The Effects Of Dl-3n-butylphthalide In Mice Of Vascular Dementia On The Molecular Pathogenesis

Objectives:As an acquired syndrome of intelligent impairment, vascular dementia (VD), which is a kind of cerebral dysfunction caused by various kinds cerebral vascular diseases, demonstrats mainly as learning and memory dysfunction, accompanied with the possible disorder of tongue, motion, direction and personality. With the increase of the proportion of the elderly in the population and that of the curing rate of cerebral vascular disease, morbidity of VD mounts continuously and hence brings about heavy loads to the society and family. However, both the pathogenesis of VD and its specific treatment remain unknown up to now. It is therefore significant to study the pathophysiological mechanism of VD and to find some effective treatments of VD.As two advanced functions of human brains, learning and memory are two closely associated neurobiology procedures despite their differences. Learning is a acquirement of experiences and skills. Memory is conservation and playbacks of experience and skills. It is now well known that the hippocampus is correlated with learning and memory. The pathological changes of neurons in the hippocampus have great effects upon the forming and maintance of learning and memory. Many results have confirmed that hippocampus long-term potentiation(LTP) is closely correlated with the process of learning and memory. It has been moreove proved that LTP as a synapse model of memory is produced and maintained with the participation of Glu and its receptor.Glutamic acid, an important excitabily neurotransmitter in central nervous system, is mainly distributed in the area of cerebral cortex, hippocampus etc, and has important effect upoin learning, memory and synaptic plasticity. It participates in many physiological functions of cerebral cortex and hippocampus, such as learning, memory, motion and sensus. In addition, it helps to nourish trophics nerve, promote growth of neuron and axon. The receptor of glutamate mainly includes the receptor ofα-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA) and that of N-methyl-D-aspartate (NMDA), both are found in postsynapse-dense area. AMPA receptor mainly induces fast synaptic transmission which is necessary for normal information transfer of nervous system. NMDA receptor, once activated, mainly triggers different types of synaptic plasticity, including induction of LTP.The intracellular ion concentration of calcium ([Ca2+]i) in neurons has been accepted as the second signal which participates in the mechanism of learning and memory. The intracellular ion concentration of calcium ([Ca2+]i) and calcium/calmodulin dependent protein kinaseⅡ(CaMPKⅡ) has been the focus of the researchers on the mechanism of ischemia-hypoxia damage. Many studies on cerebral ischemia animal model show that calcium over- loading and elevation of CaMPKⅡin neurons might participate in the development of ischemic damage and the Ca2+ channel antagons might hold back calcium over-loading and alleviate the ischemic damage in the neurons.Cyclic AMP(cAMP)-responsive element binding protein(CREB) in neurons, the third signal in intranuclear and a key ingredient of many intracellular signal circuit, has extensive biological functions, including adjustment of the learning and momory by the signal system of Ca2+-CaM- CaMPKⅡand further forming of the transmission circuit Ca2+-CaM- CaMPKⅡ- CREB. According to some related researchers, both the CREB and the expression of glutamate acid receptors in postsynapse membrane of neurons increas after the ischemic damage for their possible participatation.dl-3n-butylphthalide(NBP), a yellow compound isolated from the seed of celery, is a new drug of Class I of P.R.China and never reported abroad up to now. In China, FengYipu has made lots of related researches which proves it as an anticerebral- ischemic drug which has multitarget effects upon, the prevention of the cerebral ischemic damage, the improvement of both chondriosome function and the level of NO and PGI2 of brain blood vessel endothelium, the restraints of glumatic acid release, the degrades of the intracellular concentration of calcium and the level of arachidonic acid, the restraints of free radicle and improves the activity of antioxidase, the lightenments of cerebral edema and the reduction of area of cerebral infarction. In a word, it is proved that NBP can increase cerebral blood flow and improve brain cell energy metabolism, afunction and dysmnesia. However there is no scientific report the drug in VD at present.Based on what has been mentioned above, we attempted to study the therapeutic effect of NBP on VD and therefore explore the mechanism of action by observing GluR circuit and pathogenetic mechanism of VD. In this study, the change of AMPA receptor, NMDA receptor on neuron cellular membrane, and the influence of [Ca2+]i, CaMPKⅡ, CREB in neurons and the the contribution on learning and memory were observed as well. The study results provide us with some clues of further exploration of glutamate receptor circuit and calcium signal transduction mechanism in VD animal model. In the mean while, it is expected that our study may offer some evidence of treating VD with NBP.Methods:1 The VD model of