Lung Bx analysis, page 1
PEARLS / The art and science of BRONCHSOCOPY
Histological analysis of lung biopsies and airway smooth muscle cell culture
Michael Fayon 1 2 MD PhD
1 Université de Bordeaux, Centre de recherche Cardio-Thoracique de Bordeaux, INSERM U1045 ; 2 CHU de
Borde aux, Paediatrics Department, Centre d’Investigation Clinique (INSERM CIC 0005), F-33076
Bordeaux, France.
Correspondence to : Pr Michael Fayon, Service de Pneumologie Pédiatrique, Hôpital Pellegrin-Enfants,
Place Amélie Raba Léon, 33076 Bordeaux Cedex, FRANCE.
Tel : +33 5 56 79 56 43 ; Fax : +33 5 56 79 61 13; e-mail : michael.fayon@chu-bordeaux.fr
Introduction
Airway remodeling is observed in a variety of pediatric conditions such as asthma [1-7], bronchopulmonary dysplasia [7], cystic fibrosis [2, 8]. and COPD [2, 7]. It includes a number of structural changes, such as epithelial detachment, basement membrane (BM) thickening, smooth muscle hypertrophy, and new vessel formation. These changes contribute to thickening of airway walls and, consequently, to irreversible airway narrowing, bronchial hyper-responsiveness, airway edema and mucous hypersecretion. Airway remodeling is associated with poor clinical outcomes among asthmatic patients [9]. Early diagnosis and prevention of airway remodeling has the potential to decrease disease severity, improve control and prevent disease expression [9]. We hereby summarize means currently used for the assessment of airway remodeling, which may further our understanding of the relationship between structural changes and clinical and functional abnormalities.
Techniques for the assessment of remodeling in tissue
The usual assessment of remodeling is by histological examination of bronchial tissues. Tissue processing, visualisation and quantification have been well described [10]. Airway remodeling is evaluated after histochemical and immunohistochemical staining. In general, the assessment of tissue structure is performed with haematoxylin and eosin staining on paraffin tissues. Sirius red, van Gieson or Masson-
Trichrome stain the total collagen. Periodic-acid shiff staining is used to visualise the mucus glands.
Immunohistochemistry allows detection of specific proteins such as ECM proteins. Specific primary antibodies directed against a variety of cytokines are available. After staining, the slides are analyzed under light microscopy [2].
Reticular basement membrane thickness (RBM)
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Increased bronchial epithelial RBM thickness has been proposed as one of the main features of airway remodeling [1, 3-6]. Measurements of RBM thickness in individuals follows a log-normal distribution. For a precision of approximately +15%, 31-45 measurements are required in adults. It is recommended that RBM be measured at 20 mm intervals over a 1 mm reticular basement membrane length [11]. This methodology has been successfully applied in preschool and school-age children [12].
Inflammatory and Structural Cells Counts
It is recommended that a zone beneath the RBM of at least 5 mm of RBM should be included for counts of inflammatory cells [12]. The results are usually expressed as the number of cells per area [2].
Increased airway smooth muscle mass is present in fatal and non-fatal asthma. The study of the cellular mechanism (i.e., hyperplasia vs. hypertrophy) may add useful information regarding the functional consequences of airway smooth muscle remodeling [13]. For measurement of myocyte number and mean volume, cell nuclei are counted to enumerate myocytes under the assumption that these cells have only one nucleus. The volume of ASM may be measured using point and line intersection counts as previously described [8, 10].
Epithelial integrity
Epithelial integrity is expressed as a percentage of length of the RBM with intact epithelium on 2 biopsies, if possible [14].
Tissu density quantification
-Surface area
A simple stereologic technique of point counting may be used to determine surface area and structural composition ( ie , the percentage of the biopsy specimen composed of epithelium, RBM, subepithelial stroma, smooth muscle, submucosal glands, and other features) [10, 15]. A grid with points is superimposed on the tissue section, and the number of points overlying the tissue of interest and other subepithelial tissue (for normalization) is recorded. The results are usually expressed as the area occupied by the tissu of interest on the area of the chosen subepithelial tissu within the same biopsy specimen.
As an example, in one study, the areas of the structures of interest were determined at a magnification of x
200 with the aid of an eyepiece graticule containing 100 points, and the data were expressed as a percentage of the whole biopsy specimen area. The biopsy specimen area was calculated as follows: area
(in square millimeters) = number of points counted x 0.0016 [15].
