Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging. Mitochondria are both the primary source of organismal energy and the major source of cellular reactive oxygen species (ROS) and oxidative stress during aging . Aged caWhile more attention has been placed on mitochondrial electron leak and consequent free radical generation, proton leak is a highly significant aspect of mitochondrial energetics, as it accounts for more than 20% of oxygen consumption in the liver  and 35–5c peroxidase activity and release has been proposed as its major basis of activity (SS-31 (elamipretide), a tetrapeptide , binds to cardiolipin-containing membranes  and impractivity . SS-31 iactivity , heart factivity , neurodeactivity , and metactivity . In agedactivity  and braiactivity  and has activity . We haveactivity . HoweverIn this report, we investigated the effect and underlying mechanism of action of SS-31 on aged cardiomyocytes, especially on the mitochondrial proton leak. Using the naturally aged rodent model we provided direct evidence of increased proton leak as the primary energetic change in aged mitochondria. We further show that the inner membrane protein ANT1 mediates the augmented proton entry in the old mitochondria. Most significantly, we demonstrate that SS-31 acutely prevents the excessive mitochondrial proton entry and rejuvenates mitochondrial function through direct association with ANT1 and stabilization of the ATP synthasome.To examine whether SS-31 restores aging mitochondrial function, we applied the Seahorse mitochondrial stress assay to intact primary cardiomyocytes in a non-working state (not paced for contraction). The Seahorse assay revealed higher mitochondrial basal respiration in cells from old mice than that in young mouse cells ; howeverThe evaluation of mitochondrial proton leak by Seahorse assay is indirect, as it is based on the oxygen consumption rate. Thus, to directly investigate the reduction of mitochondrial proton leak in old cardiomyocytes by SS-31, we expressed the protein mt-cpYFP, a mitochondrial matrix-targeted pH indicator  in the rIn search of the source of the uncoupled mitochondrial proton leak in the aged cells, we examined possible involvement of proton leakage through ATPase and mitochondrial UCPs. As expected, the ATPase inhibitor Oligomycin A failed to inhibit the proton leak in pH challenged permeabilized aged cells . Levels Recently, the inner membrane protein ANT1  was identified as the major site of proton leak in mitochondria of multiple tissues , and wasConsidering that the inorganic phosphate carrier might be a potential source of the elevated proton entry in old mitochondria, we examined the proton permeability of old mitochondria to pH stress in a buffer that did not contain phosphate. In this buffer, the proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria stressed in the presence of inorganic phosphate .2/100 s in young cells, n = 28–88, p<0.05). Confirming this, we detected an increase in mitoflash activity in Langendorff perfused intact aged hearts from mt-cpYFP transgenic mice  opening , we evalTo further investigate the mechanism of SS-31 protection on the proton leak, we used biotinylated SS-31 to evaluate whether SS-31 directly interacts with the ANT1 protein. Hearts were disrupted by douncing, after a low speed spin to remove fragments, mitochondria were collected by high speed spin and disrupted in digitonin, to create lipid rafts containing their associated proteins, a protocol commonly used to prepare mitochondrial supercomplexes . This prIn this report, we have shown direct evidence of increased proton leak in the aged mitochondria as a primary energetic disturbance and that the increased proton entry in old cardiomyocytes likely takes place through ANT1. Moreover, we demonstrated that SS-31 prevents the proton entry to the mitochondrial matrix and rejuvenates mitochondrial function through direct interaction with ANT1 and stabilization of the ATP synthasome. During aging, the pathological augmented and sustained basal proton leak burdens the mitochondrial work load, resulting in a decline in respiratory efficiency. Blocking this pathological proton leak induced by aging benefits the mitochondria and the heart . We suggANT1 appears to mediate the pathological mitochondrial proton leak in the aged mouse heart. Although an increased mitochondrial proton leak in the aged heart was previously suggested by indirect oxygen consumption measurement , the sitMost interestingly, for the first time, we showed that a novel drug, SS-31 (elamipretide), now in clinical trials, prevents the augmented mitochondrial proton leak, rejuvenates mitochondrial function, and reverses aging-related cardiac dysfunction. Mechanistically, we found that SS-31 directly interacts with ANT1 and stabilizes formation of the ATP synthasome. This would seem surprising, given the prior belief that SS-31 affects mitochondria via binding to cardiolipin. However, the notion that SS-31 prevents the proton leak by direct interaction at the pore ‘pocket’ of ANT1 is supported by recent observations based on cross-linking ‘interactome’ mass-spectroscopy that showed that SS-31 is in intimate proximity to two lysine amino acid residues in the water filled cavity of the ANT1 protein . MoreoveIt has recently been shown that SS-31 alters surface electrostatic properties of the mitochondrial inner membrane . The conThe restoration of membrane potential by SS-31 in the old mitochondria  can be aIn summary, our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes and that SS-31 acutely and directly interacts with the transporter, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes. The improved mitochondrial function leads to complex secondary changes to effect enhanced diastolic function in the aged heart. These findings provide a novel insight for better understanding of the mechanisms of cardiac aging and establish the novel concept that decreasing the pathological proton leak in the aging heart restores mitochondrial function, ultimately reversing cardiac dysfunction in aging.All the animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington and conform to the NIH guidelines . The use of mice and rats have been approved by IACUC under animal protocols 2174–23 and 2654–03. Young (4–6-month-old) and aged (24–26-month-old) C57BL/6 mice (Charles River colony) and F344 rats  were obtained from the National Institute of Aging Rodent Resource. The mt-cpYFP transgenic C57BL/6 mice were housed until reaching the age described.Single ventricular myocytes were enzymatically isolated from mouse and rat hearts as described previously . The rodThe XF24e Extracellular Flux Analyzer (Seahorse Bioscience) was used for measuring oxygen consumption in intact resting cardiomyocyte, with XF assay medium containing 5 mM glucose and 1 mM pyruvate. Oligomycin A , carbonyl cyanide-p-trifluoromethoxyphenylhydrazone , and antimycin A  plus 1 μM rotenone (Rot) were added in three sequential injections, as reported .4, 1.2 KH2PO4, 5 Glucose, 1 CaCl2, pH 7.4). For mitochondrial flashes, mt-cpYFP expressing cells were exposed to alternating excitation at 405 and 488 nm and emission collected at >505 nm. Time-lapse 2D images were collected at a rate of 1 second per frame. For mitochondrial superoxide quantitation, we used the ratio of MitoSOX Red  to mitoTracker Green . For mitochondrial membrane potential measurement, JC-1 was excited at 488 nm and emission collected at 510–545 nm and 570–650 nm. For photon triggered mPTP opening, the cells were loaded with 120 nM Tetramethylrhodamine methyl ester (TMRM) and line scanned at 1 Hz as described previously  or Leica SP8  for confocal imaging at room temperature. The cells were placed in modified Tyrode’s solution  and centrifuged at 800 g for 10 min. The supernatants were centrifuged at 8000 g for 15 min to purify mitochondria. Digitonin was added to the mitochondria at a ratio of Digitonin: protein = 6:1 to break down the membrane system. Treatment drugs were added 30 min before addition of 10 μM biotin-SS-31 (Biotin-D-Arg-dimethyl Tyr-Lys-Phe-NH2) or biotin control . Streptavidin Agarose beads  were added and incubated for 2 hrs at room temperature. The beads were washed with MIB three times and then eluted by 50 μM SS-31. The eluates were boiled with LDS protein loading buffer  and loaded on NuPAGE for gel electrophoresis and Western blotting with antibody for ANT1 . In some experiments, after electrophoresis, gels were silver stained using a Pierce Silver Stain Kit .2O: Methanol: Acetic Acid = 5:4:1) 5 times at 20 min intervals. For native blotting, gels were transferred to PVDF membranes at 25 V in 4°C overnight and incubated with ATP5a antibody , followed by anti-mouse secondary antibody.Mitochondria from mouse hearts were isolated as described previously . Mitocho2/CO2-bubbled KHB solution  at 37°C. To minimize motion artifact during imaging, 10 µM (-)-Blebbistatin  was included. During imaging, the left ventricle was gently pressed to further suppress motion artifact. Mitoflashes were imaged using the procedure described above.mt-cpYFP transgenic mice were anesthetized with pentobarbital (150 mg/kg). The heart was removed, cannulated via the ascending aorta, and put on a modified perfusion system and in a custom made chamber on the confocal stage as previously reported . The perData are shown as mean ± SEM. For the multiple groups comparisons one-way ANOVA was applied followed with appropriate test. Student’s t-test was used to determine the statistical significance between two group comparisons. p<0.05 was considered statistically significant. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your work is novel and a clear demonstration of the potential role of mitochondrial proton leak in cardiac aging and dysfunction and the protective effect of SS-31.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Benjamin Miller (Reviewer #2).Thank you for submitting your article "Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at In this manuscript Zhang et al. examine the effects of the compound SS-31, a compound that has shown intervention potential in other systems, on increased mitochondrial proton leak in cardiac myocytes from aged rats and mice. The data are important contribution to the mechanism of action of SS-31, which shows great promise for cardiac aging and the interaction of SS-31 with ANT1. ANT1 is the major isoform of ANT expressed in the heart, and it has been shown previously to contribute to proton leak in cardiac mitochondria. The authors also analyze the effects of SS-31 on mito-flash and mitochondrial permeability transition pore opening. From the work presented here the authors conclude that proton leak is an important underlying mechanism of cardiac dysfunction in aging and provide evidence for the effect of SS-31 in modulating this effect.While the work was deemed interesting, however the manuscript should be improved by more clarity in the presentation of the data and their interpretation in general. There are several significant concerns that need to be addressed:Essential revisions:eLife showing that "SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts." They also demonstrated improved diastolic function. .1) This work does not appear to be a major advance beyond the author's recent publication in 2) Authors cannot claim that they have "identified ANT1 as mediating the increased proton permeability of old cardiomyocytes". ANT2 is also expressed, albeit at lower levels than ANT1, in the heart, and is also inhibited by CATR and BKA  – and thus could also contribute to the effects of SS-31. Authors state that Bertholet et al., 2019, found that ANT1 was the major site of proton leak; while this is true, they also found that ANT2 can contribute to leak, especially FA- induced leak. Thus authors cannot claim, as they have in the their summary statement that their "study reveals that ANT1 is responsible for the elevated proton leak in old cardiomyocytes and that SS-31 directly interacts with ANT1, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes." Indeed, data in Figure 1 show that SS-31 inhibition of proton leak is significantly less than the inhibition caused by BKA; BKA also inhibits ANT2.3) Moreover there are other mechanisms contributing to proton leak that could be relevant here: NNT, UCP3 and the inorganic phosphate carrier. See for example the recent work on NNT and its contributions to leak and ROS . Why have the authors not shown blots of ANT2 and other possible