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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2010 Jun;91(3):267–275. doi: 10.1111/j.1365-2613.2009.00694.x

A quantitative study of lung dysfunction following haemorrhagic shock in rats

Hiroaki Sato 1, Toshiko Tanaka 1, Toshiro Kita 1, Noriyuki Tanaka 1
PMCID: PMC2884094  PMID: 20041965

Abstract

Haemorrhagic shock occasionally causes an episode of lung dysfunction, the severity of which appears to correlate with fatal outcome. Our previous study indicated that proinflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β, played a key role in the development of lung dysfunction through recruitment of activated neutrophils by causing pulmonary endothelial cell damage. Here, we examined this issue quantitatively by grading four groups of severity of bleeding in rats. As the amount of bleeding increased, the expression of mRNA for TNF-α and IL-1β in the lung tissue and the pulmonary serum levels of both cytokines increased progressively up to 5 h, and the frequency of activated neutrophils increased likewise. The lung dysfunction indices serum lactic dehydrogenase-3 isozyme (LDH-3), partial pressure of arterial oxygen (PaO2) and alveolar-arterial oxygen tension difference (AaDO2) in blood deteriorated as the amount of bleeding increased. The frequency of activated neutrophils in the lung correlated well with the LDH-3 level 5 h after haemorrhagic shock. The present results demonstrate that the increase of proinflammatory cytokines and the recruitment of activated neutrophils into the lung following haemorrhagic shock are quantitatively related to progression of lung dysfunction as the amount of bleeding increases.

Keywords: IL-1β, lung dysfunction, neutrophil, quantitative, TNF-α

Haemorrhagic shock, when followed by an episode of lung dysfunction, is a predictor of high morbidity and mortality. The degree of functional deterioration that is in practice in autopsy cases is usually difficult for forensic pathologists to establish exclusively by postmortem morphological findings. If the relationship between the extent of lung dysfunction and haemorrhagic shock was elucidated quantitatively, pathophysiological developments could be explained definitely, and the cause of death diagnosed accurately. Several studies have demonstrated that the severity of lung dysfunction became worse as the amount of bleeding increased (Croce et al. 1999; Martel et al. 2002; Bulger et al. 2007). However, the precise mechanisms responsible for increasing lung dysfunction in response to the degree and the progression of haemorrhagic shock have not been clearly elucidated thus far.

Previously, we have shown that proinflammatory cytokines, such as TNF-α and IL-1β played an important role in the development of lung dysfunction through the recruitment of activated neutrophils in the rat lung following haemorrhagic shock (Sato et al. 2008). In this study, we examined this issue quantitatively at different degrees of bleeding. Our results provide a novel protocol to demonstrate the pathogenesis of lung dysfunction progression in forensic cases of haemorrhagic shock.

Materials and methods

Animals

Male Wistar rats, weighing 270−340 g, obtained from Seiwa Experimental Animal Co. (Oita, Japan), were allowed to acclimatize to their new surroundings, 22 °C and 12 h light–dark cycles, for 2 weeks, with ad libitum intake of water and standard rat feed. Before the experiments, the animals were refrained form eating food overnight, but were allowed to drink water ad libitum. The Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan, approved all requests for animals and the intended procedures of this study according to the guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH publication No.85–23, revised in 1996).

Haemorrhage procedure

Animals were anaesthetized with a mixture of urethane (ethyl carbamate) (470 mg/kg body weight) and α-chloralose (23 mg/kg body weight) by intraperitoneal injection, and were maintained under anaesthesia by additional injections of the mixture throughout the experimental period. The animals were maintained on their backs in a temperature-controlled surgical board (37 ± 1 °C) and were allowed to breathe spontaneously. After the induction of anaesthesia, the right common carotid artery was cannulated under aseptic conditions with a 3 Fr polyethylene catheter (Atom Medical Co., Tokyo, Japan) connected to a blood pressure transducer (G-1000; Nihon Kohden, Tokyo, Japan). The cannula was filled with heparinized saline (700 U/ml) beforehand. After cannulation, heparinized saline (700 U/kg body weight) was injected to prevent systemic coagulation. The mean arterial blood pressure (MBP) was measured from the catheter and monitored by polygraph (LEG-1000; Nihon Kohden). Electrocardiogram in lead II was also recorded on a polygraph through needle electrodes attached to the limbs. The heart rate (HR) was computed automatically by R-R interval of the ECG record in the polygraph. The mean values of MBP and HR were calculated every 30 s, and recorded on the polygraph continuously as a data file. The data values were extracted from the file and statistically analysed every 20 min throughout the experiment. After the equilibrium period, the rats were bled via the catheter using a syringe at a constant rate for 20 min.