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Surface area may also be determined by an automated cell recognition system based on colour analysis.
Quancoul# software (Quant'Image, 1997; Bordeaux, France) is a tool which calculates three independent criteria i.e., optic density (OD), hue density (HD), hue (H) from the three primary colours i.e., red, green and blue [16] for its colour recognition.
- RBM – Smooth muscle distance
As airway smooth muscle (ASM) increases in size and ASM cells migrate, the distance between an area stained by ASM bundles and the RBM is reduced. This distance is can be determined by at least 10 measurements at 50 μm regular intervals for each section [14].
Overall remodeling scores
Most of the structural changes are not characteristic of one specific airway disease, but are shared by all airway diseases (table 1, [2]). It may be tempting to adopt an overall semi-quantitative score summarizing the entire remodelling process. However, a specific weight should be atributed to each individual component.
Table 1. Features of airway remodelling in airway diseases [2]
Asthma
COPD
CF
Epithelial
Alteration
Detachment
Metaplasia
Densely ciliated
RBM thickness
+++
+
++
Subepithelial fibrosis
+++
++
++
Mucus gland hyperplasia
++
+++
+++
Smooth muscle mass
+++
++
++
Angiogenesis
+++
+
++
Each + represents the degree of association with disease. RBM: reticular basement membrane; COPD: chronic obstructive pulmonary disease; CF: cystic fibrosis.
Smooth muscle cell cultures
In asthmatic airways, recent reports indicate that the increased mass of ASM plays a critical role. ASM is the critical effector cell modulating airway tone - its contraction induces airway narrowing. Increased ASM mass is due to a simultaneous increase in size (hypertrophy) and number (hyperplasia) of ASM cells. Primary
ASM cells can successfully be isolated from human airways for cell culture [17-19], thereby providing in vitro remodeling models.
It has been shown that adult ASM cells are not only structural cells. They also contribute to bronchial inflammation by secreting a range of inflammatory mediators (secretory phenotype), recruiting and activating inflammatory cells, such as mast cells or T-lymphocytes. Such an increase has been related to a deposition
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Lung Bx analysis, page 4 of extracellular matrix proteins [20]. Chemokines have the ability to induce human ASM cells migration to the subepithelial area of the asthmatic airways and to increase their contractility in vitro [21].
ASM cell proliferation (proliferative phenotype) in severe asthma implicates a gallopamil-sensitive calcium influx and the activation of calcium-calmodulin kinase IV leading to enhanced mitochondrial biogenesis through the activation of various transcription factors (PGC-1 α, NRF-1 and mt-TFA). The altered expression and function of sarco/endoplasmic reticulum Ca(2+) pump could play a role in ASM remodeling in moderate to severe asthma. Additionally, aberrant communication between an injured airway epithelium and ASM could also contribute to disease severity [22, 23].
Studies of ASM cells in children remain extremely scarce . We have shown that stimulated non-asthmatic infant ASM cells may contribute to airway remodeling to a greater extent than adult ASM cells [24]. DNA synthesis in 10% fetal calf serum of infant ASMC was significantly enhanced (> 5-fold increase vs. ITS control medium) compared to adults (2-fold increase). PDGF-AA induced a response of lesser magnitude :
2.6-fold increase in infants vs. 1.5-fold in adults (p < 0.05). Newer data suggests that a mitochondrial biogenesis related mechanisms may be involved, similar to adult asthmatics. All the above suggests that
ASM cells may be prone to non-specific remodeling. Paediatric ASM cells also have the ability to synthetize inflammatory mediators and cytokines. TNF

-stimulated immature ASMC produce more LIF (leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine)) mRNA and protein than adult ASMC [19]. The secretion of LIF by stimulated immature human ASM cells potentially contributes to neuroimmune airway inflammation and subsequent remodeling.
Thus, ASM culture may help us to further understand the mechanisms of airway remodeling, the effect of current asthma treatments on airway remodeling, potential pharmacological targets for the treatment of airway remodeling in asthma, the mechanisms of airway remodeling, the effect of current asthma treatments on airway remodeling and the potential pharmacological targets for the treatment of airway remodeling in asthma [25].
Perspectives
Further studies involving ASMC cultures from asthmatic children are warranted. The interaction between
ASM cells and other structural (e.g. epithelial) and inflammatory cells should also be explored.
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