Individuals with high sensory processing sensitivity (SPS) (“highly sensitive persons”) are thought to be easily excitable and overwhelmed, highly attentive to aesthetic impressions, and particularly sensitive to sensory stimulation. Public discourse suggests that those who describe themselves as highly sensitive see themselves as fundamentally different from others, and view their personality as a gift and a burden. From a clinical personality perspective, high sensitivity could be considered to have substantial overlaps with hypersensitive narcissism, or generally vulnerable narcissism.n1 = 280, n2 = 310).We investigated the associations and shared nomological networks between high sensitivity and hypersensitive narcissism in two studies using convenience and representative samples (r ≤ .54) as well as vulnerable narcissism (.44 ≤ r ≤ .54), associations were not attributable to general neuroticism. Nomological networks were similar and pointed to a neurotic‐introverted personality profile with reduced personality functioning. Latent class analyses further pointed to substantial and practically relevant person‐level covariance.There is evidence for replicable associations between SPS and hypersensitive (.53 ≤ Sensory processing sensitivity and hypersensitive narcissism are substantially related constructs. For clinicians, this points to the importance of being attentive to narcissistic self‐regulatory strategies in individuals presenting as highly sensitive. This naself‐importance and entitlement . These characteristics appear to be phenomenologically similar to two central characteristics of narcissism, namely We believe that investigating potential overlaps between the constructs of high sensitivity and hypersensitive narcissism is important for gaining a more complete picture of both. This seems particularly important in the light of the public discourse, which tends to see high sensitivity as something all‐positive, and narcissism as something all‐negative  to be a personality trait characterized by a heightened depth of stimulus processing at a neural level. Highly sensitive persons are assumed to display high SPS in that they are highly aware of subtleties in their environment and easily overstimulated. The authors argued that SPS can yield evolutionary advantages in terms of a higher responsiveness to environmental stimuli , aesthetic sensitivity , and low sensory threshold  dimensions, Aron and colleagues  assumed Beyond these general aspects pointing to reduced personality functioning , which encompasses items such as “I can become entirely absorbed in thinking about my personal affairs, my health, my cares or my relations to others” or “I feel that I am temperamentally different from most people” , hiding the self (not disclosing own needs and perceived weaknesses), and devaluing , this differentiation has a connotation of specialness or uniqueness, and might thereby serve a self‐aggrandizing motive. Although specialness is not explicitly covered by item content of the HSPS, it may be discovered between the lines . We hypothesize that all HSPS factors cover this specialness aspects to some extent and should thus relate not only to vulnerable but also to grandiose narcissism , albeit weaker. We expect the strongest effects for the AES factor, because its item content appears to be most evaluative.While our hypotheses so far targeted the identification of narcissism aspects within the high sensitivity construct, a complementary perspective might be to look for aspects of high sensitivity in narcissism; particularly hypersensitive narcissism. Here, it has repeatedly been noted that high stimulus‐reactivity, thought to originate from biological differences , not only shapes narcissistic personality configurations in the vulnerable direction High sensitivity (as assessed by the HSPS) should be related to hypersensitive narcissism (assessed by the HSNS), and, to a lesser extent, to general vulnerable narcissism (assessed by the PNI).a. Effects should be strongest for the EOE and LST factors of the HSPS(2)The EOE factor of the HSPS should relate to psychological entitlement, particularly vulnerable‐based entitlement.(3)High sensitivity (HSPS) should relate to grandiose narcissism (PNI) to a small extent.a. Effects should be strongest for the AES factor of the HSPS(4)High sensitivity (HSPS) and hypersensitive narcissism (HSNS) should display similar nomological networks in FFM dimensions and indicators of personality functioning and psychological adjustment.(5)Hypotheses 1–4 should hold when controlling for neuroticism, but effects are expected to become weaker.A crucial test of our hypotheses regarding the overlap between high sensitivity and hypersensitive as well as vulnerable narcissism concerns the specificity of the effects. In particular, shared variance can be assumed to relate to neuroticism in terms of the general tendency towards reactivity, negative affect, and proneness to psychopathology . Finally, to investigate whether the scales of interest would form common or distinct factors, we conduct joint factor analyses of the HSNS and HSPS/HSNS and PNI scales across both studies. We hypothesized to find a common factor representing a general tendency towards reactivity, negative affect, and psychopathology (closely resembling FFM neuroticism) and additional specific factors. Since results, however, showed that items of the inventories factor together without forming common factors , we complement the item‐based data reduction procedures by person‐based latent class analyses. These can inform about whether there are groups of people with similar characteristics.https://osf.io/3s6e4/.The procedure was approved by the university's ethics committee (EK 236052019). The hypotheses tested here were not preregistered, but we declare that we differentiate confirmatory from exploratory analyses throughout the manuscript. Study data are available on the Open Science Framework: 22.12.1.1N = 280 individuals  with a mean age of 30.47 years (SD = 13.60). We acquired data in an online study (administered via LimeSurvey) and invited participants via the faculty's participant pool for psychological research, complemented by social media announcements . The targeted minimum sample size was N = 200 with an equal sex distribution, to detect small effects (r = .20) at a power of 1 − β = .80 . To obtain a sample with equal numbers of women and men, we determined the final sample size by the number of male study participants; datasets from additional women were discardedThe final sample consisted of 2.1.22.1.2.1ease of excitation (EOE), low sensory threshold (LST), and aesthetic sensitivity (AES). EOE describes the tendency to feel overwhelmed easily, AES captures aesthetic awareness, and LST reflects high sensitivity to and arousal following from external stimulation , α = .57 (AES), and α = .91 (LST). The internal consistency of the overall scale was α = .91.High sensitivity was assessed using the 26‐item German adaptation of the Highly Sensitive Person Scale , and vulnerable narcissism was assessed using the Brief Pathological Narcissism Inventory  was measured as the average of three subscales of the B‐PNI: contingent self‐esteem , hiding the self , and devaluing . We also used the B‐PNI to assess grandiose aspects of narcissism. Grandiose narcissism  was measured as the average of the subscales entitlement rageα = .75), exploitativeness , grandiose fantasy , and self‐sacrificing self‐enhancement . It is important to note that the PNI grandiose scales “are closer to the entitlement‐vulnerability side of the [narcissism] spectrum”  here.Personality functioning was assessed using the German 16‐item version of the Inventory of Personality Organization .For the assessment of symptom load, we used the Brief Symptom Inventory , followed by the LST factor (r = .42 and r = .35). AES displayed weaker correlations (r = .23 and r = .19). A multivariate outlier detection test yielded no evidence for influential data points (p < .001) among the variables of interest  ≤21.21).As hypothesized, high sensitivity was correlated substantially with both hypersensitive narcissism (HSNS) and vulnerable narcissism (PNI) , followed by contingent self‐esteem (r = .33). Among the grandiose aspects, correlations of the overall HSPS were strongest with entitlement rage (r = .33) and grandiose fantasy (r = .22). Among the HSPS factors, in line with our expectations, it was the AES factor which displayed the strongest overall correlation with grandiose aspects of narcissism (r = .24). As described above, since the PNI grandiose factor is closer to the vulnerable side of the narcissism spectrum than this is the case in other measures, we also removed variance of the vulnerable scales from the grandiose scales . Taken together, high sensitivity is associated with different aspects of vulnerable narcissism to a sizeable degree, and partially also with grandiose narcissism, but not with entitlement. The psychological entitlement scale used in Study 1, however, does not differentiate grandiose‐based from vulnerable‐based entitlement . Both were also moderately negatively correlated with extraversion  and unrelated to conscientiousness. High sensitivity was slightly correlated with openness to experience (r = .15), whereas hypersensitive narcissism was not. These patterns of results are largely in line with prior studies on both constructs  than high sensitivity (r = .40). A similar pattern was evident for symptom load . Finally, both were correlated with participant‐reported diagnoses of mental disorders to a similar extent .We observed generally similar nomological networks of high sensitivity and hypersensitive narcissism, as evident in correlations with measures of FFM personality dimensions, personality functioning, and symptom load. As Table r = −.36). Taken together, the result pattern shows that high sensitivity and hypersensitive narcissism have highly similar nomological networks in terms of a neurotic‐introverted personality style characterized by lowered personality functioning (though more pronounced for narcissism), elevated symptom load, and a higher likelihood of mental disorder diagnoses. With these similarities on the one hand, on the other, high sensitivity—contrary to hypersensitive narcissism—is not generally associated with feeling entitled to special treatment (see above) or disagreeable behavior.We also observed notable differences in the nomological networks: high sensitivity was not correlated with agreeableness, whereas hypersensitive narcissism was—as predicted by theory . This indicates that high sensitivity and hypersensitive as well as vulnerable narcissism are substantially associated beyond shared neuroticism variance.To investigate the extent to which the above‐reported relations would be due to shared neuroticism variance, we controlled correlations involving the HSPS for neuroticism . As expected, correlations between high sensitivity and hypersensitive/vulnerable narcissism decreased, but remained significant , sex , and ethnicity according to 2011 census data. Participants were recruited via Prolific (www.prolific.co) and received monetary compensation of £6.25. 