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Keywords:

  • hip fracture;
  • aged;
  • cigarette smoke exposure;
  • inflammation;
  • lung injury

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The aim of this investigation was to examine the influence of hip fracture on systemic inflammation and lung injury in aged chronic cigarette smoke exposed rats. Male Sprague Dawley (SD) aged rats (22–25 months old, 460–570 g) were used. Animals were subjected to either chronic cigarette smoke (CS) or air exposure for 12 weeks. These animals then underwent a sham procedure or hip fracture. Endpoint was 24 h. Systemic inflammation was assessed by TNF-α, IL-6, and IL-10 levels. Pulmonary function, inflammatory cell counts and protein concentrations in BAL, pulmonary pathological changes and scores were obtained to assess lung injury. And TLR4 mRNA expression in lung tissue was determined. The indices mentioned above were unchanged in air-exposed rats after hip fracture. However, CS-exposed animals were found to have increased serum levels of TNF-α, IL-6, and IL-10, impaired pulmonary function, increased inflammatory cell counts and protein concentrations in BAL, and intensified pathologic changes and scores. In addition, lung tissue harvested following CS-exposure demonstrated increased TLR4 mRNA expression. Our results indicate that systemic inflammation and lung injury in aged CS-exposed animals were further aggravated by hip fracture. The overexpression of TLR4 mRNA induced by CS exposure may, at least in part, involve in this process. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:24–30, 2014.

Hip fracture is a common injury in the elderly associated with high rates of mortality and perioperative complications, largely due to the poor physical condition (co-morbid diseases)[1] and the immune dysfunction induced by surgical trauma and anesthesia.[2] Chronic obstructive pulmonary disease (COPD) is one of the most common co-morbid diseases in the elderly hip fracture patients,[3] and it has been reported that patients with COPD had a 60–70% increase in mortality at 1-year following hip fracture.[4] However, the reasons hiding behind remain unclear.

It has been well established that posttraumatic systemic inflammation may result in remote organ damage of the lung.[5-8] Our previous study has shown that the systemic inflammatory response play an important role in postoperative organ dysfunction in elderly hip fracture patients.[9] However, little is known about the influence of hip fracture on the aged COPD individuals. Thus, we hypothesized that the existing systemic inflammation and lung injury in aged COPD individuals might be aggravated by hip fracture. We therefore analyzed the systemic inflammatory response and associated pulmonary injury in an aged rodent model of COPD combined with hip fracture.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Experimental Animals

Experiments were performed according to the guidelines for experimental animal care and use approved by Beijing Military General Hospital. Male Sprague Dawley (SD) aged rats (22–25 months old, 460–570 g) were used in this study. Animals were purchased from the Haiwang Laboratory animal center (Beijing, china) and kept on a 12-h:12-h light: dark cycle with free access to food and water. All rats were housed for at least 1 week prior to the experiments. Rats were randomly divided into four groups: Animals exposed to room air and subjected to sham operation or hip fracture (AIR + sham group, n = 8; AIR + HP group, n = 8), animals exposed to cigarette smoke (CS) and subjected to sham operation or hip fracture (CS + sham group, n = 8; CS + HP group, n = 8). Animals from sham group were subjected to the similar experimental procedures, including anesthetization, fluid resuscitation, and pain control but no hip fractures were performed. All animals were sacrificed at 24 h after hip fracture or sham operation.

Hip Fracture Model

Animals were anesthetized by an intraperitoneal (i.p.) injection of xylazine (25 mg/kg) and ketamine (75 mg/kg). The rat was placed onto the base of a blunt guillotine ramming apparatus[10] in a prone position with one of the legs fixed by a rubber band attached to a screw. The position of the proximal femur was determined and marked under C arm fluoroscopy guidance. The weight of the dropped steel was 500 g, and the average drop height was 14 cm. The force of the descending weight resulted in a closed hip fracture with mild soft tissue injury. Animals were radiographed immediately to verify that hip fractures had been produced (Fig. 1). Following injury, animals were resuscitated with an i.p. injection of 5 ml of sterile saline solution and were allowed to eat and drink freely in cages. The animals were administered Buprenex (buprenorphine; 0.1 mg/kg) every 10–12 h for pain control.