proteins involved? Regarding UCP2 blots, authors should show a positive control  in the western blots to convince readers that this band really is UCP2. The antibodies for UCP2 are very poor, and UCP2 protein expression is extremely low, if detectable at all in the heart.4) We felt that the authors over-interpreted their mt-cp-YFP results. Authors conducted these analyses in the absence of mitochondrial activity, and permeabilized the cardiomyocytes in a buffer that contained no substrates, ATP, or ADP. If authors had found effects under physiological conditions, this would be meaningful. Moreover, the control of the pH of the mitochondrial matrix is not via mitochondrial proton leak; there are many other ion transport mechanisms in the mitochondrial inner membrane and there are many mechanisms in the matrix itself. Authors need to conduct analyses under physiological conditions in the presence of energy substrates.5) Authors describe how oligomycin failed to inhibit proton leak, but it is well known that it does not inhibit proton leak. Authors need to revise this section.6) Authors use the well-known inhibitors of ANT, ie. CAT and BKA in non-permeabilized cells, and it is generally thought that they are not cell permeable (PMID: 26950698). Authors need to demonstrate that the inhibitors are traversing the cell membrane.7) Seahorse data for the single ventricular myocytes of rats and mice are expressed in units per 800 cells. How did authors assess the number of cells that actually adhered and thus that were actually measured in these assays?8) Why is MitoSOX normalized to mitochondrial content? Are there differences in mitochondrial content in the different experimental groups (old vs young)?9) Models are important for interpretation. The initial experiment specifies primary cardiac myocytes whereas subsequent studies indicate cardiac myocytes. I think it is important to know for sure what model was used.10) There are multiple statements of "rejuvenation" "restoring" "more youthful" etc. In a strict sense, the data compare young to old and to old+SS-31. These groups are similar or different. It is not possible to say that the a given group of cells changed with age and with treatment were made like their young self again as implied by the use of these words.11) How do the authors justify using t-tests? Within each experiment were multiple comparisons so t-tests do not seem justified.12) The presentation of the figures bounces around quite a bit and are not in sequence. This makes the study hard to follow. In addition, the figures lack detail such as what the abbreviations are and what the error bars are. Although these seem like minor quibbles, when added up, it made it difficult to assess the data.13) Why was normalization used in many of the assays? Was there a lot of variability at baseline? And if so, why?14) What was the justification for carrying out experiments in Figure 2 at pH 5.3? This pH does not seem relevant for general mitochondrial function. Do you see the same results at a pH close to actual mitochondria pH?15) The Discussion made some conclusions that were maybe a bit too much speculation. For example, the proton leak is identified as pathological, although this was not directly demonstrated in the study. Also, protein conformation was inferred as well.16) It should be emphasized that the proton leak studied is in myocytes in the resting, non-working state oxidizing endogenous substrates. The lack of a resting defect in OCR in this state is therefore plausible compared to the extensive literature describing key defects in OXPHOS with aging.17) The myocyte yield of the isolation procedure should be discussed. Are only the "best" aged myocytes obtained?18) The pH dependence of 405 excitation is of some concern. Also, calibration curves should be shown for pH 5.3 and 6.9; which are critical to the work.19) The proton leak shown is of interest since state 4 rates in isolated cardiac mitochondria have minimal change with age. This should be discussed and reconciled.20) The similarity of BKA and CAT to block proton leak is somewhat puzzling since the response of MPTP to the inhibitors is of course different. Is there evidence of MPTP opening in the permeabilized cells used for the measurement of mitochondrial pH with the buffer used as the artificial "cytosol"?21) The differences in Figure 2A are modest; the "normalized" data in Figure 2B and Figure 3C are more reasonable. How were fluorescence data normalized?22) The dynamic range and reproducibility of the cp-YFP assay should be described in greater detail. It seems puzzling that some groups apparently required n=19 for valid results.23) Is there a quality control assessment of mitochondrial inner membrane integrity in the permeabilized cells at low pH? This is critical to the conclusion of the study. Essential revisions:1) This work does not appear to be a major advance beyond the author's recent publication in eLife showing that "SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts." They also demonstrated improved diastolic function. .eLife we showed that chronic SS-31 8-week osmotic minipump treatment decreased the proton leak in aged mice and improved diastolic function. However, the mechanism for this effect was unclear; in fact, the long-term in vivo treatment could even have been produced by indirect compensatory changes, including proteomic or metabolic remodeling, as opposed to a possible direct effect on the cardiomyocyte. Moreover, the underlying mechanism of the excessive proton leak remained elusive. The present study showed that SS-31 directly and acutely decrease excessive proton leak; by directly evaluating the physical properties of the inner membrane in the absence of any active metabolic process we were able to determine the mechanism of the increased proton leak and how SS-31 prevents it in the aged mitochondria. We specified the novelties in the revision as “SS-31 acutely prevents the excessive mitochondrial proton entry”; “In this report we have shown direct evidence of increased proton leak in the aged mitochondria as a primary energetic disturbance and evidence that the increased proton entry in old cardiomyocytes takes place likely through ANT1. Moreover, we demonstrated that SS-31 prevents the proton entry to the mitochondrial matrix and rejuvenates mitochondrial function through direct interaction with the adenine nucleotide transporter and stabilization of the ATP synthasome.”; and “our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes and that SS-31 acutely and directly interacts with the transporter, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes”.We wish to respectfully disagree with this interpretation, as the difference between a functional observation (prior work) and elucidation of mechanism (present work) is large. In the recent publication in 2) Authors cannot claim that they have "identified ANT1 as mediating the increased proton permeability of old cardiomyocytes". ANT2 is also expressed, albeit at lower levels than ANT1, in the heart, and is also inhibited by CATR and BKA  – and thus could also contribute to the effects of SS-31. Authors state that Bertholet et al., 2019, found that ANT1 was the major site of proton leak; while this is true, they also found that ANT2 can contribute to leak, especially FA- induced leak. Thus authors cannot claim, as they have in the their summary statement that their "study reveals that ANT1 is responsible for the elevated proton leak in old cardiomyocytes and that SS-31 directly interacts with ANT1, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes." Indeed, data in Figure 1 show that SS-31 inhibition of proton leak is significantly less than the inhibition caused by BKA; BKA also inhibits ANT2.Together with points raised in item #3 below, we agree that the possibilities of other sites as the proton leak contributor, including ANT2, should be considered. However, of these, the crosslinking interactome data recently published by our collaborators identified only ANT1 as interacting with SS-31 (PMID: 32554501). Thus, we are more confident to say ANT1 is involved in the proton leak in the aged mitochondria. As we agree that we cannot with certainty rule out the others sites of proton entry, we have revised the Abstract “our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes” and the Discussion to state that “our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes”.3) Moreover there are other mechanisms contributing to proton leak that could be relevant here: NNT, UCP3 and the inorganic phosphate carrier. See for example the recent work on NNT and its contributions to leak and ROS . Why have the authors not shown blots of ANT2 and other possible proteins involved? Regarding UCP2 blots, authors should show a positive control  in the western blots to convince readers that this band really is UCP2. The antibodies for UCP2 are very poor, and UCP2 protein expression is extremely low, if detectable at all in the heart.Specific to inorganic phosphate carrier, we checked the potential involvement of the mitochondrial phosphate carrier (PiC) in proton entry by applying the pH stress to mitochondria in a buffer without phosphate. The proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria in buffer containing phosphate. This data is added as Figure 3—figure supplement 2, with the statement in the text “Considering that the inorganic phosphate carrier might be a potential source of the elevated proton entry in old mitochondria, we examined the proton permeability of old mitochondria to pH stress in a buffer that did not contain phosphate; in this buffer, the proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria stressed in the presence of inorganic phosphate .”Sreekumaran Nair lab demonstrated that the lack of age-related change in UCP2 transcript levels .With respect to UCP2, we confirmed the UCP2 WB in the heart samples with a UCP2 standard. The band we detected was confirmed as UCP2. This blot confirms the results now included as Figure 3—figure supplement 1E and referred to in the text. Moreover, our data is consistent with previous work from 4) We felt that the authors over-interpreted their mt-cp-YFP results. Authors conducted these analyses in the absence of mitochondrial activity, and permeabilized the cardiomyocytes in a buffer that contained no substrates, ATP, or ADP. If authors had found effects under physiological conditions, this would be meaningful. Moreover, the control of the pH of the mitochondrial matrix is not via mitochondrial proton leak; there are many other ion transport mechanisms in the mitochondrial inner membrane and there are many mechanisms in the matrix itself. Authors need to conduct analyses under physiological conditions in the presence of energy substrates.We strongly believe that performing the pH stress experiments in the absence of energy substrates is the clearest and most informative protocol. In the presence of energy substrates, the mitochondria will actively pump protons out of the matrix, making it more difficult and complicated to evaluate proton entry. Thus, the buffer without substrates allows us to evaluate the fundamental process of proton entry to the mitochondrial matrix. In this fashion, looking at the physical properties of the inner membrane in the absence of metabolic activity is highly informative and reduces many possible complications. Also, we do not claim that proton leak is the primary method of regulating pH, only that the age-related excessive leak is via ANT and is prevented by SS-31, and that this is correlated with improved behavior of MPTP opening, membrane potential and mitochondrial flashes in aged cardiomyocytes.5) Authors describe how oligomycin failed to inhibit proton leak, but it is well known that it does not inhibit proton leak. Authors need to revise this section.Thank you. We changed the description to “As expected, the ATPase inhibitor Oligomycin A failed to inhibit the proton leak”6) Authors use the well-known inhibitors of ANT, ie. CAT and BKA in non-permeabilized cells, and it is generally thought that they are not cell permeable (PMID: 26950698). Authors need to demonstrate that the inhibitors are traversing the cell membrane.We believe that this question is based on a misunderstanding – in the assay used in this report the plasma membrane is permeabilized by saponin. However, it may be of interest to point out that in cardiomyocytes both BKA  and CAT  have been shown to be effective in non-permeabilized cells. This