due to the lower level and impaired binding activity of cell - surface receptors on monocytes , a poorly controlled diabetic state increases susceptibility to infections such as endotoxemia . in the diabetic patients , infection is more serious and difficult to eradicate . however , the severity of cardiopulmonary dysfunction in type 1 diabetes during systemic endotoxemia remains unclear . as a systemic infection , endotoxemia is mainly caused by the endotoxin ( lipopolysaccharides ( lps ) from gram - negative bacteria . lipopolysaccharides binds to cd14 receptor with lps - binding protein when entering the mammalian bloodstream . as a result , nuclear factor-b is activated to induce monocytes , macrophages , and endothelial cells to release cytokines such as tumor necrosis factor - alpha ( tnf- )   and causes tissue damage . also , arterial pressure is decreased due to the dilation of peripheral vessels in rats during endotoxemia . tnf- plays an important role in this endotoxin - induced shock because serum tnf- level increases during lethal endotoxemia [ 9 , 10 ] . in addition , acute respiratory distress syndrome , which is caused by acute lung injury , is another complication of endotoxemia . a great number of monocytes and macrophages are present in alveoli and release tnf- , which damages pulmonary vessels and increases lung vascular / epithelial permeability . hence , septic patients may suffer from critical shock and acute respiratory distress syndrome as a group experience high mortality . the mortality from cardiovascular disorders in type 1 diabetes is 4-to-37 times higher than that in the general population . severely uncontrolled diabetic state may initiate pathologic events leading to the capillary leak of acute respiratory distress syndrome as well . therefore , we hypothesized that the cardiopulmonary dysfunction induced by systemic endotoxemia will be more marked in type 1 diabetes . in the present study , we used type 1-like diabetic rats to investigate hemodynamic dysfunction and lung injury after intravenous lps infusion . also , the changes in tnf- in serum and lung lavage fluid were determined . ninety - six adult male wistar rats ( 320  20  g ) were purchased from the animal center of national cheng kung university medical college ( tainan city , taiwan ) . they were housed in groups of four at an ambient temperature of 24  1c and maintained under a normal light - dark cycle ( 14:10  h ; lights on at 6:00  am ) . all protocols were approved by the institutional animal care and use committee of national cheng kung university , tainan , taiwan . all experimental procedures were conducted in compliance with the national institutes of health 's  guide for the care and use of laboratory animals . adequate anesthetic level was maintained to abolish the corneal reflex and pain reflexes induced by tail pinching throughout the course of all experiments ( approximately 8  h each ) after a single intraperitoneal dose ( 1.4  g / kg ) of urethane . animals were randomly assigned to four groups ( 24 rats for each group ) : ( a ) normal rats receiving 0.9% normal saline administration ( ns ) , ( b ) normal rats receiving lps administration ( nl ) , ( c ) diabetic rats receiving normal saline administration ( ss ) , and ( d ) diabetic rats receiving lps administration ( sl ) . an animal model of type 1 diabetes , streptozotocin-(stz- ) treated rats , was prepared by intravenously injecting rats with stz ( sigma , st . louis , mo ) ( 60  mg / kg ) via the femoral vein to irreversibly destroy pancreatic  cells after the animals had fasted for 72  h . rats with hyperglycemia ( blood sugar > 300  mg / dl ) and polyuria were considered diabetic . at the 5th week after stz administration , survival time , and fresh solutions of lps ( from escherichia coli 0111:b4 , sigma chemical co. ) were prepared in phosphate buffered saline ( ph 7.40 ) at a concentration of 10  mg / ml for infusion ( 15  mg / kg ) into the femoral vein to induce endotoxemia . different subgroups of animals ( 8 rats for each subgroup ) were used for each of the four experiments : ( i ) determination of survival rate in normal and diabetic rats after receiving an injection of lps or saline ; ( ii ) determination of the change of hemodynamic parameters during endotoxemia and lung edema at 180  min after lps or saline injection ; ( iii ) determination of arterial blood gas , lung protein leakage , tnf- level in serum and bronchoalveolar lavage , and lung histopathology at 180  min after lps or saline injection . animals were anesthetized with an intraperitoneal injection of urethane ( 1.4  g / kg ) and the right femoral artery was cannulated using polyethylene catheters ( pe-50 ) . mean arterial pressure ( map ) and heart rate ( hr ) were recorded using a polygraph ( mp35 , biopac systems inc . , goleta , ca , usa ) every 20 minutes from the arterial tube in the femoral artery throughout endotoxemia .   in order to determine arterial ph , arterial partial pressure of o2 ( pao2 ) , co2 ( paco2 ) , and o2 saturation ( so2 ) of rats , 0.4  ml of arterial blood was sampled from the femoral artery 3  h after administration of lps using a syringe rinsed with heparin . venous blood samples were taken from rats at 3  h after administration of lps or saline . blood samples were allowed to clot for 30 minutes at room temperature and then were centrifuged for 20 minutes ( 2000  g , 4c ) . the supernatants were harvested and stored at 70c until measurements were taken . at the same time , bronchoalveolar lavage samples were collected by perfusing saline from the endotracheal tube . the concentrations of tnf- were determined using a double - antibody sandwich elisa ( r&d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions . the optical density of each well was determined by microplate photometers ( multiskan ex , 110120  