Experimental protocol

Rats were divided into four groups according to the bleeding volume. In preliminary experiments, we established that the 5-h-mortality-rate was 50% after the rats lost up to 33% of their total blood volume (LD50). Taking this result into consideration, we designated 17.5% bleeding of total body blood as a mild haemorrhagic shock, 25% as moderate and 33% as severe (Sato et al. 2004, 2005, 2007, 2008). The decrease of MBP and the deterioration of lung dysfunction became more severe as the amount of bleeding increased (described below), so we believe that the present methodology is appropriate to examine lung dysfunction of different degrees of severity following haemorrhagic shock. Rats that were not bled were used as negative controls (sham group). Rats that underwent haemorrhage of up to 17.5% of their total body blood volume (which is 6.5 ± 0.1 ml/100 g body weight) (Bitterman et al. 1991), i.e. 1.1375 ml/100 g body weight, constituted the 17.5% group, while rats that underwent haemorrhage up to 25% (1.625 ml/100 g body weight) or 33% (2.145 ml/100 g body weight) made up the 25% group and 33% groups respectively.

After recording haemodynamic values (HR, MBP) and analysis of arterial blood gas, the rats of each group were killed by exsanguination from a cannulated catheter, and pulmonary blood and lungs were collected after thoracotomy at 1, 3 or 5 h after the bleeding (n = 5, in each group). Serum of pulmonary blood was separated by centrifuging the collected blood at 3000 rev/min for 20 min. The serum and tissue samples were stored at −80 °C until assayed.

Assessment of TNF-α and IL-1β mRNA expression in the lung

Total RNA was extracted from lungs using ISOGEN (Nippon gene, Toyama, Japan). One microgram of total RNA was reverse-transcribed using RNase H-reverse Transcriptase and random hexamers (Invitrogen Corp, Carlsbad, CA, USA). To assess the amount of TNF-α and IL-1β mRNA in each sample, the polymerase chain reaction (PCR) was performed for TNF-α, IL-1β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a constitutively expressed housekeeping gene. PCR was performed in a total volume of 50 μl consisting of 1.35–5.0 μl of RT sample (equivalent to 1.0–5.0 μg of total RNA) using the PCR Core Kit (Takara Bio Inc, Tokyo, Japan). PCR products were separated by 1% agarose gel electrophoresis containing 0.3 μg/ml ethidium bromide to visualize DNA bands, and the gels were scanned on Mupid-Scope WD (Advance, Tokyo, Japan). Densitometry was performed with the Adobe Photoshop software package (Adobe Systems Inc, Mountain View, CA, USA). The densities of the bands were quantified with Scion Image Beta 4.02 (Scion Corporation, Frederick, MA, USA), and the ratio of TNF-α or IL-1β/GAPDH mRNA was calculated. The obtained values were then divided by the average value of the sham group at each time point, and are expressed as the ratio relative to the sham group.

Pulmonary serum TNF-α, IL-1β and LDH-3 concentration

The pulmonary serum concentrations of TNF-α and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA; Rat TNF-α or IL-1β, immunoassay kit, BioSource International, Inc, CA, USA). LDH-3 was determined by electrophoresis using cellulose-acetate membrane and enzymatic staining.

Arterial blood gas analysis

Arterial blood gas samples (2 ml) were collected via catheter cannulated in the right common carotid artery at 1, 3 and 5 h after bleeding, and were analysed using the ABL 520 Blood Gas System (Radiometer A/S, Copenhagen, Denmark). We analysed the gas exchange capacity, which had been demonstrated to be decreased following inflammation-related lung injury (Hashimoto et al. 2002), using the partial pressure of PaO2 and AaDO2. The latter was calculated using the formula: AaDO2 (mmHg) = PiO2 − PaCO2/0.8 − PaO2, PiO2 = (760–47) × FiO2. In this equation, PiO2 is the partial pressure of oxygen at inhalation, and FiO2 is the fractional inspired oxygen concentration, which was estimated to be 0.21 units in room air.

