Cardiopulmonary Profile in Streptozotocin-Induced Type 1 Diabetic Rats during Systemic Endotoxemia

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-α) 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-α 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.


Introduction
Due to the lower level and impaired binding activity of cell-surface receptors on monocytes [1], a poorly controlled diabetic state increases susceptibility to infections such as endotoxemia [2]. In the diabetic patients, infection is more serious and difficult to eradicate [3]. 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 gramnegative bacteria [4]. 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 factoralpha (TNF-) [5] and causes tissue damage [6]. Also, arterial pressure is decreased due to the dilation of peripheral vessels in rats during endotoxemia [7]. Consequently, a fatal syndrome of irreversible cardiovascular collapse and septic shock may follow [8]. 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. Such an occurrence contributes to lung edema and poor lung compliance. Hence, septic patients may suffer from critical shock and acute respiratory distress syndrome as a group experience high mortality.
e mortality from cardiovascular disorders in type 1 diabetes is 4-to-37 times higher than that in the general population [11]. Severely uncontrolled diabetic state may initiate pathologic events leading to the capillary leak of acute respiratory distress syndrome as well [12]. erefore, 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 aer intravenous LPS infusion. Also, the changes in TNFin serum and lung lavage �uid were determined.

Animals.
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). ey were housed in groups of four at an ambient temperature of 24 ± 1 ∘ C and maintained under a normal light-dark cycle (14:10 h; lights on at 6:00 am). Pelleted rat chow and tap water were available ad libitum. 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 re�ex and pain re�exes induced by tail pinching throughout the course of all experiments (approximately 8 h each) aer a single intraperitoneal dose (1.4 g/kg) of urethane.

Experimental
Groups. 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 aer the animals had fasted for 72 h [13]. Rats with hyperglycemia (blood sugar >300 mg/dL) and polyuria were considered diabetic. At the 5th week aer STZ administration, survival time, and cardiovascular parameters were continuously monitored in animals receiving intravenous infusion of LPS or saline. 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 aer receiving an injection of LPS or saline; (II) determination of the change of hemodynamic parameters during endotoxemia and lung edema at 180 min aer 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 aer LPS or saline injection.

Measurement of Hemodynamic Parameters.
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). Aer cannulation, animals were stabilized for 1 h without data collection. 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.

Analysis of Arterial Blood Gas.
In order to determine arterial pH, arterial partial pressure of O 2 (PaO 2 ), CO 2 (PaCO 2 ), and O 2 saturation (SO 2 ) of rats, 0.4 mL of arterial blood was sampled from the femoral artery 3 h aer administration of LPS using a syringe rinsed with heparin. Samples were analyzed by blood gas analyzer (Synthesis1725, Diamond Diagnostics Inc., Hollinton, MA, USA).

Determination of TNF-in Serum and in Bronchoalveolar
Lavage Fluid. For determination of TNF-levels, venous blood samples were taken from rats at 3 h aer 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, 4 ∘ C). e supernatants were harvested and stored at −70 ∘ C until measurements were taken. At the same time, bronchoalveolar lavage samples were collected by perfusing saline from the endotracheal tube. e concentrations of TNF-were determined using a double-antibody sandwich ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. e optical density of each well was determined by microplate photometers (Multiskan E�, 110�120 V, ermo Fisher Scienti�c Inc., Waltham, MA).

Detection of Lung Vascular/Epithelial Permeability.
Albumin content of the bronchoalveolar lavage �uid was determined to assess the damage to endothelial cells of the lung capillaries. e lavage samples were analyzed using a Bio-Rad protein assay system (Bio-Rad Hercules, CA) with bovine serum albumin (BSA) as the standard. e albumin content in the bronchoalveolar lavage �uid was calculated by dividing the albumin by dried weight of lung to evaluate pulmonary capillary/endothelial cell permeability.

Measurement of Lung
Edema. ree hours aer administration of LPS (or saline), the entire lungs were dissected, weighed (wet weight), and dried (at 63 ∘ C for 48 hours). Dry weight was also recorded. e wet/dry weight ratio of lung tissue was calculated to evaluate lung edema.

Histopathologic
Change of Lung. Rats were perfused with saline followed by 4% paraformaldehyde buffer at 180 min aer LPS administration. Serial sections (4 m) of right upper lobe were stained with hematoxylin and eosin for microscopic evaluation. e characteristics of lung damage include vascular congestion, hemorrhage, polymorphonuclear leukocytes (PMN) in�ltration, and edematous changes of alveolar wall [14]. 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 in�ltration, which was assessed as the PMN/alveoli ratio, can stand for lung damage.
We selected 10 randomly areas at high-power �eld (HPF, 400x) of each blind sample. en alveoli and PMNs in each area were counted and PMN/alveolus ratio was expressed by dividing the sum of PMNs in 10 HPF �elds by that of alveoli.

Statistical
Analysis. Data are expressed as mean ± S.E.M. for each point. Statistical analysis was conducted using analysis of variance (ANOVA) for factorial experiments, and repeated-measures ANOVA (followed by Scheffe aer hoc test) for comparing hemodynamic parameters. e Wilcoxon rank-sum test was used for analysis of survival time the �aplan-Meier test for survival rates. Signi�cant differences between groups were assumed to be present at values of .