v , thermo fisher scientific inc . , albumin content of the bronchoalveolar lavage fluid was determined to assess the damage to endothelial cells of the lung capillaries . the lavage samples were analyzed using a bio - rad protein assay system ( bio - rad hercules , ca ) with bovine serum albumin ( bsa ) as the standard . the albumin content in the bronchoalveolar lavage fluid was calculated by dividing the albumin by dried weight of lung to evaluate pulmonary capillary / endothelial cell permeability . three hours after administration of lps ( or saline ) , the entire lungs were dissected , weighed ( wet weight ) , and dried ( at 63c for 48 hours ) . the wet / dry weight ratio of lung tissue was calculated to evaluate lung edema . rats were perfused with saline followed by 4% paraformaldehyde buffer at 180  min after lps administration . serial sections ( 4  m ) of right upper lobe were stained with hematoxylin and eosin for microscopic evaluation . the characteristics of lung damage include vascular congestion , hemorrhage , polymorphonuclear leukocytes ( pmn ) infiltration , and edematous changes of alveolar wall . each characteristic was scored ( 0 : normal ; 1 : mild ; 2 : moderate ; 3 : severe ) by a pathologist and overall lung injury was further calculated according to the sum of the score . moreover , the degree of leukocyte infiltration , which was assessed as the pmn / alveoli ratio , can stand for lung damage . we selected 10 randomly areas at high - power field ( hpf , 400x ) of each blind sample . then alveoli and pmns in each area were counted and pmn / alveolus ratio was expressed by dividing the sum of pmns in 10 hpf fields by that of alveoli . statistical analysis was conducted using analysis of variance ( anova ) for factorial experiments , and repeated - measures anova ( followed by scheffe after hoc test ) for comparing hemodynamic parameters . the wilcoxon rank - sum test was used for analysis of survival time the kaplan - meier test for survival rates . significant differences between groups were assumed to be present at values of p < 0.05 . blood glucose in diabetic rats was substantially ( approximately 5-fold ) and significantly ( p < 0.001 ) higher than that in normal rats ( 361  9  mg / dl versus 73  3  mg / dl ) . body weights in stz - treated rats were significantly lower than those in controls ( 262  10  gm versus 385  9  gm , p < 0.05 ) at the 5th week after injection of stz . after administration of lps , the survival time of stz - treated rats was markedly shorter than that of control rats ( 180.0  14.6  min versus 224.4  17.9  min , p = 0.027 ) . also , the survival rate of diabetic rats was significantly lower than the rate for control rats ( figure 1 ) . figure 2 shows that tnf- levels in serum and bronchoalveolar lavage fluid were significantly raised by administration of lps in both normal and diabetic rats . actually , the tnf- levels for normal or diabetic rats were undetectable with saline treatment . moreover , the lps - induced increases of tnf- levels in serum and bronchoalveolar lavage fluid were statistically higher in diabetic rats as compared with control normal rats . the time course for changes in cardiovascular parameters in normal and stz - treated rats after the administration of saline or endotoxin is shown in figure 3 . mean arterial pressure was higher in diabetic rats than that in normal rats with saline administration . moreover , heart rate and mean arterial pressure were significantly decreased in diabetic rats than those in control ones . the ratio of wet lung weight to dry lung weight and the albumin content in lung increased significantly after lps administration , and these parameters were indistinguishable between nl and sl groups ( figure 4 ) . histopathologic examination of the lungs demonstrated several changes after 3 hours of lps administration ( figure 5 ) . the interstitial spaces of aveoli became thickness with polymorphonuclear leukocytes infiltration as well as edematous changes of alveolar walls were showed in both normal and diabetic groups . quantitative analyses of histology were shown in figure 6 . the lung injury score and pmns / alveoli ratio were significantly increased in nl and sl groups compared with that of ns and ss groups . however , the values of lung injury score and pmns / alveoli ratio are indistinguishable between sl and nl group . baseline values for pao2 and sao2 for diabetic rats were significantly lower than those for control rats ( table 1 ) . during endotoxemia , paco2 diminished significantly , whereas pao2 and sao2 were significantly greater in control and diabetic rats . in the present study , we demonstrated alterations in cardiopulmonary function in both control and type 1 diabetic rats during systemic endotoxemia . we found higher tnf- levels in serum and bronchoalveolar lavage fluid in diabetic rats during systemic endotoxemia . shorter survival times and the hemodynamic abnormalities observed in diabetic rats were worse after lps challenge . however , the difference is minor from normal rats . a previous report showed a lower survival rate with a higher tnf- level during endotoxemia . multiple organ injury and irreversible cardiovascular collapse are induced in animals receiving recombinant human tnf . conversely , monoclonal antibodies against tnf- and agents which antagonize lps - induced production of tnf- are effective in preventing lps - induced lethality in vivo and in vitro [ 9 , 17 , 18 ] . thus , tnf- level seems to be related to survival rate . in the present study , the finding of a high level of tnf- in the serum and bronchoalveolar lavage fluid of diabetic rats appears to be responsible for the higher mortality rate associated with lps treatment . regarding hemodynamic parameters , our data show that the value of mean arterial pressure is higher in diabetic