WO2008065464A2

WO2008065464A2 - A method for the pulmonary aerostasis and a device for its application

Info

Publication number: WO2008065464A2
Application number: PCT/IB2006/003382
Authority: WO
Inventors: Paolo Villoresi; Stefano Bonora; Silvia Roman; Fiorella Calabrese; Cinzia Giacometti; Francesca Lunardi; Federico Rea; Giuseppe Marulli; Marco Schiavon
Original assignee: Universita' Degli Studi Di Padova
Priority date: 2006-11-28
Filing date: 2006-11-28
Publication date: 2008-06-05
Also published as: WO2008065464A3

Abstract

Disclosed is a lung tissue aerostasis method which comprises coaptation of the superficial alveolar walls and the formation of bonds by irradiation with a light source in the presence of a protein or peptide substrate associated with a chromophore.

Description

A METHOD FOR THE PULMONARY AEROSTASIS AND A DEVICE FOR ITS APPLICATION

TECHNICAL FIELD

The purpose of this invention is to offer a solution to one of the most frequent complications encountered during and after lung surgery.

Specifically, the invention relates to a technique which allows safe, effective repair of the pleuropulmonary lesions which occur during pulmonary resection and are responsible for air leaks, thus ensuring complete aerostasis of the lung.

BACKGROUND OF THE INVENTION

The lungs consist of millions of alveoli; these are tiny air sacs consisting of a thin wall of epithelial cells, called pneumocytes, supported by a collagen membrane. Their structure ensures that they only adhere to the collagen base and the adjacent perimeter cells. The "airtightness" of lung tissue is guaranteed by the pleural membrane, an elastic sac consisting of a single layer of epithelial cells with a high degree of cohesion resting on a basal membrane, under which there is a tangle of elastic fibres and collagen in which fibroblasts and blood capillaries are immersed. It is approximately ten times as thick as the alveoli.

In numerous lung diseases, especially tumours, the first-line treatment is still surgery. Lung resections require the use of electrosurgical units and electrocautery forceps to isolate the anatomical elements (bronchi, blood vessels and nerves); the extent of the lung resection can range from removal of a fragment (wedge resection) to anatomical removal of a segment, a lobe or the whole lung.

During pulmonary isolation and dissection operations, damage is often caused by microlacerations of the surface of the visceral pleura and the alveolar walls, which are responsible for air leaks and bleeding of varying extents.

If major air leaks are insufficiently repaired before the chest is closed, the persistence of this problem can be further complicated by failure of the parenchyma to re-expand, atelectasis (lung collapse), bronchial and parenchymal fistulas, respiratory failure, with a consequent high risk of morbidity and mortality, and lengthy postoperative hospitalisation. To avoid these complications it is essential to perform thorough haemostasis (closing of blood vessels) and aerostasis (sealing of all areas where air leaks occur) of the lung.

In classic surgical treatment, if a small portion of lung is removed (atypical resections or enucleoresections), the parenchyma can be stitched after haemostasis of the blood vessels has been performed with the electrosurgical unit; alternatively, staplers which cut and simultaneously stitch the lung parenchyma and apply titanium microclips can be used. However, the visceral pleura which surrounds the lung parenchyma is very thin, and air leaks from the stitches are frequent.

Loss of aerostasis may also occur in sites where the parenchyma adheres to the chest cavity, or when the various lobes are not distinctly separated at the sulcus between them, and need to be surgically separated. In these cases, depleuralisation of the lung may occur.

Another event is surgical reduction in the volume of emphysematous lungs. In this case, the emphysematous bullae are removed with the aid of staplers which compress and simultaneously stitch the lung tissue, causing forced cohesion of the alveolar walls; however, the altered structure of the emphysematous lung parenchyma prevents the suture from being completely sealed, with the risk of parenchymal fistulas that cause lengthy air leaks.

In order to reduce these air leaks from suture lines, the use of strips of bovine pericardium or other synthetic materials (teflon or polydioxanone pledgets) positioned on the two branches of the stapler has been introduced. The additional tissue thus inserted between the metal clips and lung parenchyma prevents widening of the holes around the clips during lung reventilation when the suture line is stretched. However, although this technique is effective in some cases, it does not provide the definitive solution to the problem.

