in previous studies , we demonstrated that meconium exposure in neonatal rabbit lungs leads to significant cell death and expression of inflammatory cytokines . in clinical situation , the most deleterious effect of mas is the development of both increased pulmonary vascular resistance ( pvr ) and associated pulmonary hypertension . the cause of increased pvr is believed to be related primarily to hypoxia and by the meconium - induced lung injury ; however , the relative contribution of other mechanisms and mediators are unknown . high oxygen exposure is also associated with adverse pulmonary effects [ 5 , 6 ] . in clinical meconium aspiration syndrome ( mas ) ( 3 ) during treatment , lungs are exposed to high oxygen concentration , which may also cause cellular injury . thus , all these mechanisms may play a major role in meconium - induced lung cell apoptosis and increased cytokine expression . however , it is not known whether hypoxia and hyperoxia will independently induce lung injury similar to meconium . we hypothesized that lung injury associated with meconium may be due to cellular responses to hypoxia and hyperoxia seen in mas . in order to test our hypothesis , we designed a study to determine the independent effects of meconium , hypoxia , and hyperoxia and inert substances such as saline and milk in inducing lung cell apoptosis and inflammatory cytokine response . in addition to the above studies , we conducted in vitro experiments using a549 cells to study the independent effect of meconium and saline in the presence of hypoxia and hyperoxia in inducing apoptotic cell death . in this paper , we present our findings of these studies . fifteen first - pass human meconium samples were obtained from full - term , healthy neonates . one  g of fresh newborn infant meconium was homogenized on ice in a blender with 9  ml of 0.9% nacl to a 10% ( weight / volume ) final concentration followed by spin down at 5.000  rpm for 20  min at 4c to separate supernatant and pellet ( debris ) . then supernatant filtered via a glass filter followed by sterilization via 0.2  m filter ( both filters were from millipore co. , bedford , ma ) and used for instillation in the lungs . meconium of higher then 10% concentration in our experiments caused death of many rabbit pups . their breathing was slower , followed by complete apnea . because of this reason , we decided to use a 10% meconium solution for our studies . we used two - week - old new zealand white rabbit pups ( lsp industries , union grove , wi ) as animal models for our study . the animal care and use committee at michael reese hospital , chicago , illinois , approved this study . for a few days preceding the experiment , animals were housed with their mother in stainless steel rabbit cages . the mothers were fed with regular purina rabbit chow ( scientific animal feed co. , arlington heights , il ) . we used six rabbits in each group and for each time period ( the minimum allowing for obtaining statistically significant data ) . five groups of animals were studied : group i : saline - instilled ( subgroup a : only saline ; subgroup b : saline in hypoxia ; subgroup c : saline in hyperoxia ) ; group ii : exposed to hypoxia ( fio2 : 10% ) for a period of 15 minutes ; group iii : exposed to hyperoxia ( fio2 : 95% ) for a period of 15 minutes ; group iv : instilled with 10% meconium solution ( subgroup a : only meconium ; subgroup b : meconium in hypoxia ; subgroup c : meconium in hyperoxia ) ; group v : instilled with milk  similac  with iron , infant formula , ross pediatrics . saline , milk , or meconium was instilled in the volume of 300  g per 200  g of body weight . animals did not tolerate hypoxia of fio2 less than 10% for longer than 15 minutes . the groups subjected to saline , meconium , and milk instillation ( groups i , iv , and v ) were studied at 0- , 4- , 8- , and 24-hour time periods from insult and then they were sacrificed . before instillation , we anesthetized rabbits with 10  mg / kg ketamine and 1  mg / kg xylazine , given intraperitoneally . a small midline incision was made on the ventral aspect of the neck to expose the trachea ; saline , meconium , or milk was then instilled into lungs followed by 5  ml of bolus of air to spread meconium into the lungs . the skin incision was closed with 40  nylon suture ; the rabbits were allowed to breathe room air spontaneously for 0 , 4 , 8 , and 24 hours and then were euthanized with nembutal ( 100  mg / kg , i.p . ) . immediately after sacrifice , the chest was opened by a midline incision , lungs were isolated and lung lavage was obtained as described below . we