The expression of CD36 in vessels with blood-brain barrier impairment in a stroke-prone hypertensive model.

AIMS: The class B scavenger receptor CD36, the receptor for oxidized low-density lipoprotein, mediates free radical production and brain injury in cerebral ischaemia. Free radical production is known to be involved in the remodelling of the cerebral vasculature of stroke-prone spontaneously hypertensive rats (SHRSP). Accordingly, we examined whether the expression of CD36 is increased in the vasculature with blood-brain barrier (BBB) impairment and collagen deposition of SHRSP. METHODS: The gene and protein expression of CD36 was examined in the vessels of the hippocampus of SHRSP with BBB impairment and those of Wistar Kyoto rats without the impairment, by real-time RT-PCR, Western blotting and immunohistochemical techniques. RESULTS: The gene and protein expression of CD36 was increased in the hippocampus of SHRSP compared with that of Wistar Kyoto rats. Confocal microscopic examination revealed CD36 immunoreactivity in perivascular microglial cells immunopositive for ED1. Immunoelectron microscopic examination revealed that the immunosignals for CD36 were located mainly in the cytoplasm of perivascular cells in vessels showing increased vascular permeability and a few in the cytoplasmic membranes of endothelial cells. CONCLUSIONS: These findings indicate that the expression of CD36 was increased in vessels with BBB impairment in the hippocampus of SHRSP and was mainly seen in the cytoplasm of perivascular microglial cells, suggesting a role of CD36 in cerebrovascular injury.

[Carinoplasty].

Recently, there may be a few patients for the candidates of crinoplasty. However, general thoracic surgeon must have the skills of carinoplasty. Probably, important points of carinoplasty are 1) to make adequate and complete exposure of operative fields, 2) to avoid ischemic damage of anastomotic sites, and to reduce the tension of anastomotic sites.

[Materials for reconstruction and reinforcement].

The recent development of thoracic surgery has depended on the progressive advances of surgical materials. For the reconstruction of the pleura, the chest wall, the diaphragm, the pericardium and other cardiac structures, materials of high quality are indispensable, and their appropriate use is also important.

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

[Tracheoplasty for tracheal cuff stenosis post long-term tracheostomy].

We encountered a case of tracheal stenosis post tracheostomy, so-called cuff stenosis. A 43-year-old man with ventricular septal defect (VSD) was treated by oral endotracheal intubation because of heart-failure and pneumonia, and tracheostomy was performed. He was placed on artificial ventilation for almost 3 months. Four months after discharge, he complained of dyspnea and was treated by oral endotracheal intubation again. At that time bronchofiberscopy demonstrated severe circumferential stenosis of the trachea 4 cm from the tracheostomy spot and that was compatible with the cuff site. Although the stenotic portion was dilated by an endotracheal tube, 47-days after dilation, the portion was restenosed to almost 7 mm in diameter. Therefore, surgical treatment was necessary and tracheoplasty was performed by end-to-end suture after 2 cm (4 ring) resection of trachea. Tracheoplasty is the most reliable method of treating cuff stenosis after tracheostomy.

[Combined large cell neuroendocrine carcinoma and squamous cell carcinoma of the lung; report of a case].

An 80-year-old man was admitted to our hospital because a routine chest X-ray had revealed a nodular shadow in the right lower lung field. Transbronchial lung biopsy (TBLB) failed to give at definitive diagnosis, therefore open lung biopsy was performed because of suspected lung cancer. Rapid intraoperative pathological examination diagnosed the tumor as large cell carcinoma. However, bloody pleural effusion was classified as class V. It was judged difficult to perform a curative operation, so the operation was interrupted. Pathological diagnosis was combined large cell neuroendocrine carcinoma and squamous cell carcinoma. Pleurodesis was done, and the patient is under observation at 7 months after the operation.

Application of laser Doppler velocimetry to lung transplantation.

