Study Of The Protection And Mechanism On Insulin-like Growth Factor-1 Treatment Dementia Model Rats

Alzheimer disease is an age-related neuro-degenerative disease. Clinicalmanifestation are progressive dementia, cognitive handicap, nerves or emotiondisturbance. The important pathology characters of AD are senile plaques, bifilarhelix fibril tangles and the loss of neuron and synapse. Above all, the basalforebrain and hippocampus suffer severely, where the neurons decreased 47%,the quantities of ChAT and Ach is decreased and the synthesis is deficiency.With people ageing quickly, the morbidity and mortality of AD rise fast. Sostrength the study of AD pathogenetic and finding effective treatment method ofAD have important economic and society significance.In recent years, the effect of insulin like growth factor-1 on cognitionfunction was being payed close attention. A lot of studies about the relationshipbetween IGF-1 and ageing correlated cognitive handicap focus on clinicobservation in abroad. The mechanism of IGF-1 treatment AD is not be provedin abroad. We didn't find the basic and clinic study on IGF-1 treatment AD inour country.To make sure whether IGF-1 can treat AD or not, we study the protectionmechanism of IGF-1 on central nervous system through the cholinergic nervesystem, calcium ionic balance, and apoptosis protein, the hypothesis of AD, toafford the basic theory for IGF-1 therapia AD in clinic.1 The establishment of dementia model rats and the effect of IGF-1 onethology and histological anatomy of rats1.1 Transecting hippocampal fimbria-fornix of rat to set up AD model ratsMale Wistar rats was selected whose age was from 3 to 4 months, and thebody weight of them were among 200~250g, breeding them under normaltemperature, sun light and free food. We Select swift, activated rats by MorrisWater Maze and divided them into five groups: normal control group, shamgroup, dementia group, low dose IGF-1 group, large dose IGF-1group. Everygroup divided into four experimental time points, which is 3d, 7d, 14d, 21dgroups. Every experimental time point have 10 rats.The normal control group accept no treatment. The other four groups wereanesthetized by an peritoneal injection 10% chloral hydrate(0.33ml/100g) andput them on the stereotaxis instrument to choice ambihippocampal fimbria-fornix,and transect. Ambihippocampal fimbria-fornix of sham group were not cut offafter the skull being drilled. Rats in the low dose IGF-1 group receivedintracerebroventricular infusion of recombinant human IGF-110μl (0.2μg/μl).Rats in the large dose IGF-1 group received intracerebro-ventricular infusion ofrecombinant human IGF-1 10μl(0.5μg/μl). Rats in the low large dose IGF-1group received intracerebroventricular infusion of recombinant human IGF-110μl(0.5μg/μl). Rats in the sham group and dementia group receivedintracerebroventricular infusion of saline 10μl.1.2 Determination of learning and memory ability of ratsLearning and memory ability of each group rats were determined by Morriswater maze and the escape latency were recorded. The escape latency are shorterthe learning and memory ability of rats are better. At the same time, we recordspanning platform times of rats. The more times of rats span the platform, thebetter learning and memory ability they have.Experimental results show that the escape latency and the spanning platformtimes of normal control group and sham group compared with dementia groupreveals significant difference. Student's t-test reveals no significant difference inthe escape latency and the spanning platform times between normal group andsham group, which demonstrate that operation result in the damage of centralcholinergic system and caused the learning and memory ability of rats decreased.The low dose IGF-1 group and the large dose IGF-1 group compared with thedementia group shows the escape latency decreased and the spanning platformtimes increased, have significant difference, which indicates that the learning andmemory ability of dementia group rats increase significantly. The low doseIGF-1 group and the large dose IGF-1 group comparing with the normal controlgroup demonstrated that the escape latency and the spanning platform times havesignificant difference. It shows that the learning and memory ability ofdementia rats can't reach the normal levels. The low dose IGF-1 group comparedwith the large dose IGF-1 group shows the escape latency decreased and thespanning platform times increased, which indicate that the low dose IGF-1 groupis better than the large dose IGF-1 group.1.3 HE dyeing and electron microscope results in hippocampus and frontallobe of ratsHE staining of normal control group shew frontal lobe and hippocampalpyramidal cell line up in order, uniformity, cellular structure was integrity.Electron microscope shows cellular membrane was integrity, and cellularnucleus was large and ellipse.Nucleole was clear, Neuropil was normal,Pro-membrane and post-membrane of synapse was clear. Synaptic vesicle wasabundance. Neuron morphosis was no deference between normal control groupand sham control group.HE staining of dementia group shew meshwork like malacoma focus infrontal lobe and pyramidal cell was derangement and rarefaction. Some neuronin hippocampus was lost. Electron microscope shew most of neurons wereintumesce and some of them were cavitative. Neuropil was cavitation too. Proand post-membrane was coalesce. Synaptic vesicle was decreased.HE staining of low dose IGF-1 group shew there was a reactivate zone infrontal lobe where some newborn nerve cell were found. The number of celllayer in hippocampus was increased, Array of the neuron was disturbed. Electronmicroscope shows nuclear matrix cavitate lightly, and we can see roughendoplasmic reticulum, chondriosome and lipofuscin in intracytoplasm. Somenerve plexus dissolved in axis cylinder. Synaptic cleft is not clear. Morphous ofnerve cell have no significant difference.The experimental results confirmed that transecting hippocampalfimbria-fornix can