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Figure 1. Radiograph made after injury, demonstrating the typical intertrochanteric fracture of femur caused by the blunt guillotine.

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Cigarette Smoke Exposure Model

We used a sidestream smoke exposure system that provided for whole body exposure as described previously.[11, 12] Briefly, smoke from Commercial filtered cigarettes (trade name, Zhong Nan Hai from the Beijing cigarette Factory, China) containing 14 mg tar (equivalent to 1.5-fold of tar quantity in the Kentucky Reference Cigarette 2R4F) and 1.4 mg nicotine (equivalent to 1.4-fold of nicotine quantity in the Kentucky Reference Cigarette 2R4F) per cigarette was generated by the smoke apparatus consisted of four major parts including a cigarette burner box, two circulation fans and an inhalation plexiglass chamber (volume of 20 L). Animals were exposed to 6 cigarettes twice a day with 2 h smoke-free intervals, 5 days a week for 12 weeks (chronic exposure). Levels of carboxyhemoglobin in the blood did not exceed 10%. The control animals were exposed to air without smoke. In our previous study, we have confirmed that the COPD model could be well established using this approach.[12] After the last exposure, the right leg of the rats was subjected to hip fracture on the basis of the method described above.

Pulmonary Function Test

At 24 h after fracture, pulmonary function tests were performed using the AniRes2005 lung function system (Bestlab, version 2.0, China) according to the manufacturer's instructions. The animals were re-anesthetized with an i.p. injection of 1% sodium pentobarbital and tracheostomized. An endotracheal tube was inserted and connected to the outlet of the ventilator. After 30 normal respiratory cycles, the inspiratory resistance (Ri), expiratory resistance (Re), compliance of the respiratory system (Crs) and the ratio between the Forced Expiratory Volume at 0.2 s and the Forced Expiratory Volume (FEV0.2/FEV) were recorded.

Analysis of TNF-a, IL-6, and IL-10 in Serum

Soon after the pulmonary function tests had finished, thoracotomy was performed and blood samples were obtained via cardiac puncture. Blood samples were centrifuged at 5,000 rpm for 10 min and the serum was collected and stored at −20°C until analysis. The concentrations of TNF-α, IL-6, and IL-10 in serum were quantified with ELISA kits (R&D, Minneapolis, MN) according to the manufacturer's instructions.

BAL Cell Counts and Protein Concentrations

The lungs were removed from thoracic cavity, bronchoalveolar lavage (BAL) was collected by slowly instilling and withdrawing 1 ml of HBSS 7–10 times through the cannula. The first milliliter of BAL fluid was tested for protein concentrations. Then the BAL fluid was centrifuged at 3,000 rpm × 3min and cell pellets were resuspended with 1.0 ml of PBS. Using a hemocytometer and cytospins stained with Hema3 (Biochemical Sciences, Beijing, China), the total cell counts and differential cell counts were determined.

RT-PCR for TLR4 mRNA Expression in Lung

The lungs were harvested and the total RNA was extracted using the Qiagen RNeasy Midi Kit (Qiagen, Valencia, CA) and 3 µg of total RNA was reverse transcribed into cDNA using transcriptor first strand cDNA synthesis kit according to the manufacturer's instructions. The following PCR primers were used: The forward and reverse primers for TLR4 were 5′-GAGGCAGCAGGTCGAATTGT-3′ and 5′-TTTCCATCCAACAGGGCTTT-3′, respectively; PCR amplification was performed by the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA) using BioEasy SYBR Green I Real Time PCR Kit Manual (Applied Biosystems) in accordance with the manufacturer's instructions. The fluorescence was measure at the end of elongation. The average Ct value was obtained by using the instrument's software and we used the average Ct value of GAPDH for normalization with the ΔΔCt method. Results are expressed as 2−ΔΔCt.