is indeed in contrast to the report cited by the reviewer (PMID: 26950698) which found that BKA and CAT had a milder effect on intact T98G cells than permeabilized cells.7) Seahorse data for the single ventricular myocytes of rats and mice are expressed in units per 800 cells. How did authors assess the number of cells that actually adhered and thus that were actually measured in these assays?4/ml. Then the cells were diluted to 8000 cell/ml and 100ul (containing ~800 cells) were plated in each Seahorse well. We had previously coated the plate wells with laminin (50ug/ml) to allow the cells to adhere to the bottom of the plate. The cells were allowed to attach to the plate for at least 2 hours before changed the culture medium to Seahorse Assay medium without disturbing the cells. In initial experiments Seahorse wells were examined by microscopy to confirm plated numbers and equality between young and old cardiomyocytes. As expected, no differences were seen between young and old cells. This time-consuming secondary confirmation was not continued in later experiments.For the cell number determination, we counted the cell numbers using hemocytometer after cell isolation. Only the rod shaped cells were counted as live cells. Usually the starting cell concentration was ~4*108) Why is MitoSOX normalized to mitochondrial content? Are there differences in mitochondrial content in the different experimental groups (old vs young)?The reason that the ratio of MitoSOX to MitotrackerGreen is analyzed is to reduce the cell to cell variability in analysis, which is primarily due to differences in cell size and dye uptake between cells . This ratiometric approach is exceedingly common in flow and image cytometry. There were no significant differences seen in average Mitotracker Green intensity between old vs young cells.9) Models are important for interpretation. The initial experiment specifies primary cardiac myocytes whereas subsequent studies indicate cardiac myocytes. I think it is important to know for sure what model was used.We appreciate this reminder. All experiments were performed with primary cardiac myocytes. For pH stress and mitochondrial flash experiments, rat cardiomyocytes were used; mouse heart and cardiomyocytes were used in all other experiments. We have revised the language used in the manuscript to clearly note this accordingly.10) There are multiple statements of "rejuvenation" "restoring" "more youthful" etc. In a strict sense, the data compare young to old and to old+SS-31. These groups are similar or different. It is not possible to say that the a given group of cells changed with age and with treatment were made like their young self again as implied by the use of these words.We are grateful for bringing this to our attention. In the revised manuscript we are careful to state only that the phenotypes of the treated cells are closer to that of young cells.11) How do the authors justify using t-tests? Within each experiment were multiple comparisons so t-tests do not seem justified.Thank you for bringing this to our attention. We did the t-tests as analysis of differences between the young vs. old and between old vs. a single treatment. In this revision, we applied one-way ANOVA analysis to all multiple group comparisons then followed this with student t-tests to look at pairwise comparisons, allowing readers to see both results. We updated the statistical analysis in the Materials and methods part in this revision.12) The presentation of the figures bounces around quite a bit and are not in sequence. This makes the study hard to follow. In addition, the figures lack detail such as what the abbreviations are and what the error bars are. Although these seem like minor quibbles, when added up, it made it difficult to assess the data.Thank you for this observation, which we have now corrected. The Figure 3C which showed the method of 488/405 ratio slope calculation was previously placed after the slope statistical analysis of in Figure 2D, which was hard to follow. We moved Figure 3C to become new Figure 2—figure supplement 3 and mentioned this before the statement of Figure 2D to make it easier to follow. Also, we added the full spellings for the abbreviations. All the error bars are standard errors of the mean and this information is added to the figure legends.13) Why was normalization used in many of the assays? Was there a lot of variability at baseline? And if so, why?Thank you for this question. The reasons for each normalization are as following:For the pH stress assay, we normalized the cpYFP 488/405 ratio to the starting value. This is required due to inter-experimental variation in relative intensities of the two wavelengths due to variability in confocal laser powers, detector settings and efficiency.For the superoxide evaluation, we normalized the mitoSOX signal to the MitotrackerGreen signal. The advantage of this normalization is described in response to query number 8, above.For the biotin-SS31 pulldown ANT1 blot data, we normalized bands to the Biotin-SS31 group in each experiment. This eliminated the variation due to differences in sample loading amounts and blotting procedures.For the ANT1 and UCP2 Western Blot data, we normalized the blotting bands to the total protein content in that lane, as detected by reversible protein stain. This eliminated variation from differences in sample loading amounts.14) What was the justification for carrying out experiments in Figure 2 at pH 5.3? This pH does not seem relevant for general mitochondrial function. Do you see the same results at a pH close to actual mitochondria pH?We appreciate this question. The mitochondrial intermembrane pH is around 6.9. While we did see a similar phenotype for proton entry at pH 6.9 , differences between proton permeability of young vs old inner membranes and between SS-31 treated and untreated were larger and less variable at pH 5.3. We believe that the observations at pH 5.3 are more clearly indicative of physical chemical differences in inner membrane permeability, in spite of this being a non-physiologic pH.15) The Discussion made some conclusions that were maybe a bit too much speculation. For example, the proton leak is identified as pathological, although this was not directly demonstrated in the study. Also, protein conformation was inferred as well.We thank the reviewer for this comment and have revised the language used. We have restricted the use of the term pathological to exclusively refer to previous observations of diastolic dysfunction of the aged hearts, which is not a healthy condition.The conformational states in the ANT pore conferred by BKA and CAT treatment are based on prior work by others: PMID: 30611538 and PMID: 14603310, which are generally well accepted. We have changed the wording to make this reference clearer.16) It should be emphasized that the proton leak studied is in myocytes in the resting, non-working state oxidizing endogenous substrates. The lack of a resting defect in OCR in this state is therefore plausible compared to the extensive literature describing key defects in OXPHOS with aging.Thank you for this constructive suggestion. We emphasized the resting and non-working myocyte states in both the Materials and methods and Results sections.17) The myocyte yield of the isolation procedure should be discussed. Are only the "best" aged myocytes obtained?Each experiment, only preparations having at least 80% rod shape cells (which means live cardiomyocytes) were used. Thus, while we cannot completely exclude the possibility that there was some selection present, this would have to have been a small effect that could not have appreciably affected the reported results. For the aged myocytes, we did observe that the cells were on average larger, compared with the young cells, as has been previously documented by imaging of heart tissue sections by us and others.18) The pH dependence of 405 excitation is of some concern. Also, calibration curves should be shown for pH 5.3 and 6.9; which are critical to the work.Thank you for this suggestion. We added the pH calibration results of both 488 and 405 excitations in Figure 2—figure supplement 1. Also, we replaced the previous pH 4.5 and 6.0 panels with the calibration results at pH 5.3 and 6.9 in Figure 2—figure supplement 1.19) The proton leak shown is of interest since state 4 rates in isolated cardiac mitochondria have minimal change with age. This should be discussed and reconciled.An accurate State IV is difficult to achieve since there may be contaminating ATPases in the crude mitochondrial preparation which convert newly formed ATP back to ADP, and thus prevent a true “ADP exhausted” state. Thus, most investigators use oxygen consumption in the presence of oligomycin as a surrogate of state IV. This is the parameter that we have previously shown to be elevated in aged cardiomyocytes and reverse by SS-31 (PMID: 32648542).20) The similarity of BKA and CAT to block proton leak is somewhat puzzling since the response of MPTP to the inhibitors is of course different. Is there evidence of MPTP opening in the permeabilized cells used for the measurement of mitochondrial pH with the buffer used as the artificial "cytosol"?The discrepancy of BKA and CAT effect on proton leak and mPTP opening is reasonable, as ANT is only a regulator of the mPTP, while the ATPase itself, especially the "c" ring is thought by many to be the pore of the mPTP. Thus, while we hypothesize that both BKA and CAT change the conformation of the ANT pore in such a way as to inhibit proton entry, only one of these conformational states regulates the mPTP to slow its opening. In fact, this difference is a strong argument against the mPTP pore itself being the source of excessive proton leak in old cardiomyocytes, as CAT, which has no delay on mPTP opening times , would not be expected to alter proton permeability, which it does.21) The differences in Figure 2A are modest; the "normalized" data in Figure 2B and Figure 3C are more reasonable. How were fluorescence data normalized?The differences seen by viewers in the fluorescence image are dependent on the observer’s display settings (for example γ) and may vary among viewers. On our displays and printed copies, the variation in intensities appear consistent with the quantified data, but the reviewer’s comment is an important reason for not relying on the visual image. To quantify the fluorescence intensity we first subtract the signal intensities in the background that does not contain any cell . Then, for each image area that contains a cell  the integrated fluorescence value at the 488nm detection channel is divided by the corresponding value at from the 405nm detection channel. We set the ratio of 488/405 at the beginning as one (see query 13 above) and all later values are normalized to this starting value.22) The dynamic range and reproducibility of the cp-YFP assay should be described in greater detail. It seems puzzling that some groups apparently required n=19 for valid results.Thank you for the suggestion. We put the cp-YFP assay spectral characteristics at pH 5.3 in the Figure 2—figure supplement 1. The reproducibility of the data comparing old vs. young cells and old SS-31 treated cells at pH 5.3 for example , yields a mean of 0.59, a SD of ± 0.10 and a range of 0.46 to 0.77 for the old compared to the starting pH 7.5 value. This range is non-overlapping with the mean of either young or old-SS-31 treated cells at pH 5.3. This is added to the Figure 2C legend to give a better understanding of the range and reproducibility of the assay.The number n=19 arises because we used the results of multiple experiments to validate and confirm the data. It does not require a sample size of 19 to reach a significant difference in the statistical test.23) Is there a quality control assessment of mitochondrial inner membrane integrity in the permeabilized cells at low pH? This is critical to the conclusion of the study.As the mt-cpYFP indicator  is still held within the mitochondrial matrix, this indicates that at the very least, the inner membrane does not break down enough to let this size molecule escape. Furthermore, and more to the point, while the reviewer is correct in thinking that a generalized breakdown of inner membrane integrity would prevent an informative answer as to the source of proton permeability, the fact that SS-31, BKA and CAT prevent excess proton permeability is a very strong indication that there is not a general loss of inner membrane integrity.


