| Background and ObjectivesAlzheimer Disease (AD) is the killer for the health of human, especially for the oldpeople's health. To date, the mechanism for this disease remains unclear. Strong relationshipbetween chronic brain hypoperfusion, β-amyloid deposition, neurovascular unit damage andAD has been indicated in the previous literatures. However, little information was reported onthe relationship among chronic brain hypoperfusion, β-amyloid deposition, and neurovascularunit. Therefore, the first aim of the present study is to investigate the relationship of thesethree facets through creation of rats model of chronic brain hypoperfusion.3-n-butylphthalide, the major pharmacological component isolated from celery seed, iswidely distributed in many traditional Chinese medicine and natural plants, including danggui,chuanqiong and chaqiong. It has been demonstrated to exhibit multiple pharmacologicalactivities including anticonvulsant and antiasthmatic action. The therapeutic effect of3-n-butylphthalide towards AD has been frequently reported, but the detailed mechanisms remain tobe clarified. Therefore, the second task of the present study is to preliminarily discuss themechanism through studying the intervention effect of3-n-butylphthalide towards relationshipamong chronic brain hypoperfusion, β-amyloid deposition, and neurovascular unit.Materials and Methods1. Animal Treatment. The rats were randomly divided into three groups: controlgroup (sham-operated), ischaemic group, and the the ischaemic rats+drug treatmentgroup using computer-generated random number. Under10%chloral hydrate (350mg/kg,intraperitoneally) anaesthesia, a ventral midline incision was made and the bilateralcommon carotid arteries were exposed and gently separated from the carotid sheath andvagus nerve. During the surgical procedure, the body temperature of the rats was keptstable at37°C using a heating pad. In the rats assigned to the ischaemic groups, eachartery was double ligated with5-0silk suture. The rats in the control groups (sham-operated)received the same operation without ligation. Each group consisted of48rats with identical mean body weights (234±14g). Ten days after the surgical procedure,oral administration of dl-NBP2mg/kg (Shijiazhuang Pharma Group NBP PharmaceuticalCo. Ltd., Shijiazhuang, China)or the equivalent volume of saline water (vehicle)wasgiven once daily for7days.2. Morris Water Maze Task. The rats were gently placed into the water, facing theside walls of the maze from one of the four pre-planned starting position (east, west,south, or north). Swimming paths of the rats were monitored by a video camera linked toa computer through an image analyzer. For each training trial, the latency to escape ontothe hidden platform and the pathlength were recorded. The rats were given a maximum of60s to find the hidden platform. If the rat failed to find the platform within60s, thetraining was terminated and a maximum score of60s was assigned. The rat was thenguided to the hidden platform by hand and allowed to stay on the platform for10s beforebeing removed from the water.3. Western blotting analysis. Proteins were extracted from cerebral cortex andcerebral hippocampal tissue (100-200mg)in rats using radioimmunoprecipitation lysisbuffer. Samples containing150μg protein were separated by10%sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred tonitrocellulose membranes. The membranes were blocked with1×phosphate-bufferedsaline (PBS), pH=7.4,buffer containing3%bovine serum albumin for2h at4oC. Themembranes were then incubated with primary antibodies and secondary antibodies.Horseradish peroxidase-streptavidin conjugate and enhanced chemiluminescence (ECL)detection reagents were used to visualize the protein bands. The expression of theseprotein was normalized with actin.4. Immunohistochemical staining. Tissue sections (5μm) were prepared fromformalin-fixed, paraffin-embedded rat specimens, deparaffinized and rehydrated. Afterblocking with goat serum, the sections were incubated with primary antibodies andbiotinylated horseradish peroxidase goat antimouse secondary antibody. Theimmunohistochemical staining was then examined and photographed at a magnification of400using an Olympus CX42light microscope. The immunohistochemistry signal was scoredusing the so-called "Allred Score."Results1. Treatment with dl-NBP significantly improved the learning and memory ability of hypoperfused rats. Western blotting analysis indicated that, in comparison with the sham-operated control group, levels of APP and MMP-2protein were significantly increased inthe cerebral cortex of hypoperfused rats. Furthermore, treatment with dl-NBP couldprevent increases in APP and MMP-2induced by hypoperfusion. Immunohistochemicalanalysis showed that β-amyloid40(Aβ40) and MMP-2were deposited in veinendothelial cells on day7, and in artery endothelial cells on day14after hypoperfusion.2. The volume of brain hippocampal zone decreased after chronic brainhypoperfusion for3months. Compared with control group, the volume of brainhippocampal zone at3,4and5months exhibited significant difference (p<0.05). Thealtered volume of brain hippocampal zone and cortex induced the dilation of cerebralventricle and subarachnoid. After ischemia for1month, the neurons in CA1region ofbrain hippocampal zone decreased, and glial cells proliferated. The reduction ofcognitive function of rats with chronic brain hypoperfusion might be related with ischemiain brain hippocampal zone.3. Evans blue dye was used to evaluate the damage of blood brain barrier. Comparedwith sham-operated rats, the levels of Evans blue in brain tissue of ischemia group ratssignificantly increased, which suggested the damage of blood brain barrier. The treatmentof dl-NBP towards ischemia rats prevented the penetration of Evans blue through thebrain tissues. Electron microscope scanning results also showed that dl-NBP exerted theprotection towards damage of blood brain barrier.ConclusionsThe present study clearly demonstrates that chronic brain hypoperfusion in rats canresult in the deposition of Aβ and damage of blood brain barrier. Some molecularmechanism was preliminarily clarified, including the alteration of the expression of MMP-2.Additionally, dl-NBP can alleviate the influence of chronic brain hypoperfusion on the thedeposition of Aβ and damage of blood brain barrier. All these results are beneficial forfurther understanding of mechanism of AD and therapeutic potential of dl-NBP towards AD. |
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