Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss. This study reveals the presence of the cholesterol turnover enzyme cholesterol 24-hydroxylase (CYP46A1) in cells of the inner ear and shows that increased levels of CYP46A1 in aging is accompanied by cholesterol loss in these cells. Pharmacological activation of CYP46A1 reproduces this effect and leads to impaired outer hair cells function, which can be reversed by administration of phytosterols. Functional deterioration of the nervous system is frequently observed in the elderly, with a progressive decline in cognitive capacities . Age-relCholesterol is an important component of neural cell membranes, vital to normal function of the cell and their membrane-associated proteins. Recent studies have shown that cholesterol is abundant in synaptic membranes and modulates a number of protein complexes by directly binding to and/or conditioning the conformation, dynamics, and biophysical properties . Within OHCs are sensory cells implicated in the mechanical amplification of sounds and in the frequency selectivity in mammals . OHCs reAll of these evidences led us to hypothesize that cholesterol deficiency might play a role in the associated cochlear pathologies that occur during aging. However, the role of cholesterol homeostasis in the physiopathology of the inner ear has not been studied . The aimt test, p < 0.0001 at the apical/medial and p < 0.01 at the basal end). In those surviving OHCs in 24-month-old mice, we also found an increase in CYP46A1 fluorescence intensity along the whole cochlea . In the SCs’ area, we quantified the CYP46A1 fluorescence intensity by counting all the positive-labeled cells surrounding both IHCs and OHCs. We found a significant increase in CYP46A1 expression in SCs of 24-month-old mice, but it was statistically significant only in the medial and basal region of the cochlea  (Cholesterol hydroxylation into 24(S)-hydroxycholesterol by CYP46A1, and elimination of this oxysterol through the blood–brain barrier, is the main mechanism for cholesterol removal from the brain . Thus, t region) . Accordi region) , and S1B region)  and S1A.t test, p < 0.0001). Although aged cochleae showed an evident OHCs loss, filipin staining was quantified along the diameter of surviving OHCs to unequivocally determine the cholesterol content per cell, as indicated by the dashed pink line in the magnified inset in t test, p < 0.0001).It has been previously shown that cholesterol content is a critical player in the modulation of hearing in mice due to its important role in the proper distribution of prestin within the lateral plasma membrane of OHCs ,47. To ap > 0.05 at all the frequencies). There were no differences in ABR thresholds between controls at 8 and 12 weeks . Suprathreshold ABR peak 1 amplitudes were not modified after efavirenz administration, indicating no drug-related changes in cochlear neural function . However, we observed a 20- to 30-dB SPL elevation of DPOAEs thresholds at the mid-frequency cochlear region in mice treated with efavirenz compared to controls , indicating that OHC function is degraded after efavirenz treatment. The mean data for the emission amplitude-versus-level functions for the DPOAEs at 16 kHz was reduced after efavirenz treatment . Quantification analysis of filipin staining in IHCs and SCs also showed a reduction in the cholesterol content in these cells at the 3 regions of the cochlea   . As expectively)  led to actively) . At the ctively)  and calrctively)  that lab(ad libitum) in addition to the efavirenz treatment.Phytosterols are plant naturally occurring compounds structurally and functionally similar to cholesterol in mammals . In contp > 0.05 at all the frequencies). However, there was a decrease, although not statistically significant, in DPOAEs thresholds in mice treated with efavirenz together with phytosterols compared to the ones treated with efavirenz alone .As shown in nz alone . Importap < 0.0001 at all the regions) , the increase in filipin staining in the efavirenz plus phytosterol-treated group indicates that phytosterols can partially restore the sterol content in the OHCs membrane (p > 0.05). Altogether, these results suggest that phytosterols’ supplementation in the diet partially rescues the OHC function in efavirenz-treated mice.To evaluate whether phytosterols can restore the sterol content in the OHC membrane, cochleae were dissected and stained with filipin. Previous reports have shown that filipin is able to recognize phytosterols in eukaryotic membranes . Represeregions) . Althougmembrane . Quantifp < 0.0001 at all the regions) showing a higher presence of prestin in the cytoplasm compared to the membrane. This effect was reverted in the animals treated with phytosterols in which the ratio was 1.09 ± 0.03. This ratio was not significantly different compared to control mice . Finally, we computed the mean prestin fluorescence intensity at all the regions of the cochlea  in the 3 groups of mice by drawing a line through the middle of 3 consecutive OHCs . Importantly, addition of phytosterols to the diet produces a significant increase in the pixel intensity at the 3 regions compared with mice treated with efavirenz alone . Although the increase in prestin immunostaining after addition of phytosterols did not reach the control levels at the medial and basal region of the cochlea, there was a significant increase compared with mice treated with efavirenz alone. Altogether, these data indicate that cholesterol content in OHCs decrease with the activation of CYP46A1 with efavirenz leading to a reduction of the motor protein prestin in the plasma membrane resulting in elevations in DPOAEs thresholds. Notably, we could partially restore the OHCs’ function by phytosterols supplementation.Taking into account that prestin is a critical component of the OHCs lateral membrane and is essential for their electromotility, we wondered if elevation in DPOAEs thresholds by reduction in the cholesterol content could be explained by alterations in the distribution of prestin within the plasma membrane. Prestin levels in OHCs from control, efavirenz-treated, and efavirenz plus phytosterols-treated mice were evaluated by immunohistochemistry using antibodies against prestin and calretinin to visualize hair cells. As seen in the representative confocal images of whole mounts of the organ of Corti from the medial region , there wive OHCs . The redMammalian hearing function depends on the active motility of OHCs to amplify the basilar membrane motion ,62. OHCsPrevious works have shown that aging is associated with alterations in cholesterol metabolism in the CNS, mainly in the brain, which is dependent on the region studied. In the hippocampus, aging triggers a mild but consistent cholesterol loss from neurons due to the enhanced activity of the cholesterol 24-hydroxylase or CYP46A1, the enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol  or by phWe found for the first time by immunofluorescence that CYP46A1 is expressed in the inner ear in 3 different populations of cells  in young C57BL/6J mice. Interestingly, CYP46A1 levels are significantly increased in 24-month-old mice in these 3 populations. Paralleling the CYP46A1 increase, we found a significant reduction in the cholesterol levels in sensory cells as identified by filipin staining of whole mounts organ of Corti in 24-month-old mice. To test if there is a direct cause–effect relationship among CYP46A1 activity, cholesterol loss and the auditory function, we treated 2-month-old mice with efavirenz, an anti-HIV drug able to specifically activate CYP46A1. We choose to start the treatment at 2 months to avoid the auditory threshold elevations with age developed by the C57BL/6J strain, which starts at 8 to 10 months of age . SupportThen, we tested the possibility that altered OHC function due to cholesterol loss could be rescued by dietary supplementation of phytosterols to efavirenz-treated mice. We analyzed the effect of phytosterols based on previous evidence showing that cognitive impairments due to cholesterol loss in hippocampal neurons in an aging mouse model were rescued by dietary phytosterol supplementation . We founLooking for the possible cause by which cholesterol reduction in efavirenz-treated cells would lead to OHC dysfunction, we assessed the prestin levels in OHCs of mice from the control, efavirenz, and efavirenz plus phytosterols groups by immunostaining. Prior studies have shown the critical role of cholesterol to tune the membrane-based motor to function at maximal gain in the OHC receptor potential range . Prestinstria vascularis, sensory hair cells, spiral ganglion neurons, and fibrocytes. Accumulation of reactive oxygen and reactive nitrogen species  as well as intracellular calcium homeostasis have been proposed as contributory factors making OHCs, especially those in the high-frequency region of the cochlea, most vulnerable to aging [ARHL is a multifactorial progressive disease produced by different factors . Damage to aging ,79. Indeto aging . It has to aging  and in bto aging ,83. In sto aging , and thito aging ,86. Our to aging ,35. InteAs stated above, efavirenz treatment leads to a reduction in the cholesterol levels in OHCs that was reflected by an elevation in DPOAEs thresholds. Several studies have reported an increased incidence of auditory dysfunction among HIV/AIDS patients treated with highly active antiretroviral therapy as a side effect ,88. NowaOur results provide valuable insights into the relationship between cholesterol homeostasis and the physiopathology of the inner ear. However, there are 2 major limitations in this study that should be addressed in future research. The first limitation is the strain used in the study. C57BL/6J mice are widely used for aging studies due to its low aggressivity, which makes them suitable for long-term studies. However, this strain is known to exhibit early-onset hearing loss. Therefore, it is crucial to replicate the same experiments in a different mouse strain without this predisposition. These experiments are fundamental to determine whether the observed effects are specific to C57BL/6J mice or can be generalized to other mouse strains or even humans. Thus, our future research direction will be to explore the effect of aging and efavirenz administration in cholesterol levels in the cochlea using other strains of mice, like CBA/CaJ or BALB/cJ, and to test if phytosterols supplementation can ameliorate aged- and/or antiretroviral-related hearing loss. Second, although our results show that in aged cochlear tissue there is a reduction in cholesterol content in sensory cells of the inner ear—assessed by filipin labeling—and that this reduction is correlated with an increase in the immunofluorescence of CYP46A1, it will be important to test alternative methods for