There are some situations when it is impossible to use staplers because the depleuralised area is too large, and the joins would be subject to excessive tensions, while blood would seep into the interior.

This applies to critical lobectomy operations, in which the sulcus between the two lobes is difficult to divide, and bleeding may occur on the lung surface; much of the visceral pleura in the sulcus is damaged, as are the alveolar walls (which remain open).

An attempt is usually made to cauterise some areas with electrosurgical units, but this does not entirely seal the air leaks, which cause multiple problems at the postoperative stage, with a reduction in respiratory function and the risk of lung collapse. The same kinds of problems are found in the case of segmentectomies, namely anatomical resections which involve complete depleuralisation of the resected margin, or pleural decortication when empyemas (infections of the pleural cavity) have become chronic.

All these complications involve longer hospitalisation periods, high medical costs and the risk of infections associated with long-term use of chest drainage.

Hence the need to research new methods which guarantee complete aerostasis, with a view to improving the postoperative outcome.

This research is directed towards the study of new methods, including biological adhesives. The first use of an adhesive in chest surgery dates back to 1955, when Eder instilled in the pleural cavity an adhesive consisting of autologous plasma, thrombin and calcium chloride, to seal small bronchopleural fistulas after pulmonary resections.

Next came fibrin adhesive, a haemostatic, adhesive, waterproofing, tissue-repair promoting, filling and resorbable substance; however, it has not yet proved able to guarantee complete aerostasis of the lungs.

Collagen has been added to the fibrin adhesive to increase the strength of the seal [Hiroaki Nomori et al., Ann Thorac Surg,(70):1066__70, 2000.]; other attempts have been made with the use of collagen adhesive (GAO) [Boris A. et al., Cardiothoracicsurgery, (17):8_13, 2000.], PLGA [Yuto Otani, et al., Ann Thorac Surg, (67):922_6, 1999] and GRPG, but none of these techniques has yet fully and permanently solved the problem of air leaks.

GRFG provides good aerostasis and has much greater adhesive strength than fibrin adhesive, but as it is based on formaldehyde (FR), it is toxic, carcinogenic and causes series necrotic and inflammatory lesions of the lung parenchyma that comes into contact with it [Hiroaki Nomori et al., Ann Thorac Surg, (67):212_6, 1999]. Even when it was replaced with GR-DIAL, significant fibrosis was observed.

PLGA Gelatin-Poly (L-Glutamic Acid) Hydrogel Adhesive also has much greater adhesive strength than fibrin adhesive, adheres well to lung tissue, and its removal is very difficult, unlike fibrin adhesive. However, there is no interaction with the underlying tissue; in other words, it acts as a kind of "stopper" which hermetically seals the wound, but there is a risk that it will become detached [Yuto Otani, et al., Ann Thorac Surg, (67):922_6, 1999].

Moreover, adhesives are applied to the entire lung surface, which can cause necrosis or secondary reactions even in the healthy tissue surrounding the lesion. Hence the need for a minimally invasive system restricted to the damaged area which allows the lung tissue to be sealed, thus ensuring complete aerostasis.

The use of lasers in lung surgery has already been studied. The lung parenchyma consists of 80% water, has a very low thermal capacity and density (0.15 g/cm³, only 1/5 of that of liver parenchyma), and a variable air content. It is therefore the ideal organ for photothermal laser applications.

The first use of lasers in lung surgery dates back to 1967, when Minton used one of the first Nd:YAG lasers (1064 nm) in pulsed mode to resect and vaporise lung metastases in rabbits. This technique was not adopted due to lack of fibres, ideal applicators and sufficient power.

The debate on the use of lasers in chest surgery was reopened in 1985, after the adoption of the Nd: YAG 1064 nm laser for standard endobronchial operations, by LoCicero, who experimented with the use of CO₂ (10600 nm) on the lung parenchyma.