induce the damage of neuron in hippocampal and frontal lobeand result in the learning and memory ability of rats decreased. IGF-1 canameliorate the learning and memory ability of rats.2 Effection of IGF-1 on ChAT in hippocampus and frontal lobe of dementiamodel ratsWe used hybridization in situ technique and image analysis system toobserve the changes of ChAT in hippocampus and frontal lobe of dementiamodel rats. Immune reaction intensity of ChAT was decreased in hippocampusand frontal lobe of dementia model rats. IOD value of which was lower thannormal group and sham group, student's t-test revealed significant differencecompared with normal control group and sham group. IOD value of low doseIGF-1 group and large dose IGF-1 was increased obviously compared withdementia model, the deference is significant by student's t-test. It shows thatIGF-1 can increase ChAT in hippocampus and frontal lobe of rats. Student's t-testsuggests that ChAT was increased significantly in low dose IGF-1 groupcompared with large dose IGF-1 group. It indicated significant differencecompared with large dose IGF-1 group, which manifested that low dose IGF-1group was better than large dose IGF-1 group. The experimental results showsIGF-1 can increase the expression of ChAT in hippocampus and frontal lobe ofrats, and ameliorate the function of central cholinergic system.3 Effection of IGF-1 on synaptophysin in hippocampus and frontal lobe ofdementea model ratsWe used immunohistochemistry and image analysis system to observe thechanges of synaptophysin in hippocampus and frontal lobe of dementia modelrats. Immune reaction intensity of synaptophysin was decreased in hippocampusand frontal lobe of dementia model rats after being operated for 3 days, IODvalue of which was lower than normal control group and sham group, student'st-test revealed significant difference compared with normal control group andsham group. IOD value of low dose IGF-1 group and large dose IGF-1 wasincreased obviously compared with dementia model, the deference wassignificant by student's t-test. It shows that IGF-1 can increase synaptophysin inhippocampus and frontal lobe of rats.Student's t-test suggests that synaptophysin is increased significantly in lowdose IGF-1 group compared with large dose IGF-1 group. It indicates significantdifference compare with large dose IGF-1 group, which shows that low doseIGF-1 group is better than large dose IGF-1 group.4 Effects of IGF-1 on the expression of Caspase-3 P20 and Bcl-2 inhippocampus and frontal lobe of dementia model rats.The expression of Caspase-3 P20 and Bcl-2 in hippocampus and frontallobe of rats in each group were observed, which optical density value weredetermined by immunohistochemistry and image analysis system. Caspase-3 P20didn't express in hippocampus and frontal lobe of rats in normal control groupand sham control group. Caspase-3 P20 was strong expression in hippocampusand frontal lobe of rats when hippocampal fimbria-fornix was transected afterthree days, and keep up to 14d. But it express decreased at 21d. Neurons whichexpressed Caspase-3 P20 was brown or buffy and its cell body analosised., Lowdose IGF-1 group and large dose IGF-1group compared with the dementia groupwhose intensity of cell positive response was decreased, student's t-test revealssignificant difference in cell IOD value. Low dose IGF-1 group comparing withlarge dose IGF-1group its IOD value was decreased obviously reveals significantdifference.Expression of Bcl-2 was strong in hippocampus and frontal lobe of ratswhen hippocampal fimbria-fornix was transected after three days, it continued to14d. To 21d it came down. IOD value of Low dose IGF-1 group and large doseIGF-1group compared with the dementia group was decreased obviously, whichwas determined by student's t-test revealed significant difference. Student's t-testsuggests that the IOD value increased significantly in Low dose IGF-1 groupcompared with large dose IGF-1group. It indicated that Low dose IGF-1 groupwas better than large dose IGF-1group on the expression of Bcl-2.The experimental results manifest that IGF-1 can decrease the expression ofCaspase-3 P20 at the same time increase the expression of Bcl-2 in hippocampusand frontal lobe of rats and depress cell apoptosis in dementia model rats.5 Effection of IGF-1 on calcium ion in brain cell of dementia control ratsWe use confocal laser scanning microscope to detecte calcium ion whichwas loaded by Fluo-3/AM in brain cell. Calcium ion in brain cell was increasedobviously in the dementia group compared with the normal group and shamgroup, student's t-test reveals significant difference. Calcium ion in brain cellwas decreased obviously of low dose IGF-1 group and large dose IGF-1groupcompared with the dementia group. Student's t-test suggests that Calcium ion inbrain cell have no significant difference between low dose IGF-1 group andnormal group. The difference of Calcium ion in brain cell of low dose IGF-1group was significant compared with large dose IGF-1group. It indicated that thecurative effect of low dose IGF-1 on keeping the balance of Calcium ion in braincell was better than large dose IGF-1.The results show that IGF-1 can keep the balance of Calcium ion in braincell, and inhibit cell apoptosis, ameliorate the learning and memory ability ofrats.In conclusion, IGF-1 can protect the central nervous system through manypathways. First, IGF-1 can increase the expression of synaptophysin and ChATto protect and nourish the central cholinergic system. Second, IGF-1 can alsokeep the balance of Calcium ion in brain cells as well as decrease the expressionof Caspase-3 p20 and increase the expression of Bcl-2 in hippocampus andfrontal lobe to depress cell apoptosis in rats through which ameliorate thelearning and memory ability of rats.The experimental results indicate that IGF-1 can protect central cholinergicsystem through many pathways to ameliorate the learning and memory ability ofrats and offord new target for the clinic treatment AD.