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We studied the expression of mismatch repair genes (MMRs)-mutS protein homolog 2 (MSH2), PMS2, MutL homolog 1 (MLH1), and yH2AFX in diabetic rat kidneys. Streptozotocin-induced diabetes mellitus type 1 rat model (DM1) was used. Renal samples were collected 2 weeks and 2 months after DM1 induction and immunohistochemical expression of MMR genes in the renal cortex was analyzed. Diabetic animals showed lower MSH2 and higher yH2AFX kidney expression both 2 weeks and 2 months after DM1 induction. MLH1 expression significantly increased 2 weeks after DM1 induction (P < 0.0001). The most substantial differences were observed in the period 2 weeks after induction, with lower MSH2 and higher MLH1 expression in the proximal convoluted tubules and distal convoluted tubules (DCT) of diabetic animals (P < 0.001). yH2AFX expression significantly increased in the DCT of diabetic animals at both time points (P < 0.001 ; P < 0.01). PMS2 expression changed only in the glomeruli, where it significantly decreased 2 months after DM1 induction (P < 0.05). We concluded that the most substantial changes in renal expression of MMRs are happening already 2 weeks after diabetes induction, predominantly in the proximal convoluted tubules and DCT. Moreover, DCT could have a critical role in the pathophysiology of diabetic nephropathy (DN) and might be a future therapeutic target in this condition. The obtained results point to the MMRs as a potential factor in the development and progression of DN, as well as the possible link between DN and renal carcinogenesis.

The purpose of this study was to determine the changes in the expression of sigma 1 receptors (σ1Rs) in the kidney of diabetic rats, which could indicate their possible role in the pathogenesis of diabetic nephropathy (DN). Sprague-Dawley rats were were given intraperitoneal injection of 55 mg/kg streptozotocin (STZ) in order to induce type I of diabetes (DM1). Control and diabetic rats were sacrificed 2 weeks or 2 months after DM1 induction. Expression of σ1Rs was determined in kidneys of the experimental rats, using immunohistochemistry. The most prominent expression of σ1Rs was found in distal tubuli (DT). Results have shown significant increase in renal σ1Rs section percentage area of rats 2 months after DM1 induction, compared to both control group at the same age and diabetic group 2 weeks after induction (P < 0.01 both). Similarly, a number of immunoreactive DT increased in diabetic group 2 months after induction, compared to DM1 group 2 weeks after induction (P < 0.001). We also found a decrease of a number of immunoreactive DT 2 weeks post DM1 induction (P < 0.01). However, the same was found during maturation of the control rats (P < 0.001). In addition, a strong co-expression of σ1R and proinflammatory factor TGFβ was seen in vacuolated DT. The results indicate to the potential role of σ1Rs in postnatal maturation of the rat kidneys and in distal tubular damage in the pathogenesis of the diabetic nephropathy. We conclude that σ1Rs could be potential target in treatment of the diabetic nephropathy.

Background Diabetic nephropathy is a progressive condition which develops for many years. We analyzed expression of Snail and serum response factor (SRF), epithelial-mesenchymal transition (EMT) regulatory transcription factors with a key role in renal fibrosis, in different renal areas of diabetic rats during ageing. Methods Male Sprague–Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). DM group received insulin weekly to prevent ketoacidosis. After 2 weeks, 2, 6 and 12 months kidney samples were collected and analysed in different renal areas. Results Snail expression was located within cortex in proximal convoluted tubules, in control and DM groups, in the cytoplasm. Percentage of Snail-positive cells in control groups was high and decreased with time, whereas in DM groups the highest percentage was after 2 weeks. In all time points, smaller percentage of Snail expression was seen in DM groups compared to controls. SRF expression was mostly located in the proximal convoluted tubules, always in the cytoplasm. In control groups SRF was expressed in all time periods in proximal convoluted tubules, with decrement after 12 months. Percentage of SRF-positive cells was higher in control groups compared to DM in all time points, with the exception of 12 months. To a smaller degree, SRF expression was seen in the glomeruli and distal convoluted tubules, with more SRF positive cells in DM compared to their control groups. Conclusions While Snail expression remained lower in diabetic tissues, compared to controls, expression of SRF increased in diabetic tissues in the second part of the year. These changes may need long time to develop, and, in line with earlier reports, it is possible that insulin treatment of DM rats once a week reduces possibility of EMT and development of renal fibrosis even in the long term.

Background It is believed that tubulo-interstitial fibrosis and atrophy in diabetic patients are directly associated with the progression of chronic kidney disease, CKD. AIF is one of the crucial factors responsible for mitochondrial apoptosis, however, it can also promote cell survival independently from its role in apoptosis, and therefore can be potentially used as a tool in prevention of the onset of CKD in diabetic patients. Our aim was to investigate the significance of AIF expression in the development of CKD by observing the expression of AIF in 2 weeks’ and 2 months’ kidneys of diabetic rats compared to their controls. Methods Male Sprague–Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). After 2 weeks and 2 months kidney samples were collected and analysed in different renal areas. Results Characteristic morphologic changes were found between the 2 months’ control and 2 months’ diabetic groups. Those changes, including fibrosis and possible replacement of podocytes with connective tissue were mainly present in the glomeruli. AIF expression was seen in the both cortex, and in the collecting ducts of the medulla. Strong intensity of AIF expression was seen in proximal and distal convoluted tubules in both diabetic groups. In the control groups the glomeruli showed no AIF staining but moderate staining was seen in both diabetic groups. Overall, the percentage of AIF positive cells in the glomeruli was the lowest. The greatest rise in cell positivity was displayed from the 2 weeks’ control group to 2 weeks’ diabetes group (38 %) in glomeruli. The cell positivity of the 2 weeks’ diabetic group is significantly reduced to 18 % in the 2 months’ diabetic group in glomeruli. A similar pattern was seen in the proximal tubular cells (92 % positivity 2 weeks diabetic groups; 89 % positivity 2 months diabetic groups), as well as in the distal tubules. The highest percentage of AIF positive cells was seen in the collecting ducts, more than 80 % in all groups. Conclusions Our study provides insight into AIF expression pattern during short term diabetes model, confirming possible dual role of AIF, not only in apoptosis but also in cell function and homeostasis, and proving AIF as potential therapeutic target and marker of advancement of CKD.

Changes in sensory and sympathetic innervation during diabetes mellitus (DM) can be a predictor of arrhythmias, silent myocardial ischemia, and chronic heart failure, but knowledge about these changes is still unsatisfactory. We analyzed whether prolonged DM induces changes in density of sensory and sympathetic nerve terminals of rat's heart and whether it contributes to cardiomyopathy during aging. DM was induced by i/p injecting 55 mg/kg streptozotocin to male Sprague-Dawley rats, while a control group received a citrate buffer. DM in the rats was validated by measuring blood glucose level. Animals were sacrificed after 2 weeks, 2 months, 6 months, and 12 months. Five areas of cardiac sections were analyzed. Antibodies raised against tyrosine hydroxylase (TH) and neurofilament 200 kDa (NF 200) were used to detect sympathetic and sensory fibers. TH immunoreactive fiber density increased in DM groups 2 weeks after induction, reaching a peek after 2 months, while in the later stages of DM (6 and 12 months), there was no significant difference compared to control. NF 200 immunoreactive fiber density increased 2 weeks after induction compared to control. There was no consistent pattern of change during the given period in both the DM or control groups. In the DM group, we found thickening of the left ventricle wall (P

