Thorax trauma-induced experimental lung injury
DISSERTATION
zur
Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
des Fachbereiches für Biologie an der
Universität Konstanz vorgelegt
von
Katja Eichert
Konstanz, im Juli 2003
Dissertation der Universität Konstanz Datum der mündlichen Prüfung: 16.10.2003 1. Referent: Prof. Dr. Albrecht Wendel 2. Referent: Prof. Dr. Klaus Schäfer
Table of Contents
Table of Contents
1. Introduction... 1
1.1 The lung ... 1
1.1.1 Lung morphology and aerodynamics... 1
1.1.2 Physiological parameters of the lung ... 2
1.2 Pulmonary oedema ... 3
1.2.1 Alveolar liquid clearance... 4
1.3 Mechanisms of host defence ... 5
1.4 Multiple trauma... 11
1.4.1 Impact of trauma on patients and society ... 11
1.4.2 Blast injury... 11
1.4.3 Chest trauma... 12
1.4.4 Molecular injury ... 15
1.4.5 Management of pulmonary blast injury ... 16
1.4.6 Immunomodulation after trauma... 16
2. Aims of the Study ... 18
3. Materials and Methods ... 19
3.1 Animals... 19
3.2 Chemicals ... 19
3.2.1 Chemicals used in the isolated perfused rat lung experiments ... 19
3.2.2 Chemicals used in the in vitro experiments... 20
3.2.3 Other chemicals ... 20
3.2.4 Solutes ... 21
3.2.5 Anaesthesia and analgesic... 21
3.2.6 Cell culture material... 21
3.3 Laboratory equipment and technical devices ... 21
3.4 The in vivo blast wave thorax trauma in rats ... 22
3.4.1 The blast wave generator apparatus ... 22
3.4.2 Pressure wave monitoring ... 23
3.4.3 Protocol of anaesthesia and analgesic ... 24
3.4.4 Experimental protocol... 25
3.4.5 Determination of an injury score and validation ... 26
3.5 The isolated ex vivo perfused and ventilated rat lung... 27
3.5.1 Isolated perfused rat lung preparation ... 27
3.5.2 Experimental Setup ... 27
3.5.3 Experimental design of the perfused lung studies... 28
3.6 In vitro stimulation of primary rat alveolar macrophages ... 29
Table of Contents
3.6.1 Preparation and culturing of rat alveolar macrophages... 29
3.6.2 Stimulation with dead, fluorescent bacteria... 30
3.6.3 Stimulation with endotoxin... 30
3.6.4 Fluorescence labelled E. coli/ S. aureus phagocytosis assay ... 30
3.6.5 Assay for superoxide production ... 31
3.6.6 Preparation of washed and haemolysed erythrocytes and haemolysat... 32
3.6.7 Lysis of erythrocytes... 32
3.6.8 Experimental design of the in vitro experiments... 33
3.7 Lung lavage ... 33
3.8 Determination of the alveolar haemorrhage... 34
3.9 Total and differential cell counts and cell viability ... 34
3.10 Determination of total protein content... 34
3.11 Cytokines determinations... 34
3.12 Measurement of eicosanoids ... 35
3.13 Determination of NO production ... 35
3.14 Measurement of lactate dehydrogenase ... 36
3.15 Measurement of CINC-3... 36
3.16 Gelatin zymograpy ... 36
3.17 Lung Wet/ Dry-Ratio... 37
3.18 Histological examinations ... 37
3.19 Statistics... 37
4. Results... 38
4.1 System characteristics ... 38
4.1.1 Variability and influence of physical parameters ... 38
4.2 Functional changes of alveolar macrophages and interaction with blood in vitro after trauma ... 43
4.2.1 Trauma-induced impairment in host defence mechanisms ... 43
4.2.2 Role of the trauma-induced alveolar haemorrhage in impaired macrophage functions... 48
4.2.3 Influence of different blood components on the macrophage function ... 50
4.2.4 Summary of the in vitro findings ... 53
4.3 Functional consequences and endogenous modulation in vivo... 55
4.3.1 Blast injury-induced disruption of the alveolar capillary barrier... 55
4.3.2 Blast injury-related mediator release ... 60
4.3.3 Infiltration of inflammatory cells ... 64
4.4 Ex vivo lung perfusion: Trauma-induced changes in lung function... 69
Table of Contents
4.4.2 Time-dependent recovery of the lung function after trauma... 73
4.4.3 Pharmacological intervention... 76
5. Discussion... 83
5.1 A laboratory model for studying primary blast injury ... 84
5.1.1 Rationale for the development of the blast thorax trauma rat lung model ... 84
5.1.2 Standardization and monitoring of the blast pressure wave... 84
5.1.3 Standardization of the blast thorax trauma in rats... 86
5.2 Combination of the in vivo thorax trauma model with ex vivo lung perfusion 86 5.2.1 Match of physical and biological impact... 87
5.2.2 Time-dependence of the lung dysfunction ... 88
5.2.3 Pharmacological modulation of the injury ... 89
5.3 Thorax trauma-related pathophysiological changes in vivo over time ... 91
5.3.1 Structural integrity of the lung tissue after blast injury... 91
5.3.2 Mediator release in response to primary blast injury... 93
5.3.3 Thorax trauma-related chemokine release and inflammatory cell infiltration ... 97
5.3.4 Oedema generation and resorption after thorax trauma: Comparison of the ex vivo and in vivo results... 98
5.4 Thorax trauma-related impairment of alveolar macrophage function in vitro. 98 5.4.1 Alveolar macrophage population after trauma ... 98
5.4.2 Phagocytic capacity, microbial killing and TNF release... 99
5.4.3 Involvement of trauma-related alveolar haemorrhage in impaired macrophage functions... 103
6. Summary ... 105
7. Deutsche Zusammenfassung ... 107
8. References ... 110
9. Abbreviatons ... 120
Introduction
1. Introduction
1.1 The lung
The lung is the central organ for gas exchange of land-living animals, including mammals. The respiratory system is one of the primary interfaces between the organism and the environment. As a result of air circulating through the respiratory system, the latter represents a target for toxic gases, airborne particulates, and infectious agents. Because the entire output of the right heart also passes through the vascular bed of the lung, this organ is also a target for blood-borne toxicants and pathogens. Therefore, the lung also has many non-respiratory functions, such as metabolism, mediator synthesis, host defence and the clearance of substances from the pulmonary circulation. The respiratory system, especially the conducting airways and gas exchange area of the lungs, is organized in a highly polarized fashion. As a consequence, most of the pathological responses within the lung tend to be highly focal and generally target one or more small subpopulations of the over 40 different cell types found in this organ. Thus, the cellular composition and architectural organization of airway and gas exchange tissue have major impacts on the pathological responses within the system. This chapter will only focus on physiological aspects and metabolic functions pertinent to the present study.
