate ectopic discharges and become an important source of hyperexcitability and increased nociceptive signaling (Sapunar et al., 2005; Xie et al., 2006). Therefore, targeted delivery of pharmacological agents into the DRG could prevent development of ectopic discharges (Sapunar et al., 2012) and reduce neuroinflammation (Sapunar et al., 2011; Kostic et al., 2013). Previous results indicate that intraganglionic (i.g.) injection per se is a secure procedure because injection of saline into the DRG did not result in a significant inflammatory response and it did not cause increase of pain-related behavior (Puljak et al., 2009a; Fischer et al., 2011). Calcium/calmodulin protein kinase II (CaMKII) is a serine/threonine protein kinase, which has four distinct isoforms: a, b, c and d (Tobimatsu and Fujisawa, 1989). It plays an important role in molecular memory and excitability of neurons (Eshete and Fields, 2001) by turning transient changes in calcium concentration into changes of cell activity (Morris and Torok, 2001). Autophosphorylation of CaMKII enables continued and efficient activation of the enzyme and of downstream pathways, even after the calcium influx, caused by injury for instance, subsides (Saitoh and Schwartz, 1985; Lucic et al., 2008). Previous findings have indicated that CaMKII is localized in specific subpopulations of sensory neurons, and that DRG neurons containing CaMKIIa may be involved in the processing of nociceptive information. As such, it has been suggested that CaMKII is critical for modulation of nociceptor activity and plasticity of primary sensory neurons (Carlton and Hargett, 2002). CaMKII has been implicated in the pathophysiology of many types of neuropathic pain, specifically in injury model (Kawano et al., 2009; Kojundzic et al., 2010) and in inflammation model (Carlton, 2002). Studies about the role of CaMKII in diabetic neuropathy are scarce. In our recent studies we have found that CaMKII may have a role in pathophysiology of neuropathic pain in diabetes mellitus. Changes in the expression of CaMKII in DRG neurons were accompanied with an increased
Laboratory for Pain Research, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia
Abstract—Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transmission of nociceptive input in diabetic neuropathy. The aim of this study was to test whether intraganglionic (i.g.) injection of CaMKII inhibitors may alleviate pain-related behavior in diabetic rats. Diabetes was induced in Sprague–Dawley rats using 55 mg/kg streptozotocin intraperitoneally. Two weeks after diabetes induction, CaMKII inhibitors myristoil-AIP and KN93 were injected directly into the right L5 dorsal root ganglion (DRG). Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2 and 24 h after the i.g. injection. The expression of total CaMKII and its alpha isoform in DRG neurons was analyzed using immunohistochemistry. CaMKII inhibitors attenuated pain-related behavior in a modality-specific fashion. Attenuation of nociceptive behavior was accompanied with a corresponding decrease of CaMKII alpha expression in DRG neurons on the side of injection. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons. In conclusion, our study provides evidence that CaMKII inhibitors are potential pharmacological agents that should be further explored for treatment of diabetic neuropathy symptoms. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: diabetes mellitus, dorsal root myristoil-AIP, KN-93, pain-related behavior.
ganglion,
INTRODUCTION Treatment of neuropathic pain is inadequate and a gap between understanding the mechanisms of pain and its treatment has been well recognized (Brennan et al., 2007). There are a very small number of pharmacological treatments that are effective for neuropathic pain in different disorders (Finnerup et al., *Corresponding author. Tel: +385-21-557-807; fax: +385-21-557811. E-mail addresses: [email protected] (A. Jelicic Kadic), [email protected] (M. Boric), [email protected] (S. Kostic), [email protected] (D. Sapunar), [email protected] (L. Puljak). Abbreviations: CaMKII, calcium/calmodulin-dependent protein kinase II; DRG, dorsal root ganglion; i.g., intraganglionic; mAIP, myristoylated autocamtide-2-related inhibitory peptide; PBS, phosphate-buffered saline; STZ, streptozotocin; tCaMKII, total CaMKII.
