Bipolar spectrum disorder includes Bipolar I, Bipolar II and subthreshold bipolar disorders (BD). The condition is highly prevalent, disabling and associated with high mortality. Failure of diagnosis is high. Subthreshold presentations present as 4 or more changes in polarity, are generally less responsive to standard treatment and as a result, drug combinations are often needed. High Dose Levothyroxine (HDT) has been reported to be safe and effective with this condition. Treatment response has been associated with mutations in thyroid activating enzymes and intra cerebral transporter protein carrier. Repetitive Transcranial Magnetic Stimulation (rTMS) has been shown to be effective in bipolar depression and has been proved to have neuroplastic effect. Present authors had reported clinical evidence of safe and effective use of a combination treatment protocol. Potential mechanisms of action of the combined treatment protocol and the role of mitochondria function are discussed. Bipolar Disorder (BD) is a common  and disaThe understanding of BD is evolving over the years with changes into the definition and criteria for classification. DSM 5 refers to Bipolar and Related disorders and describes BD I, II, NOS and Cyclothymic disorder , and incIn this paper we discuss the challenges and gaps in existing guidelines for the treatment of BSD, focusing on subthreshold presentations, the evidence supporting the use and the hypothesized mechanisms of action of Repetitive Transcranial Magnetic Stimulation (rTMS) and in more extent High Dose Levothyroxine (HDT). Finally, we present a new treatment protocol for the combined use of rTMS and HDT and the hypothesized mechanism of action namely restoration of mitochondrial dysfunction.Alongside the evolvement of understanding of the BSD, treatment guidelines are evolving too. Whereas there is an overall agreement across guidelines regarding the treatment of BD I, BD II, there is far less scientific evidence and certainty regarding the treatment of the rest of the categories (subthreshold presentations) .For example, there is consensus for the role of Lithium as a long-term prophylactic treatment option for BD I, the importance of withdrawal of antidepressants and elimination of lifestyle factors that might contribute to the course or have an impact on pharmacological treatment for both I and II . In geneThe National Institute for Health and Care Excellence (NICE) guidelines make recommendations for the treatment for mania (BD I) or hypomania (BD II), treatment for bipolar depression and maintenance treatment . NICE spThe American Psychiatric Association (APA) STEP-BD program proposed a three steps approach to the treatment of Rapid Cycling presentations with (i) identification and minimization or elimination of pro-cycling factors, (ii) then optimization and if necessary, add of mood stabilizers and/or second-generation antipsychotics, and (iii) invoke novel treatments such as Levothyroxine or ECT . The MauIn conclusion the understanding of diagnosis and different forms of BD has evolved over the years. It is reported that subthreshold presentations are far more common than the typical type I and II combined. We argue that there is a paradox concerning the existing treatment evidence for the disorder. The amount of evidence of existing treatment options of what is described as subthreshold presentations, including rapid cycling presentations, is limited. Existing guidelines seems not to pay appropriate attention to this knowledge gap. Existing evidence reports poor compliance with proposed treatment options due to high incidence of side effects, also possible due to the combination of several medications and medication induced mood switch. Furthermore, it is well documented that rate of relapse is high. Hence the urgent need for better understanding of the condition and the need for treatment innovation.Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive, brain stimulation therapy with growing evidence for its use in bipolar depression. A right-side DLPFC protocol was better than left side with NNT for response of 3 and response in 60 vs. 6.6% in sham 228). Low rates of treatment emergent affective switches were reported 8. Low ra, as wellThere is accumulating evidence to suggest that the effect of rTMS on brain leading to the observed clinical benefit is due to a significant neuroplastic effect , 28. NeuThe actions of thyroid hormones are mediated via two principal hormones, thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Secretory control of these hormones is mediated via Thyroid Stimulating Hormone (TSH) secreted by the pituitary gland in response to Thyrotrope releasing hormone from the hypothalamus. T3 is the most active form of thyroid hormone and approximately 20% is produced by the thyroid gland while the majority is derived by peripheral conversion of T4 to T3; Deiodinase type 1 and type 2 , convert T4 to T3 . It is tThere are two placebo controlled RCTs using supraphysiological/HDT dosage of thyroxine. A 6-week, double-blind, randomized, placebo-controlled fixed-dose (300 mcg/d) trial, assessed the efficacy and tolerability of levothyroxine as an adjunct to continuing treatment with mood stabilizer and/or antidepressant medication for patients with BD I or II, currently depressed (as per DSM-IV) . The priSeveral studies indicated the good tolerability of treatment in BSD , as far It is unclear how exactly, HDT is of benefit to patients with BSD. A Chinese case-control association study showed that compared to controls, patients with a heterozygote polymorphism of the DiO2 gene  had a 1.6-fold increased risk of BD and a 3.75-fold increased risk if they had a homozygous variant . Thr92 substitution for Ala substitution in DiO2 (Thr92Ala) is one of the most commonly studied polymorphism of DiO2 . In addiIt is interesting that DiO2 variants will produce a wide range of different phenotypic expressions in individuals. A global DiO2 knock-out mouse model extensively studied for neurological and cognitive effects demonstrated having only reduced agility . Yet, wiAdditionally, animal studies show approximately 80% of intracellular cerebral cortex T3 was derived from local, cellular conversion of T4 to T3 via DiO2 rather than from the circulation . Thus, ain vitro studies show that Thr92Ala variants are not associated with reduced activity potentially casting some doubt on the hypothesis that Thr92Ala variants exert their effect by reducing bioavailability of T3  levels and increased lactate . ThyroidIt is curious, if not mesmerizing when a clinician encounters a patient taking HDT for BSD for the first time since they are (a) taking very high doses of Levothyroxine, (b) have blood results typically seen in the thyrotoxic range [with certain caveats reported previously ] and (c)The actions of DiO3 (and D1) promptly inactivate HDT in the peripheral tissues leading to increased levels of reverse T3 (rT3). rT3 can further inhibit production of the active hormone, T3 by inhibiting DiO1 and DiO2 . ConcentOnce DiO2 converts T4 to T3, DiO2 is inactivated through a conformational change that is mediated by a ubiquitin protein. Thus, in peripheral tissues, the greater the T4 concentration, the greater the requirement for DiO2 to inactivate it which in turn leads to reduced levels of active DiO2 and ultimately, reduced levels of T3. Thus, the higher the level of T4, the greater the reduction in DiO2 activity which serves to prevent excess, harmful production of T3.The amount of intracellular T3 can vary significantly in different tissues due in part, to the actions of the deiodinase enzymes . As an eEvidence from animal and human studies show that different tissues in the body do not respond to levothyroxine in the same manner. A study published in 2011 looking at 1,811 athyreotic patients with normal TSH levels taking levothyroxine monotherapy and 3,875 euthyroid controls showed more than 20% of the group taking Levothyroxine had significantly higher free T4 and lower free T3 levels compared to euthyroid controls, despite having normal TSH levels . This suThe combination of HDT and rTMS is novel, and as the use of rTMS was recommended in the Maudsley prescribing guidelines since 2010, and HDT since 2004, the authors combined both interventions relying specifically on the statement that there is no first-choice drug or combination recommended, in the guideline. The present authors reported data on the combination of HDT with rTMS from 20 patients with rapid cycling BD (therefore BSD) who were on multiple medications at the start of treatment, and they were treatment resistant . During p < 0.001, Cohen’s d = 2.61 (95% C.I. 1.81–2.83). Eighteen patients (32.7%) scored 0 at review .In a more recent evaluation of 55 patients, from same authors, no patient had to discontinue treatment. Fifty-three patients were in remission (96.4%), with an average duration of 2.0 years . The mean duration of time to reach initial remission was 42.6 weeks and the average dose of levothyroxine was 303.7 mcg . The average number of medications prescribed including levothyroxine was 2.0 . The Sheehan Disability Scale score strongly improved from 7.33  at the start of treatment to 1.27  at the time of review (mean change 6.06 (95% C.I. 5.36–6.77), t  = 17.23,The physiological mechanism associated with the reported effectiveness of the combination of HDT and rTMS is not known. It might be associated though with intracellular and intercellular changes associated with the combination of the effect of each of the treatment components.Mitochondrial dysfunction appears to be central in the pathogenesis of mood disorders and in the treatment of mood disorders. Gimenez-Palomo extensively reviewed the evidence for this connection, and we hypothesize that the mitochondrial dysfunction, at least largely found in BPS disorders may be due to innate intracerebral thyroid deficiency in the brain tissues. They have also highlighted markers of impaired mitochondrial function such as reduced ATP and increased lactate, also in patients with BDs . ThyroidA major mechanism by which mitochondria are transported in either anterograde or retrograde directions in axons involves their energy (ATP)-dependent movement along microtubules. ATP-dependent “motor” proteins interact with the microtubules to generate the force that moves the mitochondria in anterograde (kinesin) or retrograde (dynein) directions, respectively. Several APs (adaptor proteins) mediate the interaction of mitochondria with motor proteins, including APs that interact with kinesin  and APs that associate with dynein (dynactin). In addition, in synaptic terminals and growth cones, microtubules may be moved by myosin-mediated interactions with actin filaments. Myosin V can drive short-range movements along F-actin, as well as modulate long-range transport by pulling mitochondria away from microtubules by facilitating anchorage of mitochondria to F-actin by unknown actin–mitochondrion crosslinkers [adapted from Mattson et al. ].In simpler terms, in the combination protocol of HDT/rTMS, HDT stimulates mitochondrial generation and activity whilst rTMS induces neuroplasticity. Mitochondria play an integral role in neuroplasticity, and together they enhance and sustain remission thus countering the kindling effect in mood disorders.The authors argue that there is urgent need to focus scientific attention on the understanding of physiological mechanisms associated both with the etiology of BD as well as the effect of innovative treatment approaches. A combination of HDT and rTMS seems to be a promising treatment innovation, based on valid preclinical evidence of both genetic factors, physiological mechanism underlying the condition and explaining treatment effect. Present authors argue that the focus of treatment should be on neuronal mitochondria, rather than neurotransmitters, and have reported on efficacy, safety and compliance due to low incidence of side effects and reduced risk of relapses across the bipolar spectrum with rapid cycling, a state denoting poor response and prognosis.AZ developed the treatment protocol and contributed to the literature review and writing up of the manuscript. AL contributed to the literature review. CK contributed to the literature review and had main responsibility for writing the manuscript. All authors contributed to the article and approved the submitted version.


Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, depletion of an adherens junction protein, or blocking mesoderm invagination disrupted Pins planar polarity and spindle orientation. Furthermore, directional ablations that separated mesoderm from mitotic domains disrupted spindle orientation, suggesting that forces transmitted from mesoderm to mitotic domains can polarize Pins and orient division during gastrulation. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.Spindle orientation is often achieved by a complex of Partner of Inscuteable (Pins)/LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early  Epithelial morphogenesis and homeostasis are influenced by cell division orientation, which can occur along specific axes relative to both the epithelial plane and the polarity of an organ/organism . EpithelDrosophila neuroblasts, asymmetric division and spindle orientation is dependent on Pins/LGN recruitment to the apical cortex. Apical proteins recruit Inscuteable (Insc), which in turn recruits Pins/LGN and the spindle rotation complex. Pins null mutants exhibit defects in spindle orientation in neuroblasts /LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein. In this complex, Pins/LGN acts as a linker between Gαi and Mud, which is also bound to a Dynein-Dynactin complex. Dynein’s motor activity provides the force to rotate the spindle . In Drosroblasts . In symmroblasts . Loss ofroblasts . Furtherectively .Drosophila pupal notum and Xenopus, cell shape has been shown to affect the spindle angle through the influence of molecular cues localized at tricellular junctions, which provide a ‘memory’ for interphase cell shape  expression in pockets of cells across the embryo. These pockets of cells that divide together are termed mitotic domains . MD3 is the anterior most mitotic domain and comprises around 30 cells, MD1 is the next most anterior domain and consists of two mirrored clusters of around 40 cells, and MD5 is located just anterior to the cephalic furrow and consists of two mirrored clusters containing around 30 cells  to mark cytokinetic rings and fluorescently tagged myristoylation sequence of Gap43 to mark cell membranes. Cytokinetic rings form perpendicular to the division axis. In MDs 1, 3, and 5, cytokinetic rings are oriented roughly perpendicular to the anterior-posterior (AP) axis . To furtNotably, we found that mitotic spindles are not initially oriented with the AP axis and form with no preferential orientation. Spindles then rotated up to 90° to achieve a final orientation . DespiteBecause spindles exhibited a pronounced rotation after assembly, we examined components of the spindle rotation machinery, Pins and Mud. We discovered that MDs 1, 3, and 5 all exhibit planar cell polarity of Pins. The Pins planar cell polarity is dependent on cell cycle state: mitotic cells exhibit planar polarized Pins, whereas Pins is uniformly distributed along lateral membranes of interphase cells . We quanTo ensure Pins polarization was not a consequence of expressing non-endogenous levels of Pins, we fixed and stained embryos to localize endogenous Pins. We found that similar to Pins::YFP, endogenous Pins was planar polarized in mitotic MD1 cells . We alsoIn addition to MDs 1, 3, and 5, we examined MD14, which has previously been shown to exhibit force-dependent planar polarized divisions . MitoticWe next sought to establish whether there was a connection between Pins polarity and spindle orientation. Using embryos expressing fluorescently tagged tubulin and fluorescently tagged Pins, we monitored the timing of Pins polarization and spindle rotation. We found that Pins polarization preceded spindle rotation and is abolished shortly after anaphase, when the spindle position is finalized . The corTo test the functional requirement of Pins polarity, we analyzed spindle orientation in embryos overexpressing a myristoylated Pins. Myristoylation covalently attaches a myristoyl group to a protein so the protein associates with the plasma membrane in an unregulated manner. Overexpression of myristoylated Pins, but not wild-type Pins, caused Pins to be uniformly distributed along the membrane during mitosis and abolished Pins planar polarity . ImportaDrosophila homologue of Par 3, Bazooka (Baz), did not exhibit planar cell polarity in the MDs, despite retaining apical localization during mitosis  that we have shown to inhibit force transmission without globally depolymerizing F-actin . ActomyoTo that end, we performed laser ablation experiments to mechanically isolate MDs from the rest of the tissue. We focused on MD1 and performed a series of four cuts to isolate the domain . We wereTo test whether forces transmitted from either cephalic furrow invagination (posterior to MDs) or ventral furrow  – mesoderm invagination – are required for oriented divisions, we made directional incisions to separate MD1 from these morphogenetic movements. The cephalic furrow is closest to MDs 1, 3, and 5 and could be expected to pull the MD cells along the AP axis. We made DV oriented  laser ablations in the posterior part of MD5 to separate the dorsal head from the cephalic furrow and found that cells exhibited oriented divisions, occurring with an average division angle similar to control sham ablations  . In contThe divisions in MDs 1, 3, and 5 occur during gastrulation, ventral furrow formation pulls ectoderm cells around the circumference of the embryo , and sepHere, we discovered that Pins exhibits planar cell polarity in mitotic domains of the dorsal head  and along the ventral midline (MD14). We demonstrated that Pins polarity is required for spindle orientation by overexpressing a myristoylated version of Pins that exhibits unregulated cortical localization and disrupts spindle orientation. We found that Pins planar cell polarity and AP spindle orientation depend on adherens junctions. This and our finding that actomyosin disruption also prevents Pins planar cell polarity led us to hypothesize that force transmission through the tissue orients Pins and cell divisions. We showed that forces from ventral furrow formation were critical for spindle orientation, by (1) mechanically isolating MD1 cells from all surrounding tissue . No. 1.5 coverslips coated in glue were attached to the slide to use as spacers and a No. 1 coverslip was attached on top to create a chamber. Halocarbon 27 oil was used to fill the chamber. All imaging took place at 23–25°C.For imaging fixed embryos, embryos were dechorionated in 50% bleach (Chlorox) for 2 min, washed in water, and then fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer at pH 7.4 with 50% heptane (Alfa Aesar) for 30 min and manually devitellinized (TLR mutant stains) or devitellinized by removing fixative, adding 50% methanol (Sigma Aldrich), and vortexing (Pins and Mud). Manually devitellinized embryos were stored in 0.01% Tween 20 in PBS (PBS-T and embryos devitellinized with 50% methanol were stored in 100% methanol at −20°C and rehydrated in PBS-T).Embryos were washed in PBS-T, blocked with 10% BSA in PBS-T, and incubated with antibodies diluted in PBS-T. Embryos were incubated with primary antibodies at room temperature for 2 hr. Antibodies used in this study are listed in All images were taken on a Zeiss LSM 710 confocal microscope with a 40×/1.2 Apochromat water objective lens, an argon-ion, 561 nm diode, 594 nm HeNe, 633 HeNe laser, and Zen software. The pinhole was set to 1 airy unit (au) for fixed imaging and 2.5 au for live imaging. For two-color live imaging, band-pass filters were set at ~490–565 nm for GFP and ~590–690 nm for mCherry (mCh). For three-color imaging, band-pass filters were set at ~480–560 nm for Alexa Fluor 488, ~580–635 nm for Alexa Fluor 568, and ~660–750 nm for Alexa Fluor 647.TAATACGACTCACTATAGGGAGACCAC) followed by the following recognition sequences:Primers included the sequence of the T7 promoter (AGTTTGAATCGAAACGCGAGToll 2 F: GGACACTGCACCGGATGTToll 2 R: ATCGGCCAAAAAGAGCAGTAToll 6 F: AGCAGCGTGTGCAGATTATTToll 6 R: ATGCGTACCATTTTCCTACCAToll 8 F: TTTTTACTCTTCGCTTTTTCGCToll 8 R: CGGAACCGAAACGTGACTATSnα-F: GCGGTAGTTTTTGGCATGATSnα-R: Primer pairs were used to amplify a PCR product from genomic DNA. PCR products were directly used in a transcription reaction with T7 polymerase using the MEGAscript transcription kit (Ambion). The reaction was placed in boiling water and allowed to cool to room temperature to promote annealing. RNA was extracted with phenol:chloroform, washed with ethanol, and resuspended in 0.1 M TE buffer.Stage 2 embryos were dechorionated in 50% bleach for 2 min. The embryos were mounted onto glass slides and desiccated for 4 min using Drierite. Embryos were covered with a 3:1 mixture of halocarbon 700/halocarbon 27 oils and then injected laterally. Embryos were imaged 3 hr after injecting.Embryos were dechorionated in 50% bleach for 2 min. The embryos were mounted onto glass slides and desiccated for 4 min using Drierite. Embryos were covered with a 3:1 mixture of halocarbon 700/halocarbon 27 oils and then injected laterally at the beginning of cephalic furrow formation. To disrupt F-actin we resuspended CtyoD (Enzo Life Sciences) at 0.125 mg/mL in DMSO. Embryos were imaged 3–5 min after injecting.Laser ablations were performed using a two-photon Mai-Tai laser set to 800 nm on an LSM710 confocal microscope (Zeiss) through a 40×/1.1 objective, using the Zen software (Zeiss) . For ablations, laser power was set at 20%, with a scan speed of 1.58 ms/px. Ablations were performed with four overlapping linear ROIs around one side of MD1.Sham ablations were performed with the same setup as the laser ablations except the laser power was set at 10%. This caused the ROIs to become bleached, but not ablated.http://fiji.sc/wiki/index.php/Fiji). A Gaussian smoothing filter (kernel = 0.75 pixel) was applied to all figure images. Brightness and contrast were adjusted to optimally visualize the mitotic spindle or Pins crescents in figure images. Control and experimental images were adjusted the same.All figure images were processed using MATLAB (MathWorks) and Fiji . Intensity was normalized to cytoplasmic intensity, which was measured as the mean gray value of a line drawn through the cytoplasm. Unless otherwise stated, 10 cells per embryo and 3 embryos per genotype were analyzed, averaged, and graphed as line plots with normalized intensity on the y axis and distance from start on the x axis.For statistical analysis, we found the point of maximum Pins intensity and averaged the troughs to either side of this point to get a peak-to-trough ratio for each cell and then compared this to the ratio achieved by using the corresponding points for Gap43. In experiments with control and experimental, the peak-to-trough ratio was found for the control and compared to the ratio achieved by using corresponding points in the experimental condition.Aspect ratios of interphase cells were taken using the polygon tool in Fiji and the Fit Ellipse measurement, which gives the major axis length, the minor axis, and the angle of the major axis. Aspect ratio is ratio between the major axis and the minor axis. The average aspect ratio was recorded and the angle of the major axis was compared to the axis of division. Unless otherwise stated, 10 cells per embryo and 3 embryos per genotype were analyzed.Statistical analyses were performed using the MATLAB statistics toolbox. p-Values were calculated using a two-sample Mann-Whitney test, the reference sample is the angle distribution in wild-type. Correlation coefficients r are Spearman correlation coefficient. p-Values are calculated against the null hypothesis r=0. This study provides compelling in vivo evidence that mechanical tension emanating from morphogenetic forces during gastrulation orients the spindle at distant sites. The finding that gastrulation-induced forces are required for asymmetric localization of an important and evolutionarily conserved spindle orientation factor, Pins, will be of broad interest to cell and developmental biologists. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  Reviewer #1 ):Drosophila embryo as their main model system. They find that cells in the head mitotic domains rotate and align their spindles within 30 degrees of the anterior-posterior axis of the embryo. The Pins protein, implicated in spindle orientation in other systems, is planar polarized in mitotic cells. Pins polarization precedes spindle rotation and is correlated with the division angle . Overexpression of myristoylated Pins results in uniform Pins distribution on the membrane and affects spindle orientation. α-catenin RNAi (but not canoe RNAi) disrupts Pins polarity and spindle orientation in MDs 1, 3 and 5. Low dose CytoD injections (which should disrupt force transmission) also result in defective Pins polarity and spindle orientations. Finally, mechanical isolation by laser ablation also disrupts spindle orientation. The authors find that preventing mesoderm invagination by snail dsRNA disrupts Pins polarity and spindle orientation in the head.Camuglia, Chanet and Martin investigate the mechanisms that control cell division orientation in vivo, using the mitotic domains (MDs) in the head of the Major1. Is there a certain chirality in the rotation of the spindles? From Video 1, it seems like in MDs 1 and 3 at least, a majority of spindles on the right side of the embryo rotate clockwise, while spindles on the left side rotate counter-clockwise? Is that so, and in that case, are there geometric/molecular considerations that could explain that chirality?We thank the reviewer for pointing this out. They are correct in that there is a tilt to the spindle orientation relative to the AP axis. To illustrate this tilt, we performed our spindle analysis separately on the right and left sides of MD1 and found that spindles on the left side align with an average division angle of about 30° from the AP axis whereas spindles on the right side align with an average division angle of -30° from the AP axis. To determine whether spindles on either side rotated with a certain chirality, we measured the fraction of times spindles rotated clockwise or counterclockwise on the left and right sides of the embryo and found there was no preference . We have added this data as Figure 1I-J and discussed in the Results lines 135-147.2. The authors are experts in mesoderm invagination, and understandably concentrate on the role that forces from that process may have in the orientation of head MD divisions. However, the cephalic furrow forms much closer to the head MDs, and in an orientation that might also explain the alignment of spindles in the head. Is cephalic furrow formation important for Pins polarity and spindle orientation in the head MDs?Drosophila embryo has shown that the head region of the embryo undergoes tissue movement in the direction of the cell division and that this is associated with mesoderm invagination .This was certainly a possibility, but our experimental results argued that mesoderm invagination is most relevant. For example, neither α-catenin or Snail depletion block the cephalic furrow, but do block mesoderm invagination. Both α-catenin and Snail depletion strikingly disrupted spindle orientation and Pins localization, which suggests mesoderm is most important. Furthermore, light sheet imaging of the To further test the importance of mesoderm invagination, we performed additional ablation experiments to disrupt forces transmitted to the mitotic domains from distinct embryo regions. Consistent with our original hypothesis and previous data, we discovered that separation of MD1 from the cephalic furrow region had no effect on spindle orientation, but that separation from the ventral side of the embryo had a dramatic disruption of spindle orientation. We added this data as Figure 6O-P and included Supplemental Video 7 showing the result. We added to our summary of the data and how it led us to our conclusions in the first paragraph of the Discussion and a model figure illustrating the distinct cuts and their effects .3. Does expression of myristoylated Pins afect mesoderm invagination ? From Table S1 it seems that a maternal Gal4 driver was used to express myristoylated Pins, which could affect other tissues in the embryo. So it is in principle possible that effects of myristoylated Pins on mesoderm internalization/cephalic furrow formation could affect cell division orientation much like sna loss of function does, but in a mechanism that does not depend on Pins polarity. There is definitely an effect on mesoderm invagination in α-catenin RNAi (but not in canoe RNAi) embryos, so I wonder if the effect could be consistently through defects in mesoderm invagination , and Pins polarity is really dispensable for spindle orientation. Are there head-specific Gal4 drivers that could be used to drive myristoylated Pins exclusively in the head?We apologize that we did not clarify this in our initial submission. Maternal overexpression of myrPins does not obviously disrupt mesoderm internalization or cephalic furrow formation. But, we do see that targeted disruption of mesoderm internalization via a Snail depletion affects the orientation of division. Note that our paper demonstrates the effect of force transmission on Pins polarity and division orientation, which is new and the main conclusion.Drosophila early embryo morphogenesis is more complicated and will require considerably more work to understand that is beyond the scope of this study.In response to this comment we: (1) added language in the Results that states that gastrulation proceeds in myr-Pins expressing embryos (lines 208-210), and (2) Added to the Discussion of the role of these oriented divisions to morphogenesis (lines 442-458). The role of division orientation and division in general in 4. Related to the previous point, does mechanical isolation by laser ablation  affect Pins polarity? This experiment could alleviate some of my concerns above, as it certainly does not (should not?) disrupt neither mesoderm invagination nor cephalic furrow formation.We agree that it would be useful to look at Pins polarity in laser ablated embryos. Currently, we have been unable to analyze Pins polarity after laser ablation, because the ablation to fully isolate the mitotic domain bleaches fluorescent proteins, especially our Pins::FP signal, which is weaker that other expressed fusions. Also, we have shown that Pins polarity is disrupted by (1) α-cateninRNAi, (2) low dose CytoD injection, and (3) Snail depletion, all of which are expected to disrupt force generation and transmission through tissues.In response to the reviewer comment, we have determined that Pins::FP still cannot be localized in directional laser ablations, but directional ablations that separate ventral from dorsal MDs cause similar spindle misorientation as Snail depletion, which we show has a striking effect on Pins polarity. Again, remember that myr-Pins does not affect mesoderm internalization and that Snail depletion affects Pins polarity, so we have shown a clear Pins requirement independent of effects on morphogenesis.Minor1. Figure S5: I am a bit confused about the role of Toll 2, 6, 8 in orienting spindle orientation. In Figure S5D it seems that dsRNA treatment against these genes does not disrupt spindle orientation, but Figure S5F shows quite a significant (p=0.0057) effect in triple mutants. The authors favor the idea that Toll receptors do not affect spindle orientation, but the difference with the mutant should be addressed. Furthermore, what happens in MDs 3, 5 and 14 (if the germband extension defect does not affect those divisions)? Is there a difference between dsRNA and triple mutant embryos in these other MDs?We think this is a great point. We stated in the text that TLRs are not solely responsible (line 251) for spindle orientation as they do not recapitulate the random pattern of division seen in the myr-Pins expression condition. We acknowledge the differences between the dsRNA injection and TLR triple mutant in the manuscript (lines 242-247), but our data show a greater importance for the role of force transmission. We favor the idea that other mechanisms contribute to spindle orientation because even though the TLR triple mutant had a significant effect on spindle orientation, it did not recapitulate the myristoylated Pins phenotype. In addition, we did not observe planar polarized Bazooka or myosin in the dorsal head, indicating the mechanism detailed by the Zallen lab  is likely not in play within the mitotic domains. Finally, as described above, we now show that isolating MD1 specifically from the ventral domain is sufficient to entirely disrupt spindle orientation .2. No statistical analysis is provided for any of the differences in polarity between Pins and Gap43, and this should be done to demonstrate the significance of the polarization of Pins. Also, particularly for MD14, they should compare anterior vs. posterior polarity, as based on the images in Figure 2H it is not clear that there is a difference between the anterior and posterior side of cells.We thank the reviewer for this point. We have added the statistical comparison.3. Figure 2A-D: the authors propose that Pins localizes preferentially to the posterior end of cells (instead of both anterior