13.20% of the sample reported a prior or current professionally diagnosed mental disorder. There were no missing data, all questions were mandatory. The study was part of a larger cross‐sectional research project and included additional questionnaires on personality, psychopathology, and self‐control which are not of interest and not analyzed here. The study duration was 44 min on average (SD = 18).The sample included 3.1.23.1.2.1α = .94. The internal consistencies of the subscales were: α = .90 (EOE), α = .76 (AES), and α = .86 (LST).High sensitivity was assessed using the 27‐item HSPS . Vulnerable narcissism was assessed using the PNI, this time with the original 52‐item long version . We also used the PNI for the assessment of grandiose aspects of narcissism (α = .93). The subscale assignments correspond to Study 1 (.78 < α < .94). As in Study 1, we residualized the PNI grandiose factor for vulnerability in complemental analyses, since the grandiose factor is substantially saturated with vulnerability  and one targeting grandiose‐based entitlement (α = .92).Vulnerable‐based and grandiose‐based entitlement were assessed using the Psychological Entitlement Scale—Vulnerable‐Based and Grandiose‐Based  to α = .82 (neuroticism).We assessed the FFM dimensions with three items per dimension using the Big Five Inventory‐2 XS .Self‐esteem was assessed using the 10‐item Rosenberg Self‐Esteem Scale  of the Brief Symptom Inventory , followed by the LST factor (r = .44 and r = .46), while the AES factor displayed a weaker relationship (r = .30 and r = .32). As in Study 1, multivariate outlier detection yielded no evidence for influential data points  ≤20.18).As hypothesized and as in Study 1, high sensitivity was correlated substantially with both hypersensitive and vulnerable narcissism . Among the grandiose aspects, it was again the entitlement rage subscale which displayed the strongest correlation with overall the HSPS score (r = .40). Among the HSPS factors—different from our expectations and the results of Study 1—it was now the EOE, not the AES factor which displayed the highest overall correlation with grandiose aspects of narcissism (r = .39). Again, we residualized grandiose narcissism for vulnerable narcissism. The HSPS factors were no longer positively correlated with this residualized score .Regarding the PNI subscales, again similar to Study 1, relations with high sensitivity were generally stronger for the vulnerable than the grandiose scales , but no association with grandiose‐based entitlement. At a subscale level, this effect was—in line with our expectations—most pronounced for EOE (r = .23), closely followed by LST (r = .21). The correlation between vulnerable‐based entitlement and AES was not significant (r = .10). Hypersensitive narcissism showed a substantial association with vulnerable‐based entitlement (r = .39) and was also associated with grandiose‐based entitlement to a lesser extent (r = .24), replicating previous findings and yielding additional validity evidence for the newly devised scale  in Study 1, using the PES‐V/G here, we observed a significant association with vulnerable‐based entitlement . Both were moderately negatively correlated with extraversion  and slightly negatively correlated with conscientiousness (r = −.14 and r = −.24). Openness and agreeableness differentiated between both, in the way that high sensitivity—specifically its AES factor—but not hypersensitive narcissism was related to openness (r = .44), whereas hypersensitive narcissism, but not high sensitivity, was related to agreeableness (r = −.24). Extending the nomological network towards potentially clinically relevant indicators, both high sensitivity and hypersensitive narcissism are substantially and similarly related to self‐esteem (r = −.44 and r = −.45) and symptom load (r = .49 and r = .52), and as in Study 1, both were associated with mental disorder diagnoses .As Table 3.2.3r = .34 and r = .30). The same pattern was evident in partial correlations with the PNI factors and its subscales  when controlling for neuroticism, suggesting that this association can largely be explained by neuroticism. Though we did not anticipate this a‐priori, it aligns with the finding that neuroticism itself is related to vulnerable‐based entitlement to a considerable degree , whereas the higher symptom load associated with high sensitivity could not be explained by neuroticism, pointing to a specific association. Different from Study 1, however, the association with mental disorder diagnoses did not hold when controlling for neuroticism. To sum up, results from Study 2 generally corroborate those from Study 1. In addition, Study 2 points to a significant association between high sensitivity and vulnerable‐based entitlement. This association can be in large parts explained along higher neuroticism, as the lowered self‐esteem and higher likelihood of mental disorder diagnoses associated with high sensitivity in this study.44.14.1.1N = 590 individuals  with a mean age of 39.11 years (SD = 25.73). We expected that factor analyses would yield a strong general factor, resembling a dimension similar to FFM neuroticism, and specific factors, in which the constructs under study could be represented either separately or also jointly. We conducted separate analyses for the HSPS and HSNS/HSPS and PNI vulnerable factor to have the constructs represented by an approximately equal number of items (see below).To test whether the measures of high sensitivity and hypersensitive and vulnerable narcissism would form common factors, we conducted exploratory factor analyses. We pooled samples from Studies 1 and 2, leading to a sample of Since results did not yield common factors between the inventories (see results section), likely pointing to method variance as the strongest factor, we complemented factor analyses by latent class analyses. These might help to find commonalities in the data at person‐ rather than item‐level, and might thus provide a more adequate test of our hypothesis that highly sensitive individuals would also show characteristics of hypersensitive or vulnerable narcissism.4.1.2α = .87. EOE, AES, and LST were represented by 6, 3, and 3 items, respectively, with internal consistencies of α = .85, α = .63, and α = .83.Since different German and English versions of the HSPS scale were used in Studies 1 and 2, we subjected only those items which match between both inventoriesα = .85 and α = .89, those of the vulnerable facets  were α = .84 for contingent self‐esteem, α = .79 for hiding the self, and α = .81 for devaluing.We extracted items from the 28‐item brief version of the PNI from data from both studies to pool them (the brief version used in Study 1 is a subset of the full version used in Study 2). Internal consistencies of the grandiose and vulnerable factors  were 4.24.2.1The factor analysis of the HSPS and HSNS yielded four factors  with an Eigenvalue > 1. Velicer's revised Minimum Average Partial (MAP) test , and one encompassing all hiding the self items plus the other half of the devaluing items (factor IV).The factor analysis of the HSPS and the PNI vulnerable scales yielded five factors  with Eigenvalues > 1, whereby the fifth factor was just beyond the cutoff . The revised MAP test . On the one hand, this speaks to the discriminant validity of the scales. On the other, it might also—at least partially—be an artifact of the items being presented separately rather than intermixed, and being answered on different response scales. We thus proceeded in our data analyses with complemental latent class analyses, which unveil commonalities between clusters of persons rather than items.4.2.2We used latent class analyses to unveil clusters of persons with potentially similar trait patterns. We subjected the HSPS, HSNS, the PNI vulnerable factor, and also the PNI grandiose factorSD, AES: +0.51 SD, LST: +0.22 SD), low scores on the HSNS (−0.94 SD), and average scores on the PNI factors . The second‐largest class  displayed elevated scores on the HSPS EOE factor (+0.75 SD), average to slightly elevated scores on the other factors , high scores on the HSNS (+1.12 SD), and slightly elevated scores on the PNI factors . The third and smallest group  displayed low scores on all measures  and slightly elevated scores on the HSNS (+0.43 SD).Figure ease of excitation discriminates between those who share narcissistic traits and those who do not share them.Taken together, the vast majority of individuals display either low (class I), or not very pronounced (class II) high sensitivity and narcissism. However, those with high EOE scores (class III) also show high scores on hypersensitive narcissism, and elevated scores on grandiose and vulnerable narcissism. High sensitivity and hypersensitive narcissism thus do go along at a person‐level, and 5We tested the hypothesis that high sensitivity and hypersensitive narcissism, as well as more generally vulnerable narcissism, might be closely related and overlapping constructs. Table 5.1r ≤ .54) as well as vulnerable narcissism (PNI: .44 ≤ r ≤ .54). As expected, among the HSPS factors, EOE displayed the strongest correlations with hypersensitive (HSNS: .57 ≤ r ≤ .59) and vulnerable narcissism (PNI: .50 ≤ r ≤ .57), followed by LST . This shows that an irritability through external stimuli, paired with an attitude of own fragility, which are likely covered by the EOS factor, have not only theoretical but also substantial empirical overlaps with hypersensitive/vulnerable narcissism. Among the PNI subscales, the EOS factor correlated with contingent self‐esteem, hiding the self, and devaluing, pointing to associations with all aspects of vulnerable narcissism.The results of two studies are generally in line with our hypotheses. We observed substantial correlations between high sensitivity (HSPS) and hypersensitive narcissism . However, overall SPS as well as its EOS and the LST factors were related to vulnerable‐based entitlement (Study 2), which has recently been delineated from grandiose‐based entitlement , indicating that AES does tap into grandiose narcissism to some extent. These associations became smaller, but were still evident when removing vulnerable narcissism variance from the PNI grandiose factor . This suggests that antagonistic tendencies do play a role in high sensitivity, but they are presumably not directly translated into everyday disagreeable behavior as assessed by FFM scales and might require a more specialized assessment.In line with our hypotheses, we observed similar correlations of high sensitivity (HSPS) and hypersensitive narcissism (HSNS) with FFM dimensions, self‐esteem, personality functioning, symptom load, and mental disorder diagnoses: in both studies, the two constructs were most strongly associated with neuroticism, followed by introversion. The AES factor of the HSPS was specifically associated with openness , reduced personality functioning , higher symptom load (assessed by the BSI in Studies 1 & 2), as well as higher likelihood for prior or current mental disorder diagnoses . While this pattern points to similar associations for the two constructs, associations with indicators of personality functioning and psychopathology were somewhat more pronounced for hypersensitive narcissism than high sensitivity.5.3r ≤ .35) as well as vulnerable narcissism (.24 ≤ r ≤ .30). This shows that, while large parts of the covariance are due to (nonspecific) neuroticism variance, there are also substantial portions of specific covariance between the constructs.To test to which extent associations between high sensitivity and hypersensitive/vulnerable narcissism would be due to shared neuroticism variance, we analyzed partial correlations, controlling for neuroticism. As expected, controlling for neuroticism did substantially reduce association between high sensitivity and hypersensitive , personality pathology (Study 1), or symptom load (Studies 1 and 2), indicating that these associations go beyond general neuroticism. In Study 1, even the association with mental disorder diagnoses held when controlling for neuroticism . Given that neuroticism is a good proxy for general psychopathology, this shows that high sensitivity and narcissism, as well as further specific aspects of psychopathology, are related beyond general impairments . We thus complemented the item‐level factor analyses with person‐level latent class analyses, which reduce data by revealing common response patterns among people instead of items.ease of excitation is the single aspect of high sensitivity which is most strongly associated with hypersensitive narcissism, aspects of grandiose narcissism (entitlement rage), reduced personality functioning, and symptom load. Whether or not individuals presenting or identifying as highly sensitive display strong ease of excitation (in terms of high stimulus reactivity and a pronounced tendency to avoid unpleasant situations) might thus inform about the degree of psychopathology associated with high sensitivity, including narcissistic tendencies.The analyses yielded evidence for a three‐class solution5.5The research presented here assessed the overlaps between high sensitivity and narcissism in two samples, one convenience and one representative sample. While self‐selection effects  might have been at play in the convenience sample, this seems unlikely in the representative sample, where participants were paid. Since results from both samples were highly similar , do show aspects of vulnerable narcissism and vulnerable‐based entitlement. This might not only be perceived as difficult by others, and relate to social conflicts (for instance via the entitlement rage aspect) and/or social disconnection, but it might also amplify the degree of psychopathology associated with high sensitivity. The mindset that one simply is fragile . This might amplify symptoms by means of social disconnect . This conforms to observations from the narcissism literature that hypersensitivity and reactivity play a major role in vulnerable narcissism and hinder a more reflective way of dealing with inner experiences .https://publons.com/publon/10.1002/jclp.23406The peer review history for this article is available at Supplementary information.Click here for additional data file.


Optic nerve injury is a leading cause of irreversible blindness worldwide. The retinal ganglion cells (RGCs) and their axons cannot be regenerated once damaged. Therefore, reducing RGC damage is crucial to prevent blindness. Accordingly, we aimed to investigate the potential influence of the gut microbiota on RGC survival, as well as the associated action mechanisms.Bifidobacterium, on RGC. Optic nerve crush (ONC) was used as a model of optic nerve injury. Vancomycin and Bifidobacterium were orally administered to specific pathogen‐free (SPF) mice.We evaluated the effects of microbiota, specifically Bifidobacterium promoted RGC survival and optic nerve regeneration. The administration of Bifidobacterium inhibited microglia activation but promoted Müller cell activation, which was accompanied by the downregulation of inflammatory cytokines and upregulation of neurotrophic factors and retinal ERK/Fos signaling pathway activation.Bifidobacterium‐induced changes in intestinal flora promote RGC survival. The protective effect of Bifidobacterium on RGC can be attributed to the inhibition of microglia activation and promotion of Müller cell activation and the secondary regulation of inflammatory and neurotrophic factors.Our study demonstrates that  Bifidobacterium‐induced changes of intestinal flora contributes to regulate retinal microglia and Müller cells activation, with alteration of inflammatory cytokines and neurotrophic factors. Microglia reportedly aggravates RGC damage through the release of inflammatory cytokines, such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and interleukin 6 (IL‐6).Helicobacter pylori infections.In a recent study, germ‐free (GF) mice presented with global microglia defects with altered cell proportions and an immature phenotype, which led to impaired innate immune responses.Bifidobacterium, a gram‐positive anaerobic bacterium associated with neurodegenerative diseases,In this study, we aimed to determine the protective effects of intestinal flora, as well as its associated mechanisms, on RGCs. Notably, we investigated whether the intestinal flora, specifically 22.1Female adult C57BL/6 mice, aged 6–8 weeks , were used. The water and food consumed by all mice were sterilized. Prior to all surgical procedures, the mice were injected intraperitoneally with sodium pentobarbital (50 mg/kg) as an anesthetic. Each experimental group consisted of 5–6 mice for the optic nerve crush (ONC). All animal experiments were approved by the Ethics Committee of Jiao Tong University, Shanghai, China, and were conducted in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.2.2Bifidobacterium blend  was used, which included 7 Bifidobacterium strains: Bifidobacterium infantis (Bi‐26), Bifidobacterium bifidum/lactis (Bb‐02), Bifidobacterium longum (Bl‐05), Bifidobacterium bifidum (Bb‐06), Bifidobacterium lactis (Bl‐04), Bifidobacterium lactis (Bl‐07), and Bifidobacterium breve (Bb‐03). The blend was resuspended in sterilized phosphate buffer saline (PBS), according to previous research.Bifidobacterium (1 * 109 CFU per mouse) was administered to each mouse via oral gavage. Heat inactivation was performed as previously described.Bifidobacterium was boiled at 100°C for 2 h. Live and heat‐treated Bifidobacterium was diluted in PBS and plated on reduced clostridial medium (RCM) agar to incubate in an anaerobic chamber for 3 days to test the efficacy of killing.A lyophilized Bifidobacterium was given 3 days before ONC. The precise time is also indicated in the figure legends. A total of 300 μL Lactobacillus rhamnosus LGG  or Clostridium butyricum  was orally administered to each mouse.For the ONC mouse, 2.3Total RNA was isolated from the mouse retinas with a Trizol reagent according to the RNAsimple Total Kit instructions , which was then reverse transcribed into complementary DNA (cDNA) with a reagent kit . The RT‐PCRs, using a SYBR green‐based PCR method, were performed with a real‐time PCR detection system . GAPDH was used as an internal reference to normalize the variation amounting to total cDNA. The forward and reverse primers for mouse GAPDH were 5′‐AGGTCGGTGTGAACGGATTTG‐3′ and 5′‐TGTAGACCATGTAGTTGAGGTCA‐3′. The forward and reverse primers for mouse iNOS were 5′‐GTTCTCAGCCCAACAATACAAGA‐3′ and 5′‐GTGGACGGGTCGATGTCAC‐3′. The forward and reverse primers for mouse TGF‐β were 5′‐CTCCCGTGGCTTCTAGTGC‐3′ and 5′‐GCCTTAGTTTGGACAGGATCTG‐3′. The forward and reverse primers for mouse Arg‐1 were 5′‐CTCCAAGCCAAAGTCCTTAGAG‐3′ and 5′‐AGGAGCTGTCATTAGGGACATC‐3′. The forward and reverse primers for mouse Egr1 were 5′‐TCGGCTCCTTTCCTCACTCA‐3′ and 5′‐CTCATAGGGTTGTTCGCTCGG‐3′; the forward and reverse primers for mouse Fos were 5′‐CGGGTTTCAACGCCGACTA‐3′ and 5′‐TTGGCACTAGAGACGGACAGA‐3′. Experiments were performed in three parallel wells and repeated at least twice.2.4Bifidobacterium‐treated mice. The retinas were collected and lysed. An equal amount of protein was added to 10% SDS‐PAGE gels, electrophoresed, and transferred electrophoretically onto polyvinylidene difluoride membranes. The membrane was blocked with a blocking buffer , containing 5% nonfat dry milk) for 1 h at room temperature and then incubated overnight with primary antibodies directed against GAP‐43 , GFAP , c‐fos , ERK , p‐ERK , and GAPDH . The membranes were washed with TBST for 30 min and then incubated with a secondary antibody  for 1 h at room temperature. Proteins were visualized using a molecular imaging system . The experiments were repeated three times.A western blot analysis was used to measure GAP‐43, GFAP, c‐fos, ERK, and p‐ERK expression in the retinas of the 2.5Retinas were collected, weighted, and homogenized for the indicated time points. The levels of cytokines and neurotrophic factors in the retinas were detected with ELISA kits specific for mice, according to the manufacturer's procedures (CUSABIO). Results are expressed as picograms per milliliter of medium.2.6Eyes on slides were permeated with 0.1% Triton X‐100 in PBS at room temperature for 20 min. Then, the slides were blocked ) for 1 h at room temperature, and tissues were incubated with primary antibodies against GFAP , GS , Tuj1 , pS6 , and c‐fos  at 4°C overnight. The slides were infiltrated in PBS for 30 min, followed by an hour of incubation with secondary antibodies . DAPI was used to mark the nuclei. All slides were shot and analyzed with a Leica TCS 170 SP8 confocal laser scanning microscope .2.7Bifidobacterium was administered on day 14; and ONC was performed on day 17. Mice were treated with PLX3397 from day 7 to day 31, and RGCs were observed on day 31.Pexidartinib (PLX3397) was administered to pharmacologically ablate retinal microglia . Vancomycin was administered on day 0 and lasted up to day 14; 2.8Optic nerve crush (ONC) was performed as previously described.2.9Bifidobacterium. Total bacterium was used to normalize the abundance of Bifidobacterium. The forward and reverse primers for total Bifidobacterium were CGGTGAATACGTTCCCGG and TACGGCTACCTTGTTACGACTT, and probe was 6FAM‐CTTGTACACACCGCCCGTC; the forward and reverse primers for Bifidobacterium were CGGGTGAGTAATGCGTGACC and TGATAGGACGCGACCCCA, and probe was 6FAM‐CTCCTGGAAACGGGTG.Fecal samples were collected 7 days after different antibiotics  treatment. Bacterial DNA in fecal samples was isolated by Qiagen DNA Stool Mini Kit according to the manufacturer's instructions.2.10Bifidobacterium, and feces were collected 3 days later. The collected feces were treated and resuspended according to the method mentioned in a prior studyDonor mice were fed with vancomycin for 2 weeks, then gavaged with 2.11Bifidobacterium, or fecal suspensions via oral gavage. Feces were collected at 0, 3, 6, 10 and 17 days after gavaging, respectively. The feces of 6 mice were collected in each group . The collected feces were stored at −80°C for 16 s of sequencing. In brief, microbial deoxyribonucleic acid (DNA) was extracted using a PowerFecal Pro DNA Kit (QIAamp). Samples were quantified using the Qubit 1XdsDNA HS Kit (Thermo Fisher). PCR was used to amplify the sample. The concentration of the PCR products was detected using the Qubit 1XdsDNA HS Kit (Thermo Fisher). Then, 4 nM pool samples were prepared and purified using AMPure XP beads. The purified product was subjected to paired‐end sequencing using the Illumina MiSeq platform . The meta‐sequence data were deposited at National Center of Biotechnology Information (NCBI) under Bioproject: PRJNA699743.The mice were given vancomycin orally for 2 weeks and then administered PBS, 2.12Bifidobacterium, and FMT). The RNA‐Seq procedure was carried out as previously described.Retinas were isolated and collected for RNA‐Seq at 0 and 3 days after the ONC of the three groups , and libraries were prepared based on the manufacturer's instructions. The sequencing was carried out using a BGIseq500 platform , and five biologic replicates for each type of sample were provided. Significant results were verified by RT‐PCR. The meta‐sequence data were deposited at NCBI under Bioproject: PRJNA698734.2.13t‐test, one‐way ANOVA, and two‐way ANOVA as appropriate for the specific experiment. The Shapiro–Wilk test and/or Kolmogorov–Smirnov test for normality were used to assess the data distribution. GraphPad Prism version 6.00 (GraphPad Software) was used for the analysis, and the results were presented as mean ± SD. A p value less than 0.05 was considered statistically significant .Differences between groups were analyzed with a two‐tailed Student's 33.12 (treatment vs. control), p < 0.05) and colistin (940.19 ± 106.90 vs. 843.27 ± 63.22 RGCs per mm2 (treatment vs. control), p < 0.05), which effectively act against gram‐negative bacteria. Notably, treatment with vancomycin , p < 0.05), which suggests that gram‐positive bacteria may play a critical role in RGC survival.To explore the effects of gut microbiota on RGCs, mice were fed different antibiotics (ampicillin (1 mg/mL), colistin (1 mg/mL), or vancomycin (0.5 mg/mL)) for 21 days. On day 7 of antibiotic intervention, the mice were subjected to ONC, a classic model of optic nerve injury that induces RGC death.n Figure , which ap < 0.05). We evaluated changes in retinal neurotrophic factors and observed that vancomycin reduced the levels of CNTF, nerve growth factor (NGF), and glial cell‐derived neurotrophic factor (GDNF) after ONC . We found that the relative abundance of Bifidobacterium in vancomycin group decreased significantly, whereas it in ampicillin and colistin groups did not change significantly compared with control group . No significant difference was observed between the number of RGCs in the CB and PBS groups . Approximately half of the cells in the ganglion cell layer (GCL) are RGCs.p < 0.05). In the Bifidobacterium group, we found more viable cells in the GCL of the retinas.To examine the effect of C Figure . CompareBifidobacterium group before and after optic nerve injury .To further confirm RGC survival, we measured phosphorylated S6 (p‐S6), a critical component downstream of mTOR signaling associated with RGC survival and axon regeneration.Bifidobacterium treatment using cholera toxin subunit B (CTB)‐labeling. A higher amount of regenerated fibers were found in the Bifidobacterium group . In addition, compared with PBS treatment in the retina, Bifidobacterium promoted the expression of growth‐associated protein 43 . Heat inactivation of Bifidobacterium, before oral administration, abrogated the RGC therapeutic effect on survival, which suggests that RGC protection requires live bacteria .We quantified optic nerve regeneration after 3.32 (treatment vs. control), p < 0.05).To clarify the implications of the altered gut microbiota on RGC survival, mice were treated with FMT and subjected to ONC. FMT increased RGC survival on day 14 post‐injury  gene sequencing at −3, 0, 3, 7, and 14 dpi in PBS, Bifidobacterium, and FMT groups. Bifidobacterium and FMT administration resulted in significant fecal flora differences. Specifically, there were significant differences in α‐diversity at different times between the three groups . Lactobacilli increases were first observed in intestines following FMT treatment but persisted for a shorter period. We observed that oral administration of Lactobacilli to mice increased the number of RGCs by 30% , p < 0.05).To determine the changes in intestinal microbiota after s Figure . In addis Figure . Over tis Figure . LEfSe as Figure . An incr3.4Bifidobacterium treatment. In the Bifidobacterium group, retinal slices were examined at 3 dpi, and fewer microglia cells were observed in the GCL and outer plex layer (OPL) . In the Bifidobacterium group, microglia were long and extended with more branches, whereas microglia in the PBS retinas were shrunken . Compared with PBS group, a lower rate of microglia was activated in Bifidobacterium group . These results indicate that Bifidobacterium contributes to microglia activation.We examined microglia activation and cell number in the retina following ) Figure . Microgln Figure . Sholl aBifidobacterium group . Specifically, TNF‐α in the Bifidobacterium group was 17% of that in the PBS group, and IL‐1β in the Bifidobacterium group was 19% of that in the PBS group, at 3 dpi. In addition, the levels of IL‐6 and inducible nitric oxide synthase  were lower at 3 dpi in the Bifidobacterium group. No significant difference was observed in the expression of M2 microglia markers, including transforming growth factor β (TGF‐β) and arginase 1 (Arg‐1), between the PBS and Bifidobacterium groups .Inflammatory cytokines, TNF‐α and IL‐1β, were evaluated at different time points after ONC. Following ONC, the levels of TNF‐α and IL‐1β in the PBS control group were higher than that observed in the Bifidobacterium protects RGCs via regulation of microglial cell number and activation, mice were treated with PLX3397 by oral administration. As an inhibitor of the CSF1R receptor, PLX3397 reduces tissue macrophages without affecting myeloid cells.Bifidobacterium was administered on day 14 . We further monitored RGC numbers following PLX3397 and Bifidobacterium treatment and observed that RGC survival increased in PBS and Bifidobacterium groups after PLX3799 administration . However, the number of RGCs in the Bifidobacterium group was higher than that in the PBS group.To verify that 4 Figure . The ONC3.5Bifidobacterium affects the production of neurotrophic factors in the retina, we used qPCR to quantify the levels of CNTF and NGF in mouse retinas at days 0 and 3 following ONC. The expression levels of CNTF and NGF in the Bifidobacterium group were higher than those in the PBS group 3 days following ONC , which indicates that Bifidobacterium increases the expression of neurotrophic factors.Changes in intestinal microbiota alter the hippocampal expression of neurotrophic factors.Bifidobacterium group . Notably, on day 0, when undamaged, the Bifidobacterium group also showed increased GFAP expression. CNTF or NGF expression did not differ significantly between the groups  expression between groups .Bifidobacterium induces Müller cell activation and elevates the production of neurotrophic factors but does not increase the number of Müller cells.In neurodegenerative diseases such as glaucoma, activated Müller cells produce CNTF and NGF.s Figure . Bifidob3.6Bifidobacterium regulates the retinal microenvironment, we determined differentially expressed genes in mouse retinas using bulk RNA sequencing (RNA‐seq). In the absence of injury (0 dpi group) and on day 3 following ONC (3 dpi group), there were gene expression differences in the retinal transcriptome between the PBS and Bifidobacterium groups  and Fos were higher in the Bifidobacterium group than in the PBS group . These results were further verified using qPCR analysis . Egr1, an immediate early gene, responds to stress, such as injury, and participates in cell proliferation and other activities. The Fos gene encodes proteins that dimerize with c‐Jun to form a transcription factor complex activator protein‐1 (AP‐1) heterodimer, which regulates cell proliferation, differentiation, and transformation. Notably, Egr1 and Fos are associated with retinal Müller cell activation. We further measured c‐Fos expression using immunofluorescence and western blotting . As a downstream effector of some intracellular signaling pathways, ERK/c‐Fos signaling mediates Müller cell activation and macrophage differentiation.Bifidobacterium, we measured the expression of total ERK and phosphorylated ERK (p‐ERK) at days 0, 3, and 7 post‐ONC in the PBS and Bifidobacterium groups . There was no significant difference in total ERK expression between the groups. The ratio of phosphor‐p42/total p42 to phosphor‐p44/total p44 in the Bifidobacterium group was significantly higher than that in the PBS group at each time point, which was consistent with c‐Fos expression. This indicates that Bifidobacterium triggers the ERK/c‐Fos signaling pathway.To further elucidate the mechanism by which s Figure . We idens Figure . In partg Figure . Immunof4Bifidobacterium, protects RGC survival and optic nerve regeneration. Bifidobacterium‐induced changes in the intestinal flora contribute to retinal microglia regulation and Müller cell activation, which leads to modifications in inflammatory cytokines and neurotrophic factors  found that Bifidobacterium breve MCC1274 attenuated microglial activation, consequently leading to a downregulation of pro‐inflammatory cytokines.Bifidobacterium longum CCFM1077 ameliorates microglia activity in the cerebellum of autistic‐like rats.Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 significantly suppressed the infiltration of activated microglia into the hippocampus and corticosterone in mice, and hippocampal BDNF expression was also evaluated.Bifidobacterium longum supplementation improves neurotrophic factor expression in AD.Lactobacillus was significantly depleted in the buccal microbiome of patients with uveitic glaucoma, which is associated with a broad spectrum of inflammatory diseases.Lactobacillus ameliorates chronic inflammation by decreasing TNF‐α expression in the gut mucosa.Bifidobacterium and Lactobacillus modulate the activity of microglia and the levels of inflammatory cytokines and neurotrophic factors in the CNS. In addition, we observed that the retina, an extension of the CNS, is similarly affected by Bifidobacterium and Lactobacillus. Furthermore, in our review of macroglia–microglia interactions in glaucoma, we reported that a delicate balance of glia is critical for the survival of RGCs.Bifidobacterium‐induced intestinal changes likely play a role in protecting RGCs and promoting optic nerve regeneration by regulating the activity of glial cells, neurotrophic factors, and inflammatory factors in the retina.We also observed that the administration of Bifidobacterium triggers ERK/c‐Fos signaling pathways independent of ONC. We observed that the expression of p‐ERK and c‐Fos increased after Bifidobacterium treatment, regardless of optic nerve damage. The ERK/c‐Fos pathway may mediate the activation of Müller glial cells in retinal pathology.Bifidobacterium treatment and was primarily distributed in the INL, where glial nuclei were concentrated. Therefore, Bifidobacterium likely activates the ERK/c‐Fos signaling pathway in glial cells, regulates glial cell activity, and promotes RGC survival.We propose that Bifidobacterium are not limited to glaucoma but may offer protection in many similar neurodegenerative diseases. The protective effect of Bifidobacterium has been mentioned in studies involving other neurodegenerative diseases,Bifidobacterium species were found in the feces of patients with AD than in those of healthy individuals.Bifidobacterium longum improved cognitive function and promoted BDNF expression.Bifidobacterium, can significantly improve cognitive function in patients with AD.Bifidobacterium as a well‐known probiotic, and the mechanisms we have proposed here can serve as the basis for further study of Bifidobacterium and neurodegenerative diseases. Overall, the application of appropriate probiotics is expected to become a safe and effective neurotrophic drug based on their role in promoting neurotrophic factors in neurodegenerative diseases.The anti‐inflammatory and neuroprotective effects of Bifidobacterium on RGCs. Secondly, the ONC model used in this study is essentially an optic nerve injury model, which may not fully characterize the pathogenesis of glaucoma in human. It is generally believed that intraocular pressure is one of the most important factors in the pathogenesis of glaucoma. While ONC model is independent of intraocular pressure, thus this finding could only apply to RGC injuries independent of ocular hypertension in the clinical setting. Although we have revealed the important role of Bifidobacterium in the pathogenesis of RGC loss, future studies are critical to identify the comprehensive mechanisms connecting Bifidobacterium in gut and RGCs in the retina.There are some potential limitations in this study. First of all, the exploration of the key connection between gut and retina is still lacking. Currently, it is believed that the possible pathways of communication between gut microbes and retina include: the production of neuroactive metabolites, short‐chain fatty acids and other microbial products, nerves , hypothalamic–pituitary–adrenal axis, endocrine , and immunity (immune cells and cytokines) etc. However, we have not yet explored the possible underlying mechanism of protective effects of Bifidobacterium‐induced changes. The protective effect of Bifidobacterium on RGC could be attributed to the balance between microglia and Müller cells, triggering the ERK/c‐Fos signaling pathways in the retina. Further investigation is required to determine the in‐depth protective mechanism and could illuminate the broader therapeutic potential of Bifidobacterium in the clinical treatment of neurogenerative diseases.In conclusion, our data suggest that RGC survival in mice is mediated by the intestinal microbiota, specifically, Xiaohuan Zhao, Mengqiao Xu and Zhenzhen Zhao conceived the study and designed the experiments. Xiaohuan Zhao wrote the manuscript. Xiaohuan Zhao, Mengqiao Xu and Zhenzhen Zhao performed the experiments with the help of Yang Liu, Yimin Wang and Yao Shen. Ting Zhang, Xiaoling Wan, Mei Jiang, Xueting Luo, Lei Chen, Minwen Zhou, Feng Wang and Xiaodong Sun interpreted data and contributed to discussion. All authors reviewed and concurred with the final manuscript. Xiaodong Sun and Feng Wang are the guarantor of this work and as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.This study was supported by grants from the National Natural Science Foundation of China , Science and Technology Commission of Shanghai Municipality (20Z11900400), Shanghai Hospital Development Center , the National Natural Science Foundation of China (81771739), the Program for Professor of Special Appointments (Eastern Scholar) at Shanghai Institutions of Higher Learning, the Top Young Talent Program of Shanghai, the Innovative Research Team of High‐level Local Universities in Shanghai, Shanghai Sailing Program (22YF1435400), and the National Natural Science Foundation of China (8220040737).The authors declare that they have no competing interests.All authors have read the manuscript and indicated consent for publication.Figure S1–S3Click here for additional data file.


To investigate the efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of metastatic renal cell carcinoma (mRCC) with rhabdoid (mRCC‐R) and sarcomatoid (mRCC‐S) differentiations.In this single‐institutional cohort study, we included patients with RCC with rhabdoid (RCC‐R) and sarcomatoid (RCC‐S) differentiation, who were treated with TKIs after metastasis at our institute from 2013 to 2021. Patient characteristics, treatments, and clinical outcomes were recorded and analyzed.We identified 111 patients with RCC‐R or RCC‐S differentiations, of which 23 patients were included in the final analysis. Of the 23 patients, 10 (43.5%) were grouped as mRCC‐R and 13 (56.5%) as mRCC‐S. At a median follow‐up of 40 months, mRCC‐R and mRCC‐S progressed in 7 of 10 and 12 of 13 patients, respectively. In addition, four and eight patients died in the mRCC‐R and mRCC‐S groups, respectively. The median progression‐free survival (PFS) of the two groups was 19 months  and 7 months (mRCC‐S: 95% CI 2.03–11.96), while the median overall survival (OS) was 32 months and 21 months, respectively. mRCC‐S had a worse prognosis than mRCC‐R. Based on the univariate Cox regression model, single metastasis or multiple metastasis of tumor, rhabdoid differentiation, and sarcomatoid differentiation were predictors of PFS but not OS.The efficacy of TKIs in the treatment of mRCC‐R and mRCC‐S may be different. The most common adverse reactions to anti‐PD‐1 (programmed cell death protein 1) antibody monotherapy include exertion, pruritus, nausea, and diarrhea. Adverse reactions to the combination of anti‐PD‐1 and anti‐CTLA‐4 often lead to an increase in lipase, amylase, and alanine aminotransferase activities. Anti‐PD‐1 drugs combined with anti‐VEGF drugs may cause diarrhea and hypertension. Clinically, immunotherapy in combination with targeted therapy is not suitable for all patients. Therefore, targeted TKI therapy alone is still therapeutically significant.In view of the clinicopathological characteristics of mRCC with rhabdoid and/or sarcomatoid differentiations, we collected data on patients with RCC at the Tianjin Medical University Cancer Hospital between 2013 and 2021, with the aim of investigating whether the therapeutic effects of TKIs in mRCC‐R and mRCC‐S are different. In addition, we sought to elucidate the reasons for the differences and determine individualized treatment plans for patients with different types of tumors.22.1For the cohort study, we gathered clinical and pathological information from 6500 patients with RCC who underwent surgery at Tianjin Medical University Cancer Hospital between 2013 and 2021. Of the 6500 patients, 36 presented with RCC‐R and 75 with RCC‐S. After excluding cases with interrupted/halted follow‐ups and incomplete basic information, 26 patients with RCC‐R and 44 patients with RCC‐S were included in the final analysis.Pathological diagnosis was based on the 2016 World Health Organization classification of renal tumors. Following radical nephrectomy, patients regularly went for check‐ups. TKIs treatments were administered after imaging examination showed metastasis. For patients with distant metastasis before the nephrectomy, TKIs were used after the patients recovered. Patient medication included the following: sorafenib, 400 mg twice daily, 4 weeks as a cycle; sunitinib, 50 mg once daily, 6 weeks as a cycle; axitinib, 5 mg twice daily, 4 weeks as a cycle; fruquintinib, 5 mg once daily, 4 weeks as a cycle; pazopanib, 800 mg once daily, 4 weeks as a cycle. The drug treatment was continued until it no longer produced clinical efficacy or until patients could no longer tolerate drug toxicity. Adverse reactions were evaluated after each cycle of treatment, and imaging examinations were performed every two cycles to evaluate the curative effect. Overall, 11 patients with RCC‐R and 22 patients with RCC‐S had metastasis. The medication after metastasis was unclear for one patient in the mRCC‐R group and for five patients in the mRCC‐S group. In addition, in the mRCC‐S group, two patients used traditional Chinese medicines and two used interferon‐α.Overall, the inclusion criteria for the patients were as follows: RCC with rhabdoid or sarcomatoid differentiation and postoperative metastasis, use of TKI agents after metastasis, and complete basic clinical data. The exclusion criteria were as follows: RCC without rhabdoid or sarcomatoid differentiation, no metastasis after operation, no use of TKI agents after metastasis, and incomplete basic clinical data.Finally, 10 cases of mRCC‐R and 13 cases of mRCC‐S were included in the analysis . PFS constituted the time interval between the beginning of the treatment (after metastasis) and disease progression, intolerable adverse reactions, or death from any cause. OS constituted the time interval between the beginning of the treatment (after metastasis) and death or the end of observation.2.3t‐test.SPSS v24 software (IBM Corp.) was used to conduct statistical analyses. The patient characteristics for each cohort are presented using the mean and standard deviation for continuous variables and numbers and relative percentages for categorical variables. For categorical and continuous variables, respectively, comparisons were performed using the chi‐squared test and independent samples p < 0.05.The survival results are presented using Kaplan–Meier estimates. The median PFS results were reported at a 95% confidence interval (CI). First, we used Kaplan–Meier estimates to explore the relationship between different tumor stages and tumor metastasis in the RCC‐R and RCC‐S groups, respectively. Then, we conducted univariate Cox regression analysis. For this analysis, the patients' sex, age, primary tumor location, tumor size, T stage, metastatic site, and the presence of preoperative metastasis were considered. Age and tumor size were continuous variables. In follow‐up tests, the factors that met the linear condition of the regression analysis model were included in the model in the form of an original variable. Multiple categorical variables were used as dummy variables in the analysis. Statistical significance was set at 33.1p = 0.013). However, the distant metastasis‐free survival of patients with RCC‐R in the T3 stage was better than that of patients in the T1 and T2 stages. In contrast, no significant difference between tumor stage and metastasis was observed in the RCC‐S group . Nevertheless, the metastasis‐free survival time of patients with RCC‐S in the T4 stage was significantly worse than that of patients in the other stages.The metastasis‐free survival time for 26 patients with RCC‐R and 44 patients with RCC‐S was determined through telephone follow‐up and access to the hospital system. Kaplan–Meier estimates were performed to visualize the relationship between different tumor stages and metastasis. The tumor T stage levels included T1, T2, T3, and T4. T1 and T2 stages were combined for the analysis based on the smaller number of patients and similarity in clinical characteristics Table . The res3.2Twenty‐three patients were included in this study. They were treated with TKIs after RCC relapse. Baseline data between 111 RCC (36 cases of RCC‐R and 75 cases of RCC‐S) and 23 mRCC (10 cases of mRCC‐R and 13 cases of mRCC‐S) cases were not statistically different Table . This siThe conditions of metastasis and treatment are summarized in Table p = 0.016, hazard ratio [HR] 4.00, 95% CI 1.30–12.32). Although rhabdoid or sarcomatoid pathology was related to PFS, the association was not statistically significant. . In the regression analysis for OS, no statistically significant parameters were found (Table The median PFS times of the mRCC‐R and mRCC‐S groups were 19 months (95% CI: 4.08–33.9) and 7 months (95% CI: 2.04–11.96), respectively. The mRCC‐R and mRCC‐S groups had median OS times of 32 and 21 months, respectively. In the univariate Cox regression model, the presence of single or multiple metastases was related to PFS , while that for patients using TKIs combined with immunotherapy was 10 months (95% CI: 7.43–12.57).4Both mRCC‐R and mRCC‐S have poor patient prognosis and are often associated with identifiable cancerous components. RCC with rhabdoid or sarcomatoid differentiation was defined as ISUP4 by the International Society of Urological Pathology (ISUP).p < 0.001). The overall response rate for targeted therapy with the sarcomatoid component was approximately 11.1%–19%, with progression within 1 year in 33%–57% of patients,Antiangiogenic VEGF‐TKIs have been used as a first‐line treatment option for RCC over the past 20 years and can greatly improve PFS and OS in patients with locally advanced or distant metastases.Our findings suggest that the efficacy of TKIs in the treatment of mRCC‐R was better than that in mRCC‐S. Under the same application of TKIs, PFS and OS of patients with rhabdoid pathology were better than those of patients with sarcomatoid differentiation. The mRCC‐R group had a progression rate of 70% with a mortality rate of 40%, and the mRCC‐S group had a progression rate of 92.3% with a mortality rate of 61.5%. The efficacy of pazopanib was better than that of sorafenib reported in the literature.The T stage of the tumor is closely related to prognosis. Generally speaking, the higher the stage, the worse the prognosis and the more likely it is to metastasize. Our results show that 11 patients (42.3%) had metastasis in the RCC‐R group, and 22 patients (50.0%) had metastasis in the RCC‐S group. The prognosis among different T stages was statistically different in the mRCC‐R group. However, patients with more advanced/higher stages have metastasis later than patients with lower stages. We speculate that this may be because of the small number of patients with T1 and T2 stage tumors. In the mRCC‐S group, although no statistical difference was observed, metastasis occurred significantly earlier in patients with T4 stage tumors. Overall, the prognosis of patients with mRCC‐R was better than that of patients with mRCC‐S.p = 0.01) between the two arms.In recent years, the emergence of ICIs has greatly improved the prognosis of metastatic RCC. A retrospective chart review compared the prognosis of 35 patients with metastatic RCC with a sarcomatoid component, in which 10 patients were treated with ICIs and 25 were administered other therapies. They showed that there was a statistically significant difference in OS  there may be differences in the efficacy of TKIs in mRCC‐R and mRCC‐S that need further investigation; (2) immunotherapeutic agents have a role in the prognosis of metastatic RCC.Kun Wang: Investigation ; methodology ; writing – original draft ; writing – review and editing . Pengqiang Duan: Conceptualization ; software ; visualization ; writing – original draft ; writing – review and editing . Xusheng Chen: Investigation ; project administration ; supervision . Qing Yang: Formal analysis ; validation . Guowei Feng: Investigation ; resources . Lei Diao: Funding acquisition ; resources ; visualization . Zhenting Zhang: Conceptualization ; resources . Xin Yao: Project administration ; supervision .The authors declare that there are no conflicts of interests.The study has been approved by the Medical Ethics Committee of Tianjin Cancer Hospital .Table S1.Table S2.Click here for additional data file.


This supports ongoing optimization efforts, including scale‐up evaluations of formoterol treatment in large animal models of VML.Volumetric muscle loss (VML) is associated with persistent functional impairment due to a lack of de novo muscle regeneration. As mechanisms driving the lack of regeneration continue to be established, adjunctive pharmaceuticals to address the pathophysiology of the remaining muscle may offer partial remediation. Studies were designed to evaluate the tolerance and efficacy of two FDA‐approved pharmaceutical modalities to address the pathophysiology of the remaining muscle tissue after VML injury: (1) nintedanib (an anti‐fibrotic) and (2) combined formoterol and leucine (myogenic promoters). Tolerance was first established by testing low‐ and high‐dosage effects on uninjured skeletal muscle mass and myofiber cross‐sectional area in adult male C57BL/6J mice. Next, tolerated doses of the two pharmaceutical modalities were tested in VML‐injured adult male C57BL/6J mice after an 8‐week treatment period for their ability to modulate muscle strength and whole‐body metabolism. The most salient findings indicate that formoterol plus leucine mitigated the loss in muscle mass, myofiber number, whole‐body lipid oxidation, and muscle strength, and resulted in a higher whole‐body metabolic rate ( Recently, the β2 adrenergic receptor agonist, formoterol, has shown promise for improving whole‐body and local muscle metabolism following VML injury ; in compliance with the Animal Welfare Act, the Implementing Animal Welfare Regulations, and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. Mice were housed on a 12‐h light–dark cycle (light phase begins at 06:00) with ad libitum access to chow and water.n = 72) male C57BL/6J mice  were used in all studies. While this study only included female mice, future work should evaluate any sex‐specific differences to overcome this limitation. Prior to any study procedures being performed, mice were given a one‐week acclimation period. Mice were randomly allocated to two cohorts. First, to optimize dosing, mice were randomized to one of the two pharmaceutical treatments, each separated into a high‐ (n = 8) and low‐dose (n = 8) group, or no treatment . Following optimization of skeletal muscle mass and myofiber cross‐sectional area, the second cohort of mice underwent unilateral VML surgery to the posterior muscle compartment  or served as injury naïve controls. The mice that underwent VML surgery were randomly assigned to a pharmaceutical treatment immediately following VML or no treatment. Mice were excluded from the protocol and analyses in the events of death prior to terminal harvest, weight loss ≥20% of initial total body weight, and/or failure to thrive following gavage treatment. Three mice in each cohort  were removed from the study and no data from these mice were used in the work. At the terminal time point (4 or 8 weeks), while deeply anesthetized via inhaled isoflurane (~2.0%), blood was collected via cardiac puncture and serum was stored at −20°C, and skeletal muscles were harvested, weighed, frozen, and stored at −80°C for later analyses. The extensor digitorum longus (EDL), soleus, and/or middle third of the gastrocnemius  muscles were saved for histological analyses, while the tibialis anterior (TA) and remaining proximal and distal portions of the gastrocnemius were saved for biochemical analyses. All mice were euthanized with pentobarbital . For all experimental procedures and analyses, investigators were blinded.Adult  was delivered at 6 mg/kg/day or 60 mg/kg/day via daily gavage at a volume of 0.01 mL/g body weight . Formoterol (Sigma #1286107) plus leucine was delivered via additive to the standard chow of mice at 0.3 mg/kg/day plus 8 g/kg/day or 3 mg/kg/day plus 24 g/kg/day, respectively. Doses were selected based on doses previously evaluated in models of liver and lung fibrosis, and muscular dystrophy,  as previously described .The proximal portion of the gastrocnemius muscle was homogenized in 1:10 (mg/μL) 10 mM POTwenty‐five to 50 μg of protein were loaded onto a 4%–20% Criterion TGX Stain‐Free Gel and blocked with 5% bovine serum albumin in 0.1% Tween TBS. The following primary antibodies were used at 1:1000 dilutions: Akt , MyoD1 (Proteintech 18943‐1‐AP), Myf5 (Abcam ab125078), and Myostatin (Abcam ab124721). Corresponding host‐ and isotype‐specific secondary antibodies  were used. Blots were first imaged  using the stain‐free setting for total lane protein evaluation and then imaged using the appropriate fluorescence channel to measure the protein band of interest. Image Lab software (Bio‐Rad) quantified total lane protein and band intensity. Bands were normalized to the total protein in each respective lane and compared across groups.Evaluation of inflammatory, angiogenic, and fibrotic markers in the serum of the VML cohort mice was done via multiplex. Specifically, IL‐6, IL‐10, IL‐17A, and VEGFA (Millipore MCYTOMAG‐70K) and TGFβ1‐3 (Bio‐Rad #171W4001M) were evaluated according to manufacturer's technical specifications. Plates were read using a Bio‐Plex‐200 system (Bio‐Rad). Analytes were excluded from analysis if less than 65% of the total samples were below detection; IL‐17A and VEGFA did not meet this a priori threshold and were omitted from analysis.2.8p ≤ 0.05. One‐way ANOVAs evaluated maximal torque and contractile parameters, muscle mass, myofiber number, ambulation, metabolic outcomes , and protein content. A two‐way ANOVA (group × time) evaluated body mass longitudinally. Where appropriate, Tukey's HSD post hoc determined specific differences between groups. Differences in distribution of myofiber cross‐sectional area were analyzed using a chi‐squared test and a Bonferroni post hoc (adjusted p ≤ 0.008). Data are presented as mean ± standard deviation (SD), with individual data points displayed per mouse.Data were analyzed using GraphPad Prism  and JMP software (version 16.0 SAS Institute). Dependent variables were compared across treatment groups and considered significant at 33.1p < 0.001; Figure p ≤ 0.003; Figure p ≥ 0.170). The myofiber cross‐sectional area of the EDL muscle was not different across groups, but soleus myofiber cross‐sectional area was ~31% lower with the high dose of nintedanib compared to low dose formoterol plus leucine . Although 24‐h RER and carbohydrate oxidation were not affected by treatment  except for the time to peak twitch and the average relaxation rate (−dP/dt) after achieving maximal torque . Although VML untreated mice had a similar distribution to nintedanib‐ and formoterol plus leucine‐ treated mice (p ≥ 0.030), nintedanib‐treated mice had a greater leftward shift compared to formoterol plus leucine treated mice  administered thereafter, could improve both the fibrotic and myogenic aspects of functional skeletal muscle regeneration chronically.Fibrotic formation after skeletal muscle injury replaces functional or parenchymal tissue with connective tissue Wynn, . This pr2 adrenergic receptor agonists stimulate lipolysis  and metabolism with formoterol plus leucine treatment support its use in future translational investigations of VML. Further research is warranted to explore adjunctive β2 adrenergic agonists loaded into a scaffold for local muscle administration of the drug, with or without rehabilitative exercise . In addition to the optimal dose of formoterol plus leucine identified herein, it is important to identify the optimal timing of initiation and duration of a combined regenerative rehabilitative approach to enhance functional muscle regeneration and metabolic adaptations.To continue evaluating potential therapeutic options for patients after VML, future investigations should focus on promoting myogenesis and metabolism using βExperiments were conducted in the laboratory of Sarah M. Greising. Jarrod A. Call and Sarah M. Greising were involved in conception or design of the work. Shefali R. Bijwadia, Christiana J. Raymond‐Pope, Alec M. Basten, Thomas J. Lillquist, Mason T. Lentz, Jarrod A. Call, and Sarah M. Greising were involved in acquisition, analysis, or interpretation of data for the work. Shefali R. Bijwadia, Christiana J. Raymond‐Pope, Jarrod A. Call, and Sarah M. Greising were involved in drafting of the work or revising it critically for important intellectual content. All authors approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.Funding through the Congressionally Directed Medical Research Program, Clinical & Rehabilitative Medicine Research Program: W81XWH‐20‐10,885 (JAC and SMG). SRB was supported in part by a University of Minnesota Foundation Medical Student Research Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.The authors declare that they have no conflict of interests.


The lateral periaqueductal gray (LPAG), which mainly contains glutamatergic neurons, plays an important role in social responses, pain, and offensive and defensive behaviors. Currently, the whole‐brain monosynaptic inputs to LPAG glutamatergic neurons are unknown. This study aims to explore the structural framework of the underlying neural mechanisms of LPAG glutamatergic neurons.This study used retrograde tracing systems based on the rabies virus, Cre‐LoxP technology, and immunofluorescence analysis.We found that 59 nuclei projected monosynaptic inputs to the LPAG glutamatergic neurons. In addition, seven hypothalamic nuclei, namely the lateral hypothalamic area (LH), lateral preoptic area (LPO), substantia innominata (SI), medial preoptic area, ventral pallidum, posterior hypothalamic area, and lateral globus pallidus, projected most densely to the LPAG glutamatergic neurons. Notably, we discovered through further immunofluorescence analysis that the inputs to the LPAG glutamatergic neurons were colocalized with several markers related to important neurological functions associated with physiological behaviors.The LPAG glutamatergic neurons received dense projections from the hypothalamus, especially nuclei such as LH, LPO, and SI. The input neurons were colocalized with several markers of physiological behaviors, which show the pivotal role of glutamatergic neurons in the physiological behaviors regulation by LPAG. RV‐based retrograde tracers allow us to identify neuronal presynaptic connections. we identified that seven brain structures, namely the cortex, thalamus, hypothalamus, superior colliculus, midbrain, pons, and medulla, project monosynaptic inputs to the LPAG glutamatergic neurons, in which the hypothalamus project most densely. The LPAG is involved in social,Rabies virus (RV)‐based retrograde tracers allow us to identify neuronal presynaptic connections22.1Adult transgenic mice  with Cre recombinase expression in glutamatergic neurons (Vglut2‐Cre mice) of the C57BL/6J strain and wild‐type littermates without Cre expression were used for retrograde tracing experiments. The mice were housed under an automatically controlled 12/12‐h light/dark cycle 2.212, 5 × 1012, and 2 × 108 genomic copies/mL, respectively.BrainVTA  was responsible for packaging the viral vectors. The titers of AAV‐EF1α‐DIO‐TVA‐EGFP, AAV‐EF1α‐DIO‐RvG, and an EnvA‐pseudotyped glycoprotein (RG)‐deleted and DsRed‐expressing RV (RV‐EnvA‐ΔRG‐DsRed) were 5 × 102.3n = 4 mice). At week 4, the mice were perfused.The surgical procedures were performed according to previous studies.2.4One week after injection of the RV, the mice were deeply anesthetized and perfused with phosphate‐buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer . Following perfusion, the brains were removed and post‐fixed in 4% PFA for 4–6 h at 4°C and then cryoprotected in 10%, 20%, and 30% sucrose at 4°C until they sank. Coronal brain sections (30‐μm‐thick) were cut on a freezing cryostat  into four series and were collected in 0.1 M PBS (pH 7.4).For immunofluorescence, the brain sections were rinsed three times with 0.01 M PBS containing 0.3% Triton X‐100 (PBST) and then incubated overnight at 4°C in PBST containing primary antibodies. The following primary antibodies were used: monoclonal mouse anti‐orexin‐A  and polyclonal rabbit anti‐GABA . After staining with the primary antibodies, the sections were rinsed three times with PBST again and incubated with the secondary antibodies  for 2 h at room temperature (20–22°C). Next, after three washes with PBST, the brain slices were incubated with 4′,6‐diamidino‐2‐phenylindole  at room temperature for 10 min to stain the nuclei. Finally, after washing with PBST, the brain slices were placed on glass slides and covered with coverslips.2.5Whole‐brain sections were imaged under 10× or 20× magnification with the VS120 virtual microscopy slide scanning system (Olympus). A confocal microscope was used to obtain 20× or 40× magnified images of the brain sections to obtain more details . For cell mapping of neurons, the ImageJ software was used to semi‐automatically quantify the neuronal bodies. Based on a map of the mouse brain, the boundaries of specific brain regions were depicted using ImageJ.33.1To identify monosynaptic inputs to the glutamatergic neurons in the LPAG, we used a cell type‐specific, RG‐deleted RV strategy3.2To determine the inputs to LPAG glutamatergic neurons, the brains of these injected mice were cut serially in coronal sections after adequate infection time Figure . Section3.3Next, we determined the colocalization of inputs to the LPAG glutamatergic neurons using several neuronal markers associated with important physiological functions via immunofluorescence. In the LH, the inputs to LPAG glutamatergic neurons were found to be partly colocalized with orexin  . We found that the highest numbers of inputs to LPAG were localized in the hypothalamus. The anatomical and functional connectivity of the hypothalamic‐PAG circuit, especially the ventromedial hypothalamus and ventrolateral area (VMHvl), to the LPAG has been reported previously,Next, in order to better identify the distribution of these DsRed‐labeled neurons, we performed statistical analysis after identifying the brain regions with monosynaptic inputs to LPAG glutamatergic neurons based on standard mouse brain atlases. We divided the whole brain into seven brain structures, namely the cortex, thalamus, hypothalamus, superior colliculus, midbrain, pons, and medulla. Then, we calculated the proportion of DsRed‐labeled afferent neurons of each brain region in the whole brain. We defined the brain regions in which DSRed‐labeled neurons accounted for >0.05% of the total labeled neurons as the nuclei with monosynaptic connections with LPAG glutamatergic neurons and generated a list of whole‐brain inputs to LPAG glutamatergic neurons  glutamatergic neurons have a potential role as a target for analgesia, in which the LHPV neurons‐VLPAG axonal projections play a vital role. More interestingly, an increasing number of studies have suggested bidirectional regulation of sleep–wake and pain behavior.The LPAG plays a vital role in pain‐related behavioral responses. Several clinical studies have shown that the functional connectivity of LPAG is disrupted in patients with pain.4.5LPAG is involved in fear, defensive, sleep–wake, pain and other behavioral functions. These behaviors are accompanied by changes in heart rate (HR) and respiration, which demonstrates the significant potential implications of LPAG activity for HR and respiratory control. For HR, manipulating the activity of LPAG neurons can effectively alter cardiovascular effects, including bradycardia or increases in HR.In conclusion, we mapped the monosynaptic afferents to the LPAG glutamatergic neurons and found that they received projections from other brain areas, especially the nuclei localized in the hypothalamus. This suggests a vital role of LPAG glutamatergic neurons in a wide range of physiological and pathological functions, including sleep–wake regulation, offensive‐defensive behaviors, and pain response. We also summarized the involved physiological behaviors of nuclei that input to LPAG glutamatergic neurons Table , which m5Our study confirmed that the LPAG glutamatergic neurons received dense projections from the hypothalamus, and dsRed‐labeled neurons were colocalized with several markers of physiological behaviors. The pivotal role of glutamatergic neurons in the physiological behaviors regulated by the LPAG was further confirmed based on neuroanatomical terms.YQW and ZLH contributed to the study concept and design and drafting/revising of the manuscript for content. WXM and LL contributed to running of the experiments, acquisition of the data, and figure drawing. LXK, HZ, and PCY contributed to acquisition of the data and statistical analysis. All authors have read and agreed to the published version of the manuscript.All authors declare no conflict of interest.


