Molarless Induced Changes Of Learning And Memory In KM, SAMP8 And ICR Mouse And The Effects Of Coeloglossum Viride (L) Hartm. Var. Bracteatum ( Willd.) Extract On Molarless Induced Learning And Memory

Evidence suggests chewing affects the cognitive functions of learning and memory in the hippocampus. For example, the systemic effect produced by tooth loss is an epidemiological risk factor for Alzheimer's disease, a disease that is accompanied by the loss of hippocampal neurons along with a progressive deterioration of both spatial and working memory. Therefore, studies on the effects and mechanism of the molarless condition on learning and memory may shed important insights on the prevention and intervention of learn and memory disorders.Objective: The first part is to find the molecular mechanism related with learning and memory deficit after bilateral molarless in kong ming(KM), sensecence accelerated mouse/prone8 (SAMP8) and ICR mice, which may ultimately provide experimental basis for clinical prevention of senile dementia. The second part is to study the neuroprotective effects of CE, a special extract from Coeloglossum viride (L.) Hartm. var. bracteatum (Willd.), on molarless induced learning and memory deficits in ICR mice.Methods:1 Establishment of the animal modelall mice were anesthetized via i.p. injection of 10% chloral hydrate (400mg/kg). In the sham group mice, small amounts of alveolar bone were removed with Rongeur from the toothless gap region between molars and canines in the superior alveolar ridge on the left. In the molarless group, all the bilateral maxillary molars were removed. Care was taken to extract the whole teeth; if there was root fracture, all the tooth structure visible on the gum was removed to eliminate occlusal contacts. Following surgery, animals were fed with routine pellet diet with free access to water, and observed for general conditions and body weight.2 Water maze experimentThe water maze apparatus was a bi-layer, non-transparent rectangular plastic box (80cm×50cm×20cm), We measured the number of errors entering non-exits and the time required to reach the end-point steps to evaluate their short-term memory and spatial learning and memory.3 Open field testAll animals were placed sequentially into the RD1413 open field apparatus. Mouse locomotor activities were video recorded and analyzed by the system-equipped analytical software.4 HPLCAfter water maze test, all animals were decapitated, cortex and hypothalamus were dissected to measure the monoamine neurotransmitters concentration and amino acid neurotransmitters concentration.5 Western blotting analysisFollowing decapitation, mouse brain cortex,hippocampus and brainstem were dissected, weighted, recorded and snap frozen in liquid nitrogen. proteins were extracted with Applygen total protein extraction kit. Protein concentration was normalized using Coomassie brilliant blue G-250 staining. Equal amounts of proteins were separated by SDS-PAGE on 10%(TrkB) and 12%(BDNF) polyacrylamide gel; Then proteins were transferred to PVDF membrane. After blocking with 0.1%TBST containing 5% non-fat milk at room temperature (RT) for 2 hr, primary antibody was added for overnight incubation. The membrane was washed with 0.1% TBST three times, 10 min each, and incubated with secondary antibody at RT for 2 hr. The membrane was washed as above, and color development was performed using the ECL kit. Images were acquired using the Fuji Digital Science Imager, and analyzed with Gelpro Analyzer (version 4.0) to measure the integrated optimal density (IOD) values of specific bands. 6 Hematoxylin and Eosin (HE) stainFor neuropathological observation, three mice from each group were chosen, the brains were fixed in 4% paraformaldehyde in PBS (pH 7.4), embedded into paraffin, cut into 4μm sections and stained with hematoxylin and eosin (HE). When treated with different doses of CE, This phenomenon has been markedly improved.7 Immunohistochemical procedureAfter the final maze test, the animals were anesthetized with 10% chloral hydrate (400mg/kg), then perfused with phosphate-buffered saline(PBS, PH 7.4) containing 4% paraformaldehyde in 0.1 M phosphate buffer, Following postfixation for 24h in the same buffer, then imbedded into paraffin, 4μm sections were cut on a microslicer and sections were processed through a standard immunohistochemical protocol. The sections were first rinsed with PBS, then incubated with 30% H2O2 for 10min at room temperature to quench endogenous peroxidase activity, and then blocked with 5% BSA for 20min at room temperature,followed by incubation for 2 days at 4℃with rabbit anti-BDNF antibody and rabbit anti-TrkB antibody,diluted 1:100, The sections were rinsed in PBS ,2min and 3times,and incubated for 20min at 37℃with anti-rabbit IgG, after a futher rinse in PBS, then for 20min at 37℃with ABC reagent in PBS, before being treated for about 10min at room temperature with DAB. Sections were washd with ddH2O, after the final wash, the section were using the standard procedure for light microscopy.Results:1 Body weight changes following surgery in KM,SAMP8 and ICR mice Prior to surgery, the two groups of animals showed comparable weights, Following surgery, the weight was monitored for one week, and significant decreases in body weight were observed in both groups. Relative to the sham group, the molarless group lost greater weight. seven days after surgery, body weight started to recover, and essentially reached pre-surgery levels2 Learning and memory function of KM,SAMP8 and ICR mice In the current study, following surgery, KM and SAMP8 mice were subjected to monthly water maze tests. The results indicated that both