Matija Boric

The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy

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 24h 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.

Reduced epidermal thickness, nerve degeneration and increased pain-related behavior in rats with diabetes type 1 and 2.

To examine the mechanisms contributing to pain genesis in diabetic neuropathy, we investigated epidermal thickness and number of intraepidermal nerve fibers in rat foot pad of the animal model of diabetes type 1 and type 2 in relation to pain-related behavior. Male Sprague-Dawley rats were used. Diabetes type 1 was induced with intraperitoneal injection of streptozotocin (STZ) and diabetes type 2 was induced with a combination of STZ and high-fat diet. Control group for diabetes type 1 was fed with regular laboratory chow, while control group for diabetes type 2 received high-fat diet. Body weights and blood glucose levels were monitored to confirm induction of diabetes. Pain-related behavior was analyzed using thermal (hot, cold) and mechanical stimuli (von Frey fibers, number of hyperalgesic responses). Two months after induction of diabetes, glabrous skin samples from plantar surface of the both hind paws were collected. Epidermal thickness was evaluated with hematoxylin and eosin staining. Intraepidermal nerve fibers quantification was performed after staining skin with polyclonal antiserum against protein gene product 9.5. We found that induction of diabetes type 1 and type 2 causes significant epidermal thinning and loss of intraepidermal nerve fibers in a rat model, and both changes were more pronounced in diabetes type 1 model. Significant increase of pain-related behavior two months after induction of diabetes was observed only in a model of diabetes type 1. In conclusion, animal models of diabetes type 1 and diabetes type 2 could be used in pharmacological studies, where cutaneous changes could be used as outcome measures for predegenerative markers of neuropathies.

Diabetes mellitus affects activity of calcium/calmodulin-dependent protein kinase II alpha in rat trigeminal ganglia

The activity of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The aim of this study was to investigate the immunoreactivity of phosphorylated CaMKIIα (pCaMKIIα) in subpopulations of trigeminal ganglion (TG) neurons in rat models of early diabetes type 1 (dm1) and 2 (dm2). DM1 model was induced with intraperitoneally (i.p.) injected streptozotocin (STZ) (55mg/kg). DM2 rats were fed with the high fat diet (HFD) for 2 weeks and then received 35mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and immunohistochemical analysis for detection of pCaMKIIα immunoreactivity and double immunofluorescence labelling with isolectin (IB4) was performed. Increased intensity of pCaMKIIα immunofluorescence, restricted to IB4-negative small-diameter neurons, was seen in TG neurons two months after STZ-DM1 induction. DM1 model, as well as the obesity (control dm2 groups) resulted in neuronal impaired growth while dm2 model led to neuron hypertrophy in TG. Observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. In future, innovative strategies for modulation of CaMKIIα activity in specific subpopulations of neurons could be a novel approach in therapy of diabetic trigeminal neuropathy.

Changes in epidermal thickness and cutaneous innervation during maturation in long-term diabetes

AIM Peripheral nerve fiber depletion in patients with chronic diabetes mellitus (DM) was linked to neuropathic symptoms, development of pain, foot ulcerations and lower extremity amputation. The aim of this study was to analyze cutaneous changes, including paw epidermal thickness and intraepidermal nerve fiber (IENF) density in long-term diabetes, in rats 6 months and 12 months after induction of diabetes. MATERIALS AND METHODS Epidermal thickness and IENF density were studied in Sprague-Dawley diabetic rats 6 months and 12 months after diabetes induction with streptozotocin. Epidermal thickness was evaluated using hematoxylin and eosin staining. Peripheral nerve fibers were stained with polyclonal antiserum against protein gene product 9.5 (PGP 9.5). Successful diabetes induction was validated by measuring plasma glucose and body mass regularly throughout the experiment. RESULTS This study showed that long-term diabetes, induced in Sprague-Dawley rats with streptozotocin, is characterized with significant epidermal thinning and reduction of intraepidermal nerve fibers, 6 months and 12 months after induction of diabetes. CONCLUSION Long-term studies of streptozotocin models of diabetes could be used for making normative IENF densities that can be later used as age-dependent normative values for studying new treatment modalities.

Intrathecal inhibition of calcium/calmodulin-dependent protein kinase II in diabetic neuropathy adversely affects pain-related behavior.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy.

Authors’ lack of awareness and use of core outcome set on postoperative pain in children is hindering comparative effectiveness research

AIM To analyze awareness about and acceptability of core outcome set (COS) for pediatric pain recommended by the PedIMMPACT. METHODS We invited authors of systematic reviews and randomized controlled trials about interventions for postoperative pain in children to participate in a survey. RESULTS Only a third of surveyed authors of systematic reviews and randomized controlled trials about postoperative pain in children had heard about the PedIMMPACT COS for acute pediatric pain. Problems indicated as preventing them from using the COS were lack of awareness, difficulties with implementation, and lack of resources. CONCLUSION Further discussions about the adequacy of COS for acute pediatric pain, as well as interventions to increase the uptake of COS may be warranted.

Anesthesia and perioperative pain management during cardiac electronic device implantation

Background The degree of pain caused by the implantation of cardiac electronic devices (CEDs) and the type of anesthesia or perioperative pain management used with the procedure have been insufficiently studied. The aim of this study was to analyze perioperative pain management, as well as intensity and location of pain among patients undergoing implantation of CED, and to compare the practice with published guidelines. Patients and methods This was a combined retrospective and prospective study conducted at the tertiary hospital, University Hospital Split, Croatia. The sample included 372 patients who underwent CED implantation. Perioperative pain management was analyzed retrospectively in 321 patients who underwent CED implantation during 2014. In a prospective study, intensity and location of pain before, during, and after the procedure were measured by using a numerical rating scale (NRS) ranging from 0 to 10 in 51 patients at the same institution from November 2014 to August 2015. Results A quarter of patients received analgesia or sedation before surgery. All the patients received local lidocaine anesthesia. After surgery, 31% of patients received pain medication or sedation. The highest pain intensity was observed during CED implantation with the highest NRS pain score being 8. Some patients reported severe pain (NRS >5) also at 1, 3, 6, 8, and 24 hours after surgery. The most common pain locations were surgical site, shoulder, and chest. Adherence to guidelines for acute perioperative pain management was insufficient. Conclusion Patients may experience severe pain during and after CED implantation. Perioperative pain management was suboptimal, and higher doses of sedation and intensive analgesia are required. Guidelines for acute perioperative pain management and anesthesia during CED implantation should be developed.

