The tunability of the properties of chitosan-based carriers opens new ways for the application of drugs with low water-stability or high adverse effects. In this work, the combination of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG) grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain nanocapsule grafting with PEG has been developed in this work, allowing a very good control and tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are studied and the nanocapsule systems obtained are characterized at all steps of the optimization in terms of diameter, Z potential and surface charge . Results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for future studies oriented toward drug delivery. In the last few decades, many kinds of nanocarriers have been developed for delivery and targeting of therapeutic or diagnostic agents, thanks to some important advantages that they offer depending on their physico-chemical properties ,2.Depending on the specific needs, the nanocarrier type and formulation process must be chosen on the basis of therapy goals and administration route . The mosAccording to Vrignaud and co-workers, nanocapsules are vesicular systems, composed of an oily or an aqueous core that can be considered as a reservoir in which the drug is confined to a cavity, surrounded by a polymeric shell . NanocapNatural polymers are among the most used for these kind of coatings since they usually provide a high colloidal stability in water suspensions. Active research is now focused on the use of hydrophilic biopolymers as carrier coatings because of their biocompatibility and biodegradability ,20. ChitIn this work, core-shell nanocapsules made of a nanoemulsion core and a chitosan shell were synthesized and characterized with the aim of obtaining a multipocket nano-reservoir carrier to be used in future applications for sustained release of different drugs. The secondary effects—toxicity, poor solubility, and bioavailability—of new drugs lead to the need of their encapsulation to protect them from degradation and to enhance their stability and solubility ,28.3), that links aminated PEG molecules to amino groups on nanocapsule surface. Nanocapsule behavior in different media was evaluated in terms of aggregation degree before and after grafting and the effect of PEG on their cytotoxicity was also assessed.Thanks to the presence of chitosan amino groups, the surface of the obtained nanocarrier can also be grafted with specific moieties in order to tune the net charge to introduce specific functional groups and/or improve the carrier stability in biological media and physiologic solutions for intravenous administration ,31,32,33A nanoemulsion method was developed to obtain small nanoparticles  to be used as a template for the following polymer coating and reinforcement. The aim was to obtain capsules with a lipophilic core and a hydrophilic cationic shell of chitosan hydrogel.® 85/oleic acid  to a Tween® 20  aqueous solution under stirring. Optimal ratios between components have been found adapting a method reported by Bouchemal et al. [The synthesis of the nanocapsules was carried out in two steps. The formation of the nanoemulsion template particles was carried out simply by adding a water-miscible organic solution of Spanl et al. . The parThe presence of such functional groups is responsible for the ability of the polymer to gelify in presence of multi-anions leading to the formation of hydrogel. Moreover, the amino groups exposed on hydrogel shell surface allow the easy functionalization of the nanocapsule with the desired moiety.Chitosan is directly added to the nanoemulsion that is subsequently mixed with a sodium sulfate solution to obtain the coating with a chitosan shell. This treatment has been used in several works to obtain chitosan particles ,37,38. IThe result of the synthesis is a water-stable suspension of chitosan-coated nanocapsules (CS-NCs).A schematic representation of the hypothesized nanocapsule structure is reported in The sample turned out to be composed of spherical capsules of a quite homogeneous size. The polymeric shell can be appreciated in BF-TEM and ESEM images B,C. ESEM2SO4 solution. The elimination of the sonication step in the synthesis process could represent an advantage in terms of applicability of the process for future applications to industrial production with high levels of scale-up. Nanocapsules obtained with the modified process have been characterized in terms of hydrodynamic diameter and surface charge and they have been compared with the sonicated ones. In As optimization of the process, the importance of the sonication treatment during hydrogel shell formation was evaluated by substituting it with a gentle stirring while adding nanocapsules to NaHydrodynamic diameter