Ann. Medit. Burns Club - voL VII - n. 2 - June 1994


Er-fan X., Zong-cheng Y., Li Ao (Ngao)

Institute of Burn Research, Southwestern Hospital, Third Military Medical College, Chongelin, Sichuan, China

SUMMARY. On the basis of the autofluoreseence characteristics of elastic and collagenous fibres, microspectrofluorometry was used in this study to measure the dynamic changes of the fluorescent intensity of alveolar walls in rabbit lung after smoke inhalation injury. Such changes would indicate the alteration of amounts of fibre content in the lung. The concomitant changes of arterial blood gas levels, lung water volumes and pathomorphology of lung tissue were also observed. It was found that after injury the fluorescent intensity of alveolar walls decreased progressively denoting a substantial loss of elastin and collagen in the lung. Serious lung damage and pulmonary oedema were also found. The results indicated that the disrupting effects of proteolytic enzymes on lung tissue structures may contribute importantly to lung injury after smoke inhalation. A new method, rnicrospectrofluorometry, is thus provided for the study of fibre content in the lung.


Following studies in the pathogenesis of smoke inhalation injury, many achievements have been reported regarding the damage of pulmonary and capillary endothelial cells. However, no research on the extracellular interstitium, in particular regarding the quantitative description, has yet been conducted. This study concentrated on the dynamic changes of fibre content in rabbit lung after smoke inhalation with the application of microspectrofluorometry, in order to form a complete picture of pulmonary injury.

Materials and methods

Thirty healthy adult rabbits of either sex weighing 2-3 kg were used, divided randomly into five groups. There was one control group (6 rabbits) and four experimental groups (6 rabbits each). Conscious animals inhaled smoke in a mildly hypoxic environment for a period of 7 min, according to the method previously established in our Institute (1). All the animals in the exPerimental groups were killed at respectively 2, 6, 12 and 24 h after smoke inhalation injury. Control animals underwent similar procedures except that air replaced smoke.
Arterial blood gases and pH were determined by Model IL-1303 blood gas analyser. Total lung water (TLW), extravascular lung water (EVLW) and residual pulmonary blood water (RPBW) were measured according to the method of Noble et al. (2).
The anterior chest wall was removed as soon as the animal - was killed. In addition to observation of gross pathological changes in the lung, samples taken from each low lobe of both lungs were fixed in 10% neutral buffered formalin for 48 h first, then embedded in paraffin, sectioned at 6 pm, stained with haematoxylin and cosin, and studied by light microscope for micropathological changes.
Three animals were taken from each group randomly, and two sections of each animal were used for further examinations. According to the autofluorescence characteristics of the elastic and collagenous fibre tissues in alveolar walls, alveolar wall fluorescent intensity was measured by Model MPV-111 microspectrofluorimeter (Leitz, West Germany). Twenty points were read on each section with 420 nm excitation wave length, 750 V voltage, 10 x 25 magnifying power and P 2 rim diameter of measuring diaphragm. The data were computerized.
All the experimental results were presented as mean values + standard errors. Data were compared with normal controls by analysis of variance and t-test. Results were considered significant if P < 0.05 (*) and highly significant if P < 0.01 (**).


Pa02 and S02C decreased progressively in the first 6 h after smoke inhalation injury. It then began to rise but had not returned to normal controls by the end of the experiment. PaC02 increased significantly after the injury with a peak at 6 h, and was still higher than normal level at 24 h (Table 1).



PaC02 (kPa)

Pa02 (kPa)

SO2C (%)






2 h

7.13 + 0.05**

7.00 + 1.01 **

9.21 + 1.71**

81.58 + 9.61**

6 h

7.17 + 0.09**

7.22 + 1.63**

8.25 + 1.44**

78.93 + 6.31**

12 h

7.15 + 0.07**

6.96 + 1.52**

8.76 + 1.37**

81.77 + 6.73**

24 h


5.69 + 0.88**

11.55 + 1.48*

89.15 + 6.54*

Table 1 - Results of blood gas analysis

Both TLW and EVLW increased markedly by 6 h, peaked at 12 h, and remained high at 24 h post-injury. No significant change of RPBW was found among the five groups, indicating that RPBW did not influence the changes in lung water content (Table II).