STUDY DESIGN Spine deformity and pain-related behavior after laminectomy with and without spine stabilization were investigated. OBJECTIVE We tested hypothesis that spine stabilization after extensive laminectomy can prevent spine deformation and consequent pain-related behavior. SUMMARY OF BACKGROUND DATA Various ablative procedures requiring laminectomy have been tested for prevention or reversal of pain-related behavior in studies using experimental animals. However, there is no precise description indicating how laminectomy should be performed. Lack of standardized surgical techniques makes it difficult to achieve uniformity of result reporting and to compare results of different research groups meaningfully. METHODS To test our hypothesis, extensive laminectomy with and without spine stabilization was performed in Sprague-Dawley rats. U-shaped surgical wire was used for stabilization of the spine. A validated test of mechanical hyperalgesia was used to test the development of neuropathic pain behavior after surgery. Deformity of the spine was evaluated by calculating deviation from the central axis on radiographs obtained in anteroposterior projection. RESULTS Surgical stabilization of the spine after laminectomy prevented development of spinal deformity. Laminectomy without stabilization induced hyperalgesia on the 8th and 15th days after surgery. Group with stabilized spine exhibited significant reduction in pain-related behavior on the 8th and 15th postoperative days compared with the group without stabilization. CONCLUSION Surgical stabilization of the spine after laminectomy prevented development of spinal deformity and pain-related behavior. Our results suggest that spine stabilization procedure should be used in all experimental pain models in which laminectomy is performed.

Direct injection of agents into the dorsal root ganglia (DRGs) offers the opportunity to manipulate sensory neuron function at a segmental level to explore pathophysiology of painful conditions. However, there is no described method that has been validated in detail for such injections in adult rats. We have found that 2 (µl of dye injected through a pulled glass pipette directly into the distal DRG, exposed by a minimal foraminotomy, produces complete filling of the DRG with limited extension into the spinal roots. Injection into the spinal nerve required 3 µl to achieve comparable DRG filling, produced preferential spread into the ventral root, and was accompanied by substantial leakage of injected solution from the injection site. Injections into the sciatic nerve of volumes up to 10 (µl did not reach the DRG. Transient hypersensitivity to mechanical stimulation at threshold (von Frey) and noxious levels (pin) developed after 2 µl saline injection directly into the DRG that was in part attributable to the surgical exposure procedure alone. Only minimal astrocyte activation in the spinal dorsal horn was evident after DRG saline injections. Injection of adeno-associated virus (AAV) vector conveying green fluorescent protein (GFP) transgene resulted in expression as soon as 1 day after injection into the DRG, including fibers in the spinal dorsal horn and columns. AAV injection into the DRG produced additional thermal hypersensitivity and withdrawal from the stroke of a brush and compromised motor performance. These findings demonstrate a method for selective injection of agents into single DRGs for anatomically restricted actions.

Background Diabetic nephropathy (DN) is a common complication of diabetes mellitus (DM). We studied the expression of special AT-rich sequence binding protein 1 (SATB1) and phosphatase and tensin homologue (PTEN) in the kidneys of diabetic rats during ageing. Methods Male Sprague Dawley rats were injected with 55 mg/kg streptozotocin (STZ) (DM group) or with citrate buffer (control group). Kidneys were collected after 2 weeks, 6 months and 12 months, and were analysed in three different kidney structures: glomeruli, proximal (PCT) and distal convoluted tubules (DCT). Sections were stained immunohistochemically, using SATB1 and PTEN. Results Significant differences in marker expression were observed after 2 weeks, with higher SATB1 expression and lower PTEN expression in diabetic rats. PTEN was more highly expressed in controls after 6 and 12 months. After 12 months, there was higher SATB1 expression in diabetic rats. In the glomeruli, control rats had higher PTEN expression, whereas diabetic rats had higher SATB1 expression, after 12 months. PTEN expression increased from 2 weeks to 12 months in both the PCT and DCT of control rats. SATB1 was expressed exclusively in the PCT of diabetic rats after 2 weeks, and its expression in the DCT was higher in controls. After 6 months, both the PCT and DCT showed higher SATB1 expression in diabetic rats. Conclusions The major changes in expression of SATB1 and PTEN occur after 2 weeks of DM onset, particularly in the PCT, implying an early onset of pathophysiological changes in diabetic kidneys, which would normally occur with ageing. These findings help to contribute to our understanding of changes associated with DN and guide towards possible appropriate treatment modalities.

