Multi-photon microscopy in the evaluation of human saphenous vein.

BACKGROUND: The use of conventional fluorescence microscopy to image biological systems at the cellular level is limited by its inability to spatially resolve thick tissues. We have applied the technique of multi-photon fluorescence microscopy to study the structure and function of endothelial cells in living human saphenous vein taken from patients undergoing coronary artery bypass surgery. MATERIALS AND METHODS: Vein segments were preserved for 1-4 h to determine the temporal effects of storage. The effect of pH on endothelial and smooth muscle cell viability was examined by storing segments at pH 6.0, 7.4, and 8.0. Calcein-mediated green fluorescence and ethidium homodimer-mediated red fluorescence were used to differentiate cell viability. Increases in diaminofluorescein fluorescence were used to measure bradykinin activation of endothelial nitric oxide synthase (eNOS) with or without N-nitro-l-arginine (L-NNA). Multi-photon imaging was performed with the BioRad MRC1024ES system. RESULTS: Successful imaging of endothelial and smooth muscle cells of vein segments was achieved. Cell viability was well preserved up to 3 h of storage but dramatically decreased after 4 h. Cell viability was maintained at pH 7.4, diminished at pH 8.0, and was completely lost at pH 6.0. A two- to threefold increase in eNOS activity was observed upon activation by bradykinin which was completely inhibited in L-NNA-treated samples. CONCLUSIONS: We have demonstrated the successful application of multi-photon microscopy in imaging and quantifying nitric oxide production and cell viability under various storage conditions in human saphenous veins. This imaging technique allows for the functional imaging of cellular processes and may have diagnostic potential in cardiovascular surgery for patients undergoing bypass operations.

Intraoperative metabolic monitoring of the heart: I. Clinical assessment of coronary sinus metabolites.

Numerous clinical studies have corroborated the ability of intraoperative sampling of coronary sinus blood to measure changes in myocardial metabolism induced by ischemia and reperfusion. Among other changes, cardiac arrest induces a period of obligate myocardial lactate production that persists for an indeterminate amount of time after reperfusion. Coronary sinus lactate assays have been established as a standard method to compare various myocardial protection strategies. Current methodology requires detailed sample processing, precluding real-time feedback in the operating room. Newer devices hold promise in allowing the online assessment of myocardial metabolism; however, these methods await precise validation.

Continuous perfusion of donor hearts in the beating state extends preservation time and improves recovery of function.

OBJECTIVES: Improving methods of donor heart preservation may permit prolonged storage and remote procurement of cardiac allografts. We hypothesized that continuous, sanguineous perfusion of the donor heart in the beating, working state may prolong myocardial preservation. METHODS: We developed a portable perfusion apparatus for use in donor heart preservation. Contractile, metabolic, and vasomotor functions were monitored simultaneously in an isolated swine heart. The metabolic state was monitored by myocardial tissue pH. Vasomotor function was assessed in isolated coronary ring chambers. Hearts were randomized into 3 groups: group I (n = 5), cardioplegic arrest, 12-hour storage at 4 degrees C with modified Belzer solution, and 2-hour sanguineous reperfusion in the working state; group II (n = 6), 12-hour continuous perfusion in the beating working state, 30 minutes of arrest (to simulate re-implantation time), and 2 hours of reperfusion, as above; group III (n = 7), coronary ring control hearts. RESULTS: At 2 hours of reperfusion, left ventricular developed pressure in group II was higher than in group I (mean +/- standard deviation: 90 +/- 6 mm Hg, 53 +/- 15 mm Hg, P = .005). Significantly less myocardial edema was observed in group II than in group I (73% +/- 4%, 80% +/- 1% water content, P = .01). Significantly less myocardial acidosis was noted in group II than in group I during preservation (pH 7.3 +/- 0.01, 6.1 +/- 0.03, P < .001) and reperfusion (pH 7.3 +/- 0.008, 6.8 +/- 0.05, P < .001). Coronary endothelial vasomotor function was better preserved in group II than in group I as evidenced by dose-response relaxation of coronary rings to 10(-8) mol/L bradykinin (37%, 55% delta baseline, P = .01). CONCLUSION: This new method extends the current preservation limit and avoids time-dependent ischemic injury, thereby allowing for distant procurement of donor organs.