Preterm birth (PTB) can be defined as the endpoint of a complex process that could be influenced by maternal and environmental factors. Epigenetics recently emerged as an interesting field of investigation since it represents an important mechanism of regulation. This study evaluates epigenetic impact of preterm birth on DNA methylation. Genome-wide DNAm was measured using the Illumina 450K array in cord blood samples obtained from 72 full term and 18 preterm newborns. Lymphocyte composition was calculated based on specific epigenetic markers that are present on the 450k array. Differential methylation analysis was performed both at site and region level; moreover, stochastic epigenetic mutations (SEMs) were also evaluated. The study showed significant differences in blood cell composition between the two groups. Moreover, after multiple testing correction, statistically significant differences in DNA methylation levels emerged between the two groups both at site and region levels. Results obtained were compared to those reported by previous EWAS, leading to a list of more consistent genes associated with PTB. Finally, the SEMs analysis revealed that the burden of SEMs resulted significantly higher in the preterm group. In conclusion, PTB resulted associated to specific epigenetic signatures that involve immune system. Moreover, SEMs analysis revealed an increased epigenetic drift at birth in the preterm group. Preterm birth (PTB) is defined as birth before 37 weeks of pregnancy. According to the World Health Organization the prevalence of PTB is estimated to be in a range from 5% to 18% in singleton pregnancies . Severalp-value = 0.006, p-value = 0.02 and 0.02 respectively). Results have been represented as boxplots in Clinical and phenotypic traits of pregnancy were examined in order to have an overview of the study population and to detect differences between the two groups analyzed. Results have been shown in The thick horizontal line represents the median of the distribution, while the box represents the interquartile range. Whiskers are set as the default option for boxplot function and extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Open circles represent outliers .Dimensional reduction was used to visually inspect the dataset for strong signals in the methylation values. The MDS was adopted considering methylation signals from sites and also considering genomic regions: CpG islands, promoters, genes, and tiling. Results have been reported in p-values considering multiple testing, significant differences in methylation levels between cases and controls emerged at site level. The list of probes which resulted differently methylated is reported in Differential methylation analysis was computed both at site and at region level. Blood cells composition, as well as the batch, were used as covariates to adjust the differential methylation analysis. Moreover, a surrogate variables (SV) analysis was also applied in order to correct for potential unmeasured or unmodeled confounders. After FDR adjustment of the NCOR2 and PLCH1, have been confirmed by all the studies and five genes, FOXK1, WWTR1, RASA3, PRR5L, and IGF2BP1 have been confirmed by five studies. The complete list of these genes and the genomic position involved is shown in In order to evaluate consistency among studies, results obtained in the present study were compared to those reported by five previous published EWAS that analyzed PTB. This analysis showed that among 2408 significant genes reported by six studies only 389 genes (16.1%) have been confirmed by at least two studies while 99 (4.1%) genes have been confirmed by at least three studies. The upset plot in In order to focus the analysis on a more consistent list of markers, the gene ontology classifications analysis was performed considering the list of genes confirmed by at least three studies. The result of the gene ontology analysis has been presented as TreeMap in p = 3.1 × 10−3). Moreover, the regression model showed also that both hypo and hypermethylated SEMs resulted in significantly higher in the group of preterm babies (p = 3.7 × 10−3 and 7.6 × 10−3 respectively). The median number of hypomethylated log(SEMs) was 6.3  in preterm group and 5.2  in the full term group while the median number of hypermethylated log(SEMs) was 5.8  in preterm group and 5.2  in the full-term group. Results are shown in For each subject, the burden of SEMs was calculated as described in the “Methods” section and reported in logarithmic scale. Furthermore, the burden SEMs was also divided into hypo or hypermethylated groups and compared between preterm and full-term groups. The median number of total log(SEMs) was 6.67  in preterm group and 6.0  in the full-term group. Considering the reduced sample size and the unbalanced number of cases and controls, Firth’s logistic regression model was adopted to evaluate differences in the number of SEMs between preterm and full-term babies. The Firth’s logistic regression model, considering sex, blood cellular composition and batch effect as potential confounders, indicated that this difference was significant  identification of genes that resulted epigenetically dysregulated in PTB babies in an Italian cohort; (ii) the replication of PTB-associated genes across different studies and ethnicities and identification of a reduced and robust epigenetic signatures of PTB; (iii) the identification of significant differences in blood cell composition between the two groups analyzed; (iv) the identification of a significant increase in SEMs burden at birth in PTB babies. NCOR2 and PLCH1, were replicated by all the studies, and five genes, FOXK1, WWTR1, RASA3, PRR5L, and IGF2BP1 were confirmed by at least five studies. This represents an interesting and robust list of epigenetic markers of PTB. The NCOR2 gene encodes a nuclear receptor co-repressor that mediates transcriptional silencing of certain target genes and it is a co-repressor of glucocorticoid receptor signaling pathways [NCOR2 could be disease causative [PLCH1 gene encodes an isoform of phospholipase C and plays a role in inositol signaling pathway. Notwithstanding, this gene has been reported by several studies to be differently methylated in preterm cord blood samples; at this time its role in PTB still remains largely unexplored. However, a recent paper reported that PLCH1 expression can be modulated during toxoplasma gondii infection [FOXK1 gene is a transcription factor of the forkhead box family . Hypomethylation and underexpression of FOXK1 has been recently reported to be associated with intrauterine growth restriction [WWTR1 gene is a transcriptional co-activator which acts as a regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control. Recently, it has been reported in a list of genomic biomarkers of prenatal intrauterine inflammation in umbilical cord tissue [RASA3 gene encodes a protein that binds inositol 1,3,4,5-tetrakisphosphate and stimulates the GTPase activity of Ras p21. Interestingly, recent findings showed a relation among prenatal exposure to air pollutants, alteration in RASA3 DNA methylation and PTB [PRR5L with respect to pregnancy is unknown, PRR5L is a key regulator of cellular mTORC2 in vitro, which in turn is regulated by lysophosphatidic acid (LPA) activity [LPA is implicated in the maintenance of pregnancy [IGF2BP1 encodes a member of the insulin-like growth factor 2 mRNA-binding protein family. There are several studies supporting a role of this gene in PTB describing a role of environment in its epigenetic regulation [G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) [The analysis of consistency among previous published studies was important and highlighted a relevant inconsistency among results reported so far. Studies that investigated gestational age in physiological condition were excluded from our analysis in order to reduce heterogeneity. Five studies with similar case control designs met our inclusion criteria and were selected for the analysis. We observed that these studies reported different lists of genes that to a large extent were not consistent among studies. The percentage of completely unconfirmed genes (genes that were reported only by one study) was 50% in the present study, 82.5% in the study by Knijnenburg et al., 71.5% in the study by Cruickshank et al., 64.5% in the study by Febilla et al., 34% in the study by Wu et al., and 47% in the study by Wang et al. The percentage of genes confirmed by at least two studies was only 16.1%. The identification of this inconsistency among results is interesting and reveals the presence of an important heterogeneity. A part of this heterogeneity might be due to statistical issue such as a reduced sample size and statistical power. However, we can speculate that a part of the observed heterogeneity might be due to biological differences and presence of hidden pregnancy factors able to modulate DNA methylation. It is well known for example that a great number of maternal and paternal characteristics, environmental and behavioral exposure factors can significantly modulate the epigenome of the newborn . Moreovepathways . Interesausative . PLCH1 gnfection  and thisnfection . FOXK1 gtriction . WWTR1 gd tissue . RASA3 g and PTB . Althougactivity . LPA is regnancy , uterineregnancy , and infregnancy . Finallygulation . Differegulation . Results(GM-CSF) . FurtherThe analysis of SEMs represents an innovative approach in the field of PTB and showed a significant increase in the burden of SEMs in the PTB group. SEMs have been recently defined as a biomarker of epigenetic drift and as an indicator of exposure-related accumulation of DNA damage. Moreover, SEMs have been reported to increase exponentially during life and to be associated to several pathological conditions. This result is interesting if considering that a great deal of scientific evidence indicates that survivors of PTB are more susceptible to several early on-set chronic diseases , includiA number of limitations need to be considered when interpreting our results. First, only DNA from cord blood was analyzed. Since cord blood is not necessarily representative of the epigenetic status of all tissues and cells in the newborn, additional studies on other tissues are mandatory to confirm these data. Second, the phenotypic data of the studied subjects did not include all the possible variables related to pregnancy and labor although a surrogate variables analysis was conducted to compensate for this deficiency. Third, although the number of subjects used for comparisons was of the same order of magnitude of previously conducted EWAS, we must underline that a larger sample size could better estimate normal ranges of DNA methylation in each locus. Fourth, the studies selected for the consistency analysis were similar in terms of study design and methodology. We adopted this inclusion criteria in order to reduce heterogeneity among studies; however, we cannot exclude that the list of genes confirmed by the great part of studies might be the result of persistent technical artifacts. Future studies adopting different methodological strategies are mandatory to confirm these findingsn = 72 full-term