Immunohistochemistry

For light microscopy, rats were killed 1, 3 and 5 h after haemorrhage and the lung was isolated, cut into 2-mm-thick slices and fixed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.6. Dewaxed sections were incubated in 3% hydrogen peroxide for 10 min and washed in 0.1 M PBS, pH 7.6. Rabbit polyclonal anti-rat myeloperoxidase (MPO) antibody as a marker for neutrophils (Laboratory Vision Co., CA, USA) was applied to the sections, which were then incubated at room temperature for 1 h. Products resulting from the immunoreaction were visualized by the peroxidase-conjugated streptavidin-biotin method (Simple-Stain-PO kit; Nichirei Corp. Tokyo, Japan) using 3,3′-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide. The nuclei were counterstained with haematoxylin. For the controls of each immunostaining, Tris buffer or normal sera was substituted for the primary antibodies. MPO-positive cells in 10 randomly selected fields of the lung (250 μm)2 were counted from the slides for five rats in each group. The obtained data were expressed as the number of MPO-positive cells per field. Histological analyses were performed by a pathologist without prior knowledge of the experimental conditions.

Statistical analysis

Data are expressed as mean ± SE. Differences among groups were tested for statistical significance using one-way analysis of variance and Fisher post hoc testing. A P-value of less than 0.01 denoted the presence of a statistically significant difference.

Results

Changes in blood pressure and heart rate caused by haemorrhage

MBP significantly decreased immediately on haemorrhage, proportional to the amount of bleeding (shown in Figure 1b, from 100 ± 6 to 46 ± 3 mmHg in the 17.5% group; Figure 1c, from 105 ± 6 to 36 ± 3 mmHg in the 25% group; Figure 1d, from 102 ± 2 to 30 ± 2 mmHg in the 33% group) and then recovered temporarily. In the later phase after haemorrhage, MBP tended to decrease gradually with time, again proportional to the amount of bleeding (Figure 1b–d). Similarly, HR of the haemorrhage groups decreased after bleeding, again proportional to the amount of bleeding (shown in Figure 2b, from 412 ± 5 to 312 ± 11 bpm in the 17.5% group; Figure 2c, from 424 ± 12 to 261 ± 19 bpm in the 25% group; Figure 2d, from 422 ± 7 to 268 ± 10 bpm in the 33% group), also recovering temporarily, then decreasing gradually again in the 25% and 33% groups (Figure 2b–d).

Figure 1.

Figure 1

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Time course of mean arterial blood pressure (MBP) changes. (a) Sham group (Sham), (b) 17.5% bleeding group (17.5%), (c) 25% bleeding group (25%), (d) 33% bleeding group (33%). MBP decreased progressively as the amount of bleeding increased at all times (b, c, d). Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

Figure 2.

Figure 2

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Time course of heart rate (HR) changes. The abbreviations are the same as those in Figure 1. The changes in HR were similar to MBP. Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

TNF-α mRNA expression in the lung and pulmonary serum TNF-α concentration

TNF-α mRNA expression in the haemorrhage groups increased, peaking 1 h after bleeding (2.03 ± 0.34 in the 17.5% group; 3.79 ± 0.61 in the 25% group; and 4.03 ± 0.36 in the 33% group) and tending to decrease again at 5 h. The level of TNF-α mRNA expression tended to increase as the amount of bleeding increased, at any time after haemorrhaging (Figure 3a,b). Similarly, the pulmonary serum TNF-α concentration in the haemorrhage group increased markedly 1 h after haemorrhage, again proportional to the amount of bleeding (74.4 ± 29.3 in the 17.5% group; 117.6 ± 23.4 in the 25% group; and 148.1 ± 16.1 in the 33% group) and tended to decrease once more after 5 h (Figure 3c).

Figure 3.

Figure 3

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Changes in pulmonary TNF-α expression after haemorrhage. As the amount of bleeding increased, TNF-α mRNA expression in the lung increased progressively at all times after bleeding, shown on the vertical axis in B. Similarly, the pulmonary serum TNF-α concentration paralleled the TNF-α mRNA expression at all times after haemorrhage as shown in C. Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

IL-1β mRNA expression in the lung and pulmonary serum IL-1β concentration

IL-1β mRNA expression in the haemorrhage group peaked 3 h after bleeding (3.35 ± 0.63 in the 17.5% group; 5.23 ± 0.55 in the 25% group; and 5.68 ± 0.57 in the 33% group); this increase was significant compared with the sham controls for the 25% and 33% haemorrhage groups. The increase of IL-1β mRNA expression tended to be proportional to the amount of bleeding at all the times tested after haemorrhaging (Figure 4b). Similarly, the pulmonary serum IL-1β concentration in the haemorrhage groups peaked 3 h after bleeding (126.0 ± 56.7 in the 17.5% group; 250.0 ± 53.6 in the 25% group; and 366.4 ± 63.7 in the 33% group); the difference in the latter two groups also achieved statistical significance compared with the sham control group. Here, too, the increase in the concentration of pulmonary serum IL-1β tended to be higher with greater haemorrhage at all the times after bleeding (Figure 4c).

