Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization.

There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15 consecutive cycles were performed over 32 days. Protocol B (a variation of Protocol A) ­ EDTA being added ­ with 15 consecutive cycles performed over 60 days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5 consecutive cycles being performed over 10 days. For the three protocols, hematoxylin­eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix (ECM). However, in Protocol C, more cellular components were removed in less time, making it the most efficient process. In addition, the cell tracking and viability was evaluated with chondrocytes seeding on the scaffold obtained by Protocol C, which showed an adequate cell scaffolding ability of this matrix.

5-HT Receptor Antagonism Attenuates the Ischemia-Reperfusion Injury After Rabbit Lung Preservation.

The success of lung transplantation is threatened by the appearance of ischemia-reperfusion injury, which is characterized by increased vascular permeability. 5-Hydroxytryptamine (5-HT; serotonin) is known to produce microvascular leakage in the systemic circulation, but its possible role in ischemia-reperfusion injury after lung preservation has not been reported. In this work we measured the release of 5-HT during a 24-hour rabbit lung preservation, and the effect of methiothepin (antagonist of the majority of 5-HT receptors) and SB204741 (antagonist of 5-HT2B/2C receptors) on the modified capillary filtration coefficient (mKf,c) was evaluated at the end of this period. Our results showed that the highest release rate of 5-HT occurred during the first 15 minutes after the lung harvesting and progressively decreased in the following time intervals. The baseline mKf,c greatly increased after 24 hours of lung preservation, and this increment was partially reduced by methiothepin and even more by SB204741. We concluded that 5-HT may play an important role in the ischemia-reperfusion process after lung preservation.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196°C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands.

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196 degrees C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.

[Smoking among Mexican physician. A comparative analysis with smokers who are not physicians]. / Tabaquismo en médicos mexicanos. Un análisis comparativo con fumadores que no son médicos.

OBJECTIVES: To determine prevalence, addiction knowledge and attitude on tobacco smoking in a group of smoking physicians (MF) and to compare these variables with smoking non-physicians (FNM) and non-smoking physicians (MNF) from the National Institutes of Health in Mexico (Insalud). MATERIAL AND METHODS: The results of a questionnaire among the three groups were compared. RESULTS: The prevalence of MF (22%) was significantly lower than in FNM (28%), (OR = 0.72, CI = 0.61-0.85). No significant differences regarding addiction and attitudes were found between them. The MNF had better knowledge and attitudes and agreed that their Institute should be a non-smoking area. CONCLUSION: Prevalence of smoking is lower among physicians than among FNM and the similarities between them suggest that addiction can provoke them and that a program for tobacco control is required.

Prevalence of cigarette smoking among employees of the Mexican National Institutes of Health.

OBJECTIVE: To determine the prevalence of cigarette smoking at the National Institutes of Health in Mexico (NIHM). MATERIAL AND METHODS: A survey was performed among workers who voluntarily answered a questionnaire. Smokers were identified with two specific questions, and type of employment was classified as physicians, administrative staff, investigators and support personnel. RESULTS: Total prevalence smoking was 28% (of 4,422 answered questionnaires). It was significantly higher among females, among administrative staff, and common-law and separated workers. It was significantly higher at the Mexican Institute of Psychiatry than at the remaining Institutes, even after adjusting for confounding. The prevalence was also higher among physicians from the same Institute. Of the smokers, 46% do so in their work areas and 78% of them would like to quit. CONCLUSIONS: The prevalence of smokers at the NIHM is as high as in the general population and a broad educational program for tobacco control and prevention is needed.

Transmission electron microscopy findings in the respiratory epithelium of guinea pigs exposed to the polluted air of southwest Mexico City.

We evaluated the nature and the extent of the damage to the respiratory epithelium of guinea pigs after a 4-month exposure to the mixture of air pollutants in southwest Mexico City. Guinea pigs were placed outdoors on the roof of our facility, 8 hours daily, from February to May 1995. At the same time, control guinea pigs were kept indoors breathing filtered air. Air pollutants, temperature, and humidity data were obtained from the nearest station of the Environmental Monitoring Net. The airways and lung parenchyma were analyzed after 120 days using transmission electron microscopy (TEM). During the 4-month exposure period, ozone (O3) exceeded the norm during 511 hours, and suspended particles less than 10 microm in diameter (PM10) during 52 hours. Both pollutants reached peak levels of more than twice the norm. TEM revealed no important abnormalities in the control group. In the exposed group, there was loss of cilia, detachment of epithelial cells, and eosinophil and macrophage migration toward alveolar spaces through type I pneumocytes with destruction of their basal membranes. In six guinea pigs in the exposed group, we noted bacteria along the airways, with associated inflammatory response. We explain the colonization of the respiratory epithelium by bacteria as the result of the impairment on the defense mechanism caused by the exposure to environmental O3 and PM10.

