The Expression And Meaning Of NGF,BDNF And CGRP In The Lungs Of Asthma Rats

Bronchial asthma is the most frequent chronic pulmonary disease that threats the world public health, It has draw widespread attention for its chronic recurrent attacks and the influence to asthma patients and the society. Asthma can attack any age, even the infants and young children , especially the children. The pathogenesy of asthma has not totally indentified. Airway chronic inflammation was considered to be the substance of asthma. In the past, people are prone to study the effect of inflammation mediators and pro-inflammation cytokine in asthmatic airway inflammation. Now it is considered that the nerve-immune system crosstalk participate in the development of asthma; neurotrophins were thought to connect nerve system and immune system. NANC nerve can release neuropeptides then have effects on airway smooth muscle, blood vessel, epithelium and inflammatory cells, Neurotrophins can induce growth ,development and recovery of nerve cells, also they mediate inflammation. Calcitonin gene-related peptide (CGRP) and substance P both are the main neuropeptides of NANC nerve system, they participate asthma by causing the blood vessel relaxation ,airway smooth muscle contraction and neurogenic inflammation. Thus we presume that the main members of neurotrophins nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF) and neuropeptide CGRP themselves or interact with each other participate in the development of asthma. Our experiment make the asthma rats model and treat them with glucocorticoid, observing the expression of NGF, BDNF and CGRP in the lungs of the rats in each group by immunohistochemistry, for more understanding of the pathogenesy of asthma, in order to offer therapeutic advice to asthma.Objections:45 female SD rats, aged 5-6 weeks (110±10g), divide into three groups: control group, asthma group and therapy group, there are 15 rats in each group. (1)Control group: 1ml NS by intraperitoneal injection, and inhalation NS by atomization. (2) asthma Model group: Using ovalbumin(OVA) to make asthma model. (3)Theraputic group: Each 30 minutes before each provocation injecting dexamethasone(DEX) 2 mg /kg by intraperitoneal.Making the asthma model:Each rat was injected lml 10%OVA suspension (containing lml normal saline, 100mg aluminum hydroxide, 100mg OVA) by intraperitoneal to sensitize them on the first day. Repeat the method on the eighth and fifteenth day, Using OVA for provocation from the sixteenth day. Putting the rats into a glassy box made by ourselves that is closed tightly, letting the rats inhale 1%OVA by ultrasound atomization machine. 20 times in total, and each time for 20 minute on every other day, The rats appear restlessness, accelerated breathing or immobility, abdominal muscle spasm , indicate that we make the asthma model successfully.Collecting the sample:All the rats were anesthetized by Phenobarbital sodium(100mg for each rat)then open the chest, intubate into pulmonary artery through right ventricle, using NS to lavage quickly, take the middle lobe of right lung into 10%methanal to fix them, then imbed in paraffin within 24 hours. Slicing the samples by two direction: vertical andparallel to the airway ,the thickness of each slice was 3 fin), then make HE staining andimmunohistochemistry staining.Measure the thickness of airway wallOn the HE staining slices, using computer image analysis system, chosing completed cross section of brochhi, measuring the perimeter of basement membrane BM (μm) and area of the airway wall AW (μm~2) , then calculate the thickness of the airway wall AW/BM (μm~2/μm) .Determination of the expression of NGF,BDNF and CGRPOn immunohistochemistry staining slice, using computer image analysis system, chosing five representative filed of view randomly on each slice under high power lens (400×) , selecting positive area and measuring the gray scale, then calculate the mean gray scale (OD)statistics analysisUsing SPSS11.0 statistical package to analyze, all the result were demonstrated by mean±standard deviation, using one-way ANOVA to compare the mean of each group after the test of homogeneity of variance, the correlation was tested by linear correlation analyzeResult:1. The lung sampleThe lung samples of asthma rats were much larger and hyperaemia than those of control rats, there are few fibrae cyst in mesenchyma. Campared with those of asthma rats, the change above-mentioned were less than those from therapeutic group2. HE stainingBronchi tracheal epithelium cells shedding, mucous interrupt, the plica were increased in the lung sample of asthma rats. There are lots of inflammatory cells (especially eosinophils and lymphocytes)under mucous and around airway wall. Few epithelium cells shedding, airway mucous almost complete in the lungs of therapeutic group. Thickening of airway wall and infiltration of inflammatory cells were both less than those in asthma group. There are not such changes in control group.3. Immunohistochemistry stainingNGF,BDNF expressed in the epithelium, smooth muscle cells, mesenchymal cells and inflammatory cells; the expression in asthma rats was significant higher than the control group (p < 0.01)and the therapeutic group (p < 0.01). CGRP mainly expressed in airway smooth muscle , under mucous. Also was expressed in airway epithelium and the inflammatory cells infiltrate in lung; the expression in asthma group was much higher than control group (p < 0.01) and therapeutic group(p < 0.01).The expression of NGF,BDNF were positive correlation with CGRP in asthma rats ( P<0.05 ) ; The expression of NGF,BDNF and CGRP were all positive correlation with the thickness of airway wall; The dependability between NGF and thickness of airway wall was more obviously ( P<0.01 ) .Conclusion:1. NGF,BDNF and CGRP all participate in the process of asthma2. NGF,BDNF and CGRP show synergistic action in the development of asthma.3. Corticosteriod can decrease the expression of NGF,BDNF and CGRP in the lungs of asthma rats...