Histopathological Analysis

Lung specimens were fixed in 10% formalin, embedded in paraffin, sections (4 µm) were stained with hematoxylin and eosin (H&E). Slides (200× magnification) were evaluated and scored by a pathologist blinded to the experimental groups according to the pulmonary injury scoring system.[13] The lung injury scoring was based on categories of inflammatory cell infiltration, pulmonary edema, congestion, and intra-alveolar hemorrhage graded on a scale from 0: normal, 1: mild, 2: moderate, and 3: severe injury, the maximum possible score was 12.

Statistical Analysis

Data are expressed as the mean ± SEM. Two-way ANOVA test with Tukey's multiple comparisons test were used for multiple groups' comparison. Data analysis was performed using Prizm version 6.0 (GraphPad Software, San Diego, CA). Statistical significance was defined as p ≤ 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Lung Function Measurement

Four parameters of the lung function (Ri, Re, Crs, and FEV0.2/FEV) were recorded in each group (Fig. 2A–D). Ri (p < 0.0103) and Re (p = 0.0103) values were significantly increased and the Crs (p = 0.0335) as well as the FEV0.2/FEV (p = 0.0017) values were significantly decreased in CS animals 24 h following hip fracture. However, these changes had not happened in AIR animals. The results indicate that hip fracture enhanced the impairment of pulmonary function in aged COPD rats.

image

Figure 2. (A–D) Lung function measurement. (A) Inspiratory resistance (Ri), (B) expiratory resistance (Re), (C) compliance of the respiratory system (Crs), and (D) FEV0.2/FEV were measured for analysis of Lung function. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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Serum Cytokine Levels

To assess the systemic inflammatory response following hip fracture, the circulating levels of TNF-α, IL-6, and IL-10 were measured at 24 h postinjury (Fig. 3A–D). We observed that the levels of cytokines IL-6 (p = 0.0045) and IL-10 (p < 0.0001) were significantly increased in the serum of the CS animals as compared to the AIR animals. It should be noted that the circulating levels of TNF-α, IL-6, and IL-10 had no significant increase in AIR animals when subjected to hip fracture. However, CS animals with hip fracture showed significantly increased levels of TNF-α (p < 0.0001), IL-6 (p < 0.0001), and IL-10 (p < 0.0001) as compared to sham treated CS animals. CS animals following hip fracture also had a significant increase in levels of TNF-α (p < 0.0001), IL-6 (p < 0.0001), and IL-10 (p < 0.0001) when compared to the AIR animals treated similarly.

image

Figure 3. (A–C) Systemic inflammatory response as measured by (A) TNF-α, (B) IL-6, and (C) IL-10. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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BAL Inflammatory Cell Counts and Protein Concentrations

To assess the inflammatory cell accumulation in response to hip fracture, numbers of inflammatory cells in BAL were examined (Fig. 4A–D). There was significant accumulation of total inflammatory cells (p < 0.0001), macrophages (p = 0.0068), T lymphocytes (p = 0.0216), and neutrophils (p = 0.0018) in BAL fluid in sham treated CS animals. And these inflammatory cells were further significant increased in response to hip fracture in CS animals (total cells, p = 0.0015; macrophages, p = 0.0023; lymphocytes, p = 0.0096; neutrophils, p < 0.0001). In contrast, AIR rats did not display elevated total cell numbers, nor increase in any subset of inflammatory cells obtained from BAL fluid.

image

Figure 4. (A–D) Inflammatory cell content in BAL fluid in response to hip fracture. (A) Total cell counts, (B) numbers of macrophages, (C) numbers of lymphocytes, (D) numbers of neutrophils. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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Previous work has also shown that the protein concentration in the BAL fluid is increased in animals with lung injury.[14] In this study, there is significant increase in protein concentration in the BAL fluid in response to hip fracture in CS (p = 0.0022), but not AIR rats (Fig. 5).