of chitosan–coated nanocapsules always increases with respect to the nanoemulsion template A. Nevert2 and –OH on polymer chains and Na2SO4 molecules. Moreover, it should be taken into account that the presence of salts can also promote hydrophobic interactions between polymer chains themselves. As a consequence of the establishment of such hydrophobic interactions, the exposure amino groups on the surface of the nanocapsule would be favored. To evaluate how the sonication during hydrogel formation can improve the interactions and so affect the nanocapsule surface properties, sonicated and non-sonicated nanocapsules have been compared in terms of surface potential. In particular, analysis of Z-potential and amino group spectrophotometric determination analysis are reported. Both capsules showed a positive potential when measured in a 10 mM KCl solution, being slightly more positive the surface of sCS-NC (+21.1 mV) than the surface of nsCS-NC (+13.8 mV). The observed variation in the surface potential depending on the sonication treatment could be explained in this case by the presence of a higher number of amino groups exposed on the outer surface of chitosan shell in the case of sCS-NC. This hypothesis was confirmed by the quantification of amino groups of the chitosan shell of nanocapsules by the spectrophotometric method of Orange II dye, previously reported and already optimized for inorganic nanoparticles [3− group of Orange II dye. This spectrophotometric method is simple, inexpensive, and easy, and its most important advantage over other methods for amino quantification consists in the use of a molecule with low steric hindrance—an especially important aspect for porous materials materials. Moreover, the relationship between amino groups and reactant is in this case of 1:1, allowing a direct and reliable quantification [−1) while a value of amino groups of only 0.2 μmol·mg−1 was obtained in the case of nsCS-NC.It is supposed that the hydrogel formation treatment affects the properties of the polymer-coated surface of the nanocapsules depending on the degree of the interaction that could be established between –NHarticles . Brieflyfication . In the 2 of the nanocapsules. Data from the Orange II assay were in good agreement with Z potential analysis. sCS-NC presented a number of moles mg−1 of –NH2 groups almost double with respect to the moles mg−1 of –NH2 of nsCS-NC.It should be taken into account that results from Orange II interaction only represented the amount of amino groups available for the interaction with dye molecules and that the amount of these groups could be lower than the total amount of –NHThermo Gravimetric Analysis (TGA) of chitosan-based nanocapsules sonicated or not during the synthesis process is reported in PEG coating has been chosen in this work for the further optimization of the carrier. As previously stated in the introduction and well documented in the literature, the prevention of unspecific adsorption of serum proteins on carrier surface represents a very important goal to allow a prolonged circulation time in the blood stream. From this point of view, pegylation is reported to be one of the most successful strategies ,34,35.3) (Nanocapsules obtained without sonication during the synthesis have been selected for the process of grafting of the surface due to their better stability in water suspension. The surface was grafted with α-methoxy-ω-amino poly (ethylene glycol) (5000 Da) (aminated PEG) through a previous reaction with the homobifunctional linker bis (sulfosuccinimidyl) suberate (BS3) . As the 3 has been always maintained below the total moles of functional groups of nanocapsules to reduce the possibility of crosslinking between capsules. To complete the process, the amount of aminated PEG exceeded twice the used amount of BS3 in all the experiments to assure the reaction with all the reactive groups of BS3 on the surface.The determination of the accessible amino groups was fundamental for the optimization of the grafting protocol. In fact, the method utilized here implied the use of a homobifunctional linker in which the main drawback lays in the possibility of crosslinking of amino groups from different nanocapsules and production of aggregates if the amount of reactants is not strictly controlled. For this reason, the value of accessible amino groups has been used as the starting point to adjust the amount of reactants in the following steps of the process. In particular, the amount of BSThe presence of PEG on nanocapsule surface has been evaluated by Fourier Transform Infrared Spectroscopy (FTIR) analysis  but in this case it can be supposed that BS3, which should have reacted previously with PEG, is not available to react with Tris. This hypothesis was confirmed by