CYP46A1 quantification in specific cell populations. Future studies incorporating techniques such as RNAscope or scRNA-seq are necessary to validate our findings. In conclusion, while our study sheds light on the extent to which alterations in cholesterol homeostasis contributes to the pathophysiology of the inner ear, it is important to consider the aforementioned methodological limitations. Nonetheless, the initial outcomes of our present research offer great promise as they establish the first evidence in favor of phytosterols supplementation as a potential therapeutic strategy in the prevention or treatment of hearing loss.All animal care and experimental protocols were performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (June 2013) as well as Facultad de Medicina, Universidad de Buenos Aires (UBA) Institutional Animal Care and Use Committee (IACUC) guidelines, and best practice procedures. The animal experiments were approved by the Institutional Animal Care and Use Committee of Facultad de Medicina, Universidad de Buenos Aires (UBA)  and the Institutional Animal Care and Use Committee of Instituto de Investigaciones Biomédicas, Pontificia Universidad Católica Argentina (BIOMED) .Young (2 months) and old (2 years) C57BL/6J male mice were used in this study. All experimental mice were maintained under specific pathogen-free conditions with a 12-hour light/dark cycle and open access to food and water. The temperature and humidity were set at 22 ± 1°C and 55% ± 5%, respectively.p = 0.67). Phytosterols (Vitatech) were added to standard food pellets  at a concentration of 2% (w/w) and administered ad libitum. The phytosterols treatment was performed for 3 weeks (i.e. initiated 1 week after the efavirenz treatment started). At the end of the treatments, mice exhibited similar body weights .The treatment started at 8 weeks of age (2 months), with efavirenz administered in the drinking water at a dose of 0.09 mg/kg/day as described . Efavirehttps://www.masseyeandear.org/research/otolaryngology/investigators/laboratories/eaton-peabody-laboratories/epl-engineering-resources/epl-acoustic-system). Digital stimulus generation and response processing were handled by digital I-O boards from National Instruments driven by custom software written in LabVIEW . For ABRs, needle electrodes were placed into the skin at the dorsal midline close to the neural crest and pinna with a ground electrode near the tail. ABR potentials were evoked with 5 ms tone pips  delivered to the eardrum at log-spaced frequencies from 5.6 to 45.25 kHz. The response was amplified 10,000× with a 0.3- to 3-kHz passband. Sound level was raised in 5 dB steps from 20 to 80 dB sound pressure level (SPL). At each level, 1,024 responses were averaged with stimulus polarity alternated. Threshold for ABR was visually defined as the mean between the lowest stimulus level at which a repeatable peak 1 could be identified in the response waveform and the sound intensity before that. The ABR peak 1 amplitude was computed by offline analysis of the peak to baseline amplitude of stored waveforms. The DPOAEs in response to 2 primary tones of frequency f1 and f2 were recorded at 2f1-f2, with f2/f1 = 1.2, and the f2 level 10 dB lower than the f1 level. Ear canal sound pressure was amplified and digitally sampled at 4 μs intervals. DPOAE threshold was defined as the lowest f2 level in which the signal to noise floor ratio is >1. Offline analysis of ABR thresholds and P1 amplitudes together with DPOAEs thresholds were done by 2 experimenters “blind” to each animal’s treatment condition.Inner ear physiology, including ABRs and DPOAEs, was performed in mice anesthetized with xylazine  and ketamine  and placed in a soundproof chamber maintained at 30°C. Sound stimuli were delivered through a custom acoustic system with 2 dynamic earphones used as sound sources  and an electret condenser microphone  coupled to a probe tube to measure sound pressure near the eardrum  of the apical, medial, and basal regions from each cochlea were taken using a Leica TCS SPE microscope equipped with 63×  oil-immersion lens. Image stacks were imported to Fiji software [Temporal bones were collected from young (control and treated with efavirenz and efavirenz plus phytosterols) as well as from 2-year-old mice. Cochleae were perfused intralabyrinthly with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), postfixed with 4% PFA overnight, and decalcified in 0.12 M EDTA. Cochlear tissues were then microdissected and blocked in 5% normal goat serum with 1% Triton X-100 in PBS for 1 hour, followed by incubation in primary antibodies (diluted in blocking buffer) at 4°C for 16 hours. The primary antibodies used in this study were as follows: (1) rabbit anti-CYP46A1 antibody ; (2) mouse anti-calretinin antibody ; and (3) goat anti-prestin antibody . Tissues were then incubated with the appropriate Alexa Fluor–conjugated fluorescent secondary antibodies  for 2 hours at room temperature. Finally, tissues were mounted on microscope slides in FluorSave mounting media . Confocal software  for analThe sensory epithelium was isolated and washed twice with PBS, fixed with 4% PFA for 30 minutes, and stained with the fluorescent molecule filipin  and AlexaFluor 647 phalloidin  for 60 minutes in the darkness. The samples were then washed twice with PBS and mounted on microscope slides in FluorSave mounting media. Confocal z-stacks (0.3 μm step size) of the apical, medial, and basal regions from each cochlea were taken using the Leica TCS SPE microscope equipped with 63×  oil-immersion lens. Image stacks were examined using Fiji software . Filipint test was performed . A p < 0.05 was considered statistically significant at a 95% confidence level.Data are presented as group means ± standard error (SEM) and were analyzed with R Statistical Software . KruskalS1 FigA) Representative 10× low-magnification confocal images in the xy projection of whole mounts organ of Corti from the apical/medial region immunolabeled for calretinin from young  and old  C57BL/6J mice . Insets: 40× magnification images of the selected area indicated by a white rectangle . Arrows point to OHCs and arrowheads point to lost IHCs in aged ear tissue. (B) Bar graph showing the percentage of OHCs survival in aged mice compared to young animals. At 2 years of age, there was a 70% of OHCs death along the whole length of the cochlea. Asterisks represent the statistical significance . The data underlying this figure can be found at https://osf.io/xpemk/.((TIF)Click here for additional data file.S2 FigA) Representative confocal images of whole mounts organ of Corti from the medial region stained with filipin in control, treated with efavirenz, and treated with efavirenz plus Phy mice. . Pink lines indicate the diameter of IHCs and SCs as an example on how we select cells to measure the pixel intensity. (B) Relative intensity bar graph showing the changes in filipin staining in IHCs in the 3 groups of mice at the 3 regions of the cochlea  . (C) Relative intensity bar graph showing the changes in filipin staining in SCs in the 3 groups of mice at the 3 regions of the cochlea . There was a significant reduction in cholesterol levels after efavirenz treatment in both IHCs and SCs. Treatment with efavirenz together with Phy showed an increase in filipin staining at the 3 regions compared with mice treated with efavirenz alone, except in IHCs at the apical region. However, when compared to controls, there was a significant reduction in filipin staining at the 3 regions of the cochlea in both IHCs and SCs. Group means ± SEM are shown. Asterisks represent the statistical significance . The data underlying this figure can be found at https://osf.io/xpemk/.((TIF)Click here for additional data file. 15 Sep 2022Dear Dr Gomez-Casati, Thank you for submitting your manuscript entitled "Phytosterols reverse hearing impairments caused by antiretroviral treatment. Implications in presbycusis." for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review. I should note that, while we find the topic of your study interesting, we are yet to make a firm call about whether the study provides enough cellular mechanistic insights for our Research Article format. We will be looking for some enthusiasm from the reviewers.Before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.https://www.editorialmanager.com/pbiology) within two working days, i.e. by Sep 19 2022 11:59PM.Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. 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Please don't hesitate to contact us if you have any questions or comments.Sincerely,LucasLucas Smith, Ph.D.Associate EditorPLOS Biologylsmith@plos.org-----------------------------------------REVIEWS:Reviewer #1: In the manuscript 'Phytosterols reverse hearing impairments caused by antiretroviral treatment. Implications in presbycusis.' Sodero et al., describe an increase in CYP46A1 leads to a decrease of cholesterol in the outer hair cells of aged mice and mice treated with an antiretroviral drug used in HIV/AIDS treatment. They are able to show that administrating this drug in young mice affects outer hair cell function as measured via DPOAEs and can be partially rescued by feeding phytosterols to the same animals. In general, I think this is a very interesting study that is of interest for hearing research. However, I do have some concerns with this study.1) Fig1A: The calretinin stain is very hard to see in the OHC in the young group, but very clear in the aged group. Since it is reported :517-29. doi: 10.1002/cne.903460405. PMID: 7983242.) that calretinin is not expressed in the outer hair cells in the adult (>P22) that is very surprising to me. Are you sure those are OHCs?2) The animal numbers are not completely clear to me. Did you use the same animals for the different immunostainings? Why were there only 3 animals for the filipin staining in the young group but 5 for the CYP46A1 staining in the same age group?3) While the authors do mention that C57Cl/6 mice are known to lose their hearing early it would be beneficial to discuss why they chose this mouse strain anyways vs a mouse strain like CBA/CaJ which are known to retain their hearing up to an old age. In that case the conclusion of age being the driving factor of the reduction and redistribution of cholesterol in the OHCs would have had much more merit. In the present study it is hard to prove that this effect is due to age and not also influence by the overall damage to the OHCs. This should be discussed4) In the aging study it is mentioned that an increase in CYP46A1 and decrease in filipin staining can be observed not only in OHCs but also IHCs and SCs. Was that also the case with the drug treatment?5) Fig 1A: the merged of the aged animals does not line up with the Calretinin only stain. 