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Figure 5. Quantification of protein in BAL fluid following hip fracture. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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Toll-Like Receptor 4 (TLR4) mRNA in Lung Tissue

TLR4 is involved in the systemic/local inflammatory response and associated pulmonary injury reduced by CS or tissue trauma.[15] We hypothesized that the exaggerated inflammatory response 24 h after hip fracture in CS animals would have altered TLR4 mRNA expression in lung tissue. We demonstrated that the mRNA message for TLR4 was significantly increased in CS animals compared to the AIR animals (p < 0.0001). However, both AIR and CS animals subjected to hip fracture showed no significant elevation of TLR4 mRNA in lung as compared to the sham animals (Fig. 6).

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Figure 6. Relative expression of TLR4 mRNA in lung tissue quantified by real-time PCR. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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Histopathological Analysis and Lung Injury Scoring

Morphometric analysis demonstrated evidence of increased congestions, pulmonary edema, polymorphnuclear and mononuclear cell infiltration, and disrupted alveolar structure in the CS animals with hip fracture (Fig. 7C) when compared to sham controls (Fig. 7D). However, the AIR animals subjected to hip fracture appeared to have lung histology (Fig. 7A) more similar to sham controls (Fig. 7B) but some inflammatory cells. The lung injury scores assessed by a pathologist blinded to the study were increased in CS animals with hip fracture as compared to animals who underwent sham procedures. The scores of AIR animals subjected to hip fracture were similar to control animals (Fig. 8).

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Figure 7. (A–D) Representative sections of lung from AIR and CS animals that underwent hip fracture or sham control procedures and stained with hematoxylin and eosin. (A) AIR animals after sham procedures showed normal lung histology. (B) The morphologic characteristics of AIR animals with hip fracture were similarity to the AIR animals with sham procedures but some inflammatory cells. (C) CS animals after sham procedures showed disrupted alveolar structure, increased congestions and pulmonary edema, and more inflammatory cells. (D) The CS animals after hip fracture exhibited aggravated histopathological changes as compared to the CS animals with sham procedures. Magnification is 400×.

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image

Figure 8. Lung injury scores. Lung injury scoring was based on categories of inflammatory cell infiltration, pulmonary edema, congestion, and intra-alveolar hemorrhage graded on a scale from 0 = normal, 1 = mild, 2 = moderate, and 3 = severe injury, yielding a maximum possible score of 12. Sections were graded by a pathologist blinded to the study protocol. Data are expressed as mean ± SEM of 8 animals per group. *p < 0.05 compared with the other groups.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Co-morbid COPD is a common condition which contributes to the pulmonary complications and high mortality following hip fracture in aging populations. However, what happened behind it remains unclear. To our knowledge, the present study represents the first investigation into the inflammatory response and lung injury in a combined model of COPD and hip fracture in aged rat. In the current study, the infliction of unilateral hip fractures in AIR aged rats did not result in an increase in the markers of systemic inflammation and lung injury. In contrast, animals that had a 36 weeks chronic cigarette exposure prior to injury showed an injury-associated elevation in levels of multiple circulating inflammatory markers, including TNF-α, IL-6, and IL-10. Compared with AIR animals, CS animals were found to have intensified lung injury at 24 h after hip fracture, as evidenced by impairment of pulmonary function, increases in BAL inflammatory cell counts and protein concentrations, pronounced histological changes, and scores. In addition, we found that chronic cigarette exposure resulted in significantly increased levels of TLR4 mRNA in lung tissue; however, it did not upregulate following hip fracture.