Intoduction: Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in transmission of nociceptive input in diabetic neuropathy. The aim of this study was to test whether intraganglionic injection of CaMKII inhibitors may alleviate pain-related behavior in diabetic rats. Methods: 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 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 h and 24 h after the intraganglionic injection. The expression of total CaMKII and its alpha isoform in DRG neurons was analyzed using immunohistochemistry. Results: CaMKII inhibitors attenuated pain-related behavior in a modality-specific fashion. Attenuation of nociceptive behavior was accompanied with corresponding decrease of CaMKII alpha expression in DRG neurons on the side of injection. Significant decrease of CaMKII alpha expression was seen in small and medium-sized neurons. Discussion: Studies using intraganglionic delivery of pharmacological agents have been seldomly used in the basic studies of 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. In this study, only posstreatment approach was used, since diabetic neuropathy occurs after the onset of the disease. We found that two CaMKII inhibitors, with different mechanism of action, may attenuate pain-related behavior in a modality-specific fashion.

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.

Currents through voltage-gated Ca2+channels (ICa) may be regulated by cytoplasmic Ca2+levels ([Ca2+]c), producing Ca2+-dependent inactivation (CDI) or facilitation (CDF). SinceICaregulates sensory neuron excitability, altered CDI or CDF could contribute to pain generation after peripheral nerve injury. We explored this by manipulating [Ca2+]cwhile recordingICain rat sensory neurons. In uninjured neurons, elevating [Ca2+]cwith a conditioning prepulse (−15 mV, 2 s) inactivatedICameasured during subsequent test pulses (−15 mV, 5 ms). This inactivation was Ca2+-dependent (CDI), since it was decreased with elimination of Ca2+influx by depolarization to above theICareversal potential, with high intracellular Ca2+buffering (EGTA 10 mmor BAPTA 20 mm), and with substitution of Ba2+for extracellular Ca2+, revealing a residual voltage-dependent inactivation. At longer latencies after conditioning (>6 s),ICarecovered beyond baseline. This facilitation also proved to be Ca2+-dependent (CDF) using the protocols limiting cytoplasmic Ca2+elevation. Ca2+/calmodulin-dependent protein kinase II (CaMKII) blockers applied by bath (KN-93, myristoyl-AIP) or expressed selectively in the sensory neurons (AIP) reduced CDF, unlike their inactive analogues. Protein kinase C inhibition (chelerythrine) had no effect. Selective blockade of N-type Ca2+channels eliminated CDF, whereas L-type channel blockade had no effect. Following nerve injury, CDI was unaffected, but CDF was eliminated in axotomized neurons. Excitability of sensory neurons in intact ganglia from control animals was diminished after a similar conditioning pulse, but this regulation was eliminated by injury. These findings indicate thatICain sensory neurons is subject to both CDI and CDF, and that hyperexcitability following injury-induced loss of CDF may result from diminished CaMKII activity.

Neuropeptide Y (NPY) has an important but still insufficiently defined role in pain modulation. We therefore examined the ability of NPY to modulate experimentally induced neuropathic pain by injecting it directly into dorsal root ganglion (DRG) immediately following spinal nerve ligation (SNL) injury. We have found that this application exacerbates pain-related behavior induced by SNL in a modality-specific fashion. When saline was injected after SNL, the expected increase in hyperalgesia responses to needle stimulation was present on the 8th postoperative day. When we injected NPY, hyperalgesic responses were increased in a manner similar to the SNL/saline group. To characterize NPY action, specific Y1 and Y2 antagonists were also delivered directly to DRG, which revealed that behavioral actions of NPY were abolished by Y2 receptor antagonist. We tested whether NPY effects were the result of its role in immunity by immunohistochemical staining for glial fibrillary acidic protein, in order to identify activation of DRG satellite cells and dorsal horn astrocytes. Exacerbation of pain-related behavior following NPY injection was accompanied by astrocyte activation in ipsilateral dorsal horn and with satellite cells activation in the DRG proximal to injury. This activation was reduced following Y2 receptor antagonist application. These findings indicate an important link between pain-related behavior and neuroimmune activation by NPY through its Y2 receptor.