infants , while n = 18 babies qwerw enrolled in the cases group if born spontaneously before the 37th gestational week. Samples of cord blood from both full-term pregnancies and preterm pregnancies were obtained at the time of delivery by the midwives of the Macedonio Melloni Hospital, Milano, Italy, by puncturing the umbilical vein while the placenta was in utero [This is a retrospective observational case control study. The control group consisted of in utero . The excin utero . Mean anp-value was not below the threshold (t = 0.05). In order to avoid an erroneous interpretation of stochastic epigenetic variations, probes with coordinates overlapping rare genetic variants annotated in 1000 Genomes and EXAC databases were removed [Illumina Methylation 450K raw data were analyzed using the RnBeads 2.0 analysis software package . Sites w removed . After t removed . The sigProportions of CD8 T cells, CD4 T cells, NK cells, B cells, monocytes, and granulocytes were estimated using the “estimateCellCounts” function in the Bioconductor “minfi” package , with thp-values were computed using the limma method for the site level analysis while a combined p-value was calculated from all site p-values for the region-based [Differential methylation analysis was conducted both at site and region level according to the sample groups. The on-based . Regionson-based :http://www.ensembl.org/index.html) gene definitions were downloaded using the biomaRt package. A promoter was defined as the region spanning 1500 bases upstream and 500 bases downstream of the transcription start site of the corresponding gene.Genes and promoters: Ensembl (http://genome.ucsc.edu/).GpG islands: the CpG island track was downloaded from the dedicated FTP directory of the UCSC Genome Browser  without adjustment; (ii) adjustment for batch and cell composition; (iii) adjustment for batch, cell composition and SVA. Results obtained after SVA adjustment were similar to those obtained considering only batch and cell composition as covariates. However, the strategy of also adopting the SVA adjustment was applied based on two main considerationsL (i) based on the consciousness that there are a great number of hidden potential confounding factors in pregnancy that can lead to spurious results; (ii) application of SVA adjustment had no effect on the final list of genes obtained by intersection with previous studies. SV was conducted using the function directly provided in the package RnBeads.https://toppgene.cchmc.org/help/publications.jsp).We conducted a systematic search of the literature focusing on studies that used a genome-wide approach  with the aim to compare cord blood methylation levels between full term and preterm babies. In order to reduce heterogeneity, only studies with similar study design and methodology were selected. Studies that investigated DNA methylation modifications associated with gestational age and not with a defined preterm/full term case control study design and studies that evaluated methylation by different experimental approaches were excluded. The five selected studies ,13,14,15In order to identify SEMs, we used a method previously described by our group ,32,37. BIn order to confirm the power of this analytical approach to detect epigenetic variations, two separate tests were performed on positive controls.p < 0.01) among the experiments. The duplicate samples underwent independent bisulfite conversion reaction and this suggests that epigenetic variations are not significantly influenced by bisulfite conversion errors. In the second validation step, 48 whole blood DNA samples obtained from subjects affected by imprinting diseases  who underwent diagnostic assays at Istituto Auxologico Italiano were analyzed. For these subjects, a medical report indicating the genomic position of their epigenetic alteration was already available. Briefly, after the identification of the outlier’s probes, a test for over-representation of these probes inside each gene was performed using the hypergeometric cumulative function. The analysis identified genes with enriched number of outlier’s probes  confirming the presence of the epigenetic alterations previously reported in the medical report.Three samples were analyzed in duplicates and epigenetic variations found in each of them were compared. Results showed a mean correlation of 0.99  setting the parameter “Allowed similarity” to Medium (0.7) and referring to the UniProt-to-GO mapping file [MDS charts and scatterplots were produced by “ggplot2” and “graphics” packages in R, respectively. The clustering of GO biological processes was visualized using the tool ReviGO (ing file . Upset ping file In conclusion, in the present study, we compared cord blood DNAm profiles of preterm and full-term babies. We analyzed our results together with previously published findings and obtained a more consistent list of epigenetic markers of PTB. The study described a high heterogeneity among studies and confirmed differences at immune system levels. For the first time, we reported that PTB babies have an increased number of SEMs at birth. This finding might explain why PTB babies have a higher risk of developing a vast array of chronic diseases in adult age.


Tac1, the gene encoding substance P. Finally, we show that silencing Tac1-expressing cells in the dorsal horn results in a significant reduction in reflex responses to cold and radiant heat, but does not affect withdrawal to von Frey hairs, or chloroquine-evoked itch.The tachykinin peptide substance P (SP) is expressed by many interneurons and some projection neurons in the superficial dorsal horn of the spinal cord. We have recently shown that SP-expressing excitatory interneurons in lamina II correspond largely to a morphological class known as radial cells. However, little is known about their function, or their synaptic connectivity. Here we use a modification of the Brainbow technique to define the excitatory synaptic input to SP radial cells. We show that around half of their excitatory synapses (identified by expression of Homer) are from boutons with VGLUT2, which are likely to originate mainly from local interneurons. The remaining synapses presumably include primary afferents, which generally have very low levels of VGLUT2. Our results also suggest that the SP cells are preferentially innervated by a population of excitatory interneurons defined by expression of green fluorescent protein under control of the gene for gastrin-releasing peptide, and that they receive sparser input from other types of excitatory interneuron. We show that around 40% of lamina I projection neurons express  It has ransport , with soransport . Recent ransport . In a seransport , we haveransport . In addiransport .Although Lu and colleagues  have ideAll experiments were approved by the Ethical Review Process Applications Panel of the University of Glasgow, and were performed in accordance with the European Community directive 86/609/EC and the UK Animals (Scientific Procedures) Act 1986.Cre line  and the other was the BAC transgenic Tg(Grp-EGFP)DV197Gsat in which eGFP is expressed under control of the GRP promoter  were anaesthetised with isoflurane and received intraspinal injections of one of the AAV Brainbow vectors  received injections of AAV-BB2. These were performed as described above, except that in this case injections were made into the dorsal horn of both L3 and L5 segments on one side. As before, the mice survived for 14 days and were then fixed by vascular perfusion. Parasagittal sections were cut from the injected segments and reacted with primary antibodies against TFP, eGFP, Homer and VGLUT2 , with the aperture set to less than 1 Airy unit. Z-series (0.3 μm z-separation) were obtained from as much of the dendritic tree as was visible in the section. The resulting scans were analysed with Neurolucida for Confocal software . The TFP and Homer channels were initially viewed. The cell bodies and dendritic trees were drawn, and the locations of all Homer puncta associated with the cell body or with dendritic shafts or spines were plotted. The VGLUT2 channel was then viewed and we noted whether or not a VGLUT2-immunoreactive bouton was apposed to each Homer punctum. Finally, the remaining channel  was revealed and the presence or absence of staining was noted for each of the VGLUT2 boutons that contacted a Homer punctum on the selected cell. To determine the frequency of all boutons arising from excitatory interneurons that were positive for each of these neurochemical markers, we sampled from those VGLUT2-immunoreactive boutons in the vicinity of the TFP-labelled cell. A 4 × 4 μm grid was applied within a box drawn to include the entire dendritic tree of the cell. Only the VGLUT2 channel was viewed initially and in each successive grid square, the VGLUT2-immunoreactive bouton nearest the bottom right of the square was selected. The presence or absence of NKB, neurotensin, SP or GRP-eGFP was then recorded for each of these selected VGLUT2-immunoreactive boutons. Although unmyelinated primary afferents express VGLUT2 , the expCre mice to reveal neurons that express SP  were anaesthetised with isoflurane and placed in a stereotaxic frame. They received a single injection of 300 nl 1% CTb targeted on the left LPb, as described previously  from the lateral parabrachial area (LPb), which labels the vast majority of these cells , with inpress SP . Two Taceviously . Immediaeviously . Each moeviously . Spinal Sections through the spinal injection sites were scanned with the confocal microscope through a 20× dry lens  to generate z-stacks (1 μm z-separation) of the superficial dorsal horn through the full thickness of the section. These were analysed with Neurolucida for Confocal software. Initially, the region of dorsal horn corresponding to the injection site was defined, by examining the mediolateral distribution of eGFP-positive cells. All CTb-labelled lamina I neurons within this region were then identified, and the presence or absence of eGFP was noted for each cell.Cre mice of either sex . Intraspinal injections were performed as described above, and all animals received three injections targeted on the L3, L4 and L5 segments on the right side  or control  groups. In the initial 8 TeLC experiments, we used a vector that coded for TeLC and Flag-tag (AAV.flex.TeLC-Flag). However, we were unable to visualise the Flag-tag by immunohistochemistry, and for the last 4 experiments we used a vector that coded for both TeLC and eGFP (as separate proteins).Experiments to test the effect of silencing SP cells were performed on 23 Tac1ght side . Injectight side , and aniBehavioural tests were performed before and after spinal injections. All tests and analyses were performed blind to the treatment type. Baseline tests  were performed 4–5 days before spinal injection, and these tests, together with a test for pruritogen-evoked itch, were repeated between 8 and 23 days after surgery. Mechanical sensitivity was tested with von Frey hairs. Animals were acclimatised for 1 h in a cage with a wire mesh floor and then tested by application of filaments with logarithmically incremental stiffness (starting at 0.4 g) to