The relationship between healing of a bronchial anastomosis and regional blood flow was studied in adult mongrel dogs after allotransplantation of the left lung. Animals were divided into the following three groups according to the immunosuppressive regimen. Group A: no immunosuppressive treatment (n = 7); Group B: azathioprine (5 mg/kg/day) and prednisolone (2 mg/kg/day) (n = 4); Group C: cyclosporine (20 mg/kg/day) (n = 4). The mucosal blood flow was examined serially after transplantation with laser Doppler velocimetry (LDV) at the tracheal bifurcation of the recipient (1st LDV) and at the bifurcation of the upper lobe bronchus of the transplanted lung (2nd LDV). The LDV values were expressed as a ratio to the preoperative values at the same site. Healing of the bronchial anastomosis was also assessed macroscopically with a fiberoptic bronchoscope and microscopically. The 2nd LDV value of the animals in group A at 13 days after transplantation remained low (32.9 +/- 27.6%). The 2nd LDV value in group B, although not significantly different from group A, had recovered to the preoperative level by that time (93.7 +/- 23.4%). By contrast, the 2nd LDV value in group C remained significantly higher than that in group A (119.9 +/- 23.0%, P less than 0.05). The 1st and 2nd LDV values were reduced in animals having infection at the bronchial anastomosis, even those in groups B and C. Bronchial mucosal blood flow appeared to be affected by infection and rejection of the transplanted lung, and to correlate closely with the healing of the bronchial anastomosis. This LDV method is useful to evaluate bronchial mucosal blood flow in experimental, and possibly in clinical, fields.

The effect and optimal time of administration of verapamil on lung preservation.

Calcium channel blockers have recently been shown to improve pulmonary and myocardial preservation. The effect of verapamil on hypothermic lung preservation was investigated using an isolated ventilated rabbit lung perfusion model. In phase 1, preserved lungs were not flushed prior to extraction. Four groups of five animals were studied: group 1 (no verapamil), group 2 (verapamil administration prior to extraction), group 3 (verapamil at reperfusion only), group 4 (verapamil both prior to extraction and at reperfusion). In phase 2, two groups of five animals received pulmonary artery flush with low potassium (4 mmol/L), 2% low-potassium dextran (LPD) solution; group 1 (without verapamil), group 2 (flush and reperfusion with verapamil). As in phase 1, lungs were stored for 30 hr at 10 degrees C prior to reperfusion. In phase 3, the protocol was identical to phase 2, except that the storage time was extended to 48 hr. PO2 (mean +/- SE) of effluent blood in lungs treated with verapamil prior to extraction (122.8 +/- 5.0 mmHg) was significantly increased in comparison with lungs not receiving verapamil (69.0 +/- 3.3 mmHg) or only receiving verapamil at the time of reperfusion (87.1 +/- 11.9 mmHg). Gas exchange after 30 hr storage was equivalent in lungs flushed with LPD with or without verapamil. However verapamil did provide an advantage when preservation times were extended to 48 hr (62.3 +/- 8.5 mmHg, 46.9 +/- 2.3 mmHg). Verapamil administered prior to lung extraction provides better lung function following preservation, but has benefit over LPD flush only with extended periods of preservation (48 hr).

The effect of PGE1 and temperature on lung function following preservation.