A variety of different approaches have been used to establish COPD model in animals. None of the models reproduces the exact changes seen in humans, but CS-induced disease appears to come the closest.[16] The method we used to develop the COPD rat model in the present study had been proved practicable and reliable.[9] But how to model hip fracture in rat proved to be a serious challenge. We have failed to locate any literature relating to this topic. Hip fracture is divided into three major types: Femoral neck fracture, intertrochanteric fracture, and subtrochanteric fracture. In our previous investigation, a simple blunt guillotine ramming apparatus was devised for the purpose; however, it could only give rise to a proximal femur fracture, in which the injury was more serious than hip fracture. We reckon that the reason was failure to accurately locate the greater trochanter. In this study, we used a C-arm X-ray apparatus to locate the greater trochanter before injury. Thus, the accuracy, stability and repeatability of modeling were enhanced dramatically.

In this study, for the air-exposed rats, we observed no effects of hip fracture on changes in pro-inflammatory cytokine production and makers of lung injury. This confirms clinical experience that the immunoinflammatory response and distant organ damage do not occur in a healthy host sustaining only a low-energy trauma, such as hip fracture. It is a different scenario when the host has been weakened by various factors. Of them, by far the most common compromising factor is age and age-related conditions, such as: COPD, cardiovascular problem, and osteoporosis. Low energy is only the external factor that affects the severity and outcome of the injury, whilst a multitude of preexisting internal factors can dynamically aggravate the situation. In the current study, our findings indicate that 24 h after hip fracture, the serum levels of TNF-α, IL-6, IL-10 in CS-exposed rats were remarkable increased. Meanwhile, a more severe lung injury (impaired lung function, increased pulmonary inflammation, and elevated lung injury scores) was observed.

Toll-like receptors (TLRs) are pattern recognition receptors central to the innate immune response to infection.[17] However, recent studies have shown that TLR4, the first member in the TLRs family to be characterized, also participates in the recognition endogenous molecules that are released from damaged tissues, such as heme, HMGB1, and Hsp60, which initiate sterile inflammation and lead to organ damage,[18-22] and evidence shows that the magnitudes of the inflammatory response and organ damage are related to the expression change of TLRs.[21, 22] Moreover, TLR4 is reported to be involved in the inflammation and destruction in CS-induced lung injury.[23-25] In our study, we observed that hip fracture had little effect on the pulmonary TLR4 mRNA expression both in AIR and CS animals. However, chronic cigarette exposure induced a significantly upregulated pulmonary expression of TLR4 mRNA. This result is in line with a recent report that suggests TLR4 is important in CS-induced inflammation.[15] Interestingly, when the CS animals were subject to hip fracture, the systemic inflammation and lung injury were obviously aggravated. This may be explained as the danger signals released from a second insult (hip fracture) site lead to further release of systemic inflammatory mediators through TLR4 signaling pathway.[26-28] Thus, a balance of immunoinflammatory response is broken and a more severe lung injury occurs. However, the complete pathphysiology is more complicated than this single explanation and needs further clarification.

There are several limitations to this study. Firstly, it is impossible to create a COPD model that can reproduce the exact pathophysiology processes in humans because of the massive evolution gap. If a rat of 9 months starts smoking, the length of smoking needs to reach around 15 months in order to simulate a 30 years of exposure in humans.[16, 29, 30] In our experiment, the length of cigarette smoking is only 3 months, far short of the 15 months required. Our acute experiment can hardly simulate human COPD that is slowly progressive over decades with acute exacerbations and infections. Secondly, the pathogenesis of non-infectious inflammatory response and lung injury in this combined model is complex and cannot be elucidated in a single study, let alone the first of its kind. There must be many other contributing immune receptors, such as TLR2 and TLR9.[20, 21, 31] The third limitation is that the fractured animals did not receive surgical treatment. Clinically, most pulmonary complications are more likely to occur in the postoperative period.

In conclusion, this study demonstrates that chronic CS exposure prior to unilateral hip fracture modulates the postinjury systemic inflammatory response and lung injury in aged rats. Several measures of systemic inflammatory response and pulmonary injury, including serum cytokine levels, BAL inflammatory cells and protein concentrations, and pulmonary histological analysis, were significantly increased following injury with the presence of TLR4 mRNA overexpression. Our findings may be important when quantifying the postinjury inflammatory response in the smoking aged patient with hip fracture.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES