Organic molecules such as steroids or amino acids form crystals that can facilitate the formation of ice – arguably the most important phase transition on earth. via molecular simulations that cholesterol crystals aid the formation of ice nuclei in a unconventional fashion. Rather than providing a template for a flat ice-like contact layer (as found in the case of many inorganic substrates), the flexibility of the cholesterol surface and its low density of hydrophilic functional groups leads to the formation of molecular cages involving both water molecules and terminal hydroxyl groups of the cholesterol surface. These cages are made of 6- and, surprisingly, 5-membered hydrogen bonded rings of water and hydroxyl groups that favour the nucleation of hexagonal as well as cubic ice (a rare occurrence). We argue that the phenomenal ice nucleating activity of steroids such as cholesterol  is due to (i) the ability of flexible hydrophilic surfaces to form unconventional ice-templating structures and (ii) the different nucleation sites offered by the diverse topography of the crystalline surfaces. These findings clarify how exactly organic crystals promote the formation of ice, thus paving the way toward deeper understanding of ice formation in soft and biological matter – with obvious reverberations on atmospheric science and cryobiology.Organic molecules such as steroids or amino acids form crystals that can facilitate the formation of ice – arguably the most important phase transition on earth. However, the origin of the ice nucleating ability of organic crystals is still largely unknown. Here, we combine experiments and simulations to unravel the microscopic details of ice formation on cholesterol, a prototypical organic crystal widely used in cryopreservation. We find that cholesterol – which is also a substantial component of cell membranes – is an ice nucleating agent more potent than many inorganic substrates, including the mineral feldspar . Scanning electron microscopy measurements reveal a variety of morphological features on the surfaces of cholesterol crystals: this suggests that the topography of the surface is key to the broad range of ice nucleating activity observed (from –4 to –20 °C). In addition, we show  Scanning electron microscopy suggests that the activity of these crystals as ice nucleating agents across such a wide temperature range could be due to the diverse topography of the surface of the cholesterol crystals, which are likely to offer a variety of different nucleation sites. In order to get a molecular-level insight into the mechanism of ice formation on CHL crystals, we harness enhanced sampling simulations, focusing on the hydroxylated (001) face of cholesterol monohydrate (CHLM) – the most relevant surface  in biological scenarios. We find that CHL crystals facilitate the formation of ice in a non-conventional fashion: in contrast to what has been observed in the case of inorganic substrates such as e.g. carbonaceous particlesIn this work, we bring together experiments and simulations to take an ambitious step forward in furthering our understanding of ice formation on organic crystals. We focus on CHL, due to its relevance in cryopreservation and its role within cellular membrane, unravelling microscopic motivations for heterogeneous ice nucleation likely to be shared by many other organic crystals. We find As a whole, our findings suggest that the formation of ice on CHL crystals originates from the ability of their flexible hydrophilic surfaces to trigger the formation of unconventional ice-templating molecular features. In addition, different nucleation sites potentially offered by the diverse topography of the crystal can further enhance the intrinsic ice nucleation potential of CHL surfaces. This insight could help to understand ice formation on a number of other organic compounds, from amino acid crystals22.1–1 freezing was monitored using a camera. In this way the droplet fraction frozen curve presented in The ice nucleation efficiency of CHLM was evaluated using an adapted version of the μl Nucleation by Immersed Particles Instrument (μl-NIPI) described in detail in ns, commonly used to compare the ice nucleating efficiency of different substancesn(T) is the number of droplets frozen at temperature T, N is the total number of droplets in the experiment and A is the surface area of nucleating agent per droplet. The value of A for each droplet was measured using the image analysis software imageJ, customarily used to quantify particles size in biosciences.A is 1.82 ± 0.46 mm2.In order to calculate the (surface) density of the active ice nucleation sites  is largest, the contribution of the uncertainty in surface area amounts to approximately 25% of total uncertainty in ns(T), with the Poisson uncertainty in the active site distribution accounting for the remainder of the error bars.The uncertainties associated with the values of Scanning electron microscopy (SEM) was performed on CHLM plates. These were mounted on copper tape, then coated with 2 nm of iridium. SEM was performed with an FEI Nova NanoSEM 450 in high vacuum mode, using an Everhart–Thornley Detector (ETD).2.2P1) was preserved, and we have constructed a 3 by 3 supercell with in-plane dimensions of 37.17 and 36.57 Å. We positioned 1923 water molecules randomly atop this CHLM slab at the density of the TIP4P/Ice modelThe computational setup we have used is depicted in –1. All the other atoms within the CHLM slab are unconstrained. We have verified that the thermal expansion of the crystal at 230 K  does not alter the structure nor the dynamics of the water–kaolinite interface. This setup is thus as close as we can get to the realistic (001) hydrophilic surface of CHLM within the CHARMM36 model. Implications of the flexibility of the CHLM slab are discussed in the ESI.58The CHARMM36  force fie.g.Extreme care must be taken in order to correctly reproduce the structure and the dynamics of the water–CHLM interface. The Forward Flux Sampling (FFS) simulations reported in this work rely on a massive collection of unbiased Molecular Dynamics (MD) runs, all of which have been performed using the GROMACS package, version 4.6.7. The code was compiled in single-precision, in order to alleviate the huge computational workload needed to converge the FFS algorithm and because we have taken advantage of GPU acceleration, which is not available in the double-precision version. The equations of motions were integrated using a leap-frog integrator with a time step of 2 fs. The van der Waals (non bonded) interactions were considered up to 10 Å, where a switching function was used to bring them to zero at 12 Å. Electrostatic interactions have been dealt with by means of an Ewald summation up to 12 Å. The NVT ensemble was sampled at 230 K using a stochastic velocity rescaling thermostat33.13 terminated, hydrophobic surface or a –OH terminated, hydrophilic surface. Atomic and chemical force microscopy measurements indicate that in aqueous and organic solution conditions, the hydrophilic (001) surface is most abundantly found, in the form of largely homogeneous crystalline faces.TS = 5 K).–1 and freezing monitored using a camera. In this way the fraction of frozen droplets can be determined as a function of temperature. Note that as the crystalline surface is submerged in liquid water these experiments are conducted at 100% relative humidity – i.e. in “immersion mode”.70We start by experimentally investigating the ice nucleating ability of cholesterol crystals – as a function of supercooling. CHL can crystallise into two different polymorphs, namely anhydrousns) for CHLM on the basis of the size of the contact patch of the water droplets with the CHL plates. As explained in greater detail in the Methods section, ns is a site specific measure of ice nucleation efficiency which does not take into account the time dependence of ice nucleation, on the basis that the impact of time dependence on heterogeneous ice nucleation is generally minimal.e.g. kaolinite powder,In 3.2i.e. different parts of the surface nucleate ice with differing efficiency. This is commonly known as site specific nucleation behaviour,ns as a function of temperature). As it is becoming increasingly clear that the topography of the ice nucleating agents must play an important role in the heterogeneous nucleation of ice from liquid water,e.g.As noted above the spread of freezing temperatures we report for CHLM is very broad. This behaviour suggests that the nucleation behaviour of CHLM is spatially heterogeneous, 3.3TS = 42 K) employing the CHARMM36  is in contact with the hydroxylated (001) surface of CHLM (CHLM–OH001), modelled as a single layer of CHL molecules. This surface is hydrophilic, due to the presence of amphoteric hydroxyl groups. As CHL molecules are relatively bulky and the crystal is held together by weak electrostatic interactions only, the arrangement of these –OH groups on the CHLM surface is characterised by a broad distribution of large OH–OH distances, ranging from 5.1 to 7.1 Å – as illustrated in 76e.g. water in contact with hydrophilic walls – or indeed water on kaolinite. This is because, the outer layer of the CHLM crystals is much more mobile/flexible than that of kaolinite: this is not surprising, as we are comparing a molecular organic crystal  with a  clay mineral. Importantly, it is reasonable to assume that a similar degree of flexibility characterises the majority of organic crystals containing long molecules such as steroids. This is relevant to ice formation because, as discussed in e.g.Interestingly, despite the presence of the hydroxyl groups, the density profile of the oxygen atoms of the water molecules on CHLM reported in Ih) and cubic (ice Ic) ice, and are the most abundant species in bulk water. Note that the occurrence of these rings is actually even more pronounced in the proximity of the CHLM–water interface  the formation of an ordered, ice-like over-layer of water molecules, in contrast with what is generally found in the case of idealised crystalline surfaces,n Ic see , and inve.g.We note that the emergence of these cages is the reason why we have chosen to consider as the “interfacial layer” those water molecules within 5.0 Å from the average position of the oxygens of the CHL hydroxyl groups – as illustrated by the shaded region in e.g. mineral crystals) and biological  ice nucleating agents, as they are characterised by the relatively flat and (in this case) –OH regularly patterned surfaces of the former while showing the flexibility of the latter. This is particularly relevant for CHL, which is a substantial component of animal cellular membranesvia “anchored clathrate” motifs not dissimilar to the molecular cages discussed above.Our findings thus contribute the growing body of evidence3.4e.g.λ, into a collection of interfaces λi. Here, λ corresponds to the number of water molecules within the largest ice nucleus, which can be located either in the bulk of the water slab or at the water–CHLM–OH001 interface. A diffuse crystal–liquid interface has been taken into account into the definition of λ, which relies on local bond order parameters :In order to characterise the mechanism as well as the kinetics of ice nucleation on CHLM–OH001 we have performed forward flux sampling (FFS) simulations.s see ESI, consistP(λice|λ0) for a certain MD trajectory to reach the ice basin is decomposed in a collection of (manageable) crossing probabilities which we compute by a large number (103 to 105) of unbiased MD trial runs from λi–1 to λi. Further details about the FFS algorithm can be found in the ESI.TS = 42 K) as employed previously to compute the homogeneous ice nucleation rate and the heterogeneous ice nucleation rate on kaolinite, a clay mineral of relevance to atmospheric science. As such, we can compare directly our results with those of In this way, the  total probability λ = 0) sit on top of the CHLM–OH001 surface. The calculated growth probability P(λ|λ0) as a function of lambda, together with the fraction of ice nuclei that can be found at the water–CHLM–OH001 interface are reported in the ESI  basal face of kaolinite,From the very early stages of the nucleation process, we observe a strong preference for ice to form at the water–CHLM–OH001 interface – as opposed to within the bulk of the water slab. In fact, ∼75% of the pre-critical ice nuclei we observe as natural fluctuations of the supercooled water network  progressively assume a more isotropic shape, as indicated by the evolution of the asphericity parameter α  as a function of λ. At the same time, the 1D character of the nuclei evolves toward a more compact geometry, with a significant growth along the [001] direction (z-axis) normal to the water–CHLM–OH001 interface, as demonstrated by the trend of the dimension ΔZ of the ice nuclei along that axis, also reported in TS = 42 K) has a strong anisotropic character, in stark contrast with the assumptions prescribed by classical nucleation theory (CNT).96The mechanism of ice nucleation at the water–CHLM–OH001 interface is illustrated in In fact, CNT does not take into account the molecular structure nor the “chemistry” of the substrate: these aspects are only implicitly included into the value of the contact angle of the ice nuclei with respect to the substrate. However, microscopic features such as the particular arrangement of the hydroxyl groups on the CHLM surface can influence the shape and the energetics of the ice nuclei. In the case of ice on CHLM, water molecules at the water–cholesterol interface find convenient to harness the directionality of the –OH pattern  hydroxylated surface of kaolinite also presents –OH groups at the water–crystal interface which are capable of templating the formation of ice-like structures. However, supercooled water on kaolinite forms a dense, hexagonal ordered overlayer of ice-like molecules sitting on top of the hydroxyl groups,3.5TS = 42 K), homogeneous ice nucleation results in a mixture of ice Ic and ice Ih known as stacking disordered ice Isd.Ih.Ic and ice Ih nuclei at the very early stages of the nucleation process, as depicted in the inset (left side) of Ic nuclei are three times more abundant than ice Ih nuclei at the first FFS interface (λ = 80), we would expect the formation of ice on the CHLM–OH001 surface to proceed via the growth of ice Isd. In fact, as shown in i.e. ΔT = 42 K) is dominated by ice Ic: by the time the ice nuclei have reached a post-critical size (e.g. λ = 360), the average number of Hexagonal Cages  is still about three times larger than that of DDC .At the strong supercooling considered here  on top of ice Ic crystals , as illustrated in the insets (right side) of Ic and ice Ih is thus likely to result in ice Isd crystals at strong supercooling. However, at milder supercooling ice Ih fluctuations are expected to become more relevant, and in fact experimental evidence suggests that the macroscopic crystalline habit of ice crystals grown on CHLM at ΔT = 2 K is indeed that of ice Ih.Ic and ice Ih in the case of kaolinite, where the cubic polytype is basically absent throughout the whole nucleation process.Ic or ice Ih according to the different degree of supercooling, thus contributing to unravel the strong ice nucleating ability of CHLM crystals along such a wide range of temperatures. This argument would suggest that the multi-component nature of ice nucleation on biological matter could be at least partially attributed to a greater variety of nucleation sites – as well as the specific templating effect of functional groups acting as hydrogen bond donor and/or acceptors with respect to supercooled liquid water. Moreover, we have shown in this work that some of these functional groups – such as the hydroxyl groups characterising the water–CHLM–OH001 interface – can even promote a different ice polytype at the same time, possibly according to different supercooling.Interestingly, despite the predominance of ice Finally, we note that, in agreement with previous simulations of ice nucleation,4By means of a blend of experiments and simulations, we have unravelled the origins of ice nucleation on cholesterol (CHL), a prototypical organic crystal of relevance to cryopreservation. Our results suggest that its exceptional ice nucleating activity stems from the ability of its flexible hydrophilic surface to form unconventional ice-templating structures – specifically, hydrogen bonded cages comprising 6- as well as 5-membered rings. In addition, the experimental evidence reported here suggests that the intrinsic potential of cholesterol to nucleate ice may potentially be enhanced by specific topological features of the crystalline habit. In particular, droplet freezing measurements show that cholesterol promotes the heterogeneous formation of ice across a wide range of temperatures (from –4 to –20 °C). In fact, we find that CHLM crystals nucleate ice far better than the mineral feldspar, which is one of the most effective inorganic ice nucleating agents of relevance to atmospheric science. Moreover, electron microscopy measurements suggest that the broad range of freezing temperatures we observe for CHLM crystals may be due to the coexistence of diverse structural features of the crystalline surface, which in turn can act as different nucleation sites. The microscopic structure of the latter remains to be assessed, but the possibility that different parts of the CHLM surface may nucleate ice with different efficiency suggests that surface topography can play an important role in determining the ice nucleating ability of organic crystals.e.g. Lennard-Jones crystals, which allow to rapidly explore different surface geometriese.g. the clay mineral kaolinite, where only the hexagonal polytype was observed along the whole nucleation process.TS = 42 K). Kaolinite and CHLM are both characterised by an hydrogen bond network capable of facilitating the formation of ice nuclei: thus, the substantial difference in the ice nucleating ability we observe experimentally for these two compounds is most likely rooted into their surface topography. In fact, the ns data reported in Surprisingly, we find that CHLM crystals, despite being exceptionally good ice nucleating agents, do not provide a conventional template for ice to form. Specifically, molecular simulations reveal that, as opposed to what has been reported for supercooled water in contact with simple model substrates , but the flexibility of the surface and the low density of such functional groups is typical of biological nucleating agents such as macromolecules and bacterial fragments. This is especially relevant in the case of CHL, a molecule which is not only used in crystalline form as an ice nucleating agent in cryopreservation applications, but that significantly contributes to the composition of animal cell membranes as well.In addition, the emergence of stacking disordered ice phases during the heterogeneous formation of ice has been experimentally observed,In summary, the experiments and simulations presented in this work indicate that cholesterol crystals are incredibly efficient ice nucleating agents, active across a broad range of supercooling. We show that such strong ice nucleating activity is due to the intrinsic potential of the flexible amphoteric surfaces of CHLM to form unconventional ice-templating molecular structures. It is likely that microscopic structural features of the crystals could further enhance the ability of CHLM  to form ice, by offering a diverse array of nucleating sites. In fact, we believe that for an ice nucleating agent to be very efficient, a combination of interfacial “chemistry” and surface topography is generally required. This interplay could thus be the key to understand the heterogeneous formation of ice on molecular organic crystals, and it may provide a starting point for the investigation of ice in soft and biological matter at the molecular level. In particular, tailoring the microscopic structure of the substrate and modifying the nature as well as the density of hydrogen-bonding functional groups at the water–substrate interface can be seen as two different routes to engineer the ice nucleating ability of novel cryoprotectants, the design of which, at the moment, largely relies on the high-throughput screening of whole libraries of different compounds. The absence of a proper structure-to-function paradigm is perhaps the most pressing challenge in cryobiology: this is why future work will be devoted to assess whether and how hydrogen-bonding functional groups other than hydroxyls would be equally effective to enhance the kinetics of heterogeneous ice nucleation.There are no conflicts to declare.Supplementary informationClick here for additional data file.


We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se, and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve  with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313  Synapse functionality is the result of several signaling pathways converging on intracellular kinases, which phosphorylate protein targets to perform adequate adaptive changes  were cared for in accordance with the guidelines of the European Community Council Directive for the humane treatment of laboratory animals. All the procedures realized were reviewed and approved by the Animal Research Committee of the Universitat Rovira i Virgili. Diaphragm, levator auris longus (LAL) muscle, brain and spinal cord were quickly removed and transferred to ice-cold dissection Ringer. At least three animals were used to evaluate the following techniques.Young adult Sprague-Dawley rats , rabbit anti-PKCε (Cat# sc-214 RRID:AB_2237729), goat anti-pPKCε (Ser 729) (Cat# sc-12355 RRID:AB_2171921) polyclonal antibodies and mouse monoclonal anti-GAPDH (Cat# sc-32233 RRID:AB_627679). Primary antibodies purchased from Abcam: Rabbit anti-pPKCβI (Thr 642) (Cat# RRID:AB_1310586), anti-pMunc18-1 (Ser-313) (Cat# ab138687) polyclonal antibodies. Rabbit monoclonal Munc18-1 (D4O6V) antibody was purchased from Cell Signaling Technologies (Cat# 13414) and mouse monoclonal Na+/K+-ATPase from Developmental Studies Hybridoma Bank (Cat# RRID:AB_528092).Primary antibodies purchased from Santa Cruz Biotechnology: rabbit anti-PKCβI (Cat# sc-209 RRID:AB_10015282), Rabbit anti-mouse from Sigma (Cat# A9044 RRID:AB_258431) and rabbit anti-goat from Molecular probes (Cat# R21459 RRID:AB_11180332). Immunohistochemistry was performed with antibodies widely utilized as markers of NMJ neuron and Schwann cell : mouse anti-syntaxin (Cat# S0664 RRID:AB_477483) and mouse anti-neurofilament-200 (Cat# N2912 RRID:AB_477262) monoclonal antibodies from Sigma. Mouse anti-S-100 monoclonal antibody (Cat# AM10036FC-N RRID:AB_1622661) from Acris, Germany. The secondary antibodies used were donkey anti-rabbit or anti-mouse conjugated to Alexa Fluor 488 and Alexa Fluor 647 from Molecular Probes  . Postsynaptic AChRs were detected with α-bungarotoxin (α-BTX) conjugated to TRITC from Molecular Probes  (Cat# T1175 RRID:AB_2313931).The secondary antibodies conjugated to HRP were donkey anti-rabbit from Jackson Immunoresearch Labs  for the presynaptic stimulation treatment (see below). This peptide selectively inhibits sarcolemmal voltage-dependent sodium channels (VDSCs) without altering ACh signaling . This antibody has been functionally validated as a TrkB selective inhibitor, reducing BDNF effects without binding to TrkA, TrkC nor p75NTR  obtained from BD Transduction Laboratories  in a 100-fold dilution.2+) concentration (5 mM of Ca2+) and phorbol 12-myristate 13-acetate (PMA)  and specific translocation inhibitors. The specific cPKCβI inhibitor βIV5−3 peptide  and continuously bubbled with 95/5% of O2/CO2 at room temperature. Muscles were stimulated ex vivo through the phrenic nerve at 1 Hz by the A-M Systems 2100 isolated pulse generator . The frequency of 1 Hz allows the maintenance of different tonic functions  without inducing synaptic plasticity. Visible contractions of the diaphragm muscle served to verify successful nerve stimulation resulting in contraction. Three main experiments were performed to distinguish the effects of synaptic activity from those of muscle activity .Diaphragm muscles were excised through the phrenic nerve, as it has been previously described  in lysis buffer  and protein lysates were collected. Protein concentrations were determined using the Bio-Rad DC protein assay .+/K+-ATPase immunoreactivity were used to determine the purity of the fractionation. Protein concentration was determined using the Bio-Rad DC protein assay .Membrane/cytosol fractioning required muscle samples to be homogenized before freezing and in a detergent-free lysis buffer (in mM): 150 NaCl, 20 Tris-HCl (pH 7.4), 5 EDTA, 1 PMSF, 50 NaF, 1 OrtoVaNa, and protease inhibitor cocktail (1/100). Homogenized samples were centrifugued at 1,000 g for 15 min and the resulting supernatant was further centrifuged at 130,000 g for 1 h. Afterwards, the supernatant corresponded to the cytosolic fraction and the pellet, to the membrane fraction. The membrane was resuspended in lysis buffer containing (in mM) 150 NaCl, 20 Tris-HCl (pH 7.4), 5 EDTA, 1 PMSF, 50 NaF, 1 OrtoVaNa, and protease inhibitor cocktail (1/100). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NaProtein samples of 30 μg were separated by 8 or 15% SDS-polyacrylamide electrophoresis and electrotransferred to PVDF membranes . Membranes were blocked in tris-buffered saline with Tween 20 containing 5% (W/V) nonfat dry milk or 5% (W/V) bovine serum albumin and incubated with a primary antibody overnight at 4°C and the corresponding secondary antibody for 1 h.RRID: SCR_002368). The integrated optical density of the bands was normalized in relation to (1) the background values and (2) the total protein transferred on PVDF membranes, measured by total protein blot staining . The relative variations between the experimental samples and the control samples were calculated from the same membrane image. Data are mean values ± SEM. Statistical significance of the differences between groups was evaluated under the Wilcoxon test or the Student's t-test and the normality of the distributions was tested with the Kolmogorov–Smirnov test. The criterion for statistical significance was p < 0.05 vs. the control.Blots were visualized with the ChemiDoc XRS+ Imaging System  and the enhanced chemiluminescence (ECL) kit from Amersham Life Science, Arlington Heights, IL. Sample loading and antibodies were optimized to guarantee the linear range during the first minute of exposure. The densitometry of the bands was obtained with the MetaMorph Microscopy Automation and Image Analysis Software (RRID:SCR_003070) under identical conditions of light intensity and camera gain settings. Images were assembled using Adobe Photoshop software  and neither the contrast nor brightness were modified.Whole muscles were processed by immunohistochemistry to detect and localize Munc18-1 at the NMJ. Diaphragm and LAL muscles from young adult rats were fixed with 4% paraformaldehyde for 30 min. After fixation, the muscles were rinsed with phosphate buffer saline (PBS) and incubated in 0.1 M glycine in PBS. The muscles were permeabilized with 0.5% Triton X-100 in PBS, and nonspecific binding was blocked with 4% BSA. Then, they were incubated overnight at 4°C in mixtures of three primary antibodies raised in different species  and then rinsed. The muscles were then incubated for 4 h at room temperature in a mixture of appropriate secondary antibodies. AChRs were detected with α- Bungarotoxin conjugated to tetramethylrhodamine (TRITC). As a control, primary antibodies were omitted from some muscles during the immunohistochemical procedures. These control muscles never exhibited positive staining. In double-staining protocols, omitting either one of the two primary antibodies completely abolished the corresponding staining and there was no cross-reaction with the other primary antibody. At least three muscles were used as negative controls. Localization of the Munc18-1 at the NMJ was observed with a laser-scanning confocal microscope (Nikon TE2000-E). Special consideration was given to avoid contamination between channels. In experiments involving negative controls, the photomultiplier tube gains and black levels were identical to those used for a labeled preparation made in parallel with the control preparations. At least 6 muscles and 25 endplates per muscle were studied. Zeiss LSM880 AiryScan Confocal microscope, with higher resolution, was used to image three muscles treated with PMA or εV1-2 or PBS and doubly immunolabeled to detect Munc18-1 and AChR. Images were taken with a Zeiss PlanApo × 63 1.42 NA oil objective. The fluorescence intensity of each NMJ, normalized to background, was analyzed with ImageJ  was determined in diaphragm, brain and spinal cord by immunobloting. In all tissues, the used antibodies only recognized the corresponding protein, reacting with a 70 kDa band for Munc18-1 and a 68 kDa band for pMunc18-1 as predicted by the manufacturers Figure .+/K+-ATPase immunoblotting. GAPDH was detected in the cytosol fraction and essentially undetectable in the membrane fraction. In concordance, the transmembrane protein Na+/K+-ATPase was highly enriched at the membrane and undetectable in the cytosol.Munc18-1 phosphorylation regulates its interaction and dissociation with syntaxin-1 and other binding partners in the membrane  showed a remarkable amount of Munc18-1 and a particulary high amount of its phosphorylated form on Ser-313 Figure . The spi2+, the primary intracellular trigger for exocytosis, induces Munc18-1 phosphorylation on Ser-313 in chromaffin cells  increases pMunc18-1 and decreases total Munc18-1. Accordingly, the ratio pMunc18-1/Munc18-1 increases . On the contrary, low Ca2+  only increases total Munc18-1 , decreasing the ratio pMunc18-1/Munc18-1 . Therefore, the data show that expression and phosphorylation of Munc18-1 on Ser-313 depend on Ca2+ at the NMJ in a similar way than in synaptosomes, chromaffin cells and neuron cultures . Figures Immunofluorescence staining coupled with confocal microscopy analysis was performed to determine the location of Munc18-1 at the adult NMJ. Experiments were performed in the diaphragm and LAL muscles and immunoreactivity for Munc18-1 was identical in both muscles. We colocalize Munc18-1 with NMJ markers . All pictures in Figure e Figure  and the e Figure  images se Figure . Munc18-e Figure  or Schwae Figure  in blue Stimulation (St) in the figures]. Muscle contraction includes membrane depolarization of the muscle fiber involving voltage-gated sodium channels and myofiber contraction (termed Contraction in the figures). In particular, muscle contraction was inhibited using μ-CgTx-GIIIB  and pMunc18-1  levels . Because the analysis of protein translocation is important to understand their functionality, we proceeded to evaluate whether the stimuli can move Munc18-1 between membrane and cytosol. Nerve stimulation significantly increased pMunc18-1 in the cytosol fraction  to match the effect of 30 min of stimulation. On the other hand, Munc18-1 gradually increased from 10 to 30 min  because phosphorylation is a rapid process. Surprisingly, 1 min of presynaptic stimulation did not modify pMunc18-1 levels while 10 min significantly increased them . Higher resolution NMJ images after a treatment with PBS as a control, PMA and εV1−2 peptide indicate that when all the PKC isoforms are activated by PMA, Munc18-1 levels are upregulated in the nerve terminal  and a significant increase in total Munc18-1 level . In concordance, the ratio pMunc18-1/Munc18-1 was significantly decreased  indicating that nPKCε specifically enhances phosphorylation of Munc18-1 when synaptic activity increases  phrenic nerves of muscles previously incubated with the blocking peptide . We found a significant decrease in pMunc18-1 level . We found a significant increase of Munc18-1  without changes on pMunc18-1 level . However, βIV5−3 during synaptic activity caused a significant increase in pMunc18-1 with no change in total Munc18-1. In concordance, the ratio pMunc18-1/Munc18-1 was significantly increased , indicating that the role of the cPKCβI during synaptic activity reduces Munc18-1 phosphorylation. As with nPKCε above, we compared control and stimulated samples in which cPKCβI had been blocked  with no changes in pMunc18-1  being the ratio pMunc18-1/Munc18-1 . These results indicate that cPKCβI is necessary for the increase of Munc18-1 phosphorylation caused by nerve stimulation but not for the increase of Munc18-1 levels  in both basal and synaptic activity conditions. The pnPKCε/nPKCε ratio significantly increased . Moreover, in St + βIV5−3 vs. Ctrl + βIV5−3 both nPKCε and pnPKCε significantly increased. These results indicate that cPKCβI isoform downregulates nPKCε phosphorylation and maybe its activity. Together, these data indicate that synaptic activity activates pnPKCε to phosphorylate Munc18-1 while cPKCβI decreases it, maybe inhibiting pnPKCε.Having established that nPKCε and cPKCβI oppositely regulate Munc18-1 phosphorylation, we next investigated whether cPKCβI and nPKCε regulate each other. Figure 1−2 significantly decrease cPKCβI without changing pcPKCβI levels in both basal and synaptic activity conditions. These results indicate that pnPKCε isoform enhances cPKCβI levels but does not affect its activity. Together, these results demonstrate a mutual regulatory influence between nPKCε and cPKCβI.On the contrary, Figure p < 0.05). This result indicates that endogenous BDNF-TrkB pathway inhibits Munc18-1 phosphorylation. However, exogenous BDNF  in nerve-stimulated muscles (St) did not modify the pMunc18-1 levels during stimulation  and probably its activity. Because TrkB decreases pMunc18-1 levels  with stimulated muscles that were preincubated with μ-CgTx-GIIIB (Stimulation), we found that pMunc18-1 levels decreased  and the ratio of pMunc18-1/Munc18-1 was maintained . These results indicate that muscle contraction per se prevents  the nerve activity-induced Munc18-1 and pMun18-1 increase  and declined under baseline at 30 min of stimulation-induced contraction  and raised back to the baseline after 30 min . Thus, nPKCε could phosphorylate Munc18-1 during contraction. On the other hand, we analyzed the action of cPKCβI. βIV5−3 peptide decreased pMunc18-1, increased Munc18-1 and the ratio pMunc18-1/Munc18-1 decreased   Figure . This rep < 0.05).Figure Altogether, nerve stimulation increases pMunc18-1 and muscle contraction prevents the effect of stimulation on Munc18-1 and pMunc18-1. Moreover, nPKCε phosphorylates Munc18-1 both in basal and synaptic activity conditions (with and without contraction), being higher the effect without contraction. The cPKCβI inhibits nPKCε phosphorylation activity in all the previous conditions. Interestingly, during muscle contraction, the role of the cPKCβI shifts to upregulate pMunc18-1.We described above that the continued stimulation and contraction activity caused an important fall of pMunc18-1 after 30 min. During contraction, 47/TrkB did not affect Munc18-1 but increased pMunc18-1. This result suggests that pMunc18-1 depends on TrkB signaling. Surprisingly, exogenous BDNF during contraction significantly decreased pMunc18-1 and Munc18-1 protein levels Figure . Thus, tIn the neuromuscular system, presynaptic PKCs are fundamental for the neuromuscular function. In particular, synaptic activity couples presynaptic exclusive cPKCβI and nPKCε to acetylcholine release through the BDNF/TrkB pathway  are only revealed in the absence of phosphatase activity. Therefore, during synaptic activity, the phospho-dephosphorylation balance tilts toward accumulating Munc18-1. This indicates that phosphatase activity plays a role at short stimulation times whereas prolongated stimulation activity continuously increases pMunc18-1 level to be finally sustained. This effect may be related with a positive adaptive plasticity.Altogether, our results show that stimulation increases both total and phosphorylated Munc18-1 in muscles, similarly to the PMA treatment. This suggests that Munc18-1 phosphorylation may be directly modulated by PKC during synaptic activity.nPKCε and cPKCβI are exclusively located in the nerve terminal of the NMJ, regulated by synaptic activity and involved in neurotransmitter release . Thus, in basal conditions, there is low endogenous BDNF production and TrkB receptors are free to receive exogenous BDNF. This downregulates pMunc18-1 as synaptic activity-increased endogenous BDNF does. This is in accordance with the fact that exogenous BDNF did not change cPKCβI and nPKCε phosphorylation and activation in stimulated muscles (Obis et al., As stated, synaptic activity enhances the BDNF/TrkB pathway to increase cPKCβI activity (Hurtado et al., Increasing evidence indicate that muscle contraction has a retrograde effect on the NMJ probably through BDNF (Matthews et al., There is a complex change in nPKCε, cPKCβI, and Munc18-1 interactions when synaptic activity ends up in muscle contraction. It seems that cPKCβI shifts to increase pMunc18-1 after 30 min of contraction, which is opposite to what is observed in stimulation without contraction. Muscle contraction induced similar effects on TrkB and cPKCβI than presynaptic activity, suggesting that both molecules work coordinately. Therefore, there is a similar shift in the coupling of the endogenous BDNF/TrkB signaling and cPKCβI to promote Munc18-1 phosphorylation. Thus, TrkB pathway enhances Munc18-1 phosphorylation during contraction like cPKCβI and nPKCε. Altogether, it seems that the muscle contraction-induced retrograde factor that prevents the synaptic activity-induced increase of pMunc18-1 is TrkB independent.NTR. The result also reinforces the idea that it has the same effect as contraction. BDNF effect could act through the same pathway that muscle contraction triggers or another one. Evidence support that muscle contraction induces BDNF synthesis (Matthews et al., Is thought-provoking that muscle contraction reduces Munc18-1 and pMunc18-1 but, in the same condition, cPKCβI, nPKCε, and TrkB increase pMunc18-1. Surprisingly, exogenous BDNF decreases pMunc18-1 and Munc18-1 during contraction, which indicates that exogenous and endogenous BDNF act differently. Probably, the exogenous is binding a different receptor, such as p75Figure in vivo. Here we demonstrate that the synaptic activity-dependent phosphorylation of Munc18-1 at the NMJ is regulated by the BDNF/TrkB signaling through nPKCε and cPKCβI isoforms and that muscle contraction prevents it. These results provide a molecular insight about how neuromuscular activity influences neurotrophic control to phosphorylate Munc18-1 and how this retrograde signaling regulates specific presynaptic PKC isoforms.Munc18-1 is a synaptic exocytotic molecule, phosphorylated by PKC, essential for appropriate neurotransmitter secretion, whose regulation by PKCs and synaptic activity is little known AS data collection, quantitative analysis, literature search, data interpretation, statistics. VC-M literature search, data interpretation, design graphic abstract. LJ-B literature search, data interpretation. LN, EH, and MT data interpretation. JT, ML, and NG conception and design, literature search, data interpretation, manuscript preparation.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.


Results showed that [15]aneN4S had a similar efficacy to DMSA and BAL in reversing the inhibition of MeHg over purified TrxR and Trx by looking at both the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay and insulin reduction capability. In experiments with cells, none of the chelating agents could reverse the inhibition of TrxR by MeHg, which corroborates the high affinity of MeHg to the selenol in TrxR active site. [15]aneN4S and BAL, unlike DMSA, could prevent inhibition of Trx, which allows the maintenance of downstream functions, although BAL showed higher toxicity to cells. Overall these findings highlight the potential of using [15]aneN4S in the treatment of MeHg poisoning and encourage further studies, namely in vivo. Methylmercury (MeHg) is a highly neurotoxic compound to which human populations are exposed via fish consumption. Once in cells, MeHg actively binds thiols and selenols, interfering with the activity of redox enzymes such as thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR) which integrate the thioredoxin system. In fact, it has been shown that inhibition of this system by MeHg is a critical step in the unfolding of cell death. Current clinical approaches to mitigate the toxicity of MeHg rely on the use of chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) which largely replaced British anti-Lewisite or 2,3-dimercapto-1-propanol (BAL) as the prime choice. However, therapeutic efficacy is limited and therefore new therapeutic options are necessary. In this work, we evaluated the efficacy of a macrocyclic chelator, 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN Methylmercury (MeHg) is reckoned as a highly neurotoxic compound, especially during fetal development when it can hinder neurodevelopment . In extrIn general, exposure of the human population to MeHg is much lower than in the case of the Minamata outbreak. However, populations consuming large amounts of fish—namely predatory species such as bluefin tuna, swordfish and black scabbard fish—can be exposed to MeHg levels that may cause cognitive and neuromotor deficits in children exposed in utero . TherefoThe neurotoxic effects of MeHg encompass inhibition of glutamate re-uptake at the synaptic cleft  and chanPreviously, we have shown in vitro and in vivo that Hg compounds including MeHg bind the active site of both Trx and TrxR resulting in inhibition of activity ,16,17,18Limiting the consumption of contaminated fish is the most effective strategy to reduce exposure and the risk of methylmercury-related toxicity ,19. Howe2+ inhibition but had a more limited effect over MeHg, thus mimetizing clinical findings aneN3HgCl), dithiothreitol (DTT), 3--2,5-diphenyltetrazoliumbromide (MTT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), insulin (human recombinant), BAL and DMSA were purchased from Sigma-Aldrich. Methylmercury chloride . Antibodies were all from Santa Cruz Biotechnologies, with the exception of the antibody for human thioredoxin 1 which was from IMCO.Rat recombinant TrxR1 and human Trx1 were from IMCO, Sweden  was incubated in 50 mM Tris-HCl, pH 7.5 at 37 ºC for 5 min with MeHg (200 nM), 100 µM NADPH and 40 µM of either [15]aneNur group  and corrTrxR activity was determined as previously described  by addin4S, BAL or DMSA. Afterwards, a master mix containing insulin and NADPH  was added and the change in absorbance at 340 nm (NADPH consumption) was followed in a microplate reader. Activity was calculated as the linear change in absorbance over the initial 5 min.To assess effects over the whole Trx system, activity was determined by a method previously described by Árner and Holmgren with modifications . Briefly® and cultured in a medium consisting of a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and F-12, supplemented with 10% fetal bovine serum and 1% Pen-Strep mixture in a humidified incubator at 37 °C and 5% CO2.Human neuroblastoma cells (SH-SY5Y) were purchased from ATCC4 cells/well) were cultured in 96-well plates (Nunc®) and allowed to seed for 24 h before addition of various concentrations of each chelating agent—[15]aneN4S , BAL , DMSA . MTT was added to plates at a final concentration of 400 mg/mL followed by incubation at 37 ºC for 2 h. After incubation, the medium was removed and the formazan crystals were dissolved in a 4:1 DMSO/glycine buffer (pH 10.5) with shaking for 15 min at room temperature. Cell viability was then assessed by measuring formazan absorption at 550 nm in a microplate reader . Appropriate controls for vehicle (ethanol 0.4%) and spontaneous MTT reduction by chelating agents were conducted in parallel.Cell viability was determined by the MTT assay after 24 h of exposure to each compound as described previously . BrieflyBased on viability results, test concentrations for subsequent experiments were set at 40 µM for all chelating agents. The MeHg concentration used in the following experiments (2.5 µM) was selected based on previous results by our group .6 cells/dish) in culture dishes (100 mm × 2 mm) and allowed to reach 70%–80% confluence. Fresh medium was then supplied to cells followed by the addition of MeHg (2.5 µM) and chelating agents . Coexposure to MeHg and each of the chelating agents was achieved by simultaneous addition to the culture media. All treatments lasted 24 h after which cells were harvested, washed in phosphate-buffered saline  and centrifuged  to obtain the final cell pellets which were disrupted in lysis buffer ). Before activity determinations, samples were centrifuged at 12,000× g and 4 °C for 5 min. Total soluble protein and enzymatic activities were determined in the supernatant as described below.Cells were plated , followed by transfer to a nitrocellulose membrane which was subsequently blocked with a 5% skimmed milk solution and probed with the following primary antibodies: anti-human TrxR1 rabbit polyclonal IgG , anti-human Trx1 rabbit polyclonal IgG , anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal IgG . Band intensity was quantified using the QuantityOne Software (Biorad®) and protein expression levels were normalized for protein loading on the gel, assessed either by Ponceau S staining prior to the blocking step or by quantification of GAPDH.Western blot was used to determine the effect of MeHg and chelating agents on the expression levels of TrxR and Trx. Samples (30 μg of protein) were separated under reducing conditions with SDS-PAGE on a 4%–12% Bis-Tris gel with MES running buffer (ThermoFisherp < 0.05 and very significant at p < 0.01.Results in figures are presented as mean ± standard error (SE)  to MeHg (200 nM) produced a very significant decrease in DTNB reduction (to 25% of control) a. None o4S . Exposure of cells to 40 µM of [15]aneN4S and BAL did not decrease TrxR activity, whereas DMSA caused a 20% drop in activity (p < 0.01). Most importantly, the chelating agents failed to prevent the inhibition of TrxR by MeHg (Exposure of SH-SY5Y cells to 2.5 µM of MeHg for 24 h resulted in a decrease of TrxR activity to 33% of control level ( by MeHg a.p < 0.01). Like with TrxR, the chelating agents did not affect Trx activity significantly. However, both [15]aneN4S and BAL were able to fully prevent Trx inhibition by MeHg (p < 0.05) whereas DMSA did not show any protective capability (p > 0.05) (Thioredoxin activity was less affected by exposure of SH-SY5Y to MeHg with a drop to 47% of control ( > 0.05) b. Concerning the effect of each treatment on the expression of TrxR and Trx , it is iKa (5.4), are deprotonated at physiological pH and are, therefore, preferential targets for Hg compounds such as MeHg. Indeed, TrxR is a known target for MeHg with its inhibition being a primary event in the unfolding of cell death, which involves subsequent oxidation of Trx and apoptosis initiation aneN and BAL b. This s and BAL , which ce tested .Noteworthy is the fact that DMSA, a standard chelating agent in organic Hg poisoning , could nIn this study, we evaluated the efficacy of a macrocyclic chelator in preventing MeHg toxicity by comparison with standard clinical chelating agents, BAL and DMSA. A very relevant aspect of this study is the choice of toxicity endpoints that are primary molecular targets of MeHg, namely TrxR and Trx, which are involved in the mechanism of MeHg-induced cell death.4S showed a capability comparable to that of BAL and DMSA in protecting the activity of purified suspensions of the thioredoxin system. In cells, [15]aneN4S proved less toxic than BAL and much more effective than DMSA in reversing effects over the thioredoxin system. Indeed, although experiments with cells revealed that none of the chelating agents were capable of protecting TrxR activity from MeHg, both [15]aneN4S and BAL could shield Trx activity against MeHg inhibition. Since the thioredoxin system is a highly relevant target in the unfolding of MeHg toxicity, this result encourages further research concerning the antidote capacity of [15]aneN4S, namely in vivo studies. This is especially relevant since the chelating agent [15]aneN4S presented a better compromise between efficacy and toxicity when compared with two other clinically relevant antidotes. The chelator [15]aneN


MLKL protein expression were upregulated at 48 h after injury and MLKL immunolocalised to retinal binding protein with multiple splice (RBPMS)+ RGC, inner nuclear cells and ONL cells, specifically at the retinal injury site. RIPK3 expression did not increase but RIPK3 co-immunolocalised with RBPMS+ RGC in intact and injured retinae. In vitro, a Nec-1s concentration of 0.01 pg/µL was RGC neuroprotective. In the blunt ocular injury rat model, Nec-1s prevented RGC death at the center of the impact site but did not protect against ONL thinning or provide functional restitution. RGC degeneration in our blunt ocular injury model is site-specific, with necroptosis driving death at the center of the focal impact site.Closed-globe injury can cause visual loss in military and civilian populations, with retinal cell death, including retinal ganglion cell (RGC) degeneration, leading to irreversible blindness. RGC and optic nerve (ON) degeneration after eye or head injury is termed traumatic optic neuropathy (TON). There are currently no treatments for RGC loss, therefore novel therapeutics to prevent RGC death or promote axonal regeneration are a priority. We investigated necroptotic signaling mechanisms in a rat blunt ocular injury model. After bilateral blunt trauma, protein expression and retinal localization of necroptosis pathway members  were assessed by Western blot and immunohistochemistry (IHC), and potent necroptosis inhibitor Necrostatin-1s (Nec-1s) was delivered by intravitreal injection to one eye and vehicle to the contralateral eye. RGC and photoreceptor survival were assessed by cell counting and outer nuclear layer (ONL) thickness measurements on histology. The neuroprotective effects of Nec-1s were assessed in primary retinal culture by βIII-tubulin Ocular injuries are common, occurring in 10% of all military casualties . The ligTraumatic optic neuropathy (TON) can be caused by head or eye injury and is associated with RGC and axonal degeneration, causing irreversible blindness ,4. The ONecroptosis (controlled necrosis) is a regulated cell death pathway resulting in cell leakage, organelle swelling, cytoplasmic granulation and the initiation of an inflammatory response ,20. ReceNecroptosis promotes neuronal cell death in trauma, neurodegeneration and eye disease . RIPK1 ePreviously, we have shown that RGC die by caspase-2-dependent mechanisms in the peripheral portion of the focal retinal impact site, but we did not identify the mechanisms of RGC death at the center of the impact site, where there is the greatest RGC death . Blanch + and brain-specific homeobox/POU domain protein 3A (BRN3A)+ RGC counted, and outer nuclear layer (ONL) thickness measured in retinal sections.To determine the role of necroptosis in RGC and photoreceptor death after blunt ocular injury, groups of adult female adult Lister Hooded rats were subjected to unilateral or bilateral blunt ocular injury under anesthesia, as previously described . WesternAnimal procedures were licensed by the UK Home Office, approved by the University of Birmingham’s Animal Welfare and Ethical Review Committees and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Female Lister-hooded rats, weighing 180–220 g, were purchased from Charles River Laboratories , kept on a 12 h light–dark cycle with a daytime luminance of 80 lux and fed and watered ad libitum. Surgery and ERG recordings were performed under inhalational anesthesia with 2–3% isoflurane in oxygen. Blunt ocular injury was induced as previously described . BrieflyRats were killed at 5, 24 and 48 h after bilateral blunt ocular injury, as well as uninjured intact controls, by overdose of anesthetic . Protein extraction and Western blot were performed as previously described . BrieflyNec-1s  was reconstituted at 3.6 mM in 10% dimethyl sulfoxide (DMSO) and 0.9% methyl-β-cyclodextrin (Sigma) in sterile phosphate buffered saline (PBS). Bilateral blunt ocular injury was performed and 5 µL of Nec-1s administered by unilateral intravitreal injection and 5 µL of vehicle  was injected into the contralateral eye. Injections were performed immediately after injury and at 7 dpi until culling and tissue collection at 14 dpi . Uninjured animals were culled without intravitreal injections as intact controls . Animals were intracardially perfused under terminal anesthesia and eyes processed for immunohistochemistry.TM  coated glass microscope slides, and stored at −20 °C until required.Tissues were prepared for immunohistochemistry as previously described . BrieflyFrozen sections were left to thaw for 20 min and washed 3 × 5 min in PBS, followed by 20 min permeabilization and blocking non-specific sites in PBS containing 1% Triton-X-100 (Sigma) and 3% bovine serum albumin . Sections were incubated overnight at 4 °C with appropriate primary antibodies  in 0.5% 2 over the same range. DTL fiber  corneal electrodes with pressure-molded Aclar  contact lenses were used with needle skin electrodes (Unimed). ERG traces were analyzed using the manufacturer’s semi-automated software ERG View (Ocuscience) and marker position manually verified and adjusted where necessary by a masked observer. Photopic negative response (PhNR) was recorded as a measure of RGC function.ERG were recorded  at 7 and 14 dpi and in uninjured controls and were interpreted using ERG View (Ocuscience). Animals were dark-adapted overnight and prepared for ERG under dim red light (>630 nm). Scotopic (dark-adapted) flash ERG were recorded from −2.5 to +1 log units with respect to standard flash in half log unit steps and photopic (light-adapted) flash ERG were recorded with background illumination of 30,000 mcd/mAnimals were culled at 14 dpi and eyes processed as for immunohistochemistry. ONL thickness was measured by a masked observer, as previously described, to determine photoreceptor survival . Briefly+ and RBPMS+ immunostained RGC were counted by a masked observer, in the GCL across the entire retinal section. Seven retinal sagittal sections per eye were counted: at the optic disc and center of the impact site (0 μm), and at 600, 1200 and 1800 μm to either side of this plane, and quantified as the number of BRN3A+ or RBPMS+ RGC per mm of retina.RGC survival was quantified using immunohistochemistry for BRN3A ,38 and Rv/v B27 supplement , 0.5 mM L-glutamine (Invitrogen) and 0.5% v/v Gentamycin (50 mg/mL Invitrogen), at 37 °C in 95% O2 and 5% CO2. After 1-day incubation, 200 μL of supplemented media containing various concentrations of Nec-1s was added to the wells, to make a final concentration of 0.01–100 pg/μL of Nec-1s in 500 μL of media. Cells were then fixed with 4% PFA in PBS, blocked in 0.1% Triton-X-100, incubated in primary antibody solution  counted and displayed as average number of βIII-tubulin+ RGC per mm2. Primary retinal cultures were performed in duplicate and repeated on three independent occasions.Adult 6–8 week-old Sprague Dawley rats  were killed by overdose of anesthetics and retinae dissected and dissociated into single cells with a Papain dissociation kit  . A densisolution  in antibPower calculations performed in G*Power  indicated for Western blotting, n = three animals per time point had 82% power to detect a 1-fold change in protein levels, (assuming standard deviation = 20% band intensity); for ERG assessment  eight animals had a power of 88% to detect a moderate (f = 0.25) treatment effect (correlation among repeated measures = 0.5 from past data) .+ and RBPMS+ RGC count data and ONL thicknesses were analyzed using generalized estimating equations . Data are reported as mean ± standard error of the mean (SEM).Statistical analysis was carried out using SPSS  and GraphPad Prism version 7.00. . The data were tested for normality using the Shapiro–Wilk test. Western blot data were analyzed using one-way analysis of variance (ANOVA) and post-hoc Tukey tests with p values corrected for multiple comparisons. In experiments where multiple comparisons were made, the p values were corrected with Bonferroni correction and the correction factor is stated. ERG amplitudes, BRN3AWe first determined if the expression of key necroptotic proteins, RIPK1 and RIPK3 as well as downstream MLKL, were altered in our blunt ocular trauma model by performing bilateral blunt ocular injury collecting retinal tissue for immunofluorescent staining and Western blot at 5, 24 and 48 h after injury, as well as from separate intact control animals. Protein expression of RIPK1, RIPK3, MLKL were investigated using Western blot . Representative blots are shown in p < 0.0001, ANOVA; t-test p < 0.0001). We observed that the levels of RIPK1 in the retina increased at 5, 24 and 48 h after blunt ocular injury compared to levels in intact controls by Western blot . The levels of RIPK3 remained unchanged . The number of BRN3A+ A and RBP+ , with little effect of Nec-1s on the number of BRN3A+ and RBPMS+ RGC at distances more peripheral from the impact site  to assess retinal function. Intact animals receiving no injections were also analyzed . There were reductions in the PhNR , which pathways ,47, suggThe phosphorylation of MLKL at Ser345 is catalyzed by RIPK1 and promotes MLKL translocation and accumulation at the plasma membrane, inducing membrane permeabilisation and cell lysis ,23, althFor necroptotic signaling to be activated and induce cell death, apoptotic caspase-8 must be inhibited either through endogenous or pharmacological inhibitors or remain uncleaved. This allows for the formation of complex IIb and the association of RIPK1, RIPK3 and MLKL . Blanch RIPK1 is a key mediator of necroptotic cell death, but not exclusively, and also has roles in apoptotic and inflammatory pathways . SimilarAfter ON injury, RGC death begins within 5 days and there is >90% RGC loss at 14 dpi . Adult R+ RGC compared to 67% in siEGFP-treated blunt injured eyes, suggesting that 18% of RGC in this region degenerated by a process of caspase-2 dependent apoptosis. siCASP2 had no detectable effect on RGC survival at the impact site center [+ RGC were preserved in the center of the impact site, compared to 63% after control vehicle injection, suggesting that 15% of RGC in this region undergo necroptotic cell death. We did not observe any evidence of necroptotic death outside the center of the impact site, and in our previous paper we only observed evidence of caspase 2-dependent RGC death in the region peripheral to the impact site and none at the impact site center. This implies that caspase-2 and necroptotic death pathways occur simultaneously in the same cell type but in different regions of the impact site, probably related to the severity of injury at the different locations  and RBPMS+ (cytoplasmic marker) immunostained cells in the GCL [RGC were quantified by counting BRN3A the GCL ,70. BRN3 the GCL ,71,72. R the GCL ,40, but  the GCL . Nonetheripk3 gene knockout preserved cone photoreceptors and function [Photoreceptor cell bodies populate the ONL; therefore, ONL thickness can be used to assess photoreceptor survival ,34. In tfunction , and phofunction  and a mofunction . As an afunction , suggestScotopic (dark-adapted) and photopic (light-adapted) ERG amplitudes were measured to assess retinal function. The PhNR is a common measure of RGC function; it is reduced in experimental and human glaucoma  and corr+ and BRN3A+ RGC compared to vehicle. This suggests that Nec-1s might protect a proportion of RGC, but, as there was no increase in ERG amplitudes, Nec-1s did not provide any corresponding improvement in retinal function. We have previously shown that caspase-2-dependent RGC death occurred peripheral to the impact site, which, collectively with this study, suggests that multiple cell death signaling pathways can occur in parallel in the same tissue and in the same cell type, in a single injury model.In conclusion, we have demonstrated that RGC death signaling pathways are active in a region-specific manner after blunt ocular injury, with necroptotic-dependent mechanisms likely driving a reduction in RGC numbers at the center of the impact site. The inhibition of necroptosis using pharmacological inhibitor, Nec-1s, preserved the number of RBPMS


Atlantoaxial instability (AAI)/subluxation commonly occurs in small breed dogs. Ventral stabilization techniques using screws and/or pins and a plate or, more commonly, polymethylmethacrylate are considered to provide the most favorable outcome. However, the implantation of screws of sufficient sizes for long-term stability becomes challenging in toy breed dogs (e.g. <2 kg). We herein report the application of 3D printing technology to implant trajectory planning and implant designing for the surgical management of AAI in 18 dogs. The use of our patient-specific drill guide templates resulted in overall mean screw corridor deviations of less than 1 mm in the atlas and axis, which contributed to avoiding iatrogenic injury to the surrounding structures. The patient-specific titanium plate was effective for stabilizing the AA joint and provided clinical benefits to 83.3% of cases (15/18). Implant failure requiring revision surgery occurred in only one case, and the cause appeared to be related to the suboptimal screw-plate interface. Although further modifications are needed, our study demonstrated the potential of 3D printing technology to be effectively applied to spinal stabilization surgeries for small breed dogs, allowing for the accurate placement of screws and minimizing peri- and postoperative complications, particularly at anatomical locations at which screw corridors are narrow and technically demanding. Atlantoaxial instability (AAI) has been widely described in the veterinary literature. Although a few studies have been conducted on medium to large breed dogs, AAI predominantly occurs in small breed dogs (i.e. less than 5 kg) . Most prAlthough non-surgical, conservative management is selected for immature and/or high-risk patients , surgicaAlthough current surgical techniques provide a modest success rate, surgical complications, if they occur, are associated with a high mortality , 10. MosIn order to establish a safer surgical technique for AAI in small breed dogs, we utilized 3D printing technology. We herein describe our custom-made drill guide template system, which allows for the accurate placement of screws and a custom-made titanium plate for AA fixation.This research was conducted as a prospective multi-institutional clinical study performed on client-owned small breed dogs with AAI. Surgical procedures were performed at either private hospitals with informed consent from the owners or a university teaching hospital with informed consent from the owners and under the approval of the Institutional Animal Care and Use Committee of Gifu University . All procedures were performed in accordance with the guidelines regulating animal use and ethics at Gifu University.Dogs diagnosed as AAI by means of CT and MRI between January 2014 and June 2016 and underwent atlantoaxial fixation surgery were included in this study. Dogs that had lost to follow-up by 1 year post-surgery were excluded. All cases were diagnosed with AAI based on radiography followed by computed tomography (CT)   and magnetic resonance imaging (MRI) . Age and body weight were recorded at each visit. Neurological statuses were graded as previously reported [The high-accuracy 3D CT scans described above with a slice thickness of 0.5–1.0 mm were used to obtain preoperative images of the upper cervical spine. In cases under development (defined as those < 8 months old), CT scanning was delayed until they reached at least 8 months old. CT data of other cases were obtained on the day of the first visit. Images were exported in the DICOM format to 3D/multiplanar imaging software . The trajectories of the screws in the atlas and axis were decided on reconstructed bone images viewed on several optional planes . In the Drill guide templates were designed for each case in order to achieve the accurate guidance of the screws. The design of drill guide templates involved two steps. Bone data were extracted from CT data in the DICOM format using image processing software . The range of Hounsfield units (HU) to be extracted was set to only extract bone data and this was further adjusted through visual inspections of transverse images of the atlas and axis. Bone data were then transferred to 3D modeling software , the atlas and axis were separated, and the platforms of the drill guide templates that fit the surfaces of each bone were designed . Cylindr2 <0.13% as the printing material. Electron beam melting was operated at an ambient temperature of 730°C under a vacuum of less than 2×10−2 mbar and an acceleration voltage of 60 kV. The beam diameter was approximately 300 μm and the layer thickness was 50 μm, resulting in a maximum printing accuracy of approximately ±200 μm.Using reconstructed 3D images on modeling software , a luxated AA junction was reduced to the normal position . In caseAll surgical rehearsals and actual procedures were performed by the same individual (HK). All dogs were anesthetized using a standard protocol and maintained with isoflurane in oxygen. Dogs were positioned in a dorsal recumbency on a vacuum beanbag with the neck slightly elevated and extended. The surgical area was prepared and draped, including the caudal mandible and right proximal humerus. In most cases, autologous cancellous bone was harvested from the humerus for bone grafting; however, femurs were also used in some dogs. The approach to the AA junction was performed as described previously . The lonDrill guide templates were sterilized with an ethylene oxide gas sterilizer and the titanium plates were autoclaved. Specifically, the drill guide template was firmly attached to the ventral surface of C1 or C2 by manually pressing it to the bone using a mosquito forceps, and drill holes were made using Kirschner wires . We determined the appropriate depth of the drill hole based on a preoperative CT image. The Kirschner wire was held with a wire collet at a predetermined position so that the wire only passes through the cylindrical drill sleeve and penetrates the desired depth of the bone. After all screw holes had been drilled, the autologous bone graft was placed in the AA joint space and a titanium plate was fixed with the predetermined diameters of titanium cortical screws . After our first few cases, we noted that using the drill guide templates for the center screw hole of C1 and cranial two screw holes of C2 only made the remaining procedure easier. We made the above decision for the following reasons: 1) even though the drill guide templates were custom-made and, in theory, perfectly fit the vertebral surface, there was potentially a slight deviation between the guide and bone, particularly if the target bony surface lacked asperity, which normally helps engagement of the guide to the bone, 2) slight deviations in multiple guide-bone interfaces may potentially result in marked misalignment and preclude the insertion of some screws, and 3) once the plate was secured by the first screw, the remaining screw holes may be drilled through the screw holes of the plate with high precision. The longus colli muscles were apposed over the implants, and the remainder of the wound was closed in a routine manner.The locations of the screws were examined on postoperative CT scans. Deviations in screw trajectories were assessed using image analysis software . The DICOM data of preoperative and postoperative CT were imported into the software, the atlas and axis were visually superimposed individually, and the 3D coordinates of the implanted screw locations on preoperative CT images were identified. Deviations in the coordinates of the entry points and exit points between pre- and postoperative images on the transverse, dorsal, and sagittal planes were assessed. In addition, complications related to screw insertion were recorded intraoperatively and by assessing postoperative radiographs and CT images. Specifically, major intraoperative complications were defined as vertebral fracture, the violation of vital structures , and screw/plate dislocation were recorded. We designed screw coordinates for the atlas as bicortical engagement. Therefore, the violation of the spinal canal of the atlas by the inserted screws was not considered to be a complication unless the screws impinged on the cord. We also evaluated the bone mineral density (BMD)  and cortPerioperative (2 weeks), short-term (3 months) postoperative, and mid-term (12 months) postoperative follow-up evaluations consisted of physical and neurological examinations. At each visit, lateral and ventrodorsal views of plain radiographs were taken to evaluate if any implant failure was present . In 4 cases , a telephone interview was performed in order to assess short-term and/or long-term outcomes. Neurological grading was only performed when dogs were assessed directly by veterinarians. If postoperative complications were noted, potential predisposing factors such as age, body weight, neurological grades on the day of surgery, the degree of deviations in the inserted screws, vertebral HU, and cortical thickness were statistically assessed between the complication and non-complication groups.All statistical analyses were performed with the Wilcoxon rank-sum test by JMP®10 . The level of significance was set at p < 0.05.We performed AA fixation on 18 dogs. None of the dogs were excluded from this study. Seven dogs were male, 2 of which were castrated, and 11 dogs were female, 4 of which were spayed. Median age at onset was 28.5 months , while that at surgery was 35 months . Median body weight at surgery was 2.22 kg . AA anomalies were identified on CT images in 13 dogs; a hypoplastic dens was present in 8 dogs and non-union dens in 4 dogs, and 1 dog had a hypoplastic dens that was non-union. In 4 dogs, an obvious history of trauma was present that appeared to trigger the clinical onset. Three of these dogs had a hypoplastic dens. Clinical information and the imaging findings of all cases were summarized in After the diagnosis of AAI by CT and MRI , drill gThree screw holes  were drilled using the drill guide templates. The titanium plate was subsequently placed and fixed with cortical screws with a diameter of 1.2 mm or 1.7 mm depending on the size of the target vertebra. After placement of the titanium plate, the remaining screw holes were directly drilled through the plate screw openings and cortical screws were inserted. Therefore, the accuracy of the screw locations in each case was evaluated for 3 locations in which the screws were placed using the drill guide temples and 4 locations in which the screws were placed directly through the titanium plate without the use of the drill guide templates. Two cases were excluded from analyses because pre- and post-operative CT data were not obtained under the same imaging settings (Dog No.4 and 18). Deviations in the entry point for the C1 center screw on the X dimension , Y dimension , and Z dimension  were 0.46 ± 0.31 mm, 0.41 ± 0.30 mm, and 0.69 ± 0.49 mm, respectively. Deviations in the exit point for the C1 center screw on the X, Y, and Z dimensions were 0.38 ± 0.29 mm, 0.60 ± 0.46 mm, and 0.99 ± 0.53 mm, respectively. Deviations in the entry point (both sides combined) for the cranial C2 screws on the X, Y, and Z dimensions were 0.62 ± 0.47 mm, 0.47 ± 0.38 mm, and 0.80 ± 0.51 mm, respectively. Deviations in the exit point for the cranial C2 screws on the X, Y, and Z dimensions were 0.93 ± 0.65 mm, 0.75 ± 0.59 mm, and 0.66 ± 0.44 mm, respectively. The lateral screws in C1 and caudal screws in C2 were placed without the use of the drill guide temples. The degree of deviations in these screws was similar to those placed with the use of the drill guide temples. Overall, the average ± SD deviations for all C1 entry points and exit points were 0.51 ± 0.51 mm and 0.82 ± 0.62 mm, respectively. The average ± SD deviations for all C2 entry points and exit points were 0.72 ± 0.61 mm and 0.72 ± 0.55 mm, respectively. Specific data for all screw deviations were summarized in The incidence of intraoperative complications resulting from screw insertion was assessed by intraoperative observations and postoperative CT images. No major complications occurred during the surgery. Intraoperative hemorrhage from the internal vertebral venous plexus was encountered in three cases when screw holes were drilled into the caudal axis and unintentionally penetrated the transcortex. Hemostasis was easily achieved by inserting a small volume of a hemostatic agent  and placing screws in the drill holes. Other than the above three cases, the violation of vital anatomical structures did not occur.p = 0.3024). Body weights  (p = 0.7786) and neurological grades  (p = 0.49) were also not significantly different between the two groups. Dislocated screws may have been inserted into suboptimal locations; therefore, we statistically investigated the degree of deviations in the inserted screws from their planned coordinates. Deviations (distance shown in mean ± SD mm) in the entry points for C1 in dogs with screw loosening (0.86 ± 0.47) were not significantly different from those in dogs without screw loosening (1.07 ± 0.49) (p = 0.5847), and this was also the case for deviations in exit points for C1 in dogs with screw loosening (0.93 ± 0.24) and those without screw loosening (1.48 ± 0.49) (p = 0.0452).In the first visit, neurological deficits in most dogs were considered to be moderate to severe; the median neurological grade was 3 . The conditions of 13 dogs (72.2%) improved after the application of a neck brace, and a low dose of prednisolone in 7 cases with the neurological grade of 1 or 2 at the first visit, with the median improvement of 2 grade  in a re-examination on the day of surgery . The conp = 0.3648) and cortical thickness (p = 0.8638) between dogs with and without screw loosening revealed no significant differences.Vertebral BMD and cortical thickness are predictors of screw pull-out strength , 14. TheShort follow-up outcomes were assessed in a re-examination 3 months after surgery (n = 17). The median neurological grade was 5 . In 5 dogs, the neurological grade improved from the 2-week post-surgery evaluation and 12 dogs remained at the same grade. None of the dogs deteriorated from pre-surgery or from the perioperative follow-up evaluations. Additional implant failure from the 2-week post-surgery evaluation was not detected in any of the dogs. One dog (Case No.17) was lost to the follow-up 9 months after surgery.Mid follow-up outcomes were assessed in a re-examination 12 months after surgery (n = 16). The median neurological grade was 5 . One dog (Case No.7) died 11 months after surgery from an unknown reason. Although the C1 screw loosening was noticed in this dog at the perioperative period, the neurological condition of the dog improved during the short follow-up period and remained good until a sudden deterioration in its general condition when the dog presented with repetitive hematemesis which was not considered to be associated with neurologic diseases. The cause of death was not suspected to be related to the surgery performed; however, the definite cause was not identified because necropsy was not performed. Besides this case, the neurological grades of other dogs remained the same as those in the 3-month post-surgery evaluation. Case No.18, of which C2 screws were dislocated at the perioperative follow-up period, underwent second surgery 91 days after the first surgery due to dislocation of the plate even though the dog was clinically normal throughout . RevisioWe successfully utilized 3D printing technology, which enabled the manufacture of patient-specific drill guide templates and osteosynthesis plates to stabilize the AA joint in small breed dogs. The custom-made drill guide template provided accurate and safe guidance for screw placement by which the customized osteosynthesis plate was secured to the reduced AA joint. The smallest size among the cases treated was a 1.3-kg Toy Poodle. Even with the markedly small sizes of the target structures in this dog, reduction and stabilization were safely performed. Among the 18 dogs that underwent surgery, 15 (83.3%) exhibited clinical benefits at the mid-term follow up evaluation; therefore, the success rate of our surgical technique was at least similar, if not superior, to those of previous studies utilizing other surgical techniques for AAI stabilization , 7, 10. In the present study, average deviations in the screws inserted into C1 and C2 were less than 1 mm. Drill guide templates allowed for the accurate guidance of screws and successfully aided in avoiding iatrogenic injuries to vital anatomical structures, which is of clinical importance. The only intraoperative complication encountered was hemorrhage from the internal vertebral venous plexus in three cases. This complication occurred because we unintentionally penetrated the distal cortex of the C2 and was not related to the misdirection of the drill hole. Therefore, the accuracy of our drill guide template system was considered to be clinically acceptable for the safe guidance of screw placement for AA fixation in dogs. In the present study, we estimated the accuracy of drill guide system by measuring the coordinate deviations of the entry point and exit point between pre- and post-operative CT images. In order to more precisely evaluate the accuracy of our system, the deviation of the screw trajectory needs to be evaluated in a three-dimensional manner in future study.In human medicine, patient-specific drill guide templates have been developed as an inexpensive, accurate method to guide spinal fixation screws. The concept of personalized image-based 3D templates for spinal surgery was first described by Berry et al. , Owen etSerious intraoperative and perioperative complications were not encountered in our cases. Perioperative and postoperative mortality rate is unknown with AA fixation surgery, but it has been reported that caudal brainstem trauma may be related to respiratory abnormalities and cardiac arrest in atlantoaxial subluxation repair . PostopeWe consider patient-specific osteosynthesis to provide several benefits clinically. For example, prefabricated plates are already an ideal shape, and, thus, intraoperative shaping and bending are avoided, which significantly reduces the surgical time . SurgeonWe herein demonstrated the successful application of 3D printing technology to the surgical treatment of AAI in small breed dogs. Our drill guide templates allowed for the accurate placement of cortical screws in substantially small target bones and patient-specific titanium plates effectively stabilized the AA joint without any intraoperative major complications and apparent implant-related mortality. The efficacy of the combination of a similar intraoperative guide device and custom-made osteosynthesis plate indicates its potential for application to the surgical treatment of other spinal disorders in dogs.


The indications for sacroiliac screw (SI) removal have been under debate. Data on complication rates of SI screw removal is missing in the current literature. The objective of this study was to compare the rate of intra- and perioperative problems and complications during SI screw removal to those with SI screw fixation.n = 19; age 57.3, SD 16.1 years) were included. Intraoperative technical problems, postoperative complications, duration of surgery, and radiation dose were analysed.A retrospective observational study with two interventions in the same cohort was performed. Consecutive patients who underwent both sacroiliac screw fixation for an isolated fracture of the pelvic ring and removal of the same implants between November 2008 and September 2015 (p = .021). Postoperative complications were seen in 3/19 patients after SI screw fixation and in 1/19 patients after SI screw removal (p = 0.128). The surgical time needed per screw was longer for screw removal than for implantation (p = .005). The amount of radiation used for the whole intervention (p = .845) and per screw (p = .845) did not differ among the two interventions.Intraoperative technical problems occurred in 1/19 patients (5%) during SI screw fixation and in 7/19 cases (37%) during SI screw removal (Intraoperative technical problems were more frequent with SI screw removal than with SI screw fixation. Most of the intraoperative technical problems in this study were implant-related. They resulted in more surgical time needed per screw removed but similar radiation time. Percutaneous sacroiliac (SI) screw fixation has become an established technique for posterior stabilization of pelvic ring injuries –6. ComplIn their case series on implant removal from the anterior and posterior pelvis, Stuby et al.  observedOn the topic of SI screw removal itself, there has been published only a small number of case reports and technical notes –19. ThesWhile implant removal may be well necessary in case of a symptomatic malpositioned screw, other indications for SI screw removal have been under debate. Some authors see an indication for removal only in case of complications , 20, othTo the authors’ knowledge, data on the complication rates of SI screw removal are underreported in the current literature. These would be important order to weigh the potential benefits against the risks associated with this procedure.The purpose of this study was to compare the rate of intra- and perioperative problems and complications during SI screw removal to those to be expected with their implantation – in one and the same cohort of patients.The protocol of the present study was approved by the local ethics committee  and it was assured that patients included into this analysis had not objected to use of their data for research purposes.n = 19) from a pelvic ring fracture database  and a retrospective chart review was performed. Patients younger than 18 years and those with previous surgery to the pelvis were excluded.A retrospective observational study was conducted. In order to avoid confounding factors as age, gender, co-morbidities, and the individual anatomy of the pelvis, a repeated measures design with two interventions in the same patient was chosen to compare the effect of two interventions  on the perioperative outcome in the same patient. Consecutive patients who underwent both sacroiliac screw fixation for an isolated fracture of the pelvic ring and removal of the very same implants between November 2008 and September 2015 were identified  with a washer in the pedicles of S1 and/or S2 using conventional C-arm fluoroscopy as previously described , 13. In Postoperatively, patients were allowed to mobilize with 15 kg partial weight bearing on the affected side for at least 6 weeks.Implant removal was performed in a short-term in-hospital setting. Patients with additional anterior implants underwent removal of the SI screws only. In all cases, it was aimed for a complete removal of the screws including washers. Through the scar of the previous surgery, a K-wire was introduced into the sacroiliac screw under C-arm assistance. The screw was then removed sliding a cannulated screw driver over the guide wire. The wire was left in place and served as a guide for either a hooked spoon or Kocher forceps in order to remove the washer. If this technique failed, a Kocher clamp, a nerve hook or an angulated scoop were directed towards the washer using the C-arm in different planes.Primary outcomes were intraoperative technical problems. These were defined as unexpected events that made additional measures necessary  and prolonged the intervention by 10 or more minutes per screw. Secondary outcomes were postoperative complications during the time of hospitalization, these were categorized according to the system established by Clavien and Dindo . MalposiTertiary outcomes were duration of surgery and radiation dose.p < 0.05. In case of missing data, comparisons were made only with the data available and this was reported in the results section accordingly.Statistical analysis was done by the use of SPSS for windows 22.0 . Ordinal and metric data was compared using non-parametric tests; associations between categorical data were tested using crosstabs and Fisher’s Exact test. Metric data was presented as mean with standard deviation (SD). Differences were considered significant for values of Nineteen patients  were included into the final analysis. The indication for sacroiliac screw fixation were traumatic fractures in 17 cases  and sacral fragility fractures (2 H-type) in two cases. The total number of screws was 4 screws in 2 patients, 3 screws in 1 patient, 2 screws in 8 patients and 1 screw in 8 patients. Hence, the average number of screws inserted was 1.8 (SD 1.0). Twelve received unilateral and 7 patients a bilateral fixation. In all patients, at least one screw was placed into S1, while 9 patients received an additional screw into S2. In one patient with two bilateral screws in S1 and two bilateral screws in S2, the screws in S1 were augmented with PMMA cement.Sacroiliac screw removal in the same patients was performed mean 213 days  after implantation of the screws. The indication for screw removal was screw malposition in three cases , asymptomatic screw loosening in five cases, SI joint pain in five cases and a planned pregnancy in one case. In five additional asymptomatic cases, the screws were removed only on the patients’ explicit request.Intraoperative technical problems occurred in 1/19 patients (5%) during SI screw fixation. The patient had an unexpected accumulation of bowel gas that prevented safe placement of an S2 screw.p = .021, Fig. n = 4), the need for an extended incision due to ingrowth of the screw (n = 2), and one case with a worn out screw head. A post-hoc power analysis for the primary outcome  revealed a power of 1.0 based on an observed odds ratio of 10.5 and assuming a correlation coefficient (ϕ) for exposure between matched cases and controls of 0.2 [Complete SI screw removal including the washers was achieved in all patients. No broken screws were observed. During SI screw removal, additional intra-operative measures were necessary in 7/19 cases  among the two groups, the time needed per screw was longer for screw removal than for implantation  and per screw (p = .845) did not differ among the two interventions  seen in our study population were higher than those reported in the literature –11. In cThe number of postoperative complications after SI screw removal was smaller than what would be expected from the small body of literature that reports complication rates of about 20% with implant removal after pelvic fracture stabilization .It could be shown, that SI screw removal is technically challenging and may require more OR time than screw implantation.Most of the intraoperative technical problems in this study were implant-related. A potential solution to decrease implant-related intraoperative problems is the use of advanced operative techniques. Described surgical techniques to address the difficulties associated with the retrieval of the washer include endoscopic techniques  or the uRemoving the washer seemed to be the part of the procedure that was most prone to problems. There would have been less intraoperative technical problems and less radiation exposure if we had simply left the washer inside the patients. In fact, without the problems caused by washers, the differences between the two interventions would have been negligible. Just removing the screw and leaving the washer where it is, is a frequent topic that we discuss with the patients preoperatively. However, most of the patients preferred complete implant removal and thus leaving the washer may be not a good option in these cases. In contrast, a prolonged surgical time or even an extended incision with additional surgical risk to remove a washer that is asymptomatic at the preference of the patient might be an even less preferable option.A different approach to decrease implant-related problems is the development of better implants. While the biomechanical effect of using washers to allow for better compression has been shown , their rThe limitations of this study are related to its retrospective study design and the small number of patients included. Even though the matched or repeated measure design of this study allows for ruling out age, gender, co-morbidities, and the individual anatomy of the pelvis as confounding factors, it is not possible to finally conclude that SI screw removal is more prone to complications than SI screw fixation. The time of screw removal ranged from 2 days to almost 2 years. This may have caused a bias with regard to more osseous overgrowth of the screw and washer in patients with late removal. With the numbers available, however, further subgroup analyses were not possible and could be objective of future studies.The data for radiation exposure was incomplete (13/19). However, radiation exposure was not the primary outcome but chosen as an additional secondary parameter in order to quantify the number of fluoroscopic images taken intraoperatively and to illustrate the surgeon’s strain.Further studies are needed to prove any potential benefit of SI screw removal in patients other than those with complications as symptomatic implant malpositioning. Patients who wish their implants to be removed should meticulously be informed on the risks and benefits of this procedure.Intraoperative technical problems were more frequent with SI screw removal than with SI screw fixation. Most of the intraoperative technical problems in this study were implant-related. They resulted in more surgical time needed per screw removed but similar radiation time.In light of our results, SI screw removal is an intervention with considerable radiation exposure and high rates of intraoperative problems. Even though the complications associated with this procedure rarely have long-term consequences, routine removal cannot be recommended.