Lejla Ferhatovic

Expression of calcium/calmodulin-dependent protein kinase II and pain-related behavior in rat models of type 1 and type 2 diabetes

BACKGROUND:Abnormalities in peripheral nerves and dorsal root ganglia are noticed in the early stage of experimentally provoked diabetic neuropathy. Enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) may have a modulating role in diabetic neuropathy because of its role in calcium homeostasis. METHODS:A model of type 1 diabetes mellitus (DM1) was induced with 55 mg/kg of the streptozotocin and for DM2 induction a combination of high-fat diet and low-dose streptozotocin (35 mg/kg) was used. Pain-related behavior was analyzed using thermal and mechanical stimuli. Two weeks and 2 months after induction of diabetes rats were euthanized, and the expression of CaMKII and its isoforms in the dorsal root ganglia were analyzed using immunofluorescence. RESULTS:Both types of diabetes were successfully induced, as confirmed by hyperglycemia. Increased pain-related behavior became evident in DM1 rats in 2 weeks after diabetes induction, but not in DM2 rats. The expression of total CaMKII and the phosphorylated &agr; isoform of CaMKII increased in DM1 animals concurrently with pain-related behavior. Expression of &agr;, &bgr;, &ggr;, and &dgr; isoforms in DM1 animals and expression of total CaMKII and all of its analyzed isoforms in DM2 animals remained unchanged. CONCLUSIONS:Our findings may indicate involvement of CaMKII in transmission of nociceptive input early in DM1, but not in DM2. CaMKII may be a suitable pharmacological target for diabetic neuropathy.

Intraganglionic AAV6 Results in Efficient and Long-Term Gene Transfer to Peripheral Sensory Nervous System in Adult Rats

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×109 viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

Increased vitamin D receptor expression in dorsal root ganglia neurons of diabetic rats

The effects of vitamin D on the nervous system have been studied extensively. In spite of accumulating data about the substantial changes in the vitamin D receptor (VDR) signaling system, during different types of neuroinflammatory diseases, its role in diabetic neuropathy has not been investigated in detail. To assess the role of VDR signaling in diabetic neuropathy, we examined expression of VDRs in dorsal root ganglia (DRG) neurons in a rat model of streptozotocin-induced diabetes mellitus type 1. Diabetes mellitus (DM) type 1 was induced with streptozotocin in male Sprague-Dawley rats. After two months, expression of VDRs was analyzed immunohistochemically in the cytoplasm of L4 and L5 DRG neurons of diabetic rats. Semi-quantitative analysis for the determination of staining in nuclei and plasma-membranes of DRG neurons was performed. A significant increase in VDR expression was observed in DRG neurons of diabetic rats. Expression of VDRs was increased in the cytoplasm, nuclei and in cell membranes of neurons. An increase in VDR expression occurred in all neurons, but the greatest increase of fluorescence intensity in cytoplasm was observed in neurons of small diameter. Results of the present study indicate that the VDR signaling system could be a potential therapeutic target for diabetic neuropathy.

Sex differences in pain-related behavior and expression of calcium/calmodulin-dependent protein kinase II in dorsal root ganglia of rats with diabetes type 1 and type 2

Sex differences in pain-related behavior and expression of calcium/calmodulin dependent protein kinase II (CaMKII) in dorsal root ganglia were studied in rat models of Diabetes mellitus type 1 (DM1) and type 2 (DM2). DM1 was induced with 55mg/kg streptozotocin, and DM2 with a combination of high-fat diet and 35mg/kg of streptozotocin. Pain-related behavior was analyzed using thermal and mechanical stimuli. The expression of CaMKII was analyzed with immunofluorescence. Sexual dimorphism in glycemia, and expression of CaMKII was observed in the rat model of DM1, but not in DM2 animals. Increased expression of total CaMKII (tCaMKII) in small-diameter dorsal root ganglia neurons, which are associated with nociception, was found only in male DM1 rats. None of the animals showed increased expression of the phosphorylated alpha CaMKII isoform in small-diameter neurons. The expression of gamma and delta isoforms of CaMKII remained unchanged in all analyzed animal groups. Different patterns of glycemia and tCaMKII expression in male and female model of DM1 were not associated with sexual dimorphism in pain-related behavior. The present findings do not suggest sex-related differences in diabetic painful peripheral neuropathy in male and female diabetic rats.