and posterior ends) in MDs 1, 3 and 14 (and anterior in MD 5). How is the asymmetry in the distribution of Pins along the AP axis accomplished, and is there any significance to it? This should be discussed in a bit more detail .We agree the localization of Pins to the posterior end of cells in MDs 1, 3, and 14 and anterior end in MD 5 is of great interest. We added an acknowledgment of the question and discuss possible models that could explain the result (lines 467-469). Further investigation of the mechanism of this preferential localization will require extensive molecular and genetic studies and is beyond the scope of this paper.Typos1. Line 49: "one daughter cells" should be "one daughter cell".2. Line 193: "rotation. ." should be "rotation ."3. Lines 232-237: please review.4. Line 238: "epithelia cells" should be "epithelial cells".We thank the reviewers for carefully reading our manuscript. We have fixed these typos.Reviewer #1 (Significance (Required)):This is the first study to my knowledge that demonstrates the role of mechanical forces in polarizing Pins, and provides a nice model to further investigate how mechanical forces generated in one tissue may affect cell division orientation in distant ones. The paper is clear, well written, and quantitative analysis is present for most results. I have some issues with the statistics (or lack thereof) for a couple of results, and potential alternative interpretations for some experiments that in my opinion should be addressed prior to publication. Specifically, it is not clear to me if Pins polarity is at all necessary for spindle orientation in any of the examined MDs.Reviewer #2 ):Drosophila embryo. Experimental work  lead the authors to propose that this asymmetry is driven by tissue-level tension. The findings are quite interesting and the manuscript is well-written overall.Overview: In this manuscript, Camuglia et al. show Pins/LGN, which is understood to drive spindle orientation, can localize asymmetrically (with respect to the tissue plane) in the Major Comments:The authors propose that localization is driven by tissue-level tension, but the direction of the tension isn't clear from the experimental work. For example, the laser ablation experiments cut around the entire perimeter of the mitotic domain, rather than along just one tension axis. Similarly, the finding that disruption of the ventral furrow (by Snail RNAi) interferes with spindle orientation in the head is very puzzling; the furrow is (A) outside the embryonic head and (B) runs in the parallel direction to the divisions considered. The authors need to address the directionality of tension experimentally.We thank the reviewer for this comment and agreed that better defining the direction of tension would strengthen our manuscript. We showed that blocking mesoderm invagination with Snail depletion disrupts spindle orientation, despite Snail not being required for cephalic furrow formation. Light sheet data has shown that mesoderm invagination is associated with global movements throughout the embryo . Furthermore, the ventral furrow extends into the head region just past the anterior of MD5. To address the reviewer’s comments, we: (1) Performed directional laser ablations to determine the directionality of the tension that orients the spindle, and (2) Added to our Discussion more about what is said in the literature about the movements that occur in the head during mesoderm invagination (lines 456-459).As acknowledged in the text, the asymmetric enrichment of Pins in MD14 is fairly weak. Since the cells being examined here border a divot in the tissue, and might therefore be curving relative to the focal plane, it would be good to rule out the possibility that some of the asymmetry in Pins intensity is just a consequence of cell/tissue geometry. One way this could be achieved is by showing multiple focal planes.Good point. We do not think that the asymmetric Pins enrichment in MD14 is due to tissue geometry or junction tilt. (1) MD14 divides ~10-15 minutes after mesoderm invagination is completed, so the cells do not border a divot . The cells do round up, which can be seen as gaps between cells . (2) We compared Pins to GapCh and only see an enrichment with Pins . If the enrichment was due to tissue curvature or junction orientation relative to imaging axis, we would see the same enrichment in GapCh. (3) Expression of myr-Pins randomizes spindle orientation in MD14 .In Figure 3I (and 3M?), it appears that there are fewer cell divisions in the presence of myr-Pins. Is this the case? Since cell shapes change during division, and cell shapes influence tissue tension, an increase in cell divisions could lead to a change in tissue tension. This would be important to address, since tissue tension plays an important role in the proposed model.These images are not taken at the same point of MD1 division ‘wave’, there are the same number of divisions in each condition. These mitotic domains exhibit a ‘wave’ of cell division , and so the number of divisions in each image reflect the timing at which we captured the image. Quantifications involved divisions throughout this wave, but we chose images for figures which are most representative of what we observed. We added this to the text in the final version of the manuscript (lines 207-210).The α-catenin and Canoe results are a bit confusing:– The rose plot in Figure 4D doesn't show a random distribution of spindle angles, but rather a modest change; most spindles still orient in the normal range. The p value in the figure legend (0.0012) is very different from the one in the figure (5.8284e-04).– Α-catenin is the strongest way to disrupt AJs, but (A) the epithelium appears to be intact in the knockdown condition and (B) spindle orientation is impacted but not randomized. Does this mean that the knockdown is incomplete? Or is Cadherin-mediated adhesion (in which α-catenin participates) only partially responsible for force transduction?We acknowledge that perturbation using αcat RNAi does not recapitulate the complete disruption of division orientation seen in embryos expressing myr-Pins. This is likely due to the variability in the strength of RNAi knockdown, which is observed for most of the RNAi lines that we use. To address the reviewer’s comment, we added rose plots for individual embryos showing extremes in the severity of division orientation disruption . For the main plot , we have included all the data because we obviously did not want to pick and choose which embryos were used for analysis. So Figure 4D includes all the variability.Given that previous studies implicate Canoe in Pins localization, it seems important to lock down the question of whether Canoe is participating in the mechanism described in this paper. How do the authors know the extent of Canoe knockdown? As suggested by the α-catenin results (described above), is it possible that Canoe knockdown is simply not strong enough to impact spindle orientation?Aren't there genetic nulls available?snail), (2) laser ablation to show the importance of force transmission between mesoderm and mitotic domains, and (3) RNAi to deplete Canoe so that mesoderm invagination initiates and pulls on the ectoderm, but where there is clearly compromised Canoe function. This treatment did not cause any effect on spindle orientation arguing against a role of Canoe in this case. In response to the reviewers comment, we added language to the Results to indicate that it is possible that the Canoe knockdown is not strong enough and our rationale for why we did not perform the experiment in a Canoe null (lines 278-280).We thank the reviewer for bringing these points to our attention. There are certainly genetic nulls available , but the experiment suggested by the reviewer would not establish the necessity of Canoe in mitotic domain cells. This is because Canoe nulls severely disrupt mesoderm invagination more severely than RNAi , as well as affecting junctions in the ectoderm during germband extension . Therefore, we would not be able to distinguish what effect of Canoe would be responsible for the spindle orientation using a null mutation. We did better experiments, we used (1) a gene depletion that specifically compromised mesoderm invagination .There are some discrepancies between figures, legends and text.– p-values differ between figures, legends, and/or text.– Fluorescent markers are labelled differently in figures and legend – Graphs appear to show that MD3 polarizes on posterior side, but figure legend says anterior in Figure S1. Vice versa for MD5.We thank the reviewer for catching these typos. We have fixed these issues.Ideally, multichannel image overlays should be shown along with individual channels (b/w). However, it is appreciated that the fluorescent signals are exceptionally weak in this study, presenting a challenge to presentation and to quantification.We agree the overlays would be nice. However, (1) the Pins::GFP signal is weak compared to the tubulin and Gap43 signals, (2) the merge does not provide more clarity, and (3) the figures are already quite large. Therefore, we have only included the separated the images.Graph axes depicting spindle orientation would be more clear if shown in degrees, instead of normalized or in radians.We thank the reviewer for this suggestion. We have changed the graph axes to be in degrees.Reviewer #2 (Significance (Required)):Several recent studies have demonstrated that division orientation (in the tissue plane) is governed by tissue level tension. Remarkably, it appears that diverse mechanisms link tension with spindle orientation. Here the authors provide the first in vivo evidence connecting tension to the asymmetric localization of Pins, an important and evolutionarily conserved spindle orientation factor.Reviewer #3 ):Drosophila epithelia. In response to morphogenetic forces acting on adherens junctions, PINS/LGN localises to junctions in a planar polarized fashion to orient the spindle, and de-polarization of PINS/LGN prevents planar spindle orientation. The experiments are very well performed and the findings are robust. The conclusions are well supported by the data.This beautiful manuscript uncovers a role for planar polarized PINS/LGN in orienting the mitotic spindle in Reviewer #3 (Significance (Required)):Drosophila embryo. Thus, the findings underscore the highly conserved nature and in vivo relevance of this phenomenon.These important findings mirror previous work in human cell culture, but crucially reveal that the same phenomenon occurs in vivo in the We thank this reviewer for reading the manuscript and their encouraging words.