rats than control rats during saline administration . these changes are consistent with previous studies that used the stz - induced diabetic model for chronic and continuous hemodynamic measurement [ 19 , 20 ] . those studies showed that the onset of hyperglycemia in the earliest stage of diabetes increases mean arterial pressure . moreover , studies in patients with early uncomplicated type 1 diabetes also show an increase in arterial pressure during poor glycemic control [ 21 , 22 ] . this increase is correlated with angiotensin ii , due possibly to overstimulation of the renin - angiotensin system in response to hyperglycemia . in addition , endothelial dysfunction , which can be seen in type 1 diabetic patients , was found in the rat 's aorta at the 4th week after stz injection . heart rate was reported to be increased in order to compensate for the endotoxin - induced low blood pressure , which is consistent with our results during the early stages of endotoxemia . however , later on , the animals were unable to compensate , and heart rate dropped dramatically . our data demonstrated that blood pressure drops in diabetic rats significantly than that in normal rats after lps challenge whereas the difference among lps - treated normal and diabetic groups was minor . the present results are in good agreement with those of previous findings showing that endotoxin administration to diabetic animals resulted in more severe cardiovascular dysfunction than in nondiabetic animals . high levels of tnf- were found in serum and lung in rats with endotoxemia , and more albumin was present in endotoxemic lungs than in control ones . yoshinari et al . found numerous polymorphonuclear cells in thickened alveolar interstitial tissue during endotoxemia . the tnf- released by polymorphonuclear cells damages the pulmonary vessels and increases alveolar - vessel permeability which causes lung protein leakage . some evidence has shown that albumin content in the bronchoalveolar lavage fluid increases significantly , and lung compliance is reduced after endotoxin treatment . in this study , we showed that diabetic rats have significantly higher tnf- levels in lavage fluid from the lungs , but not greater albumin content and lung edema during endotoxemia . rojas et al . have shown that tnf- and lung edema have different time courses during endotoxemia . the tnf- content was significantly higher at 24  h than at 6  h after lps injection . however , the degree of lung edema was lower at 24  h than at 6  h. there seemed to be a tnf- threshold for causing lung injury and another , much higher , threshold for inducing more serious damage . in contrast , previous studies showed that diabetes may be associated with a lower - than - average risk of acute lung injury after intratracheal instillation of lps , and the concentrations of tnf- in the bronchoalveolar lavage fluid of intratracheal lps - treated diabetic rats were markedly reduced . it is possible that intratracheal instillation of lps and intravenous lps administration cause different pulmonary responses in diabetic rats . in addition , the response to lps may be different between stz - induced and alloxan - induced diabetic rats . increases in vasodilators and vasoconstrictors that are induced by endotoxemia cause vessel dysfunction and redistribute blood flow . the insufficient blood flow for several organs slows down metabolic rate and decreases oxygen consumption . as a result , the generation of co2 decreases [ 33 , 34 ] . . the higher mortality rate of stz - treated rats may correlate with higher levels of tnf- after lps administration , but the mechanisms underlying the differences in survival rate between diabetic rats and nondiabetic rats are still unclear . to explore whether tnf- plays a critical role during endotoxemia in diabetic rats , it will be necessary to study in vivo blockade of tnf-. moreover , it is important to determine whether glycemic normalization in diabetic rats can reduce tnf- levels and improve survival after lps injection . also , further studies will be necessary to determine the responses of other inflammatory mediators ( e.g. , nitric oxide , macrophage inflammatory protein-1 , etc . ) during endotoxemia . in conclusion , the difference of cardiopulmonary dysfunction between normal and stz - induced diabetic rats was minor whereas the tnf- levels in serum and bronchoalveolar lavage fluid were increased more markedly in diabetic rats receiving an lps challenge .
this study was designed to determine the severity of cardiopulmonary dysfunction during systemic endotoxemia in type 1 diabetes . 
 thirty - two adult male wistar rats were randomly assigned to a control group or to a group treated with streptozotocin ( stz ) to create an animal model of type 1 diabetes . 
 survival time and cardiovascular parameters were continually monitored in urethane anaesthetized animals receiving intravenous infusion of endotoxin ( lipopolysaccharide ( lps ) ) or saline . 
 we also determined arterial blood gases , lung injury , and tumor necrosis factor - alpha ( tnf- ) levels in serum and bronchoalveolar lavage fluid . 
 before lps administration , the mean arterial pressure in stz rats was significantly higher than that in normal rats . 
 after lps injection , the heart rate drop significantly in stz rats than that in the control group . also , the increased levels of tnf- in serum and lavage fluid after lps treatment were significantly higher in stz rats than those in normal rats . 
 survival time in stz rats was shorter than that in normal rats after lps application . 
 albumin content , wet / dry weight ratio of lung , and lung injury were indistinguishable between stz and normal rats . 
 these results indicate that the cardiopulmonary change which occurs during lps - induced endotoxemia is minor in stz - induced diabetic rats .