Purpose: Amblyopia and strabismus affect 2%–5% of the population and cause a broad range of visual deficits. The response to treatment is generally assessed using visual acuity, which is an insensitive measure of visual function and may, therefore, underestimate binocular vision gains in these patients. On the other hand, the contrast sensitivity function (CSF) generally takes longer to assess than visual acuity, but it is better correlated with improvement in a range of visual tasks and, notably, with improvements in binocular vision. The present study aims to assess monocular and binocular CSFs in amblyopia and strabismus patients.Methods: Both monocular CSFs and the binocular CSF were assessed for subjects with amblyopia (n = 11), strabismus without amblyopia (n = 20), and normally sighted controls (n = 24) using a tablet-based implementation of the quick CSF, which can assess a full CSF in <3 min. Binocular summation was evaluated against a baseline model of simple probability summation.Results: The CSF of amblyopic eyes was impaired at mid-to-high spatial frequencies compared to fellow eyes, strabismic eyes without amblyopia, and control eyes. Binocular contrast summation exceeded probability summation in controls, but not in subjects with amblyopia (with or without strabismus) or strabismus without amblyopia who were able to fuse at the test distance. Binocular summation was less than probability summation in strabismic subjects who were unable to fuse.Conclusions: We conclude that monocular and binocular contrast sensitivity deficits define important characteristics of amblyopia and strabismus that are not captured by visual acuity alone and can be measured efficiently using the quick CSF. The magnitude of strabismus may vary with viewing distance, such that some people with strabismus are able to fuse at some viewing distances  of the fellow fixing eye remains normal or near normal in amblyopia  strabismic, anisometropic, or mixed amblyopia (AMB); (2) strabismus without amblyopia (SWA); and (3) normally sighted individuals (NSC). Inclusion and exclusion criteria were:Clinical amblyopia is often defined as at least 0.2 logMAR interocular difference in acuity with best correction. Here, we adopted the clinical definition of amblyopia for our inclusion and exclusion criteria. Strabismic amblyopia was defined as a ≥0.2 logMAR interocular difference in best-corrected visual acuity (BCVA). Strabismus was defined as angular deviation between eyes of 5–50 prism diopters at either near or far viewing distances. Anisometropic amblyopia was defined as a 2-line or greater interocular difference (≥0.2 logMAR) in BCVA with tropia ≤4 prism diopters.Strabismus without amblyopia was defined as ≤0.2 logMAR difference between the monocular BCVAs. Strabismus was defined as above. Intermittent strabismus with near fusion was defined as ≤4 prism diopters at 40 cm test distance.Normal vision was defined as BCVA ≤0.0 logMAR or uncorrected VA ≤0.2 logMAR for both eyes without any latent or manifest ocular deviation other than phorias within normal limits.Subjects with any known cognitive or neurological impairments were excluded.the non-dominant eye (NDE) and dominant eye (DE), respectively. The DE was determined by the results from clinical binocular function or acuity test (for AMB and SWA subjects) or finger pointing task (for NSC).Informed consent was obtained from subjects or (in addition to subjects’ assent) from the parents or legal guardian of subjects aged <18 years, in accordance with procedures approved by the IRB of Boston Children’s Hospital and complying with the Declaration of Helsinki. Enrolled patients underwent complete clinical examination, including best corrected visual acuity , cycloplegic refractive error, stereopsis (Titmus Fly SO-001), ocular motility, binocular fusion (a Worth 4 dot test) and cover test at near and distance fixation. The angle of any heterotropia or heterophoria was measured by prism-and-cover test at near and distance fixation. We only report the measurements made at near fixation, which is relevant to the 60 cm viewing distance of CSF assessment. Minimum participant age was 5 years and all participants were able to perform letter acuity assessment. Participant characteristics are provided in Spatial CSFs were assessed with the quick CSF method  that is based on a log-parabola S0. The parameter vector Θ has four dimensions: (i) peak gain, γmax; (ii) peak spatial frequency, fmax; (iii) bandwidth, β; and (iv) low-frequency truncation level, δ.The CSF maps a spatial frequency The quick CSF also provides two important scalar features: (i) a summary statistic, the area under the log CSF . For a finer-level analysis, we used the probability summation model, which is the simplest and most commonly used account in vision and hearing science  describes the probability of a correct response as a function of signal contrast c and threshold contrast τ:During data recording, the quick CSF was initialized with a uniform prior. After data collection, all data sets were rescored with a more informative population prior , λ is the lapse rate, β is the slope of P , and Φ is the cumulative distribution function of the normal distribution. This conversion was made as a function of spatial frequency and the final probability for each viewing condition  was the mean probability across spatial frequencies. Next, we computed the expected probability summation derived from the probabilistic summation of monocular signals from the non-dominant and dominant eyes as shown in Equation 2.where P is the probability of detecting a target signal . We then compared this expected probability summation value PExpected Binocular to the probability of binocular viewing condition PBinocular.where The posterior of the quick CSF provides a probability distribution over possible CSFs and their agreement with the data. We used the width of the credible interval, which encompasses 68.3% of the data, as a proxy to test-retest variability  for an assumed uniform prior  from normal distributions DAMB and DNSC with these parameters, and calculated how often the originally observed p-value of approximately 0.0009 would occur when comparing the observed distribution of NSC eyes against data under the null hypothesis  or a hypothesized true effect . The ratio MNSC/(MNSC+MAMB), which is influenced by statistical power of the experiment and the observed effect size and p-value, then gives us the FPR that we would see if the observed outcome was due to the null hypothesis being true; here, FPR = 0.003.For statistical tests, we used a significance level of 0.05. Because a https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/3XWZUN.Data are publicly available at The mean time to complete each quick CSF test was 170 (± 34) s, see We first analyzed the four parameter values of the quick CSF and its two summary estimates, AULCSF and CSF Acuity for the three groups.The contrast sensitivity deficits of AMB patients can be seen in p = 0.001, Wilcoxon test; FPR = 0.002) and peak gain  for the non-dominant eye. However, no significant difference was observed in bandwidth and in low SF truncation , and SWA and NSC did not show a significant difference between the two eyes for any of the parameters.More specifically, the AMB group showed a significant reduction in peak SF , respectively; two-sided t-test). AULCSF and CSF Acuity were also significantly lower in the non-dominant relative to the dominant eye  of AMB.The changes in peak SF and peak gain for the AMB group resulted in a pronounced AULCSF deficit in the non-dominant (but not dominant) eye relative to the NSC group , there was no such evidence for AMB (p = 0.14) or SWA (p = 0.09), when all subjects were included in the analysis (blue boxes).p = 0.17). The summation index was significantly greater than 1 in SWA subjects who were able to fuse . These results suggest that in strabismus, the lack of binocular summation is a direct consequence of ocular misalignment, whereas in amblyopia there is an additional fundamental developmental deficit in binocular vision.In principle, a lack of binocular summation may be due to disparate retinal correspondence that resulted from ocular misalignment of the strabismic vision rather than an absence of neural summation  at 1,000 spatial frequencies (0.64~41 cpd). Based on (Equation 1), we computed the probability of a correct response at that contrast in the monocular conditions. These probabilities were averaged across spatial frequencies. Lastly, we computed the expected binocular detection probability based on the probability summation of the monocular detection probabilities (Equation 2).p < 0.01, Wilcoxon test; FPR = 0.079) while SWA and control groups did not show any significant differences between the two eyes .Data points in p > 0.05, t-test). Binocular summation of NSC subjects, however, was significantly greater than predicted from the probability summation . For the subset of SWA subjects who were unable to fuse .For AMB subjects (with and without the ability to fuse the monocular images) and those SWA subjects that were able to fuse . Variability of logMAR acuity can be well-accounted for by log CSF Acuity (r2 = 0.64). Slightly less accountability (r2 = 0.55) was observed in the regression of logMAR acuity on AULCSF , glaucoma, and cataract. It can also be extended to other measures of visual function, such as monocular and binocular visual acuity, and monocular and binocular perimetry.In conclusion, our results suggest that monocular and binocular contrast sensitivity deficits define important characteristics of amblyopia and strabismus that are not captured by visual acuity alone. Furthermore, our results identify a key role of fusion in binocular summation. Finally, measurement of both the monocular and binocular CSFs was possible rapidly and reliably even in young children, which may allow clinicians to more accurately assess individual patients’ functional contrast sensitivity and acuity outcomes and prognosis.The datasets generated for this study are available on request to the corresponding author.This study was carried out in accordance with procedures approved by the IRB of Boston Children’s Hospital with written informed consent from all subjects or (in addition to subjects’ assent) from the parents or legal guardian of subjects aged <18 years. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the IRB of Boston Children’s Hospital.MYK, MK, KC, DH, Z-LL, and PB contributed to the conception and design of the study. AM collected the data and organized the database. MD, MYK, and LL analyzed the data. MD and PB wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.MD, LL, Z-LL, and PB have intellectual property in quick CSF assessment and equity in Adaptive Sensory Technology (AST), a company that aims to commercialize the quick CSF; LL also holds employment at AST. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Psoriasis is a common chronic inflammatory skin disease that features the abnormal proliferation of keratinocytes. This proliferation could partly result from disturbances in vitamin A metabolism. Changes in psoriasis patients of the levels of retinol-binding protein 4 (RBP4), a carrier of retinol (vitamin A); transmembrane protein stimulated by retinoic acid 6 (STRA6); and other retinol metabolic molecules have not yet been fully established. Therefore, we investigated vitamin A-related proteins in mice with imiquimod (IMQ)-induced psoriasis.Thirty mice were divided into four study groups: two groups underwent IMQ application for 3 or 6 days , and two groups underwent Vaseline application for 3 or 6 days . Blood and skin samples from both lesional and non-lesional areas of the mice were analyzed using enzyme-linked immunosorbent assays, hematoxylin and eosin staining, immunochemistry, real-time reverse transcription polymerase chain reaction, and RNA sequencing.p < 0.001); 2) retinol levels in lesional skin increased slightly (p = 0.364), but no change was evident in serum retinol levels; 3) STRA6 was upregulated in both lesional skin (p = 0.021) and serum (p = 0.034); 4) RBP4 levels were elevated in serum (p = 0.042), but exhibited only an increasing trend (p = 0.273) in lesional skin; and 5) proteins and enzymes that mediate retinoic acid formation and transformation were upregulated in lesional skin.IMQ-treated mice developed erythema, scales, and skin thickening. Compared with the control groups, IMQ-treated groups had the following changes: 1) interleukin (IL)-17A, IL-23, and tumor necrosis factor (TNF)-α levels were raised significantly in both serum and lesional skin (all As the demand for vitamin A in psoriatic mice increased, retinol underwent relocation from the circulation to target tissues. RBP4, STRA6, and the transformation from retinol to retinoic acid were upregulated, which may be part of the mechanism of psoriasis skin lesion formation. We propose that a positive feedback mechanism was formed that maintained the severity of psoriasis. Psoriasis is a T-cell-mediated, genetically determined, environmentally influenced, chronic inflammatory skin condition that affects almost 2–3% of the population . PsoriasRetinol-binding protein 4 (RBP4) is the carrier that mobilizes retinol (vitamin A) from the liver to target tissues. Humans absorb vitamin A from food and store it in liver cells as retinol . When thMoreover, patients with moderate to severe forms of psoriasis are at a greater risk of developing comorbidities such as metabolic syndrome and vascular disorders , 12. In Based on the above findings, it is clear that RBP4, retinol, STRA6, and other proteins involved in vitamin A metabolic pathways possess regulatory functions related to psoriasis and its complications. However, the ways in which the expression of these molecules is modified both in the circulation and in skin lesions of psoriasis remain unclear. More investigations are needed to explore the mechanisms of these modifications and to give us deeper insight into the pathophysiology of psoriasis.The aims of the present study were to create a psoriasis mouse model and to use it to detect the levels of RBP4, STRA6, retinol, and other vitamin A-related molecules in the circulation and in skin lesions.n = 20) were randomly divided into experimental group A, B (Aldara cream for 3 and 6 consecutive days). Mice (n = 10) were also randomly separated into two control groups: group C and D  [Male and female BALB/C mice 8–9 weeks of age obtained from the Charles River Laboratories  were used in this experiment. Aldara cream  was used to induce the psoriasis-like lesions and Vaseline cream  was used as control agents , 17. Micve days) –21. An ave days)  was usedThe scoring system described by Van der Fits et al.  was an aThe lesional and the normal skin from group A to D were excised immediately after sacrifice and were fixed in 10% formaldehyde for 48 h. The formalin-fixed skin tissue was then dehydrated in Fully Enclosed Tissue Processor  and embedded in paraffin. After embedding, the tissue was sectioned vertically at 4 μm, using microtomes . Following this, the section was dried at 72 °C  for an hour. The staining process was done with autostainer , using Harris’ hematoxylin and eosin for histological analysis.The blood was drawn from the aorta abdominalis under anesthesia. Blood serum was extracted by centrifuge  and stored in − 80 °C until use. Skins from both the lesional and non-lesional areas in all the groups were snap-frozen in liquid nitrogen and stored at − 80 °C. Harvested mouse skin tissue (approximately 0.1 g) were prepared in ice-cold buffer containing phosphate-buffered saline  (PH 7.2–7.4) for skin homogenates. The supernatant was collected after centrifugation at 2500 rpm for 20 min at 4 °C. IL-17A (XFM21581), IL-23 (XFM20248), TNF-α (XFM21527), RBP4 (XFM21121), STAR6 (XFM21632) and retinol (XFM21227) levels were detected with ELISA kits which purchased from Shanghai Xinfan Bio-Technology Co. Ltd. .Samples were snap-frozen in liquid nitrogen, and stored at − 80 °C until use. The pathologic sections were made using the same protocol as described above. Standard IHC protocol was followed to stain the skin samples using the rabbit monoclonal antibody against RBP4 , 1:100 diluted with antibody diluent . Rabbit polyclonal antibody against STRA6 , 1:1000 diluted with antibody diluent. Briefly, 4 μm sized paraffin-embedded tissue sections were de-paraffinized with xylene, hydrated through a series of ethanol concentrations. Washed with PBS , subjected to antigen retrieval using 10 mM sodium citrate  and heated with microwave . Cooled in room temperature for at least half an hour, washed with PBS. Incubated the sections in 0.5% Triton  for 15 min. Washed thoroughly with PBS. Endogenous peroxidase activity was quenched with 3% H2O2 in methanol for 15 min in a dark moister chamber. Rinsed the sections, then blocked with goat serum  for half an hour. Slides were incubated with the respective primary antibody mentioned above and kept in a moist chamber overnight at 4 °C. The next day, washed the slides with PBS several times. Then incubated with secondary antibody kit . After washing, slides were reacted with freshly made DAB (diaminobenzidine)  and immediately washed under tap water after color development. Slides were then counter-stained with hematoxylin. Slides were dehydrated through a series of alcohol and mounted with xylene. The slides were observed under a light microscope . Five high-power fields (×400) were selected at random, specimens were scored according to the intensity of the dye color and the number of positive cells. The intensity of the dye color was graded as 0 (no color), 1 (light yellow), 2 (light brown), or 3 (brown), and the number of positive cells was graded as 0 (<5%), 1 (5–25%), 2 (25–50%), 3 (51–75%), or 4 (>75%). The two grades were added together and specimens were assigned to one of four levels: 0–1 score (−), 2 scores (+), 3–4 scores (++), more than 5 scores (+++).RNA extraction was performed with TRIzol . RNA (2000 ng) was reverse transcribed using the PrimeScript™ RT reagent Kit . RBP4, STRA6, and GAPDH mRNA levels were measured by PCR analysis using the SYBR Premix Ex Taq II . GAPDH was used for normalization. The following primers were used:−ΔΔCt method by normalizing to the reference gene.GAPDH, forward primer, 5′-TGCACCACCAACTGCTTAG-3′, reverse primer, 5′-GGATGCAGGGATGATGTTC-3′. RBP4, forward primer, 5′-GACAAGGCTCGTTTCTCTGG-3′, reverse primer, 5′-AAAGGAGGCTACACCCCAGT-3′. STRA6, forward primer, 5′-AGCCAAGTCAGACTCCAAGAG-3′, reverse primer, 5′-CAGAGAGCACACTAACTTCTTTCA-3′. All the primers were purchased from Majorbio Bio-pharm Technology Co. Ltd. . The relative expression levels of the target proteins were calculated using the 2http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/), respectively.RNase H was used to remove the rRNA, DNase I was used to digesting double-stranded and single-stranded DNA in total RNA. Purified RNA from previous steps was fragmented into small pieces with fragment buffer at an appropriate temperature. Then, cDNA was generated in First Strand Master Mix by PCR. The reaction product was purified by magnetic beads, afterward, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments were amplified with adapters by PCR, and the products were purified by Ampure XP Beads. Library was validating on the Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR product from the previous step was heat-denatured and circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library, which further amplified with phi29. DNA nanoball (DNB) which had more than 300 copies of one molecular was made and loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform . All the generated raw sequencing reads were filtered to get clean reads stored as FASTQ format. Bowtie2 and HISAT were used to map clean reads to reference gene and genome respectively. Gene expression level (RPKM) is quantified by RSEM. Gene ontology (GO) and pathway annotation and enrichment analyses were based on the GO Database  and GraphPad Prism version 6 . Data were given as arithmetical means ± SEM. Statistical analysis was generated with Student’s t-test, Wilcoxon test, Kruskal-Wallis test, and ANOVA. Values of Psoriasis-like lesions with clear erythema and skin thickening were successfully induced in all mice in groups A and B, although the scales were more evident in group B than in group A. Generally, erythema appeared on day 2 and progressed until day 7. Scale formation started on day 3 or day 4 and increased gradually until day 7. Skin thickening increased from day 2 to day 4. The cutaneous changes found in groups A and B are shown in Fig. In summary, from days 2 to 3 and onward, inflammation was visible and continuously increased in severity in IMQ-treated groups A and B. In contrast, there were no signs of inflammation in Vaseline-treated group C and D.The histopathology of Vaseline-treated skin from control groups revealed a normal epidermal structure consisting of one to two layers of cells only. Normal dermis and subcutaneous structures were found in non-lesional skin Fig. a.Fig. 3In group A, the histopathological analysis showed severely thickened epidermis made up of five to 10 layers of cells. The granular layer was absent focally, corresponding to areas producing foci of parakeratosis. Severe acanthosis and moderate rete ridge formation could be seen in this group. In the dermis, there were many hair follicles. Moderate inflammatory cell infiltration (mostly lymphocytes and histocytes) was evident in the superficial layer of the dermis Fig. b. The lep = 0.273; Fig. In normal mouse skin (skin from group C and D), expression levels of RBP4 were low; therefore, we graded the control group as RBP4 negative .The RBP4 receptor, STRA6, accumulated gradually after IMQ utilization in the lesional skin. In normal skin, STRA6 is expressed in both epidermis and dermis. Because normal epidermis consists of only one or two layers of cells, STRA6 is expressed in all layers of the epidermis, mainly in the cytoplasm. In the dermis, many nonspecific expressions of STRA6 were seen in endothelial cells inside blood vessels , and LRAT. In Fig. Focusing on retinol metabolism, we further explored the changes in expression of proteins involved downstream in the vitamin A metabolism pathway, especially those that are active after retinol is transported inside target cells. Figure Table p = 0.040 and p = 0.021, respectively). In contrast, the 3 additional days of IMQ treatment in group B did not significantly increase RBP4 levels (p = 0.77), but the absolute level did increase . Serum STRA6 levels were significantly higher in group A than in controls (p = 0.010). However, only an increasing trend existed between STRA6 levels in group B and the controls (p = 0.22). There was no significant difference in serum STRA6 levels between groups A and B (p = 0.14)  Fig. e. MoreovWe demonstrated that the effects of IMQ treatment on mice closely resembled human plaque-type psoriasis with respect to erythema, skin thickening, scaling, epidermal alterations, and inflammatory cells that infiltrated the skin and were present in the circulation, consistent with previous studies . The cytThe detection of changes in serum levels of vitamin A-related molecules in psoriasis patients dates back several decades; however, the results varied between studies. Unlike the findings of the current study, Rollman and Vahlquist found a normal mean serum RBP concentration in patients with lower PASI scores and found a significantly lower serum RBP concentration in patients with more extensive disease or pustular erythrodermic psoriasis . Our resThe detection of vitamin A-related molecules in psoriatic skin lesions is very limited. Many researchers proposed that retinol metabolism was altered in psoriasis lesional skin, based on the increased synthesis of RA. Rollman et al. found no differences in retinol levels in psoriatic and normal skin . Many reWe hypothesized that additional retinols are released from the liver to the circulation to meet the increasing demand of retinol in psoriasis lesional skin. If this is correct, more RBP4 is then needed in the circulation and in lesional skin to carry more