0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.10.032 302
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pain-related behavior (Ferhatovic et al., 2013a,b) and increased CaMKII expression was present in dorsal horn neurons in long-term diabetes (Boric et al., 2013). Activation of CaMKII is a key event in calcium signaling. Others have shown that the altered calcium homeostasis may be an early molecular marker linked to the onset of diabetic sensory neuropathy, characterized with enhanced excitability of sensory neurons (Voitenko et al., 1999; Hall et al., 2001; Jagodic et al., 2007). A definite prolongation of the decay phase of the calcium current transients was observed under diabetic conditions, providing further evidence that changes of calcium signaling in nociceptive neurons may contribute to the development of neuropathy and its symptoms in the early stages of diabetes mellitus (Kostyuk et al., 2001). A number of studies have shown that intrathecal therapy with CaMKII inhibitors can attenuate painrelated symptoms in different models of neuropathic pain (Suh et al., 1997; Choi et al., 2005a, 2006a; Chen et al., 2009; Hasegawa et al., 2009; Nakayama et al., 2010; Katano et al., 2011; Shirahama et al., 2012). Systemic administration of drugs is often accompanied with unacceptable adverse events, and therefore regional approach is preferred. In our preliminary results we found that intrathecal application of CaMKII inhibitors does not alleviate pain-related behavior in diabetic rats (Jelicic Kadic et al., 2013). Therefore, we hypothesized that more direct approach using i.g. injection of CaMKII inhibitors could reduce pain-related behavior and the expression of CaMKII in the DRG of diabetic rats.
EXPERIMENTAL PROCEDURES Experimental animals Ethics Committee of the University of Split, School of Medicine approved the study. Experimental procedures and protocols followed the International Association for the Study of Pain (IASP) Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals, as well as the European Communities Council Directive of 24 November 1968 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering. A total of 28 male Sprague–Dawley rats (160–200 g) were included in the study. All rats were raised under controlled conditions (temperature 22 ± 1 °C, light schedule: 12 h of light and 12 h of dark) at the University of Split Animal Facility. After beginning of the experiment, animals were reared for 2 weeks in pairs in plastic cages with sawdust flooring. Diabetes induction and validation Rats were injected with 55 mg/kg of streptozotocin (STZ) dissolved in freshly prepared citrate buffer (pH = 4.5) after overnight fasting. Rats were fed ad libitum with normal laboratory chow (4RF21 GLP, Mucedola srl, Settimo Milanese, Italy). Plasma glucose was measured with glucometer (OneTouchVITa, LifeScan, High Wycombe, UK). The rats which showed plasma glucose
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levels > 300 mg/dl were considered diabetic. One rat was considered non-diabetic because the glucose level was lower than 300 mg/dl. Two rats died before the end of experiment. The number of remaining rats was 25. Rats were assigned to the following experimental groups: rats treated with regular saline DRG injection (N = 8), rats treated with myristoylated autocamtide-2related inhibitory peptide (mAIP) DRG injection (N = 9) and rats treated with KN-93 DRG injection (N = 8). I.g. injection One of two different conventional blockers of CaMKII, including KN-93 and more specific mAIP, was delivered into the L5 DRG using our previously described method for DRG injection in rats (Puljak et al., 2009b; Fischer et al., 2011; Sapunar et al., 2011; Kostic et al., 2013). Rats were injected with 10 lM mAIP (189482, Merck Millipore, Nottingham, United Kingdom) and 1 mM KN93 (422711, Merck Millipore, Nottingham, United Kingdom). Only one DRG was injected in each animal (right L5 DRG), and each animal was injected only with one inhibitor. I.g. injection of saline was used as a control. The injected volume was 3 lL. I.g. injection was performed as described previously (Kostic et al., 2013). An incision was made along the midline of the back and the right paravertebral region was exposed. Right paraspinal muscles were separated from the transverse process at the L5 spinal level, and connective tissue and muscles were removed by iris scissors, until the right L5 intervertebral foramen could be identified. The L5 nerve and DRG were exposed using a micro bone rongeur. A 29-gauge needle with a slightly bent beveled tip was then advanced 2–4 mm into the foramen. The DRG injection was performed with about a 60° angle relative to the spine. Our validation studies indicate a reliable and precise delivery of injectate and uniform distribution of injected volume within the DRG without leakage to adjacent tissues (Puljak et al., 2009b). Behavioral testing Behavioral testing was initially performed on the day preceding the STZ injection. On the 15th experimental day, behavioral tests were performed to confirm increased pain-related behavior following successful induction of diabetes. Rats were