Artemia salina is a brine shrimp containing high concentrations of dinucleotides, molecules with properties for dry eye treatment. For this reason, the purpose of the study was to evaluate the effect of the artificial tears based on an extract of Artemia salina in a rabbit dry eye model. (2) Methods: A prospective and randomized study was carried out. Twenty rabbits were divided into 4 groups : healthy rabbits, dry eye rabbits, dry eye rabbits treated with hypromellose (HPMC), and dry eye rabbits treated with Artemia salina. Dry eye was induced by the topical instillation of 0.2% benzalkonium chloride. The measurements were performed before and after the treatment for 5 consecutive days. (3) Results: The topical instillation of artificial tears containing Artemia salina showed beneficial effects on tear secretion, tear break-up time, corneal staining, the density of Goblet cells, heigh of mucin cloud secreted by these cells, and mRNA levels of IL-1β and MMP9 in conjunctival cells. Compared with the HPMC, there was a statistically significant improvement (p < 0.05) with the Artemia salina in all the variables under study, except for the conjunctival hyperemia, density of Goblet cells, and mRNA levels of IL-6. (4) Conclusions: The potential of artificial tears based on Artemia salina as a secretagogue agent for dry eye treatment was confirmed, opening the door for future clinical trials and studies to extrapolate the findings for dry eye patients.(1) Background:  Dry eye was defined by the Tear Film and Ocular Surface Society (TFOS) as “a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage and neurosensory abnormalities play etiological roles” . This co2 receptor, a subtype of P2Y receptors, produces chloride efflux and water movement into the extracellular medium [Dinucleoside polyphosphates are formed by two nucleotides linked by a variable number of phosphates (from 2 to 7) and play an important role in the diagnosis and treatment of dry eye . These cr medium ,8,9.2 receptor has been described in the cornea, conjunctiva, and lacrimal gland [2 receptor via topical instillation of diadenosine tetraphosphate (Ap4A) and diuridine tetraphosphate (Up4U) demonstrated its capability to stimulate aqueous [On the ocular surface, P2Yal gland . The act aqueous ,12,13,14 aqueous , and lip aqueous  componen aqueous ,17,18. A aqueous ,20 and t aqueous  and lact aqueous  in tears4U (Diquafosol®) as a secretagogue agent for the treatment of dry eye [From a clinical viewpoint, the scientific evidence supports the safety and security of the commercial form of Up dry eye ,24. The  dry eye , aqueous dry eye , evapora dry eye , dry eye dry eye , contact dry eye , and eve dry eye .Artemia salina [4G), but also others such as diguanosine triphosphate (Gp3G) and diphosphate (Gp2G), or guanosine-adenosine tetraphosphate (Gp4A) [Artemia salina was chosen in a previous study where the effect of short-term instillation of different concentrations of the brine shrimp  was evaluated in healthy rabbits [Artemia salina manifested the best results in increasing tear secretion and improving corneal epithelial damage, also confirming its safety over the ocular surface.Recently, our research group developed artificial tears based on an extract of 4% a salina , a brinee (Gp4A) ,32. The  rabbits . The 4% Artemia salina on lacrimal function and ocular surface damage and inflammation in a rabbit dry eye model. The dry eye model was induced by topical instillation of 0.2% benzalkonium chloride (BAC) for 5 consecutive days, a method previously validated [The purpose of the current study was to evaluate the effect of the artificial tears based on an extract of 4% alidated , while tArtemia salina was the only one that showed a statistically significant increase of 64.38 ± 18.41% in comparison with the rest of the groups (p < 0.05). The other groups did not show statistical differences between them nor changes in comparison with their baseline measurements (p ≥ 0.05).Concerning tear secretion, the group of rabbits with dry eye treated with p < 0.05). This deterioration was 69.13 ± 25.31% in the dry eye group, 67.78 ± 36.16% in the dry eye + HPMC group, and 37.73 ± 14.85% in the dry eye + Artemia salina group. Additionally, the treatment with Artemia salina in the rabbits with induced dry eye statistically improved the tear break-up time in comparison with the instillation of HPMC (p = 0.001).In relation to tear break-up time, there was a statistically significant deterioration in the three groups of rabbits with induced dry eye in comparison with the healthy rabbits . On the other hand, despite the rabbits with dry eye being treated with Artemia salina, presenting a deterioration in comparison with their baseline, there were no statistical differences with the healthy rabbits (p = 0.109).Regarding corneal staining, both healthy rabbits and the rabbits with induced dry eye treated with p < 0.05). Furthermore, the treatments with Artemia salina and HPMC had no effect since they showed no statistical differences with the rabbits with dry eye used as positive controls (p ≥ 0.05).In terms of conjunctival hyperemia, there was a statistically significant deterioration in the three groups of rabbits with induced dry eye in comparison with the healthy rabbits (p < 0.05). Conversely, despite the rabbits with dry eye being treated with Artemia salina they also showed a deterioration compared with the healthy rabbits, these differences, however, were not statistically significant (p = 0.117).Concerning the density of Goblet cells, the two groups of rabbits with induced dry eye treated or not treated with HPMC suffered a statistically significant deterioration of 68.03 ± 24.10% and 55.00 ± 48.29%, respectively, in comparison with the healthy rabbits . In addition, the treatment with Artemia salina in the rabbits with dry eye showed similar results to the healthy rabbits (p = 0.329).Regarding the height of mucin cloud, again the two groups of rabbits with induced dry eye treated or not treated with HPMC suffered a deterioration of 35.81 ± 5.78% and 16.31 ± 4.70%, respectively, which was statistically significant in comparison with the healthy rabbits and the rabbits with dry eye treated with Artemia salina group that was statistically significant in comparison with the healthy rabbits (p < 0.05). Additionally, the treatment with Artemia salina produced a decrease in the mRNA expression of IL-1β in comparison with the HPMC (p = 0.001).In the levels of IL-1β, the three groups of rabbits with induced dry eye showed a fold increase of 34.611 ± 35.999 in the dry eye group, 42.014 ± 27.068 in the dry eye + HPMC group, and 11.114 ± 9.537 in the dry eye + p ≥ 0.05), despite the rabbits with dry eye treated with Artemia salina and HPMC showed lower values.Regarding the levels of IL-6, there were no statistically significant differences between all the groups . The fold change was 0.162 ± 0.165 with the Artemia salina and 0.494 ± 0.477 with the HPMC.In relation to the levels of MMP9, the quantitative PCR did not detect signals for the POST measurements of the healthy rabbits. However, in the rabbit with induced dry eye, the treatment with Artemia salina in a rabbit dry eye model. The treatment with these artificial tears manifested beneficial effects on tear secretion, tear break-up time, corneal staining, the density of Goblet cells, height of mucin cloud, and inflammatory biomarkers. Additionally, the safety of these artificial tears was already confirmed in a previous study performed in healthy rabbits by our research group [The current study reported on the effect of artificial tears based on an extract of 4% ch group .Artemia salina produced an increase of 64% in the rabbits with induced dry eye  and pentaphosphate (Ap5A) act as agonists of the P2Y6 receptor, which is also expressed in corneal epithelium, decelerating corneal wound healing by inhibiting cell migration [Moreover, the treatment with artificial tears based on HPMC see . Again, and Up4U ,41,42,43and Up4U ,18. Alsoand Up4U  demonstrigration ,45.Artemia salina reduced the mRNA levels of IL-1β and MMP9 compared with the instillation of HPMC .An experimental, prospective, and randomized study was carried out. All the trials were performed before (PRE) and after (POST) the topical instillation of the different treatments: saline solution as negative control , 0.2% BAC + saline solution (dry eye group), 0.2% BAC + 0.24% hypromellose (dry eye + HPMC group), and 0.2% BAC + artificial tears containing Artemia salina, 35 µL were instilled 3 times per day , for 5 consecutive days. In total, there were 9 instillations of 0.2% BAC or saline solution per eye in each rabbit and 15 instillations of HPMC, Artemia salina, or saline solution.The 0.2% BAC was used to induce the dry eye model by instilling 35 µL, twice per day (at 10:00 and 18:00), for 5 consecutive days, except on the last day when there was a single instillation during the morning . For theOn the last day, the measurements were taken 20 min after the last instillation at 16:00, when dinucleotides produce the maximum effect on tear secretion ,14. The eyes = 40). The rabbits were randomly divided into four groups: 5 rabbits as negative control , 5 rabbits with induced dry eye as a positive control , 5 rabbits with induced dry eye and treated with HPMC , and 5 rabbits with induced dry eye and treated with artificial tears of Artemia salina .A total of 20 male New Zealand white rabbits were used in the study, including both eyes in the same experimental group . The qualitative composition of these artificial tears was as follows: an extract of 4% Artemia salina containing 10 µM of Gp4G as an active ingredient, 0.24% HPMC as a thickening agent, boric acid and borax as a buffer, and CaCl2, KCl, and MgCl2 as electrolytes. The artificial tears had an osmolality of 248 mOsm/kg, a pH of 7.1, and a viscosity of 5.6 cP.The artificial tears based on an extract of Artemia salina but with no crustacean extract. The saline solution used during the experiments was also provided by Avizor, while the BAC was provided by Merck .The treatment with HPMC had the same composition as