evaluated a timeline for the rabbits to develop signs of lung injury rather than test for their survival rate at the end of experiment . after the sacrifice of each animal and isolation of the lungs , the tracheas were cannulated and lavage was performed through the trachea and main bronchi to recover lung cells as described earlier . we collected lung lavage by 10  ml aliquot three times to collect 30  ml of lavage fluid per rabbit studies . we obtained a total protein after several times cell sonication using an ultra sound sonicator ( and then spinned down at 10,000  rpm for 10 minutes to isolate cell debris . then we precipitated supernatant - contained protein by acetone and then pellet dissolved in 50  l of ddh2o to measure protein concentration by bradford method . finally , we added 50  l of sds sample buffer to the sample and performed 10% sds gel electrophoresis . lung lavage fluid was spun down at 5,000  rpm for 10  min at 4c . then supernatant was discarded and cell pellet then was stained with 0.1  g / ml ethidium bromide and acridine orange ( sigma chemical co. , st . fluorescent microscopy of the lavage cells ( mostly macrophages , leukocytes and neutrophils ) was performed as described by us earlier live cells ( stained with acridine orange ) appeared green , while dead cells ( stained with ethidium bromide ) appeared red . the number of dead cells was counted three times , in different areas of the slide , and the results were calculated as a percentage from each 100 cells counted . the supernatants obtained after centrifugation of the samples were used for elisa assays as described earlier . we measured the il-1 , il-6 , il-10 , and tnf cytokine expressions in pictograms in milliliter ( pg / ml ) using commercially available elisa kits as described by their manufacturer ( r&d systems co. , minneapolis , mi ) . in a separate experiment , independent effects of hypoxia or hyperoxia on meconium - induced lung injury were studied using human lung epithelial a549 cells in culture . cells were treated in hypoxic or hyperoxic conditions and then 10% meconium solution was added . we are conducting in vitro studies to demonstrate meconium - induced inflammation using cultured a549 cells . the human lung adenocarcinoma cell line a549 was obtained from american type culture collection and cultured in ham 's f12 medium supplemented with 10% fetal bovine serum ( fbs ) . all cells were seeded in 24-well or 6-well chambers and all experiments were conducted at densities of 80%90% in a serum - free ham 's f12 medium . cells were treated first in hypoxia ( or hyperoxia ) for 15 minutes , and then 10% meconium was added to the plates and incubated for   eight  hrs in hypoxic or hyperoxic conditions at 37c before harvesting . for quantitation of cell death , we used acrydine orange ( live cell staining , green color ) and ethidium bromide ( dead cell staining , orange color ) stained cells . results are mean  standard deviations of at least four experiments . in morphological experiments , paired evaluations were made for experimental and control conditions within each experiment , and significance was determined by student 's t - test . saline - instilled animals ( control group ) revealed approximately same amount of lavage cells as milk - instilled lungs . meconium - instilled animals demonstrated significantly lower lavage cell number at a period of 8  hrs after meconium instillation versus milk- or saline - instilled ( 10.7  5.2  10 versus 15.2  3.7  10 versus 14.2  2.1  10 ) and 24  hrs ( 8.8  11.1  10 versus 13.8  2.6  10 versus 12.9  10 ) . hypoxia was associated with low cell count at 15 minutes treatment time similar to meconium . hyperoxia showed no differences ( table 1(b ) ) . observed cell death in lung lavage can be very significant and can reach up to 52% within 24  hrs after meconium instillation . figure 1(a ) demonstrated presence of lavage cell death in meconium , saline , and milk . the number of dead cells after meconium instillation was significantly higher than after saline and milk instillation and the differences were statistically significant ( p < .05 ) . cell death significantly increased with time in the meconium group , but not in the saline or milk groups ( figure 1(a ) ) . hypoxia resulted in 7.1%  1.3% and hyperoxia in 4.4%  0.5% of dead cells in figure 1(b ) .   on microscopic examinations we observed many inflammatory infiltrated cells and many opened alveoli . hypoxia also increases the size of alveolar septae , which shows broken alveoli ( not shown ) . the effect of hyperoxia was not significant in saline as well as meconium - instilled lungs . hyperoxic animals demonstrated sleepy behavior and its effect was seen already after   15 seconds after treatment . both hyperoxia and hypoxia animals demonstrated signs of respiratory distress ( retraction , nasal flaring ) . regardless of the noted effect of hypoxia or hyperoxia - induced damage , meconium - induced damage was significantly more destructive and meconium - induced lung injury was significantly higher than in other groups studied . meconium - instilled animals demonstrated increase of total protein content in the lung lavage ( see table 2(a ) ) . meconium - induced protein levels were significantly higher compared to levels observed in lung lavage from animals instilled by saline , milk as well as animals exposed to hypoxic or hyperoxic conditions ( table 2(b ) ) . in meconium - treated lungs , mg / ml of total protein was detected 24  hrs after meconium instillation ; 0.24  0.2  mg / ml in saline - instilled lungs ; 2.0  0.4  mg / ml in milk - treated lungs ; 2.3  0.9  mg / ml in hypoxic lungs ;   3.1  1.1  mg / ml in hyperoxic lungs . data for hyperoxic lungs also was statistically significant versus the saline group ( p < .05 ) and not significant versus the hypoxic group ( p > .05 ) . intratracheal instillation of meconium causes the dramatic expression of inflammatory cytokines in lung lavage but not in saline- or milk - instilled lungs . saline - exposed newborn rabbit lung lavage cytokine levels were similar at 4 , 8 , and 24  hrs . in this study , we demonstrated expression of tnf , il-1 , il6 , and il10 cytokines in different study groups of rabbits . the tnf and il-6 inflammatory cytokines were expressed significantly in meconium - instilled lungs ( table 3(a ) ) . both of these cytokines were expressed about four times higher compared to saline - treated than in lavage - treated lungs . milk - instilled animals did not show any significant expression of discussed cytokines versus saline - instilled , and milk - instilled and saline - instilled animals showed approximately similar , not significant levels of tnf and il-6 cytokine expression . surprisingly , il-1 was expressed about twofold higher 4  hrs after meconium - instillation ( maximal observed expression in il-1 ) than saline - instilled lungs ( see table 3(a ) for   details ) . in hypoxic or hyperoxic conditions , the levels of inflammatory cytokines tnf and il6 were increased significantly ( table 3(b ) ) . anti - inflammatory cytokine il-10 levels were not significant in any of the groups studied . we found that meconium - treated cells similar to lung lavage cells demonstrated dramatically higher percent of cell death ( figure 2 , d , e , and f ) compared to saline - treated cells ( figure 2 , a , b , and c ) . we also found that hypoxia + meconium group showed some increase in number of cell death and inflammatory cytokines expression compared to hyperoxia + meconium group ; however , this increase was not significant ( p > .05 ) . levels of hypoxia contribute to the inflammation induced by meconium as noted by increased cytokine production in hypoxic conditions . leeper - woodford and detmer showed that acute hypoxia in lungs may induce enhanced nf - kb activation in alveolar macrophages , resulting in the increased production and release of inflammatory cytokines such as tnf . hyperoxia has also been reported to cause pulmonary pathology and dysfunction , leading to death in several species [ 8 , 9 ] . in survival studies , frank et al .   showed that newborn lungs are particularly susceptible to hyperoxic exposure because of their inability to increase protective lung antioxidant enzymes , resulting in 50% mortality rate by three days . kelly et al .   reported a mortality rate of 21% in newborn exposed to 95% hyperoxia for 96  hrs . hyperoxia is associated with low inflammatory cell levels ; protease activity and levels of total protein were slightly increased compared to that of saline - instilled animals , suggesting a similar degree of pulmonary dysfunction . cell death in lungs may be induced by numerous factors , the most critical of them is   meconium and hyperoxia [ 5 , 7 , 1116 ] . previous studies have described in detail the effects of hyperoxia on the lungs in different animal models . buckley et al . examined cell death by apoptosis in ex vivo cell culture from hyperoxia - exposed rat lungs . interestingly , those human lung epithelial cells in culture were able to survive in hypoxic and hyperoxic conditions for much longer time . that is why we used a cell culture for studies in addition to a newborn rabbit model . hyperoxia is a well - known inflammatory factor , causing severe response in the lungs . it is of specific interest because of the wide use of mechanical ventilation and oxygen treatment in the management of mas . allen et al .   