Effect Of IL-1Ra With Intracerbroventricular Injection On Behavioral Depression And Hippocampal Neurogenesis In Reserpine-induced Rats

Background and objectiveIt's well known that depression disorder has become to a common clinical disease. Thehypothesis of pathophysiology mechanism about depression now consist of monoamineneurotransmitter hypothesis,disfunction of neuroendocrine system and neural plasticity.Theneurogenic hypothesis postulates that a reduced production of new neurons in the hippocampusrelates to the pathogenesis of depression and that successful antidepression treatment requires anenhancement in hippocampal neurogenesis.Abundant preclinical evidence confirm stresssuppresses hippocampal neurogenesis.Actually,stressful life events are known to precipitatedepression in vulnerable individuals.And most antidepressant treatment could block theinhibition of hippocampal neurogenesis by sress.For instance,5-HT or antidepressant drug ofSSRI group may elevate adult hippocampal neurogenesis and have antidepressanteffects.Moreover,chronic administration antidepressant treatments elevate hippocampalneurogenesis parallels the time-course of the emergence of clinical therapeutic effects.The adulthippocampal neurogenesis may be the supporter of antidepressant treatment,and have importantsignificance to the pathophysiology mechanism of depression.There is ample evidence that cytokines such as IL-1βis constitutively expressed in normalbrain and modulate central nervous system precesses.There are abundant clinical and animalresearch evidence that the alteration of cytokines such as IL-1βof brain mediate the depressiondisorder. IL-1βof brain can induce"illness behavior"in animal and human, the treat ofantidepressant drug can improve the behavioral depression rats and the remission of depressionsymptom,furthermore,decrease the level of IL-1βin rat brain. Either animal experimentindicate that peripherally or directly inject IL-1βinto the brain,could induce depressive-likesymptoms, as well as pretreatment with IL-1 receptor antagonists (IL-1Ra) have the sameeffectiveness like antidepression treatment.The brain injection of IL-1βdownregulationhippocampal neurogenesis,IL-1βmay mediate the process of hippocampal neurogenesis, furthereffect the development of depression. To examine the hypothesis that IL-1βplays a causal role in hippocampal neurogenesis ofdepression, we examined the behavioral depressive-like symptoms are reduced by reserpine inrats, and investigate the pathophysiology mechanism relationship of cytokine, hippocampalneurogenesis and depression. It could be beneficial in new target of depression treatment.Material and methodsEstablishment and detect behavioral depreesion animal modelWe choice healthy, sex maturity and clearing Sprague-Dawley rats with weight of 200-260g.After adapting the environment of 26℃, the circulation of illumination and darkness with 12:12hour, free to eat and drink for a week. The rats'intraperitoneal injection reserpine with 4mg/kginduced behavioral depression-like effect, and the control group intraperitoneal injection with thedose of reserpine solvent. The behavioral depression of rats after injected reserpineintraperitoneally were tested using Porsolt swim test,GroupThe rats were assigned to control group (C) and reserpine group (R) randomly for explorehippocampal neurogenesis at the basis of reserpine induced behavioral depression. To detect theeffect of IL-1Ra on proliferation of neural precursor cell in hippocampal of behavioraldepression rats were reduced by reserpine, the rats were assigned to control group (C) andreserpine group (R), half rats of two groups were injected IL-1Ra or PBS into lateral cerebralventricle. The total group were control group (CG), control group+IL-1Ra (C+IL-1Ra), reserpinegroup (RG), reserpine+IL-1Ra group (R+IL-1Ra).Cannula Implantation in the lateral ventricle and injectThe rats must be implanted the cannula in the experiment that IL-1Ra on proliferation ofneural precursor cell in hippocampal of behavioral depression rats were reduced by reserpine.The cannula was implanted into the lateral ventricle through the stereotaxis instrument of rat(from bregma: AP, -0.9 mm; RW, 1.5 mm; DV, -3.8 mm). The cannula was used forintracerebroventricular injection (i.c.v) was implanted into right lateral ventricle. Wait for a week,0.01M PBS and 0.25ug/ul IL-1Ra were injected (i.c.v), 4mg/kg reserpine and acetic acid solventwith the dose of reserpine were injected by i.p. The detect of hippocampal neurogenesisThe rats of all groups were injected Brdu with 75mg/kg and four times interval 2 hourintraperitoneally marked the dentate gyrus neurgenesis after reserpine 40 hour. All the rats wereperfused with saline and paraform, and then their brains were made into frozen sections whichwere used to observe the level of proliferation of hipppcampal neural precursor cell afterreserpine injected 48h, differentiation of hipppcampal neural precursor cell 72h later ofreserpine ,and the the survival of new neuron after 4 weeks.Statistics and analysis of dataThe picture from immunohistochemistry were analyzed by Image-Pro Plus 6.0(IPP).Datawere presented as mean±standard deviation ( x±SD), and statis titally analyzed by twoindependent sanmples t test or analysis of variance (ANOVA) with EXCEL 2003 and SPSS13.0.A level of P<0.05 was accepted as statistically significant, and a level of P<0.01 wasaccepted as great statistically significant.ResultPart 1 Effects of reserpine-induced behavioral depression on the hippocampalneurogenesis1. Reserpine induced rat behavioral depression-like effectThe reserpine group with reserpine (4mg/kg, i.p), control group with 2% acetic acid solvent.The mean floating time in eath group of Porsolt swim testing occurred 0, 24, 48 and 72 hoursafter injection of reserpine (4mg/kg, i.p) as the index of behavioral depression. The resultindicated that the floating time in the control group and reserpine group occurred at 24 hourswere 7.0±0.5 and 13.4±0.9, 48 hours were 7.2±0.6 and 12.9±0.7, and 72 hours were 8.0±0.8 and12.6±0.9. The floating time in the reserpine injected group significantly increased from 24 hoursto 72 hours afer injection as compared with the control group (ANOVA, P<0.01). This dataprovided evidence that acute administration of 4mg/kg of reserpine could produce behavioraldepression at least from 24 hours to 72 hours. 2. Effects of reserpine-induced behavioral depression on the hippocampalneurogenesisIn the adult mammalian brain, there are two neurogenic regions that are generally accepted,the SVZ of olfactory system and the SGZ of hippocampus. In the hippocampus, the precursorcells are found in the subgranular zone (SGZ) of the dentate gyrus. These cells arise in the SGZand migrate into the granule cell layer (GCL) where they mature into functionally excitatorygranule cells, that is, the ability to generate a multitude of different neuronal and glial lineagesThe time of neurogenesis process which includes proliferation, differentiation, migrationand survival of new neuron is 4 weeks. Multi-potent progenitors of the adult brain areproliferative keep on 14-17 hours.The neuronal precursor cells express doublecortin (DCX) thatis associated with both the initiation of neuronal differentiation and migration in 2-7 day. After7-10 day the new neuron come into maturation and expression of the neuronal markers NeuN.The reserpine group i ntraperitoneal injection 4mg/kg reserpine and control i ntraperitonealinjection 2% acetic acid solvent, and injected Brdu with 75mg/kg and four times interval 2 hourintraperitoneally marked the dentate gyrus neurgenesis after reserpine 40 hour. The rats wereperfused with saline and paraform after reserpine 48 hours, 72 hours and 4 week, then theirbrains were made into frozen sections which were in order used to observe the level of controlgroup and reserpine group were used to detect the proliferation, differentiation of neuralprecursor cell and the survival of new neuron in hippocampal dentate gyrus.2.1 Effects of reserpine-induced behavioral depression on the neuronal precursorproliferation in dentate gyrus of hippocampus.Brdu and Ki67 are used to investigate the level of neural precursor proliferation in dentategyrus of hippocampus. The Brdu and Ki67 positive cells were localized in SGZ of dentate gyrus.The results showed that Brdu positive cells of control group and reserpine group in SGZ per unitarea of dentate gyrus were 10.86±1.40 and 5.18±1.16. The results showed that Ki67 positivecells of control group and reserpine group in SGZ per unit area of dentate gyrus were 22.4±9.88and 11.38±6.71.Compared with control group, the proliferation of neuronal precursor inreserpine group had a lower number (P<0.05).It indicates that reserpine can decrease theproliferation of neuronal precursor in dentate gyrus of hippocampus. 2.2 Effects of reserpine-induced behavioral depression on the neuronal precursordifferentiation in dentate gyrus of hippocampus.Brdu immunohistochemistry was used to detect the differentiation of neural precuisor indentate gyrus of hippocampus. The Brdu positive cells were localized in SGZ of dentate gyrus.After Lg10 data transformation and in progress of K-S test, the data of Brdu positive cells ofcontrol group and reserpine group in SGZ per unit area of dentate gyru followed normaldistributed。Brdu positive cells of control group and reserpine group in SGZ per unit area ofdentate gyrus were 3.70±3.71 and 0.42±0.73. The differentiation of neuronal precursor inreserpine group had a lower number compared with control group, (P<0.05). It indicates thatreserpine can decrease the differentiation of neuronal precursor in dentate gyrus of hippocampus.2.3 Effects of reserpine-induced behavioral depression on the new neuron indentate gyrus of hippocampus.The neuronal precursor cells express doublecortin (DCX) that is associated with both theinitiation of neuronal differentiation and migration in 2-7 day. After 7-10 day the new neuroncome into maturation and expression of the neuronal markers NeuN. Furthermore, about 70%new neuron could expree the neuronal maker after 1 month.2.3.1 Effects of reserpine-induced behavioral depression on the newbornimmature neuron in dentate gyrus of hippocampus.The newborn immature neurons in dentate gyrus express doublecortin (DCX). DCXimmunohistochemistry to use detect the newborn immature neuron in dentate gyrus of the ratswhich were perfused with saline and paraform after reserpine 72 hours. The dendrites length,dendrites and the number of DCX positive cell of dentate gyrus had no difference betweencontrol group and reserpine group. Taken together, these data indicate that the reserpine-inducedbehavioral depression had no effect on the newborn immature neuron in dentate gyrus of the rathippocampus.2.3.2 Effects of reserpine-induced behavioral depression on the survival of newneuron in dentate gyrus of hippocampus.Brdu immunohistochemistry was used to detect the survival of new cell in dentate gyrus of the rats which were perfused with saline and paraform after reserpine four weeks. Brdu andNeuN double-label immunofluorescence experiments were conducted as described the newneuron. After Lg10 data transformation and in progress of K-S test, the data of Brdu and NeuNpositive cells of control group and reserpine group per unit area of dentate gyru followed normaldistributed. The survival of new neuron in dentate gyrus in reserpine group had a lower numbercompared with control group (P<0.05). It indicates that the reserpine-induced behavioraldepression can decrease the survival of new neuron in dentate gyrus of hippocampus.Part 2 Effects of IL-1βon behavioral depression induced by reserpine andthe hippocampal neurogenesis1. Intracerebroventricular injection of IL-1Ra on the behavioral depression inducedby reserpineThe four groups: C, R, C+IL-1Ra and R+IL-1Ra. 0.01M PBS and IL-1Ra weremicroinjected (i.c.v), reserpine and acetic acid solvent (i.p) infusion later. The mean floating timein eath group when Porsolt testing occurred 1, 24 and 48 hours after injection of reserpine(4mg/kg, i.p) as the index of behavioral depression. The microinjection (i.c.v) of IL-1Ra had noeffect on the floating time at 1 hour and 24 hours. But the microinjection (i.c.v) of IL-1Ra canreverse the increase of floating time at 48 hours were induced by reserpine (ANOVA, P<0.01,Newman-Keulspost-hoc Contract), and that the mean floating time of R and R+IL-1Ra groupare 12.87±0.78 and 8.68±0.76. Only microinjection (i.c.v) IL-1Ra had no effect on the floatingof the normal rats.2. Effect of IL-1Ra (i.c.v) on the proliferation of neuronal precursor cell inhippocampal of behavioral depression rats were reduced by reserpine.Brdu immunohistochemistry was used to detect the proliferation of neural precuisor indentate gyrus of hippocampus. The Brdu positive cells were localized in SGZ of dentate gyrus.After square root data transformation and in progress of K-S test, the data of Brdu positive cellsof C, R, C+IL-1Ra and R+IL-1Ra in SGZ per unit area of dentate gyru followed normal distributed. Brdu positive cells of eath group in SGZ per unit area of dentate gyrus were5.07±5.66,3.30±2.31,0.97±0.86,6.06±5.13. The results showed that IL-1Ra (i.c.v) had noeffect on the hippocampal neurogenesis in normal rat (C vs C+IL-1Ra). Reserpine (i.p) coulddecrease the proliferation of neuronal precursor in hippocampus, (P<0.05, R vs C); IL-1Ra (i.c.v)could reverse the decrease of neuronal precursor proliferation in hippocampus induced byreserpine (P<0.01, R vs R+IL-1Ra). The results indicate that simple injection of IL-1Ra had no effecton proliferation of neural progenitor cells in rat hippocampal DG. Reserpine could inhibit theproliferation of neuronal precursor in hippocampus, and that can be reversed by IL-1Ra withintracerebroventricular injection.ConclusionsReserpine induced rat behavioral depression-like effect and decreased the neurogenesis indentate gyrus of the rat hippocampus, and IL-1Ra could reverse the decrease of hippocampalneurogenesis and the depression-like effect caused by reserpine-induced behaviorval depression.The IL-1βof brain may mediate the damage of hippocampal neurogenesis in thereserpine-induced behavioral depression.Taken together, cytokine and hippocampal neurogenesismay be the pathophysiology mechanism of depression.Accordingly, it suggest that the brainIL-1βmay be the pathophysiology mechanism of behavioral depression induced by reserpine,and the disorder of hippocampal neurogenesis may be one of the pathophysiology mechanismmediated by IL-1β.