then injected i.g. with one of the CaMKII inhibitors or saline and behavioral testing was performed 2 and 24 h after i.g. injection. The experimenter who performed the behavioral tests was blinded to the treatment group of the animals. Tests used in this study were chosen based on their relevance to changes noted in clinical practice (Lindblom and Verrillo, 1979). For behavioral testing, rats were placed individually in clear plastic enclosures (10 25 30 cm) on a mesh-wire surface (3 3 mm). Tests included stimulation of the plantar skin of both hind paws of unrestrained rats. Cold sensitivity was assessed using acetone test. For heat sensitivity, analgesia meter for mice and rats model PE34 was used with maximal temperature of 50 °C (IITC Life Science, Woodland Hills, CA, USA). Mechanical
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hyperalgesia was tested by applying a noxious stimulus using a needle pin-prick test and noting simple withdrawal and a complex hyperalgesia-type response characterized by sustained paw lifting, shaking, and licking (Hogan et al., 2004). For assessment of mechanical allodynia, von Frey test was used. Furthermore, we observed animals for signs of autotomy, paw licking and chewing, and we did not observe any changes in spontaneous pain behavior between the experimental groups. Tissue collection and immunohistochemistry Rats were sacrificed 16 days after diabetes induction under general anesthetic exposure (Isoflurane; Forane, Abbott Laboratories Ltd., Queenborough, UK). The left and right L5 ganglia were removed and postfixed for 24 h in Zamboni’s fixative (4% paraformaldehyde and 0.19% picric acid in 0.1 M phosphate-buffered saline (PBS)) at pH 7.4. Tissues were left for 24 h in 0.01 M PBS. Following overnight cryoprotection in 30% sucrose, ganglia were embedded in Optimal Cutting Temperature freezing medium (Tissue Tek, Tokyo, Japan). Sections 7 lm thick were cut parallel along the long axis of the ganglia on a cryostat (Thermo Shandon Cryotome, Pittsburgh, PA, USA) and placed on glass slides. Immunohistochemical analysis was performed for detection of total CaMKII (tCaMKII) and its alpha isoform. Primary rabbit polyclonal antibodies were used in dilution of 1:100 for detection of tCaMKII (sc-9035, lot# F0304, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphorylated CaMKII alpha isoform (sc-12886-R, lot# K2305, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubation was done overnight at room temperature and PBS containing 0.3% Triton X-100 (648466, lot# B65241, Merck KGaA, Darmstadt, Germany) was used for rinsing. Secondary detection of tCaMKII and alpha isoform was performed using secondary antibody with Rhodamin red X-conjugated (Donkey Anti-rabbit IgG (H + L) Jackson ImmunoResearch, Lot No 106114, dilution 1:300). After final rinsing in PBS, all slides were mounted, air-dried, and cover slipped (Immu-Mount, Shandon, Pittsburgh, PA, USA). Staining controls included omission of primary antibody from the staining procedure, which resulted in no staining of DRG tissue.
Fluorescence intensity of neuronal cytoplasm and surface area of each neuron was measured. Further analysis was done on three separate groups of DRG neurons based on their soma diameter: small (d 6 30 lm), medium (30 lm 6 d 6 40 lm) and large (d P 40 lm). The diameter of DRG neurons was calculated using formula: diameter = (length + width)/2. Statistical analysis Comparisons between expression in ipsilateral (injected) and contralateral (non-injected) DRG neurons were analyzed using Student’s t-test. The same test was used for behavioral analyses (Statistica 7.0; StatSoft, Tulsa, OK, USA). The data were presented as mean and standard deviation (M ± SD). Any difference with p < 0.05 was considered statistically significant.
RESULTS CaMKII expression in DRG after CaMKII inhibition The expression of tCaMKII did not change significantly in ipsilateral and contralateral DRG neurons of diabetic rats after injecting saline, mAIP or KN-93 into the right L5 DRG (Fig. 1A). The expression of CaMKIIa was significantly reduced after i.g. KN-93 injection (t(7) = 2.68; p = 0.0317), but not after injection of saline and mAIP (both: p > 0.05) (Fig. 1B).
Quantitative analysis for immunohistochemistry Every fourth section of each DRG was examined with a microscope (BX61, Olympus, Tokyo, Japan). Microphotographs were taken with a cooled digital camera (DP71, Olympus, Tokyo, Japan) under the same magnification (40), exposition, binning and gain for each image. Measurements were performed using Image J (National Institutes of Health, Bethesda, MD, USA) where they were examined as monochromic microphotographs (2040 1536 pixels, 12 bits, 0–4096 gray scale), following background subtraction. Only the neurons with clearly visible nuclei were analyzed.
Fig. 1. The effect of intraganglionic injection of saline, mAIP and KN93 on the expression of total CaMKII (A) and CaMKIIa (B) in diabetic rats. White columns denote the left DRG (not injected) and black columns indicate the right DRG (injected). Data are presented as M ± SD. Asterisk denotes significant difference from the contralateral DRG (t-test; p < 0.05).