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.

Changes of calcium/calmodulin-dependent protein kinase II expression in dorsal root ganglia during maturation in long-term diabetes.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered one of the key intracellular signaling proteins for development of neuropathy. We analyzed the expression of total CaMKII (tCaMKII) and its alpha, beta, gamma and delta isoforms in dorsal root ganglia (DRG) in a rat model of Diabetes mellitus type I (DM1), 6 months and 1 year after diabetes induction. Diabetes was induced with streptozotocin and confirmed by measuring glucose levels and weight increase. Immunohistochemistry was performed for detection of tCaMKII and its isoforms in L4 and L5 DRGs. A significant decrease of CaMKII alpha and beta isoforms was noted 6 months after diabetes induction, while CaMKII gamma and delta were significantly decreased after 12 months in diabetic rats compared to controls. Analysis of neuronal subgroups based on the neuronal diameter revealed that the expression of alpha, beta and delta isoforms decreased only in small-diameter neurons. In conclusion, a significant decrease of specific CaMKII isoforms in small-diameter DRG neurons may suggest involvement of CaMKII alpha, beta and delta in the development of complex events responsible for the development of neuropathy in long-term diabetes during maturation. CaMKII is a part of the neuronal pathway that regulates the firing properties of excitable cells, especially neurons, and decreased CaMKII activity may be responsible for generation of aberrant signals, hyperalgesia and neuropathic pain.

Calcium/calmodulin-dependent protein kinase II in dorsal horn neurons in long-term diabetes.

The aim of this study was to investigate the expression of total calcium/calmodulin-dependent protein kinase II (CaMKII) and its phosphorylated &agr; isoform in the dorsal horn of the spinal cord in an animal model of long-term diabetes. Diabetes was induced in Sprague–Dawley rats using 55 mg/kg streptozotocin, and expression of total CaMKII, the phosphorylated &agr;-CaMKII isoform, and isolectin B4 was analyzed by immunohistochemical analysis in the dorsal horn of the spinal cord 6 and 12 months after diabetes induction. Results were compared with those for control rats of the same age. Increased expression of total CaMKII and its activated &agr; isoform was seen in the dorsal horn of diabetic rats 6 months after diabetes induction. The increase in CaMKII fluorescence was restored to control values after 12 months. The expression of activated &agr;-CaMKII 12 months after diabetes induction was most pronounced in laminae I–VI of the dorsal horn, not corresponding with the highest expression of isolectin B4 in laminae I–III. Increased expression of CaMKII in the dorsal horn during long-term diabetes could be involved in the development of neuropathic symptoms in diabetes. The expression pattern of CaMKII during long-term diabetes indicates that it affects the entire sensory input.

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.

Pain catastrophizing changes during the menstrual cycle

Pain catastrophizing is an important predictor of pain intensity and pain-related outcomes. Many studies have shown that the level of this phenomenon is higher in women compared to men. The aim of this study was to investigate whether there is a difference in pain catastrophizing in women during the different phases of their menstrual cycle and whether there is a difference in pain catastrophizing depending on the history of childbirth and dysmenorrhea. A prospective study was conducted among 149 healthy women aged 18–35, with a regular menstrual cycle, 80 of which were nulliparous. The participants filled a sociodemographic questionnaire at the enrollment and the Pain Catastrophizing Scale on the 1st, 12th, and 20th day of the menstrual cycle. Pain catastrophizing scores, including all the subscales, significantly varied throughout the menstrual cycle, being highest on the first day of menstrual cycle and declining subsequently. Pain catastrophizing scores were higher in nulliparous than in parous women. Higher pain catastrophizing scores on the first day of the menstrual cycle were found in dysmehorrhoic women and women who regularly use analgesics for dysmenorrhea. Knowing that pain catastrophizing varies throughout the menstrual cycle may help in creating interventions for pain prevention and treatment in cycling women.