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Pain and Analgesic Mechanisms

Expression of Calcium/Calmodulin-Dependent Protein Kinase II and Pain-Related Behavior in Rat Models of Type 1 and Type 2 Diabetes

Ferhatovic, Lejla, MSc*; Banozic, Adriana, MSc*; Kostic, Sandra, MSc*; Kurir, Tina Ticinovic, MD, PhD; Novak, Anela, MD; Vrdoljak, Luka, MD; Heffer, Marija, MD, PhD§; Sapunar, Damir, MD, PhD*; Puljak, Livia, MD, PhD*

Author Information
doi: 10.1213/ANE.0b013e318279b540
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Abstract

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. One of the most common early complications of DM is diabetic neuropathy.1 Abnormalities in the peripheral nerves and dorsal root ganglia (DRG) are apparent in the early stage of experimentally provoked diabetic neuropathy.2 Pathophysiologically significant changes with resultant axonal alterations, as well as increased cellular excitability and changes in calcium currents have been described in DRG neurons of diabetic rats.3,4 Transient increases of calcium concentration ([Ca2+]c) as a result of increased inward calcium currents can be turned into changes of activity and phosphorylation of target proteins, including autophosphorylation of neighboring subunits through activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) enzyme.5 Autophosphorylated CaMKII (pCaMKII) maintains its activity independent of the further calcium stimulation, and in this way provides a function of molecular memory of the cellular activity.6,7 The ability of the CaMKII enzyme to maintain activity long after the initial stimulus ceases and to act as a detector of frequency of neuronal stimulation is confirmed in sensory neurons.6

CaMKII is a family of 4 closely related isoforms, namely α, β, γ, and δ, which are products of 4 separate genes.8 CaMKII can be found in all tissues, but it is especially abundant in neurons. Isoforms α and β are primarily found in the central nervous system, and isforms γ and δ can be found in all tissues. Isoform α has been studied extensively, unlike the other 3 isoforms.9

CaMKIIα coexists with µ-opioid receptors in distinct pain-processing brain regions including the superficial layers of the spinal cord dorsal horn and DRG.10 In DRG, CaMKIIα was described in nociceptive small diameter primary sensory neurons.11–13 Previous studies in animal pain models have shown that changes in expression of CaMKII in DRG are associated with different symptoms of neuropathic pain, such as allodynia and hyperalgesia in models of nerve injury–induced neuropathy13–15 and visceral hypersensitivity in a model of visceral pain.16

The expression of CaMKII in a model of diabetes-induced neuropathic pain has not been studied. We hypothesized that CaMKII has a modulating role in diabetic neuropathy because of its role in calcium homeostasis. Therefore, we studied an association between CaMKII expression in DRG neurons and pain-related behavior in animal models of type 1 and type 2 DM (DM1 and DM2).

METHODS

Ethics

All experimental procedures and protocols followed the International Association for the Study of Pain Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals and were approved by the Ethical Committee of the University of Split, School of Medicine, which serves as the Institutional Animal Care and Use Committee. The study was conducted in a manner that does not inflict unnecessary pain or discomfort on the animal, as outlined by the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals (1996), prepared by the National Academy of Sciences’ Institute for Laboratory Animal Research.

Experimental Animals

Sixty-three male Sprague–Dawley rats weighing 160–200 g were used in this study. All rats were raised under controlled conditions (temperature: 22.1°C; light schedule: 12 hours of light and 12 hours of dark) at University of Split Animal Facility. We assigned animals into 4 experimental groups: model of DM1 (n = 28), a respective control for DM1 group (C-DM1; n = 12), model of DM2 (n = 12) and respective control for DM2 group (C-DM2; n = 11).

For the induction of model of DM1, after overnight fasting animals were injected intraperitoneally with 55 mg/kg of the streptozotocin (STZ) dissolved in citrate buffer (pH 4.5). STZ is toxic to the insulin-producing pancreatic β cells.17 The C-DM1 group received vehicle citrate buffer injection. These 2 groups of animals were fed ad libitum with normal pellet diet, which consisted of 27% proteins, 9% fat, and 64% of carbohydrates (4RF21 GLP, Mucedola srl, Settimo Milanese, Italy).

DM2 rats were fed ad libitum with the high-fat diet (HFD) consisting of 58% fat, 25% proteins, and 17% carbohydrates (PF 4269, Mucedola srl) for 2 weeks and then received intraperitoneally 35 mg/kg of STZ after overnight fasting.18 The C-DM2 group was also fed with HFD but received only vehicle citrate buffer solution injection. After intraperitoneal STZ or citrate buffer injection, animals were reared individually in plastic cages with sawdust and corn bedding in the ratio 3:1 and kept for 2 weeks and 2 months. Animals in the 2-month experiment received 1 unit of long-acting insulin (Lantus Solostar, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany) weekly to prevent ketoacidosis.

Body mass and plasma glucose were measured regularly. For plasma glucose measurement, blood was collected from the tail vein of rats, and a blood glucose level was determined by single touch glucometer (OneTouchVITa, LifeScan, High Wycombe, UK). In the DM1 group, 5 rats had a glucose level less than 300 mg/dL and were excluded from the study. We continued the experiment with the remaining 58 rats.