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Analysis of the tCaMKII and CaMKIIa expression after i.g. injection of saline, mAIP and KN-93 was also analyzed in neurons based on their size. The CaMKII expression analysis based on the size of neurons was conducted because of the previous findings that CaMKII is preferentially expressed in small-diameter neurons (Carlton, 2002), and that small-diameter neurons expressing CaMKII are affected after injury (Kojundzic et al., 2010), strongly suggesting that they are nociceptive neurons. The expression of tCaMKII after injection of mAIP and KN-93 was the same in all neurons, regardless of their diameter. There was no difference in the expression of CaMKIIa after injection of mAIP in neuronal groups based on their diameter. The expression of CaMKIIa was significantly reduced after injection of KN-93 in small- (t(7) = 2.65; p = 0.033) and medium-sized neurons (t(7) = 5.14; p = 0.001), but not in large neurons (data not shown). Representative tCaMKII and CaMKIIa staining of ipsilateral and contralateral DRG neurons after injection of saline, mAIP and KN-93 is shown in Figs. 2 and 3.
CaMKII inhibition and pain-related behavior Compared to baseline, diabetic rats developed increased sensitivity to cold and mechanical stimuli 2 weeks after induction of diabetes (Figs. 4A, B) but not in heat sensitivity and mechanical allodynia tests (data not shown). Sensitivity to cold was significantly increased after 2 weeks of diabetes. Sensitivity to cold was reduced after treatment with mAIP but not with KN-93 (Fig. 4A). A significant reduction in hypersensitivity to cold was observed 2 h after i.g. injection of mAIP (t(40) = 2.73; p = 0.030).
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The withdrawal response rate to mechanical stimuli was significantly increased after 2 weeks of diabetes (t(46) = 12.85; p < 0.001). I.g. injection of mAIP leads to a significant reduction of withdrawal responses 2 h (t(40); p = 0.036) but not 24 h following injection (Fig. 4B). KN-93 i.g. injection also leads to a decrease of withdrawal responses, but this result did not reach statistical significance. Diabetes induction did not change sensitivity to heat or decrease in von Frey withdrawal threshold.
DISCUSSION The aim of the present study was to explore whether DRG-targeted delivery of CaMKII inhibitors may attenuate pain-related behavior in rats with experimentally induced diabetes mellitus. Attenuation of nociceptive behavior was accompanied in a modalityspecific fashion with a corresponding decrease of CaMKII alpha expression. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons after treatment with KN-93. We found that two CaMKII inhibitors, with different mechanism of action, may attenuate pain-related behavior in a modality-specific fashion. Organic inhibitor KN-93 competitively inhibits calmodulin binding at the regulatory domain for CaMKII (Sumi et al., 1991). AIP is a highly specific membrane-permeable peptide inhibitor of CaMKII, which inhibits CaMKII by binding to its substrate-binding site for autophosphorylation, and its inhibition on CaMKII is not affected by calcium/ calmodulin (Ishida et al., 1995). Studies using i.g. delivery of pharmacological agents have been seldom used in the basic studies of nociception. It has been suggested that direct and selective DRG-targeted delivery of drugs directly may
Fig. 2. Representative images of CaMKIIa staining in the contralateral DRG of diabetic rats injected with saline (A), mAIP (C) and KN-93 (E) and in the ipsilateral DRG injected with saline (B), mAIP (D) and KN-93 (F).
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Fig. 3. Representative images of tCaMKII staining in the contralateral DRG of diabetic rats injected with saline (A), mAIP (C) and KN-93 (E) and in the ipsilateral DRG injected with saline (B), mAIP (D) and KN-93 (F).
Fig. 4. Behavioral changes in diabetic rats after direct injection of saline and CaMKII inhibitors mAIP and KN-93 into the dorsal root ganglion. Responses to cold (A), and needle (B) stimuli were recorded. One asterisk denotes a significant difference from baseline values, while two asterisks show a difference from values in diabetic rats. Legend is identical for A and B plates.