However, the CO₂ laser is not suitable for lung tissue, because its short absorption length (<20 μm) means that only the surface of the tissue is heated; the energy applied is instantly turned into tissue vaporisation (incision), without significant cauterisation. Moreover, it cannot be used with fibres, because the wavelength is too high.

The same problems are found with the Er: YAG laser and the Ho: YAG laser, which operates at 2100 nm. At this wavelength, absorption becomes dominant, and cauterisation of the lung tissue is no longer guaranteed.

Later studies conducted in the USA, Japan and Europe paved the way for experiments with the Nd: YAG 1064 nm laser in superficial lung resections. In 1988, Rolle also began testing the Nd:YAG 1328 nm laser, which absorbs 10 times more water than the 1064 nm version, on animals. The NdrYAG laser, unlike the CO₂ laser, penetrates deeply into the tissue, and combines cutting with cautery, which is essential in the lung in view of the high density of blood vessels.

However, this laser causes considerable heat damage to the underlying tissue. After laser irradiation, coagulative necrosis of the lung tissue underlying the area of application immediately takes place, with major ischaemic-necrotic damage which continues for up to 4 weeks after the operation, and subsequent destructive degeneration (necrosis) which is difficult to control, and involves deep sites distant from the contact surface.

The uses of lasers in lung surgery are described in the following literature:

Noriyoshi Sawabata et al., Ann Thorac Surg 1996;61 :164-9; Masanobu Kiriyama et al., Ann Thorac Surg 2002;73:945-9; Noriyoshi Sawabata et al., Ann Thorac Surg 1996;62: 1485-8; Tsutomu Akahane et al., Lasers Surg. Med. 23 :204-212, 1998.; Tommaso C. Mineo et al., CHEST 1998; 113:1402-07;

Jϋrgen Waldschmidt et al., Med. Laser Appl. 19: 24-31 (2004); Ton van Boxem et al., CHEST 1999; 116:1108-1112; Larkin J. Daniels et al., Ann Thorac Surg 2002;74:860-4; Federico Venuta et al., Ann Thorac Surg 2002;74:995- 8; Jan Fanta et al.

Surgical Clinic of 3rd medical Faculty of Charles University, Srobarova 50, Prague 10, CZ; AXEL ROLLE, Med. Laser Appl. 18: 271-280 (2003); Axel Rolle*,l, et al., Multimedia Manual of Cardiothoracic Surgery; Axel Rolle et al. Ann Thorac Surg 2002;74:865-9.

"Gelatin-based and Power-gel™ as solders for Cr4+ laser tissue welding and sealing of lung air leak and fistulas in organs; US2002198517; 26/12/2002" describes a method that uses a protein substrate based on gelatin and Power-gel™ with the addition of a dye associated with the radiation of a laser that emits in the near-infrared spectrum to seal the tissue and achieve lung aerostasis. In practice, the experimental medium used is unsuitable; the examples only refer to sealing of flaps of skin, with tensile and torsion tests that do not guarantee the corresponding strength of the lung tissue, which is subjected to far higher pressures during the respiration process.

However, the problem of air leaks remains as a result of the degeneration of the pleura.

In view of the composition of the lung parenchyma, which consists mainly of water, and the numerous studies of the interaction between lasers and biological tissue and the characteristics of lung tissue absorption, we tested the use of the laser to achieve coaptation and compacting of the lung parenchyma, thus guaranteeing complete aerostasis by means of a coagulative necrosis process. The type and parameters of the laser used in this case differ from ablation techniques.

SUMMARY OF THE INVENTION

It has now been discovered that radiation with a power exceeding a certain threshold, which can consequently be conveniently effected with a laser, can be used to effect aerostasis in a damaged lung by creating on the walls opened by the surgical incision a protein-cell layer able to withstand the pressures and mechanical forces caused by respiration.

The interaction mechanism is based on the absorption of the laser beam by a protein substrate (whose light absorption was increased with the addition of a chromophore) applied to the region damaged by heating, and the consequent denaturing effect directed to it and transmitted to the cytoplasmic proteins of the adjacent alveolar layer and to the collagen molecules of the basal membrane.