Behavioral Testing

Pain associated with experimentally induced DM was tested with a battery of behavioral tests for measuring sensory responses in different experimental groups over time. Behavioral testing was performed on the day preceding the injection and on the 15th, 30th, 45th, and 60th day after DM induction. Tests used in this study were chosen based on their relevance to changes noted in clinical practice.19 During all testing procedures, rats were placed individually on a mesh-wire surface (3 × 3 mm) in clear plastic enclosures (10 × 25 × 30 cm). Tests included stimulation of the plantar skin of both hindpaws of unrestrained rats, according to previously described procedures.20 Cold sensitivity was assessed using the acetone test. For heat sensitivity, a custom-made radiant heat source equipped with a potentiometer for temperature control and with a 3 × 3 mm contact probe with maximal temperature of ~42°C was used. Mechanical hyperalgesia was tested by applying a noxious stimulus using a needle pinprick test and noting simple withdrawal and a complex hyperalgesia-type response characterized by sustained paw lifting, shaking, and licking.21 For assessment of mechanical allodynia, the von Frey test was used.

Tissue Collection and Immunohistochemistry

Two weeks or 2 months after DM induction rats were anesthetized (isoflurane; Forane, Abbott Laboratories Ltd., Queenborough, UK) and perfused through the ascending aorta via the left ventricle with saline followed by 300 mL of Zamboni fixative (4% paraformaldehyde and 15% picric acid in 0.1 M phosphate-buffered saline) at pH 7.4. The left and right L4 and L5 ganglia were removed and postfixed for 2 hours in the same fixative used for perfusion. After overnight cryoprotection in 30% sucrose, ganglia were embedded in optimal cutting temperature freezing medium (Tissue Tek, Tokyo, Japan) and sectioned on a cryostat (Thermo Shandon Cryotome, Pittsburgh, PA). Sections 7-µm thick were cut parallel along the long axis of the ganglia and placed on glass slides.

For detection of total CaMKII (tCaMKII) and its α, β, γ, and δ isoforms, immunohistochemical analysis was performed. Primary rabbit polyclonal antibodies were used for detection of tCaMKII (Table 1) phosphorylated CaMKIIα isoform (pCaMKIIα) and CaMKIIβ (Table 1).

Table 1
Table 1:
Primary Antibodies Used

For CaMKIIγ and CaMKIIδ detection, antibodies raised in goat were used (Table 1). tCaMKII polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) detects all CaMKII subunits of mouse, rat, and human origin (manufacturer’s technical datasheet) and has been previously characterized. It identifies a single band of 50 kDa protein on Western blot analysis of CaMKII expression in the rat brain extract (manufacturer’s technical datasheet) corresponding to the expected molecular weight of CaMKII. An identical band was identified in mouse cholangiocytes,22 human astrocytes and neurons,23 and rat brain extract.24

The pCaMKIIα antibody (Santa Cruz Biotechnology Inc.) is a polyclonal affinity purified antibody raised in rabbit against a short amino acid sequence (Aa 282 HRQET[-phospho]VDCLK 291) containing phosphorylated Thr 286 of CaMKIIα of human origin. The sequence of immunizing peptide was determined in our previous publication.13 This antibody has also been previously characterized. In mouse brain extracts analyzed by Western blotting, this antibody recognizes a single band of approximately 50 kDa protein corresponding to the expected molecular weight of CaMKII (manufacturer’s technical datasheet). Similar bands were identified in extracts from human astrocytes23 and rat hippocampal neurons.25 Moreover, specificity of the pCaMKIIα antibody was confirmed by preadsorption control with an appropriate blocking peptide (Santa Cruz Biotechnology Inc.; Cat. No. sc-12886-P, lot D2607).

The CaMKIIβ affinity purified antibody (Abcam, Cambridge, UK) is a polyclonal antibody raised in rabbit against synthetic peptide conjugated to keyhole limpet hemocyanin derived from within residues 350 to 450 of mouse CaMKIIβ. This antibody recognizes a single Western blot band of 60 kDa in both mouse and rat brain tissue lysates (manufacturer’s datasheet). Similar bands were indentified in tissue extracts from rat nucleus accumbens and dorsal striatum.26

The CaMKIIγ (sc-1541, Santa Cruz Biotechnology Inc.) is a goat polyclonal antibody raised against a peptide mapping at the C-terminus of CaMKIIγ of human origin. It recognizes 2 Western blot bands of approximately 43 kDa and approximately 52 kDa in mouse brain extract (manufacturer’s datasheet).

The CaMKIIδ (sc-5392, Santa Cruz Biotechnology Inc.) is a goat polyclonal antibody raised against a peptide mapping within an internal region of CaMKIIδ of rat origin. It recognizes 2 Western blot bands of approximately 70 kDa in mouse brain extract. Similar bands were found on mouse tissues by other research groups.27–30 Secondary detection of tCaMKII, pCaMKIIα, and CaMKIIβ was performed using biotinylated goat anti-rabbit secondary antibody immunoglobulin (Ig)G-B (1:100, sc-2040, lot no. L0309, Santa Cruz Biotechnology Inc.) followed by Streptavidin Alexa Fluor 488 conjugate (1:500; S-32354, lot 508205, Molecular Probes, Eugene, OR). Primary antibodies for CaMKIIγ and CaMKIIδ were detected with secondary donkey anti-sheep antibody (1:100, sc-2476, lot no. K1408, Santa Cruz Biotechnology Inc.). After final rinsing in phosphate-buffered saline, all slides were mounted, air-dried, and cover slipped (Immu-Mount, Shandon, Pittsburgh, PA). Staining controls included omission of primary antibody from the staining procedure, which resulted in no staining of DRG tissue.

