Role Of Citalopram And Serotonin 1A Receptor In Transient Cerebral Ischemia In Rats

Vascular dementia (VD) is the second most common form of dementia after Alzheimer's dementia (AD) which seriously interfere the life of aged people. And till now, a restorative treatment has not been reached yet. VD is characterized by loss of neuronal cells especially in hippocampus, a region in the brain that has the ischemic injury vulnerability, and loss of executive function with milder memory loss. There is a growing body of evidence that neural stem cells(NSCs) reside in the adult central nervous system where neurogenesis occurs throughout lifespan. Neurogenesis concerns mainly two areas in the brain: the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Since the proliferation, differentiation, migration and integration of NSCs in hippocampus have close relationship with cognitive function, it provides a possible therapeutic strategy for ischemic injury repair. Although there have been consistent reports that cerebral ischemia increases the number of newly generated neurons that migrate from the SGZ into the granular cell layer(GCL) of DG and incorporate into functional synaptic circuitry in adult rats, but this still could not compensate the loss of neurons and the cognitive impairment. So it is becoming more and more important to find out a suitable microenvironment for further improve the neurogenesis triggered by ischemic injury. Fortunately, animal studies have shown strong neurogenetic function of serotonin and selective serotonin reuptake inhibitors (SSRIs) and the serotonin 1A receptor, which is abundant in hippocampus, has major effect during neurogenesis. However, it is yet not known whether SSRIs impact the proliferation and maturation of newborn neurons and the cognitive function of VD rats.Here in this study, we test our hypothesis that citalopram(CIT), one of SSRIs, could promote neurogenesis and improve cognitive functions in vascular dementia model of rats and it's action regulated by the function of serotonin 1A receptor. Our research could provide some experimental evidence for the mechanism of ischemic neurogenesis and choice for vascular dementia therapy. Objective:1. To interpret the characteristics of NSCs proliferation in vascular dementia model of adult rats.2. To identify the influence of CIT on NSCs proliferation and differentiation in different region of the brain and on cognitive function of VD rats.3. To elucidate the role of serotonin 1A receptor in ischemic neurogenesis and CIT effects in our vascular dementia model.Methods:1. Animals, drugs and establishment of rat model: Male Wistar rats (n=336) were randomly divided into five groups: normal control group(NC), sham operation control group(SC), transient cerebral ischemic group(VD), citalopram treated transient cerebral ischemic group(VD-C) and VD-CW group which treated with both citalopram and WAY100635. The later four groups were further divided into 1-, 3-, 7-, 14-, 21- and 42-days-post-operation subgroups. And in every subgroup 6 animals were involved. Citalopram(20mg/kg) and WAY 100635(0.3mg/kg) were injected intraperitoneally for 21 days before operation and the treatment proceeded until sacrifaction. BrdU(50mg/kg) was given for 7 days before sacrifaction for cell proliferation study. As for cell differentiation study, BrdU(50mg/kg) was given to 14-day-post-operation in VD, VD-C and VD-CW group, and rats were sacrificed 28 days after. The improved Pulsinelli's 4-vessle occlusion was used to establish ischemia in rats.2. Measurement for cognitive function: The water maze task was undertaken. Place navigation test and spatial probe test was to observe rats'spatial learning and memory abilities respectively. The escape latency (EL), distance percentage (DP) and times of passing through the platform were analyzed. And P3-like potential was observed too.3. Observation for NSCs proliferation: Immunohistochemical-staining was preceded with freezing coronal brain slides of 35μm thick each. After sections were incubated in 0.3%TritonX-100 and 2mol/L HCl and results were shown by SABC-Cy3 or DAB. Sections were examined with an inverted microscope.4. Observation for NSCs survival and differentiation: Double-label immunofluorescence staining was proceeded. After BrdU immunofluorescence staining, sections were incubated with Tuj1, Calbindin-D28k and GFAP respectively, to label immature, mature neurons and astrocytes, use different fluorescence coloration. And the resulting immunofluorescent signal was viewed under fluorescence microscope and confocal microscopy.5. Nissl stain was proceeded to analyze the survival of CA1 neurons.6. The expression of serotonin 1A receptor in hippocampus: After Isolation of total RNA and reverse transcription–PCR , Real-time PCR was preceded. Serotonin 1A receptor primers were 5'-GCCCCCCAAGAAGAGCCTGA-3' and 5'-GCCATCTTGCGCTTTGCTTC-3'. GAPDH was used as reference, and the primers were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'。