Humanized antibody directed to the IL-2 receptor beta-chain prolongs primate cardiac allograft survival.

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma. Murine Mik beta 1, a mAb that blocks IL-2 binding to IL-2R beta, was developed as an immunosuppressive agent. There was modest prolongation of cynomolgus cardiac allograft survival in animals treated with murine Mik beta 1 (mean survival 11.8 +/- 1.6 days compared with 8.2 +/- 0.4 days in untreated animals; p = 0.06). However, murine Mik beta 1 is ineffective in recruiting primate effector cells and is neutralized by monkey Abs directed toward the infused Ab. To circumvent these limitations, a humanized form of Mik beta 1, which is a largely human IgG1k Ab, except that murine hypervariable regions are retained, was developed. In vivo plasma survival of humanized Mik beta 1 was threefold longer than simultaneously administered murine Mik beta 1 (terminal t1/2, 104 +/- 10 h vs 37 +/- 2 h). Furthermore, humanized Mik beta 1 manifests Ab-dependent cellular cytotoxicity, an activity that is absent with the parental murine Mik beta 1. Graft survival was significantly prolonged by humanized Mik beta 1 treatment with survivals of 22, 22, 24, 27, 44, and > 300 days (p vs control < 0.01; p vs murine Mik beta 1 < 0.01). Survival was not prolonged further (p > 0.3) by the addition of humanized anti-Tac, which blocks interaction of IL-2 with IL-2R alpha subunits. There was no toxicity attributable to the use of Mik beta 1 Abs. Thus, humanized Mik beta 1 prolonged cardiac allograft survival in primates without toxicity and may be effective as an adjunct to standard immunosuppressive therapy.

Coronary arteriographic findings in black patients and risk markers for coronary artery disease.

Coronary arteriographic results are reported in 1535 black patients: 751 men (mean age 57 +/- 11) and 784 women (mean age 59 +/- 11). Among the black men 19%, 15%, 21%, and 4% had single-, double-, and triple-vessel and left main disease, respectively. Among the black women there were 12%, 10%, 15%, and 3% with similar involvement. Logistic regression models showed that most of the recognized risk factors were positively correlated with significant (at least one artery with > or = 50% stenosis) coronary disease, but a history of hypertension was not a significant independent predictor in either sex. ECG evidence of previous infarction increased the odds of detecting significant coronary disease by the greatest amount when controlling for other significant risk markers in women. In men both previous infarction and atypical pain (negative) were equally important. This study confirms but does not explain previous reports that have revealed less than expected angiographic evidence of significant coronary artery disease in black compared with white persons.

Nicardipine: myocardial protection in isolated working hearts.

The effectiveness of the calcium antagonist nicardipine in protecting the ischemic myocardium was evaluated using the hemodynamic recovery of isolated working rat hearts subjected to hyperkalemic cardiac arrest followed by ischemia at 37.5 degrees C and 10 degrees C. Rat hearts (n = 51) received 20 mL of cardioplegia and were subjected to 27 minutes of ischemia at 37.5 degrees C. Group A (control) did not receive nicardipine. Groups B through F received nicardipine in the cardioplegia with total doses ranging from 2 micrograms to 6 micrograms. Group A had 46% survival of ischemia, whereas groups C (3 micrograms) and D (4 micrograms) had survival rates of 88% and 100%, respectively (p less than 0.05). The recovery of aortic flow after ischemia was 35% in group A, compared with 76% in group B (2 micrograms) and 81% in group D (p less than 0.05). Group A had 49% postischemic recovery of cardiac output, whereas groups B and D had 82% and 85% recovery (p less than 0.05). The postischemic recovery of stroke volume was 48% in group A compared with 84% in group B, 87% in group D, and 73% in group E (5 micrograms) (p less than 0.05). Additional rats were exposed to 210 minutes of ischemia (n = 41) or 240 minutes of ischemia (n = 56) at 10 degrees C. Control groups did not receive nicardipine, whereas treatment groups received nicardipine in the cardioplegia with total doses ranging from 1.4 micrograms to 6.4 micrograms. There were no significant differences in the survival of ischemia or the recovery of function after ischemia at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylene blue guidance for simplified resection of a lung lesion.