mice were established for ischemia (20 min)- reperfusion (10 min) thrice by ligating the bilateral common carotid arteries. The changes of behavior were observed through the step-down avoidance test and water maze test. The hippocampal pathologic changes in the mice with VD was observed through HE staining.2 The changes of AMPA-GluR2 protein of hippocampus and that of the level of mRNA expression of each group of mice were measured with immunohistochemistry and reverse transcription PCR (RT-PCR).3 The expressions of NMDA receptors NR2B in neurons of hippocampus of every group mice were observed by immunohistochemistry technique. RT-PCR technique was used to measure mRNA expression of NMDA receptor in hippocampal neurons.4 The changes of [Ca2+]i of hippocampal neurons of the mice in each group were measured with flow cytometry. Western blot was applied to the measurement of the expression of CaMKII in hippocampal neurons of each mouse in the experiment. RT-PCR technique was used to measure the mRNA expression of CaMKII in hippocampal neurons of all mice in the experiment.5 The expression levels of CREB in the hippocampal of the mice in each group were analyzed with Western-blot. And RT-PCR technique was used to measure mRNA expression of CREB in hippocampal neurons of the mice in each group.Results:1 The evaluation of behavior of VD model and its pathologic features of the hippocampusThe mice were divided into the control group, sham-operated group, VD model group, NBP treating group and NBP precaution treating group. The mice were subjected for ischemia (20 min)-reperfusion (10 min) thrice by ligating the bilateral common carotid arteries to establish the VD model. Beginning on day 29 and 30 day after operation, the functions of learning and memory of each mouse in each group were tested through step-down test and water maze test.1.1 The test of learning of miceOn day 29 after operation, the learning of every group were tested by step-down test and water maze test respectively. Response time (sec), error times (number/5min) in step-down test, and swimming time (sec) and error times (number/3min) in water maze test were used to evaluate the achivements of learning.The results of step-down test revealed that:⑴The response time in learning phase of VD model group (89.41±12.08)s prolonged distinctly (P<0.01) compared with the response time of sham-operated group(48.76±9.24)s.⑵The response time of both the NBP treating group (59.68±10.27)s and the NBP precaution treating group (57.23±10.05)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of VD control group (3.74±0.42) increased notably (P<0.01), compared with the error time of sham-operated group (1.23±0.27).⑷The error times of both the NBP treating group (1.52±038) and the NBP precaution treating group (1.48±0.31) decreased notably (P<0.01), compared with VD model group.⑸The learning of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).The results of water maze test revealed that:⑴The swimming time in learning phase of the VD model group (145.80±13.29)s prolonged significantly(P<0.01), compared with sham-operated group (87.32±13.46)s.⑵The swimming time of both the NBP treating group (101.89±18.38)s and the NBP precaution treating group (99.18±11.25)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of the VD model group (34.60±5.92) increased notably (P<0.01), compared with and sham-operated group (16.88±4.19).⑷The error times of both the NBP treating group (18.71±4.35) and the NBP precaution treating group (17.29±4.82) decreased remarkably (P<0.01), compared with VD model group.⑸The learning of the sham-operated group, the NBP treating group and the NBP precaution treating group had no distinct difference (P>0.05).The results above suggest that the learning ability of VD mice reduced and NBP may improve their learning. 1.2 The test of memory of miceOn day 30 after the operations, the memory of the mice in every group was tested by step-down test and water maze test. The results of latency time (sec) and error times (number / 5 min) in step-down test and that of swimming time (sec) and error times (number / 3 min) are the memory.The results of step-down test revealed that:⑴The latency time in memory phase of VD model group (78.67±19.17)s shortened remarkably (P<0.01), compared with the latency time of sham-operated group (148.92±20.83)s.⑵The latency time of the NBP treating group (131.41±18.82)s and the NBP precaution treating group (128.57±18.29)s prolonged significantly (P<0.01), compared with VD model group.⑶The error times in memory phase of VD model group (1.72±0.13) increased notably (P<0.01), compared with the error time of sham-operated group (0.32±0.05).⑷The error times of both the NBP treating group (0.54±0.08) and the NBP precaution treating group (0.50±0.07) decreased notably (P<0.01), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).The results of water maze test revealed that:⑴The swimming time in memory phase of the VD model group (137.40±11.76)s prolonged remarkably (P<0.01), compared with sham-operated group (67.82±8.79)s.⑵The swimming time of both the NBP treating group (88.73±9.53)s and the NBP precaution treating group (83.87±8.62)s shortened significantly (P<0.05), compared with VD model group.