6) Fig 4A: in the Efavirenz+Phyto group ot seems as if Filipin is located to the cell membrane vs in the other groups where it is more central. Also, the Phalloidin intensity is reduced in this group compared to the others. And in the Efavirez group it seems to be more diffusely distributed. Please explain this.7) I am not sure about the reasoning of using the average of either two or three OHCs for the analysis in Figures 4 & 5. 8) Is there a reason why you represent different areas in the cochlea for your filipin vs prestin stain intensity?9) How did you select the two/three OHCs to measure across?Minor Comments:1) Please use the same citation format throughout the manuscripts.2) In the table with the questions from the journal you state that an ethics approval was not necessary for this study despite conducting an animal study (which was listed as one of the reasons to include an ethics statement).3) It is not always clear if the pink arrow heads are considered part of the line or if it is only the distance between the two arrow heads that is marked.4) In the results section you write that you treat for 3 weeks with the phytosterols. In the methods section it is stated that the treatment was 4 weeks. Please correct where necessary.Reviewer #2: Sodero et al. studied the role of cholesterol in hearing loss by analyzing the relationship between the decrease in cholesterol levels and outer hair cell degeneration. They detected elevation of DPOEA thresholds after administration of the mice with efavirenz, an antiretroviral which pharmacologically activates an enzyme responsible for cholesterol turnover (CYP46A1). Interestingly, the elevation in DPOAE thresholds due to efavirenz administration was prevented by dietary complementation with phytosterols. If this observation holds true, it would be of high interest as a strategy for preventing hearing loss due to age-related issues and ototoxic drugs. However, there are some important concerns about these claims since the experiments do not provide a clear mechanism of action. Also, there is no elevation of auditory thresholds (ABRs) caused by the drug administration, which dampens enthusiasm for work. Some important points to consider:- Can the authors use qRT-PCR for quantifying CYP46A1 levels? Immunostaining can be highly variable across experiments. Is the difference in CYP46A1 quantity between young and old mice stronger in the base or the apex? Only mid sections were used in Fig 1. - Could you explain how were the ROIs defined for the quantification of CYP46A1 in the supporting cells? - In Fig 2, since there is substantial hair cell loss in aged mice, the orientation of the cells is less consistent which makes it difficult to compare with younger mice that have a better-preserved Organ of Corti. How was the IHC cholesterol concentration quantification performed? In which part of the cell body was the analysis done?- Is the effect of efavirenz (and phytosterols) dose dependent? Could higher doses of the drug cause ABRs elevations? The fact that the DPOEAs thresholds are elevated with no effect on ABRs is surprising because OHC function is necessary to maintain normal ABR levels, and also this indicates that there is no evident hearing loss associated with efavirenz administration. - Could the authors perform ABR recordings of the treated mice at 2 months of age prior to the drug treatments? Then, comparisons can be made within each animal.- Line 407-409: "Studies in mice suggested a synergistic relationship between anti-HIV drugs and noise, with an increase in DPOAE thresholds consistent with drug-induced damage to OHCs" Would the efavirenz-treated animals be more sensitive to noise-exposure? - Can you show the filipin staining of IHC after efavirenz (and Phy) treatment ? - Are the newly delivered phytosterols being incorporated into the membrane? Can you radiolabel the molecules and do a quantitative analysis of these lipids incorporation? - Lines 372-373: "…with an apparent recovery of the normal prestin distribution in the lateral wall." Can you show the quantification of prestin intensity in the periphery of the cell? - Why were only male mice used in the study?Some other minor points:- Could you enhance the contrast/brightness of calretinin staining in Fig 1?- Could you also include ABR and DPOAE waveforms? - Given that efavirenz produces dyslipidemia but also dysglycemia (doi: 10.1097/MD.0000000000002385), how do you know its effect is not due to the alteration on glucose metabolism?- Does the treatment with efavirenz change the level of CYP46A1 levels in the cochlea? Reviewer #3: Numerous studies have shown that cholesterol imbalances in the development of neurodegenerative diseases, but the role of cholesterol in the physiopathology of the inner ear has not been studied. This paper characterized cholesterol homeostasis in the inner ear of young and old mice, and tested the hypothesis that cholesterol deficiency might play a role in cochlear pathologies and hearing loss by analyzing CYP46A1 levels and cholesterol content in the inner ear of young and aged mice. The authors also tested the effects of efavirenz on the auditory function. The effect of dietary supplementation with phytosterols was also tested in the inner ear of mice treated with efavirenz. Overall, their findings are new and interesting, but no histological examinations of cochlear cells/microstructures  was performed, although the aim of the study was to test the hypothesis that cholesterol deficiency might play a role in cochlear pathologies and hearing loss. Cholesterol levels were not measured in the whole inner ear tissues from the experimental groups . In addition, I have some questions about the age of the old group and the statistical method. I elaborate on these and other points below. Line 94. The authors stated that the aim of this study was to test the hypothesis that cholesterol deficiency might play a role in cochlear pathologies and hearing loss, but no histological examination of cochlear cells/microstructures  was performed in the young, old, control, efavirenz, and efavirenz + phytosterol groups. So I don't think the proposed hypothesis was thoroughly tested in the current study, and the overall finding seems inconclusive in terms of testing the hypothesis. Method. I understand cholesterol levels were measured using the cholesterol-binding fluorescent antibiotic filipin in the fixed hair cells, but I do think total cholesterol levels should also be measured in the whole inner ear tissues of all the experimental groups  to test the hypothesis that cholesterol deficiency might play a role in cochlear pathologies and hearing loss. https://www.jax.org/strain/000664), so it is not clear to me why 24 months old mice were used when they were near deaf and likely lost most of the hair cells. I think use of 3 age groups  or 2 age groups  would make more sense for the measurements of CYP46A1 and filipin labeling.Line 109. C57BL/6J mice develop early onset severe and progressive hearing loss by 12-15 months of age, with the disruption of both outer and inner hair cells, due to the Cdh23Ahl allele  the factors are 3 experimental groups and frequencies. My understanding is that Kruskal-Wallis cannot handle two way ANOVA data in the non-parametric sense. Could you clarify this? In addition, is there any reason 6 of each group was used for the ABR and DPOAE tests?Line 158. The aim of this experiment was to test the hypothesis that CYP46A1 activation might reduce the cholesterol content in young mice, leading to hearing deficits. So, I think histological examination of cochlear cells (such as hair cell counts) should also be performed at the end of the treatment (at 3 months of age) to validate the ABR test results. In addition, what is the rationale for starting efavirenz treatment at 2 months of age for just 4 weeks as opposed to starting at a later time point  for a longer period ?Lines 231 and 265. Again, what is the rationale for starting efavirenz + phytosterol treatment at 2 months of age for just 3 weeks as opposed to starting at a later time point  for a longer period ?Fig. 4, 5, 9, 10 (bar graphs). I think error bars should be added to the bar graphs. 28 Apr 2023AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 30 Jun 2023Dear Dr Gomez-Casati,Thank you for your patience while we considered your revised manuscript "Phytosterols reverse hearing impairments caused by antiretroviral treatment. Implications in presbycusis" for consideration as a Research Article at PLOS Biology. Your revised study has now been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.The reviewers largely agree that the manuscript has been strengthened in the revision, however Reviewers 1 and 2 have a number of lingering concerns and additional comments that we think should be addressed. In some cases, Reviewer 2 has requested additional data. While adding this data would strengthen the study, after discussion with the Academic Editor, we think this would not strictly be required for publication at PLOS Biology. However, in the absence of new data, you should address Reviewer 2's concerns by adding a section to the discussion outlining some of the methodological considerations and limitations of the study. In addition to addressing the reviewer comments, below, we also ask that you please address the following editorial requests: 1) TITLE: We think that the title should be edited slightly to avoid punctuation and for clarity. If you agree, we suggest you change it to something like "Phytosterols reverse age-related cochlear changes and hearing impairments caused by antiretroviral treatment in mice"https://journals.plos.org/plosbiology/s/what-we-publish#loc-short-reports2) ARTICLE TYPE: After discussion within the team, we think that your manuscript is closest in scope to our 'Short Report' article type, and so we request that you change it accordingly. Short Reports can have a maximum of 4 figures and so this will require that you condense 2 of your figures or move one to the supplement. We suggest that Figures 1 and 2 might be combined to reduce the figure number to 4. For more information about PLOS Biology Short Reports, see here: https://osf.io/xpemk/"3) DATA: Thank you for providing the underlying data for your study as a deposition on OSF. Can you please add a sentence to each figure legend  referencing this dataset? For example, you can add the sentence "the data underlying this figure can be found at We are pleased to offer you the opportunity to address the remaining points from the reviewers and the abovementioned editorial requests in a revision that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review, although might need to consult with the reviewers, depending on the nature of the revisions.plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. 