Analysis of NAD(P)H Fluorescence Components in Cardiomyocyte to Detect Allograft Rejection in Heart Transplantation
Bibliographic record
Abstract
Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart,investigate possible applications of NAD(P)H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD(P)H was studied for non-invasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy (EMB) of 14 pediatric patients with heart transplantation. Rat cardiomyocyte (n=5,13-14 week old) were also isolated by the same approach for human myocytes. Autofluorescence(AF) was recorded in living cardiomyocytes following excitation with 375 nm UV-light and detection by spectrally-resolved time correlated single photon counting (TCSPC),based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups:normoxic condition,normoxia with Rotenone,ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemia-mimicking ones,induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies,as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns,1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420-560 nm spectral range. Rat cardiomyocyte steady-state AF in ischemia-mimicking condition was significantly increased when compared normoxic ones (P0.05); application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition,however no significant difference between the two groups (P0.05).Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition(P0.05). A correlation between changes in steady-state NAD(P)H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity (P0.05),without change in the spectra shape,results can be comparable to the effect of ischemia-mimic conditions. Conclusion Our studies clearly demonstrated that spectrally-resolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones,while this activity (and thus ATP production) seems lowered during rejection process. In perspective,the advantage of this method is the possibility of its combination to multiphoton confocal microscopy,which can result in the adaptation of this approach directly to tissue biopsy,as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection,and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".