| Hypoxic-ischemic brain damage (HIBD) is one of the most common ischemic brain damage in neonate. There is no effective treatment method to Hypoxic-ischemic encephalopathy (HIE) caused by HIBD now. ATPase is an ion pump on the biomembrance, which has important functions on substance transportations and ionic balance. Nerve growth factor (NGF) has the actions of nourishment and axon growth on central and peripheric neurons. NGF is important to the survival, differentiation, growth and regeneration of nerve cells. Bcl-2 is the main anti-apoptosis protein, which is generally expressed on outer mitochondrial membrane, endoplasmic reticulum and nuclear membrane. Nissl body, normal ingredient in intracytoplasm of neuron, is an indicator of cellular defect, which is sensitive to pathological damage.Many clinical studies showed that acupuncture-therapy has positive therapeutic effect to HIE. But the animal experiment to testify the curative effect and the mechanism is still scarce. In this study, we evaluated the effect of acupuncture on HIBD and explored partial mechanism of that by the parameters of ATPase activity, HE staining, NGF and bcl-2 immunohistochemistry staining, Nissl staining, learning and memory ability.Materials and methods1. Health senven-day-old newborn Wistar rats, female or male, 12~16g, wererandomized to groups: (1) 5d group, including control group, model group, acupuncture group. Each group contained 20 rat pups. (2) 35d group, including control group, model group, acupuncture group. Each group contained 10 rat pups.2. The preparation of HIBD model: At postnatal 7 day, the neonate rats were anesthetized by Ether and the left common carotid artery was blocked permanently by double ligature. After the surgical procedure, the pups were allowed to recover for lh, The litters were placed in a chamber and then exposed to a gas mixture (8% O2 in 92% N2) for 2h. The control group subjected to neither ligation nor hypoxia.3. Protocol of acupuncture: Renzhong and Neiguan acupucture points were selected. Rats of acupuncture 5d group was acupunctured b.i.d. from Id to 5d later. Rats of acupuncture 35d group was also acupunctured after modeling. But there was a interval for 5d between lOd and 15d. Stop acupuncture after 25d. The rats were tested by maze experiment. Rats of control groups and model groups were catched and fixed in corresponding times.4. Index measurement(1) ATPase activity: Pups were killed at 5d after hypoxia and the brain was dissected out quickly. Left hippocampus was separated immediately on ice and agitated it into tissue homogenate. After enzymatic reaction and phosphonium fixing, the optical density of every tubes were tested. ATPase activity was determined according to the formula from specification.(2) Histology examination:a. HE staining: The sections was stained by HE staining and observed the pathological change under optical microscope.b. NGF and Bcl-2 immunohistochemistry staining: Calculated the positive cell on 6 random visual fields of every section under light microscope with 400-times amplifies. The positive cells of NGF and Bcl-2 expression were stained into brown-yellow in cytolymph.c. Nissl body staining: Used toluidine blue staining. The percentage and average gray scale of positive area were calculated with an image-analyzing system.(3) Learning and memory ability testing: The learning ability of pups was testedat 35d after modeling by using Y-radial maze stimulator and the memory ability was tested in the second day.5. Statistical analysis: All the empirical dates were expressed with X ± s and analyzed with SPSS11.0 software. One-way analysis of variance among groups and Mest between two groups were used. The significant testing standard was a = 0.05.Results1. ATPase activity (amolPi/mgprot/hour): The Na+-K+-ATPase activity (2.563±0.435), Ca2+-Mg2+-ATPase activity (2.858±0.524) and total ATPase activity (5.413+0.923) decreased significantly in model group compared with control group (3.991±0.569, 4.089±0.601, 8.262+1.286)(P<0.05). These all increased significantly after treatment with acupuncture in acupuncture group (3.328±0.378? 3.591 ±0.307, 7.026+0.771) than that in model group (P<0.05).2. HE staining: The shape of neurons in control groups was normal. There existed a lot of degenerated or necrotic neurons in model groups. Cellular necrosis areas can be fonund. With acupuncture therapy, the hemispheres of acupuncture groups swelled gently and the infarct size was decreased. The neuron shapes and structure were relatively normal.3. NGF immunohistochemistry (positive cell number/Hp): In model group, the quantity of NGF immune positive expression (8.76±1.00) were clearly increased than that in control group (6.31±1.17) (P<0.05), and that in acupuncture group (16.5±1.66) were significant larger than that in model group (P<0.05).4. Bcl-2 immunohistochemistry (positive cell number/Hp): In model group, the quantity of Bcl-2 immune positive expression (37.12±6.11) were clearly increased than that in control group (44.17±7.15) (P<0.05), and that in acupuncture group (59.88±5.24) were significant larger than that in model group (P<0.05).5. NissI staining: The percentages of positive area in model group (18.43±4.38) were obviously lower than those in control group (28.19±4.02) (P<0.05). And the percentages in acupuncture group (23.11+3.38) were clearly higher than those in model group (P<0.05). The gray scales of average positive area in model group(123.61±8.52) were obviously higher than those in control group (98.72±9.87) (P<0.05). And the gray scales in acupuncture group (113.90±11.21) were clearly lower than those in model group (P<0.05).6. Learning and memory ability testing: Rats of model group need more times to reach standard level in Id (90.10±7.40) and 2d(81.80+6.03) than that of control group (48.10±5.99, 34.10±5.47) (P<0.05). Rats of acupuncture group need less times in ld(56.30±6.57) and 2d(41.20±7.47) than that of model group (P<0.05). Rats of model group had poor correct rate in Id (59.70±3.40) and 2d (61.80±3.77) compared with those of control group (73.70±4.11, 79.70±2.83) (P<0.05). Rats of acupuncture group had higher correct rate in Id (68.70±3.83) and 2d (75.40±3.75) than that of model group (P<0.05).Conclusion(1) Acupuncture could enhance the enzymatic activity of ATPase and increase the expression of NGF, Bcl-2 in central nerve of neonate rats with Hypoxic-Ischemic Brain Damage.(2) Acupuncture could improve the learning and memory ability of neonate rats with Hypoxic-Ischemic Brain Damage, decrease the dissolve and disengage of Nissl body in nerve cells. The mechanism is partly related with enhancing the enzymatic activity of ATPase and increasing the expression of NGF, Bcl-2 in central nerve.
|
PDF Full Text Request