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Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,LukeLucas Smith, Ph.D.Senior EditorPLOS Biologylsmith@plos.org----------------------------------------------------------------REVIEWS:Reviewer #1, Alice L. Burghard : In the revised version of the manuscript "Phytosterols reverse hearing impairments caused by antiretroviral treatment. Implications in presbycusis." the authors have addressed most of my previous comments. However, I still have a few comments that should be addressed.1) You report group differences by stating the significance found in the ANOVA, please also include the results from the post-hoc test. 2) I am not sure what happened, but all the figures are blurry. The supplemental material is not blurry. I would recommend that you use that format throughout the manuscript.3) In Fig 3 you give a scale bar of 50 dB. Please give the reference if that is dB SPL or something else.4) When you plot distance and the pink dotted line  it is not clear immediately that the pink dashed line is representing the dashed line from panel A. Maybe instead of assigning it a level on the y-axis put it underneath the x-axis? That might also explain the 'distance' measurement a little better. Or maybe use the term width? In the figure caption the term length is used. Please make sure you keep the terms consistent throughout the manuscript. 5) Also in Fig 4, please explain the pink dashed lines already for A and not only in B.6) Page 26 line 645 and 646: I think you mean 0-2 and 6-8 and 2-6 not 2-4.Reviewer #2: In the revised version of the manuscript, Sodero et al. did not provide alternative methods for CYP46A1 quantification, so I am still not convinced that CYP46A1 increased expression is one of the mechanisms behind ARHL. Also, quantification of cholesterol using filipin staining in an intact (young) and a degenerated (aged) tissue makes comparisons between groups unreliable (I will elaborate on that). As the authors mentioned, they are exploring the effect of aging and efavirenz administration in cholesterol levels in the cochlea using other strains of mice, which would make the results stronger. Determination of a dose-response curve for the effect of efavirenz in the cochlea will be fundamental to understand the effect this drug has on patients. I do believe that the efavirenz results are very interesting since they show that localization of prestin in OHCs is affected by the drug administration and reverted by phytosterols consumption, which could explain the DPOAEs thresholds elevations and recovery respectively.Some points to work on are:* Figure 1: According to the immunostaning data, the CYP46A1 intensity level is very different in the organ of Corti of aged vs young mice and this is not just in hair cells so the fact that it was not detected through qPCR makes the staining data less reliable, even though the ages were not exactly matched with the ones shown in the paper. The authors should perform RNA Scope (or other quantitative in situ hybridization method) to resolve the discrepancy in an age where they detect the decrease in protein level in a cell-specific manner.* Figure 2: In the case of young animals, OHC are oriented more perpendicular to the plane of the photo, but this is not the case for aged animals since there is substantial hair cell death and the orientation of the cells becomes less defined. This makes comparisons between groups very inconsistent because the cell volume that is being included in the quantification zone is larger for the young group than for the aged group. * Figure 3: How did the authors call the ABR thresholds? If manually done, was it done by more than one person? Was this done blindly? An algorithm can be used for that (DOI: 10.1016/j.heares.2019.107782). In the efavirenz + phytosterols traces, it looks like another person or an algorithm could very likely pick the intensity immediately bellow the one called by the authors since there appears to be small but clear peaks 1 & 2.I agree with lines 540-545 where the authors mention that it is relevant to study this phenomenon in other mouse strains in order to confirm that the effect seen is not related to the early HL that the C57BL/6 line presents. In that sense, I believe that including an alternative method for CYP46A1 quantification will be important to make the immunostaining results more reliable, together with an antibody that can label OHC homogeneously across ages (like Myo7a).Reviewer #3: The authors have addressed all of my major concerns. 13 Jul 2023AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 18 Jul 2023Dear Dr Gomez-Casati,Thank you for the submission of your revised Short Report "Phytosterols reversal of antiretroviral-induced hearing loss and its implications in presbycusis" for publication in PLOS Biology, and thank you for addressing the reviewer and editorial requests in your revision. On behalf of my colleagues and the Academic Editor, Manuel S. Malmierca, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.Please take a minute to log into Editorial Manager at PRESSbiologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. Sincerely, Lucas Smith, Ph.D.Senior EditorPLOS Biologylsmith@plos.org


We present a multi-stage neural network approach for 3D reconstruction of coronary artery trees from uncalibrated 2D X-ray angiography images. This method uses several binarized images from different angles to reconstruct a 3D coronary tree without any knowledge of image acquisition parameters. The method consists of a single backbone network and separate stages for vessel centerline and radius reconstruction. The output is an analytical matrix representation of the coronary tree suitable for downstream applications such as hemodynamic modeling of local vessel narrowing . The network was trained using a dataset of synthetic coronary trees from a vessel generator informed by both clinical image data and literature values on coronary anatomy. Our multi-stage network achieved sub-pixel accuracy in reconstructing vessel radius (RMSE = 0.16 ± 0.07 mm) and stenosis radius (MAE = 0.27 ± 0.18 mm), the most important feature used to inform diagnostic decisions. The network also led to 52% and 38% reduction in vessel centerline reconstruction errors compared to a single-stage network and projective geometry-based methods, respectively. Our method demonstrated robustness to overcome challenges such as vessel foreshortening or overlap in the input images. This work is an important step towards automated analysis of anatomic and functional disease severity in the coronary arteries. Coronary Artery Disease (CAD) occurs when atherosclerotic plaque accumulates in the coronary arteries, leading to a local narrowing known as stenosis2. The most common imaging modality used to diagnose CAD is X-ray angiography, in which 2D X-ray images are acquired as radio-opaque dye is injected into the coronaries. This allows the cardiologist to visualize and estimate diameter reduction at regions of stenosis.The coronary circulation is divided between the right and left coronary trees, each of which supply blood to different regions of the heart muscle4. In recent years, there have been numerous efforts to derive computational estimates of those metrics or to propose new metrics such as quantitative flow ratio (QFR) that do not rely on invasive pressure wires10. A key step in generating computational estimates through modeling approaches is defining the coronary tree geometry of the patient. Creating such a 3D model is relatively straightforward when using 3D medical image data such as computed tomography angiography (CTA). However, 2D X-ray angiography requires a method to accurately reconstruct the 3D geometry from a series of images.Although anatomical assessment of stenosis severity is the most common diagnostic practice, functional metrics which take hemodynamic parameters into account through the use of invasive pressure wires placed in the coronaries, such as fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR), have led to better diagnostic outcomes25. An overview of these methods is given in "26. Furthermore, projection-based methods are susceptible to how clearly visible all coronary branches are in each angiography image. For example, overlapping branches and vessel foreshortening are two challenges which can introduce uncertainty into the reconstructed 3D geometry.Many groups have proposed coronary reconstruction algorithms using the principles of projective geometry and stereovision27 and Jiang et al.9 used neural networks to automate coronary segmentation, a key pre-processing step to identify the vessels in the angiography images. They then used projective geometry methods to reconstruct the 3D coronary tree given the segmented angiography images as input. This approach eliminated some drawbacks of previous methods by automating vessel segmentation and point correspondence; however, they are still susceptible to errors in recorded image acquisition angle or reconstruction error due to vessel foreshortening. A fully machine learning-based method has the potential to perform automated reconstruction without the need for imaging calibration. Unlike traditional projection-based methods, a deep learning method has the potential to perform well even on challenging input images with vessel foreshortening or overlap.Recently, a few groups have performed 3D reconstruction of coronary trees from X-ray angiography images using a combination of machine learning and projective geometry. Bappy et al.27 and Jiang et al.9 used machine learning for vessel segmentation; however, both relied on projective geometry for the 3D reconstruction task. Here, we have used a neural network to directly learn the 3D geometry of a coronary tree without relying on projective geometry principles. The major advantage of this method is that it does not require knowledge of precise imaging angles, thereby eliminating the need for calibration or correction algorithms to determine imaging parameters. Another advantage is that the neural network could potentially overcome limitations in the input image such as vessel foreshortening, which can hamper projection-based reconstruction. The neural network was trained using analytical 3D coronary trees from a synthetic vessel generator. The advantage of such an analytical representation of the geometry is that it makes it easy to perform parametric hemodynamic analyses. A proof-of-concept example of such analysis is also included here.The main contribution of this work is a multi-stage neural network for 3D reconstruction from 2D segmented X-ray angiographic images which outputs an analytical representation of a 3D coronary tree. To our knowledge, this work is the first purely machine learning approach for 3D coronary tree reconstruction using X-ray angiography alone. Both Bappy et al.In this