7. high-performance liquid chromatography(HPLC) was used to detect monoamine neurotransmitters in hippocampus.8. Cell counting and statistical analysis: Cells were counted under high power (20×or 40×objective) on Olympus microscope with magnifying digital camera. Data analyzed by IPP 6.0 software. All the data are presented as the means±S.E.M. of the individual values for each rat in all groups. Statistical analysis of the data was performed using one-way ANOVA for multiple comparisons.Results1. The characteristics of NSCs proliferation in vascular dementia model of adult rats.BrdU positive cells were observed mainly in dentate gyrus and SVZ and less expressed in cortex in normal rats.In DG, BrdU-positive cells were gradually increased from 3 days after operation and reached its peak 14 days after operation with the cell number triple as much as that before ischemia. And the number of newly proliferated cells in VD-C group was twice as much as that in VD group from 3 days after ischemia. And in VD-CW group, the proliferation of NSCs was significantly decreased.There was no BrdU-positive cells in CA1, CA2 and CA3 in normal rats. And NSCs cells were found in CA1 and CA2 7 days after operation, cell proliferated later and less than that in DG. By use of CIT, CA1 cell proliferation increased twice as that in VD group and CA2 cell proliferation was found 3 days after ischemia and continued 21 days after operation. But the cell proliferation was significantly down-regulated in VD-CW group compared with VD-C group.In SVZ region, cell proliferation obviously up-graded from 3-day-after operation, and it was significantly further enhanced by use of CIT. In every experimental groups, number of BrdU-positive cells 7 days post-ischemia groups was significantly higher than that in 3 days post-ischemia groups(P<0.01). But after that, cell proliferation down-regulated rapidly with significant difference between 14 days to 7days post-ischemia(P<0.01). There was no obvious difference of BrdU-positive cell numbers between VD-C group and VD or VD-CW group.Number of BrdU-positive cells began to rise in cortex PCg and Str18 area in all experimental groups after ischemia. In VD group, BrdU-positive cells emerged inⅡ~Ⅵlayer of cortex began to rise from 1~3 days post operation and obviously higher 7 days and 14 days after ischemia(P<0.01). Then the number significantly went down 21 days after operation. Number of BrdU-positive cells in VD-C group was much higher compared with VD group(P<0.01) and difference was most obvious 14 days after operation. In VD-CW group, BrdU-positive cells were higher than VD group but had no significant difference with VD-c group.2. The survival and differentiation of newly generated cells:Double labeled cells could be found in every experimental group after operation.①In DG, NSCs survival rate of VD group was lower than that in SC group but the percentage of double-labeled cell significantly higher(P<0.05). In group of VD, the BrdU-positive cells mainly differentiated into Tuj1-positive immature neurons in DG and into both immature and mature neurons with positive stain of Calbindin-D28k in CA1. And the BrdU-and-GFAP- positive cells scattered in GCL of DG and hilus. In VD-C group, newly generated cells in DG and CA1 differentiated into mature neurons obviously more than that in VD group and also had higher survival rate. And in VD-CW group, double positive of BrdU/Tuj1 and BrdU/Calbindin-D28k cell percentages were significantly decrease.②In the cortex, the BrdU-positive cells expressed relatively stable in PCg than in Str18 and PAC. NSCs'survival rate of VD group was lower than that in SC group with no significant difference. Survival rate of BrdU-positive cells in VD-C group was much higher than in VD group(P<0.05). Double-positive cell was not found in NS and SC groups but could be found in VD group of which had low percentage of BrdU/Tuj1 and BrdU/Calbindin-D28k staining and relatively high percentage of BrdU/GFAP staining. Compared with VD group, BrdU/Tuj1 double-staining percentage was similar but BrdU/Calbindin-D28k double-staining was much higher(P<0.01) in VD-C group. Although cell differentiation rate was lower in VD-CW group, there was no significant difference.3. result of RT-PCR examination of serotonin 1A receptor mRNAOne day after transient cerebral ischemia, the relative expression level of serotonin 1A receptor mRNA dropped down obviously in VD, VD-C and VD-CW groups(p<0.01). It began to rise up from 3 days after ischemia. In VD group, the expression level reached its peak 14 days after operation. In, VD-C group, the relative expression levels of serotonin 1A receptor mRNA were much higher (P<0.01) except 1-day-post-ischemia and reached its peak 7 days earlier and continued keep its ascendancy to 21 days after ischemia compared with VD group. In VD-CW group, serotonin 1A receptor mRNA expression was higher than that in VD group from 3 days after operation with significant difference in 3- and 7-day post ischemia, but lower than that in VD-C group.4. Level of monoamine neurotransmitters in hippocampus detected by HPLC①In hippocampus: The level of serotonin(5-HT) dopamine(DA) and norepinephrine (NE) were dropped 7 days after cerebral ischemia with significant difference except NE. And the monoamine neurotransmitter returned normal 21 days post-ischemia in VD group. In VD-C group, the level of 5-HT rose twice as much as that in VD group(P<0.01) but the level of 5-HT in VD-CW group rose with no significant difference vs. VD-C group. The level of DA and NE didn't change either in VD-C or VD-CW group.