[Surgical treatment of non-malignant tracheoesophageal fistula]. / Tratamiento quirúrgico de la fístula traqueoesofágica no maligna.

Tracheoesophageal fistula is a complication of endotracheal canulas with pressure balloon during mechanical ventilation, for which treatment is surgical closure. There are approximately 80 reported cases in the world literature. Here we report our experience at the National Institute of Respiratory Diseases of Mexico. We performed surgical treatment of 7 patients (4 males, 3 females, 17 to 65 years of age) with tracheoesophageal fistula from 1991 to 1995, referred from other hospitals. Six had a history of prolonged orotracheal intubation, and the seventh had a traumatic lesion of the neck. Preoperatory management varied from 1 to 8 weeks, with treatment of malnutrition and infections. Esophagus was sutured with polyglactin 000 in two layers, the inner with interrupted, and the superficial with continuous sutures. The area was covered with a muscle strap. In six patients, simple suture of trachea was performed. In the seventh, due to an extensive tracheal defect, we decided to use a second muscle strap to occlude it. Six patients have had a follow up of 15 months to 5 years with satisfactory evolution. One patient died due to abdominal sepsis. We conclude that this technique provides a good prognosis for this disorder.

Reversal of digital clubbing after lung transplantation.

Digital clubbing is a common sign in a variety of lung diseases. Although its pathogenesis remains unclear, it is known that the degree of clubbing might vary and even disappear, particularly when the underlying disease is a malignant neoplasm that has been removed. By contrast, because of the short expectancy of life in patients with pulmonary fibrosis, it is unusual to observe regression of clubbing. In this work, we report a case of reversible clubbing after lung transplantation.

[Protection of the bronchial anastomosis with omental pedicle in unilateral lung transplant]. / Protección de la anastomosis bronquial con colgajo de omento en el trasplante pulmonar unilateral.

Lung transplantation has been slow to develop due mainly to the association of complications at the bronchial anastomosis secondary to the devascularization incurred during procurement. In the present study we evaluate the importance of the protection of the bronchial anastomoses with an omental pedicle. Mongrel dogs were operated upon forming four study groups: lung allotransplantation with bronchial protection (group T1, n = 9), lung allotransplantation without protection (group T2, n = 6); the two remaining groups did not receive a transplant but instead underwent surgery with extensive dissection and transection with reanastomoses of the left main bronchus with (group B1, n = 5) and without (group B2, n = 5) bronchial protection. All animals survived the surgical procedure. There were 8 complications at the bronchial anastomosis, six of which appeared in group T2 and were directly responsible for the animals death (p less than 0.05 when comparing lethal complications with group T1). Angiographic and dye perfusion studies were performed through the right gastroepiploic artery of the omentum. Bronchial neovascularization was demonstrated macroscopically as well as with light microscopy in all animals receiving protection with omentum (groups T1 and B1). We conclude that adequate bronchial protection is achieved with an omental pedicle flap brought up to the thorax during lung transplantation. It diminishes significantly the incidence of lethal complications at the bronchial anastomosis due to the revascularization that occurs through the omentum.

Urinary thromboxane B2 as an indicator of acute rejection in lung allotransplantation.

The behavior of urinary thromboxane B2 (TXB2) during acute rejection of lung allotransplants was evaluated. Unmatched mongrel dogs were submitted to a left lung orthotopic allotransplantation (groups I and II), or a sham operation (group III). All animals had an initial significant elevation of TXB2 excretion due to surgical trauma; however, in sham-operated animals (group III) this elevation returned to basal levels after 3 days. All transplanted animals (groups I and II) had persistent TXB2 elevation with 2 important peaks on postop days 5 and 9. The elevated TXB2 excretion persisted in spite of immunosuppressive treatment with azathioprine and prednisone (group II). Rejection was followed by means of an objective grading system applied to chest roentgenograms taken on all animals. It was found that TXB2 levels correlated directly with the grade of radiographic changes seen, thus indicating degree of rejection. TXB2 can be useful as a noninvasive indicator for surveillance of lung allograft rejection.