The invention therefore provides a method of lung aerostasis with coaptation of the surface alveolar walls which are caused to adhere to one another so as to form a thicker, stronger epithelial layer due to the presence of the denatured protein substrate. This leads to instant, complete aerostasis, and allows subsequent physiological healing of the tissue by the repair systems.

To sum up, this lung tissue aerostasis method comprises coaptation of the superficial alveolar walls and irradiation with a laser source in the presence of a protein or peptide substrate associated with a chromophore.

The invention also provides a kit for obtaining aerostasis of a damaged lung, for example after lung surgery.

DETAILED DESCRIPTION OF THE INVENTION A Shrinking-Laser-Activator (SLA), based on a protein or peptide substrate preferably constituted by albumin, or alternatively by collagen, myoglobin or fibrinogen, is used to accelerate and facilitate the coaptation and adherence process.

Due to the effect of the laser radiation, the albumin monomers are polymerised, spread among the collagen fibres of the lung tissue, and merge with them. The SLA also localises and intensifies the absorption of the radiation, ensuring greater control of energy distribution in the tissue volume and protecting the underlying tissue against excessive heat damage caused by direct absorption of the laser beam. A chromophore (ICG) which restricts the interaction of the laser to the irradiated surface, without affecting the layers beneath it, was added to the SLA. This is very important, because it minimises the damage caused by coagulative necrosis, and leads to much faster tissue repair and less inflammation.

The chromophore absorbs energy from the laser beam and releases it in the form of heat, denaturing the proteins in the SLA and forming non-covalent bonds between the proteins of the SLA and the tissue collagen; a smaller amount of laser irradiation can be used to achieve the desired result, thus increasing the safety of the technique. However, to obtain an effective macroscopic process, the amount of energy delivered by the laser pulses must exceed a given threshold, which we have identified as 10 J/cm² in the case of our embodiment, which uses pulses of 200 μs. This allows coaptation of the alveolar walls, which is otherwise not guaranteed, and fusion of the albumin monomers of the SLA with the lung tissue, thus preventing merely superficial polymerisation, which would make the aerostasis non-resistant to the pressure forces to which the tissue is subjected during normal respiration.

The light source is preferably a pulsed semiconductor laser system. If required, the laser beam can be suitably guided for use in endoscopic surgery.

The fluence delivered with each pulse is between 10 and 150 J/cm², and the duration of the laser pulses is between 200 and 100,000 μs.

The SLA can be used in liquid or solid form, or in the form of a biocompatible albumin lamina. Solid SLA is easier to handle and apply, but its rigid structure makes it difficult to adapt to lung tissue, which has an irregular structure; it is also soluble, and may lose its structural properties on contact with water or other body fluids.

It was therefore decided to use the liquid form, which is easier to apply, makes the innermost areas, which cannot otherwise be reached by a solid SLA, accessible, and allows a greater coagulation depth to be reached than with a solid SLA.

The albumin or other substrate must not be very soluble, to ensure that its structural properties are not altered on contact with blood and other body fluids; this is achieved by increasing its concentration. A high concentration of albumin or substrate also causes an increase in viscosity, which allows even distribution of SLA in the tissue, preventing it from leaching in contact with the surface to be treated. The preferred concentrations range between 40% and 70%. The albumin, in freeze-dried form, is reconstituted with sterile water at the time of use.

Indocyanine green, carbon black or fluorescein was added to the SLA, preferably indocyanine green (ICG, Sigma Aldrich), which possesses good absorption at the wavelength of a diode laser (808 nm), is soluble in water, is non-toxic and is often used in clinical practice to take physiological measurements. ICG is solubilised in the same aqueous solution as used to reconstitute the freeze-dried albumin.

ICG allows the interaction of the laser with the irradiated surface only, without affecting the layers beneath. The radiation energy is thus absorbed selectively only by the target, and due to the increase in the characteristic of absorption of stained tissue, lower laser irradiation can be used to achieve the desired result, provided that it remains above a given threshold for the reasons stated above (10 J/cm² in the case of our embodiment), thus increasing the safety of the technique.