We studied the effect of a vasodilator (prostaglandin E1) as well as flush (F) and storage (S) temperatures (4 degrees C or 10 degrees C) on lung preservation in an isolated rabbit lung perfusion model. Low-potassium dextran (LPD) or Euro-Collins (E-C) solution was used as flush solution. Six groups of six animals were studied: group 1 (LPD, 4 degrees C F-S), group 2 (LPD with PGE1, 4 degrees C F-S), group 3 (E-C with PGE1, 4 degrees C F-S), group 4 (LPD, 10 degrees C F-S), group 5 (LPD with PGE1, 10 degrees C F-S), group 6 (E-C with PGE1, 10 degrees C F-S). After 18-hr preservation, left lungs alone were ventilated, and reperfused with fresh venous blood. PaO2, PaCO2, pulmonary artery pressure (PAP), tracheal pressure (Pt) during reperfusion, and wet/dry weight (W/D) ratios were measured. PaO2 after LPD with or without PGE1 was significantly higher than after E-C with PGE1 at 4 degrees C (95.8 +/- 11.5 mmHg in group 1 or 102.7 +/- 8.6 in group 2 vs. 41.8 +/- 10.5 in group 3, P less than 0.01) and at 10 degrees C (119.3 +/- 2.3 in group 4 or 131.1 +/- 6.2 in group 5 vs. 54.6 +/- 5.2 in group 6, P less than 0.01). PaCO2, PAP, Pt, and W/D ratios in the LPD groups were lower than in the E-C groups. LPD/PGE1 and LPD alone produced similar pulmonary preservation. PaO2 of lungs flushed with LPD and preserved at 10 degrees C was higher than that of lungs stored at 4 degrees C. We conclude that LPD solution is superior to E-C solution in this ex vivo rabbit lung preservation model, even when PGE1 is used. A moderate dose of PGE1 did not improve the performance of LPD as a flush solution. Pulmonary preservation with LPD at 10 degrees C is superior to preservation at 4 degrees C.

The superiority of an extracellular fluid solution over Euro-Collins' solution for pulmonary preservation.

Limited donor supply is the major factor restricting the application of lung transplantation. A uniformly reliable method of lung preservation would improve donor organ availability. At present, Euro-Collins' solution, an intracellular fluid-type solution, is most widely used in organ preservation. However, we have previously shown that initial pulmonary flush with an extracellular-type solution (low-potassium dextran solution [LPD]) provided better pulmonary preservation than Euro-C. In the present study, we used an in vitro-ventilated, blood-perfused rabbit lung model to examine whether the mechanism for this improvement was related to the effect of LPD during pulmonary flush or its effect during storage. Rabbit lungs were harvested and stored after pulmonary flush with different solutions (group 1: 400 ml of LPD; group 2: 400 ml of Euro-C; group 4: 300 ml of Euro-C followed by 100 ml of LPD; n = 5 in each group). The lungs were then preserved at 10 degrees C for 18 hr and reperfused with fresh venous blood. After 10 min of reperfusion, lungs in group 1 showed the highest PO2 (group 1: 124.4 +/- 7.7 mmHg; group 2: 46.2 +/- 9.4 mmHg P less than 0.01). Lungs in both group 3 and group 4 showed better lung function and lower wet/dry weight ratio than those in group 2. We conclude that LPD provides better lung preservation by its effects both on pulmonary flush and on storage.

Intratumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase in non-small cell lung cancer patients treated with 5-FU-based chemotherapy.

5-fluorouracil (5-FU) has been used widely, including in gastrointestinal cancer, breast cancer, and non-small cell lung cancer (NSCLC), and its efficacy has been reported to be associated with intratumoral expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). In order to clarify the clinical value of their expression in NSCLC patients treated with 5-FU, we investigated intratumoral expression of TS and DPD immunohistochemically. Of the 68 tumors studied, 40 carcinomas (58. 8%) had high TS expression, and 33 carcinomas (48.5%) had high DPD expression. The 5-year survival rate of the patients with high-TS tumors was significantly lower than that of the patients with low-TS tumors (P=0.0267), and the 5-year survival rate of the patients with high-DPD tumors was significantly lower than that of the patients with low-DPD tumors (P=0.0268). The Cox regression analysis of prognostic variables for NSCLC patients treated with 5-FU demonstrated that the simultaneous evaluation for both TS and DPD expression was found to be a significant indicator of a poor prognosis (P=0.0043). Our results demonstrated that the evaluation of intratumoral TS and DPD activity can be used to accurately predict responsiveness to 5-FU-based chemotherapy in NSCLC patients.

Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability.