retinol to target tissues. Additional retinol would then be transported inside target cells by the action of STRA6. In this study, proteins and enzymes such as CRBP1, LRAT, RDH, CRABP1, and CRABP2 were detected at high levels in IMQ-treated groups. If retinol stores are sufficient and the demand of retinol inside the body is low , retinolWe believe that the disease severity progressed from group A to group B, and that the effects on the vitamin A metabolism pathway became more severe. As a result of decreasing vitamin A levels, more retinol was needed and was subsequently transformed into its functional metabolites, which increased its functionality in group B. However, in the current study, retinol levels were not found with the expected expression pattern. It seems that there may be some redistribution of retinol in different tissues. Serum retinol levels are supposedly closely related to RBP4 levels, but in our experiments the RBP4 level was markedly elevated in the serum, but the retinol level was not. RBP4 has two forms in the circulation: holo-RBP4 and apo-RBP4. The holo-RBP4 is a complex in which RBP4 combines with retinol in a 1:1 ratio. After binding with STRA6 and detaching from retinol, holo-RBP4 becomes apo-RBP4 and returns to the circulation, where it encounters more retinol and once again becomes holo-RBP4. The paradox in our study could be explained by an increase in the total amounts of apo-RBP4 and holo-RBP4, wherein the amount of holo-RBP4 was about the same in both the IMQ and control groups, but more apo-RBP4 accumulated in the IMQ group serum. In our study, we used the same method that Mills et al. used to determine the levels of apo-RBP4 in different groups , and theThese disparate sets of results might be attributable to many factors. In human studies, the most crucial bias is related to different recruitment criteria. Patients are diagnosed with different types of psoriasis and the disease severity varies from patient to patient. Comorbidities such as obesity, diabetes mellitus, and insulin resistance affect RBP4 levels, causing bias in detection. Another factor in these variations in research findings is probably the presence of vitamin A or its derivatives in the treatments that patients received. Therefore, a major strength of our study was the application of an IMQ-induced psoriasis mouse model to detect changes in the vitamin A metabolic pathway. This approach clearly excludes all the potential influences mentioned above. To our knowledge, this was the first study on the vitamin A metabolic pathway to include both serum and skin in psoriasis. However, our study does have some limitations. First, the detection of retinol was limited by technical considerations. A unified, simple and easy detection method will be needed to further verify our hypothesis. Second, the expression of RA and its related downstream enzymes require further study to verify the RNA sequencing results. Third, a treatment group will be needed to conversely support our results. Our attempt to build such a treatment group  in a previous study failed to demonstrate any improvements in psoriasis symptoms, which we attributed to local irritation and the limited experimental period. Thus another treatment model needs to be created that would help to explain why vitamin A therapy is needed when retinol levels are already elevated in psoriasis skin. Fourth, to analyze the specific functions of RBP4 and STRA6 in mouse vitamin A homeostasis, a loss-of-function study will be needed. Many researchers have chosen the STRA6 deficiency mouse as a model to study vitamin A metabolism, but they rarely pay attention to the skin. In published reports, most concluded that, except for the eye, most peripheral tissues do not display altered retinoid levels in STRA6-deficient mice . A minor+/+) mice is due to the increase in RBP4 mRNA expression. These results tightly connect RBP4 with retinol at the level of mRNA expression. RBP4 is a key element for retinol transportation: without it, retinol can remain in the storage form, with no function in peripheral tissues.Both RBP4-deficient and RBP4 knockout models have been created in mice. Serum retinol levels in RBP4 knockout mice were reduced when compared with the wild-type controls, but there were no obvious changes in the accumulation of hepatic retinol stores . These aThe present study showed that the demand for vitamin A in psoriatic skin lesions was upregulated. In response, RBP4 and STRA6 levels increased in serum and in psoriatic skin, resulting in the transfer of more retinol to lesional skin. Retinol underwent a redistribution from the circulation to target tissues, while the transformation from retinol to RA in target tissue was accelerated, thereby inducing further synthesis of STRA6. As a result, a positive feedback mechanism was formed that maintained these psoriatic changes Fig. . This mi


Cerebrospinal fluid (CSF) flows through the brain, transporting chemical signals and removing waste. CSF production in the brain is balanced by a constant outflow of CSF, the anatomical basis of which is poorly understood. Here, we characterized the anatomy and physiological function of the CSF outflow pathway along the olfactory sensory nerves through the cribriform plate, and into the nasal epithelia. Chemical ablation of olfactory sensory nerves greatly reduced outflow of CSF through the cribriform plate. The reduction in CSF outflow did not cause an increase in intracranial pressure (ICP), consistent with an alteration in the pattern of CSF drainage or production. Our results suggest that damage to olfactory sensory neurons (such as from air pollution) could contribute to altered CSF turnover and flow, providing a potential mechanism for neurological diseases. The brain has a very high metabolic rate, but lacks a conventional lymphatic system for clearance of metabolites and waste products. It is thought that the movement of cerebrospinal fluid (CSF) plays an important role in removing these waste products . CSF is While there are lymphatic vessels in the meninges , there i2  and the total area of the plate was 9.76 ± 1.12 mm2 , similar to previous reports (2) and Swiss Webster mice (8.821 ± 0.607 mm2), no significant difference was observed for total foramina area  greatly enhance the permeability of cells to water , and theWe then examined the expression of AQP4, which is enriched around astrocyte endfeet in the cortex  and is hLYVE1 was observed in the dural lymphatics, while minimal cross-labeling with fluorescent dye (rhodamine) conjugated to phalloidin  adjacent to OSNs in the nasal cavity  was obshe brain . We alson  mice were injected with 3kDa-FITCDextran in the cisterna magna  vessels.To visualize CSF flow in the regions downstream from the cisterna magna, we injected the high-contrast Evans blue (EB)  dye intona magna . 3kDa-FIna magna , and co-ry nerve , althoug4 causes the rapid death of OSNs, and persists for months (4 (10% in sterile H2O) or vehicle (sterile H2O) was administered to the left nare. Because a loss of the olfactory nerve layer and overall reduction in the size of the olfactory bulb indicates successful ablation of OSNs by ZnSO4  and histologically examined using Nissl-thionin staining 30 days after treatment (The olfactory marker protein (OMP) antibody was utilized to histologically examine OSN damage . Two dayreatment , we obsereatment , we obsereatment . We quancle mice . A signireatment , showing4 treatment. AQP1, expressed by epithelial cells in the choroid plexus, is involved in CSF production  mice 2, 10, and 30 days after treatment, compared to vehicle controls  on the center of mass location  exhibited some non-lymphatic specific LYVE1 expression in vascular endothelial cells in the brain, there were also LYVE1+ vessels along the olfactory nerve that did not co-label with vascular perfusion markers  and that absorbed dye injected into the cisterna magna. These vessels are putatively lymphatic vessels that transport CSF out of the cranial cavity. Previous work has shown that there is transport of fluid across the CP  and Ai6 (RCL-ZsGreen)+/+ (Jackson Laboratory - Stock No: 007906) mice. ‘LYVE1-tdtomato’ mice are the heterozygous offspring of EGFP-hCre+/+Lyve1 (Jackson Laboratory - Stock No: 012601) and Ai14 (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze)+/+ (Jackson Laboratory - Stock No: 007914). This breeding scheme was used because the basal fluorescence of GFP in the EGFP-hCreLyve1 mice is low  at the Pennsylvania State University. Mice were maintained on a 12 hr light-dark cycle with ad libitum access to food and water. We used both male and female Swiss Webster , C57BL/6J , and LYVE1-GFP or LYVE1-tdtomato mice. We used a total of 294 mice [60–120 days/29.0 ± 5.8 g]. Partitioning was: Male/female (137/157), Swiss Webster (239), C57BL/6J (9) and LYVE1 mice (52). All anatomy studies were repeated in at least three mice. Mice used were ages 60–90 days (young adult). ‘LYVE1-GFP’ mice are the heterozygous offspring of e is low . No diffMice were sacrificed via isoflurane overdose and perfused intracardially with heparinized-saline followed by 4% paraformaldehyde, unless otherwise noted. In some cases, the vasculature was labeled with perfusion of dye, FITC-albumin  or DiI  using the protocols described in , respectThe animal was anesthetized with isoflurane  in oxygen. The dorsal surface of the head was elevated 8 mm above the back of the animal and the head was tilted downward at an angle of 10 degrees from horizontal. The base of the skull was exposed by an incision, and a fine glass-pipette (~20 micrometers in diameter for dye injections) or a 26G needle (for kaolin injections) was inserted into the cisterna magna using a stereotaxic instrument . Either 2.0 µL of 2% Evans blue in aCSF  or 5.0 µL of 10% solution of 3kDa-FITCDextran in aCSF was infused at a constant rate of 0.2 μL/min using a syringe pump , an infusion rate that does not raise ICP in mice . After tThe animal was anesthetized with isoflurane in oxygen . The dorsal surface of the skull was exposed by an incision, and a fine glass-pipette was inserted into the right olfactory bulb using a stereotaxic instrument . One microliter of 5% solution of 3kDa-FITCDextran in aCSF was infused at a constant rate of 0.1 μL/min using a syringe pump , an infusion rate that does not raise ICP in mice . After tA modified version of the SeeDB protocol  was usedSwiss Webster skulls were imaged with a GE v|tome|x L 300 high-resolution nano/microCT scanner and C57BL/6J skulls were imaged on an OMNI-X HD600 industrial microCT scanner . Scan settings were 100kV, 0.1mA (GE) or 160kV, 0.25 mA. Image stacks were processed and analyzed in Avizo 8. For each skull, a threshold that segmented the calcified and non-calcified tissue was chosen such that the zygotic maxillary suture, which is externally visible by eye, was clearly defined in the segmented image. To validate that the CT images accurately captured the fine details of the CP, in a subset of skulls the cranial vault was removed using a Dremel tool and the CP photographed on a Zeiss StereoDiscovery V8.4 (10% in sterile H2O) or vehicle control (sterile H2O) was administered to the left nare with a blunt 32G needle. The animal was then inverted to allow for excess fluid to exit the nasal cavity. The animals were monitored and weighed daily after treatment. Approximately 4% of animals treated with ZnSO4 died within 24 hr of administration.After the mouse had been rendered unconscious by a brief exposure to isoflurane, 20 µL of ZnSO4- and vehicle-treated mice using a Zeiss StereoDiscovery V8 with a 2000R scientific CCD camera  utilizing µManager software  and there are 35 microliters CSF in the mouse . There are 12–14 Evans blue (Evans blue: 1 mg*0.02/ (960g/mole)=2*10–8 moles Evans blue) binding sites per albumin, so ~10% of the Evans blue molecules injected will be bound to CSF albumin  utilizing µManager software  Figure  top. UsiKlotho, and AQP-1, validated on the kidney, were used to determine mRNA expression levels. Primer sequences: AQP1, 5’CATGGTGGAAGCCAGTTCCTT (FOR) and 5’CAGACCAGGGTGTGTAGTGG (REV); Klotho, 5’GTGGCCGAGAGAGTTTTGGA (FOR) and 5’GGGGTCTCACCTTTCAGAGC (REV). Primers for NF-KB and RelA were previously validated (NF-KB1: 5’GAAATTCCTGATCCAGACAAAAAC (FOR) and 5’ ATCACTTCAATGGCCTCTGTGTAG (REV). RelA: 5’CTTCCTCAGCCATGGTACCTCT (FOR) and 5’CAAGTCTTCATCAGCATCAAACTG (REV). Mouse GAPDH was used as a house-keeping gene and loading control: 5’AGGCCGGTGCTGAGTATGTC (FOR) and 5’ TGCCTGCTTCACCACCTTCT (REV) .RNA was extracted from tissue using the Trizol and chloroform method . A titanium head-bar was attached to the skull with cyanoacrylate glue and dental cement  and the Unless noted, all statistical analysis was performed using Matlab , with the Matlab function used (i.e. ‘ttest2’) is listed. All summary data were reported as the mean ± standard deviation (SD). Normality  and of the samples were tested before statistical testing. When the data conform to the normality assumption, difference between treatments were compared using unpaired t-test (ttest2). If the condition of normality was not met, parametric tests (ttest2) were substituted with a non-parametric method . For comparing the effects of treatment and duration on CSF drainage, two-way ANOVA was used. For the wheel activity data, the K-S test was used. For the ICP data, a mixed model ANOVA test was used. A result was considered significant if p≤0.05 after correcting for multiple comparisons using the Bonferonni correction. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Ronald Calabrese as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Roxana Carare (Reviewer #1); Robert G Thorne (Reviewer #2); Jeff Illif (Reviewer #3).Thank you for submitting your article "Anatomical basis and physiological role of cerebrospinal fluid transport through the murine cribriform plate" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:4 nerve ablation experiments because of the difficulty / challenge in interpreting the resulting findings. Still there is a lot of new compelling data here and a novel way of looking at the CSF system.This is a nicely executed study with some very interesting and novel findings related to olfactory nerve – cribriform plate (CP) – olfactory mucosa relationships. Overall this study provides important anatomical insight into an understudied region of the skull base which has recently re-emerged interest-wise into the neuro-immunology field owing to the recent detailed characterization of meningeal lymphatic vessels by the Alitalo and Kipnis groups. Arguably the strongest portion of the work relates to the anatomical descriptions of the nerves/vessels/lymphatics/water channels in this key interface region, previously not well described in the rodent. Somewhat weaker are the author's ZnSOEssential revisions:The reviewers were in agreement that the portion of the manuscript dealing with CSF secretion needs to be extensively rewritten. It should be possible for the authors to address the comments that all the reviewers have made while retaining much of the content, not only the anatomical description portion. Specifically, for the functional studies, the authors must account much better for other possibilities and not be so definitive in their conclusions. Particularly the conclusion that lesioning the olfactory path in some way results in a feed-back regulation of CSF secretion. The experiment performed simply isn't solid enough to definitively answer that question. The other functional question – the contribution that drainage down this pathway makes to overall drainage along the cervical lymphatics helps to place the study within the wider experimental context of the field , but this experiment is not totally definitive either and must be discussed with care.Reviewer #1:This is an elegant study encompassing novel methodology and demonstrating firstly the exact anatomical features of the cribriform plates and their penetrating structures. Secondly, the authors demonstrate that ablating the olfactory sensory neurons results in the decreased production of CSF with ICP remaining unchanged. My main question is whether the authors could be more specific about the nature of the vessels that express LYVE1- are they arteries or veins and is LYVE1 present also on capillaries.Reviewer #2:4 nerve ablation experiments because of the obvious difficulty / challenge in interpreting the resulting findings. The authors would be wise to revisit some of their conclusions, softening the description and better allowing for alternatives . Nevertheless, the results are important. So long as some of the interpretations acquire a bit more nuance and care in the strength of conclusions derived from them, the experiments described in this manuscript represent a significant advance for the rapidly growing research area concerned with lymphatic drainage from the brain.This is a nicely executed study with some very interesting and novel findings related to olfactory nerve – cribriform plate (CP) – olfactory mucosa relationships. Arguably the strongest portion of the work relates to the anatomical descriptions of the nerves/vessels/lymphatics/water channels in this key interface region, previously not well described in the rodent. Somewhat weaker are the author's ZnSO1) The authors would do well to comment upon and relate their findings to Buck and Axel's 2004 Nobel Prize winning studies (superfamily of GPCR odorant receptors) and subsequent related work better identifying zones and projection patterns from the olfactory epithelium to glomeruli in the bulb. For example, do the authors believe that these projections are organized in any special way between the major and smaller foramina that they describe in the manuscript? What do the authors think are the primary differences between the major foramina and smaller, minor foramina that are so consistently represented in the two strains of mice they examined? This is an excellent topic for speculation that the authors somewhat avoid in favor of less wise discussion later in the manuscript over their ablation experiments (see below). However, the possible significance of the anatomical relationships they describe more fully for the first time in the mouse would certainly seem to merit a bit more discussion.2) Subsection “Blood vessels traverse the cribriform plate”. The authors state in the results that 'blood vessels traverse the CP in the mouse, much like the ethmoidal arteries observed in humans (refs), linking the vascular territories of the nasal epithelia and brain.' This statement would seem to imply that the ethmoidal artery – CP relationship has not been described in mice / rodents but there are publications that previously discuss this . Of some interest and not commented upon is venous drainage. Did the authors identify veins traversing the CP? Is there any link between draining veins of the olfactory mucosa and the olfactory sinus via the CP? The authors should at minimum comment on this, citing what is known from the literature, so that their manuscript provides more complete coverage of the subject area. It is not beyond the scope of what they have studied and described in the manuscript to more fully on these important points. The vascular relationships between the nasal mucosae and brain are quite important clinically; better elaboration of these relationships in the rodent will aid in understanding the relevance (and possible limitations) of rodent models of disease.3) Subsection “Aquaporins at the olfactory nerve-bulb interface”. AQP1 is described as lining the 'foramina of the CP along the crista galli.' Several questions arise: did they mean to imply that it is only the smaller foramina and not the major foramina that had this AQP1 expression? Looking at Shields et al., 2010 that identified olfactory ensheathing cells as expressing AQP1, it is not clear if the authors agree that their findings are consistent with that reference or that their AQP1 expression is rather localized to the olfactory nerve fibroblasts that surround axon bundles . The authors should further comment on this. Most importantly, Weller et al., have described in a recent publication (2018) the expression of AQP1 on 'dural border cells' of the meninges with some discussion about the CP area . The authors of the current manuscript describe AQP1 as limited to the CP area they describe and the choroid plexus, but this description lacks the real complexity of the fuller picture described in the Weller et al. reference and some of Mollgard's prior related work. The authors should consult this literature and more fully elaboration their AQP discussion.4-treated animals and for the lack of ICP increase they observed? The authors would do well to flesh out their interpretation in this section of the manuscript far better, allowing for other possibilities. It would seem to be easily argued that the authors results do NOT definitively 'show that in response to the disruption of the nasal outflow pathway… CSF production was decreased' (subsection “OSN ablation does not affect ICP”), as the authors assert in the closing sentence, simply because CSF production was not directly measured. Inference is quite different from proof! The authors would do well to recognize the countless number of times in science where results looked straightforward in allowing a certain inference at a given time, only to be made to look naïve with subsequent future studies showing a more complex situation. Accordingly, I strongly advise that the statements and related discussion by the authors regarding interpretation of the ZnSO4 ablation experiments really need to better reflect this, more fully allowing for other interpretations and possibilities, not least that the physiology may be much more complicated than their description allows for (see comments in opening summary). Such a revision will not require a major change or rewrite, only the addition of a few more carefully and thoughtfully placed qualifiers and perhaps some further thought about alternatives. The Discussion section accomplishes this balancing act better than the Results section, as currently written. The Results section would be greatly improved by making this strongly advised change.4) The authors argue in their ablation experiment description (subsection “Ablation of OSNs decreases CSF outflow”) that 'because the space between the OSN axons provides a conduit for outflow of CSF and ISF, removing these axons should decrease outflow of CSF.' However, the opposite might be argued as well. If axons are removed from bundles surrounded by olfactory ensheathing cells, then the space may be filled in, so to speak, by fluid secretion, thus creating effectively larger volume paths for fluid outflow. Perhaps this might be an alternative explanation for the observation that EB outflow to the deep cervical lymph nodes was unchanged in ZnSOReviewer #3:The study by Norwood and colleagues uses several imaging approaches to define the anatomical landscape of the cribriform plate and nasal turbinates; then using fluorescent tracers and chemical ablation of the olfactory nerves, they demonstrate that this ablation appears to reduce CSF efflux across the cribriform plate, reportedly associated with changes in CSF secretion. Overall this study provides important anatomical insight into an understudied region of the skull base which has recently re-emerged interest-wise into the neuro-immunology field owing to the recent detailed characterization of meningeal lymphatic vessels by the Alitalo and Kipnis groups. While the anatomical descriptions of these barriers appears to be conducted well and with care, and are themselves a useful addition to the field, the functional elements of the study that seek to address what this efflux pathway is doing, and what the consequences of its lesioning are, are generally underdeveloped and should be strengthened to improve the value of the study to the field. Most critical items follow:Effect of nerve lesion on CSF secretion and reabsorption. By plotting CSF tracer distribution along the neuroaxis, the authors note that distribution shifts away from the anterior portion with olfactory nerve ligation, although distribution down the spinal cord is also reduced. From this, a reduction in CSF secretin is inferred, supported by unchanged ICP measurements in awake-behaving animals.