the glabrous skin of the hindpaw. Each filament was applied for 5 s, and the presence or absence of a withdrawal response was noted. Depending on the response, the filament with the next incremental stiffness was tested. Testing continued until a series of six filaments had been applied from the point when the response threshold was first crossed. The 50% withdrawal threshold was determined by the up-down method . Thermal−5 M (2. It detects SP and peptides containing at least the last 6 amino acids (Gln-Phe-Phe-Gly-Leu-Met-NH2), but does not cross-react with either neurokinin A or NKB (manufacturer's specification). The CTb antibody was raised against the purified protein, and specificity is demonstrated by the lack of staining in regions that did not contain injected or transported tracer.The sources and dilutions of primary antibodies used in the study are listed in −5 M . The neuFor the analysis of synaptic inputs to the SP cells from different neurochemical populations of excitatory interneurons, comparisons between the proportion of VGLUT2 boutons synapsing on the cell and those in the vicinity of the cell that were positive for each marker were made using Wilcoxon matched pairs tests. Mann-Whitney U test was used to compare the proportion of input arising from VGLUT2-positive boutons to SP and GRP-eGFP cells.post hoc when ANOVA showed significant differences in the main effects or interactions (p < 0.05).For the analysis of behavioural data in the studies involving synaptic silencing we used 2-way ANOVA (itch and Rotarod) or 3-way ANOVA (nocifensive reflex tests) followed by Tukey's multiple comparisons test The amount of Brainbow virus injected in these experiments was intentionally kept very low, such that the number of labelled cells was limited, and the native fluorescence within these cells was barely detectable. Immunostaining for TFP resulted in the appearance of scattered immunoreactive cells, with a laminar distribution that was similar to what we had previously observed . The celCre mice and 3 each from 3 Tac1Cre;GRP::eGFP mice) and details of the dendritic lengths and the total number of Homer puncta associated with each cell are given in p < 0.0001, Thirty-six TFP-labelled cells were reconstructed in full .Nine SP cells were analysed from 3 Tac1positive . For comCre mice, 9 of which were from sections that had been reacted with each neuropeptide antibody  (p = 0.018), suggesting that SP boutons tend to avoid other SP cells. In the case of the other two peptides, there was no significant difference , although there was a trend for neurotensin to be more frequently present in boutons synapsing on the SP cells than in nearby VGLUT2 boutons.We analysed 27 cells in the Tac1in, NKB) , Table 3Cre mice completely filled the parabrachial area in each case, with some extension into nearby regions, including the caudal part of the periaqueductal grey matter , but there was no difference in the response between the mice that had received intraspinal injections of TeLC or eGFP  that synaptic silencing of dorsal horn SP neurons causes a significant reduction in thermal nocifensive reflexes.Our main findings are: (1) that approximately half of the Homer puncta on the SP cells in lamina II are associated with VGLUT2-immunoreactive boutons, which originate mainly from local interneurons, (2) that axons of GRP-eGFP cells are significantly over-represented among these boutons, (3) that around 40% of lamina I spinoparabrachial neurons express Cre in adult Tac1We adapted the viral Brainbow method , by usinAs reported previously , the SP We found that GRP-eGFP axons consistently accounted for around one third of the VGLUT2-positive boutons that innervated the SP cells, but for only around 10% of the nearby VGLUT2 boutons. We have previously shown that the SP and GRP-eGFP cells in lamina II largely correspond to two of the classes defined by Many SP interneurons in lamina II have been shown to express enkephalin , and thiIt should be noted that although the approach that we used can reveal the presence of synapses between different dorsal horn neurons, electrophysiological methods, for example optogenetics, will be required to demonstrate the function of these synapses.Cre mouse. Cre mice were crossed with a tdTomato reporter line. Since most lamina I cells with high levels of NK1r are ALT neurons  are Tac1-positive and contain the precursor protein PPTA, but do not have detectable levels of SP in their axonal boutons, possibly due to lack of the enzyme peptidylglycine alpha-amidating monooxygenase, which is required for maturation of certain biologically active peptides  and projection cells in the SDH, as well as some neurons in deeper laminae, and it is not possible to determine which of these cell types are responsible for the behavioural effects that we observed. Only around 1% of SDH neurons are projection cells  and we hCre mice . We ther


Only 105 (49.8%) patients who met the indications for OAC were actually placed on OAC post-PCI. There was no significant relationship between discharge on OAC and HAS-BLED score  or CHA2DS2-VASc score . Patients younger than 65 years of age were prescribed more triple therapy  or any OAC  on discharge in comparison with patients 65 years of age or older. The older patient group had a significantly higher average CHA2DS2-VASc score  and a higher average HAS-BLED score . Ultimately, this study indicated that less than half of AF patients with an indication for OAC were placed on OAC post-PCI. There was no association between discharge on OAC and CHA2DS2-VASc score, HAS-BLED score, or any other individual risk factor, with the exception of age.A common dilemma facing physicians treating patients with atrial fibrillation (AF) who have undergone percutaneous coronary intervention (PCI) is the management of oral anticoagulation (OAC) therapy, because there is also an indication for dual antiplatelet therapy in these patients. The purpose of this study was therefore to evaluate anticoagulation patterns in this patient population in an attempt to identify patterns of risk factors that may influence OAC prescribing habits. This retrospective study entailed a review of a total of 4,648 patients from two academic hospitals who underwent PCI between 2008 and 2016. We ultimately included 211 patients who had AF and an indication for OAC. Chart review revealed patients’ risk factors, CHA Meanwhile, percutaneous coronary intervention (PCI) is one of the most commonly performed cardiovascular procedures, with more than 500,000 PCI procedures performed annually in the United States alone.2 As a result, a common dilemma faced by many physicians is the management of AF and oral anticoagulation (OAC) in post-PCI patients who also require dual antiplatelet therapy (DAPT).Atrial fibrillation (AF) is the most common sustained arrhythmia,2DS2-VASc and HAS-BLED scores, which stratify thromboembolic risk and bleeding risk, respectively.4 The 2013 What is the Optimal Antiplatelet and Anticoagulant Therapy in Patients With OAC and Coronary Stenting (WOEST) trial provided some insight into the safety of the two strategies by randomizing patients with AF undergoing PCI to triple therapy (OAC and DAPT) versus double therapy (OAC and clopidogrel).5 Results from this investigation indicated that double therapy was associated with a significant reduction in bleeding complications and led to a class IIb recommendation in the 2014 American Heart Association/American College of Cardiology/Heart Rhythm Society (AHA/ACC/HRS) AF guidelines, where it was noted that it may be reasonable to use double instead of triple therapy in this specific patient group.6A lack of strong evidence and consensus regarding the optimal combination of therapies has led to many different philosophies and treatment strategies, including some based on CHA8 In 2018, a meta-analysis comparing double and triple antithrombotic therapy in patients with AF who underwent PCI found roughly similar rates of thromboembolism but a 47% increase in thrombolysis in myocardial infarction major or minor bleeding for patients receiving triple therapy.9 Although a number of investigations have attempted to identify a regimen that can maximize thromboembolic and stent thrombosis prophylaxis while minimizing bleeding risk, the lack of corroborating evidence has largely left physicians with underwhelming guidance.Several other studies including Safety and Efficacy of Six Months Dual Antiplatelet Therapy After Drug-eluting Stenting (ISAR-SAFE) and Triple Therapy in Patients on OAC After Drug-eluting Stent Implantation (ISAR-TRIPLE) have suggested that a shorter duration of DAPT after PCI may be acceptable and could help to reduce bleeding events.10 This leads to a phenomenon of suboptimal prescription of OAC in patients with AF11 despite overwhelming evidence demonstrating associated benefits.17 Given the suboptimal rate of anticoagulation of AF patients on the whole, it is reasonable to suspect that the subset of patients who are also undergoing PCI would similarly have a suboptimal rate of anticoagulation.Regardless of the data presented, however, practitioners and patients alike are biased in their decision-making when it comes to anticoagulation in AF.In the present research, we reviewed the prescribing habits for patients with AF undergoing PCI to dissect the potential reasons for this and to identify patterns of risk factors that may influence anticoagulation prescribing habits. Our hypothesis was that there would be a correlation between stroke OAC prescription and risk, bleeding risk, or certain other risk factors.From January 2008 to April 2016, 4,648 PCIs were performed at the Los Angeles County and University of Southern California Medical Center and the Keck Hospital of the University of Southern California. A retrospective chart review was performed for each patient with special attention paid to medications and problem lists on admission and discharge. This evaluation revealed 224 patients with AF undergoing PCI and appropriate documentation of their antithrombotic regimen on admission and discharge. We then were able to create a database of these patients including their demographics, medication lists, and risk factors.2DS2-VASc score was calculated for each of the 224 patients to establish indications for OAC. Thirteen patients did not meet indications for anticoagulation and were given a score of less than two points. Of the remaining 211 patients, a HAS-BLED score was calculated to assess bleeding risk. The antithrombotic regimen of each patient on discharge was evaluated to assess for correlation with CHA2DS2-VASc score, HAS-BLED score, individual thromboembolic risk factors, or antithrombotic regimen prior to PCI, respectively. An independent samples t-test was conducted to compare CHA2DS2-VASc and HAS-BLED scores for discharge regimens with and without OAC. A chi-squared test was used to assess the relationship between discharge antithrombotic regimen and each individual risk factor.The CHA. Patient characteristics and demographics are summarized in Table 1. Of the patients who received OAC, six specifically were placed on direct OAC (DOAC) therapy.Of the 211 AF/PCI patients