1.1.1
Lung morphology and aerodynamics
Most of the thoracic cavity is occupied by the right and the left lungs, which are divided into three and two lobes, respectively. The anatomical structure of the lung combines the respiratory system and the blood circulation. During breathing, the inhaled air enters the lung via the trachea and flows towards the respiratory zone driven by pressures, generated by constriction of the inspiratory muscles. The airways divide about 25 times, depending on the species. The trachea branches out into the two main bronchi, which then further divide in the lobular bronchioles, the respiratory bronchioles and the alveolar ducts, which end blindly in the alveoli. The upper airways are stabilized by cartilage; towards the lower airways less cartilage but more contractile muscle fibers and elastic fibers are found. The total number of airways grows exponentially and with each division the total diameter increases, while the diameter of the single airways decreases. The total area of the 300 million alveoli is estimated to vary between 80 and 140 m2 (depending on the publication) in the adult lung. The alveoli are 0.2 – 0.3 mm in diameter and are surrounded by a net of capillaries with 5 µm in diameter. Here, at
Introduction
exchange occurs. This membrane consists of capillary epithelial cells, a very narrow interstitium and endothelial cells. Two different systems ensure the blood supply of the lung:
i) the pulmonary circulation (low pressure system), which starts at the right ventricle, leaving the heart via the pulmonary artery which carries the oxygen-poor blood to the lung. After saturation with oxygen, the blood leaves the lung by the pulmonary vein, which ends in the left atrium of the heart.
ii) The bronchial circulation (high pressure system) supplies the central airways and vessels as well as lung nymph nodes with blood.
The alveolar epithelium is composed mainly of cuboidal type II pneumocytes and large, flat type I pneumocytes. Alveolar type II pneumocytes synthesize, store and secrete the highly surface active pulmonary surfactant, a heterogeneous mixture of lipids and proteins, that reduces surface tension along the air-liquid interface and prevents alveolar collapse upon expiration. The main function of the type I pneumocytes is the formation of the air-blood barrier and therefore they facilitate the gas exchange. Alveolar and interstitial macrophages, eosinophils and neutrophils are also located in the lung, in order to maintain its immunological functions.
1.1.2
Physiological parameters of the lung
The functional properties of the lung, i.e. the airway and vascular mechanics, are characterized by different lung function parameters, including the tidal volume, the pulmonary compliance, the airway resistance, the vascular resistance, and the extent of extravascular lung water (oedema). The determination of these parameters is not only interesting for the diagnosis used by the medical specialist, but also for the scientist. Various experimental approaches in whole animals or in isolated organs, such as the isolated perfused rat lung, 1 have been done to investigate these lung parameters.
1.1.2.1 Airway mechanics
The tidal volume is the amount of air which enters the lung during a single breathing cycle of inspiration and expiration. It can be calculated by integrating the airflow velocity during respiration. The driving force for the airflow in the airways is the transpulmonary pressure, e.g.
the difference between the alveolar and the pleural pressure. During inspiration, the thorax expands, thus the pleural pressure decreases, and the transpulmonary pressure increases, causing inspiratory airflow from the outside into the alveoli. During expiration, the thorax relaxes forcing expiratory airflow back into the atmosphere. At a given transpulmonary pressure, tidal volume depends mainly on two parameters: airway resistance and lung compliance. The airway resistance is an index for the resistive forces against the airflow in the
Introduction
airways and depends on the diameter and the length of the airways. Because the total diameter is lowest in the upper airways, this region accounts for the major part of the airway resistance. The airway resistance can be calculated from the relation between transpulmonary pressure and airflow velocity. The airway resistance increases, as a consequence of narrowing the airways due to bronchoconstriction or obstructive processes, e.g. bronchial oedema or enhanced mucus deposition.
The pulmonary compliance is a marker for the functional stiffness of the lung and thus depends on properties of the peripheral lung tissue, such as elastic fibers. The compliance can be calculated from the relation between tidal volume and transpulmonary pressure and thus is also affected by the air volumes at which it is calculated and the lung volume history.
The pulmonary compliance decreases during restrictive pathological changes e.g. atelectasis, fibrosis, loss of elastic fibers, pulmonary oedema or disturbed surfactant secretion.
1.1.2.2 Vascular mechanics
The pulmonary blood flow is regulated by contractile elements of the pulmonary vasculature and depends mainly on the smooth muscle tone of the small arteries. The vascular resistance is calculated from the ratio between the blood pressure difference (Part - Pven) and blood flow rate. This parameter is increased as a consequence of vasoconstriction, but also of air emboli and vascular obliteration, due to thrombus formation or blood cell aggregation.
1.2 Pulmonary oedema
The term oedema denotes the presence of abnormally high amounts of water in the interstitium (interstitial oedema) or in the alveoli (alveolar oedema). Under physiological conditions, the positive hydrostatic blood pressure, interstitial pressure and oncotic forces are in balance and small amounts of excess water are drained of by the lymphatic system. Only when the lymph`s capacity is exceeded e.g. due to massive interstitial fluid accumulation or loss of the alveolar-capillary barrier integrity, water gets access into the alveoli. The consequences are impaired gas exchange and a loss of pulmonary compliance. Depending on the cause, two different kinds of oedema formation can be determined: Cardiogenic or hydrostatic oedema and non-cardiogenic or high permeability oedema. The former is due to enhanced capillary pressure, leading to subsequent water influx into the interstitial space, whereas the latter is independent of the hydrostatic pressure and is related to an impaired permeability for water and/ or proteins. Non-cardiogenic oedema and hypotension represent the hallmarks of ARDS (adult respiratory distress syndrome).
Introduction
1.2.1
Alveolar liquid clearance
Sodium and fluid transport across the intact respiratory epithelium
Although, for many years, Starling forces (hydrostatic and protein osmotic pressures) were thought to play a major role in maintaining the alveolar space free of fluid 2, there is now strong evidence that active ion transport across the alveolar epithelium creates an osmotic gradient that leads to water reabsorption both during the perinatal period 3 and in adulthood 4 (reviewed by Matthay and coworkers, 5; Sartori and Matthay, 6). Despite significant species differences in the basal rates of sodium and fluid transport 7-12, the basic mechanisms seem to be comparable. On the apical membrane of alveolar type II cells there is a sodium uptake by amiloride-sensitive cation channels, such as the amiloride-sensitive epithelial sodium channel (ENaC) as well as by several other non-selective cation channels 3,4,13. On the basolateral surface of the cell, sodium is discharged into the interstitium by the ouabain-sensitive sodium/
potassium adenosine triphosphatase (Na+/ K+-ATPase) 14,15. In addition to the sodium transport, a pathway for chloride transport, involving CFTR (cystic fibrosis transmembrane regulator chloride channel) is suggested to play an important role in fluid resorption 16. CFTR activation was recently suggested to regulate ENaC 17. Due to the osmotic gradient, water follows passively, probably through water channels (the aquaporins, AQPs) 18,19, although the presence of these water channels is not required for maximal alveolar epithelial transport in the lung 20. This process can be upregulated by several catecholamine-dependent and independent mechanisms.
Regulation of alveolar transepithelial fluid transport
Beta2-adrenergic agonists can upregulate alveolar fluid clearance in isolated perfused rat lungs 21,22, ventilated rats 8, sheep 7 and dogs 23 as well as in mice 24, partly due to cyclic adenosine monophosphate (cAMP)-dependent mechanisms 25. Furthermore β1-adrenergic stimulation is reported to be also effective in upregulating fluid resorption. Both β1- and
β2-receptors are detectable on both the apical and basolateral surface of the alveolar epithelium 26.