Quantitative Analysis for Immunohistochemistry

Every fourth section of each DRG was examined under a microscope (BX61, Olympus, Tokyo, Japan), and microphotographs were captured using a cooled digital camera (DP71, Olympus) under the same magnification (×40), exposition, binning, and gain for each image. Images were then transported into MetaMorph software (Molecular Devices, Sunnyvale, CA) where they were examined as monochromic microphotographs (2040 × 1536 pixels, 12 bits, 0–4096 gray scale). Background subtraction was performed.

Each micrograph was divided into 100 × 100 µm squares with at least 80% surface occupied by ganglion neurons. Analysis of microphotographs with MetaMorph software was done very carefully, remembering that only cells with clearly visible nuclei should be measured. Fluorescence intensity of neuronal cytoplasm (MetaMorph delineating function) and surface area of each neuron were measured. We analyzed about 150 cells per animal for every immunostaining procedure in an effort to make our analysis as objective as possible.

Statistical Analysis

Comparisons between control and DM neuronal groups were made by Student t test or 1-way analysis of variance (ANOVA) followed by a Fisher post hoc test. Behavioral test scores were analyzed with repeated-measures ANOVA (Statistica 7.0; StatSoft, Tulsa, OK). The data were statistically analyzed and are presented as mean ±SEM. Any difference with P < 0.05 was considered statistically significant.

RESULTS

Validation of DM1 and DM2 Animal Models

Concentration of plasma glucose increased markedly in DM1 and DM2 animals after the diabetes induction. Plasma glucose of DM1 and DM2 animals on the 15th, 30th, 45th, and 60th experimental day was significantly higher than baseline values, as was glycemia of control animals measured on the corresponding day (ANOVA F(4,76) = 117.2, P < 0.0001 and F(4,20) = 3.65, P < 0.05 respectively, Fisher post hoc test). After the 15th postinjection day, when plasma glucose of DM1 and DM2 animals was 380 ± 16.2 mg/dL and 378 ± 37.6 mg/dL, respectively, DM2 animals retained constant plasma glucose until the end of the experiment and DM1 animals’ plasma glucose increased continuously until it reached a plateau on the 30th postinjection day at 546±11.7 mg/dL. C-DM1 and C-DM2 animals maintained normal plasma glucose throughout the experiment that did not differ significantly from baseline levels (111 ± 4.2 and 115 ± 9.54 mg/dL, respectively; Fig. 1A).

Figure 1
Figure 1:
(A) Plasma glucose levels and (B) relative body mass increase (expressed as a percentage of the body mass at the beginning of the experiment) in all experimental groups for 2 months.

Growth of DM1 rats was dramatically impaired after diabetes induction, and after 2 months the DM1 group lost 6% ± 7.9% of its baseline body mass value, whereas C-DM1 animals doubled their baseline weight (45% ± 9.0%; ANOVA F(1,19) = 21.62, P = 0.002, Fisher post hoc test). At the end of the experiment, DM2 and C-DM2 animals gained 84% ± 6.2% and 100% ± 2.2% of their initial body mass, respectively (Fig. 1B). There were no significant differences between these 2 groups.

Experimentally Induced DM1 Increases Pain-Related Behavior

DM1 animals developed hypersensitivity to cold and heat; the number of withdrawal responses to cold stimuli (Fig. 2A) was highest on the 15th postinjection day, when it started to decline until the end of the experiment, maintaining a significant difference in comparison to the C-DM1 group (ANOVA F(4,76) = 3.6, P = 0.001). DM1 animals also exhibited significantly shortened withdrawal latency after heat stimulus (ANOVA F(4,76) = 3.58, P = 0.01; Fig. 2B).

Figure 2
Figure 2:
(A) Number of withdrawal responses to cold stimulus; (B) response latency to heat stimulus; number of (C) withdrawal responses to mechanical stimulus; (D) hyperalgesic responses to mechanical stimulus; and (E) vonFrey with drawal threshold in the type 1 diabetes mellitus (DM1) animal model. Data are presented as mean ± SEM. Asterisk denotes significant difference from baseline.

The withdrawal response rate to mechanical stimuli was significantly increased in DM1 animals throughout the experiment, compared with the C-DM1 group (ANOVA F(4,76) = 7.34, P < 0.001; Fig. 2C). The number of hyperalgesic responses to needle stimulation was increased in DM1 animals, with the highest sensitivity on the 15th day (Fig. 2D), and declining afterwards (ANOVA, F(4,76) = 2.4, P = 0.05). Mechanical allodynia was tested with von Frey fibers. Diabetes induction was accompanied by the reduction of withdrawal threshold in DM1 animals throughout the experiment, but this reduction was not significantly different comparing with the C-DM1 group (ANOVA, F(4,76) = 0.71, P = 0.59; Fig. 2E). Behavioral analysis showed that DM2 animals failed to develop hypersensitivity to mechanical and thermal stimuli during the 2-month experiment (Fig. 3). In both DM2 and C-DM2 groups, not a single hyperalgesic response was observed during the 2-month follow-up (data not shown).

