| Objective: Established the tetracycline- induced pleural adhesion and fibrosis model in the rabbit which was used to observe the changes of every character after intrapleural PNS. These characters as follows: the character of pleural effusion(glucose,leucocyte ,lactate dehydrogenase, protein ),the volume of pleural effusion, detecting the concentration of transforming growth factor (TGF)-βand vascular permeability factor (VEGF) which were from pleural effusions by enzyme-linked immunosorbent assay (ELASA ), observing pleural adhesion and fibrosis and assessing pleural adhesion points after these animals were killed at 14 days ,drawing pleural to make paraffin section and do HE, Masson staining which was used to observe pleural thickness, collagen deposition. Evaluated the effect of the PNS on tetracyclineinduced pleural adhesions and fibrosis in the rabbit by the means of these characters in order to explore the treatment of pleural adhesions and fibrosis .Materials and Methods: Sixty rabbits( Equally divided male and female, weight 2.5-3.0 Kg) were randomly divided into six groups: the control group, model group, low-dose PNS group(L-PNS), middle-dose PNS group (M-PNS), high-dose PNS group(H-PNS) and urokinase group (UK). 10 rabbits in each group, All animals were inserted a chest tube between the spine and the tip of the scapula into the right pleural space. The control group was given 3 ml Nomral saline intrapleurally via a chest tube. Each animal in others group was given solution of tetracycline hydrochloride, 35 mg / kg ,thereafter, every 12 hours,the control group was still given 3 ml Nomral saline intrapleurally via a chest tube, L-PNS group, M-PNS group and H-PNS group were respectively given PNS 20 mg / kg, 40 mg / kg and 80 mg / kg intrapleurally via a chest tube, each animal of urokinase group were given urine kinase 1500IU intrapleurally via a chest tube.A total of 8 times were given every animals until pulling out chest tube at 96 hours. Extract pleural effusion respectively at 24,48,72,96 hours via a chest tube before administrating agents ; recorded the total volume, character and took out of 3 ml using to detect the character of biochemicalion and enzymelinked immunosorbent assay. During the experiment, all animals were got water and food freely. All animals were killed at 14 days. Opened the pleural cavity of every animals in each group, observe and record pleural adhesions points . Pleural lesions obtained tissue fixation and embedding, sections for HE, Masson staining for microscopic observation.Results:1 the tetracycline- induced pleural adhesion and fibrosis model in rabbitThere were 55 animals which completed the experiment .8 animals completed the experiment in model group and two died of hemothorax; nine animals completed the experiment in PNS low-dose group and one animal died of pneumothorax because of bitting chest tube by itself; 9 animals completed the experiment in PNS high-dose group and one animal may died of pneumothorax because we found the chest tube falling off; nine animals completed the experiment in urokinase group and one died of unknown causes; all animals completed the experiment in the control group and the PNS middle - dose group .2 drainaging volume of Pleural effusionThere was no significant difference in each group (except control group) at the 24hour. There was significant difference in H-PNS and UK group at the 48 hour ,72 hour 96 hour and the volume was Significantly less than other groups. The volume reached the peak at 48 hour and less than other groups at the same moment . The volume reached the peak at 72 hour in othe groups and then than gradually decreasd3 Biochemical parameterof Pleural effusion3.1 the content of protein : The content of protein in pleural effusion gradually decreased over time in every group. There was no significant difference in each group at the 24 hour.But there was significant difference in H-PNS group at the 48 hour and less than other groups(p<0.05). There was also significant difference in H-PNS and UK group compared with other groups at the 48,72,96 hour,no significant difference was found between two groups.3.2 The number of leukocytes :There was no significant difference in each group at the 24,48 hour.But there was significant difference in H-PNS group at the 72,96 hour(P<0.05) and lower than others.With the time passing , a declining trend was found in every group.3.3 The content of LDH: No significant was found among groups at any time.But it is gradually decrease over time.3.4 The content of glucose:, Glucose from pleural effusion in each group had gradually increased over time . H-PNS group was significantly higher than otherS at the 48 hour.4 cytokine concentration4.1 concentration of TGF-β: The concentration of TGF-βin H-PNS group was higher than others at 24 ,48 hour and there was significant difference between them (P<0.05) . The concentration in H-PNS and UK group was higher than others at 72 ,96 hour and there was significant difference between them (P<0.05). The concentration of TGF-βin model and L-PNS group increased rapidly after 48 hour, while the curve of the other three groups tended more gentle.4.2 concentration of VEGF: The concentration of VEGF in H-PNS group was lower than others at the same moment and there was significant difference between them (P<0.05) .Expecting the concentration of VEGF in L-PNS group reached peak at 72 hour ,others reached peak at 48 hour ,after that it became decrease gradually . 5 Pleural cavity and the pleural adhesion score:5.1 Pleural cavity: the control group , pleural was smooth and no dhesion; model group, L-PNS and M- PNS group :It had formated extensive pleural adhesion and multiple adhesion belt, there was one cases of pleural hemothorax . H-PNS and UK group: There was a little bit of pleural adhesion.5.2 the pleural adhesion score: there was significant difference in H-PNS and UK group (P<0.05).The values were (1.56±0.53)an(1 .44±0.53)respectively.6 Pleural pathology6.1 Microscopic observations: HE staining showed that pleural was thin , mesothelial cells was flat and inflammatory cells and fibroblasts were not obvious in the control group. Masson staining showed that no or only a very small amount of collagen in control group. In model group ,L-PNS and M-PNS group,HE staining showed that pleural was more thicker ; there was a large number of inflammatory cells and fibroblasts ; cube-shaped esothelial cells are sometimes was seen . Masson staining showed that thickening of the connective tissue under mesothelial cells was blue-green and there were a large number of collagen fibers. HE staining showed pleural thickness, inflammatory cells, and fibroblasts decreased, compared with other groups. In the same groups ,Masson staining showed that the blue and green colors of the connective tissue which existed under mesothelial cells became lighter than the other groups .this suggested that collagen fibers decreased.6.2 pleural thickness: The pleural thickness in H-PNS and UK group was significant difference than other group(p<0.001),the values were (71.22±33.02)μm and(100.00±57.01)μm respectively6.3 the numbers of fibroblast : The numbers of fibroblast in H-PNS and UK group was significant difference than other groups(p<0.001);the values were(93.33±25.29)/mm2,( 113.55±35.87)/mm2,Conclusion1 Injecting 35mg/kg tetracycline hydrochloride into the thoracic cavity of rabbits cound be induced significant pleural adhesions and fibrosis. Replication animal mode successful for study the PNS .2 PNS cound reduce the volume of pleural effusion, decrease the production of TGF-βand VEGF, inhibited the generation the inflammatory cells and fibroblasts cells, inhibit collagen production. PNS is effective in reducing pleural adhesions and thickening.3 PNS 80 mg / kg injected into the pleural cavity cound effectively inhibit pleural adhesions and fibrosis in rabbit induced by tetracycline . no significant adverse reactions was found. Compared with urokinase1500IU of one rabbit there was no significant difference in treatment effect. PNS 40mg/kg, 20mg/kg did not come into significant therapeutic effect.
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