the artificial tears of Tear secretion with anesthesia was measured by performing a Schirmer’s test  for 5 min. The paper strip was positioned in the inferior lid and the rabbit’s eyes were closed to avoid the reflex secretion associated with blinking. Each millimeter of the paper strip soaked corresponded to 1 µL of tear secretion. The topical anesthesia was induced by the instillation (2 drops in 5 min) of commercial eye drops containing 4 mg/mL of oxybuprocaine hydrochloride and 1 mg/mL of tetracaine hydrochloride . Tear secretion was measured 5 min after the last instillation of anesthesia.Tear break-up time was measured during the slit-lamp examination. It was evaluated after the instillation of 2 µL of commercial 2% fluorescein sodium (Alcon Cusí) over the ocular surface. Three consecutive measurements were taken with a timer after manually forcing the rabbits to blink.The signs of ocular surface damage were examined with a VX75 slit lamp . The severity of corneal staining and conjunctival hyperemia was quantified by using the Efron Grading Scales , which cThe medical device EYEPRIM  was used to collect the superficial conjunctival cells. Two cytologies of both superior and inferior quadrants of the bulbar conjunctiva were taken. The superior cytology was used to quantify the density of Goblet cells and the height of mucid cloud of these cells, while the inferior cytology was used to quantify the mRNA levels of interleukin 1β (IL-1β), IL-6, and metalloproteinase 9 (MMP9) by polymerase chain reaction (PCR).The superior cytology was fixed in 96% ethanol at 4 °C for 24 h to be stained with the hematoxylin-periodic acid Schiff procedure posteriorly. For the visualization of the Goblet cells, a confocal microscopy system FV1200  was used, while the images were analyzed with the ImageJ software . The samples were excited by a wavelength of 559 nm and the light emission was filtered for a range between 580 and 620 nm. Magnifications of x20 and x40 were used for the quantification of the density of Goblet cells and the height of mucin cloud, respectively. The Z-stacking to visualize the three-dimensional cells was performed with a pupil diameter of 180 µm and a stack interval of 0.25 µm. All of these procedures were previously detailed by Peral and Pintor .The density of Goblet cells was quantified in 5 different regions of each sample, while the height of mucin cloud, including the cell thickness, was quantified in 15 different cells.The inferior conjunctival cytology was fixed in RNAlater  at 4 °C for 24 h. Then, RNAlater was removed and the samples were stored at −80 °C until being processed.The RNA isolation and purification of the samples were performed with the commercial kits QIAshredder and RNeasy Mini Kit , following the manufacturer’s instructions.Twenty-two µL of the total RNA were used for the first-strand cDNA synthesis that was performed with the High Capability cDNA Reverse Transcription Kit and random hexamer primers (Thermo Fisher Scientific). The quantitative PCR was carried with the QuantStudio 3 system (Thermo Fisher Scientific) by using the cDNA, Quantitect SYBR Green Kit (Qiagen), and specific primers of IL-1β, IL-6, and MMP9 . The hyp−ΔCt method.The analysis of the melting curves confirmed the specificity of the primers and the absence of primer-dimers. Finally, both the stability of the HPRT1 gene and the analysis of the quantitative PCR data were performed by the 2t-test for paired samples  or the Wilcoxon signed-rank test . Besides, the comparison of the effect of the different treatments (difference between PRE and POST measurements) between groups was performed using the Student’s t-test for independent samples  or the Mann–Whitney U test . A statistical significance of 95% (p < 0.05) was established in all the tests.The statistical analysis was performed with the SPSS 23 software . The normality of all the variables was checked using the Shapiro–Wilk test. The comparison between the measurements before (PRE) and after (POST) the instillation of the different treatments was carried out with the Student’s The analyzed variables were: tear secretion, tear break-up time, corneal staining, conjunctival hyperemia, density of Goblet cells, height of mucin cloud, and mRNA levels of IL-1β, IL-6, and MMP9. Results are reported as mean ± standard deviation (SD).Artemia salina showed secretagogue properties on aqueous and mucinous components of tears, accompanied by a protective effect against ocular surface damage and inflammation in a rabbit dry eye model. Thus, the potential of these artificial tears as a secretagogue agent for dry eye treatment was confirmed, which opens the door for future clinical trials and studies to extrapolate the findings to dry eye patients.The artificial tears based on an extract of 4% Artemia salina to treat the ocular surface. 2017.Pintor Just, J.J.; Pérez de Lara, M.J.; Huete Toral, F.; Colligris, B.; Carracedo Rodríguez, J.G. WO/2018/015582—Preparation and use of an extract of


Mechanism study indicated that FRE decreased cytosolic Ca2+ (Ca2+[c]) level rapidly and reversibly. Moreover, FRE decreased Ca2+[c] of MCs independent of plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). While, along with Ca2+[c] decrease, the increase of mitochondrial Ca2+ (Ca2+[m]) occurred simultaneously in FRE-treated RBL-2H3 cells. In the isolated mitochondria, FRE also promoted the subcellular organelle to uptake more extramitochondrial Ca2+. In conclusion, by increasing Ca2+[m] uptake, FRE decreases Ca2+[c] level to suppress MC degranulation. Our findings may provide theoretical support for the clinical application of Forsythiae Fructus on allergy and other MC-involved diseases.Mast cells (MCs) activated via IgE/FcεRI or MAS-related G protein coupled receptor (Mrgpr)-mediated pathway can release granules that play prominent roles in hypersensitivity reactions. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for allergic diseases. Although previous studies indicated that Forsythiae Fructus extract inhibited compound 48/80-induced histamine release from MCs, its effect on IgE-dependent MC degranulation and possible underlying mechanisms remain to be explored. Herein, we prepared the forsythiasides-rich extract (FRE) and investigated its action on MC degranulation and explored its underlying mechanism. Our data showed that FRE could dampen IgE/FcεRI- and Mrgpr-mediated MC degranulation  Mast cells (MCs), distributing at the host-environment interfaces, are a class of tissue-resident innate immune cells that can respond to various immunogenic stimuli in the first place. Generally, MCs can be activated via either IgE-dependent or IgE-independent pathways. In IgE-dependent pathway, MC activation can be initiated by crosslinking the IgE-FcεRI complexes with multivalent antigens, thereby resulting in MC degranulation. In IgE-independent pathway, MCs also possess unique responsiveness to the basic secretagogues , which can directly induce MC degranulation via activating the Mas-related G-protein coupled receptor (Mrgpr) (MrgprX2 for human MCs and MrgprB2 for mouse) .2+ (Ca2+[c]) and subsequent MC degranulation  Vahl (Oleaceae), is a well-known traditional Chinese medicine (known as lianqiao) that has been thought of having heat-clearing and detoxifying effects on diseases like epidemic pyrexia, carbuncle, scrofula and erysipelas  and demonstrated its inhibitory effect on MC degranulation and the underlying mechanism. Our findings not only enrich the existing understanding of the ethnomedicinal application of Forsythiae Fructus, but also exhibit FRE’s potential as an herbal preparation.TM-H4000 was from Engreen Biosystem . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from Gibco BRL . Nycodenz was from Axis-shield . Forsythiaside A (CAS#79916-77-1), isoforsythiaside (CAS#1357910-26-9) and phillyrin (CAS#487-41-2) were from Baoji Herbest Bio-Tech Co. Ltd. . All the other reagents were of analytical grade.C48/80, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, bovine serum albumin (BSA), Pluronic F-127 and thapsigargin were purchased from Sigma-Aldrich . Fluo-3 AM Ester, Calcium Green-5N and Cal-630 AM Ester were from Biotium , Invitrogen  and MKbio , respectively. Plasmid pcDNA3-4mtD3cpv was from Beijing Zoman Biotechnology Co. Ltd. . The transfection reagent EntransterPenaeus japonicus and the anti-SP serum were prepared as previously described (SP from escribed .in vitro experiments) or saline (in vivo experiments) before used, and the concentrations and doses were chosen according to the preliminary experiments.We first collected both unripe Forsythiae Fructus (UFF) and ripe Forsythiae Fructus (RFF) from different producing areas, and prepared the crude aqueous extracts for comparing their effects on β-hexosaminidase release. The obtained results showed that both effect and yield of UFF were better than that of RFF. To avoid disturbance of batch-to-batch variation, we chose UFF from Sangjiahe Zhendong Wild Medicinal Tending Base of Forsythiae Fructus  to prepare adequate FRE. Thus, UFF used in the present study was all from the same batch. Forsythiae Fructus was validated by Professor Yun Qi in Institute of Medicinal Plant Development (IMPLAD) of Chinese Academy of Medical Sciences (CAMS), China. A voucher specimen (IMPLAD-XL327) was deposited in the Herbarium of IMPLAD. Dried raw materials were shattered and refluxed in distilled water  for 2 h. The obtained solution was precipitated with ethanol-water  at 4°C overnight (>20 h). The supernatant was concentrated and passed through the AB-8 macroporous resin column, followed by gradient ethanol elution 10% elution was discarded and 60% elution was collected and lyophilized . The finA Shimadzu LC-15C HPLC system  equipped with LC solution software, a UV spectrophotometer detector  and a Syncronis C18 column  were used for HPLC analysis. And the sample (10 μl) was injected using an autosampler. The mobile phases were (A) 0.4% acetic acid and (B) acetonitrile with a gradient elution as listed in All animal care and experimental procedures were complied with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the IMPLAD