Correlation Between NF-H Expression In The Corpus Callosum And Cognitive Dysfunction In Experimental Diabetic Rat Model

Nervous system pathological changes is one of the major complicationsin type1and type2diabetes, more effort has been dedicated to study theetiology and pathology of peripheral neuropathy, but central nervous systemneuropathy has also been described. In recent years, people pay more andmore attention to central nervous system involvement caused by diabetes. Thecentral nervous system pathological changes are characterized by decreasedconduction velocity with changes in the EEG, cerebral atrophy, decrease incognition, and increased risk for stroke. Furthermore, diabetes mellitus alsohas been associated with increased risk for Alzheimer's disease and othertypes of dementia. The pathogenesis of cognitive dysfunction in diabetesmellitus is still not completely understood at present, and there is not effectivemeans of prevention and treatment.More than forty years ago, scholars proposed the concept of diabeticencephalopathy, But up to now there is still no diagnosis standard and specificaided examination means; the most common clinical symptoms are thedecrease in learning ability, memory, comprehension and expression ability etal. Diabetic cognitive dysfunction may due to sustained hyperglycemia whichdirectly damage the cerebral neurons; With the prolonging of duration of DM,various pathogenic factors will be involved in and interact, make theaggravation of cognitive dysfunction. It has been proved that the correlativefactors such as the activation of polyol pathway, oxidative stress, advancedglycation end products, activation of protein kinase C, activation ofhexosamine pathway, low grade chronic inflammation, decreased endothelialnitric oxide synthase, depletion of myo-inositols, decreased Na+,K+-ATPaseactivity, alterations in fatty acid metabolism, lack of neurotrophic factors, abnormal activities of blood coagulation and fibrinolytic system et al interactto induce neurons disorder, and result in the nervous system changes.The integrity of neural network architecture is the basis of all executivefunction. If the neural network was more complete, the amount of informationtransmission will be larger, and more beneficial to accomplish the work.Neurofilament is the cytoskeletal protein of neurons, forms the fundamentalstructure of the neuronal communication network; Corpus callosum is theassociation fibers of the left and right hemisphere neocortex, mainly is relatedwith fundamental cognition included memory, attention, language expressionand intelligence et al. Anterior corpus (frontal region) mainly relate bilateralfrontal lobe. Some data show that frontal lobes cortex is the region closelyassociated with learning and memory.Therefore, the expression of NF-H in the anterior corpus callosum wasconsidered to be changed in experimental diabetic rats, and have certaincorrelation with cognitive dysfunction.Objective: In this study, an experimental diabetic rat model wasestablished by a single intraperitoneal injection of streptozotocin (STZ). Thepurpose of the study is to investigate the expression of NF-H in the anteriorcorpus callosum and the morphologic changes of anterior corpus callosum. Toexplore the cognitive disfunction in diabetic rats at the different time and themechanism of the diabetic cognitive dysfunction.Methods:(1) Healthy adult male Sprague-Dawley rats were randomly divided into2groups: STZ induced diabetic group (DM group) and control group (CONgroup). After the model was established successfully, each group was dividedinto4subgroups:4week group,7week group,10week group and13weekgroup.(2) After fasting for12hours, DM group rats were intraperitoneal injection ofSTZ by i.p.at65mg·kg-1. After72hours, the rats that show polydipsia,polyuria, polyphagia, a non-fasting plasma glucose level≥16.7mmol/L were considered to be diabetic rats; Control group rats were intraperitoneal injectionof isodose citrate buffer solution.(3) Materials and samples preparation: Preparation for Paraffin block: At thecorresponding time point, in each group the Morris water mase, the bodyweight and the plasma glucose of rats were all measured. After the perfusionwith the4%paraformaldehyde solution with intraperitoneal injection of10%chloral hydrate, the rats were sacrificed. Pieces of selected brain tissue wereimmediately fixed in the same paraformaldehyde solution, then the sampleswere embedded in paraffin to make up paraffin block. Then, cerebrum wasobtained for optical microscopy studies with improved trichrome staining andimmunohistochemical staining; Preparation of samples for electronmicroscopy: After perfusion with the4%paraformaldehyde-2.5%glutaraldehyde solution with intraperitoneal injection of10%chloralhydrate,the rats were sacrificed. Pieces of selected anterior corpus callosum wereimmediately fixed in the4%glutaraldehyde solution, chopped to get pieces ofapproximately1mm3, and then post-fixed in1%osmium tetroxide. They werethen dehydrated in acetonum series, soaked, embedded. Semithin sectionswere stained with toluidine for orientation and identification of nerve fiber.Thin sections were stained with uranyl acetate and lead citrate. The sectionswere viewed and photographed with a OLYMPUS JEM-1230electronmicroscope.(4) The learning and memory of rats were detected with the Morris watermase, the corpus callosum pathological changes were observed with improvedtrichrome staining, the expression of NF-H was investigated usingimmunohistochemical staining, the ultrastructure changes of nerve fibers wereobserved by electron microscope.(5) Statistical analysis: Data are expressed as mean±standard deviation (x±s),statistical analysis was used SPSS19.0software. Group comparison wasdetermined by independent sampler T test, one-way ANOVA, repeatmeasured data ANOVA, size of test was α=0.05.Results: ①Significantly decreased weight gain was observed in each DM subgrouprats when compared to the corresponding controls (p<0.05). Significantlyincreased plasma glucose was observed in each DM subgroup rats whencompared to the corresponding controls (p<0.05);②Significantly increased escape latency was observed when compared tocontrol group; Significantly decreased escape latency and search strategyscore were observed in7week group and10week group of DM subgroup ratswhen compared to4week group (p<0.05); Significantly decreased searchstrategy score, frequency of crossing platform and activity time of range I-IVaround the platform were observed in DM group of7week group and10weekgroup when compared to the corresponding control (p<0.05); No significantlychange was observed in CON group rats.③(1)improved trichrome staining：nerve fibers in anterior corpus callosum ofcontrol groups seemed to be not changed, also was in DM group of4weeksubgroup rats, but in other DM groups, axon and myelin were damaged (axonssparse, myelin collapse and vacuoled).(2)Transmission electron microscopeobservation: nerve fibers in anterior corpus callosum of control groups seemedto be not changed; axons and myelin fractured, showing that myelin loosedand axons atrophied in all DM groups.④Significantly decreased expression of NF-H was observed in each DMsubgroup rats when compared to the corresponding controls (p<0.05);Significantly decreased expression of NF-H were observed in10week and13week of DM subgroup when respectively compared to4week group and7week group of DM subgroup rats (p<0.05). No significantly change wasobserved in CON group rats.⑤There is significant correlation between expression of NF-H and course (r=－0.983,p<0.05); Escape latency, search strategy score, frequency of crossingplatform and the activity time of range I-IV around the platform have nosignificant correlation (p＞0.05); there are also no correlation between theexpression of NF-H and escape latency, search strategy score, frequency of crossing platform and the activity time of range I-IV around the platform (p＞0.05).Conclusion: Diabetic rats had cognitive disfunction. There were changesof pathology of anterior corpus callosum and NF-H expression in diabetic rats.The expression of NF-H was related with the course of diabetes. There was nocorrelation between cognition and course, so was the expression of NF-H.