⑶The error times in memory phase of the VD model group (20.90±4.25) increased remarkably (P<0.05), compared with sham-operated group (9.57±2.91).⑷The error times of both the NBP treating group (11.17±3.04) and the NBP precaution treating group (10.27±2.83) decreased significantly (P<0.05), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).These results suggested that the memory of VD mice decreases and NBP may improve their memory.1.3 The pathologic changes of the hippocampus of the VD miceThe observations under the light microscope show:⑴The arrangement of the pyramydal neurons in the hippocampal CA1 area in sham-operated group were tight and in order with the nucleus being large and round and its neucleolus being evident.⑵In the VD model group, the pyramydal neurons in the hippocampal CA1 decreased and had no clear arrangement. There was empty dye area surrounding the neurons which had condensed nucleus dense cytoplast and small body. In addition, there was infiltration of inflammatory cells.⑶In the NBP treating group and the NBP precaution treating group, the condensed nucleus dense cytoplast and small body decreased apparently and there was no infiltration of inflammatory cells.2 The level of AMPA-GluR2 receptor in the hippocampal CA1 area of VD mice and the effect of NBP .2.1 Staining of immunohistochemistry of AMPA-GluR2.The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and AMPA-GluR2 was stained through immunohistochemistry. And then the statistic study of each group (surface density Sv showing their numbers) was conducted. The result revealed that:⑴The AMPA-GluR2 positive neurons of hippocampus CA1 area in VD model group (27.69±5.15) reduced distinctly (P<0.05), compared with sham-operated group (124.37±7.48).⑵The AMPA-GluR2 positive neurons of hippocampus CA1 area of the mice in both the NBP treating group (88.57±10.98) and the NBP precaution treating group (94.42±12.37) increased (P<0.05), compared with VD model group.⑶The NBP treating group, the NBP precaution treating group and the sham-operated group had no distinct difference (P>0.05).These suggested that the lower level AMPA-GluR2 of hippocampus CA1 area might participate in the pathogenesis of VD and NBP could prevent the level AMPA-GluR2 of hippocampus CA1 area of VD from decreasing and hence improve their learning and memory ability.2.2 RT-PCR of AMPA-GluR2 mRNAThe mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of GluR2 mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of AMPA-GluR2 mRNA expression in VD model (0.28±0.07) reduced notably (P<0.01), compared with the magnitude value of GluR2 mRNA expression in sham-operated group (0.96±0.21).⑵The level of AMPA-GluR2 mRNA expression in NBP treating group (0.71±0.15) and NBP precaution treating group (0.75±0.18) elevated remarkably (P<0.01), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of AMPA-GluR2 mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of AMPA-GluR2 mRNA to reduce and improve their learning ability and memory.3 The level of NMDA-2B receptor in the hippocampal CA1 area of VD mice and the effect of NBP.3.1 Immunohistochemistry of NMDA-2B receptorThe mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made and NMDA-2B receptor was dyed through immunohistochemistry; and made the statistics of group (surface density Sv showing their numbers).The result revealed that:⑴NMDA-2B receptor positive neurons of hippocampus CA1 area in VD model group (29.70±2.09) reduced distinctly (P<0.05), compared with sham-operated group (149.27±8.67).⑵NMDA-2B receptor positive neurons of hippocampus CA1 area in NBP treating group (100.53±10.00) and NBP precaution treating group (108.51±7.28) increased (P<0.05), compared with VD model group.⑶NBP treating group, NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower level NMDA-2B receptor of hippocampus CA1 area might participate in the pathogenesis of VD; but NBP could prevent the level NMDA-2B receptor of hippocampus CA1 area of VD to reduce and improve their learning ability and memory.3.2 RT-PCR of NMDA-NR2B receptor mRNAThe mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of NMDA-2B receptor mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of NMDA-2B receptor mRNA expression in VD model (0.22±0.04) reduced notably (P<0.05), compared with the magnitude value of NMDA-2B receptor mRNA expression in sham-operated group (0.71±0.18).⑵The level of NMDA-2B receptor mRNA expression in NBP treating group (0.54±0.11) and NBP precaution treating group (0.59±0.20) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of NMDA-2B receptor mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of NMDA-2B receptor mRNA to reduce and improve their learning ability and memory.4 Calcium signal transduction pathway in hippocampal neuron of mice with VD and the effect of NBP4.1 The resting [Ca2+]i in the hippocampus of VD mice and the effect of NBPThe mice were executed quickly after anaesthetized by 10% chloral hydrate solution and their hippocampuses were extracted in low temperature operation. They were separated through mechanical method. Thus the single cell suspension sample of hippocampus was prepared. We observed the change characteristic of the resting [Ca2+]i by flow cytometryanalytical technique and intervention of NBP. The result indicated that:⑴The concentration of intracellular [Ca2+]i in hippocampus of VD model group (1.92±0.35) increased notably (P<0.01), compared with the concentration of intracellular [Ca2+]i in sham-operated group (1.29±0.32);⑵the intracellular [Ca2+]i in hippocampus of NBP treating group (1.34±0.28) and NBP precaution treating group(1.33±0.41) decreased notably (P<0.01),compared with VD model group;⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the calcium over-loading in hippocampus cells of VD might participate in its pathogenesis; but NBP could restrain the change and improve the learning ability and memory.4.2 Western blot of CaMPKⅡin hippocampus of VDThe mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. The level of CaMPKⅡexpression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CaMPKⅡexpression in VD model group (0.30±0.07) reduced notably (P<0.01), compared with the level of CaMPKⅡexpression in sham-operated group (1.06±0.14).⑵The level of CaMPKⅡexpression in NBP treating group (0.94±0.13) and NBP precaution treating group (0.98±0.27) elevated notably (P<0.01), compared with the level of CaMPKⅡexpression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmight participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡto reduce and improve their learning ability and memory.4.3 RT-PCR of CaMPKⅡmRNAThe mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CaMPKⅡmRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CaMPKⅡmRNA expression in VD model (0.31±0.06) reduced notably (P<0.05), compared with the magnitude value of CaMPKⅡmRNA expression in sham-operated group (0.92±0.15).⑵The level of CaMPKⅡmRNA expression in NBP treating group (0.78±0.11) and NBP precaution treating group (0.82±0.14) elevated notably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CaMPKⅡmRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CaMPKⅡmRNA to reduce and improve their learning ability and memory.5 The level of CREB in hippocampus of VD and the effect of NBP5.1 Western blot of CREB in hippocampus of VDThe mice were executed quickly through decollation and their hippocampus were extracted in low temperature operation. The level of CREB expression in hippocampus was examined through Western blot method. The result indicated that:⑴The level of CREB expression in VD model group (0.43±0.06) reduced remarkably (P<0.05), compared with the level of CREB expression in sham-operated group (1.37±0.21).⑵The level of CREB expression in NBP treating group (1.06±0.12) and NBP precaution treating group (0.12±0.14) elevated remarkably (P<0.05), compared with the level of CREB expression in VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB to reduce and improve their learning and memory.5.2 RT-PCR of CREB mRNAThe mice were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in nitrogen liquid. The total RNA in hippocampus was distilled through Trizol method. The magnitude value of samples in 260 nm and 280 nm was measured through ultraviolet radiation spectrophotometer to confirm that there was no protein staining. Then the level of CREB mRNA expression was examined through RT-PCR half quantity method. The result indicated that:⑴The level of CREB mRNA expression in VD model (0.29±0.04) reduced remarkably (P<0.05), compared with the magnitude value of CREB mRNA expression in sham-operated group (0.98±0.17).⑵The level of CREB mRNA expression in NBP treating group (0.82±0.09) and NBP precaution treating group (0.84±0.11) elevated remarkably (P<0.05), compared with VD model group.⑶NBP treating group and NBP precaution treating group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of CREB mRNA might participate in the pathogenesis of VD; but NBP could prevent the expression of CREB mRNA to reduce and improve their learning and memory.Conclusions:1 The VD mice model which had the cognization impairment in primary of damage of learning and memory was successfully established. The VD model is the perfect animal model and feasible to the further study of VD. The hippocampus CA1 area in the VD mice model had the pathologic changes.2 The level of AMPA-GluR2 and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the reduction of the expression of AMPA receptor might participate in the pathogenesis of VD.3 The level of NMDAR-2B and its mRNA in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the lower expression of NMDA-NR2B might participate in the pathogenesis of VD.4 The imbalance of calcium signal transduction pathway resulted from the increase of the resting [Ca2+]i in VD mice hippocampus and the reduction of CaMPKⅡin VD mice hippocampus, which might be one part of the molecular pathogenesis of VD.5 The reduction of the protein level of CREB in VD mice hippocampus consistent with the decline of learning and memory suggested that the reduction of the protein level of CREB might participate in the moleculear pathogenesis of VD.6 NBP could resist the over-loading of calcium in hippocampus of VD and prevent the expression of CaMPKⅡand AMPA receptor and NMDA receptor and the level of CREB from reducing. These further improve the calcium signal transduction pathway and ameliorate the pathologic changes in hippocampual CA1 area in the mice with VD. This was in accord to their better learnig and memory. All mentioned above suggest that the medicine may improve the pathogenesis of VD.