of CAMS [SYXK (Beijing) 2017-0020]. BALB/c mice  were purchased from Beijing Vital River Laboratory Animal Services  and housed under specific pathogen-free conditions with a 12 h light/dark cycle with free access to standard diet and water. And the anesthetic and other necessary measures were used in order to reduce the animal suffering in studies.Rat basophilic leukemia cell line (RBL-2H3) was purchased from the cell bank of Chinese Academy of Sciences . Human LAD2 cell line  was a gift from Prof. Renshan Sun . And mouse peritoneal mast cells (MPMCs) were isolated from the BALB/c mice.5 cells/well) were cultured in a 96-well plate and treated with FRE (0–1 mg/ml) at 37°C for 2 h. The supernatant (70 μl) was incubated with 20 μl of lithium lactate solution  and 20 μl of INT solution . The reaction was initiated by adding 20 μl of a mixture of NAD+ and diaphorase  and incubated at 25°C for 20 min. The absorbance was measured at 492 nm.LDH assay was chosen to predict the early cell damage of FRE . Cells . The fluorescence intensity was read at λ ex 355 nm/λ em 460 nm by a fluorescence microplate analyzer .The β-hexosaminidase release assay was performed following previously described . For theFor IgE/FcεRI-mediated β-hexosaminidase release, 1% anti-SP serum sensitized-RBL-2H3 cells were pretreated with FRE for 30 min at 37°C and then co-incubated with SP (40 ng/ml) for another 1.5 h. The supernatant was collected for the β-hexosaminidase detection.2+[c] level was measured using the Ca2+-reactive fluorescent probe Fluo-3 AM as previously described  at 30°C in dark for 30 min. Probenecid was added to block Fluo-3 AM leakage. After removing the extracellular dye, the stained cells were treated with FRE at 37°C for 30 min and then challenged by 40 ng/ml of SP. Ca2+[c] level was immediately determined. For the measurement of C48/80-caused Ca2+[c] increase, LAD2 cells were loaded with Fluo-3 AM as above described and treated with FRE at 37°C for 30 min. C48/80 (10 μg/ml) was added and the Ca2+[c] level was immediately monitored.Caescribed . For det2+[c] was due to activating PMCA or SERCA, the experiments were performed by using the alkaline pH (9.0) medium or thapsigargin (5 μM) to suppress the PMCA or SERCA activity, respectively . Briefly, RBL-2H3 cells loaded with Fluo-3 AM were resuspended in Na+ solution  or Na0 solution . The cells of the two groups were then treated with FRE and the Ca2+[c] levels were immediately determined.The NCX activity assay was carried out as previously described with slight modifications . Ca2+[c2+[c] and Ca2+[m] levels in RBL-2H3 cells, the Ca2+-reactive fluorescent probe Cal-630 AM and the mitochondrial-targeted Ca2+-sensitive plasmid pcDNA-4mtD3cpv were used. Firstly, RBL-2H3 cells were transiently transfected with pcDNA-4mtD3cpv using EntransterTM-H4000  according to the manufacture instruction. Forty-eight hours later, the cells were loaded with Cal-630 AM (4 μM) in HEPES buffer containing 0.04% (w/v) Pluronic F-127 and incubated at 37°C in dark for 90 min and further incubated at room temperature for another 30 min. At the end of incubation, probenecid (4 mM) was added to block Cal-630 AM leakage. After removing the extracellular dye, cells were treated with FRE or the equal volume of vehicle. The fluorescent image was immediately taken by a laser confocal fluorescence microscopy (Nikon A1) using a 60 × oil objective .To simultaneously determine Ca2+[m] uptake of isolated mouse liver mitochondria was also measured using Calcium Green-5N according to previously described  with C48/80 (4 mg/kg), while the mice in the negative control group were received an equal volume of saline. Five minutes later, the mice were intraperitoneally injected with FRE or an equal volume of saline . Thirty minutes later, the rectal temperature was monitored by a thermal probe .The C48/80-induced hypothermia model was established as previously described with slight modifications . BALB/c i.v.) with anti-SP serum (30 μl/mouse). Twenty-four hours later, mice were pretreated (i.p.) with FRE or equal volume of saline  for 30 min and then challenged by SP  except the negative control group. Twenty minutes after SP injection, the rectal temperature was measured.Mouse PSA model was established as previously described . BALB/c post-hoc analysis was followed for between-group comparison. Statistical analysis between two groups was performed by Students’ t-test. Differences at p < 0.05 were considered statistically significant.The results were expressed as the mean ± SD. One-way analysis of variance (ANOVA) was used when more than two groups were compared using SPSS 20.0 software. The Tukey The representative standard fingerprint of FRE was shown in We first evaluated the effect of FRE on cell viability using extracellular LDH assay. After being incubated with FRE (200–600 µg/ml) for 2 h, LAD2 cells released undetectable LDH in the culture medium (data not shown). In RBL-2H3 cells and MPMCs, FRE did not increase, or rather, significantly decreased LDH leakage . These fin vitro effect of FRE on MC degranulation was investigated by detecting β-hexosaminidase release in the supernatants of LAD2, MPMCs and RBL-2H3 cells, respectively. Based on our previous observations, LAD2 and MPMCs are more suitable for the Mrgpr-mediated MC degranulation. Whereas, RBL-2H3 cells are more suitable for IgE/FcεRI-mediated MC degranulation (p < 0.01), while FRE could concentration-dependently suppressed the β-hexosaminidase release with IC50 value of 441.5 and 525 µg/ml, respectively.Next, the nulation . Therefo50 value of 540.4 µg/ml . In IgE-mediated in vivo model, injection of SP markedly lowered the rectal temperature (ΔT = 3.52 ± 0.68°C), while FRE could significantly counter the hypothermia  through SERCA  and Fluo-3 AM (λ ex 485 nm/λ em 538 nm), we deliberately chose an alternative Ca2+ fluorescent dye Cal-630 AM (λ ex 600 nm/λ em 640 nm) to avoid interfering with each other. As expected, we indeed observed an obvious decrease in red fluorescence (Ca2+[c]) concurrent with an obvious increase in yellow fluorescence (Ca2+[m]) in RBL-2H3 cells , indicating that the intra-mitochondrial Ca2+ was increased. These findings revealed that FRE decreased Ca2+[c] mainly through a direct action on mitochondria.Besides the above three ways, mitochondria uptake of Cawas used . Given tH3 cells , demonstUpon extracellular stimulation, MCs can be activated via IgE/FcεRI or Mrgpr-mediated signaling and consequently release lots of preformed granules which contain many biologically active mediators  . However2+[c] elevation, and eventually triggers MC degranulation  during Ca2+[c] increasing, thus markedly buffering the Ca2+[c] rise  complex, a Ca2+-activated Ca2+ channel, and is driven by the electrochemical gradient that exists across the mitochondrial inner membrane (ΔΨm)  . Therefo2+[m] is crucial for cellular ATP production and erobic respiration. Upon Ca2+[m] rise, the Ca2+-sensitive dehydrogenases of Krebs cycle are activated, facilitating the oxidative phosphorylation and thus promoting the ATP synthesis (2+ enters into mitochondria (Ca2+[m] overload), it can cause overproduction of reactive oxygen species (ROS), a by-product of Krebs cycle, which in turn impairs the bioenergetic metabolism and thus eventually triggers cell apoptosis (2+[m] homeostasis. Likewise, buffering Ca2+[c] through Ca2+[m] uptake to dampen MC degranulation also requires such a balance, which is to intake Ca2+ into mitochondria effectively without causing Ca2+[m] overload. In the present study, FRE was effective and non-toxic in both in vivo and in vitro models, indicating that the effect of FRE on Ca2+[m] increase was sustainable to a certain degree. Moreover, the non-toxicity of FRE under the effective concentrations might also be related to its reversible feature on reducing Ca2+[c]. In addition, the characteristic components in FRE, including forsythiaside A, isoforsythiaside, and phillyrin (Caynthesis . Howeverpoptosis . Obvioushillyrin , possesshillyrin . Indeed,2+ to buffer the IgE/FcεRI- or Mrgpr-caused Ca2+[c] rise, and consequently inhibits MC degranulation (In summary, our study reveals that FRE promotes mitochondria to uptake more Canulation . These f


Recent advances in imaging techniques have identified localized Ca2+ transients within the fine processes of the astrocytic structure, which we term microdomain Ca2+ events. These Ca2+ transients are very diverse and occur under different conditions, including in the presence or absence of surrounding circuit activity. This complexity suggests that different signalling mechanisms mediate microdomain events which may then encode specific astrocyte functions from the modulation of synapses up to brain circuits and behaviour. Several recent studies have shown that a subset of astrocyte microdomain Ca2+ events occur rapidly following local neuronal circuit activity. In this review, we consider the physiological relevance of microdomain astrocyte Ca2+ signalling within brain circuits and outline possible pathways of extracellular Ca2+ influx through ionotropic receptors and other Ca2+ ion channels, which may contribute to astrocyte microdomain events with potentially fast dynamics. Astrocytes are complex glial cells that play many essential roles in the brain, including the fine-tuning of synaptic activity and blood flow. These roles are linked to fluctuations in intracellular Ca Physiologically, astrocytes are critical for brain homeostasis . They bu2+  that are2+ ,13,14,15 GCaMP6f ,23,24,252+ transients are dictated by the resting, basal intracellular Ca2+ concentration [2+ events [2+ signal onset timescales (a few milliseconds after the start of stimulation), this astrocytic Ca2+ signalling was deemed too slow to modulate rapid processes such as synaptic activity or blood flow [2+ dynamics have recently been described within fine astrocyte structures in response to physiological stimuli in vivo [2+ signalling to play a rapid role in fine-tuning circuits as discussed below.Astrocyte MCEs are heterogenous; they vary in amplitude and duration, and occur within astrocytes at rest  ,18. The ntration , which intration . The numntration ,28,29 fontration ,18,30,31+ events ,33,34 an+ events ,18 activood flow ,33,34. H in vivo . In part in vivo . This su2+ dependent, suggesting that astrocyte Ca2+ events are a key component of bidirectional astrocyte-neuron interactions [2+ domains or more global astrocyte Ca2+ signals may modulate neuronal networks and dictate animal behaviour [Astrocytes are active contributors to brain processes through the release of gliotransmitters or vasoactive molecules that modulate the nearby neuronal activity or blood flow ,11,12. Tractions . Specifiractions ,48,49,50ehaviour ,53,54,552+ signalling and gliotransmitter release influences basal synaptic activity, excitatory and inhibitory neurotransmission, and synaptic plasticity  have repetitive [2+ events that locally regulate circuit activity, modulate the processing of information in large networks and impact animal behaviour. Fast onset MCEs evoked by neuronal activity could be of critical importance for rapidly tuning changes at single synapses that amount to alterations in activity over larger circuits. Again, future studies specifically targeting pathways that contribute directly to astrocyte MCEs will help to link MCEs to the modulation of single synapses, but will also help determine how the scaling of astrocyte Ca2+ signalling and the recruitment of MCEs influence larger neuronal networks and behaviour.During periods of neuronal activity, astrocyte Caial area ,29,30,31response . For exaresponse ,70, sincllations . Additiooeruleus ,33,71,72ormation , while mpetitive , depresspetitive , or autipetitive . Thus, a2+-dependent release of vasoactive molecules, such as arachidonic acid metabolites  [2+ influx in astrocytes [A number of mechanisms are known to contribute to localized astrocyte MCEs ,15,20,25 release  via the trocytes ,86,87,882+ events is the release of Ca2+ from the endoplasmic reticulum following inositol-1,4,5-trisphosphate receptor (IP3R) and upstream Gq-G-protein coupled receptor (GPCR) activation  when activated by synaptic glutamate (N-methyl-D-aspartate (NMDA) receptors [2+, deactivation kinetics, and Ca2+ permeability [2+ and therefore do not require membrane depolarization to be activated. These subunits are also less permeable to Ca2+ than GluN2A/2B receptors, and they have slower deactivation kinetics [Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that conduct cations . There is evidence of astrocyte kainate receptor subunit expression at the mRNA and protein levels [Astrocytes express the genes of iGluRs, albeit at lower levels than neurons. All four AMPA receptor subunits GluA1-GluA4) have been detected in astrocytes -GluA4 ha, althougtrocytes ,109,110.n levels ,112; hown levels ,116,117.2+ permeability and their contribution to Ca2+ signalling, has been controversial. Early Ca2+ imaging studies were conducted in primary astrocyte cultures  by combining Ca2+ imaging dyes with GFAP-eGFP transgenic mice to better label astrocytes [2+ events in fine structures. Using GCaMP3 and GCaMP6f, Haustein et al. [2+ events [2+ responses happened on the same time scale as neurons and preceded local vasodilation. Blockers for AMPA or NMDA receptors were applied directly to the brain and both drugs reduced fast Ca2+ events in astrocyte processes, but only CNQX reduced fast Ca2+ events in endfeet [Although iGluR agonists evoke Catrocytes ,128,129.n et al.  showed t+ events . In simi+ events ,31, whic endfeet . This su endfeet , making 2+ in the soma, limited studies have found a functional role for astrocyte AMPAR. In the cerebellum, Bergmann glia astrocytes express the GluA1 and GluA4 subunits [2+ impermeable [While it is clear that AMPA receptor activation can cause an elevation in astrocyte Casubunits . When Beermeable  or (b) termeable , structuermeable  and defiermeable . Clearly2+ stores [2+ in astrocytes through elevation of store uptake [2+ concentrations, which has implications for Ca2+ microdomain activity and their dynamics [2+ transients in cultured cortical astrocytes upregulate the Cdk5/Nrf2 pathway, a key regulator of genes for cell antioxidant machinery [Astrocyte NMDA receptors have functional roles in maintaining astrocyte Ca+ stores , antioxi+ stores , gliotra+ stores , and the+ stores . First, e uptake . Thus, Ndynamics ,27. Secoachinery . This inachinery , and conachinery . 2+ events in astrocytes, then gliotransmitter release may happen in a temporal realm that can rapidly tune synaptic activity. Finally, astrocyte NMDA receptors may also regulate synaptic strength in the hippocampus by maintaining paired-pulse ratio heterogeneity, which is seen between two presynaptic neurons that target the same postsynaptic cell [2+ transients and astrocytic NMDA receptor signalling in diversifying the presynaptic strengths and potentially elevating the network dynamics of dendrites [2+ signalling mediated by these receptors and their physiological roles.As discussed in tic cell . Paired-endrites . This tyendrites . While t1/5 [7 subunits [2+, K+, and Na+ into the cell. The subunit composition confers ATP binding affinity and Ca2+ permeability [7 receptors are only activated by high extracellular ATP levels and have been linked to pathology and astrocyte reactivity [1/5, with its higher affinity for ATP and good Ca2+ permeability, is more likely to be involved in astrocyte MCEs, particularly with a fast onset during local circuit activity. So far, the contribution of P2X1/5 activity to astrocyte MCEs has not been explored with GECIs, but P2X activation causes astrocyte Ca2+ transients (primarily somatic) in brain slices and acutely isolated astrocytes, as measured with Ca2+ dyes [Astrocytes express ionotropic P2X purinergic receptors , likely 1/5  or homotsubunits . These leability ,143. P2Xactivity ,145. Thea2+ dyes ,146.1 receptors have been linked to endfoot Ca2+ transients and capillary dilation during neurovascular coupling, suggesting that these ionotropic receptors induce the release of vasoactive molecules that specifically act on capillaries and not arterioles [Coincidently, astrocyte P2X receptor activation enhances purinergic signalling in different brain regions. In the cortex, astrocyte P2X receptors increase ATP release , which mterioles . Astrocyterioles ,147, whiterioles . These e2+, Na+ and K+ and are made up of 16 possible subunits. Astrocytes express homomeric alpha-7 nicotinic acetylcholine receptors , which has an important role in buffering intracellular Ca2+ in exchange for Na+ influx  glutamate activation of Na+-permeable ionotropic kainate or NMDA receptors [+ gradient to drive synaptic glutamate uptake [+ into the cell during GABA uptake [2+ events due to NCX reversal may also trigger Ca2+-induced Ca2+ release from intracellular Ca2+ stores, suggesting NCX reverse function amplifies agonist-induced Ca2+ events in astrocytes [Several possible mechanisms increase intracellular astrocyte Naeceptors ,162,163,e uptake ,164,165,A uptake ,166. Ca22+ may evoke gliotransmitter release, such as glutamate [2+ transients and regulate excitatory transmission. While a number of studies have attempted to model the contribution of NCX to astrocyte MCEs in fine processes [Astrocyte NCX reversal and increased cellular Calutamate ,168, ATPlutamate , and homlutamate . An incrlutamate . This isrocesses ,170,171,2+ channels (VGCCs) [2+. VGCCs do not contribute to spontaneous MCEs [+ uptake [+ and glutamate accumulate. Functionally, astrocyte VGCCs induce the release of glutamate [Astrocytes also express voltage-gated Ca (VGCCs) ,173,174, (VGCCs) . These cous MCEs , but the+ uptake . Thus, alutamate . However2+ that grant them the potential to quickly regulate local processes such as synaptic activity and blood flow. By aiding the integration of information at multiple synapses and among different neuronal types in brain circuits, astrocytes may impact neuronal activity at the population level, thereby contributing to information processing and animal behaviour. Future experiments should focus on GECI tools to better identify MCEs, particularly with a fast onset during nearby synaptic activity. Additionally, pathways of extracellular calcium influx outlined here have important implications for astrocyte calcium physiology and are gaining interest in the field. Approaches to selectively target these pathways will help to better understand their contribution to rapid onset astrocyte MCEs and their functional relevance regarding neuronal network activity.Astrocytes have localized, rapid fluctuations in intracellular Ca


Polyphosphoinositides (PPIns) and their modulating enzymes are involved in regulating many important cellular functions including proliferation, differentiation or gene expression, and their deregulation is involved in human diseases such as metabolic syndromes, neurodegenerative disorders and cancer, including Acute Myeloid Leukemia (AML). Given that PPIns regulating enzymes are highly druggable targets, several studies have recently highlighted the potential of targeting them in AML. For instance many inhibitors targeting the PI3K pathway are in various stages of clinical development and more recently other novel enzymes such as PIP4K2A have been implicated as AML targets. PPIns have distinct subcellular organelle profiles, in part driven by the specific localisation of enzymes that metabolise them. In particular, in the nucleus, PPIns are regulated in response to various extracellular and intracellular pathways and interact with specific nuclear proteins to control epigenetic cell state. While AML does not normally manifest with as many mutations as other cancers, it does appear in large part to be a disease of dysregulation of epigenetic signalling and many novel therapeutics are aimed at reprogramming AML cells toward a differentiated cell state or to one that is responsive to alternative successful but limited AML therapies such as ATRA. Here, we propose that by combining bioinformatic analysis with inhibition of PPIns pathways, especially within the nucleus, we might discover new combination therapies aimed at reprogramming transcriptional output to attenuate uncontrolled AML cell growth. Furthermore, we