Figure 3
Figure 3:
(A) Number of withdrawal responses to cold stimulus; (B) response latency to heat stimulus; (C) number of withdrawal responses to mechanical stimulus; and (D) von Frey withdrawal threshold in type 2 diabetes mellitus (DM2) animals. Data are presented as mean ± SEM. There were no observed significant differences in nociceptive responses between 2 experimental groups in all behavioral tests used.

The Expression of CaMKII in DRG Neurons 2 Weeks and 2 Months After the STZ Injection

To investigate changes in DRGs caused by diabetes induction, immunofluorescence analysis of total CaMKII and its α, β, γ, and δ isoforms was performed. Two weeks after diabetes induction, tCaMKII expression (Figs. 4A and 5, A and B) was significantly higher in DM1 animals compared with controls (52 ± 7.5 and 29 ± 3.75, respectively; unpaired t test, P = 0.03). Further analysis revealed that this overall increase of tCaMKII in DM1 animals, compared with the C-DM1 group, was due to the increase in small diameter DRG neurons (d < 30 µm), usually considered nociceptors, and medium diameter neurons (30 µm < d < 40 µm) (unpaired t test, P = 0.03). A significant increase of pCaMKIIα expression (Figs. 4B and 5, C and D) was also observed in DM1 animals but only in large (d > 40 µm) DRG neurons (unpaired t test, P = 0.03). Induction of DM1 did not cause a change in expression of β, γ, and δ isoforms in DRG neurons, regardless of the neuronal size (data not shown).

Figure 4
Figure 4:
The expression of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII; A) and phosphorylated CaMKIIα isoform (pCaMKIIα; B) in control for type 1 diabetes mellitus group (C-DM1) (white bars) and DM1 rats (gray bars) 2 weeks after induction of diabetes. Diabetes induces a significant increase in tCaMKII and pCaMKIIα in L4 and L5 dorsal root ganglia neurons (t test). Data are presented as mean ± SEM. Asterisk denotes significant difference between groups.

In the 2-week experiments, there were no significant differences in expression of tCaMKII and all of its analyzed isoforms between DM2 and C-DM2 rats (Fig. 6). The expression of pCaMKIIα increased, but not significantly, in all DRG neurons of DM2 rats, compared with C-DM2 (94 ± 15.3 and 70 ± 9.1, respectively; Fig. 6B).

Figure 5
Figure 5:
Representative example of immunofluorescence intensity of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII) in control for type 1 diabetes mellitus group (C-DM1) (A), DM1 (B) and phosphorylated CaMKIIα isoform (pCaMKIIα) in C-DM1 (C), and DM1 (D) rats. Diabetes induction causes a significant increase in fluorescence intensity of tCaMKII (all cells and small [d < 30 µm], medium [30 µm < d < 40 µm] neuronal size group analyzed separately) and pCaMKIIα (large cells, d > 40 µm) in rats L4 and L5 dorsal root ganglia 2 weeks after streptozotocin (STZ) injection. Magnification: ×40. Scale bar 100 µm (applies to all).
Figure 6
Figure 6:
The expression of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII; A) and phosphorylated CaMKIIα isoform (pCaMKIIα; B) in control for type 2 diabetes mellitus group (C-DM2; white bars) and DM2 rats (gray bars) 2 weeks after induction of diabetes. There were no significant differences in tCaMKII and pCaMKIIα between experimental groups (t test). Data are presented as mean ± SEM.

Two months after diabetes induction tCaMKII expression in all DRG neurons of DM1 animals remained significantly increased compared with the C-DM1 group (16 ± 0.9 and 14 ± 2.0, respectively; unpaired t test, P = 0.05; Figs. 7A and 8, A and B). Expression of pCaMKIIα increased significantly in all DRG neurons of DM1 animals and in each neuronal size group analyzed separately (unpaired t test, P = 0.01) (Figs. 7B and 8, C and D). The expression of β, γ, and δ isoforms did not differ between DM1 and C-DM1 animals (data not shown). There was no difference in the expression of tCaMKII and pCaMKIIα between DM2 and C-DM2 rats 2 months after the diabetes induction (Fig. 9). Likewise, the expression of β, γ, and δ isoforms did not differ between DM2 and C-DM2 animals (data not shown).