- A reduced CSF secretion would be very interesting. CSF secretion should be evaluated directly using a gold-standard ventriculo-cisternal tracer perfusion method.- ICP monitoring was conducting in waking, moving animals, while tracer studies were conducted in anesthetized animals. Either ICP monitoring should be done under the same physiological state as the tracer studies, or vise-versa.- The authors note no change in the lymphatic vasculature at the cribriform plate with Zn ablation. But that tracer movement across is reduced. Is overall CSF drainage down the cervical lymphatic trunks altered? If so, then this would support their contention that altering cribriform plate efflux alters CSF dynamics, while no change in CSF tracer efflux along the lymphatic trunks would argue that compensatory efflux along another route is occurring.- Lastly – does the nerve lesion result in any neuroinflammation – either glial changes with the parenchymal compartment, or immune cell changes in the meningeal or CSF compartments? Such changes might be predicted to alter CSF tracer dynamics and distribution and should be ruled in or out.Tracer uptake into lymphatic vessels. The presence of lymphatic vessels at the cribriform plate is described, and the results of the present study clearly are relevant to how meningeal lymphatic vessels throughout the cranium, including in the skull base and in association with dural sinuses function – what their relative contribution to CSF solute efflux are, and how they are impacted by the intervention to lesion olfactory nerves. Some questions remain on this that are important for placing the significance of this efflux pathway relative to these others.- Do CSF tracers concentrate in the cribriform plate lymphatic vessels? Or is there tracer simply permeating this structure, which also has lymphatic vessels within it? Do the authors note uptake of tracer into dural sinus-associated lymphatic vessels .- Does uptake into non-cribriform lymphatic vessels change with nerve lesion? This would be very interesting question to answer regardless of the outcome and would be related to overall lymphatic efflux issue noted above. Summary:4 nerve ablation experiments because of the difficulty / challenge in interpreting the resulting findings. Still there is a lot of new compelling data here and a novel way of looking at the CSF system.This is a nicely executed study with some very interesting and novel findings related to olfactory nerve – cribriform plate (CP) – olfactory mucosa relationships. Overall this study provides important anatomical insight into an understudied region of the skull base which has recently re-emerged interest-wise into the neuro-immunology field owing to the recent detailed characterization of meningeal lymphatic vessels by the Alitalo and Kipnis groups. Arguably the strongest portion of the work relates to the anatomical descriptions of the nerves/vessels/lymphatics/water channels in this key interface region, previously not well described in the rodent. Somewhat weaker are the author's ZnSOEssential revisions:The reviewers were in agreement that the portion of the manuscript dealing with CSF secretion needs to be extensively rewritten. It should be possible for the authors to address the comments that all the reviewers have made while retaining much of the content, not only the anatomical description portion. Specifically, for the functional studies, the authors must account much better for other possibilities and not be so definitive in their conclusions. Particularly the conclusion that lesioning the olfactory path in some way results in a feed-back regulation of CSF secretion. The experiment performed simply isn't solid enough to definitively answer that question. The other functional question – the contribution that drainage down this pathway makes to overall drainage along the cervical lymphatics helps to place the study within the wider experimental context of the field , but this experiment is not totally definitive either and must be discussed with care.4 treatment causes a decrease in CSF production. We have added text to the Discussion section discussing other possible explanations, and a statement that our experiments cannot distinguish between the possible origins of lack of an ICP increase. The Discussion section now reads:In response to the reviewer’s comments, we removed the text claiming that ZnSO4 treatment. We can think of two non-mutually exclusive possibilities that could explain the lack of ICP rise when an outflow pathway is blocked. The first is that other drainage pathways become engaged and/or reduce their outflow resistance to compensate for the blockage of the cribriform plate outflow. In addition to providing enough drainage to prevent ICP rise, the CSF flow dynamics would be altered by these outflow resistance changes such that the flow down the spinal cord is decreased. One possible location for outflow increase this could by the lymphatics at the skull base  or along the other nerve bundles that exit the skull . A re-routing of CSF flow in a more rostral direction would account for the decreased flow down the spinal cord. Some outflow pathways have thresholds for opening (Welch and Friedman 1960), such as arachnoid villi, and these might become engaged following ZnSO4 treatment. While arachnoid villi have been described in primates and other species, there are no definitive anatomical studies in the mouse. The second possibility could be a decrease in the production of CSF. CSF production is not a passive process, but rather one under active neural control . As ICP needs to be maintained within a healthy range, there are likely homeostatic processes that sense ICP, and cause compensatory increases or decreases in CSF production, similar to other mechanosensory processes in other organs of the body . A potential mechanism mediating this feedback is mechanically-sensitive ASIC3 channels, which are expressed in the ventricles , and are sensitive to changes in pressure that are physiologically relevant to ICP regulation. Pathological elevations of ICP due to trauma produce a rise in systemic blood pressure and a decrease in heart rate, known as the Cushing response , showing the brain has a way of sensing ICP , and pressure-sensitive brain regions mediating these responses have been identified . A similar response may be occurring in the choroid plexus to compensate for the decreased CSF drainage we observe after ZnSO4 treatment, thus, avoiding the rise in ICP that is normally associated with a decrease in CSF drainage . Neither of these two possibilities excludes the other, and from the experiments performed here, we are unable to differentiate between these two possibilities.”“Interestingly, instead of a rise in ICP as would be expected due to a blockage of CSF drainage into the nasal cavity, we observed no change in ICP. This could not be explained by an increase in drainage of CSF down the spinal cord pathway, as we observed a similar reduction in CSF flow down this pathway following ZnSOReviewer #1:This is an elegant study encompassing novel methodology and demonstrating firstly the exact anatomical features of the cribriform plates and their penetrating structures. Secondly, the authors demonstrate that ablating the olfactory sensory neurons results in the decreased production of CSF with ICP remaining unchanged. My main question is whether the authors could be more specific about the nature of the vessels that express LYVE1- are they arteries or veins and is LYVE1 present also on capillaries.To answer this question, we stained brain sections from LYVE1-Ai6 mice with rhodamine-conjugated phalloidin. We observed LYVE1 expression along both arteries and capillaries in the mouse cortex. Results of these experiments were added to Figure 3—figure supplement 2 and the subsection “Lymphatic vessels traverse the cribriform plate”.Reviewer #2:4 nerve ablation experiments because of the obvious difficulty / challenge in interpreting the resulting findings. The authors would be wise to revisit some of their conclusions, softening the description and better allowing for alternatives . Nevertheless, the results are important. So long as some of the interpretations acquire a bit more nuance and care in the strength of conclusions derived from them, the experiments described in this manuscript represent a significant advance for the rapidly growing research area concerned with lymphatic drainage from the brain.This is a nicely executed study with some very interesting and novel findings related to olfactory nerve – cribriform plate (CP) – olfactory mucosa relationships. Arguably the strongest portion of the work relates to the anatomical descriptions of the nerves/vessels/lymphatics/water channels in this key interface region, previously not well described in the rodent. Somewhat weaker are the author's ZnSO1) The authors would do well to comment upon and relate their findings to Buck and Axel's 2004 Nobel Prize winning studies (superfamily of GPCR odorant receptors) and subsequent related work better identifying zones and projection patterns from the olfactory epithelium to glomeruli in the bulb. For example, do the authors believe that these projections are organized in any special way between the major and smaller foramina that they describe in the manuscript? What do the authors think are the primary differences between the major foramina and smaller, minor foramina that are so consistently represented in the two strains of mice they examined? This is an excellent topic for speculation that the authors somewhat avoid in favor of less wise discussion later in the manuscript over their ablation experiments (see below). However, the possible significance of the anatomical relationships they describe more fully for the first time in the mouse would certainly seem to merit a bit more discussion.The question regarding the anatomical relationships and patterns of the projections through the major and minor foramina could be addressed by using markers  for specific olfactory receptor proteins and histologically determining if the axons of a specific receptor type exhibited a stereotyped pattern or preference for foramina. We did not check for any such relationships or patterns, but have added the following to the Discussion section:“We observed a highly stereotyped pattern in the positioning of the major foramina across mice. It is tempting to speculate that the OSN axons are predisposed to go through either the dorsal or ventral foramina depending on their receptor group and target glomeruli .”2) Subsection “Blood vessels traverse the cribriform plate”. The authors state in the results that 'blood vessels traverse the CP in the mouse, much like the ethmoidal arteries observed in humans (refs), linking the vascular territories of the nasal epithelia and brain.' This statement would seem to imply that the ethmoidal artery – CP relationship has not been described in mice / rodents but there are publications that previously discuss this .We thank the reviewer for bringing this literature to our attention. The relationship of the vascular territories of the nasal epithelia and brain were already very well documented in . This comparison was used to emphasize the similarities of the anatomy of this area between humans and mice and the usefulness and applicability of using the mouse as a model for this system. We have added the Lochhead and Thorne reference and other references, so the section now reads:“These results are consistent with previous work showing blood vessels traverse the CP in the rodent , much like the ethmoidal arteries and veins observed in humans , linking the vascular territories of the nasal epithelia and brain.”Of some interest and not commented upon is venous drainage. Did the authors identify veins traversing the CP? Is there any link between draining veins of the olfactory mucosa and the olfactory sinus via the CP? The authors should at minimum comment on this, citing what is