outline how different part of a PPIns signalling unit might be targeted to control selective outputs that might engender more specific and therefore less toxic inhibitory outcomes. Acute myeloid leukemia (AML) is a cancer of blood cells, in which myeloid progenitor cells lose their ability to differentiate while increasing their rate of proliferation giving rise to too many and/or too immature myeloid cells derivatives. These blast cells fail to differentiate into granulocytes or monocytes. Blast accumulation in the absence of proper haematopoiesis leads to impairment of the immune system and eventually to death. Although, compared to other types of cancers, AML is characterised by a low number of mutations, it is a highly heterogenous hematologic disease classified into many different subtypes, reflected in different clinical manifestations: understanding the relevance of this heterogeneity is critical to develop novel and more personalized clinical therapies . Today, 2], PtdInsP2, PtdInsP2 and PtdIns 3,4,5-trisphosphate [PtdInsP3]. PPIns can be interconverted by the activity of kinases and phosphatases whose activations can be controlled by extracellular and intracellular inputs P2 and induce a change in conformation and channel activity. PtdInsP3, which is synthesised by the phosphorylation of PtdInsP2 is also predominantly localised to the plasma membrane and can initiate many different signal cascades. In contrast PtdIns(4)P is predominantly found in the Golgi complex P complex , where iysosomes  P3 and increased levels of PtdInsP3 are sensed by specific Plekstrin Homology (PH) domain containing proteins such as the serine threonine kinase AKT/PKB. PtdInsP3 can be degraded by a number of phosphatases such as PTEN, SHIP1 or INPP5D which are essential to maintain long term hematopoietic stem cells , they have met with a very limited success as monotherapies, probably as a consequence of compensatory activation of other survival pathways . For thiin vivo. Altogether, these data show a possible benefit of PI3K/AKT/mTOR inhibition as a therapeutic approach for MLL-rearranged leukaemia.Firstly, PI3K/mTORC1/2 inhibition reached higher apoptosis rates than single inhibition or combined AKT/mTORC1 inhibition. Primary patient samples and cell lines carrying MLL rearrangements show higher sensitivity to PI3K/mTOR inhibition. In a larger cohort of MLL-AF9+ AML patients, a high incidence of additional mutations in genes involved in growth factor signalling pathways was identified, which could explain their preferential sensitivity . In THP-In the battle against AML, the effect of PI3K/AKT/mTOR inhibition can be augmented by BCL-2, which is an anti-apoptotic protein often overexpressed in several blood disorders, including AML. Increased expression of BCL-2 enhances survival by blocking apoptosis and is associated with increased chemoresistance and poor patient outcome .The selective BCL-2 inhibitor Venetoclax has shown strong cytotoxic effects combined with a safe patient profile in AML  and VeneComplete responses to BCL-2 are only observed in approximately 20% of patients suggesting that monotherapy does not reach durable responses. Thus, Venetoclax combined with inhibitors of survival pathways or classical chemotherapeutic drugs have been assessed for the treatment of AML, to increase the cytotoxic effects. Among these, the combined treatment of BCL-2 and PI3K inhibition enhanced leukemia cell death in AML cell lines, patient-derived blasts and xenograft models . The ant2 into DAG and InsP3: DAG can be bound by many proteins, i.e. protein kinase C (PKC) that transduce changes in DAG levels into phosphorylation and regulation of downstream targets, whilst InsP3 can be bound by its cognate receptor on the endoplasmic reticulum and regulate Ca2+ efflux (Phospholipases C (PLCs) hydrolyse PtdInsP+ efflux Figure 2+ efflux –35.Interestingly, PLCβ1 is involved in haematological malignancies by regulating haematopoiesis, especially in Myelodysplastic Syndromes (MDS), where it acts on both erythropoiesis and myelopoiesis –38, with2 from the plasma membrane and presents it as a substrate to PLCβ3 for hydrolysis, mediating InsP3-induced endoplasmic reticulum Ca2+ release P release , 46. Imp, subunits of the PI3K pathway , PLCβ2 and PIP4K2A . PIP4K2Alls HSCs . In AML lls HSCs . Moreovells HSCs , 52.The revelation that many different PPIns modulators are essential for AML cell growth suggest that understanding their individual mechanism of action might lead to the development of patient specific therapies that could be generated through combinations of molecules that deregulate these pathways. Importantly the enzymes that modulate PPIns are highly druggable and many inhibitors are already available. Given that many of these enzymes are also found in the nucleus where they regulate a specific pool of PPIns, which impact on transcriptional output we next describe their potential roles as epigenetic regulators in AML.2 and PtdInsP3 are localized in the nucleus P nucleus , in the d islets . In the d islets . These cd islets , 56. Sur2 beads  for its broader use as a therapeutic in AML. ATRA is highly successful in treating Acute Promyelocytic Leukaemia (APL) but has little effect in other AML subtypes. Recent studies have shown that treatment with either KDM1A or GCN5 (a histone h3K9 acetylase) inhibitors reprogram non-APL AML cells to become sensitive to ATRA induced differentiation , 84. IdeTargeting epigenetic signalling enzymes, such as methylases or demethylases, may turn out to have toxicity issues and uncovering how they are controlled would allow for further fine tuning. Remarkably it appears that nuclear PPIns interaction with downstream targets regulates selective output signalling. For example, mutations that attenuate the interaction of the PHD finger of either ING2 or TAF3 with PPIns do not completely inhibit their function but rather lead to a more selective inhibition that impacts on the transcriptional output of a subset of gene targets , 86. PPI2. Interestingly, the other two isoforms, 2B and 2C also stratify patient survival with 2C showing similar characteristics as 2A. However, PIP4K2B, which is predominantly localised in the nucleus, shows an inverse correlation with survival compared to 2A and 2C, such that low levels of 2B associate with poorer survival. This is in accord with our previous studies in breast cancer patients which revealed that low PIP4K2B is associated with poor survival  stratify patient overall survival. For example and in accordance with our previous studies , high exsurvival . SimilarAnother key aspect in targeting PPIns pathways, is that the enzymes that modulate them are highly druggable and many of them have already been under intense development for the identification of small molecular weight inhibitors. For example, there are multiple inhibitors targeting the PI3K pathway which are in various stages of clinical development. In many respects the PI3K pathway holds a special place in PPIns metabolism as flux through the pathway is generally very low but is strongly stimulated by oncogenic pathways. While this imparts a therapeutic potential, the involvement of the PI3K pathway in normal cellular processes ultimately leads to potential for on target toxicities. For example the PI3K pathway is intimately involved in insulin signalling and on target issues with glucose homeostasis are often seen in therapeutic treatments . As signExploiting PPIns signalling complexity, however, requires deep level understanding of how these interventions impact output, the gathering of which has become much more feasible with the advent of CRISPR gene editing tools. Combining this knowledge with bioinformatic network analysis using tools such as Mogrify and knowledge of patient specific transcriptional landscapes might allow the complexity within PPIns signalling pathways to be exploited by intervening at various levels to convert an AML cell to a differentiated and non-proliferative cell state.RF and ND contributed to the conception of the work. All authors contributed to the article and approved the submitted version.The 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.


Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca2+]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca2+ entry is required for the cortisol-mediated rise in ([Ca2+]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca2+]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca2+ channel transducing rapid nongenomic signalling in hepatocytes during acute stress.Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout ( GCs promote energy substrate redistribution and is essential for the metabolic adjustments to regain homeostasis in the face of stressor exposure1. This steroid action is mediated by binding to an intracellular receptor, the glucocorticoid receptor (GR), which acts as a transcription factor upon ligand-binding to regulate GC-responsive transcriptional machinery1. Consequently, this stress-mediated genomic effect of GCs takes longer to transpire compared to the sympathetic activation of the fight or flight response, which is rapid3. However, it is now increasingly clear that apart from the genomic responses, GCs also exert rapid effects, which are non-genomic and important in the acute stress adaptation7. For instance, studies have shown that GCs stimulate second messenger signalling, including rapid changes to intracellular cAMP and cytosolic free calcium ([Ca2+]i) levels, leading to downstream phosphorylation of protein kinases and the associated cellular responses6. Although, putative corticosteroid binding sites on plasma membrane were identified in vertebrates10, including teleosts11, as possible mediators of the rapid signaling, specific plasma membrane protein(s) transducing GC signals have yet to be discovered6.Glucocorticoids (GCs) are acutely released in response to stressor-mediated activation of the hypothalamus–pituitary–adrenal (HPA) axis in vertebrates2+]i)13, and studies have shown that this steroid either stimulate or inhibit basal Ca2+ levels in a tissue-specific manner14. Ca2+ is