PAMs) of AGABA receptors that cause sedation, hypnosis, and anxiolysis. Benzodiazepines bind AGABA receptors at the extracellular interface of the α and γ subunits. Within the α subunit, the benzodiazepine binding site is defined by three highly conserved structural loops, loops A‐C. Although previous mutagenesis studies have identified His102 in Loop A as important for benzodiazepine modulation of AGABA receptors, the functional roles of many of the other conserved residues in loops A‐C remain incompletely understood. In this study, we made single mutations in loops A‐C of the benzodiazepine binding‐site across all six α subunits. We used whole‐cell patch clamp recording to measure the functional effects of these mutations on midazolam potentiation. The results showed that mutating the threonine in loop B and serine in loop C (Thr163 and S206 in human α1) did not abolish the receptors’ responsiveness to midazolam, as the α1(H102R) mutation did. The loop C mutations exhibited a novel array of α‐isoform specific effects on midazolam potentiation. The α3(S230I) and α5(S209I) mutations had the largest effect on midazolam potentiation, increasing the efficacy of midazolam. Novel benzodiazepines targeting loop C may represent a future direction for designing new drugs that specifically alter the activity of α3‐ and α5‐containing AGABA receptors.Many benzodiazepines are positive allosteric modulators ( The α4 and α6 subunits have different residues  in these locations  GABAA subunits  were subcloned into pcDNA3.1+ vectors with a cytomegalovirus (CMV) promoter. The hβ2 and hα3 sequences were humanized rat (Rattus norvegicus) cDNA with amino acid substitutions made to match the human protein sequence. The α1‐3, α5, β2, and γ2s subunits were a generous gift from Neil L. Harrison . The α4 subunit was obtained from GenScript , and the α6 subunit was a generous gift from Robert L. McDonald . All point mutations , catalogue number, CRL3216. HEK293T cells were maintained at 37°C and 5% CO2 in Eagle Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum , 40 μM l‐glutamine, 100 U/mL penicillin and 0.1 mM streptomycin. Cells were passaged regularly when they reached 70% confluency using trypsin. Cells were not passaged more than 22 times. New cells were revived from frozen stocks at passage 2‐4. Cells used for in vitro electrophysiology experiments were grown on poly‐D‐lysine‐coated glass coverslips  and transfected with X‐tremeGENE  with the desired receptor subunit cDNAs at a 1:1:1 ratio to express αxβ2γ2s receptors  and with 0.5 μg green fluorescent protein (GFP) as an expression marker. The γ2s incorporation into receptors was tested with zinc inhibition assays regularly.Human embryonic kidney cells containing the SV40 T‐antigen (HEK293T) were acquired from American Type Culture Collection . Extracellular solution contained 161 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, and 6 mM D‐glucose, adjusted to pH 7.4 with NaOH (320‐330 mOsm). GABA and midazolam  were delivered using a rapid solution changer  connected to a 10‐channel infusion pump . The perfusion system was controlled by protocols written in pClamp 9 . Whole‐cell currents were recorded at ‐60 mV, filtered at 100 Hz and sampled at 200 Hz with a MultiClamp 700B amplifier  and DigiData 1322A  digitizer. GABA concentration‐response assays were performed by exposing each whole‐cell patch to eight concentrations of GABA spread over a 3.5 logarithmic decade. Each GABA exposure was for 2 seconds with 8 seconds of washout between ligand application. GABA concentrations for αxβ2γ2 receptors were: 0.3‐1000 μM (α1), 0.1‐300 μM (α2 & α3), 0.03‐100 μM (α4), and 0.01‐30 μM (α5 & α6). Midazolam concentration‐response assays were performed by exposing patches to two successive EC10 (10% effective concentration) GABA exposures and then exposing the patches to ascending concentrations of coapplied midazolam  and GABA (EC10)  and then 3 seconds of a saturating GABA concentration (100‐300 μM depending on the α subunit) with 8 seconds of washout between ligand applications. Cells were recorded with the midazolam protocol no more than two times to avoid desensitization and incomplete washout or irreversible modulation.Wildtype and mutant GABA2.4GABA concentration‐response relationship: Whole‐cell peak currents (I) were measured from GABA concentration‐response assays and fit using a nonlinear regression analysis based on the Hill equation: I = Imax*[A]nH/(EC50nH + [A]nH) where I was the peak current amplitude, Imax was maximum current amplitude, EC50 was the half‐maximal GABA concentration, A was the agonist concentration, and nH was the Hill coefficient. The maximum peak current, EC50 and Hill coefficient were estimated for assays from each cell. When Hill parameters are estimated from whole‐cell recordings, the changes in the parameters across receptor conditions can be ascribed to the following changes in receptor physiology. Changes in maximum current can be due to changes in the single‐channel conductance or the rate of desensitization. Changes in cell surface receptor expression can cause minor changes in maximum current, but are unlikely to occur in the time course of our experimental protocol. Changes in the Hill coefficient can be due to changes in altered GABA cooperativity, the loss of a GABA binding site or altered channel desensitization. More often minor changes in the Hill coefficient are attributed to the altered homogeneity in the receptor population expressed by the HEK293T cell. For example, a shallower Hill coefficient could be caused by a shift in the population of receptors from mostly αβγ receptors to a combination of both αβ and αβγ receptors. Changes in GABA apparent‐affinity can be due changes in GABA's binding affinity, gating or both for the receptor. Other explanations than the above are possible but less likely. Midazolam concentration‐response curves: The midazolam potentiation (%) of each GABA‐evoked response was calculated by the equation: Pot = (IMDZ – IG)/IG x 100%, where Pot was potentiation (%), IG was the average amplitude of peak currents from the two EC10 GABA responses, and IMDZ was the amplitude of peak currents from co‐applied GABA + midazolam. The potentiation measurements from midazolam concentration‐response curves were fit using the Hill equation: P = P max*[M]nH/(EC50nH + [M]nH), where P was potentiation, Pmax was maximum potentiation, EC50 was the midazolam concentration producing the half‐maximal potentiation response, M was midazolam concentration, and nH was the Hill coefficient. Concentration‐response relationships that were not described by a sigmoidal function were not included in our analysis (eg: no response or a linear nonsaturating response). The Hill equation was fit to each individual cell's concentration‐response curve data.Recordings were analyzed using MATLAB . 2.5α = 0.05 and β = 0.8) for a one‐way analysis of variance (ANOVA) test. Hill parameters  from concentration‐response curves (GABA and midazolam each) were compared for significant differences within each α subunit (α1‐6) and its loops A‐C mutants using a one‐way ANOVA at the significance threshold of α = 0.05. Where the results of the ANOVA were significant (P < 0.05), Dunnett's post‐hoc analysis for multiple comparisons (α = 0.05) was performed. Statistical analysis was carried out using Prism 7.0 .Optimal sample sizes (n ≥ 10 cells) were calculated beforehand from preliminary α1 mutant data using G*Power   in the α1, α2, α3, and α5 subunits. For the α4 and α6 subunits, the reverse arginine‐to‐histidine mutation was made. GABA concentration‐response assays revealed only modest changes in loop A mutant receptors  mutant receptors (α5(H105R)β2γ2 = 9.84 ± 3.29 μM (n = 10); α5β2γ2 = 3.18 ± 0.71 μM (n = 10), P = 0.0093) and twofold increase in the GABA EC50 for the α2(H101R) mutant receptors (α2(H101R)β2γ2 = 16.25 ± 2.20 μM (n = 11); α2β2γ2 = 8.29 ± 0.78 μM (n = 40), P = 0.0003). Midazolam assays showed that the α1(H102R), α2(H102R), α3(H126R), α5(H105R) mutations abolished the ability of receptors to respond to midazolam potentiation, and Hill fits could not be performed on this data (Table 50: α4(R100H)β2γ2 = 73.99 ± 3.44 nM (n = 8) and α6(R100H)β2γ2 = 41.88 ± 6.02 nM (n = 7), Figure 4β2γ2 and α6β2γ2 receptors showed no notable midazolam potentiation, and no meaningful Hill parameters could be estimated  in α1‐3, which we predicted would reduce the receptor's responsiveness to midazolam. The opposite mutation (proline‐to‐threonine) was made in the α4‐6 subunits. No significant (P < 0.05) shifts in GABA apparent‐affinity were seen for any α1‐6 loop B mutations  changes in GABA activation were modest changes in the amplitude of the maximum whole‐cell current evoked by GABA for α1(T163P), α2(T162P), and α4(P161T) mutated receptors. The GABA EC50 values remained unaltered for these mutants , α2(T162P), and α3(T187P) mutants remained unchanged relative to the wildtype receptors β2γ2 receptors had a significantly lower maximum potentiation compared to wildtype α1β2γ2 receptors (α1(T163P)β2γ2: 133.8 ± 19.51%, n = 11; α1β2γ2: 203.0 ± 17.6%, n = 7, P = 0.0092). The α5(P166T) mutation produced little change in midazolam potentiation, either maximum potentiation or midazolam EC50 . The presence of a threonine residue failed to confer midazolam responsiveness to α4(P161T)β2γ2 and α6(P161T)β2γ2 receptors  had little effect on GABA apparent‐affinity but more noticeable effects on the magnitude of the midazolam potentiation of αxβ2γ2 GABAA receptors. Five of the six loop C mutations failed to significantly (P > 0.05) alter the receptor's apparent‐affinity for GABA, Table 50: α6(N204I)β2γ2 = 0.421 ± 0.061 μM (n = 14); α6β2γ2 = 0.703 ± 0.078 μM (n = 11), P = 0.0001) β2γ2 = 135.8 ± 23.8% (n = 6); α1β2γ2 = 203.0 ± 17.6% (n = 7), P = 0.0403). The α2(S205I) mutation reduced the maximum midazolam potentiation by approximately 31% (α2(S205I)β2γ2 = 116.4 ± 23.0%, n = 8) compared to wildtype receptors , but this result was not significant (P = 0.416). The α3(S230I) mutation had the largest alteration in midazolam potentiation β2γ2 = 436.0 ± 39.4% (n = 7); α3β2γ2 = 267.8 ± 20.3% (n = 7), P = 0.0004), and it increased the midazolam EC50 by approximately 63% (α3(S230I)β2γ2 = 73.6 ± 1.8 nM; α3β2γ2 = 46.4 ± 7.4 nM, P = 0.0014). Similarly, the α5(S209I) mutation increased the maximum degree of midazolam potentiation by approximately 63%, although this difference was not statistically significant (α5(S209I)β2γ2 = 175.1 ± 26.6% (n = 6); α5β2γ2 = 107.9 ± 20.3% (n = 7), P = 0.1067). The α4(I204S) and α6(N204I) mutations failed to convey any notable midazolam potentiation to the receptors and no meaningful Hill parameters for midazolam concentration‐response curves could be estimated (Table 1(S206I)β2γ2 and α2(S205I)β2γ2 receptors had a decreased maximal midazolam potentiation and the α3(S230I)β2γ2 and α5(S209I)β2γ2 receptors had an increased maximal potentiation.The loop C mutations  and serine (loop C) residues failed to dramatically abolish the ability of α4A receptors by altering the GABA binding steps,A receptors. Understanding how different parts of the benzodiazepine site interact with modulators will help us better define the precise molecular mechanisms of these drugs.Midazolam is a benzodiazepine used to induce sedation and anesthesia.A receptor by midazolam. The histidine‐to‐arginine loop A mutation provided an example of how a single residue mutation can dramatically alter the efficacy of midazolam potentiation. The loop B threonine and loop C serine are highly conserved across α subunits, except in α4 and α6 subunits which are generally insensitive to classic benzodiazepines.xβ2γ2 GABAA receptors by midazolam. Overall, the mutation of the conserved threonine‐to‐proline in loop B had subtle effects on midazolam potentiation. The serine‐to‐isoleucine mutation in loop C altered the efficacy of midazolam potentiation, especially for α3‐ and α5‐containing receptors.In this study, we examined the role of the histidine in loop A, threonine in loop B, and serine in loop C within the α subunit and how these residues affected the allosteric potentiation of the GABAAcross the 18 mutations made in loops A‐C within the benzodiazepine site, only subtle changes were seen in GABA apparent‐affinity. Since the mutation was away from the GABA binding site, it is unlikely the mutations caused a structural rearrangement of the extracellular domain that affected the channel's activation. The α6(N204I) mutant increased