Results
Blood glucose in diabetic rats was substantially (approximately 5-fold) and signi�cantly ( ) higher than that in normal rats (361 ± 9 mg/dL versus 73 ± 3 mg/dL). Body weights in STZ-treated rats were signi�cantly lower than those in controls (262 ± 10 gm versus 385 ± 9 gm, ) at the 5th week aer injection of STZ.

Survival Time and TNF-Levels.
Aer 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, ). Also, the survival rate of diabetic rats was signi�cantly lower than the rate for control rats (Figure 1). Figure 2 shows that TNF-levels in serum and bronchoalveolar lavage �uid were signi�cantly 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 �uid were statistically higher in diabetic rats as compared with control normal rats. Figure 3. Mean arterial pressure was higher in diabetic rats than that in normal rats with saline administration. Aer LPS challenge, heart rate increased slightly and then dropped dramatically. Moreover, heart rate and mean arterial pressure were signi�cantly decreased in diabetic rats than those in control ones.

Lung
Injury. e ratio of wet lung weight to dry lung weight and the albumin content in lung increased signi�cantly aer LPS administration, and these parameters were indistinguishable between NL and SL groups ( several changes aer 3 hours of LPS administration (Figure 5). e interstitial spaces of aveoli became thickness with polymorphonuclear leukocytes in�ltration 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. e lung injury score and PMNs/alveoli ratio were signi�cantly 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 PaO 2 and SaO 2 for diabetic rats were signi�cantly lower than those for control rats (Table 1). During endotoxemia, PaCO 2 diminished signi�cantly, whereas PaO 2 and SaO 2 were signi�cantly greater in control and diabetic rats.

Discussion
In the present study, we demonstrated alterations in cardiopulmonary function in both control and type 1 diabetic rats during systemic endotoxemia. We found higher TNFlevels in serum and bronchoalveolar lavage �uid in diabetic rats during systemic endotoxemia. Shorter survival times and the hemodynamic abnormalities observed in diabetic rats  were worse aer 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 [15]. Multiple organ injury and irreversible cardiovascular collapse are induced in animals receiving recombinant human TNF [16]. 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]. us, TNF-level seems to be related to survival rate. In the present study, the �nding of a high level of TNFin the serum and bronchoalveolar lavage �uid 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. ese changes are consistent with previous studies that used the STZ-induced diabetic model for chronic and continuous hemodynamic measurement [19,20]. ose 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]. is 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 aer STZ injection [23].
Heart rate was reported to be increased in order to compensate for the endotoxin-induced low blood pressure , compared with the SS group. [24], 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 signi�cantly than that in normal rats aer LPS challenge whereas the difference among LPS-treated normal and diabetic groups was minor. e present results are in good agreement with those of previous �ndings showing that endotoxin administration to diabetic animals resulted in more severe cardiovascular dysfunction than in nondiabetic animals [25]. 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 [26]. Yoshinari et al. found numerous polymorphonuclear cells in thickened alveolar interstitial tissue during endotoxemia [27]. e TNF-released by polymorphonuclear cells damages the pulmonary vessels and increases alveolar-vessel permeability which causes lung protein leakage. erefore, the lung injury is associated with elevated cytokines. Some evidence has shown that albumin content in the bronchoalveolar lavage �uid increases signi�cantly, and lung compliance is reduced aer endotoxin treatment [28]. In this study, we showed that diabetic rats have signi�cantly higher TNF-levels in lavage �uid 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 [29]. Aer 6 hours of LPS administration, increased TNF-brings about lung injury. e TNF-content was signi�cantly higher at 24 h than at 6 h aer LPS injection. However, the degree of lung edema was lower at 24 h than at 6 h. ere seemed to be a TNFthreshold 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 aer intratracheal instillation of LPS [30], and the concentrations of TNF-in the bronchoalveolar lavage �uid of intratracheal LPS-treated diabetic rats were markedly reduced [2]. 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.
e present results show that PaCO 2 decreases and PaO 2 increases signi�cantly during endotoxemia. A previous study showed that endotoxemia causes large changes in the respiratory system [31]. Endotoxin upregulates reactive oxygen species, TNF-, and many other mediators. ese are harmful mediators and are associated with respiratory muscular dysfunction [32]. Increases in vasodilators and vasoconstrictors that are induced by endotoxemia cause vessel dysfunction and redistribute blood �ow. e insu�cient blood �ow for several organs slows down metabolic rate and decreases oxygen consumption. As a result, the generation of CO 2 decreases [33,34].
ere are some limitations to our study. e higher mortality rate of STZ-treated rats may correlate with higher levels of TNF-aer 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 TNFlevels and improve survival aer LPS injection. Also, further studies will be necessary to determine the responses of other in�ammatory mediators �e.g., nitric oxide, Macrophage in�ammatory 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 �uid were increased more markedly in diabetic rats receiving an LPS challenge.