The Covid-19 pandemic has not affected the population evenly. This must be acknowledged when it comes to understanding the Covid-19 death toll and answering the question of how many life years have been lost. We use level of geriatric care to account for variation in remaining life expectancy among individuals that died during 2020. Based on a linkage of administrative registers, we estimate remaining life expectancy stratified by age, sex, and care status using an incidence-based multistate model and analyze the number of years of life lost (YLL) during 2020 in Sweden. Our results show that remaining life expectancy between individuals with and without care differs substantially. More than half of all Covid-19 deaths had a remaining life expectancy lower than 4 years. Yet, in a 1-year perspective, Covid-19 did not seem to replace other causes of death. Not considering the differences in remaining life expectancy in the affected populations overestimated YLL by 40% for women and 30% for men, or around 2 years per death. While the unadjusted YLL from Covid-19 amounted to an average of 7.5 years for women and 8.6 years for men, the corresponding YLL adjusted for care status were 5.4 and 6.6, respectively. The total number of YLL to Covid-19 in 2020 is comparable to YLL from ischemic heart disease in 2019 and 2020. Our results urge the use of subgroup specific mortality when counting the burden of Covid-19. YLL are considerably reduced when the varying susceptibility for death is considered, but even if most lifespans were cut in the last years of life, the YLL are still substantial.The online version contains supplementary material available at 10.1007/s10654-022-00915-z. It is clear that a large share of Covid-19 deaths have occurred among individuals with preexisting health problems . This haAlmost all countries experienced excess mortality during the pandemic , 10. TheIn Sweden, life expectancy (LE) in 2020 has been estimated to be reduced by 0.6 years for females and 0.9 years for males compared to 2019 . ConsideAn alternative perspective on the impact of the pandemic on mortality are years of life lost (YLL) –18. YLL Most studies that quantify the mortality impact of Covid-19 do not account for health status, and implicitly assume that individuals dying from Covid-19 had the same remaining life expectancy as the total population. However, in many countries including Sweden, the majority of deaths with Covid-19 took place among individuals receiving formal care, and thus, within a subpopulation, which has on average a poorer health status and lower remaining life expectancy compared to the total population , 22, 23.In Sweden, municipalities are legally obliged to provide publicly funded geriatric care to older individuals in need. These services are allocated according to individuals’ needs. Most commonly individuals are offered home care, and only when a person’s needs can no longer be met in their own home do they move into a care home. This makes care status an important stratification criterion when analyzing life years lost in the context of Covid-19.In this study, we investigate the death toll caused by the Covid-19 pandemic in Sweden along the dimensions of age, sex, and care status. We use life expectancy estimates derived from an incidence-based multistate model stratified by age, sex, and care status to analyze the questions of how remaining life expectancy differs between individuals with and without formal care, and how many life years have been lost due to Covid-19 when taking this aspect into account. To put the YLL into context, we additionally compare the YLL from Covid-19 to the YLL by other causes of death.Our study relies on different administrative population registers, which include all individuals registered in Sweden during the observation period  identified through the Total Population Register. Information on care status—distinguishing between receiving no formal care, receiving formal home care, and living in a care home—was derived from the Social Service Register (SSR). The SSR is an administrative register that collects monthly information on home care and care home residence . Death dInformation on care status was only available for ages 70+. However, to provide a full picture of the Covid-19 death toll, we also incorporated deaths at ages below 70 into our analysis, for which we used aggregated annual death counts by age, sex and cause of death provided by Statistics Sweden . For theCare-specific life expectancy was calculated based on an incidence-based multistate model with rewards, described in more detail elsewhere . This apAge- and birth cohort-specific probabilities of transitions between care states and of remaining in a care state were derived in intervals of three months for the range between ages 70 and 105 . The short interval allowed to capture several transitions of an individual within a short period. In the final multistate model, we used the median estimate from a set of 2000 smoothed transition probabilities that we obtained from fitting a Generalized Additive Model (GAM) with a P-Spline, an offset, and Poisson distribution to resample numbers of transitions . To ensuWe used Markov chains with rewards to calculate the remaining life expectancy conditional on being in a specific state at a given age x. This methodology is based on the idea of assigning a specific reward to transitions, such as one if individuals survive an interval. From the respective transition and reward matrices, we calculated care-specific life expectancy following the methodology of Caswell and van Daalen . To contWe estimated YLL for deaths with Covid-19 as underlying cause of death  and all excess deaths, stratified by age, sex, and care status. In contrast to Covid-19 deaths that are based on death certificates, excess deaths indicate the net effect on mortality, including both additional and averted deaths during the pandemic. Deaths below age 70 were only available in 5-year age groups. We therefore used the Penalized Composite Link Model (PCLM) to ungroup the data and estimate age-specific counts , 33.Excess deaths were defined as the difference between the observed deaths and deaths derived from baseline mortality. Baseline mortality—the hypothetical mortality level in absence of the pandemic—was estimated using a GAM with quasi-Poisson distribution to account for overdispersion. The model had three main components: (1) a component for the change in mortality over time, (2) a component for seasonality, and (3) an offset to incorporate changes in population size over time. The baseline mortality was estimated independently for each age interval (5-year age groups), sex, and care status (only at ages 70+), and the PCLM was used to obtain age-specific excess deaths. YLL were then calculated by multiplying the respective life expectancy and number of deaths at each age.Figure Figure Figure The figure shows that for both men and women almost 60% of all deaths occurred among individuals with a remaining life expectancy less than four years. Among women, 7.5% of all Covid-19 deaths occurred among individuals with an expected remaining lifetime of 1–2 years. Among men the corresponding number is 16%. Less than 10% of all deaths for women and around 15% of all deaths for men had a remaining life expectancy of 12 years or more. Note that most deaths with unknown care status are deaths that occurred between ages 60 and 70 and most of them would likely belong to the no care group. With around 37.5% for men and 55% for women, a large share of deaths occurred in care homes.Table When adjusting for care status, the total number of YLL declines from 32582 to 23576 among women, and from 43,521 to 33,510 among men. Thus, not considering the differences in remaining life expectancy in the affected populations overestimated YLL by 40% for women and 30% for men. Consequently, the average YLL per death are around 2 years lower when care status is considered. Based on the adjusted average YLL per death and the distribution shown in Fig. For a woman who received no care and died from Covid-19, the estimated average YLL was 11 years, while the corresponding numbers were 5 and 3 years for women with home care or in a care home. For men, these numbers were 9 years, 4 years, and 2 years. The YLL were very similar for excess mortality.To put the YLL from Covid-19 in context, Fig. We investigated the death toll of the Covid-19 pandemic in Sweden during 2020 along the dimensions of age, sex, and care status. We used care status-specific life expectancy in combination with confirmed Covid-19 deaths and excess deaths to estimate YLL in 2020 and compared it to other causes of death in 2019 and 2020. The comparison allowed us to put into context the amount of expected remaining life years that have been lost during the pandemic.Our analysis revealed four key findings. First, remaining life expectancy varies greatly across care states. Second, the total amount of YLL from Covid-19 is reduced by 30% when care-specific life expectancy is considered, which translates to a reduction of around 2 years in the YLL per death. Third, more than 50% of all Covid-19 deaths had an average remaining lifetime of less than 4 years. Fourth, our results suggest that Covid-19 deaths did not replace other causes of death in 2020 but came on top of expected deaths. YLL from Covid-19 rank among the highest in comparison with other causes of death, also when considering care status.Previous studies have documented a close link between existing comorbidities and an elevated risk for a severe progression and death from Covid-19 . Our anaIn Sweden as well as in other countries Covid-19 mortality has been particularly high among individuals in care homes , 22, 23,The high number of YLL among men with no care is a worrisome finding. However, these numbers must be interpreted in the context of the gender differences in institutionalized care. Previous studies documented that living together with a potential caregiver considerably reduces the risk of admission to a care home , 35. ThiThe observation that Covid-19 deaths closely resemble the distribution of excess deaths over age speaks towards that Covid-19 did not replace other causes of deaths but rather added deaths on top of the expected “normal” mortality. This is also confirmed when comparing the relative share of different causes on all deaths in 2019 and 2020 when excess deaths have been removed . The similar age distribution also shows that the pandemic particularly increased the mortality levels of frail individuals that already have a high death risk. The higher number of excess deaths compared to Covid-19 deaths may also suggest that cause-of-death-based death counts underestimate the true death toll of the pandemic, a problem that has been pointed out previously for many countries worldwide .In most cases, Covid-19 deaths cut individual lifespans in the last years of life, as for instance, indicated by the finding that more than 50% of all deaths had an expected remaining lifetime of less than 4 years. This has implications for future mortality trends in Sweden. First, the concentration of deaths near the end of life suggests that potential mortality displacement may only have a short-term effect. We can thus assume that any adverse consequences following for instance from reduced cancer screening, or postponed care that may affect the total population will likely result in additional excess mortality. Second, increased mortality during 2020 will likely not result in a deviation of future mortality trends in the long run, even if the overall number of YLL caused by the pandemic remains high. Moreover, considering the variation in remaining life spans between affected groups, and the fact that many deaths had a remaining life expectancy well below the average number of YLL per death, this measure does not serve as a good summary indicator and should be used with caution in the case of estimating YLL during the Covid-19 pandemic for total populations.Our paper has the strength of nationwide high quality administrative register data. This allowed us to use both cause of death statistics from medically signed death certificates, as well as excess mortality, an indicator for the adjusted net effect on mortality. We had access to monthly information about care status and could therefore model transitions in very short intervals, which is an advantage given the dynamics of transitions between care states and death. However, even with this approach, not all short-term transitions have been captured. Such transitions are mostly transitions into a state with higher care demand and into death soon after. Particularly remaining life expectancy for care home residence may thus be slightly overestimated. Moreover, we only had care status for the population aged 70 years and older. For ages below 70, we thus used total life expectancy as expected remaining lifetime, which may also lead to some overestimation of the remaining lifetime for some subgroups. There is likely also variation in death risk within different care status, which we did not consider in our analysis. With care-specific life expectancy, we thus capture variation between the different subgroups but not the variation within subgroups. This could result in an overestimation of YLL as individuals that died during 2020 may had a higher mortality risk compared to their subgroup peers. We also did not address the variation in the exposure to the virus within and across subgroups, which likely explains part of the differences in deaths and YLL between the different care groups. Individual health status and mortality risk would have captured even more variation in remaining life expectancy, yet also increased the complexity of the analysis. Overall, considering the large variation we observe in remaining life expectancy across the three care groups, we believe that our stratification of deaths and our estimates provide a more realistic picture of the YLL compared to previous estimates that stratify along age and sex only.The large differences in remaining life expectancy between individuals with and without formal care, and the fact that most Covid-19 deaths in 2020 in Sweden occurred in care homes or among individuals receiving home care illustrates the need for subgroup specific mortality rates when estimating the burden of Covid-19 on overall mortality. Nevertheless, despite the shorter life expectancies of many individuals that died from Covid-19, the data are in line with the view that Covid-19 did not replace other causes of death but were additional deaths that came on top of expected mortality, and thus, reduced lifespans in all groups. Even after taking care status into account, YLL to Covid-19 in 2020 remain comparable to YLL from ischemic heart disease.Supplementary file1 (PDF 1047 KB)Below is the link to the electronic supplementary material.