the GABA's apparent‐affinity, but this was not sufficient to make the receptor any more responsive to midazolam than the wildtype α6‐containing receptors. On the whole, our results were consistent with mutations that had minimal effects on GABA's normal actions at the mutated receptor.HNG) of the α subunit is important in determining the molecular xβ2γ2 receptors. These results provided an example of how a single residue mutation could dramatically alter the efficacy of midazolam potentiation across receptors containing α1‐6 isoforms.It is well‐established that the conserved histidine present in loop A  and serine  mutations had more subtle effects on midazolam potentiation than the α1(H102R) mutation. Our loop B results showed that only the α1(T163P) mutation decreased the maximum amplitude of midazolam potentiation as predicted. Of the other loop B mutations, α3(T187P) only slightly decreased the maximum potentiation, while α5(P166T) slightly increased it. Our results were consistent with the threonine in loop B conferring slightly higher midazolam efficacy to the receptor than the proline. This is consistent with a proline‐to‐threonine mutation in α5 and α6 that moderately increased zolpidemIn this study, the threonine (GSYAYP < 0.05) altered midazolam's EC50. In the case of an allosteric modulator, an altered EC50 might be caused by changes in the modulator's ability to bind and interact with the receptor or the modulator's ability to alter GABA's binding and gating of the channel.The loop C mutation had more obvious changes in the efficacy of midazolam potentiation. The wildtype α1, α2, α3, and α5 subunits all contain the homologous Ser206 (human α1) that we predicted would reduce midazolam potentiation when mutated to an isoleucine. Surprisingly, the results did not follow the predicted pattern. In the α1(S206I) and α2(S205I) mutants, the isoleucine decreased midazolam's maximum potentiation by 31‐33%, but in α3(S230I) and α5(S209I), it increased midazolam's potentiation by approximately 63%. Only α3(S230I) significantly ,A receptors,xβ2γ2 receptors provide a reasonable estimate of benzodiazepine efficacy in the brain. Based on our results, loop C might be a potential target for developing novel drugs that specifically modulate α3‐ and α5‐containing GABAA receptors using PAMs targeting the allosteric benzodiazepine site.In our results, the greatest increase in midazolam's efficacy was seen with the α3S230I) loop C mutation. The wildtype α3‐containing receptors were the most sensitive to modulation by midazolam with the lowest midazolam EC30I loop Participated in research design: O.A. Moody, A. Jenkins. Conducted experiments and data analysis: O.A. Moody. Wrote or contributed to the writing of the manuscript: O.A. Moody, A. Jenkins.None declared. Click here for additional data file.


The intricate neuronal architecture of the striatum plays a pivotal role in the functioning of the basal ganglia circuits involved in the control of various aspects of motor, cognitive, and emotional functions. Unlike the cerebral cortex, which has a laminar structure, the striatum is primarily composed of two functional subdivisions  arranged in a mosaic fashion. This review addresses whether striatal compartmentalization is present in non-mammalian vertebrates, in which simple cognitive and behavioral functions are executed by primitive sensori-motor systems. Studies show that neuronal subpopulations that share neurochemical and connective properties with striosomal and matrix neurons are present in the striata of not only anamniotes (fishes and amphibians), but also amniotes (reptiles and birds). However, these neurons do not form clearly segregated compartments in these vertebrates, suggesting that such compartmentalization is unique to mammals. In the ontogeny of the mammalian forebrain, the later-born matrix neurons disperse the early-born striosome neurons into clusters to form the compartments in tandem with the development of striatal afferents from the cortex. We propose that striatal compartmentalization in mammals emerged in parallel with the evolution of the cortex and possibly enhanced complex processing of sensory information and behavioral flexibility phylogenetically. The striatum is a key component of the basal ganglia circuit, which is involved in normal motor, cognitive, and emotional functions. Dysregulation of striatal function contributes to a variety of neurological and neurobehavioral disorders . AccuratLittle is known about striatal compartmentalization in non-mammalian vertebrates with primitive motor functions and behaviors. In this review, we focus on whether the mosaic organization of the striatum is unique to animals with well-developed cerebral cortices. We postulate that clear compartmentalization in the striatum emerges parallel to the phylogenetic development of the cerebral cortex. Do neurochemically-identified striosome and matrix neurons exist in non-mammalian vertebrates or anamniotes? Neurochemical markers to distinguish striosome and matrix compartments were extensively reviewed by Crittenden and Graybiel . For exaAlthough the structure of the basal ganglia in early vertebrates has been extensively studied ,13,14,15In lamprey, a very ancient anamniote, subpopulations of calbindin-negative striatal neurons projecting to structures other than the globus pallidus internus (GPi)/globus pallidus externa (GPe) and CB-positive neurons projecting to the GPi/GPe  are presPrevious reports demonstrated the presence of a nucleus in the ventral telencephalon composed of neurons positive for SP  and GABAProtopterus annectens, (a living species that represents an extinct member that gave rise to an amphibian), were immunohistochemically examined to study fish—amphibian transition [The telencephalic structures of the lungfish, ansition ,19. A clansition , and NPYansition  were fouansition . In Afriansition . It is pXenopus laevis expressed the transcription factor gene Distal-less-4 of Xenopus  [Pax6, which plays an important role in neuronal differentiation during mammalian development, was shown to be expressed in the Xenopus striatum [Rana perezi showed that there were clearly demarcated areas labeled with these markers in the basal telencephalon [Rhinella arenarum tadpoles, showed that immunoreactivity for NPY, a matrix marker, was present in the developing striatum but decreased in number and staining intensity after metamorphosis into adult forms [The striatum constitutes a major part of the basal telencephalon in amphibians . The strse Dlx2)  in both se Dlx2) . A transstriatum , suggestcephalon . Any comlt forms . We founFernandez et al.  studied Caiman crocodilus showed that the ventrolateral telencephalon, a homolog of the striatum, was divided into two distinct “small-celled” and “large-celled” fields [Chrysemys scripta, SP-positive neurons were preferentially found in the small-cell field [Anolis carolinensis, the gene expression of serotonin (5-HT) receptors was not confined to a mosaic pattern although expression patterns of 5-HT receptor subdivisions in the telencephalon resembled those reported in mammals [A number of comparative anatomical studies have demonstrated the presence of homologies between reptilian and mammalian striata in their structural organization, connectivity, and neurochemical properties ,11,31. T” fields ,34. SP-p” fields  were fou” fields . These s mammals . While cThe avian striatum is divided into three components called the medial striatum, lateral striatum, and accumbens . These sMelopsittacus undulates, and the non-vocal learner bird, Colinus virginianus, in which heavily-labeled cells were scattered amidst the weakly-labeled cells [Columbia livia. They showed a gradient of staining intensity of NPY and ENK, both of which are matrix markers, in the rostro-caudal and ventro-dorsal axis of the striatum, and found the globus pallidus “interwoven” into the striatum with finger-like structures in the rostral sections. The low levels of calbindin and high levels of parvalbumin in the medial part of the lateral striatum in pigeons resembled those in the matrix of the mammalian caudo-putamen [The expression of matrix markers was reported in the avian striatum. CB, a calcium-binding protein preferentially expressed in the matrix compartment in mammalian striatum, was found in neurons of the striatum of male zebra finch . A diffeed cells . The caled cells  studied -putamen .Gallus domesticus [Taeniopygia guttata [The expression of striosome markers was also reported in the literature. The low calbindin and parvalbumin, and high SP levels in the medial part of the striatum raise the possibility that the area corresponded to the striosome compartment ,42. The mesticus . The medmesticus ,45. The  guttata . However guttata ,47,48,49The compartment structure of the striatum is considered to be evolutionarily conserved in mammals including humans ,50. NeurIn the mammalian striatum, two types of projection neurons can be identified based on different neurochemical and functional properties. Neurons expressing SP project to the GPi and SNc. This is called the direct pathway and is involved in promoting planned movement. Neurons expressing ENK project to the GPe. This is a part of the indirect pathway, which finally projects to the GPi via a projection to the subthalamic nucleus. Striatal projection neurons are derived from the lateral ganglionic eminence (LGE) ,66,67. TA traditional view is that the striatum can be subdivided into three functionally distinct parts called the sensori-motor, associative, and limbic domains , approxiJohnston et al.  performeThere have been reports suggesting that different thalamic nuclei send thalamo-striatal axons differentially to the striosome and matrix in the mammalian brain . The parThe neocortex is considered to first appear as a uniform, six-layered sheet consisting of radially deployed neurons in the early small mammals that evolved from their reptilian ancestors during the transition of the Triassic/Jurassic periods  for rev. In amphBrain size expanded dramatically during mammalian evolution ,95,96. WThere are known to be two different kinds of cortico-striatal projection neurons depending on their clearly distinctive patterns of targeting ,98. IntrAccumulating evidence shows that late-born matrix neurons receive their dominant input from the neocortex, whereas early-born striosomal neurons connect with components of limbic circuits such as the amygdala ,101,102 Recent reports show that there is considerable similarity between neuronal populations that form the striatum among vertebrates, including—to some extent—anamniotes with regard to neurotransmitter contents, physiology, and connectivity. However, the clear compartmentalization in the striatum exclusively develops in the mammalian brain, which has a six-layered cerebral cortex. During the evolution of mammals, the number of cortical neurons and corticofugal fibers increased extensively and the striatum subsequently received massive inputs from the cerebral cortex. By gathering information from the external world via sensory organs such as the eyes, nose and ears, the brain maps multiple streams of information onto a single axis of value ,106 and