We previously reported that cryopreservation of tracheas for 1 month was possible in a canine tracheal autotransplantation model with use of a preservative solution containing trehalose. Realizing that the allogenicity of many organs is decreased by freezing, we examined the possibility of immunosuppressant-free canine tracheal allotransplantation after long-term cryopreservation. Six to 10 rings of the trachea were removed from donor dogs (n = 12), immersed in the preservative solution, and cryopreserved in a deep freezer at -85 degrees C for 285 +/- 28 days (cryopreservation group). Five rings of the mediastinal trachea of recipient dogs were removed. The cryopreserved tracheas were thawed and transplanted to replace the excised mediastinal tracheas. In a control group (n = 6), the graft was preserved in Euro-Collins solution at 10 degrees C for 16 to 17 hours. Allotransplantation of tracheas was done as in the cryopreservation group. The anastomotic site and graft were covered with an omental pedicle in both groups. In the cryopreservation group, every animal, except one that was killed for pathologic examination, survived more than 2 months. All the grafts of this group were viable, and no stenosis or tracheomalacia was observed. In the control group, most of the animals died within 1 month of tracheal stenosis caused by rejection. These findings reveal that immunosuppressant-free canine tracheal allotransplantation was consistently possible after long-term cryopreservation of the graft in a preservative solution containing trehalose. This simple method could solve both donor shortage and immunosuppression problems.

Inhibition of reperfusion injury by human thioredoxin (adult T-cell leukemia-derived factor) in canine lung transplantation.

Human thioredoxin, which was previously recognized as adult T-cell leukemia-derived factor, has many physiologic activities, one of which is a radical scavenger effect. Its ability to reduce reperfusion injury was assessed in vivo in a canine lung transplantation model. In 19 dogs, left lung allotransplantation was performed after 100 minutes of warm ischemia. The function of the transplanted lung was assessed after clamping of the contralateral pulmonary artery. In the human thioredoxin group (n = 6), human thioredoxin 30 mg/kg was given to the recipients during reperfusion. In the N-acetylcysteine group (n = 5), N-acetylcysteine 150 mg/kg, known as a radical scavenger, was given in the same manner. In both groups, arterial oxygen tension was significantly higher than in the control group (n = 8). In the human thioredoxin group, peak inspiratory pressure was significantly lower than in the control group. Macroscopic and microscopic examinations showed an almost normal appearance of the lung tissues in the human thioredoxin and N-acetylcysteine groups, in contrast to the abnormal findings in the control group. Thus it would appear that human thioredoxin has a protective effect on transplanted lungs, as does N-acetylcysteine, and that its action may be a radical scavenger effect.

Effect of preoperative irradiation on wound healing after bronchial anastomosis in mongrel dogs.

To determine the endurable radiation dose before bronchial anastomosis, the bronchial circulation of mongrel dogs was assessed after bronchoplastic surgery conducted after various single doses of radiation. Plastic surgery of the right main bronchus was carried out 1 week after cobalt 60 irradiation to the right hilar region. Bronchial blood flow was measured with laser Doppler velocimetry 7, 14, and 30 days after operation. Animals were killed 30 days after operation and the region of the bronchial anastomosis was examined histologically. The animals were divided into five groups: group A (control group without irradiation), group B (8 Gy irradiation), group C (16 Gy), group D (19 Gy), and group E (24 Gy). Wound healing of the anastomosed bronchus was excellent in groups A and B. In group A, bronchial blood flow did not drop below 80% of the preoperative level throughout the postoperative observation period. In group B, bronchial blood flow on day 7 after operation was only 65% of the preoperative level, but blood flow had returned to the preoperative level by day 30. In groups C, D, and E, bronchial blood flow was less than 60% of the preoperative level on day 30. There was a high prevalence of radiation-induced morbidity in the region of anastomosis in groups C, D, and E. Postoperative recovery of bronchial blood flow and wound healing of the anastomosed bronchus were thus delayed in dogs that had received high doses of radiation before operation. We conclude that the safe dose of preoperative radiation in clinically used fractionation is 36 Gy or less (corresponding to a single dose of 16 Gy or less in dogs).