Figure 7
Figure 7:
The expression of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII; A) and phosphorylated CaMKIIα isoform (pCaMKIIα; B) in control for type 1 diabetes mellitus group (C-DM1; white bars) and DM1 rats (gray bars) 2 months after induction of diabetes. Diabetes induces a significant increase in tCaMKII and pCaMKIIα in L4 and L5 dorsal root ganglia neurons. Data are presented as mean ± SEM. Asterisk denotes significant difference between groups (t test).
Figure 8
Figure 8:
Representative example of immunofluorescence intensity of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII) in control for type 1 diabetes mellitus group (C-DM1; A), DM1 (B) and phosphorylated CaMKIIα isoform (pCaMKIIα) in C-DM1 (C), and DM1 (D) rats. Diabetes induction causes a significant increase in fluorescence intensity of tCaMKII (all cells) and pCaMKIIα (all cells and small, medium, and large neuronal size groups analyzed separately) in rats L4 and L5 dorsal root ganglia 2 months after STZ injection. Magnification: ×40. Scale bar 100 µm (applies to all).
Figure 9
Figure 9:
The expression of total Ca2+/calmodulin-dependent protein kinase II (tCaMKII; A) and phosphorylated CaMKIIα isoform (pCaMKIIα; B) in control for type 2 diabetes mellitus group (C-DM2; white bars) and DM2 rats (gray bars) 2 months after induction of diabetes. There were no significant differences in tCaMKII and pCaMKIIα between experimental groups (t test). Data are presented as mean ± SEM.

DISCUSSION

In this study, we demonstrated that the expression of tCaMKII and pCaMKIIα increases in DRG neurons of a STZ-induced rat model of DM1. Increased CaMKII expression was observed at 2 weeks after diabetes induction, and it remained increased over the 2-month period. Changes in CaMKII expression in DRG neurons corresponded to the changes in pain-related behavior and development of mechanical and thermal hyperalgesia in DM1 rats. The DMZ animals, induced with a combination of a HFD and low-dose STZ, did not exhibit significant changes in CaMKII expression or pain-related behavior during the experiment.

The STZ DM1 rat model exhibits early neurological dysfunction,31 including altered pain sensation,32 that suggests the early involvement of small nociceptive sensory neurons.33,34 Results of behavioral testing in STZ DM1 rats vary in different experiments, depending on the rat species, baseline body mass, duration of the experiment, and STZ dose. Our findings are in agreement with the results of previous studies lasting 2 weeks35 and 2 months,36 where mechanical and thermal hypersensitivity were also observed in STZ DM1 rats.

Compared with DM1 models, diabetic neuropathy in DM2 models is usually less pronounced.37 Distinct differences in the functional, structural,37,38 and metabolic39 expressions of neuropathy in the 2 types of diabetes have been clearly delineated by investigators. The STZ DM2 models are expensive and lengthy experiments are rarely performed.18,38,39 In comparison with other experimental models of DM2 currently used, the model used in our experiment offers significant advantages in replicating the natural history and metabolic characteristics of human conditions.18,40 DM2 animals in our study developed less intense hyperglycemia than DM1 rats, which might be a reason why these animals did not exhibit changes in CaMKII expression and pain-related behavior during the 2-month follow-up. Yang et al. used a combination of HFD, high-sucrose diet and STZ injection (30 mg/kg) to induce DM2. They observed an increase in the plantar pain withdrawal latency 10 weeks after the diabetes induction. The probable source of pain-related behavior in diabetic animals in this study might have been the high level of sucrose which was added to their diet.41 It has been proven that prolonged ingestion of sucrose in adult rats leads to profound hyperalgesia.42

Systemic hyperglycemia is the most obvious symptom that 2 types of diabetes have in common. Various pathways activated by hyperglycemia converge in generation of excess reactive oxygen species (ROS), which ultimately leads to oxidative stress and proinflammatory conditions in the tissue.43 Chronic local in vivo perfusion of rat lumbar DRG with a hyperglycemic solution causes hyperalgesia in the hindlimb innervated by perfused ganglion, but not in the contralateral limb.44

Experimental diabetes changes expression of voltage gated potassium and sodium channel activity in DRG neurons, and this has been linked to the pathophysiology of diabetic neuropathy.45 Biessels and Gispen have suggested that disturbed calcium homeostasis is involved in the pathogenesis of diabetic neuropathy.46 CaMKII converts cytoplasmic calcium signals into diverse responses through phosphorylation of various substrates involved in exocytosis, transcriptional, and translational processes.47 Voltage-dependent calcium channels (VDCCs) are overexpressed in primary sensory neurons in diabetic rats.48 Intracellular calcium levels are increased in most diabetic tissues, and altered intracellular calcium metabolism may be a common, underlying abnormality linking the metabolic, cardiovascular, ocular, and neural manifestations of the diabetic disease process.49,50 In addition, it has been shown that central sensitization mechanisms are calcium dependent.51 There is ample evidence that infers a close relationship among sustained increase in calcium currents, the production of ROS, ischemia, and neuronal cell death.50 Alteration in expression of selective P/Q-type blocker of VDCCs, especially in the small and medium diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.52

CaMKII has been linked to many pathophysiological changes in STZ-induced diabetic rats. ROS and CaMKII expression increased in hearts of STZ-induced diabetic rats, and this increase can be inhibited with CaMKII antagonist.53 Furthermore, signal transduction involving CaMKII contributes to the development of abnormal vascular reactivity and renal dysfunction during simultaneous occurrence of hypertension and diabetes.54

STZ-induced diabetes resulted in a pronounced increase of CaMKII activity in rat brain. The total amount of enzyme also showed an increase. Insulin treatment resulted in recovery of enzyme activity near control values from the majority of the brain regions studied. The expression of CaMKIIα activity correlates with the enzyme activity in the diabetic rat brain.55

An overall increase in tCaMKII expression is the result of the individual increases in all of its isoforms. Although pCaMKIIα expression in large cells increased significantly in DM1 animals 2 weeks after diabetes induction, the expression of other CaMKII isoforms did not change significantly enough for this difference to be evident in tCaMKII expression. Likewise, a significant increase in tCaMKII expression in small diameter size neurons probably results from small but statistically insignificant increases in expression of its other isoforms.