known from the literature, so that their manuscript provides more complete coverage of the subject area. It is not beyond the scope of what they have studied and described in the manuscript to more fully on these important points. The vascular relationships between the nasal mucosae and brain are quite important clinically; better elaboration of these relationships in the rodent will aid in understanding the relevance (and possible limitations) of rodent models of disease.Whether the vessels traversing the cribriform plate were arteries or veins cannot be definitively concluded from our experiments. The vessels were identified by FITC-albumin or DiI cardiovascular fills, neither of which provides any contrast between arteries and veins. The section on the cribriform plate traversing vessels now reads:“We first looked at whether blood vessels traversed the CP by either filling the vessel lumen with FITC-albumin , or labeling the endothelial cells by perfusing the vasculature with the fluorescent lipophilic dye DiI . Note that we cannot distinguish between arteries or veins using these methods.”3) Subsection “Aquaporins at the olfactory nerve-bulb interface”. AQP1 is described as lining the 'foramina of the CP along the crista galli.' Several questions arise: did they mean to imply that it is only the smaller foramina and not the major foramina that had this AQP1 expression?We did not observe any AQP1 expression lining the major foramina of the cribriform plate, and have clarified the relevant section to read:“We observed high levels of AQP1 expression on the olfactory nerve layer, down to the glomeruli, and in the lamina propria of the neuroepithelium at both the medial  and dorsal  junctions of the olfactory bulb and nerve, but not lining the major foramina.”Looking at Shields et al., 2010 that identified olfactory ensheathing cells as expressing AQP1, it is not clear if the authors agree that their findings are consistent with that reference or that their AQP1 expression is rather localized to the olfactory nerve fibroblasts that surround axon bundles . The authors should further comment on this.The localization of expression and pattern of staining of AQP1 along the olfactory bulb observed would support previous findings that olfactory ensheathing cells along the periphery of the olfactory bulb express AQP1 . Expression of AQP1 in fibroblasts was checked by staining with Vimentin (expressed by fibroblasts) in the foramina of the cribriform plate but co-localization of AQP1 and vimentin expression was not observed. Based on these results, we believe the cells expressing AQP1 that surround the axon bundles might be olfactory ensheathing cells (OECs). We have added figures showing AQP1 and Vimentin antibody staining in the cribriform plate to Figure 2—figure supplement 1J-K.Text below was added to the Results section:“We observed high levels of AQP1 expression on the olfactory nerve layer down to the glomeruli, as observed previously , and in the lamina propria of the neuroepithelium at both the medial  and dorsal  junctions of the olfactory bulb and nerve. AQP1 was also observed lining the foramina of the CP along the crista galli  but did not co-label with Vimentin-positive fibroblasts . Based on previous reports  and our own results, we hypothesize that these cells might be olfactory ensheathing cells (OECs).”Most importantly, Weller et al., have described in a recent publication (2018) the expression of AQP1 on 'dural border cells' of the meninges with some discussion about the CP area . The authors of the current manuscript describe AQP1 as limited to the CP area they describe and the choroid plexus, but this description lacks the real complexity of the fuller picture described in the Weller et al. reference and some of Mollgard's prior related work. The authors should consult this literature and more fully elaboration their AQP discussion.Reviewer #2 brings up a good point about AQP1 expression in development that we overlooked. Embryonically, AQP1 expression begins around E13 (in the rat) and its pattern and sequence of development is maintained throughout many mammals, including humans . Interestingly, AQP1 expression was also observed in dural border cells of the embryonic rat dura . Text below was added to the Results section:“We first looked at the expression of aquaporin-1 (AQP1), as it has been shown that expression in the CNS begins early , and is required in adulthood to maintain CSF production by the choroid plexus . Interestingly, previous work has also shown that AQP1 is expressed along the periphery of the olfactory bulb in neonatal mice , and there is a high level of expression of AQP1, 3, and 5, within the nasal cavity .”4-treated animals and for the lack of ICP increase they observed?4) The authors argue in their ablation experiment description (subsection “Ablation of OSNs decreases CSF outflow”) that 'because the space between the OSN axons provides a conduit for outflow of CSF and ISF, removing these axons should decrease outflow of CSF.' However, the opposite might be argued as well. If axons are removed from bundles surrounded by olfactory ensheathing cells, then the space may be filled in, so to speak, by fluid secretion, thus creating effectively larger volume paths for fluid outflow. Perhaps this might be an alternative explanation for the observation that EB outflow to the deep cervical lymph nodes was unchanged in ZnSO4 treatment . An alternative explanation to the unchanged EB outflow to the deep cervical lymph nodes is that the EB dye may be entering other outflow pathways at the skull base, such as the optic nerves or lymphatics in the spinal column .There are two possible outcomes from our ablation experiment: either ablation of the OSNs creates a hole, or the removal of the OSNs leads to sealing of the opening. Since we did not observe flow into the nasal cavity, there is unlikely to be a hole. Additionally, larger volume paths were not observed, since there were large decreases in EB and fluorescein in the nasal cavity after ZnSOThe authors would do well to flesh out their interpretation in this section of the manuscript far better, allowing for other possibilities. It would seem to be easily argued that the authors results do NOT definitively 'show that in response to the disruption of the nasal outflow pathway… CSF production was decreased' (subsection “OSN ablation does not affect ICP”), as the authors assert in the closing sentence, simply because CSF production was not directly measured. Inference is quite different from proof!The reviewer brings up a very good point and we have addressed this above.4 ablation experiments really need to better reflect this, more fully allowing for other interpretations and possibilities, not least that the physiology may be much more complicated than their description allows for (see comments in opening summary). Such a revision will not require a major change or rewrite, only the addition of a few more carefully and thoughtfully placed qualifiers and perhaps some further thought about alternatives. The Discussion section accomplishes this balancing act better than the Results section, as currently written. The Results section would be greatly improved by making this strongly advised change.The authors would do well to recognize the countless number of times in science where results looked straightforward in allowing a certain inference at a given time, only to be made to look naïve with subsequent future studies showing a more complex situation. Accordingly, I strongly advise that the statements and related discussion by the authors regarding interpretation of the ZnSOWe have rewritten the Discussion section to cover alternate possibilities .Reviewer #3:The study by Norwood and colleagues uses several imaging approaches to define the anatomical landscape of the cribriform plate and nasal turbinates; then using fluorescent tracers and chemical ablation of the olfactory nerves, they demonstrate that this ablation appears to reduce CSF efflux across the cribriform plate, reportedly associated with changes in CSF secretion. Overall this study provides important anatomical insight into an understudied region of the skull base which has recently re-emerged interest-wise into the neuro-immunology field owing to the recent detailed characterization of meningeal lymphatic vessels by the Alitalo and Kipnis groups. While the anatomical descriptions of these barriers appears to be conducted well and with care, and are themselves a useful addition to the field, the functional elements of the study that seek to address what this efflux pathway is doing, and what the consequences of its lesioning are, are generally underdeveloped and should be strengthened to improve the value of the study to the field. Most critical items follow:Effect of nerve lesion on CSF secretion and reabsorption. By plotting CSF tracer distribution along the neuroaxis, the authors note that distribution shifts away from the anterior portion with olfactory nerve ligation, although distribution down the spinal cord is also reduced. From this, a reduction in CSF secretin is inferred, supported by unchanged ICP measurements in awake-behaving animals.- A reduced CSF secretion would be very interesting. CSF secretion should be evaluated directly using a gold-standard ventriculo-cisternal tracer perfusion method.While this has been used as a gold-standard for measuring CSF secretion, the results are often times variable, even in much larger animals than the mouse. Based on this variability and the challenging nature of this method in the mouse, we did not perform these experiments. We hope that future technical innovations make these experiments more feasible in a mouse model.- ICP monitoring was conducting in waking, moving animals, while tracer studies were conducted in anesthetized animals. Either ICP monitoring should be done under the same physiological state as the tracer studies, or vise-versa.The reviewer brings up a good point. In response to the reviewer’s point, we have added ICP measurements made at the end of the surgery in the two minutes after the cessation of isoflurane but before the animal regained consciousness . The concentration of isoflurane in the mouse during this period will still be very high. In general, the ICP was higher during this period, consistent with previous results that show isoflurane increases resting ICP . We find that there is no difference between the ICP of the different groups immediately after isoflurane. While this is not the optimal experiment, it is the best we can do without repeating this series of experiments in anesthetized animals. The text below was added to the Results section:4 treated mice at rest or during locomotion as compared to the vehicle controls  measured 10, 30, and 60 days after treatment (Supplementary file 2). Additionally, there was no difference between the groups’ ICP immediately after isoflurane cessation and before the animal regained consciousness .”“No statistically significant difference was observed in ZnSO- The authors note no change in the lymphatic vasculature at the cribriform plate with Zn ablation. But that tracer movement across is reduced. Is overall CSF drainage down the cervical lymphatic trunks altered? If so, then this would support their contention that altering cribriform plate efflux alters CSF dynamics, while no change in CSF tracer efflux along the lymphatic trunks would argue that compensatory efflux along another route is occurring.We were unable to quantitatively measure the Evans blue signal in the lymph nodes in the neck optically. Future experiments using radioactive tracers and collection of lymphatic fluid flow should be better positioned to quantify any changes.- Lastly – does the nerve lesion result in any neuroinflammation – either glial changes with the parenchymal compartment, or immune cell changes in the meningeal or CSF compartments? Such changes might be predicted to alter CSF tracer dynamics and distribution and should be ruled in or out.4 treatment. However, no changes in any of the inflammation markers was observed. This has been added to Figure 6—figure supplement 2, and the following was added to the Results section:To address the reviewer’s question, we assayed glial changes in the cortex and inflammation marker mRNA expression in the choroid plexus both 2 and 10 days after ZnSO4 treatment. AQP1, expressed by epithelial cells in the choroid plexus, is involved in CSF production . Klotho is a protein involved in suppressing aging and is significantly elevated in the CSF of Alzheimer’s disease patients . The NF-κβ pathway is involved in the choroid plexus acute-phase response to peripheral inflammation , and RelA is required for NF- κβ activation (Chen and Greene 2004). GFAP is a hallmark marker for astrocytes and is upregulated as part of an inflammatory response . However, we found no appreciable changes in mRNA levels or GFAP expression  2 or 10 days after ZnSO4 treatment indicating there is no inflammatory response in the cranial cavity following treatment.”“We then assayed mRNA levels in the choroid plexus and GFAP expression in the cortex to look for immune responses to ZnSOTracer uptake into lymphatic vessels. The presence of lymphatic vessels at the cribriform plate is described, and the results of the present study clearly are relevant to how meningeal lymphatic vessels throughout the cranium, including in the skull base and in association with dural sinuses function – what their relative contribution to CSF solute efflux are, and how they are impacted by the intervention to lesion olfactory nerves. Some questions remain on this that are important for placing the significance of this efflux pathway relative to these others.- Do CSF tracers concentrate in the cribriform plate lymphatic vessels? Or is there tracer simply permeating this structure, which also has lymphatic vessels within it?+-vessels in it. Though the dye co-localizes within these vessels, it does not concentrate in it. We have clarified this in the text:We observe dye throughout the tissue in the major foramina, which has LYVE1“3kDa-FITCDextran was observed draining into the nasal cavity along the olfactory nerve , and co-localizing with LYVE1+ vessels along the olfactory nerve , though the dye did not appear to be concentrated in the LYVE1+ vessels.”Do the authors note uptake of tracer into dural sinus-associated lymphatic vessels .- Does uptake into non-cribriform lymphatic vessels change with nerve lesion? This would be very interesting question to answer regardless of the outcome and would be related to overall lymphatic efflux issue noted above.4-treated mice. The presence of the dye in the dural likely depends on the specifics of the injection and perfusion protocol , so we are hesitant to generalize our results.In preliminary experiments in untreated mice, we did not see cisterna magna-injected dye in the dural sinus-associated lymphatic vessels or other locations in the dura using our dye-infusion protocol. We did not check the dural sinus-associated lymphatic vessels in ZnSO


Staphylococcus aureus (S. aureus), coagulase-negative staphylococci and streptococci but infection with Corynebacterium striatum (C. striatum) is also becoming prominent. We present the case of a 65-year-old female with a recent history of the coronary artery bypass graft with bioprosthetic aortic valve replacement. The surgery was complicated by sternal wound dehiscence with methicillin-sensitive S. aureus (MSSA) for which she was treated for six weeks with intravenous antibiotics. Two months later, she was found to have C. striatum which was treated. A transesophageal echocardiogram was done as well which did not show any vegetation. She presented to the hospital with vomiting, cough, fever, and shortness of breath. She had pyuria on urinalysis and was started on empiric antibiotics after taking blood cultures. She decompensated soon after admission and was transferred to the intensive care unit where she had a pulseless ventricular tachycardia and was resuscitated but required vasopressor support. The blood cultures from admission started growing C. striatum again. Daptomycin was added to the empiric antibiotics and supportive care was continued, but the family decided to make her 'do not resuscitate - comfort care only'. The support was withdrawn and she passed away.Infective endocarditis is typically caused by  Staphylococcus aureus (S. aureus), coagulase-negative staphylococci and streptococci are the biggest offenders, Corynebacterium striatum (C. striatum) is beginning to appear more frequently as the causative agent [C. striatum was published in 2002, which was found in a 72-year-old female with an aortic valve replacement [Infective endocarditis (IE) is an infection of the endocardial surface, which reflects the infection of the heart valves or infection of the intracardiac devices. Outcomes of infectious endocarditis are grim. Mortality rates in patients with a prosthetic or native valve IE reach as high as 20% to 25% . Prosthelacement .We present a 65-year-old female with known coronary artery disease (CAD), diabetes mellitus (DM) type II, chronic diastolic heart failure, and a recent history of coronary artery bypass grafting (CABG), along with bioprosthetic aortic valve replacement (AVR). She presented from a nursing home with a five-day history of vomiting, cough, fever, shortness of breath and reports of low pulse oximeter readings. On admission, the leukocyte count was 16,000 , the international normalized ratio (INR) was 8.5 , and her urinalysis revealed pyuria. Blood cultures were drawn and intravenous ciprofloxacin was empirically started for the suspected urinary tract infection (UTI) and sepsis.S. aureus (MSSA) bacteremia. A peripherally inserted central catheter (PICC) line was used for six weeks of intravenous vancomycin administration. Two months later, she was hospitalized with recurrent falls and her blood cultures grew C. striatum. The PICC line was removed and she was discharged after 10 days of intravenous vancomycin. She returned a month later with necrotizing fasciitis in the right lower limb, which culminated in an above-the-knee amputation. Once again, blood cultures showed the growth of C. striatum. Transesophageal echocardiography done at that time was unremarkable and did not show any vegetation. The blood cultures turned negative after another four weeks of intravenous vancomycin and the PICC line was removed.Chart review revealed a protracted infectious history after the CABG and AVR. The patient’s postoperative course was complicated by sternal wound dehiscence and methicillin-susceptible C. striatum yet again for which daptomycin was added to her antibiotic regimen. Repeated echocardiography still did not reveal any vegetation. The patient’s family changed her code status to 'do not resuscitate - comfort care only', and soon thereafter, she was pronounced dead. Within hours of admission, the patient developed acute tachypnea, diaphoresis, and high-grade fever. Her antibiotic regimen was switched to intravenous ampicillin-sulbactam and she was moved to the ICU. A few hours later, she had pulseless ventricular tachycardia in the intensive care unit. After two cycles of cardiopulmonary resuscitation, including defibrillation and intubation, there was a return of spontaneous circulation. She was severely hypotensive, and hemodynamic support with vasopressors was instituted. Blood cultures drawn at admission grew C. striatum as an infectious agent were limited to native valve infective endocarditis [C. striatum is playing a bigger role than previously presumed [Previous findings of carditis . Howeverpresumed -9.C. striatum is a common flora of the skin and mucosa of the nasopharynx, it is an opportunistic pathogen in immunocompromised patients [C. striatum bacteremia in patients with prosthetic valves and initial imaging results is shown below in Table Although patients . HoweverC. striatum but no vegetations on TTE initially. He was then treated for pneumonia but was eventually readmitted due to the recurrence of fever. Upon the second admission, the patient grew recurrent C. striatum blood cultures and was found to have a 10-mm mobile mass on his prosthetic mitral valve on TTE imagining. The case report by Houghton et al. [C. striatum infective endocarditis by de Arriba et al. [C. striatum in patients with a history of prosthetic valve placement should be treated with urgency. Negative findings on TTE/TEE should not rule out the prosthetic valve IE. Positive cultures should be tested for antibiotic resistance. The initiation of intravenous vancomycin has shown favorable outcomes in patients. Follow-up imaging should also be performed due to the possibility of IE development after initial treatment, as was found in our case as well as others [As with our patient, several of the cases had positive blood cultures with a negative transthoracic echocardiogram (TTE) or transesophageal echocardiogram (TEE) findings -8. The 6n et al. , a 72-yea et al. , the patC. striatum, recurrently grew this organism in her blood cultures during the nine months between her valve replacement and her death. Her bacteremia resolved on two instances with intravenous antibiotics only to recur within months, suggesting a consistent source for seeding this pathogen. Despite the lack of vegetations on echocardiography, the association between recurrent bacteremia with this organism and recent prosthetic valve placement was too obvious to ignore. Due to the previous infrequency of C. striatum caused by IE in the literature, our suspicion of prosthetic valve IE as the source for the positive blood cultures was low. The current literature is suggestive of adding C. striatum to the differential in prosthetic valve IE patients. Additionally, as in the case of our patient, the diagnosis of prosthetic valve IE was elusive as the echocardiography in the first published case report was negative for vegetation [The Corynebacterium species, commonly considered contaminants in cultures, deserve attention if they grow in a patient who is immunocompromised or has a prosthesis. Our patient, with no previous history of infection with getation .C. striatum in the blood can be indicative of endocarditis which may not manifest as vegetations on echocardiography and mandates early and aggressive intravenous antibiotics with screening for recurrence.In a patient with a recently placed prosthetic heart valve, the growth of