work, we demonstrate smaller reconstruction errors compared to a single-stage neural network "" and tra28.We now present an overview of 3D coronary reconstruction from single or biplane X-ray angiography images. Typically, two or three 2D X-ray angiography images are acquired from different angles to visualize the coronary arteries. First, the position of the X-ray sources and image planes must be located in 3D space using the parameters recorded during image acquisition. Then, the 3D geometry of the vessels can then be recovered using one of two broad classes of methods: centerline projection-based methods and deformable model-based methods. A comparison of methods from the last 10 years is given in Table 21. Many of these algorithms employ segmentation or Hessian-based filtering of the X-ray angiography images as a preprocessing step to identify the 2D vessel centerline points more easily. Meanwhile, the second class of methods projects a template 3D vessel onto the angiographic images and uses an energy function such as generalized gradient vector flow to iteratively deform the 3D template until it aligns with the projected images25. The initial template is often a cylinder representing a single vessel, which is projected onto each of the input images. An energy function is used to deform the 2D projection to match the angiography images, and the forces from the 2D images are projected and applied to the 3D template. This iterative process is repeated for each branch.In the first set of algorithms, epipolar geometry or other constraints are used to identify corresponding vessel centerline points or segments in the 2D angiography images. These points or segments are projected into 3D space to recover the shape of the vessels27 developed a method for automatic coronary reconstruction using biplane angiograms acquired 90° apart. First, a multi-resolution U-Net was used for fast coronary segmentation. Vessel centerlines were extracted and automatically paired using branch endpoints and features, then projected into 3D space. Jiang et al.9 used a combination of X-ray angiography and IVUS images for their reconstruction method. Their method involved directly extracting vessel centerlines from the angiography images by calculating the first and second derivatives of the image, and using a deep neural network to segment vessel contours from the IVUS images. Centerlines were projected using point-to-point correspondence to generate the 3D centerline, while IVUS images were re-aligned and used to accurately reconstruct the vessel lumen. While these methods used neural networks for segmentation, they still relied on projective geometry for 3D reconstruction. We aim to improve upon previous work by using neural networks for the 3D reconstruction task itself. In addition to eliminating the need for image calibration procedures or algorithms, a deep learning method would improve the automation and speed of coronary reconstruction.More recently, Bappy et al.20, wave-propagation12, spline-fitting to user-defined control points24, or active contour methods25 are typically used to delineate the vessels or their centerlines. Therefore, the input to our network is two or three segmented angiograms for a given coronary tree, with a Euclidean distance transform applied to create a smooth field that implicitly encodes both vessel centerlines and radii  and radii (stage 2) for each branch in the coronary tree. We refer to the network as multi-stage because it has two separate regression heads, or multilayer perceptrons (MLP), to learn the 3D vessel centerline coordinates and radii. This approach is in contrast to a single-stage network with a single regression head to learn both the centerline coordinates and radii. The centerline and radius stages both shared a convolutional neural network backbone to learn relevant features of the coronary tree from the input images. In this work, we chose to use ResNet 101In the centerline stage, the final fully connected layer of the backbone network was replaced by a multilayer perceptron (MLP) with Rectified Linear Unit (ReLU) activation and batch normalization between layers. The MLP was composed of 4 hidden layers, where the first 3 layers had 1024 neurons and the last layer had 512 neurons. The output of the centerline MLP was a Meanwhile, the radius stage replaced the final layer of the backbone with a separate MLP for each branch, for a total of The outputs of both stages were concatenated to form an mentclass2pt{minimThe radius stage was trained using the mean squared error as the loss function:An Adam optimizer with learning rate 5e−4 and weight decay (L2 regularization) was used to train both stages. Batch size was set to 8 and the multi-stage network was trained for 300 epochs. For the radius MLPs, the backbone was frozen after the initial 300 epochs and each branch MLP was trained for an additional 50 epochs. We did not observe a notable improvement in accuracy when retraining the backbone network for the centerline and radius tasks.We now present the single-stage counterpart to our multi-stage network for comparison. The single-stage network architecture was composed of the backbone network and a single MLP which outputs an mentclass2pt{minimHere, 29 could be used to convert clinical angiograms obtained during routine patient care in the future into a suitable input for our network.To train the proposed multi-staged neural network, we require hundreds or thousands of ground truth 3D coronary trees and their corresponding segmented 2D angiograms. In practice, this means that we must identify thousands of patients with both 3D CTA data and 2D X-ray angiograms, which is typically not feasible in single-center studies such as ours. Another challenge of using clinical image data as input is that the coronaries deform in each frame of an X-ray angiography series due to the contraction of the heart. This necessitates temporal registration of frames from multiple angiographic series to create a valid set of input images for 3D coronary tree reconstruction. To produce a large enough dataset and eliminate external sources of error such as temporal registration, we devised a method to produce a sufficiently large training dataset consisting of 5000 static 3D coronary tree geometries and their corresponding sets of 2D projections. While we have used synthetic data to train and validate our 3D reconstruction network, the use of synthetic projection images as input does not preclude future clinical application. A segmentation algorithm or neural network such as AngioNet31 makes reconstruction more challenging. The method includes two steps: (1) a synthetic 3D coronary tree generator, and (2) a projection algorithm to create sets of segmented angiograms. A brief description of these steps is provided next.This work focuses on 3D reconstruction of the right coronary tree as the large anatomical variation in the left coronary arteriesStep 1: Synthetic 3D coronary tree generator: Fig. 32 software.Vessel centerline splines and radii are manually extracted from 3D CTA images using Cardiovascular Modelling and Simulation (CRIMSON)The range of values for centerline control points and vessel radius . The dataset was thus composed of 5000 total synthetically generated coronary trees, each defined by an In this section, we present several methods to evaluate the performance of the proposed method. We first compared the reconstruction error of our multi-stage network against a single-stage neural network to demonstrate the advantages of the multi-stage approach. We then performed a head-to-head comparison between our neural network reconstruction method and a projection-based method. Both comparisons were performed on single vessel geometries for simplicity. Next, we examined the performance of the method in reconstructing right coronary trees. Lastly, an analysis of how geometric reconstruction error affects hemodynamics is presented.A key design feature of our network is its multi-stage nature, which considers the tasks of centerline and radius reconstruction separately. The importance of this feature is illustrated in this section via a comparison between the single- and multi-stage networks. The performance of both networks was evaluated using 10 distinct RCA synthetic vessels. For each synthetic vessel, stenosis between 20 and 90%, with increments of 5%, were introduced, for a total of 150 unique vessel geometries. Reconstruction error was assessed for each geometry. An example of a vessel geometry with 70% stenosis and its single- and multi-stage reconstructions is shown in Fig. As for radius reconstruction accuracy, mean absolute error (MAE) at the stenosis was evaluated instead of RMSE in the whole vessel due to the importance of accurately predicting stenosis severity Table . For bot11 and Vukicevic et al.12. Briefly, a point cloud approach11 was used to automatically identify up to 10 possible corresponding points for each centerline point in the reference projection image. The set of possible corresponding points was further refined using the reprojection error and ordering constraint cost function proposed in12. Matched points from both images were then back-projected and interpolated with a b-spline to create a 3D centerline.We now compare the performance of our multi-stage neural network against a projection-based method in the simplest case of a single vessel centerline reconstruction from 2 projection images. In this example, the projection-based approach was a combination of the algorithms proposed by Banerjee et al.The dataset of synthetic projection images for this comparison was defined as follows. 