If the chromophore concentration is varied, generally between 0.30 and 0.50 ng/mL, the depth of the thermal coagulation in the tissue substrate will vary. More specifically, increasing the ICG concentration increases the coefficient of absorption of SLA and reduces the depth of optical penetration. The majority of the laser energy is deposited on the surface of the SLA; depending on the exposure time, radiation and intensity of irradiation, this leads to excessive coagulation on the surface of SLA and lack of interaction between SLA and tissue. This uneven denaturing along the thickness of the SLA causes unstable bonds between SLA and tissue. However, if the ICG concentration is too low, the irradiation times needed to reach the protein denaturing temperature of SLA (70⁰C) will have to be increased, leading to an increase in heat damage to the underlying tissue.

In view of these factors, it has been found that the most uniform absorption is obtained with an ICG concentration of 0.42 mg/ml of SLA, which represents an excellent compromise between even distribution of energy in the SLA and rapid reaching the 7O⁰C temperature required for the protein denaturing process.

The albumin can be modified to increase the number of bonds under certain conditions, and other proteins based on collagen or fibrinogen can be used as SLA.

The chromophore is chosen according to the laser used. Various materials can be added to the SLA before the laser procedure, and/or administered afterwards. Examples of these substances are proteins, polysaccharides, vitamins, synthetic organic molecules which maintain their biological characteristics if exposed to a temperature of up to 80⁰C for 10 sec - 2 min; enzymes, haemostatic agents such as thrombin and fibrinogen, vasoconstricting agents which reduce bleeding in the area to be repaired, antiinflammatories, bacteriostatic and bactericidal agents such as antibiotics, which prevent or cure infections.

The SLA is administered before performing laser irradiation, e.g. with a small spatula or syringe, in view of its highly viscous consistency due to the high albumin concentrations.

The method according to the invention offers the following advantages:

• it is a rapid, simple, minimally invasive procedure;

• it uses biocompatible materials;

• it reduces reactions to foreign bodies;

• it prevents suture- and needle-induced trauma;

• it can be used in areas difficult to reach with other techniques (such as the pulmonary hilus region);

• it improves and accelerates the healing process, consequently reducing hospitalisation time; • it produces perfect, elastic aerostasis, resistant to the pressure forces to which the tissue is subjected during normal respiration.

The lung aerostasis technique according to the invention can be used in: Critical lobectomy operations: in this case the sulcus between the two lobes is difficult to divide, and the lung surface may be damaged; a large part of the visceral pleural membrane is injured, and the walls of the alveoli are damaged. Staplers cannot be used in this case, because the depleuralised area is too large. An attempt is usually made to cauterise some areas with electrosurgical units, but this does not completely seal the air leaks.

Segmentectomies: anatomical resections involving complete depleuralisation of the resected margin. They present the same problems as critical lobectomy operations.

Atypical resections or enucleoresections: the area removed is small, and the parenchyma is closed with tobacco-pouch sutures after performing haemostasis of the blood vessels with an electrosurgical unit or stapler. However, the parenchyma is very thin, and air leaks often occur from the stitches.

Adherence of parenchyma to chest cavity: in this case, depleuralisation of the lung can occur at the intra- operative stage, leading to loss of aerostasis.

Operations to reduce the volume of emphysematous lungs: the emphysematous bullae are removed with the aid of staplers; however, the altered structure of the emphysematous lung parenchyma prevents the suture from being completely airtight, with the risk of parenchymal fistulas which cause lengthy air leaks.

Any lung surgery that damages the visceral pleura, as a result of which measures need to be taken to achieve aerostasis.

The invention is illustrated in greater detail in the following examples, wherein a pulsed diode laser is used which emits at a wavelength of 808 nm, with peak power of 140 W and 400 or 600 μm fibres.