who met indications for OAC, 105 (49.8%) were placed on OAC post-PCI. Overall, 97 (46.0%) patients were placed on DAPT only, 85 (40.3%) were given triple therapy, and 19 patients (9.0%) were given a combination of OAC and a single antiplatelet agent 2DS2-VASc scores  or HAS-BLED score . These results suggest that neither CHA2DS2-VASc score  nor HAS-BLED score have an effect on whether or not patients were discharged on OAC. Regarding the individual risk factors, no association was found between discharge on OAC and congestive heart failure, systemic hypertension, vascular disease, female gender, or diabetes mellitus . Although there was a trend toward association, a history of stroke or transient ischemic attack was also not significantly associated with discharge on OAC (p = 0.14).There was no significant difference between discharge regimens with and without OAC for either CHA2DS2-VASc score  and a significantly higher HAS-BLED score . Expectedly, if a patient was admitted on OAC, they were more likely to be discharged with OAC (p < 0.01).Patients who were younger than 65 years of age (average age: 57.9 years) were more likely to be anticoagulated than patients who were 65 years of age or older (average age: 76.2 years). Furthermore, the younger patients were prescribed more triple therapy  or any OAC  on discharge. Conversely, the older patient group had a significantly higher average CHA18 The mainstay of stroke risk management remains anticoagulation; however, the inherent risk of bleeding is a concern that has consistently led to the inconsistent management of cerebrovascular accident (CVA) risk. Nowhere is this more evident than in those patients with AF who are post-PCI and who require DAPT and OAC.The most feared and debilitating complication of nonvalvular AF is stroke. The rate of stroke in this population averages approximately 5% per year, or two to seven times the rate in those without nonvalvular AF.2DS2-VASc score, or HAS-BLED score correlated with the decision to discharge patients on OAC. In fact, the only two factors that had a statistically significant association with OAC on discharge were whether a patient was already on OAC prior to PCI and if they were younger than 65 years of age. These findings are in line with what’s expected in a community of physicians who lack a consensus on how to manage such individuals. Moreover, these findings seem to confirm that physicians continue to base decisions on clinical gestalt rather than on strong data supporting OAC in the context of either triple or double therapy. This is due in part to weak direction from guidelines.The current study has shown that only half of the study participants who met the indication for OAC for AF following PCI were actually placed on OAC. We found no correlation when we delved into whether clinical risk factors such as a history of CVA, CHA19 A review by Bungard et al. in 2000 found that physicians generally base their decision to anticoagulate older AF patients on the perceived risk of embolism and hemorrhage.10 Some surveyed physicians, however, noted that they have withheld anticoagulation in patients based on the belief that patients may refuse treatment or may not be compliant, although there is evidence that, when given the choice, most patients would comply with treatment.20 Nevertheless, a reduced stroke risk generally far outweighs the risk of major hemorrhage while on anticoagulation and most concerns about bleeding are generally unfounded.21 Simply put, the oldest patients with AF are those who have the highest risk of stroke and the most to gain from anticoagulation. While the risk of falls is in many cases the documented reason for withholding anticoagulation, several analyses have shown that warfarin still has a significant net protective effect against composite outcomes of out-of-hospital death or hospitalization for stroke, myocardial infarction, or hemorrhage.22 Evidence points to focusing on maintaining international normalized ratio (INR) values of 2.0 to 3.0 (therapeutic range) and ensuring proper blood pressure control as a means of mitigating intracranial hemorrhage risk, as opposed to withholding anticoagulation.24Our study found that younger AF patients had a lower stroke risk and were more likely to be anticoagulated than older AF patients, which confirms prior findings.25 A meta-analysis of four existing major randomized control trials for DOACs showed a significant reduction in stroke, intracranial hemorrhage, and mortality  in comparison with warfarin.26 In real-world use, compliance rates are generally similar between warfarin and DOACs; however, warfarin requires INR monitoring, which allows physicians to directly see where noncompliance may be an issue.27 The highest thromboembolic risk appears to be among patients who are not compliant with DOAC therapy. Nevertheless, with more AF patients being prescribed anticoagulation in the post-DOAC era, it will be of interest to see whether long-term rates of thromboembolism show strong improvement.Our data were not powered enough to detect a difference in prescribing habits between warfarin and DOACs, as only six of the 105 patients who received OAC received a DOAC specifically. We have seen an increase in the use of DOACs as compared with warfarin; some studies have even shown a resulting increase in the overall percentage of AF patients receiving OAC.9 Thus, a limitation of this study relates to the fact that trends in prescribing habits are expected to change with double therapy regimens being increasingly utilized. This study was also limited due its retrospective design and the quality of the data retrieved from electronic records being beholden to the accuracy of the documenting physician. Future studies in this arena could also elicit more explicit information as to why providers chose one regimen over another or why patients were not able to tolerate OAC; this may provide a clearer picture of the reasons for the suboptimal prescription of OAC in patients with AF.The findings of this study were based on patient data from 2008 to 2016; however, in more recent years, landmark trials have shown that treatment with OAC and a P2Y12 inhibitor may perhaps be safer and noninferior in terms of efficacy.2DS2-VASc score, HAS-BLED score, or any other individual risk factor, with the exception of age. This study ultimately presents three observations, as follows: (1) there are major gaps in treating physicians’ perceptions of the scientific consensus on how to treat AF patients undergoing PCI; (2) there is a lack of established guidelines to direct community physicians; and (3) there remains a stigma regarding treating older patients with anticoagulation.Less than half of patients who met indications for OAC for AF after PCI were discharged on appropriate therapy. Patients were more likely to be discharged on OAC if they were already on OAC at the time of admission or if they were younger than 65 years old. There was no association between discharge on OAC and CHA


This paper aimed to develop and validate a deep learning (DL) model for automated detection of the laterality of the eye on anterior segment photographs. Anterior segment photographs for training a DL model were collected with the Scheimpflug anterior segment analyzer. We applied transfer learning and fine-tuning of pre-trained deep convolutional neural networks  to develop DL models for determining the eye laterality. Testing datasets, from Scheimpflug and slit-lamp digital camera photography, were employed to test the DL model, and the results were compared with a classification performed by human experts. The performance of the DL model was evaluated by accuracy, sensitivity, specificity, operating characteristic curves, and corresponding area under the curve values. A total of 14,468 photographs were collected for the development of DL models. After training for 100 epochs, the DL models of the InceptionV3 mode achieved the area under the receiver operating characteristic curve of 0.998 (with 95% CI 0.924–0.958) for detecting eye laterality. In the external testing dataset , the DL model achieves an accuracy of 96.1% (95% CI 91.7%–100%), which is better than an accuracy of 72.3% (95% CI 62.2%–82.4%), 82.8% (95% CI 78.7%–86.9%) and 86.8% (95% CI 82.5%–91.1%) achieved by human graders. Our study demonstrated that this high-performing DL model can be used for automated labeling for the laterality of eyes. Our DL model is useful for managing a large volume of the anterior segment images with a slit-lamp camera in the clinical setting. There is an increasing need for evaluation of corneal, glaucoma, and lens disease on DL algorithms with anterior segment imagery in telemedicine approach or clinical practice6.Anterior segment imaging has largely improved documentation and understanding of anterior segment structures and pathological processes. With the rapid advancement of deep learning (DL) in healthcare, it is now possible to perform automated detection of several anterior segment eye diseases, such as pterygium9. Compared to fundus photos, it is more challenging and time-consuming for an investigator to identify the eye side without automatic eye side labeling in anterior segment photography.Due to the different types and stages of ocular diseases that are often present in the right and left eyes, it is critical to identify the eye at the laterality level to evaluate the disease characteristics. In previous studies, several authors have proposed automated labeling of the eye laterality on the color fundus photograph using DL models with promising resultsIn this study, we developed a DL system to automatically distinguish the sides of the eyes (left and right sides) from anterior images that were captured by the Scheimpflug camera. We also investigate the performance of this model to predict eye laterality in photographs taken by a slit-lamp digital camera in the clinical setting.The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Shanghai Aier Eye Hospital (IRB: SHAIER2020IRB10). Informed consent was waived because of the retrospective nature of the fully anonymized images.In this cross-sectional study, anterior segment photographs recorded with a Pentacam HR  were extracted from clinical databases at Shanghai Aier Eye Hospital between June 2018 and December 2019. We selected 8,593 subjects, including 5,386 normal subjects ; 1446 subjects with cataracts; 1,201 subjects with dry eyes; and 560 subjects with strabismus. The diagnoses were based on the evaluations of the subjects’ electronic medical records. The eye laterality was labeled by the pre-defined examination sequence (right eye first and then left eye) and then confirmed by the operators in the field during the examination course. All photographs were manually reviewed by a post-graduate medical student (L.H) and reviewed again by the supervising cataract specialist (Z.C) to ensure correct categorization. Only the images with the quality parameter (QS) of the Pentacam HR that were marked "OK" were included in the study.A total of 15,091 photographs were collected. Among these photographs, 623 photos (4.1%) were excluded because of corneal scar, corneal degeneration, pterygium, or any history of eye surgery; 14,468 photographs (7170 right eye photographs and 7298 left eye photographs) remained for the dataset. This dataset was randomly divided into three sets: a training set  to develop the DL model; a validation set  to tune the parameters of the trained model and a testing set  to evaluate the performance of the developed model. To test the generalization ability of the DL model, we further assess one external testing set: 76 primary gaze photographs taken of 76 cataract subjects before cataract surgery by a Topcon DC-3 8-megapixel digital camera attachment for the SL-D7 slit lamp . All patient-specific information  was removed from the datasets. To simulate eye laterality detection in clinical scenarios, we cropped the original images to remove the media and lateral canthal areas to perform further image preprocessing. Diagrams of the grading workflow of the anterior segment photographs are shown in Fig. 10, Inception-V3 with 159 layers 12, and MobileNetV2 with 88 layers 13. All CNNs were pretrained on approximately 1.28 million images (1000 object categories) from the 2014 ImageNet Large Scale Visual Recognition Challenge12. However, as the recognition task of this work largely differed from that of ImageNet, we further applied transfer learning and fine-tuning of the original CNNs weights for the 2-class eye laterality classification. Further training was performed by initially unfreezing the last 2 layers. The global average pooling layer of the models is then followed by a batch normalization layer, drop-out layer (50%) and 2 dense layers (512 nodes and 1 node). The image pixels were rescaled to values of 0 to 1 and interpolated to fill a 224 × 224 matrix to match existing pre-trained networks. Data augmentation was performed to enhance the dataset, including random horizontal flipping and adjustments to saturation, brightness, and contrast. A minibatch gradient descent of size 32 was employed for training, with an Adam optimizer learning rate of 0.0001 for better convergence. All the DL models and strategies were implemented in the TensorFlow framework  with Keras API (v.2.2.4). The models were trained on an Ubuntu 16.04 operating system with Intel Core i7-2700 K 4.6 GHz CPU, 128 GB RAM, and NVIDIA GTX 1080 Ti 12 GB GPU.In this study, we trained the model by fine-tuning the pre-trained convolutional neural networks (CNNs). The applied CNNs were VGG16 with 23 layers To better understand and demonstrate the DL model, we applied the gradient-weighted Class Activation Mapping (CAM) to highlight the area in which the DL model may focus on eye laterality detection. CAM uses the class-specific gradient information that flows into the final convolutional layer of a CNN to produce a coarse localization map of the important regions in the image. This technique is crucial for interpreting network output and validating whether the network learned meaningful features.14. t-SNE is a tool for visualizing high-dimensional data, such as the DL features in this study. The tool converts similarities between data points and minimizes the Kullback–Leibler divergence of the joint probabilities between the low-dimensional embedding and the high-dimensional data.Moreover, t-distributed stochastic neighbor embedding (t-SNE) was employed to intuitively analyze the characteristics of the extracted hierarchical features (512 features from InceptionV3 model) from the DL modelWe also validated the DL model by performing human–machine comparisons. The external testing dataset (76 primary gaze photographs) was employed to compare the DL models’ referral decisions with the decisions made by human graders. Three ophthalmologists with different levels of clinical experience  who were blinded from the data set collection were instructed to independently make a decision about each testing image. Using CAM that is generated from the external testing dataset, we also reviewed and categorized the misclassified photographs according to the three most commonly viewed features: (1) eyes with coexisting eye conditions, such as involutional ptosis, trichomegaly or misdirected lashes; (2) eyes with other photo conditions, such as overexposure or low exposure and off-center; and (3) other features (incorrect labeled).15. The area under the receiver operating characteristic curve (AUC) metrics were utilized to determine the performance of our DL model.All statistical analyses were performed using the Python (version 3.6) and Scikit_learn modules . The applied performance metrics included accuracy, sensitivity, and specificity with 2-sided 95% CIs. The formulas for calculating the accuracy, sensitivity, and specificity were defined asThe details of the eye laterality distribution of the datasets are shown in Table CAM was developed during the training phase to determine which regions of the anterior segment photographs were being paid the most attention. Figure Figure Table In this study, we developed a robust and highly accurate DL model to distinguish the eye laterality on anterior segment photographs that were captured by the Scheimpflug imaging system. This DL model was also validated by slit-lamp photography, an imaging modality which is commonly employed in daily work. To our knowledge, there is no DL system for identifying the laterality of anterior segment photography. We believe that this DL model is highly reliable in clinical practice for anterior segment images with different pathologies and as a large-scale telemedicine approach without eye laterality labels, especially after the worldwide coronavirus disease 2019 (COVID-19) crisis.8. Even for an experienced ophthalmologist, the accuracy of the eye side labeling is lower in the anterior segment images than labeling in the fundus images. The optic nerve and retinal vessel are significant indicators that are used to identify the laterality of the fundus images, but the cornea, iris, pupil, and even the conjunctival vessel are often similar in both eyes. Previous studies have shown that DL can acquire an additional biomarker or new knowledge from medical images by an optimized algorithm and recognize signals of disease that medical experts may not detect by their experience or knowledge17. Because the medial and lateral canthus can be the reference of the eye side, we purposely cropped anterior segment images with only the limbus area and part of the eyelids during image preprocessing. Note that the algorithm actually identifies the eye side by activating the nasal iris area in images during CAM analysis. Using 3D swept-source anterior segment optical coherence tomography, Invernizzi et al. reported that the iris’s morphology varied by iris sector and the iris color was associated with differences in iris volume and thickness18. Sidhartha et al., determined that the iris thickness was correlated with the iris surface characteristics, such as the color and number of crypts19.In this study, our DLS module achieved a sensitivity of 94.7% (95% CI 89.7%–99.7%) and a specificity of 97.4% (95% CI 93.8%–100.0%) in eye laterality identification, which is similar to the performance of the algorithm for the fundus laterality detectionDL has made substantial progress in medical image analysis and achieved excellent performance that exceeds traditional diagnosis by human experts. Even though most ophthalmic devices can record the eye laterality in their operation system or display the laterality label on images in related software, the original image and laterality information are separately stored in the databases of these devices. Manual labeling of the eye side in the image dataset is labor-intensive and time-consuming. It is necessary to develop a DL system for the application of eye laterality classification.20. We tested the DL by using the color photography taken by a slit lamp digital camera. Although the DL algorithm was initially developed from the image dataset collected by the Scheimpflug camera, we proved it also has excellent accuracy with an independent image dataset, where the images were collected from primary gaze photographs by a digital camera. For this dataset, the manual label method had an accuracy that varied from 72.3 to 86.8%, while our module was able to detect eye laterality with an accuracy of 96.1% (95% CI 91.7%–100%). The results showed that the performance of DL was significantly better than that of the manual method, which proved its reliability in anterior segment image management.The dataset images were initially captured by an infrared (IR) camera with the Pentacam. Black-and-white (B/W) IR images were generally employed in diagnosis and treatment in ophthalmology, such as in the Verion or Callisto eye system, which uses anterior segment IR images to register the iris for guiding toric intraocular lenses alignment. However, these two image guide systems still need to manually match the eye lateralityOne of the key purposes of this study was to validate DL models for automated eye laterality detection. In any screening or telemedicine program, misclassification results create unnecessary referral and burden to the healthcare system. To further validate the performance of the DL model, we also evaluated misclassification of human graders by evaluating the CAM. In this study, note that the DL model not only shows high accuracy but also highlights locations that are informative for human graders, which can potentially serve as a training tool for clinicians.One limitation of this study was that images were captured in B/W mode, which provided lower information than the color images. It is possible for the DL system to be trained with color and high-resolution photography. However, the DL system required a vast amount of datasets and longer training times, which was impractical for this study. We also consider that our DL system would be more clinically useful because a color photograph can be easily converted to B/W mode for analysis and is more convenient for multiple clinical sets.21), dry eye, and strabismus (a common eye disorder that affects children22), further research is warranted amongst different eye diseases, including corneal scar, corneal degeneration, pterygium, etc. In this study, we excluded 623 images from subjects with rare corneal diseases based on the PRISMA diagnostic test accuracy guideline23. How to detect eye laterality with rare corneal is still a challenge. Our DL system cannot achieve satisfactory performance in this exclusion dataset [an accuracy of 64.3% (95% CI 53.5%–75.1%)]. Other machine learning techniques, such as few-shot learning24, may be helpful to improve the results.Future work will involve more devices for eye laterality determination, such as a surgery recorder, topography, biometer, or mobile phone camera. In busy clinical settings, eye laterality recognition for a patient eye is more important and meaningful than for a healthy eye. Although we included images from subjects with cataracts (a leading cause of blindness in the worldThe DL system can be used by clinicians for eye laterality recognition. Further studies are needed to investigate where this technology could be best utilized within clinical and research settings.