Hormones like epidermal growth factor (EGF) 27, keratinocyte growth factor (KGF) 28 or TGFα
29 are capable of inducing a catecholamine-independent increase of the alveolar fluid clearance, primarily by stimulating alveolar type II cell proliferation.
Introduction
Sodium and fluid transport across the respiratory epithelium in acute lung injury
A profound injury to the alveolar epithelium can disrupt the integrity of the alveolar barrier and/
or downregulate ion transport pathways, thus reducing net alveolar fluid reabsorption and enhancing the extent of alveolar oedema. Endogenous catecholamines in experimental rat models of hyperoxia 30, haemorrhagic shock 31, septic shock 32 and in a canine model of neurogenic pulmonary oedema 33 were reported to stimulate the alveolar fluid clearance.
Alternatively, catecholamine-independent stimulation of the fluid transport may occur due to a mechanism involving the lectin-like domain of TNF 34,35.
Pharmacological stimulation of the alveolar fluid clearance
Beta2-agonists are attractive therapeutic agents because of their minimal side effects even in critically ill patients 36. They accelerate not only the resolution of experimental induced alveolar oedema, e.g. aerosolized salmeterol in sheep 37, moderate injury due to hyperoxia in rats 38, but also increase the secretion of surfactant and perhaps exert an anti-inflammatory effect 39, thus helping to restore the vascular permeability of the lung 40. With regard to accelerate re- epithelialization of the alveolar barrier in the case of acute lung injury 41, benefits with KGF pre-treatment 42 have been achieved. The combination of KGF and β2-agonist treatment results in an additive upregulation of fluid clearance 43. This suggests that there may be mechanisms for providing both short-term (β2-agonist) and long-term (growth factors) upregulation of fluid transport, that might hasten the resolution of clinical pulmonary oedema.
1.3 Mechanisms of host defence
The respiratory tract is situated at the interface between the environmental air and the internal tissues of the organism. During normal ventilation or as a result of aspiration, noxious materials, including infectious agents, are deposited on mucosal surfaces of the airways and penetrate into the depths of the lung parenchyma. Foreign material encounters a highly integrated system of natural and acquired defence mechanisms, that prevent injury, infections, and invasion of host tissue 44-46. This system of host defence includes mechanical (filtration, cough, mucociliary clearance), molecular (airway secretions), phagocytic, and antigen-specific immune mechanisms (B- and T-cell-mediated reactions) of resistance (reviewed in 47).
Introduction
1.3.1.1 The alveolar macrophage
The alveolar macrophage (AM) is the primary resident respiratory cell responsible for maintaining the sterility of the alveolar space and thus represents the first line of defence. As a mobile cell and representative of the mononuclear phagocyte system, the AM is believed to be the central modulator, as both regulator and effector of the degree of inflammation and anti- inflammation within the alveolar space 48. This metabolically highly active cell scavenges particulate matter, removes macromolecular debris, kills microorganisms, functions as an accessory cell in immune response, maintains and repairs the lung parenchyma and provides surveillance against neoplasms 49. Since the pulmonary alveolar macrophages are enveloped by surfactant in vivo, and since their lysosomes contain hydrolytic enzymes necessary for surfactant degradation 50, it is believed that alveolar macrophages are also involved in the clearance of surfactant from the alveoli. When confronted with a particularly large inoculum of microorganisms, the AM supplements its direct antimicrobial capabilities by recruiting and activating polymorphonuclear leukocytes (PMN) from the bloodstream, which may represent the second line of defence. Approximately 40% of the body’s PMN’s are marginated within the microvasculature of the lungs, facilitating the recruitment to the alveolus, or to other sides of the body 51.
1.3.1.2 Location and origin
Pulmonary macrophages constitute the most abundant non-parenchymal cell type in the lung
52. Macrophages are present in the alveoli (alveolar macrophages, AM), interstitial spaces (interstitial macrophages), intravascular spaces (intravascular macrophages), conducting airways (airway macrophages), pleura (pleural macrophages), and lymph nodes (lymph note macrophages) 53,54.
Alveolar macrophages initially encounter materials, that reach gas exchange units and subsequently perform phagocytosis and microbial killing. Interstitial macrophages serve as precursors for physiologic renewal of alveolar macrophages and for their expansion during pulmonary inflammation 53. Adhering to the pulmonary endothelial cells, intravascular macrophages remove circulating particles, but may also contribute to pathogenesis during sepsis 48,55. Airway macrophages are responsible for the reactivity of the conductive airways in response to inhaled stimuli in patients with asthma 47.
The ultimate source of pulmonary macrophages are monocyte progenitor cells in the bone marrow 56, which enter the lung from the vascular bed, and adapt to the local environment by maturation into tissue macrophages. Maturation results in an increase in cell size, in the number of cytoplasmatic organelles, lysosomal enzyme activity, phagocytosis capacity, and expression of Fc, complement and cytokine receptors on surface membranes 54. Alveolar
Introduction
macrophages vary considerable in size and morphologic features. They represent both phenotypically and functionally a heterogenous population of cells 57. Pulmonary macrophages live for weeks to months and possess some limited replicative potential 58. It is believed that the effector arm of the cell-mediated immune response depends on both the activation of resident macrophages as well as on the recruitment of circulating monocytes into the alveoli
58. However, the proportion of monocyte influx and local replication during the pulmonary inflammation has to be defined.
Chemotactic factors like complement component C5a, transforming growth factor beta (TGF- β), formylated peptides and monocyte-chemotactic peptide (MCP-1), stimulate the influx of monocytes into the lung (reviewed in 50). To exert their effector functions, alveolar macrophages have to be either activated non-specifically by adherence 59 or usually through ligand-to-receptor coupling. Among a large number of receptors expressed on alveolar macrophages 57, immunoglobulin (Ig) and complement (C) receptors (CR1, CR3) participate in receptor-mediated phagocytosis. The former bind to the Fc portion of Ig molecules (IgG, IgA, IgE), whereas the latter are important natural opsonins for microbial organisms and particulates 60. Macrophages further express C5a, a potent chemotactic, cell-activating and pro-inflammatory molecule, as well as various cytokine receptors for interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon γ (INF-γ), growth factor receptors and cell activation factors e.g. lectins, lipoproteins and glucocorticoids (reviewed in 50,57).
CD14, a 55-kDa phosphatidylinositiol-anchored membrane glycoprotein expressed on the surface of monocytes, macrophages, and, to a lesser extent on neutrophils 61 has been identified as an important receptor for LPS, in association with LPS-binding protein (LBP). The stimulation of CD14 triggers a concentration-dependent macrophage activation 62. This activation results in the release of TNF, IL-6 and IL-8 63,64 and has been described as both LBP-dependent and -independent 63. Haugen and coworkers reported a higher expression of CD14 on monocytes (MO) than on AM, with AM of different size and maturity having different CD14 expression levels. The expression of CD14 can be modulated in response to LPS, but the LPS binding capacity of AM and MO does not correlate with their CD14 levels 65. Indeed, several other macrophage receptors that function as LPS receptors have been described 66.