CaMKII expression also increases in some other models of neuropathic pain in addition to the model used in our study. CaMKII is critical in maintaining aberrant dorsal horn neuron hyperexcitability in the neuropathic pain condition after spinal cord injury. In the spinal cord injury model, pCaMKII expression was significantly upregulated in the dorsal horn neurons and treatment with the CaMKII antagonist significantly attenuated mechanical allodynia.56 In our previous study, we found that spinal nerve ligation causes a decrease of CaMKII expression in DRG neurons and an increase of pain-related behavior in rats.13 The pathophysiology of pain in diabetes is somewhat different from the spinal nerve ligation neuropathic pain model, because in diabetes calcium levels increase in many tissues,49 VDCCs are overexpressed in diabetic DRG neurons,52 and voltage-dependent calcium currents are enhanced in DRG neurons,48 which results in intracellular calcium increase. The calcium overload may trigger CaMKII overexpression in DRG neurons and result in abnormal firing of DRG neurons. CaMKII signaling in neurons may promote cell death and result in degradation of neuronal tissue.57

Of all the CaMKII isoforms studied in our experiment, CaMKIIα is the most abundant in neurons of the central nervous system,9 and it has been implicated as playing a critical role in the modulation of the nociceptor activity and plasticity of primary sensory neurons.12 Of all isoforms of CaMKII analyzed in our study, only CaMKIIα became overexpressed after DM1 induction indicating that this isoform alone may be involved in the pathophysiology of abnormal perception of pain in diabetes.

Enzyme CaMKII is a downstream effector of multiple signaling pathways designed to convert cytoplasmic calcium signals into diverse cell responses,47 and increased intracellular calcium levels were proven to be connected with various abnormalities in the diabetic disease process.49,50 This abnormal calcium signaling most probably induced an increase in CaMKII expression and caused impaired nociception in DM1 animals in our study.

Other research teams have also reported differences in severity of neuropathic disturbances between the 2 types of diabetes.37–39 We assume that DM2 animals in our study failed to develop abnormal nociception because CaMKII levels in their DRG neurons remained unchanged during the 2-month follow-up. Furthermore, the level of glycemia of DM2 animals was lower compared with DM1 animals.

In conclusion, induction of DM1 caused an increase in CaMKII expression, corresponding to the characteristic nociceptive dysfunction, which may indicate an involvement of this enzyme in transmission of nociceptive input. This phenomenon should be explored further to determine whether CaMKII inhibition could result in improvement of nociceptive responses in DM1 rats.

DISCLOSURES

Name: Lejla Ferhatovic, MSc.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Lejla Ferhatovic has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Adriana Banozic, MSc.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Adriana Banozic has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sandra Kostic, MSc.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Sandra Kostic has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Tina Ticinovic Kurir, MD, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Tina Ticinovic Kurir has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Anela Novak, MD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Anela Novak has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Luka Vrdoljak, MD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Luka Vrdoljak has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Marija Heffer, MD, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Marija Heffer has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Damir Sapunar, MD, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Damir Sapunar has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Livia Puljak, MD, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Livia Puljak has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Jianren Mao, MD, PhD.

REFERENCES

1. Gooch C, Podwall D. The diabetic neuropathies. Neurologist. 2004;10:311–22
2. Price SA, Zeef LA, Wardleworth L, Hayes A, Tomlinson DR. Identification of changes in gene expression in dorsal root ganglia in diabetic neuropathy: correlation with functional deficits. J Neuropathol Exp Neurol. 2006;65:722–32
3. Kishi M, Tanabe J, Schmelzer JD, Low PA. Morphometry of dorsal root ganglion in chronic experimental diabetic neuropathy. Diabetes. 2002;51:819–24
4. Jagodic MM, Pathirathna S, Nelson MT, Mancuso S, Joksovic PM, Rosenberg ER, Bayliss DA, Jevtovic-Todorovic V, Todorovic SM. Cell-specific alterations of T-type calcium current in painful diabetic neuropathy enhance excitability of sensory neurons. J Neurosci. 2007;27:3305–16
5. Morris EP, Török K. Oligomeric structure of alpha-calmodulin-dependent protein kinase II. J Mol Biol. 2001;308:1–8
6. Eshete F, Fields RD. Spike frequency decoding and autonomous activation of Ca2+-calmodulin-dependent protein kinase II in dorsal root ganglion neurons. J Neurosci. 2001;21:6694–705
7. Molloy SS, Kennedy MB. Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices. Proc Natl Acad Sci USA. 1991;88:4756–60
8. Tobimatsu T, Fujisawa H. Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem. 1989;264:17907–12
9. Lucchesi W, Mizuno K, Giese KP. Novel insights into CaMKII function and regulation during memory formation. Brain Res Bull. 2011;85:2–8
10. Brüggemann I, Schulz S, Wiborny D, Höllt V. Colocalization of the mu-opioid receptor and calcium/calmodulin-dependent kinase II in distinct pain-processing brain regions. Brain Res Mol Brain Res. 2000;85:239–50
11. Carlton SM. Localization of CaMKIIalpha in rat primary sensory neurons: increase in inflammation. Brain Res. 2002;947:252–9
12. Carlton SM, Hargett GL. Stereological analysis of Ca(2+)/calmodulin-dependent protein kinase II alpha -containing dorsal root ganglion neurons in the rat: colocalization with isolectin Griffonia simplicifolia, calcitonin gene-related peptide, or vanilloid receptor 1. J Comp Neurol. 2002;448:102–10
13. Lovric-Kojundzic S, Puljak L, Hogan QH, Sapunar D. Depression of calcium/calmodulin dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation. J Comp Neurol. 2010;518:64–74
14. Hasegawa S, Kohro Y, Tsuda M, Inoue K. Activation of cytosolic phospholipase A2 in dorsal root ganglion neurons by Ca2+/calmodulin-dependent protein kinase II after peripheral nerve injury. Mol Pain. 2009;5:22
15. Kawano T, Zoga V, Gemes G, McCallum JB, Wu HE, Pravdic D, Liang MY, Kwok WM, Hogan Q, Sarantopoulos C. Suppressed Ca2+/CaM/CaMKII-dependent K(ATP) channel activity in primary afferent neurons mediates hyperalgesia after axotomy. Proc Natl Acad Sci USA. 2009;106:8725–30
16. Qin HY, Luo JL, Qi SD, Xu HX, Sung JJ, Bian ZX. Visceral hypersensitivity induced by activation of transient receptor potential vanilloid type 1 is mediated through the serotonin pathway in rat colon. Eur J Pharmacol. 2010;647:75–83
17. Weir GC, Clore ET, Zmachinski CJ, Bonner-Weir S. Islet secretion in a new experimental model for non-insulin-dependent diabetes. Diabetes. 1981;30:590–5
18. Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao P. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol Res. 2005;52:313–20
19. Lindblom U, Verrillo RT. Sensory functions in chronic neuralgia. J Neurol Neurosurg Psychiatr. 1979;42:422–35
20. Sapunar D, Vukojević K, Kostić S, Puljak L. Attenuation of pain-related behavior evoked by injury through blockade of neuropeptide Y Y2 receptor. Pain. 2011;152:1173–81
21. Hogan Q, Sapunar D, Modric-Jednacak K, McCallum JB. Detection of neuropathic pain in a rat model of peripheral nerve injury. Anesthesiology. 2004;101:476–87
22. Francis H, Glaser S, Demorrow S, Gaudio E, Ueno Y, Venter J, Dostal D, Onori P, Franchitto A, Marzioni M, Vaculin S, Vaculin B, Katki K, Stutes M, Savage J, Alpini G. Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway. Am J Physiol, Cell Physiol. 2008;295:C499–513
23. Song JH, Bellail A, Tse MC, Yong VW, Hao C. Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. J Neurosci. 2006;26:3299–308
24. Rose AJ, Hargreaves M. Exercise increases Ca2+-calmodulin-dependent protein kinase II activity in human skeletal muscle. J Physiol (Lond). 2003;553:303–9
25. Tai Y, Feng S, Ge R, Du W, Zhang X, He Z, Wang Y. TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway. J Cell Sci. 2008;121:2301–7
26. Reissner KJ, Uys JD, Schwacke JH, Comte-Walters S, Rutherford-Bethard JL, Dunn TE, Blumer JB, Schey KL, Kalivas PW. AKAP signaling in reinstated cocaine seeking revealed by iTRAQ proteomic analysis. J Neurosci. 2011;31:5648–58
27. Croatian National Bank. . General information on croatia —economic indicators. Available at: http://www.hnb.hr/statistika/e-ekonomski_indikatori.htm. Accessed April 3, 2008
28. Yamagata Y, Kobayashi S, Umeda T, Inoue A, Sakagami H, Fukaya M, Watanabe M, Hatanaka N, Totsuka M, Yagi T, Obata K, Imoto K, Yanagawa Y, Manabe T, Okabe S. Kinase-dead knock-in mouse reveals an essential role of kinase activity of Ca2+/calmodulin-dependent protein kinase IIalpha in dendritic spine enlargement, long-term potentiation, and learning. J Neurosci. 2009;29:7607–18