KM and SAMP8 mice did not display any significant deficits in learning and memory in the first two months after complete molarless. However, after three months, the molarless group started to exhibit significantly longer latency than the sham group (P < 0.05) in SAMP8 and ICR mice, and after two months, the molarless group in KM mice showed significantly longer latency than the sham group, indicating significantly impairment in learning and memory functions under the molarless condition. compare with DM group, the escape latence was significantly decreased in DML (DM+CE2.5mg/kg) and DMM (DM+CE5mg/kg), suggest that CE could amiliate molarless induced learning and memory impairment.3 Open field testOur results suggest that the molarless animals were indistinguishable from their control counterparts of locomotor functions in both KM and SAMP8 mice.4 HPLCMonoamine neurotransmitters results suggest that molarless induced changes of DA and 5-hydroxytryptmine(5-HT) in KM mice, especially in mice cortex and hypothalamus, in mice cortex, where levels of DOPAC/DA and 5-HIAA/5-HT were increased 39.8%,29.7%,(P<0.05 vs. sham). In mice hypothalamus,we also observed that nor-adrenalin(NA),5-hydroxyindole acetic acid(5-HIAA) and HVA increased(P<0.05 vs. sham). Amino acid neurotransmitters results suggest that molarless induced glutamate(Glu) decreased in cortex and hypothalamus(P<0.05 vs. sham). However, there isn't any change in Gly and GABA.5 Western blot analysisWe examined expressions of BDNF and its receptor TrkB in the cortex,hippocampus and brainstem of KM, SAMP8 and ICR mice.The results indicated that following 2 months in KM mice, BDNF expression was significantly decreased in mice cortex(P<0.05 vs. sham), where its receptor TrkB was not significantly decreased,we also detected CREB and Bcl-2, there isn't any changes in cortex. Following 3 months in SAMP8 mice, BDNF expression was significantly decreased in mice cortex(P<0.01 vs. sham) and hippocampus(P<0.05 vs. sham),and its receptor TrkB wasn't decreased。Following 3 months in ICR mice, BDNF expression was significantly decreased in mice cortex(P<0.01 vs. contro), hippocampus(P<0.05 vs. contro) and brainstem(P < 0.05 vs. contro),and its receptor TrkB was significantly decreased in cortex(P<0.01 vs. contro), not in hippocampus and brainstem. Compared with DM group, the expression of BDNF was significantly increased in brainstem of the low dose (DML) group, and BDNF expression was significantly increased in cortex of the high dose (DMH) group. in medium dose (DMM) group, the expression of BDNF was significantly increased in both cortex and brainstem, while TrkB expression was significantly increased in cortex.7 Hematoxylin and Eosin (HE) stainHE staining showed that cells in the hippocampus CA3 area arranged in irregular, disordered, reduce the number of cells, cell body shrinkage, cytoplasmic eosinophilic change, condensation nuclei can be seen, but at the same time, the amygdala is also observed in cortex and decreased significantly Cytoplasmic eosinophilic change, When treated with different doses of CE, This phenomenon has been markedly improved.8 ImmunohistochemicalCortex part of the immunohistochemistry results showed that after unilateral and bilateral molarless, the expression of BDNF in cortex were significantly decreased, statistical analysis showed P <0.01; TrkB results showed that the cortex of TrkB expression was significantly decreased in bilateral molarless group, and P <0.01. Hippocampus (P <0.05), After the cortex amygdale (P <0.05) and spinal trigeminal nucleus(P <0.01) showed that BDNF expression was significantly decreased. while the unilateral molarless group in the above brain regions, there were no significant changes in the expression of BDNF. Compared with the DM group, the number of BDNF positive cells significantly increased in cortex and the spinal trigeminal nucleus in DML group. And the number of BDNF immunopositive cells were also significantly increased in cortex, amygdala after the cortex and spinal trigeminal nucleus in DMH group. There were significantly differences between the control and DMM group, the number of BDNF positive cells significantly increased in cortex, amygdala after the cortex and spinal trigeminal nucleus, and the expression of TrkB was also significantly increased in cortex. CE could increase the expression of BDNF in both spinal trigeminal nucleus and the cortex with its related organizations, and also increasing the expression of TrkB in the cortex, which re-start the BDNF-TrkB signaling pathway, play their importment role in learning and memory.Conclusions: Molarless could induce chewing functions decrease, so that the spinal trigeminal nucleus activetity was decreased, because chewing activities uploaded to the spinal trigeminal nucleus through the trigeminal nerve, then the spinal trigeminal nucleus project through the brain stem reticular formation stimulation to reduce cortical activity. Furthermore activity dependent BDNF expression was decreased in Cortex, hippocampus, spinal trigeminal nucleus and cortical regions after amygdala, and its receptor TrkB expression also activity-dependent decreased in the cortex, disturb the balance of BDNF-TrkB to maintain the excitability of neuronal activity and Inhibitory activity, causing a series of reactions downstream pathways, which ultimately lead to learning and memory decline. CE could increase the expression of BDNF in the spinal trigeminal nucleus and the cortex, in addition, CE could also increase the expression of TrkB in the cortex, which re-star the BDNF-TrkB signaling pathway and the effect of protectivity in molarless induced learning and memory disability.