1.3.1.3 Secretory function
Macrophages are pluripotent cells. The range of products secreted by alveolar macrophages is broad, with over 100 molecular species having been identified, including reactive oxygen species, proteases and anti-proteases, bioactive lipids, cytokines as well as growth factors (reviewed in 50).
Introduction
Reactive oxygen radicals are generated via the “respiratory burst”. Together with several proteases and other enzymes that function intracellularly, reactive oxygen species are involved in microbial killing. Secreted proteinases (metalloproteinases, serine proteinases) affect fibrinolysis and extracellular matrix remodelling 50. Stimulation of alveolar macrophages with IgG or IgE immune-complexes leads to the production of both cyclooxygenase-derived (thromboxane A2 and prostaglandin E2 and D2) and lipoxygenase-derived (leukotriene B4) compounds 67. In the IgG immune complex animal model of acute lung injury 68, bronchoalveolar lavage fluids contain large amounts of biologically active TNF and IL-1. These
“early response cytokines” function in a pro-inflammatory manner, by playing a key role in the upregulation or induction of adhesion molecules in the lung vasculature, such as ICAM-1 and E-selectin, which are critically necessary for neutrophil recruitment. In this regard, the α- chemokine family (IL-8 or its family relatives such as MIP-2, CINC) are described to have chemotactic activity primarily for neutrophils and to a lesser extent for T-cell subsets, while the β-chemokines (e.g. MIP-1a, b; MCP-1, 2 and 3) are thought to be predominantly chemotactic for monocytes and lymphocytes. In contrast, there are various anti-inflammatory cytokines such as IL-10, IL-4, IL-6 or IL-13 produced by alveolar macrophages to suppress the cytokine production and therefore regulate the intensity of the inflammatory response (reviewed in 69).
In addition to cytokines, also lipid mediators such as leukotrienes (LT) upregulate directly the antimicrobial potential of macrophages as well as PMN’s from different species against both bacteria and fungi (reviewed in 70). Leukotriene levels have been shown to be elevated in bronchoalveolar lavage fluid from patients with bacterial pneumonia and also in lung tissue in animal models of pneumonia 70.
1.3.1.4 Phagocytic function
Macrophages remove materials present in the local environment through both pinocytosis and phagocytosis. In contrast to pinocytosis, phagocytosis is an energy-dependent process, characterized by adherence through receptors and engulfment, leading further to internalisation of the particulates and finally to digestion in phagolysosomal vacuoles. Efficient phagocytosis depends on opsonization. Opsonins defined in the lung include IgG1, IgG3, C3b, surfactant protein A/ D (SP-A/ D) 71,72, mannose and lipopolysaccharide-binding proteins 60. Furthermore, SP-A has been reported to enhance the phagocytosis of C1q-coated particles by alveolar macrophages 73. However, binding of inhaled environmental particles must be accomplished without the benefit of opsonization by specific antibodies. The identities of receptors on AM’s that mediate unopsonized particle binding are not fully known, whereas the role of some scavenger receptors (SR) have been recently reported 66,74,75.
Introduction
1.3.1.5 Microbicidal function
The production of reactive oxygen species (ROS) upon receptor-mediated stimulation of phagocytosis was first recognized in phagocytes such as macrophages, a process determining their microbicidal activity and referred to as respiratory burst. This process results from the assembly and activation of the nicotinamide dinucleotide phosphate (NADPH) oxidase, a multicomponent enzyme that catalyses the one-electron reduction of molecular oxygen to superoxide 76, which is subsequently converted to other oxygen-derived species, by either spontaneous or enzyme-mediated reduction. ROS generated in an inflammatory milieu act in an autocrine and paracrine manner, in order to rapidly amplify the effector potential of Fcγ R on quiescent phagocytes, by means of altering signal transduction (reviewed by Pricop and Salmon, 77). Controlled upregulation of oxygen generation is important, since dysregulation or overproduction results in acute tissue injury 78,79. With regard to this, it has been reported that during the respiratory burst of phagocytic cells in vitro, the superoxide anion production per cell shows an inverse correlation with the cell density, a phenomenon described as autoregulation 80. Furthermore, it has been shown that the decrease in individual cell response is due to a significant increase in the amount of basal responses of the macrophages, thus, concomitantly, the number of reactive cells remains unchanged, irrespective of the cell density of the population 78.
In vitro, the respiratory burst can be triggered e.g. by certain soluble (PMA) or particulate opsonized zymosan (OZ) agents. Both agents produce an intense oxidative burst, although mediated by different signal transduction pathways: PMA is soluble and associated to cell membrane perturbations that is dependent on protein kinase C (PKC) activation, whereas particulate OZ reflects processes related to PKC-independent phagocytic mechanisms 81. Moreover both stimuli utilize more than one activation pathway to stimulate NADPH-oxidase
82, revealing that different stimuli produce the same reaction in terms of respiratory burst autoregulation at the single cell level. Together with the LPS-induced oxidative burst (CD14 receptor-dependent) these pathways depend on ERK 1/ 2 activation. Activated ERK kinases also control the production of TNF in both LPS and PMA membrane activation of human alveolar macrophages. Whereas LPS activates NFκB nuclear translocation, PMA does not, but rather activates alveolar macrophages through the ERK 1/ 2 MAP kinase pathway. It has been shown that IgA, a predominant Ig isotype of respiratory secretions, regulates LPS-and PMA-induced oxidative burst and TNFthrough both dependent and independent modulation of ERK pathways, revealing a role of IgA in both lung protection and inflammation 83. The LPS- related oxidative burst, TNF and IL-10 release of human monocytes can also be modulated by IL-9, through an upregulation of TGF-β 84. In contrast, ADP stimulates the respiratory burst,
Introduction
The physiological generation of ROS production has now been clearly implicated in the activation of signalling pathways, resulting in a broad array of physiological responses ranging from cell proliferation to gene expression and apoptosis 79,86. In this regard, and as reviewed by Forman and Torres, 79, it has been suggested that: 1. hydrogen peroxide and superoxide act as second messengers; 2. anti-oxidant enzymes are implicated in the “turn-off” phase of signal transduction; 3. the primary physiological role of the respiratory burst in macrophages may be redox signalling, rather than microbicidal activity.
In contrast to neutrophils, oxygen-dependent killing by macrophages is myeloperoxidase–
independent because they lose this enzyme during maturation 47.
In macrophages from rodents, a second oxidant-generating pathway features inducible nitric oxide synthase (iNOS), that is inducible by TNF, IL-1, and interferon-γ 69.