Figure 4.

Figure 4

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Changes in pulmonary IL-1β expression after haemorrhage. The mRNA expression and serum concentration in the lung were similar to TNF-α. Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

Serum LDH-3 values

LDH-3 values in the haemorrhage group increased with time and were significantly greater in the 25% and 33% groups than the sham group at 5 h (13.0 ± 2.1 IU/l in the 17.5% group; 32.6 ± 8.5 IU/l in the 25% group; and 47.4 ± 12.6 IU/l in the 33% group). The increase in the LDH-3 values also tended to be greater proportional to the amount of bleeding at all times (Figure 5).

Figure 5.

Figure 5

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Changes in lactic dehydrogenase (LDH)-3 isozyme values. The increase was proportional to the amount of bleeding at all times after bleeding. Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

Arterial blood gas analysis

In the haemorrhage group, PaO2 decreased with time and was significantly lower in the 25% and 33% groups than in the sham group, 5 h after bleeding (77.1 ± 3.5 mmHg in the 25% group; and 61.9 ± 4.4 mmHg in the 33% group). The degree of decrease in PaO2 was also greater at all times tested proportional to the amount of bleeding (Figure 6a).

Figure 6.

Figure 6

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Arterial blood gas analysis. The degree of decrease in PaO2 was greater as the amount of bleeding increased, as shown in (a). The increase in AaDO2 was similar to PaO2, as shown in (b). Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

AaDO2 in the haemorrhage group tended to increase with time and was significantly greater than that in the sham group at 5 h after bleeding in the 25% and 33% groups (11.0 ± 3.1 mmHg in the 17.5% group; 18.4 ± 2.7 mmHg in the 25% group; and 26.0 ± 10.7 mmHg in the 33% group). Here, too, the degree of increase in AaDO2 tended to be greater in relation to the amount of bleeding at all times (Figure 6b).

Immunohistochemical examination

A clustered appearance of reddish brown precipitates in the interstitial space or vascular cavity indicated the presence of activated neutrophils in the histological analysis. The interstitial space was found to be moderately and diffusely oedematous 5 h after bleeding (Figure 7b–d), contrasting with the few activated neutrophils and oedema observed in the sham group (Figure 7a). The presence of activated neutrophils tended to increase with both the amount of bleeding and time after haemorrhage (Figure 8). In the 25% and 33% haemorrhage groups, the accumulation of activated neutrophils at 5 h was significantly greater in all groups (2.21 ± 0.9 counts/fields in the 17.5% group; 3.44 ± 1.01 counts/fields in the 25% group; and 5.07 ± 1.93 counts/fields in the 33% group) compared with the sham group.

Figure 7.

Figure 7

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Light micrograph of MPO-immunoreactivity in the lung 5 h after haemorrhage. (a) Sham group (Sham), (b) 17.5% group (17.5%), (c) 25% group (25%), (d) 33% group (33%). Some neutrophils (reddish brown precipitate) and diffuse oedema in the interstitial space were present in the haemorrhage groups (b, c, d). However, these histological changes are not observed in the Sham group (a). Bar = 50 μm.

Figure 8.

Figure 8

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Presence of activated neutrophils in the lung (250 μm)2 after bleeding. The degree of increased accumulation of activated neutrophils was greater as the amount of bleeding increased 5 h after bleeding. Data are shown as mean ± SE. n = 5/group. *P < 0.01 and **P < 0.001 vs. Sham.

Individual data of Figure 8 are plotted and evaluated in Figure 9. The frequency of activated neutrophils in each animal was strongly correlated with its serum LDH-3 value (r = 0.96) 5 h after bleeding (Figure 9).

Figure 9.

Figure 9

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Correlation between the amount of activated neutrophils and serum LDH-3 5 h after bleeding. A significant positive correlation was observed (r = 0.96).

Discussion

Many studies have investigated haemorrhagic shock following massive bleeding (50% of total blood volume) (Ahmadi-Yazdi et al. 2009; Sakai et al. 2009) or in artificial continuous hypotension models (Liu et al. 2008; Chen et al. 2009; Douzinas et al. 2009; Wang et al. 2009a,b;). In practical forensic cases, however, lung damage following haemorrhagic shock is often observed even after mild to moderate bleeding. Comparative studies of the mechanisms resulting in lung dysfunction had not been reported; therefore in this study we investigated the impact of lesser degree of haemorrhage to more adequately model actual forensic practice cases.