TLW (ml/g)

EV1-W (Ynllg)

Fl (N=120)



3J2 + 0.21

161.57 + 43.23

2 h



157.30 + 35.67

6 h

4.58 + 0.42**

4.12 + 0.49**

143.40 + 43.11

12 h

5.22 + 0.77**

4.88 + 0.68**

134.45 + 34.67**

24 h


3.76 + 0.41*

128.58 + 35.26**

M*: Fluorescent intensity of alveolar walls

N: Numbers of samples in each group

Table II - Changes of lung water volumes and Fl*

Gross examination of the specimen revealed congestion, swelling and petechial and/or patchy haemorrhages of lung tissues. Microscopically, haemorrhagic and oedematous cuffs were observed around small blood vessels and branchioles. A large number of phagocytes aggregated and infiltrated the lung. The alveolar spaces were flooded with oedematous fluid. Fluorescence microscopy showed bright yellow-green elastic fibres ranged regularly in normal alveolar walls. After injury these fibres were disintegrated and disrupted, and even became indiscernible in some alveolar walls.
The fluorescent intensity of the alveolar walls decreased gradually throughout the entire experimental period after smoke inhalation. It was already significantly lower than normal control levels at 6 h, and the lowest value occurred at 24 h (Table II).


The causes of damage of smoke inhalation injury are very complicated. Besides the direct destructive actions of noxious chemical substances and particulate materials, the effects of local andlor systemic factors, including neural, humoral and cellular factors, etc., are also very important after injury. Among these factors, the roles of proteolytic enzymes, such as elastase, collagenase, etc., released from polymorphonuelear leukoeytes (PMN) and alveolar macrophages (AM), have received growing attention.
Elastin and collagen are the major components of lung tissue structural proteins. Elastic and collagenous fibres participate in the formation of alveolar walls, blood vessel walls and basement membranes. Elastase and collagenase can decompose clastin and collagen respectively. Elastase can also hydrolyse lung type 111 (interstitium) and type IV (basement membrane) collagen, proteoglycan and microfibril constituent (3). Once these tissues are disrupted, the structural integrity of the lung will be affected, the permeability of the alveolar capillary membrane will increase, and pulmonary oederna will develop. Our previous studies illustrated that the clastase released from PMN and AM might play an important role in the development of lung injury after smoke inhalation (1). But the direct evidence of lung tissue damage caused by proteases are not yet sufficient. Although the results of morphometry showed that the total length of all the parenchymal elastic fibres (LT) in rabbit lung decreased significantly 24 h after smoke inhalation injury compared to control (4), this method has the following shortcomings:

  • the measuring process is complicated and hard to observe dynamically;
  • the results may be affected by many factors, especially subjective interference;
  • the lung specimen cannot be used for other pathomorphological studies, because it is necessary to perform perfusion fixation of the whole lung (in order to measure inflated lung volume); and
  • only elastic fibres can be measured in this method, and a substantial loss of elastin in the lung may have occurred, considering the decrease of fibre LT.