1.3.1.6 Immune function
Macrophages contribute importantly to the induction, expression and regulation of immune responses 47. Alveolar macrophages possess the capacity to serve as antigen presenting cells (APC’s) and secrete co-stimulatory cytokines, both required for T-cell activation. In contrast to resting macrophages, alveolar macrophages activated by chronic pulmonary inflammation or by T-lymphocyte-derived cytokines are more effective in antigen presentation 48. Furthermore, pulmonary macrophages are important effector cells for T-cell mediated immunity and regulate immune responses. Thus IFN-γ, secreted by activated T-lymphocytes potently activates resting macrophages and upregulates these host defence functions 87. The balance between macrophage-derived enhancing and -suppressive signals determines the degree with which antigen presentation and induction of immune responses occur. Therefore, not only T-cells, but also dendritic cells (DCs), another antigen-presenting cell, are maintained in a down- modulated state in the airways, tightly controlled by high numbers of nearby macrophages 47. Immuno-modulatory factors involved in this process include among others: AM-derived nitric oxide (NO) 88, prostaglandin E2 (PGE2) 89,90, TGFβ91 and IL-10 92.
Introduction
1.4 Multiple trauma
1.4.1
Impact of trauma on patients and society
Trauma is generally the third leading cause of death in the United States and specifically the leading cause of death in men under 40 years of age 93, due to accidents and military causes.
Speed limits combined with improvements in seatbelts, airbags, and vehicle constructions lead to a decline in the number of accidental deaths, but also result in an increase in the number of accidental victims suffering from blunt chest trauma. Trauma further represents the second leading cause of life-threatening acute lung injury, i.e. in its severe form the Adult Respiratory Distress Syndrome (ARDS) 41,94. Prevention and intervention are thus both a socio-ethical and economic necessity. After initial, non-fatal injury thoracic trauma is considered to be an important clue to single or multi-organ failure, itself making the patient more susceptible to infections 95-97. With exception of changing the ventilation strategy 98, none of the numerous interventions made it into clinical practice. This emphasizes the need for further basic understanding of both pathogenesis and consequences of intervention.
1.4.2
Blast injury
Blast injuries are a special form of blunt trauma, with serious internal injuries, often without evidence of external lesions. Blast injury occurs following a sudden change in environmental pressure, originating from an explosion. The process of the explosion is equivalent to the energy release which is then transmitted as a radial pressure wave, called shock-wave, from the source into the surroundings 99,100. After a short travelling distance, a new shock front with lower pressure peak and initial velocity is formed in the air, called the blast wave. It travels supersonically and exerts its effects at comparably long distances from the explosion. The simple blast wave, described as the Friedlander wave form, rises very rapidly, then decays slowly (the vacuum phase) and may even drop below the previous ambient pressure. If the blast is confined by reflective surfaces, then a complex wave-form is formed. Complex blast waves contain multiple overpressure peaks 101. These wave-forms are usually expressed by their impulse (integral of pressure changes over time, P/ dt) rather than by their maximum peak overpressure value alone. Several factors can alter the response to the blast, such as peak overpressure, duration of the overpressure, impulse (in case of complex wave forms), the surrounding medium (e.g. water is a dense medium), and the distance from the explosion, because the effect is inversely proportional to the distance and the body orientation towards the blast wave front (reviewed by Elsayed, 102). Blast injuries are divided into 4 groups 103,104:
Introduction
interfaces, 2. secondary blast injury resulting from flying debris, 3. tertiary blast injury due to victims being thrown against an object, and 4. miscellaneous blast injuries including exposure to dust, thermal burns or fire by the blast.
The pressure wave of the primary blast injury, encountering media of different densities in the body, is reflected, causing turbulence and cavitation. The phenomenon termed “spalling”
describes the process where fluid is thrown from a more dense to less dense medium.
Another effect is the “secondary” explosion or implosion, as a consequence of compression and re-expansion of gas pockets in the organs by the traversing blast wave. As the blast wave reaches the surface of the victim’s body, a pressure difference develops between the outer surface of the body and the internal organs. In air-containing organs like the lung, the air in the alveoli is easily compressible and thus rupture of the alveolar capillary membrane occurs, with blood driven from the capillaries into the alveolar space, due to the pressure differences created. Finally, inertia is the shear created when the pressure wave moves tissues of different densities at different speeds 105,106.
1.4.3
Chest trauma
The organs most affected by blast injuries are the hollow, gas-filled organs such as the ears, the lungs, the gastro-intestinal tract 103,104,107-110, and to a lesser extent the cardiovascular and central nervous system 111. The lungs are almost always affected by blast injuries 103,107-109. Postulated explanations are: 1. The positive pressure is transmitted through the airways into the lung, causing rupture of the alveoli and the capillaries, 2. the longer lasting negative phase produces the alveolar capillary disruption, and 3. direct compression of the lung.
1.4.3.1 Pacemaker function of thorax trauma
Thoracic trauma seems to be an important trigger of morbidity and mortality in polytraumatic patients and may lead itself to severe lung injury 93. Indeed, the lethality of polytrauma patients without thorax involvement is 4%, as compared to 23% with simultaneous thorax trauma 112. Therefore, thorax injury is a major negative determinant for the long-term consequences after trauma 104,113. One of the major problems for clinical evaluation of the early phase after trauma is the missing of clinical signs of pulmonary dysfunction 114,115. This means that the severity of the consecutive injury is unpredictable at this stage.
Introduction
1.4.3.2 Pathology of pulmonary blast injury Pulmonary contusion and haemorrhage
Experimental lung injury from air blast 116,117 showed that the most consistent lesion was bilateral traumatic haemorrhage, variable in extent and distribution. Furthermore, oedema is usually prominent 116-118. Post-mortem data of fatal blast injury in humans is rare.
Ultrastructural examination of lungs 24 hours after blast exposure, reveals haemorrhagic areas in the lung contralateral to the side of blast exposure which initially microscopically appeared only slightly congested 118. Loss of structure of the alveolar epithelium and changes to the type II pneumocytes 118 and thus loss of surfactant production may be important in the development of respiratory insufficiency.
Haemopneumothorax and air embolism
Tension pneumothorax has been observed in rats exposed to air blast under laboratory conditions 116. Air embolism, pulmonary contusion and haemorrhage account for the majority of immediate and early death 104,110,119,120. Whether air emboli are important in survivors is unclear, but early and persisting neurological deficits might be the result of this phenomenon
103. Whether air-embolism is caused by mechanical ventilation is still a matter of debate 121. However, recently it has been shown that air embolism, present in blast victims, is not a mere ventilation-induced artefact 119.
1.4.3.3 Acute physiologic responses to primary blast
The reported physiologic responses in blast-injured patients vary considerably, largely as a result of varying observation times and the influence of other injuries. The most consistent physiological data is derived from animal experiments from which common cardiorespiratory and haemodynamic changes can be determined.
Pulmonary response
Moderate to severe blast exposure in animals is usually followed by an immediate, variable period of apnoea, lasting from a few seconds to up to more than one minute 122. This apneic period is followed by a fast and shallow breathing for a variable time before returning to pre- blast values 122. Blast-induced effects on gas-exchange and ventilatory function are variable throughout different animal experiments. The reduction of arterial blood oxygen tension
Introduction
of severity. Carbon dioxide tension (PaCO2) however is not affected and has been related to a ventilation-perfusion mismatch, due to a massive pulmonary haemorrhage 116.
Cardiovascular response
The most consistent finding of experimental blast injury on the heart rate appears to be an immediate bradycardia, which is more severe with higher intensity blast waves 116. Bomb blast survivors are occasionally found in profound shock and hypoxia without external signs of injury
115. An immediate fall in the mean arterial pressure (MAP) to less than 50% has been shown in various animal species following blast exposure 116. This response to blast wave injury is sometimes bimodal 116,117 and graded with higher intensity blasts, resulting in lower MAP and slower recovery. Recovery time to pre-blast values ranges from two hours 122 to up to several days 123. Concomitant with the reduction in MAP, a fall in the cardiac index (CI) can be observed. The CI response to injury, like MAP, is lower with exposure to higher intensity blast pressure waves. Despite hypotension and low CI, no compensatory peripheral vasoconstriction is detectable 116. This triad of apnoea, bradycardia, and hypotension 116,122 is only seen in animals in which just the thorax is exposed to the blast, but not in animals undergoing abdominal blast exposure 122. These changes are attributed to air embolism and direct cardiac injury 120 and probably mediated by vagal reflexes 124,125. Experimental animal studies 126 and clinical observations 127 following blunt chest trauma reveal a variety of EKG disturbances, from ventricular extracystoles to ventricular fibrillation, that are usually temporary but might account for some fatalities following blast injury and may exacerbate the triad of apnoea, bradycardia, and hypotension.
Progressive pulmonary insufficiency (PPI)
Several different mechanisms, in addition to the pressure wave-induced disruption of the lung architecture, are considered to be responsible for the various presentations of blast-induced pulmonary insufficiency (“blast lung”) and delayed respiratory insufficiency. Respiratory failure occurring 24 - 48 hours after blast exposure is unlikely to be caused solely by the primary blast 103,128. The pulmonary results of injury, namely the combination of blast effects, inhalation injury, tissue injury, and fluid resuscitation are frequently diagnosed as the Adult Respiratory Distress Syndrome (ARDS) 41,94. The late mortality after trauma is related to multi-organ failure (MOF), as a consequence of shock or sepsis 97. The pathophysiology of injury-induced organ MOF is poorly characterized, but has been linked to systemic inflammation, as a result of infection (either obvious or occult) or massive tissue injury (systemic inflammatory response
Introduction
inflammatory response syndrome (CARS), also contribute to the development of MOF (reviewed by Cobb and coworkers, 129), 95,130.
1.4.4
Molecular injury
Recent information indicates that there is a complex cellular and molecular generic response to injury, leading to multi-organ failure.
First of all, there is the direct tissue injury leading to interstitial or alveolar haemorrhage and oedema formation. The intensity of pulmonary oedema was positively correlated with the length of the survival time 119. As a result, hypoxemia and pulmonary air embolism may occur
120. Such events can also cause oxidative stress in the lung, which is characterized by an antioxidant depletion (water soluble and lipid soluble), an increase in lipid peroxidation, an increased methaemoglobin (metHb) content 131, and an inhibition of ATP-dependent Ca2+
transport 132. Supplementation with vitamin E and lipoic acid, but not vitamin C, increases the Hb oxygenation 133, however the pro-oxidant action of water soluble antioxidants via redox cycling of oxyHb and metHb may promote oxidative stress rather then prevent it 102,131.
Due to tissue trauma itself, as well as due to blood components and cell debris, an activation of host defence mechanisms occurs, including soluble plasma factors (complement and clotting cascades) and immunocompetent cellular components (neutrophils, monocytes, macrophages, and endothelial cells). Such activated cells in turn produce potentially toxic host mediators, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as chemical species such as kinins, eicosanoids, platelet-activating factor (PAF), and nitric oxide. This sequential increase of the inflammatory response may variably lead to shock, multiple organ failure and death. Eicosanoids have been shown to play an important role in the pathogenesis and modulation of pulmonary oedema associated with blast injury. Significantly elevated plasma levels of the prostanoids, thromboxane A2 (TXA2), prostacycline (PGF1a), PGF2a and PGE2 are reported for polytrauma patients with thorax involvement 134,135 and after surgery induced lung tissue injury 136, implying a relative prognostic value of these early mediators with regard to the intensity and the pattern of the injury. Concomitantly elevated plasma levels of type II a phospholipase A2 (PLA2) are suggested to bear some prognostic value 137,138. Unchanged levels of prostaglandin M (PGM) indicate an unaltered pulmonary metabolic capacity 136. Pharmacological inhibition of 5-lipoxygenase, immediately prior to blast exposure, reduces oedema formation, accumulation of neutrophils and generation of lipid peroxidation products in injured rabbit lungs 139 and permits respiratory compensation of metabolic acidosis in the general circulation, in spite of increased hypotension and acidosis in the venous
140
Introduction
collapse observed in this setting 141. Unlike TNF and IL-1, increased IL-6 levels are commonly detected during disease 135,142, thus IL-6 serum concentrations received more attention as a prognostic indicator for both severity and outcome.
1.4.5
Management of pulmonary blast injury
Success or failure of the treatment may depend on the judicious use of resuscitative fluids and respiratory support 128. In trauma victims, increased interstitial and intra-alveolar oedema impairs gas exchange. The initial ventilator settings in the trauma victim are somewhat different from those used in other patients, because the decreased functional residual capacity, due to oedema formation requires a lower tidal volume. Thus, modern ventilator strategies include a low tidal volume with a positive end-expiratory pressure (PEEP) and high inspired oxygen concentrations, in order to recruit alveoli and thus avoid atelectasis. However, the beneficial effects of supplementary oxygen and mechanical ventilation remain controversial and depend on the extent of the respiratory insufficiency 143. Concomitant respiratory acidosis is thereby compensated by therapeutic bicarbonate application. Together with pharmacological manipulations that increase the cardiac output, the function of both the heart and the lung, as well as other organs, can be augmented. The outcome of pharmacological interventions manipulating the reflex mechanisms and therefore altering haemodynamic functions remains unclear 128.
Various attempts have been done to interrupt the interaction of the neutrophils, endothelial cells, cytokines, and free radicals 133,144. Despite limited clinical and experimental success, this approach may prove to be worthwhile in the future for treating the severely injured patient 145.
1.4.6
Immunomodulation after trauma
Besides clinical characterization, recent research data indicate that the prognosis of trauma patients is strongly associated with a post-traumatic imbalance of the immunological system
146. For the outcome of patients with multiple injuries, it seems very important to gain an inflammatory mediator homeostasis as fast as possible. Overwhelming immune activation, however, can result in a systemic inflammatory response syndrome (SIRS) and septic shock.