Over the last decade, striking progress has been made in the field of organ transplantation, such as better surgical expertise and preservation techniques. Therefore, organ transplantation is nowadays considered a successful treatment in end-stage diseases of various organs, e.g. the kidney, liver, intestine, heart, and lungs. However, there are still barriers which prevent a lifelong survival of the donor graft in the recipient. Activation of the immune system is an important limiting factor in the transplantation process. As part of this pro-inflammatory environment, the complement system is triggered. Complement activation plays a key role in the transplantation process, as highlighted by the amount of studies in ischemia-reperfusion injury (IRI) and rejection. However, new insight have shown that complement is not only activated in the later stages of transplantation, but already commences in the donor. In deceased donors, complement activation is associated with deteriorated quality of deceased donor organs. Of importance, since most donor organs are derived from either brain-dead donors or deceased after circulatory death donors. The exact mechanisms and the role of the complement system in the pathophysiology of the deceased donor have been underexposed. This review provides an overview of the current knowledge on complement activation in the (multi-)organ donor. Targeting the complement system might be a promising therapeutic strategy to improve the quality of various donor organs. Therefore, we will discuss the complement therapeutics that already have been tested in the donor. Finally, we question whether complement therapeutics should be translated to the clinics and if all organs share the same potential complement targets, considering the physiological differences of each organ. Organ transplantation is the gold standard treatment for end-stage diseases in various organs, including the kidney, liver, intestine, heart, and lungs . The fieBrain death is the irreversible, total loss of brain function. Due to artificial ventilation and cardiovascular support, the circulation remains intact. Therefore, DBD donors do not have a nominable time of warm ischemia. The occurrence of brain death is an important risk factor for organ quality since brain death is associated with a cascade of hemodynamic, hormonal and immunologic events that become operational after brain injury, or/and brain death . IntracrCurrently, deceased donors represent the primary source for transplanted organs. However, the increasing demand for organ transplants mandates for expansion of the donor pool. Therefore, alternative strategies are deployed, such as an increase of the number of living donors, extension of the criteria for deceased donation and improvements in donor management and procurement . Still, 2O). Factor B is recruited and cleaved by factor D to form the AP C3 convertase (C3bBb). These convertases are intrinsically unstable, but their half-life is lengthened by interaction with factor properdin (P), a positive stabilizing regulator of the AP , lectin pathway (LP), and the alternative pathway (AP). The CP was the first pathway discovered and is activated by antigen-antibody complexes. These complexes are recognized by C1q, inducing a cascade via C2 and C4, leading to the production of CP C3 convertase (C4b2b). The same result is achieved when pattern recognition molecules of the LP  bind to their MBL-associated serine proteases (MASP) to cleave C2 and C4. The AP is spontaneously activated by hydrolysis of C3 to C3. As a serine protease inhibitor,  pathway –42. Pre- pathway . C1-INH  pathway .Besides C1-INH, more complement therapeutics are already tested in the deceased donor in experimental setting. Soluble complement receptor 1 (sCR1) was given to DBD rats and treatment with sCR1 before and after confirmation of brain death led in both cases to significantly improved renal allograft function. In addition, treatment with sCR1 led to reduced renal gene expression of IL-6, IL-1β, and TGF-β. These results provide proof that complement inhibition in the donor is effective, even after the confirmation of brain death .ex vivo to deceased donor kidneys. The trial, called EMPIRIKAL, is still ongoing and aims to evaluate the efficacy of Mirococept in reducing the incidence of DGF in renal transplants from deceased donors  are performed in Europe per year . In geneThe number of studies that investigated the role of the complement system in deceased donation for LiTx is scarce. Of interest, since the liver is responsible for the biosynthesis of 90% of plasma complement components and soluble complement regulators . Other tThe role of the complement system in the deceased liver is mainly investigated in the DBD donor. Rebolledo et al. were the first to show complement activation in DBD donor livers. In this study, rats were subjected to brain death for a period of 4 h. Results showed that DBD donor livers had a significant increase in C3 mRNA levels compared to the control group. Next, rats were pretreated with prednisolone 30 min before induction of brain death as a proof of principle. Pretreatment with prednisolone resulted in a reduced pro-inflammatory state, reflected by lower mRNA levels of IL-6, IL-1β, TNF-α, and MCP-1. In contrast, mRNA levels of C3 were upregulated in the prednisolone treated rats . In an aSo far, only one study investigated the effect of complement inhibitors on the donor liver before transplantation. Bergamaschini et al. studied the potential of C1-inhibitor (C1-INH) treatment during preservation. Porcine livers were removed from donors and perfused with University of Wisconsin (UW), with or without addition of C1-INH, and stored statically at 4°C for 8 h. To assess liver function, livers were subsequently reperfused for 2 h with pig blood on an extracorporeal circuit. Results demonstrated less complement activation, reflected by normal levels of complement haemolytic activity and absence of C3 activation production in both plasma and tissue at time of reperfusion. Morphological analysis of the livers showed significantly decreased inflammation as shown by only a mild increase in portal and lobular inflammatory infiltration, compared to necrotic lesions in the untreated group . Taken tImportant to take into account in LiTx is the process of liver regeneration. Liver regeneration is a process in the liver that allows mature hepatocytes to re-enter the cell cycle, proliferate, and eventually replace lost or damaged hepatocytes . Liver rIntestinal transplantation (ITx) is the least common form of organ transplantation. The field of ITx is small with only 6 ITx reported in 2017 by Eurotransplant . ITx is Hardly any studies are performed to unravel the pathophysiological processes in the intestine of a deceased donor, probably since the number of ITx performed per year is low. However, based on the few studies there are, it is likely that the deceased donor state causes significant alterations in the intestine and affects intestinal barrier function. The degree of permeability already changes in response to a low level of pro-inflammatory cytokines, which results in the translocation of intestinal bacteria. The intestine contains microbial LPS, which is thought to be one of the most potent activators of the AP of the complement system. Although studies showed that LPS from different bacterial strains interact in qualitatively different ways with complement, LPS from gram-negative bacteria indeed induce consumption of complement . A studyThe technique for heart transplantation (HTx) was already developed in 1967. Nevertheless, it took more than a decade before immunosuppressive treatment strategies improved this technique to such an extent, that HTx became the gold standard treatment for end-stage heart diseases . In 2017in situ normothermic regional perfusion for thoracic organs are explored in order to convert from a DCD to a DBD-type procurement  in an attempt to test and repair discarded donor lungs  configuration, had a higher risk for chronic rejection after LuTx. Under normal circumstances CD59 acts as a regulatory protein, which suppresses MAC formation by binding C9 to C5b-C8 complexes, which results into inhibited cell lysis. The presence of a CD59 SNP configuration affects CD59 expression and sensitivity to complement-mediated cell lysis, which increases the risk for rejection in recipients who receive a CD59 SNP donor lung . These rDespite the limited amount of research performed on the topic of DBD-related lung injury, it is suggested that the complement system is already involved from the first step of the process, namely the donor. Of particular interest, given the observation that lungs locally produce complement proteins. Pulmonary alveolar type II epithelial cells generate proteins of the CP and AP, in particular C2, C3, C4, C5, and factor B . BesidesThe complement system regained new interest in the field of transplantation and the amount of acquired knowledge is increasing. The complement system was first studied in RTx, revealing its potential role in reperfusion injury and survival. However, new studies elucidated that the complement system already plays a role from the first step of the transplantation process, in the donor . Other sOne of the questions that remains unanswered regarding complement-targeted interventions is the optimal timing of drug delivery to the donor graft. Different timing possibilities are (1) treatment in the organ donor, (2) during preservation, or (3) after implementation in the recipient. The main benefit of treating the organ donor is the opportunity to target the immunologically active state of the organs, nearly directly after the damage has occurred. However, it should be considered that all organs will be subjected to the same type and dose of treatment. An important disadvantage of this strategy is the risk that not all donor organs benefit from the same treatment. One organ might benefit from certain therapy, while the other organ might be even negatively affected. This is of particular importance for the liver, given the fine balance between injury and regeneration in this organ , 64.ex vivo perfusion systems. Various strategies in ex vivo perfusion are being practiced such as different perfusion solutions and perfusion temperatures (ex vivo perfusion strategies blood flow through the organ is mimicked, thereby reducing ischemia times. Besides, oxygen and other additives can be supplemented to the perfusate solution in order to preserve or improve the quality of the organ, which provides opportunities for complement therapeutics as well. In the lungs, even ex vivo inhalation of therapeutics can be considered as route of administration. An important benefit of treating the organ in an isolated manner is that a lower treatment dose might be required, especially in in organs with little metabolic activity. This might lower costs of complement-targeted therapies. However, little is known about the approach of treatment during preservation, especially with regards to the effect of treatment on donor-related injury. The few experimental studies that have been performed have mainly focused on preventing or diminishing IRI, by treating unharmed, “healthy” organs with complement inhibitors during the preservation phase (The preservation state, however, provides a window of opportunity to treat the organ in an isolated manner. Various approaches are herein possible, which depends on the method of preservation. Cold static storage is the preservation technique with the longest history and is mostly used . Therapeeratures . Neverthon phase , 56, 105on phase .Finally, treatment of the organ after implementation in the recipient should be considered as a possible timing of drug delivery. It should be emphasized that donor-related graft injury is amplified by the inevitable event of IRI . BeneficAnother unanswered question is the translatability of results from one organ, to other organ systems. Especially since most knowledge on the involvement of complement is gained from research in deceased donor kidneys, it is questioned whether those findings apply to other organs as well. In order to answer this, more studies are needed that focus on unraveling the mechanisms of complement activation, specifically per organ and donor type. Especially, given the physiological dissimilarities between organs and the differences in mechanisms of pathophysiology between DBD and DCD donors. The role of the complement system has been underexposed mostly in DCD donation. Yet this will be of growing importance, since DCD donors are increasingly deployed in an attempt to expand the donor pool.In conclusion, it has become evident that the complement system plays an important role in the donor, affecting all potential donor organs. To answer the question whether complement therapeutics should be clinically applied in the multi-organ donor, several uncertainties need to be elucidated first. These uncertainties include the timing, route of drug delivery and optimal target of complement therapeutics, complicated by dissimilarities in the pathophysiology of organs and differences between donor types. A tailor-made approach for each donor organ is pursued, aiming to improve the quality of donor grafts that possibly leads to an enlargement of the donor pool and improved outcomes after transplantation.JvZ and NJ contributed to the conception and design of the work, drafting the work, and agree to be accountable for all the aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. MD, ME, HL, and MS revised the work critically for important intellectual content and provide approval for publication of the content.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.


