Effects of crocin on hippocampus and learning in STZ-induced diabetic rats

Chronic hyperglycemia in diabetes leads to neurological complications. It has been reported that there are cognitive disorders in diabetic patients and that antihyperglycemic drugs have a positive effect on memory problems. It is reported that saffron and crocin among its components have antioxidant, anti-inflammatory, memory and learning enhancing and antidiabetic effects. In STZ-induced diabetes, cognitive disorders, behavioral tasks related to the hippocampus, and spatial memory impairment occur. It has been reported that crocin improves cognitive functions by reducing oxidative stress in diabetes-related memory disorders. In our study, we aimed to investigate the effects of crocin application on possible memory disorders in the diabetes model created with STZ and how it affects the relevant parts of the hippocampus. Adult 44 maleWistar albino rats were separated to 4 groups as Control (0.9% FTS), Diabetes (single dose 65 mg / kg STZ), Crocin (50 mg / kg), Treatment (single dose 65 mg / kg STZ, 50 mg / kg crocin). Blood glucose levels and weights of the animals were measure dregularly. Substance applications were performed for 21 days from the 4th day of the STZ injection. Passive avoidance test was performed to determine the short and long-term memory changes in diabetes, and the experiment was terminated after wards. Hippocampus of 7 animals wereused in biochemical stıdies, hippocampus of 4 animals were used in histological and immunohistochemical studies. Superoxide dismutase (SOD) in hippocampus and blood samples, insulin level, brain derived neurotrophic factor (BDNF) for learning and memory, neuronal nuclear antigen (NeuN), tumor necrosis factor-alpha (TNF-α) important in inflammatory processes, cytochrome-c level in relation to apoptotic pathway was measured by biochemical method. Histologically, hippocampus tissue was examined by Nissl staining method, and immunohistochemically, NeuN expression was examined and inter group distribution was determined by H-Score method for CA1, CA3 and dentate gyrus which are sub-sections of hippocampus. The fact that glucose values measured on the 15th and 25th days as a result of crocin (50 mg / kg) application for twenty-one days in diabetic rats were significantly decreased compared to the glucose values of diabetic animals in our study supports the hypoglycemic effects of crocin. At the end of the experiment, while the weight of the animals increased in the control and crocin groups; decreased in diabetes and treatment groups due to diabetes. It was determined that with crocin application which lasted 21 days, weight loss was less in diabetic animals compared to the diabetic group. In our passive avoidance test results, 50 mg / kg administered crocin for 21 days led to a statistically in significant increase in latency times, but this suggests that it has no significant effect on short and long-term memory. It was determined that plasma insulin levels were significantly decreased in the diabetes and treatment groups compared to the control group. This finding confirms that we have created a type 1 diabetes model with STZ. In addition, BDNF and NeuN levels did not show any difference in plasma and hippocampus tissue in our biochemical findings. Longer time is required for the translation of these molecules that are effective in learning to be affected. Our study suggests that the hippocampus insulin, BDNF and NeuN levels are not affected because diabetes lasts for 25 days. In the hippocampus tissue homogenate samples, it was determined that the SOD level increased significantly only in the crocin group compared to the control group. It has been shown that administration of 50 mg / kg crocin to diabetic rats increased SOD levels in crocin group in our hippocampus homogenate and plasma samples. The increase in SOD level in plasma and homogenate samples of crocin group indicates that this may have been caused by antioxidant properties of crocin. In our study, it was determined that the TNF-α levels decreased in the hippocampus tissue homogenate samples of the treatment group, in which 50 mg / kg crocin was administered for 21 days after diabetes was induced, compared to diabetes group. Crocin administration decreased TNF-α in the treatment group, while increasing the plasma TNF-α level. This effect of crocin suggests that it may have different effects under different physiological conditions. In our histological examinations, neuronal loss and neuronal changes were observed in sections of hippocampus CA1, CA3 and DG regions of the diabetes group. Degenerated neurons are distinguished by their dark stained cell and shrunken cytoplasm. It was determined that the percentage of degenerated neurons in the CA1, CA3 and DG regions of the diabetes group increased significantly when compared to the control group. These histological findings support the structural disorder that develops with diabetes. In addition, it was determined that crocin applied to diabetic rats significantly decreased the percentage of degenerated neuron only in the DG region of the hippocampus compared to the diabetes group. It has been shown that in the hippocampus tissue examined by immunohistochemistry, the application of crocin increased the decreased NeuN expressiononly in the CA1 region of diabetic individuals. It has been shown that crocin shows this effect only in the CA1 part, and interestingly, NeuN expression decreased in the hippocampus CA3 region in the crocin group. As a result, it was confirmed that the administration of 50 mg / kg / ip crocin to diabetic rats had an antidiabetic effect and reduced weight loss effects. The lack of effect on memory may have occurred depending on the duration of diabetes, because no significant difference was found in hippocampus homogenates in terms of insulin and cytochrome-c levels, BDNF, and NeuN. Interestingly, crocin applicational one increased the plasma and hippocampus SOD levels and plasma TNF-α levels. It was determined that the percentage of degenerated cells increased in the CA1, CA3 and DG sections of the hippocampuses of only crocin-applied groups. In addition, crocin showed a decreasing effect of NeuN expressions in CA1 region, while decreasing effect of NeuN expressions in CA3 region. In order to see more pronounced possible effects of crocin on hippocampus tissue, studies involving different doses of crocin can be performed in a longer-term diabetes model.