A 60-year-old patient returned 1 year after right pneumonectomy with a new primary squamous cell carcinoma of the left lower lobe. Using fluoroscopic guidance, the lesion and the shortest track to the surface were marked by methylene blue preoperatively. The lesion was easily excised by wedge resection without the need for manipulation or deflation of the lung.

Brain protection during circulatory arrest.

Previous nuclear magnetic resonance studies in this laboratory have shown a beneficial biochemical effect of antegrade cerebroplegia (CP-A) during hypothermic circulatory arrest. This study compared CP-A with other methods of cerebral protection during hypothermic circulatory arrest to assess the clinical utility of this technique. Twenty-three sheep were divided into four groups: systemic hypothermia alone (SYST) and systemic hypothermia combined with external cranial cooling (EXTNL), retrograde cerebroplegia (CP-R), or CP-A. Cardiopulmonary bypass was started, and the sheep were cooled to 15 degrees C and subjected to 2 hours of circulatory arrest. Cardiopulmonary bypass was restarted, and the animals were rewarmed and weaned from cardiopulmonary bypass. Serial neurological examinations were performed and hourly scores assigned until the animals were extubated. Postanesthetic neurological scores improved in all groups throughout the 6-hour recovery period except the CP-R group. The improvement over time for these scores was similar for the EXTNL and CP-A groups and significantly better than for the SYST or CP-R groups (p = 0.004). The CP-A group had 5 of 7 animals with deficit-free survival despite the similarity in recovery of baseline brainstem function. We conclude that both antegrade infusion of cerebroplegia and external cranial cooling confer distinct cerebroprotective effects after a protracted period of hypothermic circulatory arrest when compared with the other methods studied.

Cardiovascular surgery in blacks.

The available information concerning cardiovascular surgery in blacks is very limited and incomplete. Those few reports that do exist seem to show that the usual indications for surgery, preoperative findings, and final results can be expected. Acquired heart disease is as important a cause of mortality in African Americans as in others and is amenable to surgical intervention. Surgical mortality is easily attributable to the usual iatrogenic variables or comorbid disease. At present it is uncertain whether the incidence of inoperable disease or problems of access to health care are responsible for the low rate of utilization by blacks. A more accurate account will become available when large-volume programs begin to report their results.

Blunt chest trauma in the elderly.

Significant differences were identified between a group of elderly patients (65 years and older) and a nonelderly group both with blunt thoracic trauma. There was a lower incidence of elderly patients presenting in shock; however, cardiopulmonary arrest at arrival was more frequent in this group. Although the types of complications were similar in both populations, the morbidity and mortality rates were higher in the elderly. A high index of suspicion must be generated for an elderly patient who has sustained blunt chest trauma. An aggressive diagnostic and therapeutic approach may lead to a decrease in the high morbidity and mortality rates in the elderly.

Aorta-right atrial fistula: an unusual complication of ascending aortic dissection.

Ascending aortic dissection complicated by aorta-right atrial fistula is a very rare occurrence. The patient may present with atypical clinical findings such as a continuous murmur or atrioventricular block, making the diagnosis difficult. Surgical repair is straightforward unless the correct diagnosis is missed, in which case there may be difficulties during the initial period of cardiopulmonary bypass. This report deals with the successful management of a 67-year-old man with this condition.