used cultured respiratory epithelial - like cells treated with hyperoxia alone , tnf alone , or both factors to show their effects on il-8 gene expression . he demonstrated that hyperoxia alone has a minimal effect on il-8 expression , but synergistically increases it in the presence of tnf. ben - ari et al . base their studies on neonatal rats , showing that the exposure of lungs to 100% oxygen causes the production of pulmonary tnf on the third day and il-6 on days 69 of hyperoxia [ 19 , 20 ] . there is a similar degree of pulmonary dysfunction in hypoxic and hyperoxic animals . in our studies , meconium - instilled animals demonstrated significantly higher levels of cell death and higher cytokine expression , compared to other treatments . hypoxic and hyperoxic animals produce initial lung inflammation ( polymorphonuclear cells and inflammatory cytokines increase ) . we compared the effects of milk instillation to meconium instillation studies as reported before . to the best of our knowledge , there is no data about the effects of milk instillation on lung cell apoptosis or cytokine expression in the literature available , making our study unique in this area . finally , another manifestation of lung injury is capillary leak resulting in an increase of total protein concentration in lavage fluid , which was high in meconium group ( table 3 ) . in contrast , the total amount of protein in bal in milk - instilled animals was comparable to saline - instilled animals . in the present study , we compared the inflammatory effect of meconium with effects of milk instillation . we also presented a data about the effect of hypoxia and hyperoxia on lung cell death and expression of inflammatory cytokines . our data seems to prove that none of the irritants causing acute lung injury contributed to lung cell death or cytokine expression to the extent seen by the aspiration of meconium . hypoxic and hyperoxic insults , even if treated for only 15 minutes , resulted in minimal changes , whereas milk and saline caused even less injurious effect . hypoxia , always a factor in meconium - induced lung injury , does not seem to be responsible for its injury and apoptotic effect . more studies are needed to find the role of mechanical stress of ventilation or the characteristics of meconium such as particle size , viscosity , and so forth on lung cell injury , including a longer time of treatment , as 2 , 4 , 8 , and 24  hrs of hypoxia or hyperoxia treatment . in summary , we demonstrated that hypoxia and hyperoxia resulted in no significant increase in cell death and inflammation in newborn rabbit pups . our present studies demonstrate that aspiration of milk and saline into the lungs will not cause a significant lung injury . overall , our data demonstrate that meconium - induced lung injury may not be explained by the concomitant presence of milk , hypoxia , or hyperoxia in mas . meconium with its multiple compounds is likely the major agent , which induces lung inflammation and lung cell death .
background . 
 we previously   showed   that meconium   cuases lung   cell death by apoptosis and inflammatory cytokine expression . 
 whether this is due to   meconium   exposure itself , or meconium related hypoxia remains unclear . 
 objectives . to   elucidate the effects of   meconium , 
 saline , milk , hypoxia and hyperoxia induced lung injury . 
 design / methods . 
 we studied 5 groups of rabbit pups :   ( i ) normal saline ;   ( ii ) milk ;   ( iii ) 10%   solution of meconium ;   ( iv ) only to 15 minutes of hypoxia ( 10%   o2 ) , and   ( v ) 5 minutes of hypoxia   ( 95%   o2 ) . 
 after exposure lung lavage cells were used for   apoptotic cell count and cytokine expression .   in vitro   response    of human   a 549 epithelial cells to   meconium - and milk exposure was also studied . 
 results . 
 there was no difference in   cell death between saline and milk   groups . 
 however , meconium   caused a significant cell loss compared to    saline and milk  inflammatory cytokines increased   significantly in meconium group   compared to saline or milk group . although hypoxic and hyperoxic lungs showed increased inflammatory reaction compared to saline - treated lungs , this injury was not significant compared to meconium group . 
 studies with    a549 cells also showed similar results . 
 conclusions . 
 we conclude that   lung cell injury in meconium aspiration   is maily   from meconium itself .