5 synthetic RCA vessels were first created. We considered a spherical coordinate system As seen in Fig. In this section, we measure the accuracy of the proposed method for coronary tree reconstruction, using the validation set of 500 synthetic coronary trees defined in "entclass1pt{minimaWe now consider the error in vessel radius along the centerline for all 2000 branches (500 trees with 4 branches each). The RMSE of the vessel radius was 0.16 ± 0.07 mm, which corresponds to sub-pixel resolution. The error in radius was larger when comparing minimum stenosis diameter (MAE = 0.27 ± 0.18 mm), particularly for severe stenoses greater than 70% diameter reduction, consistent with the behavior reported for single vessel reconstruction in "33, a smaller backbone network than ResNet 101, and ViT (Vision Transformer)34, a larger network and one that uses attention modules instead of convolutional layers for image feature extraction. Table In this work, we chose to use ResNet 101 as the feature extraction backbone network; however, other feature extraction networks can also be used in our multi-stage network framework. Here, we investigate the performance of using EfficientNet v2The three networks produce similar errors on centerline reconstruction and overall radius reconstruction, however ResNet 101 achieves the smallest error on stenosis radius reconstruction. Figure 35.In the previous examples, we established the performance of the multi-stage neural network to reconstruct centerline and radius of vessels in coronary artery trees. However, as stated in the introduction, functional assessment of vessel disease has recently received significant attention. In this section, we investigate the quality of our proposed multi-stage neural network from a functional standpoint. Towards that end, we simulate the physics of blood flow and pressure in the reconstructed coronary trees using computational fluid dynamics (CFD) simulations and compare the results against known ground truth CFD data. Specifically, we compare distributions of pressure down the RCA vessel in ground truth coronary trees (known synthetic geometries) and their reconstructions. The pressure field is the basis to calculate well-established functional metrics of CAD such as FFR, iFR, and QFR32. Two examples are discussed: (1) a healthy coronary tree without stenosis, and (2) a diseased tree with one stenosis. Details of simulation parameters  are given in Appendix B. Results are summarized in Fig. CFD simulations with consistent inflow and outflow boundary conditions were run using the validated open-source software CRIMSONDPGT = 9.8 mmHg and DPR = 8.4 mmHg, resulting in a difference between pressure gradient estimates of Results for the healthy coronary tree are given in the top panel of Fig. GT = 18.8 mmHg and DPR = 14.3 mmHg, resulting in an error of In the diseased coronary tree, the pressure gradients over the ground truth and reconstructed RCA vessels are DPThe larger pressure error in the stenosis case compared to the healthy case can be attributed to the radius reconstruction error at the stenosis. Given a vessel with radius Reconstructing 3D geometries from sets of 2D image data is a challenging task that often necessitates accurate knowledge of imaging parameters such as angles and distances during image acquisition. Uncertainty of these parameters poses a large challenge for projective geometry approaches to reconstruct the coronaries from X-ray angiography images; therefore, we have developed and tested a novel multi-stage neural network method for 3D coronary reconstruction.The most salient feature of the proposed network is its multi-stage nature, which enables accurate reconstruction of centerline paths and vessel radii. When considering the single-stage approach to reconstructing vessel geometry, we observed that the network was able to accurately capture the trend of linearly decreasing radius along the vessel but could not capture the stenoses "". This iThis observation motivated going one step further for the coronary trees and using a separate MLP to learn the radii for each vessel. This was necessary since not all vessel branches contained stenoses; if the network was trained to predict the radii of all branches at the same time, the stenoses would once again make up less than 2% of the The size of the MLPs for the centerline and radius tasks was determined by testing networks with a varying number of hidden layers and neurons per layer. Networks with 128, 256, 512, 1024, 2048, and 4096 neurons in each of 1–4 hidden layers were evaluated for both centerline and radius accuracy. For the centerline MLPs, the minimum error was achieved when using 4 hidden layers. The number of neurons per layer did not greatly affect mean squared error, which ranged from 2.72 to 2.91 mm in the validation set of 500 synthetic coronary trees. Therefore, a network with 1024 neurons per layer was chosen as it was of the same order of magnitude as the network output size , smaller networks performed better at regions of stenosis. The MAE at the stenosis for a network with 128 neurons was 0.27 mm, compared to 0.40 mm and 0.42 mm for networks with 2048 and 4096 neurons, respectively. Thus, we determined that the optimal size for the radius network was 3 hidden layers with 128 neurons each.25. Despite their success, projection-based methods have limitations such as the need to identify corresponding features in the input images, and precise information on the image acquisition angles. Overlapping branches and foreshortening are two key challenges leading to uncertainty in the reconstructed geometry using projective geometry methods.3D reconstruction of coronary trees from 2D X-ray angiographic images has been generally performed using projective geometry and stereovision techniquesThe key contribution of this paper is a neural network for reconstructing 3D coronary trees that aims to overcome the limitations of projective geometry methods. Our network was trained over a wide range of angles between images The higher robustness to foreshortening and overlap in the input images of the neural network method is further supported by the qualitative examples in Fig. The main limitation of the neural network compared to projection methods is that it does not capture all the bends in the vessel centerline path despite accurately following the average path. The lack of tortuosity leads to a shorter reconstructed vessel than the ground truth, despite the length regularization term in the loss function Eq. . This beThe neural network was trained using a MSE loss, which only accounts for point-wise differences between ground truth and reconstructed centerlines. A loss function that incorporates more global shape information such as curvature in addition to the vessel length regularization could improve the centerline path accuracy.When applying the multi-stage neural network to reconstructing a whole coronary tree, the reconstructions captured the average path of all branches in the tree but under-estimated their tortuosity, similar to the behavior observed for single-vessel reconstruction. This appears to correspond to a well-known curve-fitting regression phenomenon: when the network is not large enough to capture the full complexity of a dataset, it tends to predict its average behavior. We hypothesized that increasing the complexity of the neural network by increasing the size of the MLP may alleviate this issue. We tested this hypothesis by performing a scaling study in "As seen in "Another challenge of coronary tree reconstruction compared to single vessel reconstruction was to accurately predict stenoses radius. As described in "While changing the network backbone had little effect on centerline coordinate reconstruction, a much larger effect was observed in stenosis radius reconstruction. In Fig. The last application example explored the impact of geometric reconstruction errors on hemodynamic indices such as pressure. The simple test examples demonstrated that gradients of pressure (a surrogate of FFR) compared well between ground truth and reconstructed geometries, even though the neural network centerlines were not as tortuous as the ground truth.The error in radius had a much larger impact on the pressure drop than the centerline error, particularly errors in stenosis radius. As seen in the stenosis example, the neural network approximated two serial stenoses (51% and 57% radius reduction) as a single stenosis with 53% radius reduction. Despite only a 0.07 mm difference in stenosis radius, there was a noticeable difference in pressure profiles across the stenosis between the 2 geometries. However, the gradients of pressure across the stenosis were relatively similar between the 2 cases (less than 1% difference). We hypothesize that accurately capturing the overall vessel lengths and stenosis radii contributed to the accurate estimation of gradients across the vessels. However, at higher levels of stenosis, even small errors in reconstructed radius can lead to large differences in pressure gradients across the vessels.38.Given the high sensitivity of vessel resistance to the reconstructed vessel radius (Future work will focus on increasing the scope of the dataset to match real-world data more closely. In terms of synthetic data generation, we will expand our tree generator to incorporate further variations in the number and types of branches in the right and left coronary trees. One assumption of our synthetic dataset is that the patient is not moved between image acquisitions; however, in the catheterization lab, it is common practice to move the patient table during image acquisition to follow the flow of injected dye. Since the motion is controlled via a joystick and not often recorded, clinical angiograms are challenging to use as inputs for projection-based reconstruction methods because they require precise knowledge of image acquisition parameters. Neural networks may therefore provide a robust alternative for 3D reconstruction of coronary trees when those parameters are unknown. We can mimic the clinical workflow in our synthetic data generation by randomly translating the synthetic 3D coronary tree while generating binary angiography images. This would help our method to generalize better to clinical data since we would eliminate the assumption that the patient is not moved between image acquisitions. Radius reconstruction, particularly at the stenosis, can be improved by alternate strategies to encode the 3D coronary geometry and stenoses. Although we found that training a separate MLP to learn the radius along each branch led to better stenosis reconstruction, this strategy may become infeasible in tree structures with many branches. Changing the matrix representation of the coronary tree to emphasize stenoses could lead to better performance using one radius MLP. For example, the vessel radii could be encoded in a vector containing the proximal radius, distal radius, and parametric position and severity of the stenoses for each branch.39 could benefit our multi-stage network as well.As discussed in "Besides the synthetic projection images considered in this paper, our multi-stage neural network can be applied to clinical X-ray angiograms that have been processed with a segmentation algorithm to create binary input images. Clinical angiograms bring additional challenges, such as the motion of the coronary tree during the cardiac cycle. Care must be taken to identify angiographic frames acquired at the same point in the cardiac cycle to reconstruct a valid 3D coronary tree from clinical images.In conclusion, in this work we have presented a proof-of-concept of a multi-stage neural network that can be used for 3D reconstruction of coronary trees from sets of uncalibrated X-ray angiography images, with sub-pixel resolution of vessel radius. We have demonstrated that reconstruction error is at an acceptable level to accurately model hemodynamic quantities such as pressure gradients across a vessel.Supplementary Information.