a ubiquitous second messenger and an important regulator of the physiological actions of cells, including rapid hormonal regulation of glucose output during acute stress15. Rapid modulation of ([Ca2+]i) may be due to alterations in either the intracellular Ca2+ release from endoplasmic reticulum (ER) and/or extracellular Ca2+ entry across the plasma membrane through specific channels, including the voltage-gated calcium channels (VGCC) and/or calcium release-activated calcium (CRAC) channels18. GCs have been shown to stimulate CRAC channel gating leading to increased Ca2+ concentration in rat L6 muscle cells in culture19. Also, we recently showed that cortisol rapidly increased ([Ca2+]i) in the developing trunk muscle of zebrafish (Danio rerio), suggesting a possible role for CRAC channel activation20.A key mode of action for rapid physiological effects of GCs involve changes in , to interact with the CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel, and regulate channel gating23. Studies have shown that GCs modulation of ([Ca2+]i) may involve CRAC channel gating20, but this has not been studied from the standpoint of liver stress response. While cortisol rapidly increased ([Ca2+]i) in trout hepatocytes4, the mechanism leading to this rapid surge in Ca2+ was unknown. Also, cortisol rapidly increased the phosphorylation of protein kinase A (PKA), Akt /protein kinase B (PKB) and protein kinase C (PKC) in rainbow trout (Oncorhynchus mykiss) hepatocytes24, suggesting possible activation of second messenger signaling via putative membrane receptor(s)25. Given the important role of Ca2+ in the liver stress response15, we hypothesized that a membrane receptor transducing rapid GC signal may involve rapid modulation of ([Ca2+]i) in hepatocytes. Here, using freshly isolated hepatocytes from rainbow trout, we investigated whether cortisol, the principal GC in humans1 and teleosts2, directly activate plasma membrane Ca2+ channels to modulate ([Ca2+]i). GCs have been shown to either increase or decrease basal ([Ca2+]i) in a tissue-specific manner14, but the effect of this steroid in modulating Ca2+ channels activity in hepatocytes is less clear. Using targeted pharmacological approaches, including Ca2+ channel blockers, we investigated the mechanism by which cortisol modulates ([Ca2+]i) in trout hepatocytes. Our results indicate that cortisol rapidly increases ([Ca2+]i) in hepatocytes, and, for the first time, suggests a possible direct role for this steroid in CRAC channel gating.Liver is a key target for the metabolic actions of GCs to cope with stress, but most studies focussed on the genomic actions with little mechanistic insights into the nongenomic mode of action for this steroid-ol-3-one), corticosterone and β-estradiol , dexamethasone, EGTA (Ethylene glycol bis (β-amino ethyl ether)-N,N,N′,N′-tetraacetic acid), nifedipine, ryanodine, trypan blue and cadmium chloride were purchased from Sigma-Aldrich . Pluoronic-F 127 , BAPTA-AM  ethane-N,N,N′,N′-tetraacetic acid), Xestospongin C, H-89, U73122 and Fura 2-AM were purchased from Life Technologies, USA. Cortisol conjugated BSA was purchased from EastCoast Bio, USA. All hormones utilized during the experiment were dissolved in ethanol, the final concentration of which never exceeded 0.01%, and the control had the same amount of ethanol. All other chemicals were of analytical grade and were purchased from local suppliers.Bovine serum albumin (BSA), Leibovitz L-15 medium were obtained from Invitrogen life technology. Collagenase (Type IV), cortisol (hydrocortisone), testosterone (4-androsten-17βJuvenile rainbow trout (~ 100 to 200 g body mass) were obtained from the Allison Creek Brood Trout Hatchery Station  and maintained at the Animal Facility, Department of Biological Sciences, University of Calgary. The fish were maintained in a 500 L tank supplied with a constant flow of aerated de-chlorinated water with temperature maintained at 12 ± 1 °C and photoperiod set at 12 h light:12 h dark. Trout were fed commercial trout feed  to satiety once daily. The fish were acclimated to this condition for at least 2 weeks, and were food deprived for 24 h prior to their use for hepatocyte isolation. All experimental protocols were approved by the Animal Care Committee at the University of Calgary and were in accordance with the Canadian Council on Animal Care guidelines, and the ARRIVE guidelines.26. In brief, fish were always sampled in the morning (0900-1000) euthanized with an overdose of 2-phenoxyethanol , followed by bleeding the fish to maximize blood drain and minimize red blood cell contamination during hepatocyte isolation. The fish was dissected, and the portal vein cannulated to perfuse the liver with Medium A  for 15–20 min to remove blood, followed by Medium B  digestion for 20–30 min. Buffers used for cannulation and enzymatic digestion were kept on ice throughout the process. After the perfusion, liver was transferred to a petri dish with Medium A, finely minced, and the suspension was filtered through two nylon filters (250 and 75 µm). The cell suspension was centrifuged 3 times  at 200×g for 5 min at 11 °C with Medium A, after which the pellet was re-suspended in Medium C (Medium A with 1.5 mM CaCl2 and 1% BSA) and centrifuged as mentioned above. The pellet was re-suspended in 25 ml of L-15 medium  and allowed to settle down on ice for 30 min. The medium was aspirated, and the settled cells were re-suspended in 10 mL of L-15 medium followed by cell counting using a haemocytometer. Cell viability was checked using trypan blue (Sigma) dye exclusion test27 and the viability was > 95%. The cell suspension at a concentration of 0.75 × 105 million cells per ml L-15 medium were incubated in 15 mL falcon tube at 11 °C on a slow tube rotator for 24 h prior to treatments and calcium imaging.Hepatocytes were isolated following collagenase digestion as described previouslyg for 5 min followed by trypan blue test. The cells were washed with Medium C and incubated with fura2-AM28  and 5 µL of 20% pluronic-F127 (Thermo Scientific)29 followed by washing and centrifugation at 200×g for 5 min. Then, the cell pellet was re-suspended in medium C for 30 min for the cell esterases to carry out de-esterification of fura2-AM to allow binding of fura2 to calcium, followed by imaging. Briefly, cell suspension (50 µL) was mounted on a coverslip (of approx. 18 mm) attached to a petri dish of 28 mm diameter28. This whole set up was then mounted on an inverted microscope (Qimaging RETIGA EXi FAST 1394) for imaging. All experiments were conducted after 30 min post-fura2-AM incubation to ensure deesterification. The hormones (or hormone agonist) added were cortisol (hydrocortisone), testosterone (4-androsten-17β-ol-3-one), corticosterone, 17β-estradiol and dexamethasone dissolved in 100% ethanol, with the final concentration of ethanol in all treatment groups being < 0.01%. The Ca2+ chelators (EGTA (2 mM)30, BAPTA (10 µM)31), plasma membrane Ca2+ channel inhibitors  and drugs to block intracellular pathways, including IP3 receptor [Xestospongin C (10 µM)34], ryanodine receptor (ryanodine(20 µM)35], ER Ca2+ ATPase  and phospholipase C [U73122 (10 µM)39] were added to the medium 30 min prior to cortisol addition as described previously20. The cortisol concentration used for the experiments were either unstressed (5 and 10 ng/mL) or physiologically stressed levels of cortisol (100 ng/mL) reported in trout. Other steroid concentrations used were all kept at the cortisol concentration (100 ng/mL) to avoid any membrane biophysical changes due to varying steroid concentrations as a factor for the observed response. ORAI-1 rabbit polyclonal antibody 42 was used to block ORAI1 protein on the hepatocytes, while protein A purified mouse monoclonal cortisol antibody (1:100)43 was used to sequester cortisol, and anti-plant alpha tubulin antibody (1:200)44 was used as a control for these studies. The antibody dilutions for cortisol (1:100) and ORAI1 (1:250) were based on previous studies45. Cells were imaged immediately after cortisol and other steroid additions for 10 min, with the images captured at 10 s intervals.After overnight incubation cells were subjected to washing and centrifugation at 200×2+]i) was expressed as a ratio of fluorescence intensities at 380 nm  and 340 . Hence, settings were made on lambda DG 4 for filters switching from 340 to 380 nm wavelengths, respectively, followed by exposure and gain settings. Light emitted from a 75-W xenon arc lamp  passed through an excitation filter set (Chroma) to generate ultraviolet monochromatic wavelengths of 340 and 380 nm. With the aid of a computerized filter wheel , the cells in the chamber were alternately exposed to the two wavelengths through an objective (40×/340/0.90 N.A.). All image acquisition was computer-controlled by Northern Eclipse . Images acquired were corrected for background fluorescence and shading across the field of view before calculating the ratio of the fluorescent emission intensities at each excitation wavelength (340/380 nm). Images were acquired at 10 s intervals to reduce photo bleaching. The exposure parameters for 340 and 380 nm were kept unchanged throughout the experiment. All measurements were made at room temperature (~ 20 °C).Cytosolic free calcium  for overnight at 4 °C. Both the ORAI and caveolin-1 antibodies have been tested in zebrafish previously20. Secondary antibody donkey anti-mouse Alexa 594 conjugate  was carried out the next day after washing. DAPI (100 ng/mL) staining was done to identify nuclear localization prior to mounting. Coverslips were mounted to clean slides using DABCO  followed by sealing the periphery using transparent nail paint. This whole mounting step lasts for an hour to air dry. Post drying, slides were wrapped in aluminium foil and stored at 4 °C until imaging. This step is crucial in keeping the staining stable for longer period with less photo bleaching.Isolated hepatocytes were cultured on 22 mm coverslips in 6 well plates  for 24 h prior to treatment exposure. After exposure to cortisol (100 ng/mL) for 5 min, the cells were fixed using ice cold methanol and stored at 4 °C. The coverslips with the fixed cells were tapped on paper towel to remove excess methanol followed by air-drying. Hydrophobic pen was used to draw a barrier around the coverslip boundary to secure the central area for antibody application. Coverslips with cells attached were then incubated with a permeabilization buffer (0.1%TritonX-100 in Medium A) for 10 min followed by washing with Medium A (with 0.1%Tween-20) 3 times at 5 min interval. The permeabilized cells were incubated with a blocking solution (Medium A with 2% BSA) for 3 h at 37 °C followed by overnight incubation with anti-ORAI antibody at 4 °C. The next day cells were then probed with the secondary antibody, which consisted of goat anti-rabbit Alexa 488 conjugate . Double antibody staining was carried out by incubating the cells with caveolin-1 mouse monoclonal IgG26. Whole trout liver was dissected out from terminally anesthetised trout. The liver was homogenized in TCD buffer , and the homogenized mixture was subjected to multiple centrifugation steps at 4 °C for separation of cellular fractions as described previously47. The membrane fractions of trout liver were prepared using ultracentrifugation at 100,000×g as described previously26. Membrane fraction enrichment and any cytosolic contamination was detected by measuring the activities of 5′AMP nucleotidase48 and lactate dehydrogenase (LDH)49.Trout liver homogenate for western blotting was prepared as described previously50 as described previously47. At room temperature, membranes were blocked for 1 h in 5% skim milk, followed by an overnight incubation with rabbit polyclonal ORAI-1primary antibody (1:250) at 4 °C. Membrane were further washed and incubated with secondary goat anti rabbit IgG (H+L) horse radish peroxidase  (1:3300 diluted in 5% skim milk) for 1 h at room temperature. Blots were washed followed by protein band detection using chemiluminiscence substrate ECLplus incubation according to manufacturer’s instructions and imaged using Syngene G-Box Imager .Protein concentration was measured using bicinchoninic acid (BCA) method. Membrane fractions and liver homogenate were diluted in Laemmli’s buffer . A total of 40 μg protein lysate was resolved on a 10% SDS-PAGE, transferred onto a 0.45 μm nitrocellulose membrane Drosophila melanogaster) was downloaded from RCSB protein data back in PDBQT format. Further, metal ions were removed from the structure to avoid insignificant binding. Polar hydrogens were added to the PDB file using AutoDockTools and converted to PDBQT file. Hydrogen addition mimics a more realistic environment for docking. To find the entire binding site on CRAC, the grid was modified to cover the entire protein (CRAC), as we are unaware of the binding sites. The ligands, including cortisol, corticosterone, estradiol, testosterone and dexamethasone were used from ZINC database51. The Drosophila melanogaster CRAC channel (4HKR.pdb) was used as a receptor. The receptor protein coordinates of CRAC channel with PDB id of 4HKR.pdb were considered to study binding sites. The structural integrity of the binding was assessed by analyzing the root mean square deviation (RMSD) between interacting molecules. RMSD values are used to validate protein–ligand binding in terms of binding energy and interaction established between protein and ligand. CRAC channel structure is a hexameric assembly of four transmembrane helices (M1–M4) and helix extension of M4 extending into the cytosol. The channel pore is made up of six M1 helices to form the inner pore. M2 and M3 together form the outer lining for M1 helices and separate them from M4 helices. M4 helices are the peripheral outer ring subunit of CRAC that interacts with STIM for channel gating. Studies confirm that STIM binding to Leu319 or Ile319 at the M4 extension A and B is critical for channel activation. The best results containing 8 coordinates of each ligand (as mentioned above) are considered for further analysis. PyMOL was used to predict the orientation of amino acids. The metal ions, known to bind the core of the receptor, were removed at the beginning of the docking process to avoid non-specific binding predictions. The exhaustiveness value was set to default (8) and a local computer (with 8 core CPUs) was used for the docking. During the docking process, the receptor was treated as a rigid molecule and the ligands were flexible in the binding site. One single best score-binding site of each ligand was considered for further analysis.The docking was done using AutoDock Vina v1.1 program. Input files for AutoDock Vina were prepared using AutoDock tools . File preparation involved changing atom type, removal of water molecules and addition of polar hydrogen atoms. Structure files were saved in PDBQT format. CRAC channel structure , and this may be a mechanism for the rapid nongenomic actions of GCs on liver function24. Our results for the first time suggest CRAC channels as a potential cortisol-gated calcium channel in trout hepatocytes.GCs are key stress hormones and are important in re-establishing homeostasis after stressor exposure2+]i) in isolated hepatocytes  was significantly greater (p < 0.001) with stress levels (100 ng/mL) of cortisol  responses were also mimicked by the membrane impermeable cortisol-BSA  wave  in different cell types21 our results indicate the greatest ([Ca2+]i) increase with cortisol (> 60%) in hepatocytes compared to the other steroids tested  rapidly increases  rise by manipulating the extracellular Ca2+ availability. In the absence of extracellular Ca2+ cortisol did not elicit a ([Ca2+]i) rise in trout hepatocytes  rise  compared to the control suggests that cortisol may stimulate Ca2+ release also from ER  increase in cells pre-treated with the membrane permeant Ca2+ chelator BAPTA-AM  and more on CRAC channel for hormone-mediated ([Ca2+]i) modulation18. To test this, we treated hepatocytes with the L-type VGCC inhibitor nifedipine, which blocked only 10% of the cortisol-induced ([Ca2+]i) response  rise. However, cadmium, a non-specific blocker of both VGCC and CRACC56, completely abolished cortisol-induced ([Ca2+]i) rise , PKA (H-89), inositol 1,4,5, triphosphate receptor (IP3R) (Xestospongin C) and RYR (ryanodine) inhibitors. While the inhibition of the PLC-IP3R and PKA-RYR pathways blocked cortisol-induced biphasic ([Ca2+]i) rise by only 5–20%  rise. These results led us to propose that activation of PKA-RYR pathway may be one possible mechanism by which cortisol rapidly stimulates ([Ca2+]i) rise in trout hepatocytes. However, the plasma membrane receptor involved in this activation by cortisol remains elusive. Also, whether activation of the PKA-RYR pathway partly accounts for the CRAC gating by cortisol remains to be determined.While several studies have showed GCs to modulate intracellular calcium levelstes Fig. d. While llular Ca+ contribllular Ca+ contribllular Ca+ contribllular Ca+ contribllular Ca+ contribllular Ca+ contribllular Ca+ contrib2+ stores with thapsigargin (Tg), a sarco/endoplasmic reticulum calcium ATPase (SERCA) inhibitor, that facilitates STIM-mediated CRAC channel gating17. As expected, Tg treatment induced an increase in ([Ca2+]i)  rise in hepatocytes  rise  expression in trout liver , and cortisol stimulation rapidly recruited ORAI1 to the membrane  wave in hepatocytes  Fig. a, supporsol Fig. a suggesttes Fig. b, and thise Fig. c, underpver Fig. d, and coane Fig. e. The grane Fig. f, and th2+]i) rise, we carried out in silico molecular docking studies using the Drosophila ORAI (4HKR.pdb)58. While modulation of ion channel activity by steroids have been studied61, no study has looked at the CRAC channel activation. Here, our modelling predicts a putative cortisol-binding site in the ORAI gating domain  rise in hepatocytes  showed that the interacting amino-acid residues of ORAI1 are conserved across species . These results underscore a potential direct modulation of ORAI1 by cortisol, and complements the evidence provided by the pharmacological manipulation . While studies have shown that the PLC-IP3R pathway is a major player in increasing ER store depletion and CRAC channel gating17, our results suggest that PKA-RYR pathway may be more important in modulating CRAC channel opening in trout hepatocytes under short-term stress. However, the membrane protein(s) involved in transducing the GC signal is currently unknown. (2) A direct effect of cortisol by binding to the ORAI1 leading to CRAC channel gating causing the ([Ca2+]i) wave in trout hepatocytes. Based on our molecular simulation, and pharmacological approach, we propose that CRAC channels may function as a cortisol-gated Ca2+ channel during stress in hepatocytes. To confirm this, future studies should focus on assessing the binding characteristics of cortisol on ORAI1 proteins.Our results point to a rapid effect of cortisol on CRAC channel gating as a mechanism for the nongenomic cortisol action on hepatocyte stress responseing Fig. . (1) An 18. So the results may have far ranging implications, especially considering that GCs are widely used as therapeutics for inflammation and other immune-related disorders involving Ca2+ signalling65. Chronic usage of this steroid leads to multiple side effects because of its predominant genomic actions, including osteoporosis, fluid and mineral imbalance and Type 2 diabetes65. Our in silico determination of a binding site for cortisol regulating pore opening on CRAC channel has potential therapeutic implication in the development of drugs that specifically target this binding pocket, thereby offsetting any unwanted side effects associated with GR activation due to chronic corticosteroid use6. Also, the novel role of CRAC channel as a possible GC-gated calcium channel has relevance as a potential therapeutic target for the treatment of stress-related disorders, including ER stress, major depressive disorders and immune dysfunctions66, all of which are associated with abnormal Ca2+ dynamics, and warrants further investigations.The CRAC channels are not just limited to hepatocytes, but also are prevalent in other non-excitable cells, including immune cellsSupplementary Information 1.


An analysis of the slaughterhouse post-mortem examination records over a decade showed that for animals transported to slaughter in containers, the risk of traumatic injury was highest in laying hens (2.80%) and rabbits (1.52%), while the overall incidence of trauma was below 0.5% in other species. The results show that the current rearing conditions and/or pre-slaughter handling of poultry and rabbits have comparatively negative welfare consequences, with significantly more traumatic injuries to the limbs than on the trunk in all species studied. In poultry, traumatic findings on the trunk were orders of magnitude lower to negligible, so the focus should be on preventing injuries to the limbs. In rabbits, the difference was less pronounced with a high number of injuries found on both limbs and trunk.p < 0.01) more frequent on the limbs than on the trunk in all species studied. In poultry, traumatic findings on the trunk were orders of magnitude lower to negligible, so the focus should be on preventing injuries to the limbs. In rabbits, the difference was less pronounced, and many injuries were found on both limbs (0.83%) and trunk (0.69%). Our results emphasize the need to reconsider both housing and pre-slaughter handling methods to determine minimum standards for the protection of rabbits, which are still lacking in European legislation.The findings of traumatic injuries during post-mortem inspection in slaughterhouses reflect the level of pre-slaughter handling of animals at the farm and during transport to the slaughterhouse. The prevalence of traumatic injuries was monitored in poultry  and rabbits  originating from farms in the Czech Republic and slaughtered in slaughterhouses in the Czech Republic between 2010 and 2019. The greatest incidence of traumatic injuries was found in laying hens (2.80%) and rabbits (1.52%); while the overall incidence of trauma was less than 0.5% in other species and categories. The results show that the current rearing conditions and/or pre-slaughter handling of poultry and rabbits particularly affect the limbs; traumatic findings were significantly ( Post-mortem injuries on the carcasses of poultry and rabbits detected during inspection at the slaughterhouse reflect the quality of pre-slaughter handling of the animals at the farm and during transport to the slaughterhouse. The advantage of using inspection of slaughtered animals for welfare assessment is the ability to assess injuries individually, on each slaughtered animal, even after removal of feathers or skin, making any lesions that may be commonly overlooked on the farm visible . TraumatA large proportion of traumatic injuries detected by inspection in slaughterhouses come from pre-slaughter handling. Meluzzi et al.  evaluateRabbits are also transported to slaughterhouses in containers. The high number of traumatic findings in rabbits reported by Valkova et al.  correspoIn order to list the aetiological factors that contribute to the findings of traumatic injuries in slaughterhouses, it is necessary to mention the factors that originate in the husbandry of the animals in question. Both very high stocking density and low stocking density may lead to weaker birds being victims of injurious attacks causing trauma such as bruising ,31. FurtThe aim of this study was to assess the welfare of poultry  and rabbits in relation to the prevalence of traumatic findings detected post-mortem at the slaughterhouse. The incidence of traumatic injuries was further evaluated in the categories and species of animals studied with respect to their localization (limbs and trunk).The welfare of poultry and rabbits slaughtered in slaughterhouses was assessed in terms of the prevalence of traumatic lesions in animals detected during veterinary post-mortem inspection in slaughterhouses in the Czech Republic during the ten-year period from 2010 to 2019.The Czech Republic is a landlocked country in Central Europe with an estimated population of 10,6 million, about three-fourths of the population being urban. The Czech Republic has a hilly landscape that covers an area of 78,871 square kilometers with a temperate climate. The contribution of agriculture to the GDP in the Czech Republic is about average for the EU. Plant production predominates over animal production in the Czech Republic. Milk production forms almost one half of the total animal output. The highest portion of animal farming is pig farming, followed by cattle, which are approximately on the same level as poultry. Egg production accounts for 5% of the total animal production. About 80% of egg-laying hens in commercial production in the Czech Republic are kept in enriched cages. Meat rabbits are almost exclusively housed in cages. As a member state of the EU, the Czech Republic is obliged to follow EU animal welfare legislation. However, some stricter measures have been maintained or established, namely a ban on force-feeding of ducks and geese, a maximum 8-hour journey limit for animals transported for the purpose of being slaughtered and a requirement of secondary education in the field of butchery for persons carrying out slaughter of animals in the slaughterhouse.The prevalence of traumatic injuries was monitored retrospectively in poultry and rabbits originating from farms in the Czech Republic and slaughtered in slaughterhouses in the Czech Republic. In the period under review, a total of 1,089,406,687 broilers, 20,030,744 laying hens, 1,181,598 turkeys, 37,690 geese, 28,579,765 ducks and 1,876,929 rabbits were slaughtered in slaughterhouses in the Czech Republic. Only animals that were slaughtered for human consumption and thus underwent slaughterhouse veterinary inspection were included in the analysis. Animals that died on farms or during transport were not included in the analysis.The assessment of traumatic injuries in animals was carried out by official veterinarians of the State Veterinary Administration, who recorded the number of traumatic findings. Inspections were carried out after slaughter and defeathering or depelting. The classification of findings was based on the methodology for postmortem examinations of animals at slaughterhouses. All animals slaughtered at slaughterhouses (their carcasses) are inspected by certified veterinary inspectors who received uniform training and certification of competence to perform this kind of veterinary slaughterhouse inspections. The legal requirements and specification of veterinary postmortem examination are laid down by European Union legislation . The resDuring postmortem examinations, veterinary inspectors differentiated between injury to the body (including keel bone fractures) and injury to the limbs (including wings). Wounds at various stages of healing in skin and underlying tissues, haematoma in the hypodermis and muscles, bruises, dislocations, fractures (open and closed) and other changes that may arise as a consequence of the housing system used, incorrect handling and interactions between animals on the farm, during transport or during lairage before slaughter were included among findings of trauma. However, veterinary inspectors did not aim to classify the cause of the antemortem injuries. They only distinguished injuries that occurred ante- and postmortem on the basis of their appearance . Postmortem injuries (technology-related damage following stunning) were not included among findings of trauma analyzed in our study.Gallus gallus), differences between adult (end-of-lay hens) and meat birds (broiler chickens) were also assessed. For other species, the difference between adult and fattened animals was not evaluated, because adult animals other than laying hens are not commonly slaughtered for human consumption in slaughterhouses in the Czech Republic.For the purposes of the study, the prevalence of traumatic injuries to the trunk and limbs in poultry  and in rabbits relative to the total number of birds and rabbits slaughtered was analyzed. Differences in the prevalence of traumatic injuries between limbs and trunk within each species, and differences in the prevalence of traumatic injuries in limbs and trunk between species were compared. In domestic chickens . Differences in frequency of traumatic injuries among various species/categories of animals and differences in frequency of traumatic injuries to the limbs and trunk in the monitored animal species and categories were tested on the basis of a chi-square test within the r × c and 2 × 2 contingency table procedures . In the p < 0.01) between adult (end-of-lay hens) and fattened birds (broiler chickens) in both the number of traumatic findings on the limbs and the number of traumatic findings on the trunk. In both cases, a significantly higher (p < 0.01) number of injuries were recorded for laying hens than broiler chickens. The results show that the highest number of traumatic injuries was found in laying hens (2.7957%) and rabbits (1.5184%), while the lowest number of traumatic injuries was found in ducks (0.0031%). In domestic chickens, there was also a difference  difference in the number of traumatic findings on the limbs between all species  difference between all species except laying hens and geese, and geese and broiler chickens  than on the trunk , whereas the heavier meat birds (geese and turkeys) sustained more damage. Substantially more trauma was recorded for end-of-lay hens and rabbits. Levels of injury are seldom reported in the literature but Gerpe et al.  recordedThe records analyzed in our study could not distinguish between injuries originating on the farm and those occurring during transport or at the slaughterhouse. However, evidence suggests that pre-slaughter handling of animals is the main factor influencing the occurrence and frequency of traumatic injuries ,4. AssumOur comparison of the position of traumatic injuries in poultry and rabbits post mortem shows that traumatic injuries to the limbs occurred significantly more often than traumatic injuries to the trunk in all species studied. This can be related to pre-slaughter handling, which damages the limbs more than the trunk of the animals, as the limbs are also the main part of the body by which poultry and rabbits are caught and carried when handled during catching, loading, unloading and hanging at the slaughterhouse ,5,26. ThWhen comparing traumatic injuries between broiler chickens and laying hens, we found a statistically significant difference both in the limbs  and in the trunk . Not only the period of intensive egg production but also the housing system has an impact on the quality and strength of the musculoskeletal system of poultry. Cage systems in which laying hens are still predominantly housed in the Czech Republic limit the movement of laying hens, and thus increase their susceptibility to the development of disuse osteoporosis . Aviary In laying hens, traumatic limb injuries were the most frequent (2.78%) compared to other species and categories studied. Rabbits ranked second in terms of frequency of limb trauma (0.83%) but also in terms of total number of traumas irrespective of location 1.52%). In addition, rabbits were found to have the highest incidence of trunk traumatic injuries, namely 0.69%, while the incidence of trunk trauma was an order of magnitude lower in poultry, ranging from 0.00% (ducks) to 0.05% (turkeys). When being unloaded, rabbits are manually removed from cages, carried and placed in transport containers. Verga et al.  found th%. In addGeese had the third highest incidence of traumatic injuries, predominantly to their limbs (0.43%), while injuries to the trunk were rarely detected (0.01%). Similarly, for turkeys, limb injuries (0.21%) significantly outnumbered trunk injuries (0.05%). A possible explanation for this is the larger size of geese and turkeys relative to transport cages compared to the much smaller broiler chickens and ducks, which had the lowest incidence of injuries . Larger animals may experience more frequent traumatic injury to limbs performing escape behaviors during loading into and unloading from transport containers. When comparing different handling methods for loading turkeys into transport containers, the method of herding turkeys to the loading site and having them self-board using a ramp into a transport container placed on the transport vehicle was assessed to be the least stressful and cause the least injuries, compared to manually carrying and pushing them into transport containers .The limitations of the study include inability to quantify risk factors leading to injuries as the extent of data collected during routine post-mortem slaughterhouse inspections and archived on the national level is limited, i.e., more thorough analysis of the origin of traumatic injuries recorded and the origin of injured birds was not possible. However, since all birds and rabbits slaughtered in the Czech slaughterhouses over a ten-year period were included in the study, the results show overall trends concerning prevalence of traumatic injuries in slaughtered poultry and rabbits reared in intensive housing systems.An analysis of the slaughterhouse post-mortem examination data recorded over a decade showed that the risk of traumatic injury was highest in laying hens (2.80%) and rabbits (1.52%), while the overall incidence of trauma was below 0.5% in other species. The results show that the current rearing conditions and/or pre-slaughter handling of poultry and rabbits have negative welfare consequences, with significantly more traumatic injuries to the limbs than on the trunk in all species studied. In poultry, traumatic findings on the trunk were orders of magnitude lower to negligible, so the focus should be on preventing injuries to the limbs. In rabbits, the difference was less pronounced with a high number of injuries found on both limbs and trunk. Our results thus confirm the need to re-assess the farming methods and pre-slaughter handling used and ideally to legislate minimum standards ensuring the protection of rabbits, which are still lacking in European legislation . More detailed post-mortem inspection carried out routinely in the slaughterhouses would be beneficial not only to identify specific risk factors in general but also to compare individual farmers and transporters, i.e., to exercise closer scrutiny when the number of injured animals exceeds some level.