Recent clinical trials on proliferative diabetic retinopathy (PDR) show that presenting visual acuity can be stabilised with panretinal photocoagulation (PRP) within 2 years despite the need for supplementary PRP or anti-vascular endothelial growth factor therapy for concomitant diabetic macular oedema (DMO). It is unclear whether similar results can be obtained in daily clinical practice. Here, we query the probability of vision loss in patients with treatment-naïve PDR who have attained stability after PRP and its predictors.Retrospective cohort study at a tertiary eye centre between 01 January 2015 and 31 December 2019, wherein 2336 eyes met study criteria with first record of stable PRP-treated PDR in at least one eye. Kaplan-Meier and Cox proportional hazards modelling were used to report the probability of vision loss of at least five Early Treatment Diabetic Retinopathy Study (ETDRS) letters.The probability of losing at least five ETDRS letters was 50% at 3.32  years after achieving first stability post PRP in treatment-naïve PDR. The mean decrease at this event was 14.2 (SD 13.0) ETDRS letters irrespective of the presence of DMO. The strongest risk factor for vision loss was a history of DMO at baseline , p<0.001).One in two patients with stable treated PDR lose a line of vision by 3.5 years. This resulted in 15% of patients losing their eligibility to drive. Notably, 13% of the cohort died during the follow-up period. Proliferative diabetic retinopathy (PDR) is treated with panretinal photocoagulation, but it is unclear whether visual loss can occur after disease stabilisation in real-world data.Despite achieving stability for PDR, the majority of patients proceed to lose clinically meaningful vision.Patients with PDR require further management even after achieving ‘stability’ and macular ischaemia as a contributory cause for deterioration of vision loss ought to be further researched.Proliferative diabetic retinopathy (PDR) is characterised by the growth of abnormal new vessels on the retina and/or optic disc. These clinical features alone are not usually associated with visual impairment unless there is coexistent diabetic macular oedema (DMO).5–7The main cause of severe visual loss in PDR is due to complications of untreated or suboptimally treated PDR such as vitreous haemorrhage and tractional retinal detachment. The landmark clinical trials, Diabetic Retinopathy StudyRecent studies such as Protocol S and the CLARITY trial, designed to examine the role of anti-VEGF therapy as an alternative therapy to PRP for active PDR not requiring vitreoretinal surgery at baseline, showed that eyes treated with PRP maintain presenting visual acuity (VA) over 1–2 years.17The aim of this study was to assess the probability of visual loss over time of patients from first documentation of stable PDR after PRP in routine clinical practice and to determine the predictors of visual loss. We considered outcomes in eyes with and without DMO at presentation.This retrospective cohort study was conducted at Moorfields Eye Hospital National Health Service Foundation Trust, a tertiary centre in London, UK. The study was conducted in compliance with the Declaration of Helsinki and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology reporting guideline. Informed consent from the study cohort was not required as per the standard when using retrospective, de-identified data for research within the UK National Health Service.The cohort comprised consecutive patients with a first record of stable PDR (coded R3S) after treatment with PRP with or without history of previous vitrectomy in at least one eye between 1 January 2015, and 31 December 2019. Patients were required to have at least two records of VA beyond 3 months of first R3S record. Stability was defined by the retinal specialist after clinical examination, which may or may not include retinal photographs. In clinical practice, stability is achieved if the neovascularisation has completely regressed or partially regressed compared with previous visit or remain unchanged despite adequate laser coverage of the retina. The cohort included both phakic and pseudophakic eyes, as well as eyes with and without DMO. Baseline DMO status was subcategorised as eyes with absence of DMO (coded M0), active DMO on anti-VEGF treatment (coded M1A) and eye with stable DMO under observation (M1S). The M1S eyes may have parafoveal oedema or centre-involving oedema with good VA or post-treated DMO that is persistent but stable. If both eyes had a record of stable treated PDR, inter-eye correlation was acknowledged and considered by selecting one eye at random using the sample function of base R software, V.3.6.2 . Exclusion criteria were study eye with: active PDR or any other retinopathy grades in the study eye that have not achieved stability; incomplete baseline records; fewer than two post-baseline records of VA; less than 3 months of follow-up data; ungradable maculopathy status.The nationwide diabetic eye screening programme invites all patients with diabetes aged 12 years or over to annual primary care-based screening. Here, two-field fundus photography (one image centred on the macula and a second image centred on the optic disc) is acquired and graded according to the English Screening Programme for Diabetic Retinopathy standards.The primary outcome was time to a decrease of at least five ETDRS letters (one Snellen line) from baseline recorded at two consecutive visits post stability for the whole cohort. These outcomes were also stratified based on DMO status . Secondary outcomes included: (a) predictors of a loss of five letters and (b) incident ineligibility to meet the UK legal limit of driving after achieving stability of PDR  and (c) outcome of vitrectomised eyes and (d) patients with missing data defined as loss to follow-up. As recent treatment of DMO or vitrectomy might influence final visual outcome, we repeated the primary outcome analysis only in eyes who did not have records of anti-VEGF therapy or supplementary PRP or vitrectomy at least 6 months prior to two consecutive records of five-letter loss. The data of a random sample of 100 patients with loss of at least five letters and the optical coherence tomography (OCT) scans were examined to ensure reliability of the electronic medical records.All data analyses were carried out with R (V.3.5.1).For covariable effects, multivariable Cox proportional hazards models22Due to the post-hoc analysis methodology of this study, it was not appropriate or possible to involve patients or the public in the conduct of our research.Between 01 January 2015 and 31 December 2019, a total of 2336 patients with stable R3S recorded for at least one eye met the inclusion criteria see . A total10.1136/bmjophth-2022-001068.supp1Supplementary dataA total of 1236 (52.9%) eyes recorded loss of five ETDRS letters or more from baseline confirmed at two consecutive visits during the observation period, with the median decrease in VA from baseline being nine letters . Of noteWhen we consider the DMO status in the fellow eyes, 55.3% had no DMO, 14.1% had active DMO (M1A) and 25% stable DMO (M1S) (data not shown). Maculopathy status was not available in 5.6%. In addition, 66% had PRP treated PDR in the fellow eyes. When both eyes are considered together, 15.6% of this study cohort would not be eligible to meet UK driving standards due to the loss of one line of vision in the study eye . AdditioKaplan-Meier modelling demonstrates that the median survival time to loss of five ETDRS letters or more was 3.32 . Those with active DMO (M1A) at baseline are more likely to experience this event ) than those with stable DMO ) and without DMO ) . A sensiLoss of VA due to cataract, DMO, vitreous haemorrhage, reactivation of proliferative diabetic retinopathy was considered by querying whether cataract surgery, anti-VEGF or macular, vitrectomy and PRP—respectively—had taken place within 6 months of losing five or more ETDRS letters . Only 7.The distribution of last hospital appointments for our patient cohort is shown in The results of this retrospective study show that there is a 50% likelihood for a real-world cohort under follow-up after achieving stability post PRP for treatment naïve PDR to maintain their presenting VA for at least 3.5 years. However, in the 50% that lost at least one line of vision, the median loss was nine ETDRS letters, irrespective of the presence of DMO at baseline. A 10-letter loss is a clinically meaningful difference as these changes exceed measurement variability.1420 The baseline predictive factors of 5-letter losers included older age, higher VA and presence of any previous, active or stable DMO. Treating DMO aggressively will likely enable more patients to retain vision that meets the legal limits of driving.A five-letter loss is particularly important in a person whose baseline VA is around 70 letters, when the VA in the other eye is already below 70 letters. Although at baseline, 65% had VA of ≥70 ETDRS letters  in the study eye, only 34% maintained this vision at the point when the study eye lost at least a line of vision, highlighting that stable treated PDR may continue to affect the quality of life of patients. When we consider the VA of both eyes together, this loss of at least one line of vision in the study eye disqualifies approximately 15% of the study cohort from driving based on VA criterion alone.et al). Although we did not have access to systemic data for our patient cohort, our results also indicate that previous or coexistent DMO is the highest risk for decline in VA in real life. We could not explain why circa 50% of eyes without DMO (676/1312) also experienced a median drop of nine letters. Although 70% of the eyes with no DMO had 70 or more letters at baseline, only 30% of patients in this group had VA of 70 letters or better despite absence of DMO at baseline. New onset DMO that did not meet the central subfield thickness of 400 µm, a requirement to start anti-VEGF in the National Health Service in England and Wales, may be a reason but the database does not capture this data. Other factors such as diabetic macular ischaemia were not quantified by OCT-A in this study. With the advent of OCT-A, it is likely that we will be better informed of its contribution to vision loss in people with PDR. A proportion of patients with PDR also develop disorganisation of the inner retina and this feature is associated with poor visual outcomes.28Another