29. Akimoto T, Ribar TJ, Williams RS, Yan Z. Skeletal muscle adaptation in response to voluntary running in Ca2+/calmodulin-dependent protein kinase IV-deficient mice. Am J Physiol, Cell Physiol. 2004;287:C1311–9
30. Peng W, Zhang Y, Zheng M, Cheng H, Zhu W, Cao CM, Xiao RP. Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70. Circ Res. 2010;106:102–10
31. Coppey LJ, Davidson EP, Dunlap JA, Lund DD, Yorek MA. Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve. Int J Exp Diabetes Res. 2000;1:131–43
32. Romanovsky D, Hastings SL, Stimers JR, Dobretsov M. Relevance of hyperglycemia to early mechanical hyperalgesia in streptozotocin-induced diabetes. J Peripher Nerv Syst. 2004;9:62–9
33. Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. J Biol Chem. 2005;280:618–27
34. Craner MJ, Klein JP, Renganathan M, Black JA, Waxman SG. Changes of sodium channel expression in experimental painful diabetic neuropathy. Ann Neurol. 2002;52:786–92
35. Romanovsky D, Cruz NF, Dienel GA, Dobretsov M. Mechanical hyperalgesia correlates with insulin deficiency in normoglycemic streptozotocin-treated rats. Neurobiol Dis. 2006;24:384–94
36. Malcangio M, Tomlinson DR. A pharmacologic analysis of mechanical hyperalgesia in streptozotocin/diabetic rats. Pain. 1998;76:151–7
37. Sima AA, Sugimoto K. Experimental diabetic neuropathy: an update. Diabetologia. 1999;42:773–88
38. Sima AA, Zhang W, Xu G, Sugimoto K, Guberski D, Yorek MA. A comparison of diabetic polyneuropathy in type II diabetic BBZDR/Wor rats and in type I diabetic BB/Wor rats. Diabetologia. 2000;43:786–93
39. Li F, Abatan OI, Kim H, Burnett D, Larkin D, Obrosova IG, Stevens MJ. Taurine reverses neurological and neurovascular deficits in Zucker diabetic fatty rats. Neurobiol Dis. 2006;22:669–76
40. Chen D, Wang MW. Development and application of rodent models for type 2 diabetes. Diabetes Obes Metab. 2005;7:307–17
41. Yang XY, Sun L, Xu P, Gong LL, Qiang GF, Zhang L, Du GH. Effects of salvianolic scid A on plantar microcirculation and peripheral nerve function in diabetic rats. Eur J Pharmacol. 2011;665:40–6
42. Mukherjee K, Mathur R, Nayar U. Nociceptive responses to chronic stress of restraint and noxious stimuli in sucrose fed rats. Stress Health. 2001;17:297–305
43. Dobretsov M, Romanovsky D, Stimers JR. Early diabetic neuropathy: triggers and mechanisms. World J Gastroenterol. 2007;13:175–91
44. Dobretsov M, Hastings SL, Stimers JR, Zhang JM. Mechanical hyperalgesia in rats with chronic perfusion of lumbar dorsal root ganglion with hyperglycemic solution. J Neurosci Methods. 2001;110:9–15
45. Cao XH, Byun HS, Chen SR, Cai YQ, Pan HL. Reduction in voltage-gated K+ channel activity in primary sensory neurons in painful diabetic neuropathy: role of brain-derived neurotrophic factor. J Neurochem. 2010;114:1460–75
46. Biessels G, Gispen WH. The calcium hypothesis of brain aging and neurodegenerative disorders: significance in diabetic neuropathy. Life Sci. 1996;59:379–87
47. Fink CC, Meyer T. Molecular mechanisms of CaMKII activation in neuronal plasticity. Curr Opin Neurobiol. 2002;12:293–9
48. Hall KE, Sima AA, Wiley JW. Voltage-dependent calcium currents are enhanced in dorsal root ganglion neurones from the Bio Bred/Worchester diabetic rat. J Physiol (Lond). 1995;486(pt 2):313–22
49. Levy J, Gavin JR 3rd, Sowers JR. Diabetes mellitus: a disease of abnormal cellular calcium metabolism? Am J Med. 1994;96:260–73
50. Shutov L, Kruglikov I, Gryshchenko O, Khomula E, Viatchenko-Karpinski V, Belan P, Voitenko N. The effect of nimodipine on calcium homeostasis and pain sensitivity in diabetic rats. Cell Mol Neurobiol. 2006;26:1541–57
51. Stanfa LC, Dickenson AH. The role of non-N-methyl-D-aspartate ionotropic glutamate receptors in the spinal transmission of nociception in normal animals and animals with carrageenan inflammation. Neuroscience. 1999;93:1391–8
52. Umeda M, Ohkubo T, Ono J, Fukuizumi T, Kitamura K. Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice. Life Sci. 2006;79:1995–2000
53. Nishio S, Teshima Y, Takahashi N, Thuc LC, Saito S, Fukui A, Kume O, Fukunaga N, Hara M, Nakagawa M, Saikawa T. Activation of CaMKII as a key regulator of reactive oxygen species production in diabetic rat heart. J Mol Cell Cardiol. 2012;52:1103–11
54. Yousif MH, Akhtar S, Walther T, Benter IF. Role of Ca2+/calmodulin-dependent protein kinase II in development of vascular dysfunction in diabetic rats with hypertension. Cell Biochem Funct. 2008;26:256–63
55. Bhardwaj SK, Kaur G. Effect of diabetes on calcium/calmodulin dependent protein kinase-II from rat brain. Neurochem Int. 1999;35:329–35
56. Crown ED, Gwak YS, Ye Z, Yu Tan H, Johnson KM, Xu GY, McAdoo DJ, Hulsebosch CE. Calcium/calmodulin dependent kinase II contributes to persistent central neuropathic pain following spinal cord injury. Pain. 2012;153:710–21
57. Coultrap SJ, Vest RS, Ashpole NM, Hudmon A, Bayer KU. CaMKII in cerebral ischemia. Acta Pharmacol Sin. 2011;32:861–72
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