Expression of adult T-cell leukemia-derived factor in bronchoalveolar lavage cells after canine lung transplantation.

Lung transplantation is now an accepted therapeutic option for patients with end-stage lung disease, and an early diagnosis of rejection is essential in the management of these patients. Adult T-cell leukemia-derived factor (ADF), known as a human homolog of thioredoxin, has been shown to be induced by a variety of stresses. In this study we examined ADF expression in lung tissues and bronchoalveolar lavage cells after canine lung transplantation to determine whether it could be induced by allogenic stimulations and could be used to diagnose early rejection. Allotransplantations were performed in adult mongrel dogs, and immunosuppression was performed from the day of operation to the fifth postoperative day. No immunosuppressant was given from the sixth to the tenth postoperative days. Animals were put to death on the tenth postoperative day. Bronchoalveolar lavage was performed on the fifth and tenth postoperative days, and the lavage cells and lung tissues were examined immunohistochemically with anti-ADF antibody. The grades of rejection were as follows: grade 1 in two animals, grade 2 in three animals, and grade 3 in two animals. The percentages of ADF high-producer cells in bronchoalveolar lavage cells on the fifth and tenth postoperative days were 4.29% +/- 2.65% and 26.6% +/- 3.99%, respectively (p < 0.01). The percentages of ADF high-producer cells in normal healthy dogs and in those with grade 1, grade 2, and grade 3 rejection were 3.00% +/- 1.64%, 20.5% +/- 9.00%, 25.5% +/- 6.06%, and 34.5% +/- 6.50%, respectively. The percentage in each rejection group was significantly higher than that in normal healthy dogs (p < 0.05). These results suggest that examination of bronchoalveolar lavage cells with ADF staining may be useful in the early diagnosis of rejection.

Reliable cryopreservation of trachea for one month in a new trehalose solution.

We previously reported that trehalose, a reduced disaccharide, was effective in the preservation of lungs. In this study, we investigated the possibility of prolonged cryopreservation of tracheas in a preservative solution containing trehalose. Five rings of cervical trachea were removed and immersed in the preservative solution. The harvested tracheas were then cryopreserved and stored in a deep freezer at -85 degrees C. One month later, five rings of mediastinal trachea were removed. The cryopreserved cervical tracheas were thawed and autotransplanted in place of the excised mediastinal trachea (n = 6). The anastomotic site and graft were then covered with an omental pedicle. All six animals survived for more than 6 months. All grafts survived without any evidence of atrophy or stenosis. Microscopic examination of the grafts showed that the integrity of the tracheal tissues was maintained. Our findings show that consistent cryopreservation of the trachea for 1 month is possible in a preservative solution containing trehalose.

The immunosuppressive effect of FK 506 on canine lung transplantation.

The immunosuppressive potency of FK 506 was studied after left lung transplantation in adult mongrel dogs in comparison with cyclosporine. Fiberoptic bronchoscopy, bronchial mucosal blood flow measurement with laser Doppler velocimetry, and chest x-ray and pathologic examinations were performed. Group A had no immunosuppression (n = 5); group B received FK 506 (0.10 mg/kg/day intramuscularly (n = 5); group C received cyclosporine (20 mg/kg/day orally) (n = 5). In group A four dogs died of rejection on the seventh to the twenty-first postoperative days. Another one was killed on the fourteenth postoperative day because bronchial dehiscence occurred at the anastomosis. In group B one died of alveolar rejection on the seventh postoperative day. The remaining four survived 28 days and were put to death. In group C all five dogs survived 28 days and were put to death. In group A bronchial stenosis or dehiscence at the anastomosis was found in every one during the early postoperative period. In group B stenosis did not develop in any of the dogs, including the one that died on the seventh postoperative day. In group C slight stenosis was seen in one dog, severe narrowing in another, and good healing in the remaining three. The transplanted lungs were almost normal histologically in four animals of group B, and one showed alveolar phase rejection. In all animals of group A severe rejection was observed, and in group C two of five animals showed vascular phase rejection, two latent phase, and one fibrosis. Histologic examination of the bronchial anastomosis in group B showed almost normal bronchial epithelium and slight submucosal infiltration of mononuclear cells. In group A there was desquamation of epithelium and mild to moderate mononuclear cell infiltration. In group C hyperplasia of the epithelium was observed in two animals, an abscess at the site of anastomosis in one, and mild to moderate mononuclear cell infiltration in all five. With use of laser Doppler velocimetry, bronchial blood flow in group B was found to be the same as in group C. Laser Doppler velocimetry values reached preoperative levels by the twenty-eighth postoperative day in both groups. Although diarrhea developed in two dogs of group B, no other significant side effect of FK 506 was seen.