important observation noted in the study is the visual outcomes of patients with stable treated PDR with no baseline DMO. Only 70% of these eyes had a VA of 70 letters or better. Other causes of impaired VA in patients with treated PDR include uncorrected refractive errors, visually significant cataract and DMO.Another important finding from this study was the loss to follow-up proportions. Non-English speakers, increasing age, multiple comorbidities and lack of health insurance cover, longer distance travelled for clinical appointment and failure to attend other non-eye-related appointments have all been reported as factors associated with lost to follow-up.This study on a large multiethnic cohort of patients with stable treated PDR in a real-world setting in the National Health Service in the UK is generalisable as the cohort comprises patients with and without concomitant DMO, vitrectomised eyes, both type 1 and 2 diabetes and varying age groups. Due to variability of VA recordings in clinical settings, we mandated 5-letter drop to be confirmed in two consecutive visits. A key assumption implicit to time-event analyses is that censored patients have the same chance of experiencing an event as those still under observation. Survival analyses make use of all available data up until censorship to calculate probabilities of surviving each interval and thereby estimate event probability at any time point. For patients with missing data at a given time point , the calculated time-event probability can be thought of as a combination of the patient’s own available data and imputation from other patients in their cohort with more data. Another assumption is that time of study enrolment is assumed not to affect event probabilities. It is also important to note that Cox modelling can identify associations between variables.Limitations of observational studies of real-world clinical data are well recognised.10.1136/bmjophth-2022-001068.supp2Supplementary data


It is acknowledged that the definition of CR-POAP remains an area of evolution3 and for this study the authors elected to use the initial definition proposed by my previous work  more than180 mg/dl day 1–2)2. Importantly it should be noted that somatostatin analogues and hydrocortisone were selectively used for patients defined as having a high-risk remnant, with some being enrolled in a randomized clinical trial comparing the aforementioned agents. No information was provided regarding external pancreatic stent use. Complications were reported by use of the comprehensive complication index (CCI). Patients were split into those with high (CCI score 33.7 or higher) and low (CCI less than 12.3) morbidity. Of the 614 patients who met inclusion criteria, 106 (17 per cent) were excluded due to missing data regarding CR-POAP. No information is provided regarding the outcomes of those patients. Neoadjuvant therapy was administered to 106 (21 per cent) of included patients. Data on patients receiving hydrocortisone or somatostatin analogues are not provided and do not seem to have been considered as potential variables in the outcome analyses. In addition, it is difficult to determine the distribution of patients across the fistula risk-score zones, although data on the individual components are provided. In terms of outcomes, patients with CR-POAP made up 39 per cent of patients with high morbidity, but only 4 per cent of those patients with low morbidity, thus confirming the importance of this complication. Of the 64 patients diagnosed with CR-POPF, 46 (72 per cent) had underlying CR-POAP, but only 4 patients with CR-POPF had CRP more than 180 ng/dl on day 2 and a normal day 1 amylase. The updated alternative fistula risk score4 seemed to best predict both CR-POPF and CR-POAP with an area under the curve (95 per cent c.i.) of 0.819 (0.771 to 0.867) and 0.834 (0.794 to 0.873) respectively.Postoperative acute pancreatitis (POAP) has become a point of interest among pancreatic surgeons with the increasing recognition of its aetiological role in major postoperative morbidity following pancreatoduodenectomy (PD). Bonsdorff 5 In addition, external stenting, and omission of somatostatin analogues are considered to be the optimal mitigation strategy for high-risk anastomoses6. Thus, it is possible such interventions will ‘down stage’ the severity of the complication, thus biasing assessment, or validation of the definition. The increasing use of neoadjuvant therapy is also another contributing factor, as this almost certainly modifies the underlying pancreatic remnant. Such patients rarely develop CR-POAP, yet the overall complication burden may well be higher due to the more extensive surgery; in this series alone, 3 per cent of patients underwent arterial resection. Thus, it may become important when validating and refining the burden of POAP that the analysis is stratified by the state of the underlying pancreatic remnant. It is highly likely that the underlying inflammatory response and risk of pancreatic necrosis is highly variable between such patients.This paper adds further weight to the increasing body of evidence that POAP is a major aetiological factor in pancreatic specific morbidity following pancreatoduodenectomy. However, this paper also highlights that while the definition of POAP continues to be refined or validated, it will be important to consider how modifying interventions may alter the outcomes and thus alter the accuracy of the assessment of the definition in question. There is now evidence that hydrocortisone attenuates postoperative inflammatory response and morbidity.Disclosure. The authors declare no conflict of interest.


Median heart rate recovery (HRR) increased from 10.5 (IQR 7.5–14) to 15.5 (IQR 11–20) bpm; p = 0.24. There was a significant drop in distress (median 3 (IQR 0–5) to 1 (IQR 0–2); p = 0.04) and a small drop in anxiety (median 3 (0–5) to 2 (0–3); p = 0.22). There was no difference in the postoperative complication rate and length of hospital stay between the two groups. Discussion: This study has shown that digital prehabilitation can be delivered effectively to patients with OG cancer, with high engagement and retention rates. We observed improvements in some physical and psychological parameters with the digital service, with comparable clinical outcomes to the in-person service. Background: Home-based and supervised prehabilitation programmes are shown to have a positive impact on outcomes in patients with oesophago-gastric (OG) cancer. The primary aim of this study was to establish the feasibility of delivering a digital prehabilitation service. Methods: Patients undergoing treatment for OG cancer with curative intent were recruited into the study. During the COVID-19 pandemic, patients were offered a digital prehabilitation service. Following the lifting of COVID-19 restrictions, patients were also offered both a hybrid clinic-based in-person service and a digital service. Implementation and clinical metrics from the two prehabilitation models were compared. Results: 31 of 41 patients accepted the digital service (75%). Of the people who started the digital programme, 3 dropped out (10%). Compliance with the weekly touchpoints was 86%, and the median length of programme was 12 weeks. Twenty-six patients enrolled in the in-person service. Two patients dropped out (10%). Average compliance to weekly touchpoints was 71%, and the median length of programme was 10 weeks. In the digital group, sit to stand (STS) increased from 14.5 (IQR 10.5–15.5) to 16 (IQR 16–22);  Around 17,000 patients are diagnosed with oesophago-gastric cancer in the UK each year, of which a third undergo radical treatment. This includes neoadjuvant chemotherapy (NAC), with or without radiotherapy, followed by a major surgical procedure. In total, 60% of patients have at least one complication, and the median length of stay is around 11 days . The reaPrehabilitation, a relatively new concept, is the process of using the time prior to surgery to improve the functional capacity of an individual so they can better withstand the stress of surgery . There iDue to the challenges of delivering the programme during the COVID-19 pandemic, we sustained the service through a digital prehabilitation model. Through this initiative, it was also anticipated that we could provide access to prehabilitation to patients across our partner cancer network, as prior to this programme patients from the network did not receive prehabilitation. This study aims to firstly explore the feasibility of implementing a digital prehabilitation service in cancer patients, secondly to measure the impact of digital prehabilitation on physical fitness and psychological wellbeing, and finally to compare the impact of digital prehabilitation on postoperative outcomes against in-person clinical service. Patients undergoing treatment for OG cancer with curative intent between September 2020 and September 2021 were recruited into the study. Referrals were received from North-West London Network  and the Bedfordshire and Hertfordshire network (2 site). In the period from Sept 2020 to Feb 2021, patients were only offered the digital service due to staffing constraints in the in-person service as a consequence of the COVID-19 pandemic. The in-person service resumed in February 2021 with the lifting of the COVID-19 lockdown restrictions. From then on, patients were first offered the digital arm. If they were unwilling or unable to access the digital service, they were referred to the in-person service . However, clinical teams still had the option of referring patients to the hospital physiotherapist directly, as she regularly attended outpatient clinics. The digital prehabilitation service (DPS) is a behaviour change health coaching programme for people undergoing and/or preparing for cancer treatment. The features available in the app during the study period consisted of a screening questionnaire , video platform for consultations, messaging, and wearable device connectivity. All patients undergoing the digital service were offered a Fitbit Inspire 2 device . The digital service was offered through an app and a non-app pathway. App-pathway patients downloaded the app  from the Apple and Google app stores. Non-app-pathway patients received the digital service through e-mail, a web browser for surveys, web-based video conferencing, and the wearable technology manufacturer’s app.The digital service was delivered by a team of behaviour change