We found that MBP decreased with bleeding, but recovered temporarily (Figure 1). Autonomic reflexes, such as tachycardia, vasoconstriction and interstitial fluid shift into vessels, may be induced immediately after the fall of MBP, leading to its re-elevation. In the later phase, MBP tended to decrease gradually with time in all haemorrhage groups, with the degree of MBP depression being proportional to the amount of bleeding, as well as the gradual aggravation of pulmonary function. Hypoxemia resulting from the lung dysfunction and circulatory deterioration following haemorrhage seem to worsen the symptoms during the later phase.

Both mRNA expression and serum levels of TNF-α in the lung peaked at 1 h and remained increased until 5 h after bleeding. Similarly, IL-1β peaked at 3 h and also remained elevated until 5 h after haemorrhage. Chaudry et al. demonstrated that the expression of TNF-α and IL-1β is activated following the induction of tissue hypoxia affected by organ ischaemia (Zingarelli et al. 1994). In this study, the initial depression of MBP immediately after bleeding may cause pulmonary blood flow decrease and ischaemia, and induce the significant expression of TNF-α and IL-1β observed in the lung. A gradual decrease in systemic blood pressure at the later phase would cause an additional ischaemic state in the lungs, which could be the leading cause maintaining significant elevation of these pro-inflammatory cytokines 5 h after bleeding. The pulmonary expression of pro-inflammatory cytokines was elevated proportional to the degree of bleeding, as reported here. Pulmonary blood flow decreases following haemorrhagic shock, correlating with the bleeding volume according to some reports (Tiefenbrun & Shoemaker 1971; Dubin et al. 2000). Thus, a correlation between the degree of haemorrhage and cytokine expression was confirmed in this study.

Our results showed that the accumulation of activated neutrophils in the lung rose as the amount of bleeding increased. Previously, we demonstrated that pulmonary endovascular recruitment and infiltration of activated neutrophils are promoted by the local expression of pro-inflammatory cytokines, such as TNF-α and IL-1β in the lung (Sato et al. 2008). TNF-α and IL-1β are important mediators affecting adhesion of vascular endothelial cells and neutrophil infiltration into the organs (Zerwes et al. 2002; Brown et al. 2006; Togbe et al. 2007; Kita et al. 2008). These studies and our results suggest a general quantitative relationship between the amount of bleeding, cytokine expression and the frequency of neutrophils in the lung.

Lung dysfunction was evaluated by serum LDH-3 levels and gas exchange capacity using PaO2 and AaDO2, which deteriorated following haemorrhagic shock as shown in our previous study (Sato et al. 2008). In this study, these parameters were shown to deteriorate gradually with time as the amount of bleeding increased. Activated neutrophils are known to induce substantial tissue damage through the release of numerous active substances such as proteolytic enzymes, reactive oxygen species and vasoactive substances (Boehme et al. 2002; Brown et al. 2006). All of these factors can damage both the endothelial layer and adjacent tissue (Boehme et al. 2002; Fujimi et al. 2003; Brown et al. 2006). These factors cause pulmonary endothelial cell damage resulting in increasing microvascular permeability, which leads to the formation of interstitial and alveolus oedema in the lung (Windsor et al. 1993; Haslam et al. 1997; Orfanos et al. 2004). The pulmonary phenomena affected by aggregated neutrophils included LDH-3 isozyme, PaO2 and AaDO2 levels, the values for which worsened as the amount of bleeding increased (Figures 5 and 6). The increase of activated neutrophils correlated with the progression of lung dysfunction (Figure 9). These results suggest that progressive lung dysfunction was because of increased expression of proinflammatory cytokines and the recruitment of activated neutrophils into the lung following haemorrhagic shock.

In conclusion, our results suggest that the increased expression of proinflammatory cytokines and the recruitment of activated neutrophils into the lung cause progressive lung dysfunction as the amount of bleeding increases. In addition, the frequency of activated neutrophils recruited into the lung may be a quantitative morphological marker of the severity of lung dysfunction following haemorrhagic shock. These results may help forensic pathologists to diagnose the extent of antemortem lung dysfunction in perplexing haemorrhagic cases.

Acknowledgments

This work was supported by a grant-in-aid for scientific research (No. 21790615) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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