There is limited literature on paediatric donors in endothelial keratoplasty.This study investigated the efficacy of and appropriate paediatric donor age for Descemet’s stripping endothelial keratoplasty (DSEK).Retrospective and observational case series.Thirty‐eight consecutive patients underwent DSEK with paediatric donor corneas.The age of the donors ranged from 32 weeks gestation (premature neonate) to 3 years old. All donor consents were obtained from the parents. The causes of donor death included traffic accident, congenital heart disease and neonatal respiratory distress syndrome.The outcome measures included best‐corrected visual acuity, endothelial cell loss and complications.2. The mean endothelial cell density of grafts was 3977 ± 556 cells/mm2 at 18 months postoperatively. Three lenticules from premature neonate donors exhibited severe contraction postoperatively. The edge of six lenticules from donors <1‐year‐old exhibited contraction in the early postoperative period and gradually flattened spontaneously. Graft detachment occurred in one patient.Best‐corrected visual acuity at last follow‐up was >20/40 in 28 of 38 eyes (73.7%). The mean preoperative endothelial cell density of donor corneas was 4682 ± 520 cells/mmDSEK with paediatric donor corneas can achieve good clinical outcomes. The corneal lenticules from 1‐ to 3‐year‐ old donors are suitable for DSEK while those from donors <1‐year‐old are less suitable due to the possibility of severe postoperative graft contraction. Descemet’s stripping endothelial keratoplasty (DSEK) is the preferred treatment for corneal endothelial dysfunction. However, graft dislocation and excessive endothelial cell loss (ECL) over time are the major limitations. Although the graft dislocation rate has been reduced with the advancement of surgical techniques, ECL is inevitable.ECL is affected by storage methods, surgical stress, postoperative elevation of intraocular pressure and inflammation and subsequent intraocular procedures.et al. reported a 50.4% decrease in ECD in the foetus, an annual decrease of 17.73% in the early postnatal period between 0 and 2 years, and a 0.56% yearly reduction thereafter. The rapid decrease in cell density observed in utero and during the first 2 years of life is proportionally related to the degree of corneal growth.et al. recently reported an increased rate of ECD reduction during the first 2 years of life, followed by a decreased ECD reduction rate between 2 and 5 years of age, which is similar to the reduction rate reported in adults, who have stable corneal diameters.A statistically significant relationship between age and endothelial cell density (ECD) was reported in numerous previous reports.Previous reports have suggested that paediatric donor grafts are not suitable for penetrating keratoplasty due to increased allograft reactions, smaller diameters and reduced corneal thicknesses, rendering unpredictable refractive results.Using an increased number of cases, the following study investigated the outcomes of DSEK using paediatric donor grafts and the influence of donor age on graft outcomes.We retrospectively analysed the outcomes of all patients undergoing DSEK with paediatric donor corneas in Peking University Third Hospital from June 2008 to December 2013. DSEK was performed on 38 eyes of 38 consecutive patients  from 3 to 81 years old (mean age 49±22.6 years). Indications for surgery are presented in Table n = 7), congenital heart diseases (n = 10) and neonatal respiratory distress syndrome (n = 2). All corneas were donated after cardiac death by the parents of the deceased donors.Thirty‐eight donated corneas were obtained from 19 donors ranging in age from 32 weeks gestation (WG) (premature neonates) to 3 years (mean 0.75±0.82 years) . The ECD of all donor corneas was quantified by a certified technician at our eyebank using an EB‐3000 XYZ Eyebank specular microscope  before DSEK. The preoperative cell counts were obtained using an apex digitized method with the manufacturer’s calibrations for magnification. The apices of at least 100 cells were counted from the endothelial images of each cornea. Three donor groups were divided for analysis by donor age .The redundant tissue surrounding the eyeball was carefully removed, including the conjunctiva, fascia and muscles. The eyeball was steeped in tobramycin saline solution twice for 15 min per time. Under sterile conditions, a suitable trephine was used to strip the sclera posterior cornea 3–4 mm. Then, the corneoscleral buttons were obtained and stored in Optisol‐GS  at 4In this study, all procedures were performed by a single experienced surgeon (JH) at the Peking University Third Hospital from July 2008 through December 2013.Before the preparation of the 38 lenticules, the preparation of two other lenticules was attempted by using a Moria automated microkeratome. However, both cuts penetrated the centre because the paediatric corneas were small, thin and soft. Therefore, all 38 lenticules were prepared by deep manual dissection. The anterior lamella of 70% to 80% stromal thickness was bluntly dissected. The posterior lamellar tissue was punched from the endothelial side with an 8.0‐mm trephine. The donor corneal lenticules were then immediately covered by viscoelastic  until transplantation.n = 4), removal of an anterior chamber (AC)‐IOL and placement of an IOL in the sulcus with suture fixation (n = 6), and suture fixation of a PC‐IOL in aphakic eyes (n = 2). A 4‐mm temporal sclera tunnel incision was made. The AC was filled with viscoelastic. Further paracentesis wounds were made at the limbus at the 6‐o’clock position and each side of the main incision. Descemet’s membrane and the dysfunctional endothelium were scored with a blunt‐tip hook using the epithelial mark as a guide, stripped off using a stripper and removed with forceps. An AC irrigator was inserted via the left paracentesis beside the main incision.According to the status of the recipient eyes, at the time of DSEK, some patients underwent combined surgeries, such as phacoemulsification cataract extraction and posterior chamber intraocular lens (PC‐IOL) implantation . The lenticule was pulled into the glide opening with forceps by holding the leading stromal edge, and a 10–0 nylon suture was placed to pull the lenticule into the AC. The suture was knotted and inserted through the main incision into the AC and then pulled from the 6‐o’clock paracentesis. The AC viscoelastic was removed. The glide was inverted and positioned at the entrance of the corneal tunnel. The lenticule was pulled into the AC by the suture via the 6‐o’clock paracentesis. The suture was then removed. The wounds were closed with sutures. The grafts were pressed up against the recipient cornea with filtered air. The graft position was verified with the slit lamp on the microscope. Interface fluid was squeezed out.The clinical outcome of DSEK was assessed by best‐corrected visual acuity (BCVA), ECD and complications at 1, 3, 6 months postoperatively and every 6 months thereafter. Graft attachment and contraction were assessed with anterior segment optical coherence tomography . ECD was measured by laser confocal scanning microscopy . The same certified ophthalmic technician performed all postoperative testing of patients using the same microscope.P < 0.05 was considered statistically significant.Statistical analysis was performed with SPSS v16.0 . The main outcome measure was ECD at different times. Donor age and ECD associated with different donor age groups were assessed using analysis of variance (ANOVA). All tests were 2‐tailed, and n = 4), amblyopia (n = 5), macular scar (n = 2) and optic nerve dystrophy (n = 1) due to trauma. Postoperative refractive astigmatism was within three diopters (D) in all patients. Myopic shift was 0.85 ± 1.34 D. The mean corneal astigmatism was 2.47 ± 1.50 D. The mean true‐net power was 41.2 ± 1.0 D. The mean thickness of the central grafts was 110 ± 65 μm at the last follow‐up .The average follow‐up time was 26 months (range 18–45 months). BCVA at the last follow‐up was >20/40 for 28 of 38 patients (73.7%). In some patients, pre‐existing conditions limited visual acuity and included advanced glaucoma  occurred on the first postoperative day in a buphthalmic eye. Postoperatively, the intraocular pressure of four eyes was elevated and was controlled by medical treatment. Of these, three eyes had a history of glaucoma and one eye newly developed glaucoma following DSEK. No graft rejection was observed.n = 2) and 34 WG (n = 2), 1 day (n = 1), 3 days (n = 1), 3 months (n = 1) and 7 months (n = 2). All donors were <1‐year‐old. Three grafts from premature neonate donors  developed full peripheral circular contraction which progressed daily. Of these, two grafts had to be replaced with adult grafts at 10 days and 3 months postoperatively because the patients experienced worsening visual acuity . The mean ECD values for groups 1, 2 and 3 were 5100 ± 115, 4771 ± 450, and 4186 ± 542 cells/mm2, respectively , suggesting a significant decrease in donor ECD after age 1 year. In association with the reduction in ECD, the size of ECs increased as donor age increased  at 18 months postoperatively, representing a mean EC loss postoperatively of 14.0 ± 11.5% . The mean EC losses for groups 1, 2 and 3 were 16.9% (n = 1), 13.3±8.5% (n = 25), and 16.3 ± 20.1% (n = 7), respectively. The EC density was increased in 3 cases . ECD data could not be collected for five patients because two received replacement grafts as mentioned previously, one died 2 months after surgery, and the remaining two declined the ECD assay. No significant differences were noted between groups 1 to 3.The mean ECD of the grafts was 3977 ± 556 cells/mmn = 3), 6 (n = 2) and 12 (n = 2) months, postoperatively.In addition, the ECD in seven patients increased over time Table , as demoIn this study, using corneas from paediatric donors, BCVA at the last follow‐up was >20/40 in 28 of 38 patients (73.7%). DSEK with paediatric donor corneas can achieve good clinical outcomes. The lenticules from donors aged 1 to 3 years are ideal, while those from donors <1‐year‐old contract easily and are not suitable for DSEK.DSEK is an effective treatment for endothelial dysfunction. Theoretically, if a graft harbours more ECs preoperatively, the graft will have a better survival advantage. Given that the number of ECs decreases with age, grafts from paediatric donors with a higher ECD may provide such a benefit.2 (range 3407–5366 cells/mm2), whereas the value for adult donor corneas is typically from 2270 to 4209 cells/mm2.2 at 18 months, representing a mean ECL of 14.0 ± 11.5%. This reduced ECL rate may contribute to improved graft longevity, which remains to be verified in future studies. In our study, one graft underwent initial reverse implantation, which was corrected during surgery, and re‐bubbling had to be performed twice, postoperatively. The ECD of this graft was 3171 cells/mm2 at 18 months, equating to a 41% ECL following DSEK. In our experience, grafts that undergo this operative manipulation likely become dysfunctional in 18 months. However, because the donor age was 3 months and the preoperative ECD was 5366 cells/mm2, the graft survived well, and the patient achieved a BCVA of 20/50 at 30 months (last follow‐up).In this study, the mean preoperative ECD in paediatric donors was 4576 ± 617 cells/mmWe identified no significant difference in the ECL rate in grafts between all donor groups in this study. This result illustrates a similar vitality of the ECs in donors <3 years old. Although the ECD of grafts from 1‐ to 3‐year‐old donors was significantly lower, the ECL rate was not significantly increased compared with that in <1‐year‐old donors. Grafts from 1‐ to 3‐year‐old donors exhibited a reduced number of ECs, but this reduction did not seem to be a disadvantage overall.In vivo human corneal endothelial cells are thought to be arrested at the G1 phase of the cell cycle; thus, proliferation is inhibited.In addition, the number of ECs increased over time in seven patients. Such a result may indicate that the ECs may multiply in grafts from donors <3 years old. vs. 19%).In this study, one graft dislocation occurred in an eye with congenital glaucoma. The diameter of the recipient cornea was 14 mm, and the AC was deep. The posterior corneal surface topography was irregular because the patient had undergone two DSAEK procedures with adult donor corneas. The first graft failed 2 years postoperatively, whereas the second graft failed 6 months postoperatively. The corneal curvature was steeper in the buphthalmic eye. In addition, the patient had previously undergone multiple surgeries for glaucoma. However, overall, in this study, the graft dislocation rate was lower than that reported previously in our hospital  that is not commonly observed in eyes implanted with adult grafts. All nine grafts with contraction were from donors <1 year old. Three of these were from premature neonates who died of respiratory distress syndrome, and the contractions were very severe. Two grafts had to be replaced with adult grafts. Six grafts exhibited partial contraction in the early postoperative period but flattened spontaneously at 2 and 6 months postoperatively. This finding may be explained as follows. First, the development of paediatric donor corneas may be incomplete, and tissue elasticity is greater. An infant’s eyeball at birth is approximately 80% of the adult size.This study is subject to the limitation of the small sample size in the 1–3‐year‐old group. But it can be readily concluded that DSEK with donor corneas from children <3 years old can achieve good clinical outcomes. The corneal lenticules from 1‐ to 3‐year‐ old donors are suitable for DSEK while those from donors <1‐year‐old are less suitable due to the possibility of severe postoperative graft contraction.