Natalija Filipović

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.

Effects of isoflurane postconditioning on chronic phase of ischemia-reperfusion heart injury in rats

INTRODUCTION The application of isoflurane in a postconditioning manner, during early reperfusion of ischemic myocardium, reduces the infarct size. Its favorable effect on highly vascularized granulation tissue formation is very important considering the fact that increased genesis of blood vessels in peri-infarct zone reduces the infarct size and improves cardiac function. Taking into consideration the influence of isoflurane on the subacute phase of infarct healing, by using different immunohistochemical markers, we wanted to explore whether isoflurane postconditioning influences the chronic phase of healing. METHODS The size of infarcted region was measured, and comparisons between isoflurane-treated and control animals were made. Quality of infarcted area was assessed by detecting vascular endothelial growth factor (VEGF), platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) as a marker of angiogenesis, and nestin as a marker of immature progenitor cells, and de novo formed blood vessels (vasculogenesis). RESULTS There was no difference between the control and isoflurane-treated groups in VEGF and PECAM-1/CD31 expression. However, a large reduction in infarct size was found (68.1% of control). Also, a marked decrease of nestin expression in immature progenitor cells, along with a marked increase of the same marker in cardiomyocytes, (signs of myocardium regeneration), was found in experimental animals when compared to control animals that did not receive isoflurane treatment. CONCLUSIONS Based on our results, we can emphasize two morphologically detectable benefits of isoflurane postconditioning: a marked reduction in infarct size along with a more mature-looking infarct area in the chronic phase of infarct healing.

Long-term streptozotocin diabetes impairs arachidonic and docosahexaenoic acid metabolism and ∆5 desaturation indices in aged rats

We have investigated the long term effects of insulin dependent diabetes mellitus (IDDM) on the fatty acid profile of tissues in aging rats. For this purpose, a rat model for IDDM was established by streptozotocin application. The rats were randomly divided into four groups of 8 animals each: CON 6 (control group sacrificed after 6 months of the experiment), CON 12 (control group sacrificed after 12 months of the experiment), DM 6 (streptozotocin treated and sacrificed after 6 months of diabetes) and DM 12 (streptozotocin treated and sacrificed after 12 months of diabetes). The periods of 6 and 12 months were taken to observe the changes in lipid metabolism for chronic, long-term diabetes. Fatty acid profiles of the liver and skeletal muscle total lipids and phospholipids as well as desaturation indices for ∆6 desaturase (D6D), ∆5 desaturase (∆6D), ∆9 desaturase (∆9D) and de novo lipogenesis index (DNL) were estimated. Additionally the long-term effects (12 months) were tested in the brain, perirenal fat and bone marrow. The fatty acid composition of lipids was altered in IDDM rats in all tested tissues. The desaturation indices revealed the expected significant decrease in ∆9D and ∆5D indices in tested tissues, while indices for ∆6D were not influenced by diabetes. DNL revealed the strong inhibition of de novo lipogenesis in the liver tissue. Values for arachidonic C20:4n6 (arachidonic acid) significantly decreased in liver total lipids in DM 6 and DM 12 groups and in phospholipids in the DM 12 group. Surprisingly, values for C20:4n6 were also significantly lower in the brain tissue in the DM 12 group. Accumulation of C20:4n6 precursors (C18:2n6 and C20:3n6) was visible in all tissues. Docosahexaenoic acid (C22:6n3) significantly decreased in liver total lipids, liver phospholipids and in the brain phospholipids of the DM 12 group. The present results show that age could exacerbate the expected decrease in the liver synthesis of C20:4n6 in IDDM. Moreover, long-term diabetes could impair C22:6n3 synthesis in the liver and muscle, and incorporation of both important fatty acids into brain phospholipids. In conclusion, numerous changes in fatty acid composition are caused by long-term diabetes in aged rats. These changes could be involved in the pathogenesis of senile and diabetes-induced damage. The results could have clinical significance due to the increasing age of diabetic patients.

Serum fructosamine concentrations in relation to metabolic changes during late pregnancy and early lactation in mares.

The changes in blood serum fructosamine concentrations as indicators of glycaemia during a longer period of time were investigated in mares during late pregnancy and early lactation, as well as their relationship to the changes in the concentration of biochemical indicators of energetic status. The samples were taken from eleven mares on 60 +/- 10 and 20 +/- 10 days before foaling, and 20 +/- 10 and 60 +/- 10 days after foaling. Concentrations of serum fructosamine, non-esterified fatty acids and triglycerides were higher during late pregnancy (from P < 0.05 to P < 0.0005), while the concentrations of beta- hydroxybutyrate increased significantly (P < 0.00001) during early lactation. A significant correlation between the concentrations of fructosamine and metabolic parameters was observed. The results indicate a relationship between energetic status and serum fructosamine in mares. Higher concentrations of fructosamine are related to the adaptation to late pregnancy. Therefore, the measurement of the serum fructosamine concentrations could serve as an indicator of energetic status of mares during pregnancy and lactation.

Gender and gonadectomy influence on neurons in superior cervical ganglia of sexually mature rats.

Gonadal hormones have a significant influence on both the number of neurons and the density of synapses in the superior cervical ganglion (SCG) during the early postnatal period. There are no studies reporting influence of the absence of these factors in sexually mature animals, although changes in SCG-neurons of the rat were observed up to 6 months of age. Hence, we investigated whether gonadectomy of sexually mature rats influences morphological properties of neurons in the SCG of the rat and if so, would it have a specific effect on neurochemically distinct subpopulations. Male and female Sprague-Dawley rats were gonadectomized at the age of two months. After 30 days, they were sacrificed and SCGs were harvested and processed immunohistochemically. The mean diameter of NPY- neurons was greater in male rats, in comparison to all other groups (p<0.05). The number of NPY+, NPY- and total neurons per section area was significantly higher in female than in male, orchidectomized or the ovariectomized animals (p<0.05). The share of the different neuronal populations in the SCG that were encircled with calretinin-positive baskets (c.b+) or c.b.- (NPY+ or NPY-) was significantly influenced by the gender of the animals and gonadectomy, with significantly more c.b.+ in male animals (p<0.05). Results of the present study indicate that substantial changes in the SCG neurons of the rat occur after reaching sexual maturity, and are influenced by the gonadectomy.

Tissue fatty acid composition and estimated ∆ desaturase activity after castration in chicken broilers fed with linseed or sunflower oil

The aims of this study were to investigate the influence of the short-term addition of sunflower and linseed oil and castration on fatty acid composition and desaturation indexes in chicken broilers. Forty-eight male Ross 308 chicken broilers were supplemented with 5% of sunflower or linseed oil. The four experimental groups were linseed oil supplementation and castration (LC), linseed oil without castration (LN), sunflower oil and castration (SC) and sunflower oil without castration (SN). There was no significant influence of castration or oil supplement on live weights, weight gain, feed intake or feed conversion. Castration resulted in an increase in polyunsaturated fatty acids (PUFA), total n3, n6, measured desaturation indexes and a decrease in the saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) content of abdominal fat. In breast muscle, castration increased PUFA and 18:3n3 values, while in the liver tissue, castration did not influence the parameters measured. Linseed oil supplementation significantly increased 18:3n3, n3 long chain polyunsaturated fatty acids (LC PUFA), total n3 and decreased total n6, n6/n3 ratio, and 20:4n6 content. Values for 20:4n6 were the highest in SC and the lowest in the LC group. Linseed oil also significantly decreased ∆5 and ∆4 desaturation indexes in the thighs and ∆5 and ∆5, 6 in abdominal fat and the liver. These results suggest that short-term supplementation of basal diet with 5% of linseed oil could significantly increase n3 LC PUFA and decrease n6/n3 ratio content in the edible tissues of chicken broilers, without adverse effects on growth performance. Meanwhile, castration only improved fatty acid profile in abdominal fat, which is not nutritionally important. The interactions observed between basal diet, supplemented oil, sex hormones and other non-nutritional factors must be elucidated in future trials in order to correctly predict the nutritional value of linseed-fed poultry.

Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serous carcinoma.

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologically analyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immunohistochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the 15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cells in week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expression characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildly to moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle where IGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the tertiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausal human ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were strongly positive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausal ovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indicate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovarian cell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSC as well.

Expression of PTHrP and PTH/PTHrP receptor 1 in the superior cervical ganglia of rats.

PTHrP and its receptor PTHR1 are found in the CNS and peripheral nervous system. The presence of PTHrP mRNA has been detected in the superior cervical ganglion (SCG), but there are no data on the cellular distribution of PTHrP and PTHR1 in the SCG. Although it is known that ovarian activity and reproductive status influence sympathetic activity, and the PTHrP/PTHR1 system is influenced by estrogens in different tissues, it is not known whether these factors have a similar effect on expression of PTHrP and PTHR1 in the nervous system. Hence, we investigated the presence and distribution of PTHrP and PTHR1 in neurons and glia of the SCG of rats, as well as the influence of ovariectomy on their expression, by using immunohistochemistry. PTHrP and PTHR1 immunoreactivity was observed in cytoplasm as well as in nuclei of almost all neurons in the SCG. In male rats, intensity of PTHrP fluorescence was significantly higher in cytoplasm of NPY-, in comparison to NPY+ neurons (p < 0.05). In female rats, 2 months post-ovariectomy, significantly lower intensity of PTHrP fluorescence in cytoplasm of the SCG neurons was observed in comparison to sham operated animals (p < 0.05). In addition to neurons, PTHrP and PTHR1 immunoreactivity was observed in most of the glia and was not influenced by ovariectomy. Results show the expression of PTHrP and its receptor, PTHR1, in the majority of neurons and glial cells in the SCG of rats. Expression of PTHrP, but not PTHR1 in the cytoplasm of SCG neurons is influenced by ovarian activity.

Effects of treatment with sucrose in drinking water on liver histology, lipogenesis and lipogenic gene expression in rats fed high-fiber diet

We studied the influence of sucrose in drinking water on liver histology, fatty acid profile and lipogenic genes expression in rats maintained on high-fiber. The experimental groups were: control group (water) and sucrose group (sucrose solution in drinking water, 30% w/v). Liver histology of sucrose treated rats revealed steatosis and increased number of αSMA immunoreactive cells without the signs of fibrosis. Sucrose treatment increased de novo lipogenesis, lipid peroxidation and MUFA content and decreased PUFA content, C18:2n6 and C20:4n6 content in total phospholipids and phosphatidylethanolamine and C18:2n6 content in cardiolipin. RT-qPCR revealed increase in Δ-9-desaturase and SREBP1c gene expression and decrease in the Δ-5-desaturase and elongase 5 expression. Treatment with sucrose extensively changes fatty acid composition of hepatic lipid and phospholipid classes including cardiolipin, increases oxidative stress and causes pathological changes in liver in rats maintained on high-fiber diet.

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.