To control the potentially harmful pro-inflammatory response, the immune system, as a mechanism of counterregulation, releases anti-inflammatory mediators inducing the so-called anti-inflammatory response syndrome (CARS). In addition to the autoregulatory pathways of the immune cells, the systemic immune response is controlled by two neuroimmune pathways: 1. the hypothalamic-pituitary-adrenal (HPA) axis and 2. the sympathic nerve system leading to glucocorticoid and catecholamine release, respectively, that affect primarily
Introduction
monocytes and macrophages 130. However, if the anti-inflammatory mediators predominate, immunosuppression (“immunoparalysis”) with ineffective eradication of microorganisms and septic complications may follow 147. In this context, the early presence of soluble TNF receptors (sTNFRs) 142 and interleukin 1 receptor antagonist (IL-1ra) in the circulation of trauma patients has been reported, even with a strong correlation to mortality 148,149. Traumatic injuries cause the liberation of various mediators such as IL-6, IL-10, and PMN elastase, with more or less an association between anatomic injury and the pattern of mediator release
135,142. For IL-6, an anti-inflammatory role in controlling the levels of other pro-inflammatory cytokines 150 and by inducing the release/ formation of sTNFRs has been suggested 151. In addition, PMN elastase is thought to increase the sTNFR release by proteolytic mechanisms
152. Since PGE2 is significantly elevated in the plasma of injured patients and since the immune cells (primarily the macrophages) from traumatized and burn patients hypersecrete PGE2 upon stimulation 153, this potent immunosuppressive agent is regarded to be a pivotal mediator in the post-trauma immune dyshomeostasis 154. Not only increased synthesis of PGE2, but also decreased removal and degradation have been reported in burn injury and trauma (reviewed in 89). Furthermore, it has been shown that trauma results in delayed macrophage hypersecretion of inflammatory mediators, such as TNF, IL-6 and end products of the respiratory burst, such as H2O2, that is associated with persistent functional macrophage defects in antigen presentation 155. The paradoxical combination of suppressed macrophage function and hypersecretion of inflammatory mediators, simultaneously renders the host susceptible to both infectious complications and the immune-mediated sequelae of SIRS and MOF. However, despite immunosuppression immediately after injury, a recovery from an early impairment occurring 3 to 6 days after trauma is also reported 156.
Aims of the Study
2. Aims of the Study
The blast trauma model is based on a rodent model originally developed by Irwin et al. 1998,
117.
The objective of this study was to use and to improve this model, in order to reproduce the clinical spectrum of injuries seen in blast victims, for studying the pathophysiology and potential treatment approaches of thorax trauma in rats.
The first aim was to standardize the blast pressure wave and its application, with regard to assessing characteristical physical blast properties, in order to study its biological correlate.
The second aim was to asses and to characterize the physiological and functional changes after in vivo trauma by means of:
I. Assessing lung physiological parameters over time in order to characterize trauma- induced changes, by combining our primary blast injury model with our ex vivo isolated perfused rat lung model. These studies include the investigation of pharmacological interventions, as to their therapeutic potential.
II. Assessing pro-and anti-inflammatory mediator release, oedema generation and resorption, as well as pulmonary infiltrate formation in vivo over time.
The third aim represented the study aspects of local and general immunodeficiency after thoracic trauma, that might clinically result in a higher number of infections. Due to their key role as modulator of pulmonary inflammation, macrophage functions will be assessed in vitro, as to their phagocytic capacity, respiratory burst and mediator release.
Materials and Methods
3. Materials and Methods
3.1 Animals
Female Wistar rats (230 ± 20 g) obtained from Harlan Winkelman GmbH (Borchen, Germany) and kept under controlled conditions (24°C, 55% humidity, 12 hours day/ night rhythm) on a standard laboratory chow and water ad libitum were used as lung donors for experiments with isolated perfused rat lungs, for the isolation of primary alveolar macrophages and for the in vivo experiments.
3.2 Chemicals
3.2.1
Chemicals used in the isolated perfused rat lung experiments
Substance Producer Solute in Concentration Storage
Amiloride Sigma-Aldrich, Deisenhofen,
Germany
DMSO *, NaCl 10-4 M freshly prepared
Bovine serum albumin fraction V, receptor-grade;
Lot No. 12353
Serva,
Heidelberg, Germany buffer 2% freshly
prepared
Formoterol AstraZeneca, Zug, Switzerland saline, 0.05%
Na2S2O5
1 nM freshly prepared Propranolol Sigma-Aldrich,
Deisenhofen, Germany
saline 10-4 M freshly
prepared
Terbutaline Sigma-Aldrich, Deisenhofen,
Germany
NaCl 10-4 M stock at
-20°C
Table 3.1 Substances used in the isolated perfused rat lung experiments. * 0.03% DMSO.
Materials and Methods 3.2.2
Chemicals used in the in vitro experiments
Substance Producer Solute in Concentration Storage Escherichia coli (K-12 strain)
tetramethylrhodamine conjugate
Molecular Probes, Eugene, OR, USA
PBS 250 µg/ ml 20 mg/ ml stock at 4°C
Liquemin Hoffmann-La Roche,
Grenzach-Wyhlen, Germany
NaCl (1:10) 8 Units/ ml Heparin
4°C
LPS from
Salmonella abortus equi Metalon, Wusterhausen, Germany
PBS 100 µg/ ml –
10 ng/ ml 1 mg/ ml stock 4°C LTA from
Staphylococcus aureus LS Wendel,
University of Konstanz, Germany
PBS 100 µg/ ml –
2 µg/ ml 5 mg/ ml stock -70°C SOD
(superoxide dismutase)
Sigma-Aldrich, Deisenhofen, Germany
HBSS * 1000 U/ ml freshly prepared
Staphylococcus aureus
(wood strain without protein A), tetramethylrhodamine
conjugate
Molecular Probes, Eugene, OR, USA
PBS 250 µg/ ml 10 mg/ ml stock at 4°C
WST-1
(C19H11IN5NaO8S2, tetrazolium salt)
Probior, München, Germany
HBSS * 500 µM freshly
prepared
Table 3.2 Substances used in vitro. * Hanks balanced salt solution.
3.2.3
Other chemicals
Brij, Tween 20, 0.4%Trypan blue, 3,3´,5,5´-tetramethyl-benzidine liquid substrate solution, LPS from Salmonella abortus equi, sodium dodecylsulfate (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany); HEPES (ICN Biomedicals, Ohio, USA); OptEIATM, biotin labelled polyclonal anti mouse/ rat TNFα (Pharmingen, Hamburg, Germany); Hoechst 33342, Sytox green (Molecular Probes Europe BV, Leiden, Netherlands); rabbit anti-TNFα- serum, K533 (LS Wendel, Biochemical Pharmacology, University of Konstanz, Germany); Streptavidin- peroxidase (Jackson Immuno Research, West Grove, PA, USA); MMP-2, matrix metalloproteinase 2, human rheumatoid synovial fibroblast, MMP-9, matrix metalloproteinase 9, human neutrophil granulocyte, Eosine-methylene blue, Azur-eosine-methylene blue (Merck KGaA, Darmstadt, Germany); Coomassi brilliant blue R250 (Serva, Heidelberg, Germany);
Materials and Methods
Temed (Biorad, München, Germany); APS (Amersham, Little Chalfont, UK); Pierce BCA protein assay reagent (Bender & Hobein GmbH, Heidelberg, Germany); ELISA/ EIA: IL-6, IL- 10 (Biosource Europe SA, Nivelles, Belgium); TXB2, 6-keto-PGF1α (BioTrend GmbH, Köln, Germany); CINC-3, mouse sTNFR1, sTNFR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany). All standard chemicals were purchased from established companies, primarily Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany).
3.2.4
Solutes
Distilled water; Dimethylsulfoxid, DMSO (Riedel de-Haen, Seelze, Germany); Ethanol; NaCl 0.9% (Delta-Pharma Boehringer-Ingelheim, Germany); Phosphate-buffered saline solution, PBS (PAA, Germany); Triton X-100 (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany);
EDTA, ethylendiamine tetraacetic acid (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
3.2.5
Anaesthesia and analgesic
• Halothane, 2-bromo-2-chloro-1, 1, 1-trifluoroethane (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany)
• Pentobarbital sodium (Narcoren), (Wirtschaftsgenossenschaft deutscher Tierärzte, Hannover, Germany)
• Buprenorphin (Temgesic), (Tierforschungsanlage, Universität Konstanz, Germany)
3.2.6
Cell culture material
RPMI 1640: with L-Glutamine, with/ without phenol red (PAA, Pasching, Austria); Hanks balanced salt solution (PAA, Pasching, Austria); Penicillin-Streptomycin (Gibco BRL Life Technologies, Eggenstein, Germany); Fetal calf serum (FCS) (Boehringer Mannheim GmbH, Mannheim, Germany); Cell culture plates (Greiner, Frickenhausen, Germany)
3.3 Laboratory equipment and technical devices
Centrifuges: Eppendorf 5417R (Netheler & Hinz, Hamburg, Germany); Minifuge RF (Heraeus Instruments, Hanau, Germany); ELISA reader: SLT Spektra Rainbow Photometer (SLT Labinstruments, Grailsheim, Germany); Microplate Fluorescence Reader FL 600 (Deelux Labortechnik, Gödenstorf, Germany); Isolated perfused rat lung (Harvard Apparatus, March-
Materials and Methods
USA); Eppendorf ACP 5040 (Netheler & Hinz, Hamburg, Germany); Microscope: Zeiss Axiovert 35 (Zeiss, Oberkochen, Germany)
3.4 The in vivo blast wave thorax trauma in rats
3.4.1
The blast wave generator apparatus
The blast wave generator was originally designed and constructed by Irwin and colleagues in 1998 117 and optimized by Liener and coworkers to discharge a global blast pressure wave in a safe laboratory environment. We have modified this setting to our specific needs in order to improve the pressure wave monitoring and the reproducibility of pressure wave exposure. The blast wave generator (Wissenschaftliche Werkstätten, Universität Konstanz) consists of three parts: a pressure reservoir, a blast nozzle, and a 190 A µm Mylar polyester film (Strohmeier, Rheda-Wiedenbrück, Germany) that separates the two compartments. Compressed air from the storage tank was slowly delivered by a pressure reducer to fill up a 0.75 l compressed air bottle, that is located close to the pressure reservoir of the generator. When a working pressure of 18 bar was reached the air supply was stopped. The valve (opening time: ≈ 25 ms) (HEE-D-MINI-24, Festo AG & CO, Esslingen, Germany) between the compressed air bottle and the pressure reservoir was triggered by the computer. Opening of the valve resulted in a fast pressure build-up in the reservoir. As soon as the burst strength of the Mylar diaphragm was reached, a blast pressure wave was discharged towards the rat by the nozzle.
Figure 3.1 shows the blast wave generator.
Figure 3.1: Schematic diagram of the blast wave generator (front view). Dimension is in millimetre (mm).
Materials and Methods 3.4.2
Pressure wave monitoring
Different sensors at varying positions offered an exact monitoring of the exposed blast pressure wave (Erne B., Abteilung Elektronik, Wissenschaftliche Werkstätten, Universität Konstanz):
1. A pressure transducer (PR-6ST-80400.XX-20, sensitivity: 39.7 mV/ bar, excitation: 4 mA, natural frequency: < 30 KHz; Keller AG, Winterthur, Switzerland) in the pressure reservoir determined the cracking pressure meaning the rupture of the polyester film. The measured time course of the pressure wave curve indicated the velocity of the pressure increase followed by the decay. The fast pressure build-up and pressure decline is a requirement for explosively discharging of the pressure wave (Fig. 3.2).
2. Two pressure transducers (2 MI PAA 110-050-020, sensitivity: 8.03/ 7.99 mV/ bar, excitation: 1 mA, natural frequency: > 400 KHz; Keller AG, Winterthur, Switzerland) on both sides of the rat measure the pressure peaks on rat thorax level (Fig. 3.2).
3. Another transducer (Halleffekt-IC 634-SS2, sensitivity: 7.5 – 10.6 mV/ mT; RS Components GmbH, Mörfelden-Walldorf, Germany) records the breathing frequency of the animal.
Therefore a string connected with the breathing sensor was placed above the thoracic cage.
Via this transducer the valve was triggered automatically depending on the breathing situation (e.g. inspiration, expiration or resting expiratory position) (Fig. 3.2). In addition the breathing frequency of the anaesthetized animals, before and after the pressure wave exposure was recorded.
All the measured pressure wave signals by the sensors were transmitted through a high accuracy measuring instrumentation amplifier Burr-Brown (Texas Instruments GmbH, Freising, Germany) either direct to the computer (cracking pressure transducer) via an A/ D- converter (MAX 186, Maxim Corporate Headquarters, Sunnyvale, CA, USA) or first (pressure peak transducer) to the HAMEG oscilloscope HM 407 (HAMEG instruments, distributor VELMA, Großkrotzenburg, Germany). Then pressure wave data were analysed by a special developed software (Heine G., Abteilung Elektronik, Wissenschaftliche Werkstätten, Universität Konstanz) for cracking pressure of the polyester film (Rp), wave form, pressure peaks (Pp(r), Pp(l)), impulse or area under the curve (AUC(r), AUC(l)), duration (t(r), t(l)) and pre-and post breathing frequency (Fpre, Fpost). For calculation of these parameters the following equations were used:
Materials and Methods
pressure peaks (Pp(r), Pp(l)):
2 1
2
1
t t
U P
m i
m i
i
=
∑
= −= (U = voltage; t = time), whereas the voltage U is proportional to the pressure P (cracking pressure transducer: 0.5 bar = 1 bar, pressure peak transducer: 0.1 bar = 1 bar);
area under the curve (AUC(r), AUC(l)):
∑
==
= 2
1 t i
t i
Ui
AUC ,
and pre-and post breathing frequency (Fpre, Fpost): breath/ min.
As an index for variations between different experiment the variation coefficient CV was calculated using the equation: CV
[ ]
% = medianSD ⋅100.Figure 3.2: The blast wave generator including pressure transducers at different positions. A cracking pressure transducer in the pressure reservoir, two overpressure sensors on the left and right side of the rat and a transducer for recording the breathing frequency.
3.4.3
Protocol of anaesthesia and analgesic
The rats were anaesthetized in a bell jar flooded with a Halothane®/ O2 gas mixture through a Halothane-evaporator (flow: 2 L/ min oxygen, 4% Halothane). By the time the breathing frequency was slow enough the animals were weighed and placed on a special immobilized