limit influences of drugs on other neuronal populations and provide basis for development of novel therapies (Kostic et al., 2013). Unlike i.g. route of delivery, intrathecal application of CaMKII inhibition has been used in various pain models. These studies indicate that CaMKII has a role in abnormal nociception in neuropathic conditions. However, one can observe a significant methodological diversity in previous studies on i.t. delivery of CaMKII inhibitors in rodent models. Different pain models, doses of CaMKII inhibitors and limited behavioral tests that were used leave plenty of room for further studies on the effects of CaMKII inhibition on nociception. Most of the studies are using only one CaMKII inhibitor (KN-93) and one-time assessment. Pretreatment, posttreatment, as well as combination of both were studied (Choi et al., 2005b, 2006b; Dai et al., 2005; Luo et al., 2008; Chen et al., 2009; Hasegawa et al., 2009; Katano et al., 2011;
Shirahama et al., 2012). In this study, only posttreatment approach was used, since diabetic neuropathy occurs after the onset of the disease (Veves et al., 2008). AIP binds to the autophosphorylation site of the enzyme (Ishida et al., 1995) and blocks both Ca2+dependent and Ca2+-independent activities (Lisman et al., 2002). KN-93 only blocks CaMKII activation but it does not inhibit CaMKII autonomous activity (Sumi et al., 1991). Our results suggest that inhibitors of activated/phosphorylated CaMKII are more effective in the condition following the changes that occur in diabetic neuropathy, and this may be the reason why KN-93 was not as successful as mAIP in alleviating pain behavior. We have also observed modality-specific behavioral response after CaMKII inhibition. It is not uncommon to encounter the results of behavioral analysis which show modality-specific changes. The
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possible explanation is that experimental manipulation affects specific cell phenotype responsible for affected sensory modality. An abnormality in CaMKII expression in DRG neurons of STZ-induced diabetic male rats indicates a possible important role of this enzyme in the pathophysiology of abnormal pain perception in diabetes (Ferhatovic et al., 2013a,b). Changes in the expression of CaMKII in DRG neurons corresponded to the changes in pain-related behavior and development of mechanical and thermal hyperalgesia in rat models of diabetes type 1. The animal model of diabetes type 2, induced with a combination of low-dose STZ and high-fat diet, did not exhibit changes in CaMKII expression or pain-related behavior. The two animal models of diabetes differed significantly in the level of hyperglycemia (Ferhatovic et al., 2013a). In studies of pain-related behavior, authors frequently use limited battery of behavioral tests, analyzing reactions either mechanical or thermal stimuli. In our study, we have used a full range of behavioral analyses, with hot and cold thermal stimuli and two types of mechanical stimuli, therefore providing data about the effect of CaMKII inhibition on several modalities of nociceptive behavior. Cellular mechanisms responsible for the development of hyperalgesia and allodynia in neuropathic pain are still poorly understood. It has been suggested that remodeling of ion channels can increase excitability of sensory neurons and thus contribute to development of neuropathic symptoms (Coderre et al., 1993; Woolf, 2004). Disturbed calcium homeostasis has been implicated in the pathogenesis of diabetic neuropathy (Biessels and Gispen, 1996). Pathophysiologically important changes in calcium currents, with increased cellular excitability and resultant axonal alterations, have been described in DRG neurons of diabetic rats (Hall et al., 2001; Kishi et al., 2002; Jagodic et al., 2007). As the end result, intracellular calcium overload may lead to CaMKII overexpression in primary afferent neurons and cause abnormal firing of DRG neurons (Coultrap et al., 2011). CaMKIIa is the most abundant isoform of CaMKII in neurons (Lucchesi et al., 2011). The present study shows that CaMKII inhibition via i.g. injection causes ipsilateral reduction in CaMKIIa fluorescence, providing further evidence that CaMKIIa is involved in nociception across different pain models. Furthermore, a significant reduction of CaMKIIa was found only in small- and medium-sized neurons. These small to medium DRG neurons are known to respectively give rise to C and Ad fibers, which are responsible for transmission of noxious stimuli (Julius and Basbaum, 2001).
CONCLUSION I.g. administration of CaMKII inhibitors in STZ-diabetic rats attenuated pain-related behavior in a modalityspecific fashion. Our study provides further evidence that CaMKII is a potential pharmacological agent that can be tested further for attenuation of pain-related behavior and nociception.
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AUTHOR CONTRIBUTIONS The experiments were performed in the Laboratory for Pain Research at the Department of Anatomy, Histology and Embryology of the University of Split School of Medicine. Conception and design of the experiments were performed by L.P. and D.S. Collection, analysis and interpretation of data were performed by A.J., M.B., S.K., L.P. and D.S. Drafting the article was performed by L.P. All authors discussed the results, commented on the manuscript and approved the final version to be submitted. Acknowledgements—The study was funded by the Croatian Foundation for Science (HRZZ) grant no. 02.05./28 awarded to Livia Puljak.
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(Accepted 14 October 2013) (Available online 23 October 2013)