②In cortex: The level of 5-HT dropped 7 days after cerebral ischemia with no change of NE and DA and returned normal 21 days post-ischemia. In VD-C group, the level of 5-HT and NE had no statistical difference but the DA level was significantly higher compared with that in VD group(P<0.05). In VD-CW group, the 5-HT level largely rose up, almost triple as much as that in SC group, the level of DA decrease obviously vs. VD-C group(P<0.01), and NE level didn't change much.5. Cell survival in CA1The comparisons indicated that this effect was due to significantly lower cell densities in the ischemic animals as compared to sham-operated (p < 0.001). As compared to sham-operated animals, almost 90% of CA1 pyramidal cells showed signs of neuronal damage 7 days after operation and only regained nearly 65% of CA1 cell number 42 day post-ischemia. In contrast, CIT treated rats had 25% more CA1 neurons than non-treated ischemic rats. But this protective effect was blocked by co-administration of WAY 100635, a specific 5-HT1A receptor antagonist. These results provide evidence that CIT can induce a long lasting (i.e., 6 weeks) hippocampal protection against global cerebral ischemia. And this effect of CIT might be regulated by the function of 5-HT1A receptor.6. cognitive function examined by Morris water maze testResults from data analysis indicated that the average escape latency(EL) of all rats in every group was gradually decreased by the increase of trials in the place navigation tests. The EL in the three groups of VD,VD-C and VD-CW were significantly longer than that of the NC and SC groups(P<0.01). The group of VD-C existed longer escape latency(P<0.05), while the EL in this group of 14 days and 21 days after operation were significantly decreased compared with the VD group(P<0.05).In the contrast, after the administration of the WAY100635,he specific inhibitor of 5-HT1AR, the VD-CW rats showed significant longer escape latency.To test the spatial memory ability, the spatial probe test was performed after the removal of the platform. The data showed that the DP spent in target quadrants had no difference between the NC and SC rats, and the DP of the VD,VD+C and VD+W were respectively decreased significantly. Furthermore, the administration of Citalopram made a better performance in memory compared with other groups(P<0.05) and almost returned to normal 42 days after ischemia. In contrast, the DP decreased sharply compared with VD-7d group(P<0.05) and VD-14, 21, 42d group(P<0.01). There was similar result of times of passing through the platform according to DP.7. Observation of P3-like latency(P3L)Except VD-C-42d group, the P3L prolonged significantly in all ischemic groups vs. SC group. The P3L extended largely 3 days than 1day and 7 days than 3 days after ischemia. These extensions had no statistic significance in VD and VD-C groups but really sharply prolonged in VD-CW group. After 7 days of ischemia, the degree of P3L extension extenuated as time goes on with differences in distinct groups. The significant decurtation of P3L did not emerge until 42 days after ischemia in both VD and VD-CW groups. But in VD-C group, the P3L ameliorated from 21 days post-operation and keep on improving to almost normal level 42 days post-operation.Conclusions1. Adult brain neurogenesis could be triggered by transient global ischemia. Three days after cerebral ischemia and reperfusion (CIR), enhanced neurogenesis emerged in hippocampus, cortex and SVZ with the most obviously enhancements in DG and SVZ. We found that neurogenesis in CA1, CA2 and PCg could be triggered by ischemia that had seldom been reported. The neurogenesis reached its peak 14 days after operation and decreased from 21-day-post-ischemia till 42 days after ischemia in hippocampus and cortex. And in SVZ, neurogenesis reached its peak 7 days after operation. There was no correlation between changes of cognitive function evaluated by Morris water maze test and P3-like detection and proliferation of BrdU-positive cells in hippocampus and cortex.2. Chronic administration of CIT (20mg/kg) could obviously enhance BrdU-positive cell proliferation in different brain region of VD rats, areas including DG, CA1, CA2 and cortex, but barely affected the proliferation of newly generated cells in SVZ. Meanwhile, newly generated cells in DG, CA1 and cortex differentiated more into neurons by treatment of CIT and it had close relationship with the curative facts of CIT on cognitive impairments of VD rats. The effects of CIT mentioned above could be reversed by co administration with WAY 100635.3. The mechanism of neurogenetic effect of CIT in VD rats might be concerned with its function of elevating the 5-HT level in synaptic space in the central nervous system (CNS) and/or of promoting the expression of 5-HT1A receptor mRNA. CIT could ameliorate the cognitive impairment via modulate the CNS levels of monoamine neurotransmitters within 21 days after operation and via neuroprotective effect or promote hippocampus and cortex newly generated cells differentiate into neurons 42 days after ischemia.