To determine the relationship between anterior chamber depth (ACD) and percent endothelial cell loss (ECL) after Descemet Stripping Automated Endothelial Keratoplasty (DSAEK).t test. Eyes were then separated into 2 groups based on ACD, group 1 with ACD < 3mm and group 2 with ACD ≥ 3mm. Students t test was then performed to compare group 1 and group 2 ECL at 6 months, 1, 2, 3, and 4 years postoperative.In 78 eyes receiving triple procedure (DSAEK combined with cataract extraction and posterior chamber intraocular lens (PCIOL) implantation), ACD was measured preoperatively with an intraocular lens (IOL) Master and ECL was calculated with specular microscopy at 6 months, 1, 2, 3, and 4 years postoperatively. ACD and ECL from all 78 eyes were compared using correlation analysis and students P > .05). At 2 and 4 years postoperative, mean ECL was 32.6% ± 16.1% and 43.0% ± 23.2% in eyes with ACD < 3mm and 25.3% ± 13.0% and 29.6% ± 18.2% in eyes with ACD ≥ 3 mm (P = .041 at 2 years and .008 at 4 years).Mean ACD for all 78 eyes was 2.93 ± 0.43 mm. Mean ECL was 32.7%, 27.6%, 29.6%, 32.5%, and 37.2% at 6 months, 1, 2, 3, and 4 years. No significant correlation between ACD and ECL was observed at any time point for the combined analysis of 78 eyes (ACD and ECL were not directly correlated; however, there may be a threshold ACD in which shallower anterior chambers preoperatively result in greater donor ECL over time. Although Descemet Membrane Endothelial Keratoplasty (DMEK) has been rapidly evolving, Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) is still the most commonly performed technique for EK. donor age, donor tissue storage time, and donor tissue preoperative endothelial cell count. No significant correlations were found between overall graft survival and any of these factors.Many previous articles have studied the factors that may affect the graft survival in DSAEK, including donor tissue size,,7There are currently many DSAEK techniques for donor tissue insertion and manipulation. Insertion of the graft using various forceps remains a common method of tissue insertion, and a shallow anterior chamber can make this maneuver difficult. In addition, once the DSAEK graft is delivered to the anterior chamber, a shallow chamber can make complete unfolding and positioning more difficult. The greater the manipulation of donor tissue, the greater the risk of endothelial damage.In this report, we studied the relationship between anterior chamber depth (ACD) and donor Endothelial Cell Loss (ECL) for the short and long-term postoperative periods in a group of DSAEK patients who underwent triple procedures. To our knowledge, this would be the first article that studies this relationship.22.1This is part of a retrospective series of the EK patient registry at Devers Eye Institute, and a continuation of our prospective study of EK. A Legacy Health Institutional Review Board approved and Health Insurance Portability and Accountability Act compliant clinical protocol and surgical consent form was developed and enrollment was initiated for patients with endothelial dysfunction.2, any age between 4 and 75 years old, and death to transplantation time of up to 12 days were accepted.No specific requests were made of the eye bank to provide tissue for EK with any different characteristics than what is normally requested for full thickness PK tissues. Donor tissues with an endothelial cell density (ECD) more than 2000 cells/mmPatients receiving a triple procedure for Fuchs’ endothelial dystrophy and cataract were considered eligible for the study.Among the 78 triple procedure cases performed between October 20, 2005 and February 27, 2008, 65 cases in 45 patients had complete datasets available for analysis.Eyes with a history of age-related macular degeneration, cystoid macular edema, controlled glaucoma, and other comorbidities were not excluded as visual acuity was not an outcome of the study.Cases with glaucoma drainage devices, retained anterior chamber intraocular lens (IOLs), graft dislocation or other possible causes of endothelial damage were identified and excluded from analysis.The ACD for all patients were measured preoperatively with the IOL Master instrument.2.2The vast majority of donor tissue came from Lions VisionGift in Portland, Oregon. Specular images were obtained with an EB-3000 XYZ Eyebank specular microscope . The preoperative cell counts were obtained using an apices digitized method and the manufacturer's calibrations for magnification. The apices of at least 100 cells from the endothelial images of each cornea were counted. Preoperative donor tissue specular microscopy was performed by 3 trained eye bank technicians, all of whom had at least 1 year of experience with this technology.Postoperative specular microscopy measurements of ECD were acquired at Devers Eye Institute using the Konan SP4000 noncontact specular microscope  at 6 months, 1 year, then annually after surgery. A certified ophthalmic technician (COT) performed all postoperative testing of patients using the same specular microscope each time.These postoperative cell counts were obtained using the manufacturer's calibrations for magnification and were counted with a fixed-frame method with the protocol requiring the marking of at least 50 to 100 cells for each image. Specular microscopy measurements with insufficient quality of the image were not included in the analysis. Insufficient quality of the image was determined subjectively by the examining physician based on the edge clarity of the individual cells. Approximately 8% of specular images are rejected in the authors’ clinical program, and approximately 10% of the time central specular images cannot be obtained despite a crystal-clear graft. Clarity of the graft was not an issue with any rejected specular image. Analysis of endothelial pleomorphism and polymegathism was not performed in this study.2.3In all cases of our triple procedures, the phacoemulsification and intraocular lens implantation were done before DSAEK through the same 5 mm temporal scleral tunnel, but with only a 2.8 mm keratome opening into the AC. This opening was subsequently enlarged to the full 5 mm for the insertion of the donor tissue.,9 In all cases, the tissue was precut by an eye bank technician and then provided to the surgeon, usually within 26 hours of precutting.,8,10 A video of the DSAEK technique using precut donor tissue is available online (https://www.youtube.com/watch?v=mtu8dxZUCx0) from a previous report on the use of precut tissue in DSAEK.DSAEK was performed with a standard technique as previously published.In brief, the standard DSAEK procedure involves placement of 2 limbal 1-mm wide paracentesis incisions on either side of a 5-mm temporal scleral access wound. This wound is placed approximately 0.5 mm peripheral to the temporal limbus with a scleral– corneal tunnel of approximately 2 mm until entry into the anterior chamber. After filling the anterior chamber with cohesive viscoelastic, a circular template mark is placed on the surface of the cornea to delineate the area of stripping, and this determines the same size for the donor tissue. The donor diameter chosen is individualized for each patient and is based on the largest diameter circle that can be fit on the individual cornea without overlap of the edge of the tissue over the 2 paracentesis sites or the corneal portion of the main insertion wound. After stripping of the recipient Descemet's membrane and scraping of the recipient bed with a Terry Scraper , the Healon  viscoelastic is removed from the eye with standard automated irrigation and aspiration technique. The pupil is constricted with Miochol-E , the microscope is moved to the donor table, and the donor tissue is trephinated with the same size diameter as the recipient template mark. After preparation, the donor tissue is grasped and then inserted with non-coapting Charlie II insertion forceps  in a taco configuration with the 60% edge placed anteriorly in the chamber. The tissue is unfolded with deepening of the anterior chamber with balanced salt solution and injection of air to complete unfolding of the tissue into position. After surface sweeping with the Cindy Sweeper  to remove interface fluid while the anterior chamber is filled with air, the procedure is completed with complete removal of the air from the chamber and then reinjection of a specified amount of air to yield only a 5 or 6 mm residual bubble for minimal graft support. This small air bubble is preferred to avoid the event of pupillary block that can occur with larger air-bubble retention at the conclusion of surgery.Surgery was performed by 5 corneal surgeons at Devers Eye Institute, with the senior surgeon (MAT) performing 63% of the cases. All surgeons used the exact same surgical technique for every case.2.4t test and Chi Square test as appropriate. All statistics were performed using SPSS v. 23 .Postoperative ECD and percentage ECL was recorded at 6 months, 1 year, 2 years, 3 years and 4 years. The preoperative ACD and the postoperative ECD at each postoperative time point were first compared using Pearson correlation analysis to see if there was any direct relationship between the preoperative ACD and ECL at different time intervals. Then the cases were divided into 2 groups based upon the ACD. Group 1 had a preoperative ACD < 3mm (45 eyes) and group 2 had a preoperative ACD ≥ 3 mm (33 eyes). Baseline and intraoperative characteristics were compared between groups using independent samples 3The 78 eyes in this retrospective study were from 49 patients with a mean age at surgery of 63.5 ± 8.7 years (range 42–83 years). 54 eyes (69.2%) were from females. All required DSAEK surgery due to Fuchs’ endothelial dystrophy.P = .848), tissue preservation time , preoperative ECD , or donor diabetes .Baseline characteristics that were evaluated and compared include mean donor age, tissue preservation time, preoperative ECD, and number of tissues that came from donors with a history of diabetes. Group 1 and group 2 were not statistically different for mean donor age   was 2.93 ± 0.43 mm range 2.10–4.02 mm). The mean percent ECL from baseline for the combined groups was 32.7% ± 13.0% at 6 months, 27.6% ± 14.3% at 1 year, 29.6% ± 15.2% at 2 years, 32.5% ± 18.8% at 3 years, and 37.2% ± 22.1% at 4 years. The ACD for all eyes did not correlate significantly with ECL at any postoperative time point . The difference between the 2 groups at 2 and 4 years was statistically significant (P = .041 and .008). Figure 2Mean ACD was 2.65 ± 0.25 mm for Group 1 and 3.32 ± 0.29 mm for group 2. Mean percentages of ECL for group 1 were 34.5% ± 13.1% at 6 months, 27.4% ± 14.4% at 1 year, 32.6% ± 16.1% at 2 years, 35.4% ± 20.6% at 3 years and 43.0% ± 23.2% at 4 years. For group 2, the percentages were 30.0% ± 12.5%, 27.9% ± 14.4%, 25.3% ± 13.0%, 28.5% ± 15.6%, 29.6% ± 18.2% at 6 months, 1 year, 2 years, 3 years and 4 years, respectively. Table 4One of the aims of corneal grafts, in general, is to provide the patient with the best possible visual acuity for the longest period of time. Early and late graft failures are reported complications of EK that usually require repeat surgery.Delivering the largest number of healthy donor endothelial cells into the recipient's eye is the main goal during the DSAEK surgery. This is usually accomplished by choosing donor tissues with an excellent endothelial cell count and by the meticulous handling of the donor tissue during the surgery. No significant correlation has been found between the preoperative endothelial cell count and the graft survival, given that all donor tissues had ECD higher than 2000 cell/mm2.The relationship between preoperative donor endothelial cell count and the overall graft survival has been previously studied.,12 The immediate endothelial loss in a wet lab experiment performed at the eye bank was found to be about 18% with a significant central cell loss related to tissue grasping and folding. The earliest postoperative specular microscopy done in our center is at the 6 months postoperative time point. This count represents a combination of both the intraoperative ECL as well as the cell loss in the first 6 months postoperatively.The immediate ECL after DSAEK has also been studied.In this study, the ECL was similar between the 2 ACD comparison groups at the 6 month and 1 year postoperative visits. However, by 2 years postoperatively, the difference in ECL was statistically significant and this difference increased even more by year 4. It may be that the surgical trauma due to dealing with a shallow ACD at the time of surgery has a delayed and deleterious effect on the long-term survival of the donor endothelium.One of the limitations of this study is that in combined phacoemulsification, IOL placement, and DSAEK surgery, the measured preoperative ACD may differ from the postoperative ACD. However, with the exception of severe nuclear sclerosis eyes (which none of this series of cases contained), it was found that deeper ACD before cataract surgery is associated with deeper ACD after surgery, which keeps the relative difference in ACD between the 2 groups.As a part of the management in patients that will undergo triple DSAEK procedures, taking note of the preop ACD may be an important factor in predicting the long term ECL of the DSAEK graft.Conceptualization: Asem A. Alqudah.Data curation: Alex J. Bauer.Formal analysis: Alex J. Bauer.Investigation: Asem A. Alqudah, Alex J. Bauer.Methodology: Alex J. Bauer.Software: Asem A. Alqudah, Alex J. Bauer.Supervision: Michael Straiko, Mark A. Terry.Writing – original draft: Asem A. Alqudah.Writing – review & editing: Asem A. Alqudah, Alex J. Bauer, Mark A. Terry.


P<0.001). The poor outcomes distribution across the RBP4 quartiles ranged between 9.3% (first quartile) and 60.8% (fourth quartile). In multivariate models comparing the second(Q2), third, and fourth quartiles against the first quartile of the RBP4, RBP4 in Q3 and Q4 were associated with poor functional outcome, and increased risk of poor functional outcome by 144% : 1.22–5.03) and 602% , respectively. Interestingly, RBP4 improved the NIHSS score (area under the curve (AUC) of the combined model, 0.79; 95% CI: 0.74–0.85; P<0.001). The data showed that elevated serum levels of RBP4 at admission were associated with severity and prognosis of AIS, suggesting that vitamin A metabolism or impaired insulin signaling could be involved.The aim was to investigate the relationship between retinol-binding protein 4 (RBP4) levels and short-term functional outcome, and to determine its possible role in acute ischemic stroke (AIS). In a prospective observational study, 299 first-ever AIS who were admitted to our hospital were included. Serum levels of RBP4 were assayed and severity of stroke was evaluated with the National Institutes of Health Stroke Scale (NIHSS) score on admission. The prognostic value of RBP4 to predict the poor outcome within 3 months was compared with the NIHSS and with other known outcome predictors. The median age of the included patients was 66 (interquartile range (IQR): 55–77) years and 155 (51.8%) were women. A poor functional outcome was found in 88 patients (29.4%), and significantly higher RBP4 values were found in poor outcomes rather than good outcomes patients ( CirculaStroke is the second leading cause of death and the leading cause of serious, long-term disability worldwide . HyperteThis was a prospective cohort study at Department of Emergency in our hospital. From January 2016 to December 2017, all patients with first-ever AIS were included. All patients were admitted within 24 h of experiencing a new focal or global neurological event. A baseline MRI scan was performed in all patients at admission to verify the diagnosis. The patients with the following criteria were excluded: (i) malignant tumor, (ii) and a history of recent surgery or trauma during the preceding 2 months; (iii) liver and kidney function insufficiency; (iv) acute and chronic inflammation; metabolic abnormalities (diabetes not included), (v) febrile disorders and autoimmune diseases; (vi) other neurological diseases .In the present study, 150 age and gender-matched healthy volunteers were assigned to the normal group. The median age of control cases was 65 (interquartile range (IQR): 55–77) years and 52% were women. The study was approved by the Ethics Committee of First Affiliated Hospital of Xinxiang Medical University The patients or their relatives gave written informed consent prior to entering the study.At baseline, demographical and clinical data including: age, sex, body mass index (BMI), temperature, systolic blood pressure, conventional vascular risk factors  were obtained. In addition, the information about pre-stroke therapy  and acute treatment  were also recorded. All patients received treatment according to current guidelines. Stroke severity was assessed on admission using the National Institutes of Health Stroke Scale  by a neurologist . Stroke The functional outcome was determined in month 3 according to the modified Rankin Scale   blinded n=73), 6–12 (n=98), 12–24 (n=92), and 24–48 (n=36) h from symptom onset). After centrifugation, the serum samples were immediately stored at –80°C before assay. The information about time from onset to blood collection was recorded. Concentrations of serum RBP4 were batch analyzed using a commercially available Quantikine® ELISA kit for human RBP4 . The lower detection limit for RBP4 was preset as 0.60 ng/ml and the detection range was 1.6–100 ng/ml. Inter-assay and intra-assay coefficients of variation were 5.0–9.0% and 4.0–7.5%, respectively. Due to the high levels of RBP4 (μg/ml) in the blood sample, 1:100 dilution was used. Other blood biomarkers, for instance triglyceride, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), high-sensitivity C-reactive protein (Hs-CRP), fasting blood glucose (FBG) were tested using an enzyme cycling method by BS-2000M . All those tests were done in duplicates and samples with a CV exceeding 10% were reanalyzed.Fasting serum samples were drawn from the antecubital vein at the first morning after admission and within 48 h of symptom onset (within 0–6 (2 test (proportions) or Mann–Whitney U-test .Results are expressed as percentages for categorical variables and as medians (quartiles) for the continuous variables. Spearman’s rank correlation was used for bivariate correlations. The difference between groups was assessed by χThe influence of RBP4 on PSD was performed by univariate and multivariate binary logistic regression analysis. In the multivariate analysis, significant confounding factors which were tested in the univariate analysis were adjusted. Results were expressed as adjusted odds ratios (OR) with the corresponding 95% confidence interval (CI). For a more detailed exploration of the RBP4 and outcome, we also used multivariate analysis models to estimate adjusted OR and 95% CIs of PSD for RBP4 quartiles (with lowest quartile as reference).Further, a receiver operating characteristic (ROC) curve analysis was used to identify the cut-off point on the serum levels of RBP4 on admission with the greatest sensitivity and specificity to predict poor outcomes at the 3-month follow-up. Area under the curve (AUC) was calculated as measurements of the accuracy of the test. The influence of elevated levels of RBP4 (≥cut-off) on poor outcomes was also performed. Furthermore, to test whether the RBP4 level improves score performance, we considered the two nested logistic regression models with RBP4 and the established risk factors as compared with the established risk factors only.P<0.05.At last, we conducted analyses separately amongst cases who experienced with and without diabetes. All statistical analysis was performed with SPSS for Windows, version 22.0 , the ROCR package (version 1.0-2), and GraphPad Prism 5.0. Statistical significance was defined as In our study, 299 AIS patients completed 3-month follow-up and were included in the analysis. The median age was 66 (IQR: 55–77) years and 155 (51.8%) were women. In those patients, the median NIHSS score at admission was 9 points (IQR: 5–13). The median time from stroke onset to blood collected was 14.5 (IQR: 8.0–18.0) h. In addition, the number of tissue plasminogen activator-treated patients was 41 (13.7%). Basal characteristics of patients with AIS were provided in P<0.001). Serum levels of RBP4 increased with increasing severity of stroke as defined by the NIHSS score. There was a correlation between levels of RBP4 and NIHSS score (r(Spearman) = 0.437, P<0.0001). There was still a significant positive correction between RBP4 serum levels and NIHSS score, using ordered logistic regression after multivariate adjustment for possible confounders (P=0.015). Similarly, there was a positive correlation between levels of RBP4 and the infarct volume . In addition, serum levels of RBP4 were also correlated with Hs-CRP , FBG , BMI , and triglycerides . Furthermore, significantly higher RBP4 values were found in women rather than men patients (30.5 (IQR: 21.9–40.1) μg/ml compared with 24.5 (IQR:13.4–32.2) ug/ml; P<0.001). We also examined the relationship between serum RBP4 levels and stroke subtypes. The median RBP4 levels were significantly greater for atherosclerosis than for the other stroke subtype groups (32.8 (IQR: 22.8-42.7) μg/ml compared with 25.4 (IQR: 17.5–35.5) μg/ml, respectively; P<0.001).The serum levels of RBP4 were significantly higher in stroke patients as compared with normal cases (27.3 (IQR: 18.6–36.4) μg/ml compared with 17.6 (IQR: 11.8–23.5) μg/ml; P<0.001; P for trend <0.001.A poor functional outcome was found in 88 patients  with a median mRS score of 4 (IQR: 3–6). Significantly higher RBP4 values were found in poor outcomes rather than good outcomes’ patients (37.2 (IQR: 25.8–46.2) μg/ml compared with 24.8 (IQR: 15.7–31.8) μg/ml; P<0.001).In univariate logistic regression analysis, we calculated the OR of RBP4 levels as compared with the NIHSS score and other risk factors as presented in P<0.001; P<0.001), FBG , and age , while was in the range of NIHSS score . Interestingly, RBP4 improved the NIHSS score . This improvement was stable in an internal five-fold cross-validation that resulted in an average AUC (S.E.M.) of 0.73 (0.032) for the NIHSS and 0.79 (0.028) for the combined model, corresponding to a difference of 0.07 (0.004). In addition, a significant difference in the AUC between the established risk factors alone and the addition of RBP4 concentrations was observed (difference: 0.02 (95% CI: 0.01–0.03); P=0.033).Based on ROC curves, the optimal cut-off value of serum RBP4 level to diagnose the poor outcomes was 37.4 μg/ml, which yielded the highest sensitivity and specificity  and 258% , respectively . Interesn=133) rather than other patients (31.8 (IQR: 21.3–42.5) μg/ml compared with 25.9 (IQR: 16.8–34.9) μg/ml; P<0.001). In multivariate logistic regression analysis, we calculated the prognostic value of RBP4 levels in diabetic stroke patients and diabetes-free stroke patients. The data suggested that for each 1 μg/ml increase in serum level of RBP4, the association was stronger amongst diabetic patients  compared with non-diabetic patients .We conducted analyses separately amongst cases who experienced with and without diabetes. Significantly higher RBP4 values were found in diabetic stroke patients (P<0.0001). After adjusting for all other significant outcome predictors , RBP4 level remained an independent predictor for mortality with an OR of 1.19 (95% CI: 1.08–1.33). We also used multivariate analysis models to estimate adjusted OR and 95% CIs of mortality for RBP4 quartiles (with first quartile as reference). In multivariate models comparing the Q2, Q3, and Q4 against the Q1 of the RBP4, RBP4 in Q2, Q3 and Q4 were associated with mortality, and increased risk of poor functional outcome by 88% , 198%  and 708% , respectively.At 3 months, 31 patients (10.4%) had died, RBP4 levels in those patients were more than two times greater as compared with patients who survived . With an AUC of 0.81, RBP4 presented a significantly greater discriminatory ability than Hs-CRP , age , and NIHSS score . Similarly, RBP4 improved the NIHSS score .Based on ROC curves, the optimal cut-off value of serum RBP4 level to diagnose the mortality was 37.5 μg/ml, which yielded the highest sensitivity and specificity  compared with non-diabetic patients ; (iv) RBP4 showed a significantly greater discriminatory ability to predict poor functional outcomes as compared with other biomarkers, such as Hs-CRP, FBG, and age.Adipose tissue secretes many types of adipokines  that play a role in the progression of stroke . The prePrevious studies had suggested that different variables recorded on admission were associated with poor outcome in stroke patients, for example insulin resistance , vitaminP=0.09) amongst men with type 2 diabetes mellitus in a 22-year prospective study. Different populations, sample sizes, geographical regions, health status, RBP4 ELISA testing kits, and ethnicity may justify the conflicting results observed in those studies.Consistent with our findings, Prentice et al.  reportedin vitro inflammation in endothelial cells through stimulating expression of proinflammatory molecules [The possible mechanism of increased levels of RBP4 with poor outcome in acute stroke in patients has not yet established. Two important pathophysiological mechanisms involved in ischemic stroke were inflammation and oxidative stress . Plasma olecules , and anoolecules . Second,olecules  and a neolecules . Furtherolecules . Becauseolecules . The involecules . A previolecules . At lastolecules . Chiba eolecules  found tholecules . Thus, en=299) and single center might limit the generalization of the results of the present study. Second, without serial measurement of the circulating levels of RBP4, the present study yielded no data regarding when and how long biomarkers were elevated in these patients. In addition, the present study measured RBP4 in serum, not in cerebral spinal fluid (CSF). It was still uncertain whether peripheral RBP4 levels reflect similar changes in the central nervous system (CNS). Third, since the majority of patients with classical cardiovascular risk factors  were already treated, we cannot rule out the plausible effects of pharmaceutical agents (e.g. statins) on RBP4, leading to underestimation of its predictive power. In addition, the binding of RBP4 to retinol and transthyretin (TTR) was affected by several factors  [Some limitations should be considered. First, the relatively small sample size n=29 and siniseases) . FurtherThe data showed that elevated serum levels of RBP4 at admission were associated with severity and prognosis of AIS, suggesting that vitamin A metabolism or impaired insulin signaling could be involved. Further studies should be carried out with respect to what was the cause of the increased serum levels of RBP4 and the role in the pathology of the stroke outcomes.


Extracellular vesicles (EVs) are nano-sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. Here, we characterize the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND).n = 10) or without (n = 10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 h to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes , inflammation (CRP), and stress responses  were evaluated in EVs released by U87 cells following induction of oxidative stress and in CSF EVs from HIV+ patients by immunoblotting.CSF EVs isolated from 20 HIV+ subjects with (Mass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heat-shock proteins and a subset of neuronal, astrocyte, oligodendrocyte, and choroid plexus markers, in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood-brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs from subjects with HAND and were also detected in EVs released by U87 cells under oxidative stress.These findings suggest that CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammation-related, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery. While the introduction of combination antiretroviral therapy (ART) has dramatically improved overall health and life expectancy of HIV-positive (HIV+) individuals, HIV-associated neurocognitive disorders (HAND) remain a major cause of morbidity. HAND, consisting of asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV-associated dementia (HAD), affects up to 20–50% of HIV-infected individuals in the era of combination ART –3. The iCerebrospinal fluid (CSF) is in part derived from brain parenchyma and serves as an important indicator of neurological diseases. Elevated CSF neurofilament light chain (NFL), neopterin, soluble CD14 (sCD14), inflammatory cytokines, and chemokines are biomarkers frequently associated with HAND in HIV patients, particularly those with non-suppressed plasma viremia –14. HoweExtracellular vesicles (EVs) have been identified as biomarkers for neurological diseases including Alzheimer’s disease , 22, ParEVs are proposed to play important roles in HIV pathogenesis –34. HIV CSF samples from 20 HIV+ subjects were collected between 1998 and 2013 by the National NeuroAIDS Tissue Consortium (NNTC)  and stored at − 80 °C. All subjects were enrolled with written informed consent and institutional review board (IRB) approval at each study site. HAND clinical diagnoses were determined using established criteria  based on2. To induce oxidative stress, U87 cells were treated with increasing concentrations  H2O2 for 4 h in serum-free medium. After 4 h treatment, cells were washed with phosphate-buffered saline (PBS) and cultured in exosome-depleted medium for 72 h. Control and H2O2-treated U87 cells and culture supernatants were harvested for immunoblotting and EV isolation.Human glioblastoma U87 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% exosome-free fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO×g for 15 min to remove cellular debris. Supernatants were immunoglobulin (Ig) depleted by incubation with protein A/G agarose beads and protein-L beads at room temperature for 1 h. Twelve common abundant proteins  were depleted using Proteome Purify-12 immunodepletion resin (R & D Systems) at room temperature for 30 min. Abundant protein-depleted CSF samples were then incubated overnight at 4 °C with ExoQuick exosome precipitating reagent . The mixture was centrifuged at 1500×g for 30 min and EV pellets were resuspended in 20 μl PBS. EV-depleted CSF was concentrated 5-fold by passing through a 10K Amicon filter and stored at − 80 °C until further processing. EVs from control and H2O2-treated U87 cells were isolated from 150 ml cell culture media by ultracentrifugation. In brief, cell culture media was centrifuged at 300×g at 4 °C for 10 min to remove floating cells. Supernatants were passed through a 0.2 μm filter to remove contaminating apoptotic bodies, larger microvesicles, and residual cell debris. The flow through was centrifuged at 150,000×g at 4 °C for 90 min to pellet exosomes and smaller vesicles. The supernatant was removed and EV pellet was resuspended in 35 ml cold PBS and then centrifuged at 150,000×g for 90 min. The resulting EV pellet was resuspended in 100 μl PBS.CSF samples (400 μl) were centrifuged at 3000CSF and U87 EV concentrations and sizes were measured by nanoparticle tracking analysis (NTA) on a ZetaView instrument . For electron microscopy, CSF EVs isolated from 300 μl CSF were suspended in 10 μl PBS containing 1% paraformaldehyde. EV samples were adsorbed for 1 min to a formvar/carbon coated grid and fixed for 5 min in 1% glutaraldehyde. EVs were washed on a drop of water and stained with 1% uranyl acetate for 30 s. EV morphology was analyzed with a Tecnai G2 Spirit BioTWIN transmission electron microscope (TEM) equipped with an AMT 2 k CCD camera at the Harvard University TEM core.CSF EV and corresponding EV-depleted CSF samples were mixed with an equal volume of radioimmunoprecipitation assay (RIPA) lysis buffer . U87 cells and EVs were also lysed with RIPA buffer and protein concentration was estimated by BCA assay. Modified Laemmli 4X sample buffer was added to lysed EVs and EV-depleted CSF samples and boiled for 5 min. Equal volumes of CSF EVs and corresponding EV-depleted CSF samples were loaded on 4–12% gradient polyacrylamide gels. Forty micrograms of U87 cell and EV lysates were loaded in each lane. After electrophoresis, protein bands were transferred onto PVDF membranes for 1.5 h at room temperature. The membranes were blocked with 4% non-fat milk for 1 h and probed at 4 °C for human IgG  CD9 (Santa Cruz Biotechnology), CD81 (System Biosciences), heat-shock protein 70 (HSP70) (System Biosciences), flotillin-1 (FLOT-1) (BD Biosciences), glial fibrillary acidic protein (GFAP) (Abcam), glutamine synthase (GLUL) (Abcam), parkinsonism associated deglycase (PARK7) (Abcam), and C-reactive protein (CRP) (Abcam). After secondary antibody treatment, blots were developed with enhanced chemiluminescence (ECL). Images were captured using the Bio-Rad ChemiDoc™ Imaging System. Densitometric quantification was carried out with ImageJ software.To isolate CSF EVs for proteomics, CSF samples were pre-cleared using Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) and Proteome Purify™ -12 kit (R&D systems). Immunodepleted CSF samples were characterized by SDS-PAGE and silver staining before EV isolation  for 4 h in the dark at 37 °C. The resultant blue formazan crystals in cells were dissolved in dimethyl sulfoxide. Optical density in each well was determined at 570 nm using a microplate reader.U87 cells were seeded in 96-well plates at a density of 5 × 102DCFDA) reagent. Cells were treated with 50, 100, or 250 μM H2O2 for 4 h. Mock and H2O2-treated cells were loaded with 20 μM H2DCFDA for 45 min at 37 °C. Following two washes with pre-warmed PBS, fluorescence was measured at 485ex/535em nm or analyzed with a fluorescence microscope. Data were normalized to corresponding protein concentrations.Intracellular ROS was measured using dichlorodihydrofluorescein diacetate (Hgenexplain.com/transfac) and PANTHER (pantherdb.org). Proteins identified by ≥ 2 peptide count were included for analyses. Heatmaps were generated using log2 transformed peptide intensities from LC-MS analyses using R (version 3.5.1). Immunoblot bands were quantitated using ImageJ software and graphical representations plotted in GraphPad prism version 7.0. Densitometric values represent mean + SEM. Differences in CSF EV concentrations and number of proteins detected in CSF EV between groups were analyzed by Mann-Whitney U test. Differences between HAND and control subjects, or between control and H2O2-treated U87 cell lysates and EVs, were analyzed by one-way ANOVA. Differences in ROS induction and cell viability between control and H2O2-treated U87 cells were analyzed by t test.Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase , tetraspanins (CD81 and CD9), RAB proteins, and heat shock proteins were more abundant in EV fractions compared to EV-depleted CSF , while 16 were decreased in subjects with HAND compared to those without HAND  of which 20 were significantly altered (≥ 2 fold change and p < 0.05) in subjects with ANI or MND compared to those without HAND  or in ANI + MND and no HAND (n = 1010) with ≥ 2 peptides are shown in Additional file Next, we examined the relationship between the CSF EV proteome and HAND. CSF EV concentrations and number of proteins detected in EV fractions were higher in HIV+ subjects with HAND compared to without HAND  and biological processes (immune/inflammatory and stress responses) between and within individual HIV+ subjects, we generated supervised heatmaps using the sum of peptide intensities from 101 representative proteins Fig. . ProteinTo further investigate the role of astrocyte-derived EVs in stress responses and inflammation in HIV+ subjects with and without HAND, we selected a subset of markers for astrocytes , stress responses , and inflammation (CRP) for validation by immunoblotting. For this purpose, we used independent pools of 300 μl CSF from HIV-negative, HIV+ without HAND, and HIV+ with HAND  subjects. CSF EVs from HIV-negative subjects were included as an additional control. Mean EV concentrations from three independent CSF pools are shown in Fig. 2O2-treated astrocytic U87 cells and U87-derived EVs were used to further evaluate astrocyte, stress response, and inflammation markers detected in Fig. 2O2 for 4 h, which showed a dose-dependent effect in three independent experiments and induced 3% to 58% cell death with increasing doses  cells and EVs were characterized by immunoblotting for astrocyte , inflammation (CRP), and stress response  markers  mapped to biological functions.Additional file 5: Table S5. EV-depleted CSF proteins from HIV+ subjects (n=20) mapped to cellular components.Additional file 6: Figure S1. Depletion of abundant proteins from CSF. Figrue S2. Immunoblotting for ER membrane markers calnexin and Erp72, and exosome markers CD81 and CD9 in CSF EVs from 2 representative HIV+ subjects. Figure S3. Comparison of CSF EV concentrations and protein abundance in HIV+ non-HAND, ANI + MND, and HAD subjects. Figure S4. Unsupervised heatmap of 101 CSF EV proteins identified in 20 HIV+ subjects with (n = 10) and without (n = 10) HAND.


Background. Both percutaneous and arthroscopic techniques have been introduced in anatomic ankle lateral ligaments reconstruction. The purpose of this study was to compare these two techniques in identifying the calcaneal insertion of the calcaneofibular ligament (CFL). Methods. Fifteen fresh-frozen human ankle cadaver specimens were used in this study. Each specimen was tested in three stages. For stage 1, each specimen was evaluated under arthroscopy. After debridement was performed, the insertion of the CFL on the calcaneus was identified, and a 1.5mm Kirschner wire was drilled at the center of the insertion. For stage 2, a percutaneous technique was used to identify the center of the insertion of the CFL. A second 1.5 mm Kirschner wire was drilled through the skin marker. For stage 3, the ankle was dissected, the footprint of the CFL was identified under direct vision, and the distances between the center of the CFL insertion on the calcaneus and the two Kirschner wires were measured, respectively. Results. In the arthroscopic technique group, the mean distance from the Kirschner wire to the center of the CFL insertion in the calcaneus was 3.4 ± 1.3 mm. In the percutaneous technique group, the mean distance from the Kirschner wire to the center of the CFL insertion was 3.2 ± 1.4 mm. No significant difference was found between the two groups. Conclusion. No difference in identifying the calcaneal insertion of the CFL was found between the percutaneous and the arthroscopic ankle lateral ligaments reconstruction technique. Both techniques can be used during anatomic ligaments reconstruction in treatment of chronic ankle instability. Lateral ankle sprain is the most common sports-related injury. Thirty-four percent of patients will incur a chronic ankle instability within 3 years after the first ankle sprain . MoreoveIn the past decade, ankle arthroscopy has been more and more frequently used in sports medicine. Thereafter, arthroscopic anatomic lateral ankle ligaments reconstruction procedures have been introduced –10. The The shortage of arthroscopic procedure in drilling the calcaneal tunnel is the calcaneal insertion of the CFL is difficult to visualize . ThereafFifteen fresh-frozen human ankle cadaver specimens were obtained and approved for use by the Body Donation Center of the authors' University. The mean age of the patients at death was 62.2years (range: 49–69 years), with nine males and six female's cadaveric ankles obtained. Eight of the ankles were right and seven were left. The ankles were examined visually and radiographically to rule out major ankle pathology. The specimens were fresh frozen and stored at −20°C. Prior to the study, the cadaveric ankles were thawed at 5°C for 24 h. The moisture of the specimens was maintained with saline spray during the preparation and studying phases.Each specimen was tested in three stages. For stage 1, each specimen was evaluated under arthroscopy. After debridement was performed, the insertion of the CFL on the calcaneus was identified and a 1.5mm Kirschner wire was drilled at the center of the insertion. For stage 2, a percutaneous technique was used to identify the center of the CFL insertion on the calcaneus. A second 1.5 mm Kirschner wire was drilled through the skin marker. For stage 3, the ankle was dissected, the footprint of the CFL was identified under direct vision, and the distances between the center of the CFL insertion and the two Kirschner wires were measured, respectively.The ankle was fixed on a standard extremity holder in supine position without distraction. Standard arthroscopy equipment including 4.0mm, 30° arthroscopy, and 4.5mm shaver was used. Three portals including anterior medial, anterior lateral, and sinus tarsi portals were established, which was described in detail in previous studies , 13. DebThis technique was described specifically in previous studies , 15. TheThe dissection was then performed to expose the CFL insertion at the calcaneus . The disA senior surgeon specializing in ankle arthroscopy and sports medicine performed all the operations to avoid the bias.Each measurement was repeated 3 times for intraobserver analysis as well as for calculation of the mean values. As a test of interobserver, two of the authors (the first and the second authors) measured the specimens independently.A priori power analysis was used to calculate the sample sizes. According to previous study , the minThe intra- and interobserver reliabilities were 0.987 and 0.942, respectively. In the arthroscopic technique group, the mean distance from the Kirschner wire to the center of the CFL insertion in the calcaneus was 3.4 ± 1.3 mm. The distances varied from 1.3 mm to 5.8 mm. In the percutaneous technique group, the mean distance from the Kirschner wire to the center of t the CFL insertion was 3.2 ± 1.4 mm. The distances varied from 1.0 mm to 4.1 mm. No significant difference was found between two groups. Moreover, in the arthroscopic group, 9 of 15 Kirschner wires were anterior and 14 of 15 Kirschner wires were inferior to the center of the CFL footprint. In the percutaneous technique, 12 of 15 Kirschner wires were posterior and 13 of 15 Kirschner wires were inferior to the center of the CFL footprint.The most important finding of the present study was that there was no difference in identifying the calcaneal insertion of the CFL between the percutaneous and arthroscopic techniques. As anatomic lateral ligaments reconstruction to chronic ankle instability provides satisfactory clinical outcomes, more and more studies introduced the arthroscopic techniques to reconstruct ankle lateral ligaments –10, 14. Previous studies indicated that CFL is very important for lateral ankle stability –19. It pMany previous studies described the anatomic reference to identify the CFL footprint during anatomical CFL reconstruction. Neuschwander et al.  found thOne of the shortages of percutaneous technique was that variable insertion of the CFL has been found. The variable insertions result in variable obliquity and angles of the ligament relative to the long axis of the fibula, coronal, and sagittal planes , 27. ClaIn the current study, most Kirschner wires in the arthroscopic technique group were anterior and inferior to the center of the CFL footprint, and most Kirschner wires in the percutaneous technique were posterior and inferior to the center of the CFL footprint. Therefore, the results can help us to improve the accuracy in making calcaneal tunnel.The main limitation of the current study was that we did not measure the characteristics of the CFL, the footprint of CFL in relation to bony landmarks, and the distance between the insertion and the surrounding nerves and blood vessels. However, these characteristics were measured repeatedly in many previous studies; it is not necessary to repeat the same work. On the other hand, after arthroscopic procedure, the irrigation fluid penetrated the surrounding tissue, which made it inaccurate to measure the distance between the Kirschner wire and the surrounding nerves and blood vessels. In the future work, paired ankles may be needed to compare these two techniques.In this cadaveric study, no difference in identifying the calcaneal insertion of the CFL was found between the percutaneous and the arthroscopic ankle lateral ligaments reconstruction technique. Both techniques can be used during anatomic ligaments reconstruction in treatment of chronic ankle instability.


The presence of atherosclerosis of the common hepatic artery (CHA) in donor livers potentially contributes to vascular complications after liver transplantation, thereby most of those organs are traditionally discarded. Herein, we describe the successful outcome of three patients transplanted with grafts that had severe atherosclerosis of the donor CHA up to the level of the gastroduodenal artery (GDA).In all three cases, endarterectomies were performed by dissection between the atheromatous core and the artery intima using a dissecting spatula, allowing to secure the lumen of the vessel. The native CHA/GDA patch was aligned with the corresponding CHA/GDA patch from the graft for the arterial reconstruction. No vascular complications were seen post-operatively.Endarterectomy and anatomical reconstitution of the arterial tree, without any redundancy or kinking, allowed for the successful transplantation of organs that would be otherwise discarded. Further, the straight alignment of the arteries may enhance flow dynamics, preventing thrombosis.This report might guide future studies targeting means to increase the utility of donor livers discarded due to arterial atherosclerosis. In the Atherosclerosis’ impacts graft outcomes after LT in many ways. Usually, atherosclerosis is confined to the ostia of origin of visceral vasculature, such as the celiac axis or renal arteries, and it may cause poor organ perfusion during procurement. Extensive atherosclerosis of the common hepatic artery (CHA) can lead to vascular complications after LT, including hepatic artery thrombosis (HAT), which ranges from 2% to 9% . ProgresThe arterial reconstruction during graft implantation depends on a combination of factors, including donor and recipient artery diameters, vessel quality, donor/recipient anatomical variations and specific conditions related to the indication for LT ,14. In tThe early success and technical aspects are described herein and enlighten the readership on careful consideration of such grafts for clinical transplantation.22.1The patient was a 51-year-old, female sex, blood group O, with advanced decompensated primary biliary cirrhosis presenting with refractory ascites, sarcopenia, portal hypertension and significant jaundice. The United Kingdom Model for End-Stage Liver Disease (UKELD) score was 61  and she The graft had normal hepatic artery anatomy, but an extensive atheromatous plaque up to the GDA. During implantation, the graft GDA was divided obliquely along the main hepatic artery stem and an endarterectomy was done. A plaque free portion of the hepatic artery above the GDA was obtained for direct anastomosis to the native CHA at the GDA junction; thereafter, a short, straight and non-redundant arterial reconstruction was performed. The anastomosis width was just over 6 mm, there were good pulse waves and the resistance index was confirmed by doppler ultrasound intra-operatively.Surgical times are presented in 2.2N-acetylcysteine with antimicrobial cover and listed for super-urgent liver transplantation. A liver from a donor after brain stem death (DBD), 48-year-old, female sex, was offered for this patient  levels of 4000 IU/L, international normalized ratio (INR) of 6.0 , mental The graft hepatic artery had atheromatous plaque beyond the GDA in the proper hepatic artery (PHA). Graft hepatic artery was prepared at the GDA, and endarterectomy was performed. Recipient CHA was prepared at the GDA junction and arterial reconstruction was done with an anastomosis just over 5 mm wide. The plaque removed from the donor CHA/GDA junction had 1.2 × 0.3 × 0.2 cm and it was reported as an organised atheromatous plaque on histology. The remnants of the donor artery discarded and sent for histology showed extensive atheromatous changes with intimal thickening and infiltration of foam cells and cholesterol clefts. Focally, in one of the branches, there was at least 75% luminal occlusion. Surgery timings are presented in After the liver transplant, the patient required inotropic and ventilation support for 2 days, with subsequent uneventful postoperative course. She was discharged on POD 14. The same protocol of thrombosis prophylaxis previously reported has been followed.Biochemical values were within normal range after 3 months of the transplant . An ultr2.3A 56-year-old patient, blood group O, diagnosed with alcohol-related liver disease, presenting with a UKELD of 49 that had previously undergone laparotomy for diverticular abscess and reversal of Hartmann’s procedure, was listed for LT. He received a DBD liver from a 74-year-old, male sex donor, who died for intracranial thrombosis  and right hepatic artery (RHA) branches. This was not informed by the retrieval or bench picking teams, and therefore, was recognized only during implantation once the surgeon was ready for the arterial reconstruction following portal reperfusion. The lumen of the atheromatous proper hepatic artery was significantly narrow, and the disease was circumferential. Surgical endarterectomy of the PHA was performed and the plaques extending up in both branches were removed en-mass. The total length of the endarterectomized specimen was 1.5 cm upwards from the GDA of the graft. Distal hepatic artery, following the procedure, was soft. The native CHA was prepared at the GDA junction. Further, the hepatic arteries were aligned straight, and they were reconstructed with 7-0 prolene with a continuous suture. The intra-operative doppler ultrasound showed good flows on the right, left and segment 4 artery branches. Histological assessment reported extensive atheromatous change, markedly thickened intima with myxoid change and foam cells. Occlusion the lumen of 50% was found in some portions.This patient had an uneventful post-operative course, stayed in the ICU for one day and was discharged at POD 8. He was at a higher risk of artery thrombosis because the atheromatous plaques were extending up to the LHA and RHA branches, hence short-term anticoagulation with warfarin was undertaken post-operatively. The targeted INR was between 2.0 and 2.5 over the three initial months after transplantation. Biochemistry values were within a normal range at 3 months post-transplant , and an 3Atherosclerosis is a disease that occurs in elastic arteries, such as the aorta, or in medium-sized muscular arteries, and elevated lesions of the tunica intima called plaques characterize it. Morphologically, it is classified into four stages: fatty streaks, fibrous plaques, atheromatous plaques and complicated lesions . AtherosIn the three cases reported, the arteries chosen for anastomosis had severe atherosclerosis and, technically, unique steps were followed. In all three cases, the native CHA was prepared at the GDA level after dividing from the distal GDA and transfixing it. A wide arterial patch was created by opening the GDA patch. The key rationale was to align this native CHA/GDA with the corresponding CHA/GDA patch from the graft. This is a natural anatomical reconstitution of the arterial tree without any redundancy or kinking, which straight aligns the arteries and thus, enhances flow dynamics, preventing thrombosis. The GDA/CHA patch was created, and a wide Carrel patch was obtained from the grafts before endarterectomy. Endarterectomies were performed by dissection between the atheromatous core and the artery intima using a dissecting spatula, allowing to secure the lumen of the vessel. Preservation of the tunica media, including the elastic fibres, is crucial for maintaining the elasticity of the arteries. Residual plaque is a risk for postoperative thrombosis and thus, its total removal is considered essential .Fig. 4ScEndarterectomy was first carried out on a superficial femoral artery in 1946 by Cid dos Santos and subsequently, on the abdominal aorta by Wylie in 1951 ,22. DuriMany transplant surgeons have chosen the splenic artery patch or the CHA stem from the graft for arterial reconstruction, whereas we opted for the GDA patches. We believe that avoiding kinking and aligning the arteries contributes to the long term patency of graft vessels. Flow and pulse wave are routinely checked by intra-operative and post-operative ultra sound examinations , Fig. 2.4In conclusion, we reported three cases of severe atherosclerosis of donor hepatic arteries recognised during LT, where pragmatic and judicious decision making enabled successful endarterectomy and appropriate arterial reconstruction. All three grafts were under different circumstances, from urgent to elective transplantation, yielding successful transplantation through technical modification, demonstrating potential avenues to increase the utility of otherwise discarded donor livers.I have no financial relationships to disclose and there are not any financial and personal relationships with other people or organisations about all authors.I have no financial relationships to disclose and there is not any sponsor with funding.Our reported case here involves a sufficient ethnical level. The patient was approved to be on the published paper through our formed document at our institute. The patient showed their willingness to be reported by medical paper or presentation via our document approved by our institute.In addition, we obtained ethical certification with the approval number CARMS-14495 from ethic committee in University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital Birmingham, United Kingdom.Fully informed consent was obtained with some document. Written informed consent was obtained from all patients for publication of this case series and accompanying images. We confirm that have written consent for all patients A copy of the written consent of all patients is available for review by the Editor-in-Chief of this journal on request. Moreover, we obtained ethical certification with the approval number CARMS-14495 from ethic committee in University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital Birmingham, United Kingdom.Yuhki Sakuraoka: Literature review and writing the article.Amanda Pinter Carvalheiro da Silva Boteon: mainly managed the patient and led the patient to discharge earlier as well as they helped author collect the clinical data smoothly.Rachel Brown: the pathologist detected the specific pathological features and provided all microscopic pictures.M Thamara PR Perera: Editing the article mainly and he per-formed the surgery. He is the chief consultant surgeon of liver transplant.Our reported case here is not research study. This is a case series.Moreover, we obtained ethical certification with the approval number CARMS-14495 from ethic committee in University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital Birmingham, United Kingdom.In addition to this, we participated in Research Registry with the approval number “researchregistry4629” to be publicly accessible.Dr Yuhki Sakuraoka is the Guarantor of this report and has full responsibility to it.Not commissioned, externally peer-reviewed.