Preoperative hemoglobin (Hb) level is a predictor of in‐hospital and midterm mortality in patients undergoing coronary artery bypass grafting surgery (CABG). However, the debate about the different hazards across Hb levels and sex differences in outcome occurrence is still on the table.This is a registry‐based serial cross‐sectional study at Tehran Heart Center. Nonanemic patients who underwent CABG with complete follow‐up data were included. The Restricted Cubic Splines (RCS) in the Cox model was used to calculate the sex‐specific correlation between in‐hospital, 6‐month, and 1‐year mortalities and normal Hb levels using odds ratio for the in‐hospital and hazard ratios for 6‐month and 1‐year mortality, adjusted for all possible confounders.From 2005 to 2016, a total of 13,557 patients were included, of which 134 had in‐hospital mortality as our primary outcome. Preoperative Hb levels were significantly lower in the deceased. Moreover, dead patients had significantly higher rates of diabetes and hypertension, while lower ejection fraction. Cut‐offs for reference Hb level were higher for males compared with females. The correlation between Hb level and in‐hospital mortality was nearly U‐shaped. Quantitatively, Hb of ≥15.62 and ≤13.25 g/dL for men and that of ≥14.92 and ≤13.4 g/dL for women tended to be associated with higher in‐hospital mortality.The association between preoperative Hb level and in‐hospital mortality differs in men and women and does not follow a linear correlation among nonanemic patients. Both low and high numbers in the Hb level spectrum are at greater risk. In this study conducted at Tehran Heart Center, we investigated the association between preoperative hemoglobin levels and mortality in 13,557 patients undergoing coronary artery bypass grafting (CABG) surgery. In‐hospital mortality occurred in 134 patients. Hemoglobin cutoffs were higher for men, compared with women, and the correlation between hemoglobin levels and in‐hospital mortality was almost U‐shaped. In conclusion, both high and low hemoglobin levels accounted for higher mortality, which can have clinical implications. In addition, we determined the sex‐specific prevalence of all‐cause mortality in patients with different levels of Hb.22.1This is a registry‐based serial cross‐sectional study conducted between 2005 and 2016 at Tehran Heart Center (THC). THC is a major tertiary referral center in Tehran, Iran, dedicated to the treatment of heart diseases.2.2Patients' Hb levels were recorded before the operation as a part of the complete blood count (CBC) test, reported in grams per deciliter (g/dL). Demographics of the patients, including age, sex, weight, height, smoking status, opium consumption, and family history of coronary artery disease, were obtained before admission. Past medical history of previous myocardial infarction (MI), previous CABG, previous percutaneous coronary intervention (PCI), cerebrovascular accident (CVA), chronic obstructive pulmonary disease (COPD), and complete drug history were also recorded. Fasting blood glucose (FBS), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), total cholesterol, and triglycerides were measured during hospitalization. Finally, left ventricular ejection fraction (LVEF) was measured using echocardiography, and used as a risk estimator for surgery. Total intensive care unit (ICU) time, total ventilation time, and total admission days were recorded in our databank as postoperative variables.2.3Patients were evaluated 6 months and 1 year after CABG and then annually after surgery by direct clinic visits. Patients who did not attend the clinic were followed by telephone interviews. The primary endpoint was in‐hospital mortality while 6‐month and 1‐year mortality were secondary endpoints in the current study.2.4t test for normally distributed and Mann–Whitney U test for skewed distributed variables. The χ2 test was applied to compare categorical variables.Normally and non‐normally distributed continuous variables were presented as mean with standard deviation (SD) and median with 25th and 75th percentiles (interquartile range [IQR] boundaries), respectively. The Kolmogorov–Smirnov test was used to test for the data normality.p value of <.05 was considered statistically significant.To eliminate the gender effect on mortality, data were stratified into “male” and “female.” For the in‐hospital mortality, a logistic regression model calculating odds ratio (OR) with its 95% confidence interval was used. The adjusted Cox proportional hazards model was used to evaluate the Hb effect on in‐hospital and mid‐term mortality. Hb relationship with in‐hospital, 6 months, and 1‐year mortality hazards were estimated by entering Hb as a continuous variable in the adjusted model. The Restricted Cubic Splines (RCS) in the Cox model allow prospecting for a nonlinear relationship of Hb level with the “Odds Ratio” of in‐hospital mortality and “Hazard ratio” of 6 months and 1‐year mortality, estimated from the Cox regression model adjusted for all possible confounders. We applied five knots (df = 4), applied at the 5th, 25th, 50th, 75th, and 95th percentile of Hb levels. A cutoff point was identified to investigate the independent prognostic role of Hb level using the optimal equal‐hazard ratio (HR) method, which selects optimal cut‐points of continuous predictors that have U‐shaped relationships with log(λ) in survival analysis. The statistical details are beyond the scope of this study and are mentioned elsewhere.33.1p < .001). Lower preoperative LVEF and lower estimated glomerular filtration rate (eGFR), in addition to higher diabetes, hypertension, and renal failure rates, were also recorded in dead patients. The baseline characteristics of patients are summarized in Table Of the 24,328 recorded patients, we excluded 10,771 patients due to various reasons, including having anemia, as described comprehensively in Figure 3.2As the normal Hb range for men and women differs, the analyses were divided based on patients' sex. Table 3.33.3.1p = .925; women: OR = 2.06 [95% CI: 0.93–5.67], p = .073). Moreover, although not significant, a higher incidence of in‐hospital mortality was observed with Hb lower than 13.4 g/dL in women . However, men with Hb below 13.25 g/dL had significantly higher mortality than the reference range .Calculated gender‐specific ORs for mortality based on baseline Hb levels are presented in Table 3.3.2p = .014 for both). This was also the case for Hb levels higher than 13.6 g/dL, however, HR was only significant for 1‐year mortality . Another difference was that the nadir of the mortality HRs moved up to higher Hb levels in men, but it decreased in women compared with in‐hospital results. Also, compared with in‐hospital mortality, 6‐month, and 1‐year estimated HRs in women tend to decrease in higher Hb levels (Figure Mortality HRs were almost identical in 6‐month and 1‐year follow‐ups Table . In womes Figure .4The findings of the present study suggest that higher preoperative Hb levels have a clear tendency toward higher mortality in both men and women. In addition, patients with lower limits of normal Hb levels were at greater risk compared with mid‐normal values. Although anemia was thoroughly discussed in the literature as an important risk factor for in‐hospital mortality after CABG,Our study found that there is a higher mortality in females, compared with males. This is in line with other studies assessing CABG mortality.Kullier et al. reported a dose‐dependent significant increase in adverse outcomes including mortality and cardiac complications, with decreasing Hb levels compared with patients with Hb>14.Aside from enrolling nonanemic patients, major differences between our study compared with studies evaluating the effect of Hb level independent of anemia were the longer follow‐up period for mortality and gender‐specific reference range and results. We also estimated gender‐specific cutoffs of baseline Hb for post‐CABG mortality. Certainly, this concept is novel and other studies are required to elaborate further on this matter.4.1We did our best to include and adjust all important confounding parameters. However, more data are needed to evaluate etiologic pathways in addition to other possible confounders which were not included. In assessing blood loss in the operation room, the number of transfused packed cells is among the important variables. The association between transfusion and post‐CABG mortality has been established before, and its effect is independent of the presence of preoperative anemia and Hb levels. To decrease the effect of this confounder, we enrolled only nonanemic patients in our study. Although not able to omit this factor and as reported before that most anemic patients who underwent CABG needed blood transfusion during surgery, our inclusion criteria could overcome this issue to some extent.Mid‐ and long‐term outcomes are associated with much more parameters and cannot be justified merely with preoperative baseline characteristics. Unfortunately, we could not capture changes in risk factor profile after CABG; hence postdischarge results should be assessed with caution.5Simple one‐value cut‐offs in quantitative parameters are insufficient and sometimes misleading. Patients at the highest end of Hb levels and in lower limit normal showed a tendency toward higher in‐hospital mortality after CABG. Moreover, there is a clear gender difference in Hb cut‐offs and effects. This study was hypothesis‐generating and needs confirmation with a more detailed analysis in a larger sample size.Study conception/data analysis/drafting the manuscript/revision: Amirmohammad Khalaji, Ali Ajam, Ali Sheikhy, Amir Hossein Behnoush, Aida Fallahzadeh. Drafting the manuscript/revision: Jamshid Bagheri, Soheil Mansourian, Shahram Momtahen, Farzad Masoudkabir. Study conception/drafting the manuscript/critical revision: Kaveh Hosseini.The authors declare no conflict of interest.