Split transplantation of the trachea: a new operative procedure for extended tracheal resection.

Before tracheal transplantation can be applied clinically, several problems must be solved: immunosuppression, blood supply to grafts, and reliable long-term preservation of grafts. We have conducted experiments on tracheal transplantation to solve these problems. In the present study, we tried a new operative procedure to accomplish reliable revascularization of transplanted tracheal grafts. It has been reported that transplantation of a 10-ring length of trachea is difficult even with omentopexy. Long tracheal allografts can be transplanted with use of direct revascularization, but this technique is extremely troublesome. Thus we developed a new operative procedure, "split tracheal transplantation," in which grafts are divided at the midportion and covered with omentum, after demonstrating that the blood supply to tracheal grafts can be reestablished around the suture lines. Two groups of dogs were used. In group A (control, n = 4), a 10-ring length of trachea was autotransplanted. The anastomotic sites and grafts were covered with omental pedicles. In group B (split tracheal transplantation, n = 10), tracheal grafts 10 rings long were autotransplanted. These grafts were divided at the midportion, a piece of omentum was inserted between the two halves, and the midportion was sutured. Grafts were observed regularly by bronchoscopy and examined histopathologically after the animals died or were killed. In some animals, microangiography of the bronchial circulation was done. In the control group, necrosis, stenosis, or malacia of the grafts was observed in three of the four animals. In the split transplantation group, all animals survived for at least 2 months, all grafts were incorporated, and none showed ischemia, stenosis, or malacia. Microscopic examination and microangiography revealed that neovascularization of the graft was promoted by omentum inserted at the midportion of the graft. Split transplantation of the trachea is an easy and reliable way to extend tracheal resection.

Importance of intrapulmonary lymph nodes in the differential diagnosis of small pulmonary nodular shadows.

STUDY OBJECTIVE: The objectives of the present study were to evaluate the importance of intrapulmonary lymph nodes (IPLNs) in the differential diagnosis of small pulmonary nodules and to review the CT findings of IPLNs. DESIGN: Retrospective analysis of patient records. SETTING: Chest Disease Research Institute Hospital, Kyoto University. PATIENTS: Between January 1991 and May 1996, we examined 26 patients with pulmonary nodular shadows smaller than 1 cm in diameter that could not be diagnosed before surgery. All patients (19 men, 7 women) underwent chest CT (28 to 72 years old; mean, 52.3 years). RESULTS: The pathologic diagnoses were IPLNs in 46.2% (12/26), pulmonary hamartoma in 23.1% (6/26), lung cancer in 11.5% (3/26), pulmonary tuberculoma in 11.5% (3/26), and metastatic lung tumor in 7.7% (2/26). IPLNs were located in the lower lobe in 72%. The characteristic CT findings of IPLNs were a clear border and location close to the pleura. Two of them resembled lung cancer. The CT features in these two IPLNs and in three small lung cancers overlapped. CONCLUSIONS: In the present study, we investigated small nodular shadows <1 cm in diameter and found that IPLNs located underneath the pleura are important to consider in the differential diagnosis of lung cancer. The CT scan findings of IPLNs were not necessarily specific and sometimes resembled those of lung cancer. Because of their location, video-assisted thoracic surgery is useful in making a definite diagnosis.