Tuberous sclerosis complex is a rare autosomal dominant genetic disorder that affects multiple organ systems, primarily affecting the central nervous system. It develops with a pathogenic mutation in tumour suppressor genes i.e. Tuberous Sclerosis Complex 1 or Tuberous Sclerosis Complex 2 which codes for protein hamartin and tuberin leading to unopposed hyperactivation of the mammalian target of the rapamycin signalling pathway. It presents with a triad of facial angiofibroma, intellectual disability, and epilepsy. We present a case of a 17-month female toddler with abnormal body movement with loss of consciousness and later developing into generalised jerky movements. On magnetic resonance imaging, a diagnosis of tuberous sclerosis was made. The patient underwent symptomatic management with anti-epileptic. As seizures in these cases are subtle, they remain undiagnosed for a long time leading to delays in management and developing refractory seizures. The informant (her mother) also gave a history of loss of consciousness and unresponsiveness to vocal or tactile stimuli with clenching of teeth and urinary and stool incontinence. After a few min, abnormal body movements generalised to the right upper limb and bilateral lower limbs in the form of jerky movements that lasted for more than 30 min. She was managed with phenytoin and levetiracetam injections and then referred to our hospital for paediatric intensive care unit (PICU) admission. She also had a past history of abnormal stiffening of the bilateral upper limb with focal motor seizures of fingers since 12 months of age. She had a family history of seizure disorder with her cousin and uncle.On examination, the general condition was sedated and febrile. The pulse rate was 132 beats per min with irregular character, respiratory rate 32 times per min, body temperature 101.2°F, oxygen saturation 98% measured in the fetal monitor, and capillary refill time of fewer than 3 seconds. Hypopigmented macules were present over the forehead, right side of the face, right knee, left arm, and back .Routine laboratory tests were unremarkable. Computed tomography (CT) scan of the head showed subcortical hypodensities in the bilateral frontal, temporal and parietal lobes with calcified nodules along the margin of bilateral lateral ventricles .Magnetic resonance imaging (MRI) of the brain revealed cortical tubers with cystic changes in the grey matter of bilateral cerebral hemispheres along with sub-ependymal nodules adjacent to bilateral lateral ventricles. Few of those nodules are calcified. Ultrasound of the abdomen and pelvis reported normal findings. Electroencephalogram (EEG) showed an interictal record of 7-10 Hz alpha activity with generalised slowing to theta and delta range. Intermittent spike slow wave complexes are seen mainly arising from the right side.Supportive management of uncontrolled seizure was done with two anti-epileptic drugs (injection levetiracetam and injection phenytoin). CT head was done suggestive of tuberous sclerosis. The ophthalmological evaluation showed no papilledema or any retinal haemorrhages. Oral antibiotic syrup cefpodoxime was added for persistent fever and raised c-reactive protein (CRP). The patient was shifted to the ward on the third day and again shifted to PICU for 2-3 episodes of seizure. Injection sodium valproate was added. After 2 days of seizure-free episodes, the patient was shifted to the ward and discharged on the eighth day of admission.At the time of discharge, the patient was seizure free and afebrile for more than 3 days and was tolerating oral feed without abdominal distension and vomiting and is passing stool and urine normally She was recovering well with a normal state of health as stated by investigation reports. She was prescribed syrup cefpodoxime 2.5 ml twice a day for 2 days, syrup vitamin B-complex 5 ml once a day for 2 weeks, and advised to continue syrup sodium valproate 3 ml, tablet phenytoin 40 mg and syrup levetiracetam 3 ml per oral twice a day. And to use aerosol midazolam 2 puffs intranasal during seizure attacks. The patient was then discharged with advice for follow-up in the pediatric outpatient department (OPD) after 4 days with EEG and MRI brain report and tapering of phenytoin on follow-up. Counselling was done with a proper explanation of danger signs to the informant. On follow-up, an MRI brain report showed cystic changes in bilateral cerebral hemispheres for which she was advised to drug continuation. The patient party was advised for follow-up in the duration of 3 months regularly. According to the follow-up, she is within a normal state of health and is in continuation of the drugs prescribed to date.4 Mostly TSC shows a triad of facial angiofibroma, intellectual disability, and epilepsy.5 It develops with a mutation in tumor suppressor genes i.e. TSC1 (chromosome 9q34)6 and TSC2 genes (chromosome 16p13.3)6 which codes for hamartin and tuberin respectively that leads to permanent activation of the mammalian target of the rapamycin (mTOR) pathway affecting cellular proliferation4 which later results into dysfunction of affected organs with hamartomas and focal dysplastic lesions formation.7 TSC2 genes are more common to be involved with more severe neurological dysfunction as compared to TSC1 gene which can be identified by genetic testing.7 Almost two-thirds of all cases of TSC are sporadic type but in our case, it is familial in origin.1TSC is also known as epiloia or pringle-bourneville phacomatosis.8 As according to the International tuberous sclerosis complex consensus conference 2012 diagnostic criteria for TSC comprises two major features or one major feature with two or more minor features.9 Similar to that in our case the patient presented with three major features i.e. more than three hypomelanotic macules which were greater than 5 mm in diameter, cortical tubers with cystic changes in bilateral cerebral hemispheres along with bilateral sub ependymal nodules adjacent to bilateral lateral ventricles and was diagnosed of tuberous sclerosis.Most patients present with TSC as a newborn or as a young child as seen in this case but sometimes they develop elusive symptoms which are not diagnosed till adulthood.10, which might progress into status epilepticus (SE) or refractory seizure.11 Different risk factors are associated with progressions like non-compliance to treatment, fatigue, infections, intellectual disability, and symptomatic epilepsy.11 SE and refractory seizure are usually associated with increased neurological and behavioural morbidity and mortality requiring urgent medical care.10 However, there is very little literature on TSC-associated SE, and among those very few were of non-convulsive SE (NCSE). Two cases of TSC-associated NSCE were reported in 2019.11 In our case the patient presented with NCSE initially which later on progressed to generalized convulsive seizure.The most prevalent presentation of TSC is epilepsy 12 Immediate treatment with anti-epileptic after seizure-onset in children decreases the risk of neurodevelopmental complications but about 50-60% of them may develop intellectual disability further.12 A case of epilepsy-associated TSC was reported in 2022 where levetiracetam was given for the management of epilepsy.13 Similar to that, in our case patient was treated with levetiracetam along with phenytoin as soon as possible to prevent developing complications. Counselling of the patient party was done about the risk of seizures and suggested to visit a neurologist when seizures occurred. Each lesion or symptom is highly variable with respect to age,7 onset, and severity of organ involvement so the management of TSC patients requires life-long follow-up.7Antiepileptic treatments are recommended after two unprovoked clinical seizures or after one seizure in patients at high risk of recurrent seizures.With the absence of the facility of genetic testing, we had limitations that could not distinguish the mutated gene (i.e. TSC 1 or 2) in our patient. As there might be subtle symptoms presented in patients so it can be underdiagnosed till adulthood when the seizures could develop into a refractory type. So during investigations, the seizure presented should be assumed to be repeated, and the history of previous seizure episodes should be asked thoroughly for appropriate treatment.