Both elastic and collagenous fibres have their own autofluorescence characteristics (5). Under fluorescence microscopy, the fluorescent light of elastic fibres is yellow-green, strong and concentrated, while that of collagenous fibres is pale, weak and homogeneous. These characteristics were adopted in the present study. The autofluorescent intensity of alveolar walls was measured by microspectrofluorometry, the values of which indicated the amounts of elastic and collagenous fibre content in the lung. The results showed that the autofluorescent intensity of alveolar walls decreased gradually after smoke inhalation injury, and was already apparently lower than that in normal controls 6 h post-injury. Pa02 and S02C dropped, P.C02 was raised, TLW and EVLW increased, and serious lung damage was also observed in pathological examinations. These changes suggested that the fibre content of alveolar walls decreased markedly in the early post-injury stage, and a large amount of elastin and collagen was digested, thus causing the destruction of lung tissue structures. Twenty-four hours after injury, Pa02, PaC02, TLW and EVLW showed some improvement, but did not return to normal levels. However, the fluorescent intensity of alveolar walls became even weaker, indicating continuation of hydrolysis of proteases on fibre content. This was consistent with the results of our previous studies, i.e. elastase activity in bronchoalveolar lavage fluid was still significantly higher than normal control levels 24h after smoke inhalation injury (1). Starcher (6) also suggested that elastase entering the lung interstitium continues to attack the elastin fibre for several days at least. Total clastin content can be replaced within a month, although severed alveolar walls clearly cannot be rebuilt.
The results of this study further demonstrated that the disrupting effects on lung tissue structures of proteolytic enzymes released from PMN and AM might contribute importantly to lung injury after smoke inhalation. The study also provided a new quantitative determination method, icrospectrofluorometry, for the study of fibre content in the lung. The method has the following advantages:

  • the autofluoreseence of elastic and collagenous fibres can be maintained at length without distinct changes;
  • section preparation does not need any special staining and staining conditions have no effect;
  • the measuring process is simple, rapid and accurate;
  • morphological observation and morphometry can be performed at the same time, and this is convenient both for dynamic analysis and for studying functions combined with forms.

RESUME. Sur la base des caractéristiques de l'autofluorescence des fibres élastiques et collagènes, les auteurs de cette étude ont utilisé la microspectrofluorométrie pour mesurer les changements dynamiques de l'intensité fluorescente des parois alvéolaires dans le poumon de lapin après la lésion d'inhalation de fumée. Ces changements indiqueraient l'altération des quantités de contenu fibreux dans le poumon. Les auteurs ont aussi observé les changements concomitants des niveaux du gas hématique artériel, des volumes du liquide pulmonaire et de la pathomorphologie du tissu pulmonaire. Ils ont trouvé que, après le lésion, l'intensité fluorescente des parois alvéolaires diminuait progressivement, ce qui indiquait une perte considérable d'élastine et de collagène dans le poumon. Ils ont aussi trouvé de graves dommages pulmonaires et l'oedème pulmonaire. Les résultats indiquaient que les effets perturbateurs des enzymes protéolytiques sur les structures tissulaires pulmonaires peuvent contribuer en manière importante à la lésion pulmonaire après l'inhalation de fumée. En cette manière les auteurs fournissent une nouvelle méthode, la microspectrofluorométrie, pour l'étude du contenu fibreux du poumon.


  1. Xie E.F., Li A. (N), Yang Z.C. et al.: Dynamic balance changes between elastase and antiprotease in the early stages after smoke inhalation injury. Burns, 18: 362-7, 1992.
  2. Noble W.H., Obdrzalek J., Kay J.C. et al.: A new technique for measuring pulmonary edema. J. Appl. Physiol., 34: 508-12, 1973.
  3. Spragg R.G., Cochrane C.G.: Human neutrophil elastase and acute lung injury. In: Avenue M. (Ed.): "Acute Respiratory Failure (Lung Biology In Health and Diseases)", 24: 379-405, New York, 1985.
  4. Xie E.F., Li A. (N), Yang Z.C. et al.: Changes of length of elastic fibers in rabbit lungs after smoke inhalation injury. Chin. Med. J., 104: 191-3, 1991.
  5. Hrapchak R.J.: Immunohistochemistry. In: Sheehan D.C., Hrapchak B.B. (Eds): "Theory and Practice of Histotechnology", 2nd ed., 3 10- 1, Missouri, The C.V. Mosby Company, USA, 1980. Starcher B.C.: Elastin and the lung. Thorax, 41: 577-85, 1986.


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