For the SLA we used 50% bovine albumin (BSA, minimum 98%, A7030, Sigma Aldrich), with the addition of indocyanine green (0.42 mg/ml of SLA) used as chromophore (ICG, Sigma Aldrich)

To define the ideal laser parameters (power, frequency and pulse duration) we conducted in vitro and in vivo tests on porcine lung tissue. EXAMPLE 1 - In vitro tests

The in vitro tests for the air-leak sealing study were conducted on bipulmonary pig specimens, including the trachea and main bronchi. The lungs were insufflated (first the right, and then the left lung) using a ventilation tube inserted in the trachea and connected to a fan calibrated at pressures suited to the size of the animal.

The lung was then immersed in a basinful of water to ensure the complete absence of air leaks (docimastic test).

Linear and bowl incisions were made with a classic mechanical system (scalpel with cold blade). After each incision the lung was insufflated to check for air leaks from the wound. The lesion was then treated: after the application of the SLA and laser irradiation, and the airtightness of the treated lung tissue was checked to evaluate aerostasis; the lung was immersed in water again and insufflated. Finally, for the histological evaluation, pulmonary biopsies were performed in the areas in which the aerostasis tests had been conducted, to obtain samples of lung tissue measuring between 2 cm and 3 cm, including the fibrous lesions. All fragments were then fixed in neutral formalin buffered to 10% and taken to the Padua Hospital's Institute of Pathological Anatomy. The biopsy fragments of lung tissue fixed in formalin were sampled extensively in correspondence with the laser-treated area, so that it could be embedded in toto. The samples thus obtained were embedded in paraffin wax, cut into sections 4-5 μm thick, stained with haematoxylin-eosin and observed under an optical microscope.

The current intensity was varied from 60 to 10OA, the frequency from 20 to 100Hz, and the duration of the pulses from 200 μs to 2 ms.

No coaptation took place at currents below 6OA. When the current exceeded IOOA and the pulse duration was 2 ms, the optical power applied proved excessive, as it caused necrosis and carbonisation of the tissue, even after very short exposure times. At frequencies <100Hz, uneven coaptation took place due to the hand movement, which does not guarantee continuity.

The best results were achieved with a current of IOOA, pulses lasting 200 μs at the frequency of 20 Hz, and a 400 μm spot. To obtain stronger aerostasis it is necessary to repeat the procedure (SLA+irradiation) 2-3 times and to irradiate the area 2-3 mm from the edge of the incision, including an intact edge of visceral pleura, to ensure that no small damaged areas which may cause further air leaks have been overlooked.

In the case of linear incisions, the SLA must be applied with the wound wide open so that it adheres in depth; this prevents merely superficial interaction between SLA and tissue, which is not very resistant to the strong tensions to which the tissue is subject during ventilation.

To accelerate the process of sealing air leaks and achieve perfect aerostasis in half the time taken in the earlier tests, the laser lenses were changed (f=13 mm; f=22 mm) and a 1 mm spot was obtained.

The laser parameters were reset to: IOOA current, 20 Hz frequency and 1 ms pulses.

Histological evaluations of in vitro tests

On histological examination, an area of 100% superficial and deep fibrous repair was found, mainly supported by thickening of eosinophilic material, which involved the most superficial area damaged by the laser, and the alveolar spaces beneath it. The lung parenchyma surrounding and below the damaged area treated did not exhibit any significant necrotic alterations. No areas of atelectasis caused by laser/tissue interaction were found.

"Ideal" repair (100%) was defined as repair with no histologically apparent areas of damaged lung parenchyma which opened outwards, causing failure of aerostasis.

EXAMPLE 2 - In vivo tests

The in vivo tests for the air leak sealing study were conducted on the lungs of small pigs (25 Kg).

Linear or bowl incisions were made with a classic mechanical system (cold scalpel) by thoracotomy. The air leak from the wound made was checked after each incision. When the laser procedure had been completed, the airtightness of the treated lung tissue was checked to assess whether aerostasis had been achieved.