The aim of this study is to find whether prescribed drug therapy was associated with the increased risk of death from asthma and life-threatening attacks (LTA). The “case” group comprised those under 35 years of age who expired or experienced LTA from January 1994 through December 1996. For each case, an age and sex matched control was selected from asthma patients. Hospital records were reviewed to obtain information on the prescribed drug therapy and clinical asthma severity for the cases and controls. Bivariate analysis with conditional logistic regression models for matched data sets were used to estimate the severity-adjusted odds ratios for each asthma medication. Twenty-four fatal cases and 54 LTA cases were observed. The crude odds ratio of clinical severity  was larger than unity and with statistical significance. After adjusting for clinical severity, the odds ratios computed for all β2-agonists delivered by metered dose inhaler (MDI) increased  from that of crude analysis. Among those subjects under 20 years of age, the clinical severity-adjusted odds ratio for the use of all β2-agonists by MDI  was higher than that of all subjects. The prescription of β2-agonists by MDI increased the risk of asthma death after taking clinical severity into account. Although not statistically significant, our results suggested that β2-agonists administered by a MDI might have increased the risk of asthma death and LTA in Japan because the magnitude of the effect was similar to that reported in other countries.Sales of inhaled β In the 1980s, a relation between inhaled fenoterol and increased risk of death from asthma was reported from New Zealand4-7). However, these findings might be due to a tendency to prescribe the drug more frequently for patients with severe asthma8).In the 1960s, asthma mortality was alluded to during an epidemic on the basis of an ecologic study in England and Wales4-11). Opinion is still divided: one is that fenoterol was not associated with severe life-threatening attacks (LTA) after controlling severity8,10); and the other is that the association between fenoterol and asthma death was significant, but after adjustment for markers of asthma severity was made, there was no difference in the magnitude of the association between fenoterol and asthma death4-7,9,11). In this paper, we report the results of a case-control study on prescribed drug therapy, severity, and the risk of death from asthma and LTA in Japan.Many investigations were conducted to elucidate uncertainty about the confounding between severity and fenoterol useIn January 1998, a questionnaire on the clinical courses of asthma patients (LTA or death from asthma) was mailed to the departments of internal medicine, respiratory medicine, allergy and collagen diseases, and pediatrics of hospitals with 100 or more beds throughout Japan. The criteria for eligible cases were: 1) asthma patients aged under 35 with LTA or death from asthma during a three-year period from January 1994 through December 1996 and 2) under asthma therapy at the department of the hospital during a one-year period before LTA or death. LTA is defined as requiring airway intubation or assisted ventilation and resulting in a loss of consciousness due to asthma. Of the 8,454 departments, 2,711 responded and 466 reported that they had treated qualifying cases.In January 1999, physicians working in the 466 department that have treated qualifying cases were asked whether they had also treated eligible controls. A control was defined as one that required asthma treatment from the department of the hospital during the one-year period but who had never experienced LTA until the time the case subject experienced it or expired. If the department had both eligible cases and controls, one eligible control was selected for each eligible case. The control was matched for age (within 3 years) and sex. If there were two or more eligible controls in regard to age, the nearest age control was selected. If a suitable control could not be found, the selection period was extended to include 1994 through 1996. If a suitable control was still unavailable, a control of the opposite sex was selected.2-agonists delivered by a metered-dose inhaler (MDI). Exposure to a certain drug is defined as whether a drug was prescribed for the patient during the one-month period prior to the date of his or her demise or the development of LTA.Regardless of the case-control status, hospital records were reviewed to obtain information on the prescribed drug therapy and clinical asthma severity by the physicians who had reported the eligible cases and controls. The principal risk factor examined was the use of inhaled β12). In this study, clinical asthma severity was dichotomized into ‘severe’ and ‘non-severe’ groups. The former included those under 20 years of age and with a history of major asthma attacks during the one-year period prior to the date of death or LTA. The group also includes those 20 years or older who experienced exacerbation of asthma affecting physical activity or sleep, and whose activities of daily living were restricted prior to the same period. The subjects with no experience of a major asthma attack during the period were placed in the ‘non-severe’ group. Those who were asymptomatic or between exacerbations during the period were also placed in this group.To assess clinical asthma severity, criteria that consisted of clinical features and that were based on the International Consensus Report on the Diagnosis and Treatment of Asthma were chosen2-agonists by MDI, 2) β2-agonists by nebulizers, 3) oral β2-agonists, 4) oral theophyllines, 5) sodium cromoglycate, 6) inhaled corticosteroids, and 7) oral corticosteroids. The risk of particular β2-agonists  was estimated independently. Data were analyzed with the aid of the SAS statistical package (Version 6.12). Initially, univariate analyses were carried out to estimate the crude effect of each variable. Next, bivariate analyses were performed to estimate the severity-adjusted odds ratio for each asthma medication. The possibility of effect modification by age was assessed by calculating the odds ratios for two subgroups categorized by age. One group consisted of patients under 20 years of age and the other, those whose ages were 20 years or older.Conditional logistic regression models for matched data sets were used to estimate the odds ratios for the effects of asthma medications. The categories of asthma medications are : 1) βTwenty-four fatal cases, 54 with LTA, and 78 controls were observed. 2-agonists by MDI were prescribed for 51% of the cases and 41% of the controls. Fenoterol by MDI was prescribed for 17% of the cases and 15% of the controls. The crude odds ratios indicated that β2-agonists by MDI  did not augment the risk of an asthma death or LTA with statistical significance.The frequency of administering asthma medications and clinical severity are shown in an unmatched format for the cases and the controls, together with the calculated crude odds ratios . β2-agon2-agonists by a nebulizer were prescribed for 38% of the cases and 29% of the controls. Procaterol by a nebulizer  increased the risk of an asthma death or LTA more than twice; but the increase was not statistically significant. More than half of the subjects had oral β2-agonists (55% of the cases and 62% of the controls) and oral theophyllines (69% of the cases and 81% of the controls) prescribed. Those drugs-oral β2-agonists  and oral theophyllines –reduced the risk but the data were not statistically significant. Sodium cromoglycate was prescribed for 40% of both cases and controls but it did not change the risk . The proportion of subjects who had inhaled the prescribed corticosteroids was higher than for those on oral corticosteroids among both cases  and controls . Inhaled corticosteroids  showed a null value for the risk of an asthma death or LTA. Oral corticosteroids  increased the risk but the result was not statistically significant. The proportion of cases with clinically severe asthma was higher (49%) than the controls (17%). The odds ratio for clinical severity  was higher than unity, with statistical significance.β2-agonists by MDI. The odds ratio of oral corticosteroids decreased from 2.00 to 0.65 when the clinical severity was adjusted. Oral β2-agonists  and oral theophyllines  reduced the risk with statistical significance. Salbutamol administered by a nebulizer  reduced the risk but without significance. Among β2-agonists delivered by MDI, the severity-adjusted odds ratios of fenoterol  and salbutamol  were higher than the crude odds ratios.The severity-adjusted odds ratios for each asthma medication, using bivariate analysis, are shown in 2-agonists , fenoterol administered , and salbutamol, all delivered by MDI, were greater than those for all of the subjects, although the differences were not statistically significant. Salbutamol administered by a nebulizer  decreased the risk but the difference was not statistically significant.The severity-adjusted odds ratios of each asthma medication given to the subjects under 20 years of age were estimated by bivariate analysis. The frequency of asthma medications and severity-adjusted odds ratios are shown in 2-agonists , oral theophyllines , sodium cromoglycate , and inhaled corticosteroids  appeared to have decreased the risk. The odds ratios for all β2-agonists, all administered by MDI , fenoterol , and salbutamol  were greater than unity, but lower than those of subjects under 20 years. Salbutamol given by a nebulizer  did not decrease the risk, as observed in the subjects under 20 years.2-agonists delivered by MDI are related to the risk of asthma deaths and LTA after controlling for the clinical severity of asthma. The results suggest that self-administration of β2-agonists via MDI inhalation increases the risk of asthma death after accounting for the clinical severity, especially in subjects under 20 years of age.The aim of this study was to determine whether a prescribed drug therapy was associated with the risk of death from asthma and LTA. The starting hypothesis was that β4-7,10,11,13,14), the use of three or more categories of drugs4-7,10,11,13,14), and the prescription of oral corticosteroids4-7,9,10,11,13,14). In this study, the clinical severity was defined in terms of asthma symptoms and the frequency of exacerbation. Only clinical severity was adjusted in bivariate analysis with conditional logistic regression models for matched data sets. Because the management of asthma is considered to control its symptoms, adjusting the clinical severity of asthma and the pattern of asthma drug administration may simultaneously cause problems in multicolinearity. In discussing the relationship between β2-agonists administered by MDI and asthma death, it is necessary to consider severity15). In this study, the crude odds ratio for oral corticosteroids decreased when adjustments were made for clinical severity. This indicates that the unadjusted findings were probably largely affected by the confounding effect of clinical severity.In other studies, there were three types of parameters for clinical severity of asthma were given: hospital admission in the 12 months prior to asthmatic death of LTA2-agonists given by MDI were not statistically significant, although they were larger than unity. It was reported that the entire class of selective β2-agonists was associated with an elevated asthma mortality16). There were differences among β2-agonists administered by MDI vis-à-vis the risk of death from asthma and LTA. After adjusting for the severity, an increased risk with fenoterol4-7) and albuterol9,16), no relationship in salbutamol4,6), salmeterol13) and albuterol17), and a decreased risk with salbutamol5) were reported. In this study, salbutamol when administered by MDI, increased the risk, but when the drug was delivered by a nebulizer, the risk generally decreased. It was reported that salbutamol given by MDI decreased the relative risk of having a near-fatal attack of asthma; but administration of the same drug  via a nebulizer increased the risk13). The overall increase in risk for patients on fenoterol administered by MDI  was similar to the results obtained from an investigation conducted in New Zealand4-7). It is necessary to find out if each β2-agonist administered by MDI increases the risk of asthma death and LTA.The severity-adjusted odds ratios of β2-agonists and theophyllines decreased the risk. The findings on oral β2-agonists have been inconsistent, according to several studies4-7,10). Most do not generally show an increased risk4,5,7,10). However, an increased risk was observed in a cohort analysis9), and among subjects who were taking oral corticosteroids at admission6). Similar findings were reported for oral theophyllines4-6,14). Some have shown an increased risk14), some have not4-6). Further investigation is required with respect to the way these drugs are used.After adjusting for clinical severity in this study, oral β2-agonists inhaled by nebulizer were inconsistent among age groups. For β2-agonists given by MDI, it was reported that higher risks were observed in subjects under 20 than in their older counterparts4,5). These results are consistent with those obtained in this study. The findings may be due to differences in treatment compliance18). It has been reported that compliance to asthma treatment varies with age19).The findings for oral corticosteroids, inhaled corticosteroids and β4-7) although the results are not statistically significant. The results of a test for statistical significance rely on both the sample size and magnitude of the effect; therefore a statistically significant finding does not always provide substantial evidence in medical or public health practice. A lack of statistical significance does not provide evidence for the absence of an effect20). Our results may support the hypothesis that the self-administration of β2-agonists given by MDI increases the risk for asthma death even when clinical severity is taken into account.The findings of this study are consistent with the case-control studies conducted in New Zealand