Analysis of perioperative pain management in vascular surgery indicates that practice does not adhere with guidelines: a retrospective cross-sectional study

Background Inadequate treatment of pain related to surgery may be associated with complications and prolonged recovery time and increased morbidity and mortality rates. We investigated perioperative pain management in vascular surgery and compared it with the relevant guidelines for the treatment of perioperative pain. Methods We conducted a retrospective study on 501 patients who underwent vascular surgery at the University Hospital Split, Croatia. We collected the following data from patients’ charts: age, gender, premedication, preoperative patient’s physical status, type of surgery, duration of surgery and anesthesia, type of anesthesia, postoperative analgesia, and need for intensive care. We examined departmental procedures to assess adherence to guidelines for perioperative pain management. Results None of the 501 patients’ charts recorded information about perioperative pain intensity, 28% of patients did not receive any medication the night before their elective surgical procedures, and 17% of patients did not receive premedication immediately before the procedure. Most patients (66%) did not receive any pain medication in the operating room after surgery. Following surgery, 36% of patients were monitored in the intensive care units, while the rest were released to the ward. Some patients (17%) did not receive any analgesia after surgery. Procedures at the department did not adhere to the current recommendations for perioperative pain management. Conclusion The study indicates that management of surgery-related pain in complex vascular procedures at this hospital did not follow guidelines for the management of acute perioperative pain. Our finding that most patients did not receive appropriate analgesia after vascular surgery leads to the conclusion that the institution would benefit from developing guidelines for the management of acute perioperative pain, which should be applied in all cases.

The expression of calcium/calmodulin-dependent protein kinase II in the dorsal horns of rats with type 1 and type 2 diabetes

The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) has been proposed as a key factor in chronic pain development. This study therefore aimed to investigate the expression of CaMKII in the dorsal horn in a rat model of early phase diabetes mellitus (DM) types 1 and 2. Sprague-Dawley rats were used. DM1 was induced using streptozotocin (STZ) (55mg/kg injected intraperitoneally (i.p.)). DM2 was induced using a combination of a high fat diet (HFD) and STZ (35mg/kg i.p.). Controls received an i.p. injection of pure citrate buffer solution. DM2 animals and their controls also received HFD 2 weeks prior to the i.p. injection. Rats were sacrificed 2 weeks and 2 months after diabetes induction. The expression of tCaMKII, pCaMKIIα and IB4 in the dorsal horns was quantified using immunohistochemistry. Increased expression of tCaMKII and pCaMKIIα was seen in the dorsal horns of DM1 animals 2 weeks and 2 months after diabetes induction. In DM2 animals, similar changes in the expression of tCaMKII and pCaMKIIα were observed after 2 weeks, but not after 2 months. The expression of pCaMKIIα was most pronounced in laminae I-III. No difference in IB4 expression was observed between the groups. These results suggest a potential role for CaMKII in diabetic neuropathy development. Inhibition of CaMKII signaling pathways should be further explored as a potential treatment target in painful diabetic neuropathy.

Cutaneous expression of calcium/calmodulin-dependent protein kinase II in rats with type 1 and type 2 diabetes.

Changes in calcium-calmodulin protein kinase II (CaMKII) have been well demonstrated in nervous tissue of diabetic animal models. Skin shares the same ectodermal origin as nervous tissue and it is often affected in diabetic patients. The goal of this study was to analyze expression of CaMKII in rat foot pad 2 weeks and 2 months after induction of diabetes type 1 and 2. Forty-two Sprague-Dawley rats were used. Diabetes mellitus type 1 (DM1) was induced with intraperitoneally (i.p.) injected 55 mg/kg of streptozotocin (STZ) and diabetes mellitus type 2 (DM2) with a combination of high-fat diet (HFD) and i.p. injection of low-dose STZ (35 mg/kg). Two weeks and two months following diabetes induction rats were sacrificed and skin samples from plantar surface of the both hind paws were removed. Immunohistochemistry was performed for detection of total CaMKII (tCaMKII) and its alpha isoform (pCaMKIIα). For detection of intraepidermal nerve fibers polyclonal antiserum against protein gene product 9.5 (PGP 9.5) was used. The results showed that CaMKII was expressed in the skin of both diabetic models. Total CaMKII was uniformly distributed throughout the epidermis and pCaMKIIα was limited to stratum granulosum. The tCaMKII and pCaMKIIα were not expressed in intraepidermal nerve fibers. Two weeks after induction of diabetes in rats there were no significant differences in expression of tCaMKII and pCaMKIIα between DM1 and DM2 compared to respective controls. In the 2-month experiments, significant increase in epidermal expression of tCaMKII and pCaMKIIα was observed in DM1 animals compared to controls, but not in DM2 animals. This study is the first description of cutaneous CaMKII expression pattern in a diabetic model. CaMKII could play a role in transformation of skin layers and contribute to cutaneous diabetic changes. Further research on physiological role of CaMKII in skin and its role in cutaneous diabetic complications should be undertaken in order to elucidate its function in epidermis.