coaches with a background in dietetics, physiotherapy, and nursing. Patients were allocated a coach who covered all aspects of prehabilitation . Any specialist services, such as specialist dietetic support and clinical psychologist, were provided through the hospital services. This model is aligned to Macmillan’s tiered framework for prehabilitation delivery .The exercise aspect of the digital service is similar to in-person service , but theThe details of the in-person prehabilitation model have previously been published . In brieEngagement with digital prehabilitation was estimated by calculating the proportion of people for whom the feature was available to those who engaged with the feature. The different features measured in this study were the screening questionnaire  and wearable device connectivity. This was defined as data being available for >3 days of the week for >50% of the time in the programme. Feasibility measures included attendance at the baseline (T1) and postprogramme (T2) physical performance assessment sessions (as below); compliance, which was defined as a ratio of the weekly contact/touchpoints attended per week to the total weeks in the programme; and retention/dropout rate, defined as the proportion of patients who completed the programme. In the digital programme, patients self-reported physical activity levels during the programme using a modification of the International Physical Activity Questionnaire (IPAQ). Patients were asked to quantify the time spent per week in moderate and vigorous physical activity. METs mins per week were calculated as the product of time spent multiplied by 3.5 METs for moderate intensity activity and 6 METs for vigorous intensity activity . Both moCardio-respiratory fitness assessed by performing the 1 min sit-to-stand test and measuring the heart rate at rest and at the end of the test and 1 min after recovery . Heart rLower limb muscle strength assessed using the 30 s ‘sit-to-stand test’ .A virtual physical performance assessment was undertaken at the beginning and completion of the neoadjuvant treatment period (T1 and T2), which consisted of a short physical assessment battery. This is a modified version of the Short Physical Performance Battery  for remoPsychological wellbeing was assessed by self-report of Anxiety and Distress using the Emotional Distress Scale . Within the in-person programme, physical fitness was assessed using the Chester Step Test to calculate predicted VO2max. Details of this assessment have previously been published . Lower limb muscle strength was also assessed using the 30 s ‘sit-to-stand test’ .Postoperative outcomes were recorded for both the digital and in-person groups. Complications were defined according to the Esophagectomy Complication Consensus Group (ECCG) guidelines  and gradAll patients in the digital programme gave informed consent to sharing their data as per GDPR regulations. Ethical approval for the retrospective analysis of data for publication purposes was granted by the UK Health Research Authority (Ref 268837).Changes in health outcomes  during prehabilitation were assessed using the Wilcoxon signed-ranks test and the chi-squared test where appropriate. T tests were used, depending on distribution. Two-tailed tests were used throughout, with a significance level of p < 0.05. Statistical analysis was performed using SPSS version 25 . Baseline characteristics and postoperative outcomes were compared between digital and in-person using chi-squared tests for categorical outcomes. For continuous outcomes, either Mann–Whitney or unpaired Recruitment and retention to the digital programme is shown in n = 3), lack of confidence in technology (n = 2), and feeling overwhelmed (n = 2). No reason was given by the other three patients.Of the 10 patients who declined, 5 declined prehabilitation completely and 5 started the in-person service. Reasons for declining the digital programme included language problems  a median of 4 weeks after starting the programme. Compliance to the weekly touchpoints was 86%. In total, 100% of patients attended the T1 assessment and 55% attended the T2 session. The median time in the programme was 12 weeks.Twenty-six patients started the in-person service. Twenty-one patients were direct referrals to the service. Two patients dropped out of the programme (10%) after a median of 4.5 weeks from starting the programme. Both patients had also declined the digital service. Average compliance to weekly touchpoints was 71%. Attendances at the T1 and T2 assessments were 46% and 15%, respectively. Median time in the programme was 10 weeks.The Cancer Health Checker was available to all patients and was completed by all 31 patients (100%). The connectivity of wearables was available to all patients, but reliable data were available for only 17 of the 31 patients (55%). This improved from 4/16 (25%) in the first half of the study period to 13/15 (86%) in the second half of the study period. Health improvement data could only be analysed in the digital arm due to low rates of completion of the T2 assessment in the in-person arm (14%). p = 0.02). There was also a trend for an increase in HRR during the programme: median HRR increased from 10.5 (IQR 7.5–14) to 15.5 (IQR 11–20) bpm; p = 0.24. p = 0.64) or the average step count per day (5179 (3204) to 4550 (3061); p = 0.55). There were no changes in the mean self-reported physical activity (346.5 (sd = 362) METs min per week to 407 (sd = 400); p = 0.04) and a small fall in anxiety (median 3 (0–5) to 2 (0–3); p = 0.22). There was no significant change in depression scores (1 (0–3) and 1 (0–2); p = 0.41).There was a significant fall in Distress ((median 3 (IQR 0–5) to 1 (IQR 0–2); Baseline characteristics were comparable between the two groups , with thIn total, 26 patients (83%) in the digital group and 17 patients (65%) in the in-person group proceeded to surgical resection. Five patients and nine patients in the respective groups had disease progression during the preoperative period, which precluded resection.The postoperative complication rate was 50% versus 64%, the postoperative pneumonia rate was 23% versus 41%, and the postoperative hospital stays were 10.5 versus 17 days for the digital and in-person arms, respectively. There was no significant difference between the two groups. This pilot study demonstrated the feasibility of delivering a fully virtual cancer prehabilitation service. Digital recruitment was 75%. Of these patients, nearly 40% did not possess mobile technology or have confidence in the use of apps, and consequently a non-app pathway was implemented using a web browser. The findings are consistent with the finding that nearly 22% of patients in the NHS do not have basic digital skills . The higJust 5 (8%) patients chose not to do prehabilitation altogether, and a small number of patients (10%) in both arms dropped out of the programme. We believe that this reflects that with clinical endorsement and support, a high number of patients do start and complete prehabilitation programmes. The high programme completion (84%) and compliance rates (86%) in the digital arm, seen in our study, most likely reflect the element of professional support offered to patients throughout the 12-week programme. This is in comparison with self-management apps that are associated with high drop-out rates . The finWhile just over half of the patients attended the end of the programme’s virtual health assessment, this was only 14% in the in-person cohort. The reasons for this are manifold but include conflicting clinic appointments and last-minute scheduling of cancer operations. One of the challenges of hospital-based behaviour change interventions is the low uptake among patients due to difficulties with travel and with having to fit in their therapy sessions around other hospital appointments ,26. The We showed maintenance or improvement in physical activity, physical fitness, and psychological measures. Even maintenance of physical activity would be beneficial, as it is contrary to the drop traditionally seen in patients undergoing chemotherapy . The sliWhile this study demonstrated improvements in the physical assessment measures, we faced a number of challenges in undertaking the assessment remotely. We either considered or implemented other tests, such as the Six Minute Walk Test (6MWT) and the Chester Step test (CST). During the co-design of our remote programmes, our patient user group, as well as clinical team, were of the view that the 6MWT and the Incremental Shuttle Walk Test would be challenging for many patients due to the lack of even a 10 m distance   in most We also showed a maintenance of postoperative outcomes in the patients who underwent the digital programme when comparing the outcomes in this study with our previously reported findings . AlthougIn addition to being able to sustain the programme during the pandemic, we were also able to extend the programme to patients from our partner network who did not have access to prehabilitation due to the distance involved in travelling to our centre. Digital solutions allow us to ‘democratise’ behaviour change interventions and address the ‘postcode’ lottery that can exist in the delivery of patient support services . A centrThis study is a pragmatic real-world implementation of a digital prehabilitation programme, which replicated our previous hybrid clinic–home-based programme. There are a number of limitations. We did not have a control group who did not receive prehabilitation, but we compared prehabilitation processes and outcomes with a parallel group of patients who underwent the in-person programme. The engagement with the latter was most likely impacted by the pandemic, and the results of which were likely influenced by a selection bias. There was also a difference in the median number of weeks in the programme. This was likely a result of there being more people who either dropped out of the programme or who had disease progression in the in-person cohort. We also faced a number of technology issues, especially with the interoperability of the wearable device with the DPS, which could have affected the physical activity measurement. During this study, we only implemented the features that facilitated the remote delivery of our programme. We developed further behaviour change features that were only available to the last four patients recruited in this study. Future research will explore the impact of these new features.