Some of the pigs were killed at time 0 to evaluate the extent of the heat damage caused to the tissue by the laser, analyse the thickness of the coapted tissue and establish histologically whether aerostasis had been achieved (check for cut alveoli which were still open and/or histological modifications). Other pigs were killed after 7 days to study the body's response to the tissue damage (level of inflammation and granulation tissue which determines the repair process).

For the histological evaluation, lung biopsies were conducted in the areas in which the air leak seal tests had been performed, and samples of lung tissue measuring between 2 cm and 3 cm, including the fibrous lesions, were obtained.

All fragments were then fixed in neutral formalin buffered to 10% and taken to the Padua Hospital's Institute of Pathological Anatomy.

In order to maintain a standard operating protocol, we kept the laser parameters constant in all the tests: frequency 50Hz₃ current IOOA and 400 μs pulses.

The air leaks were sealed in all lesions. After 7 days the area treated with the laser and SLA resembled the rest of the normal tissue. No adherences were observed in the area of the repaired lesion, whereas numerous adherences were found in the area where the thoracotomy had been performed. Histological examination conducted after 7 days showed the fibrous repair area, characterised by granulation tissue with a considerable inflammatory and fibroblastic component. The granulation tissue extended continuously over the entire damaged surface to the adjacent intact pleura, partly encapsulating it. The average thickness of the repair area ranged from 1 mm on the edge of the lesion to 2.5 mm at the centre of the damaged area. The lung parenchyma surrounding and below the lesion did not exhibit significant necrotic and/or inflammatory alterations. No areas of emphysematous alteration caused by the repair process, or atelectasis due to collapse of the alveolar structures caused by interaction between the laser and the tissue, were found.

Claims

1. A lung tissue aerostasis method which comprises coaptation of the superficial alveolar walls and the formation of bonds by irradiation with a light source in the presence of a protein or peptide substrate associated with a chromophore.

2. A method as claimed in claim 1, wherein the light source consists of a semiconductor laser which emits a series of pulses.

3. A method as claimed in claim 1 or 2, wherein the laser beam is suitably guided for use in endoscopic surgery.

4. A method as claimed in any of claims 1 to 3, wherein the fluence delivered with each pulse is between 10 and 150 J/cm².

5. A method as claimed in any of claims 1 to 4, wherein the duration of the laser pulses is between 200 and 100,000 μs.

6. A method as claimed in any of claims 1 to 5, wherein the protein or peptide substrate is selected from among albumin, collagen, myoglobin and fibrinogen.

7. A method as claimed in any of claims 1 to 6, wherein the aqueous concentration of the protein substrate is between 40% and 70%.

8. A method as claimed in any of claims 1 to 7, wherein the chromophore is chosen from among indocyanine green, carbon black and fluorescein.

9. A method as claimed in claim 8, wherein the chromophore is indocyanine green.

10. A method as claimed in any of claims 1 to 9, wherein the chromophore concentration is between 0.30 ng/mL and 0.50 ng/mL.

11. A lung tissue aerostasis kit consisting of a light source, a protein or peptide substrate and a chromophore.

12. A kit as claimed in claim 11, wherein the light source consists of a pulsed semiconductor laser.

13. A kit as claimed in claim 11 or 12, wherein the beam of the laser source is suitably guided for use in endoscopic surgery.

14. A kit as claimed in claim 11, 12 or 13, wherein the fluence delivered with each pulse is between 10 and 150 J/cm².

15. A kit as claimed in any of claims 11 to 14, wherein the duration of the pulses of the laser source is between 200 and 100000 μμs.

16. A kit as claimed in any of claims 11 to 15, wherein the protein or peptide substrate is selected from among albumin, collagen, myoglobin and fibrinogen.

17. A kit as claimed in any of claims 11 to 16, wherein the aqueous concentration of the protein substrate is between 40% and 70%.

18. A kit as claimed in any of claims 11 to 17, wherein the chromophore is chosen from among indocyanine green, carbon black and fluorescein.

19. A kit as claimed in claim 18, wherein the chromophore is indocyanine green.

20. A kit as claimed in any of claims 11 to 19, wherein the chromophore concentration is between 0.30 ng/mL and 0.50 ng/mL.