comparing FTIR spectra of nanocapsules after the incubation with BS3  but they completely disappeared in hdCS-NC spectrum.To determine if the grafting with PEG successfully occurred, the FTIR spectrum of the intermediate step of nanocapsules after the incubation with BSreported B. It showith BS3 B and spe−1. In this region the C-O-C peak (1105 cm−1) presents an increased intensity, compared to other peaks in the same region, especially in the hdCS-NC spectrum. Moreover, in this sample peak at 1055 cm−1 is lost. This peak is supposed to refer to R-SO3− group that is released from BS3 in the linking reaction. In the BS3 intermediate step the peak is still appreciable and it can be observed as a low intensity peak in ldCS-NCs too. It is possible that in these samples not all BS3 reactive groups are quenched after Tris-HCl incubation and that some of them are still present on the surface. In any case this state did not represent a problem for the quality of the sample since any appreciable crosslinking and consequent aggregation are observed.Differences in the spectra could also be appreciated, comparing peaks and intensities ratios between 1150 and 1030 cmThe successful grafting on nanocapsule surface was proved also by measuring the Z potential of the material surface before and after the modification with PEG. Using both a low amount and high amount of PEG, a decrease in the potential was observed, indicating a decrease in the free amino groups on the surface and an increase in the total electronegativity of the surface.3+ groups would be hampered by the presence of the polymer chains since, in the case of low-density PEG coverage, it can be supposed that polymer molecules stand in a conformation that lays around nanocapsule surface, probably thanks to a possible hydrogen bond interaction of the PEG chain with positively charged amino groups on the nanocapsule surface  registered between low-density and high-density PEG-covered nanocapsules. The measured amounts of amino groups are 0.1 and 0.08 μmol/mg respectively, corresponding to 50% and 40% of the amounts of amino groups on ungrafted nanocapsules. These values are considered as the apparent disappearing of amino groups probably due to a screening effect for the presence of PEG molecules on the surface. Orange molecule interaction with –NHface see . The tenThe effect of the grafting on the behavior of nanocapsules in physiological medium was further evaluated by measuring the degree of aggregation of grafted and not-grafted nanocapsules in water and phosphate saline buffer (PBS) by means of hydrodynamic diameter measurement. Ungrafted nanocapsules are sensitive to the presence of salts in the medium and they tend to aggregate during incubation in physiological media like PBS. The grafting with PEG chains is a common strategy to improve the stability of nanoparticles in physiological and in vitro culture media . Both phIn To further demonstrate the stabilizing effect of PEG grafting on nanocapsule surface, an aggregation test has been carried out by measuring the hydrodynamic diameter of grafted and ungrafted nanocapsules in the presence of increasing concentrations of Bovine Serum Albumin (BSA). As in the case of the above reported stability assay in presence of PBS, the high density of amino groups on nanocapsule surface can be considered responsible for the adsorption of proteins and aggregation observed. The comparison of the behaviors of ungrafted nanocapsules, ldPEG-CS NC and hdPEG-CS NC, has been reported in Data reported in the graph demonstrated without any doubt the strong stabilizing effect of PEG on nanocapsules in the presence of proteins. The grafting with PEG, and so the strong decrease of the free amino groups on the surface lead to a very good stability of the nanocapsules in a protein-rich medium. Both ldPEG-CS NC and hdPEG-CS NC showed a significant increase in the hydrodynamic diameter only at very high concentrations of proteins (higher than 0.1 mg/mL). Comparatively, the concentration of BSA one order of magnitude lower is enough to produce the same increase in the diameter in the case of ungrafted NCs.The cytotoxicity of chitosan-based nanocapsules before and after the grafting was tested on Vero cells through MTT spectrophotometric assay. Cells were incubated for 24 h with different concentrations of nsCS-NC, selected as ungrafted nanocapsules over the sonicated ones due to their better stability in water, and with the same concentrations of ldCS-NC and hdCS-NC. In It can be observed from Using an inverted microscope, it can be observed that non-sonicated nanocapsules lead to bigger vesicles inside cells than both types of grafted nanocapsules , which cThe test proved not only that aminated PEG was definitively linked to nanocapsule surface, but also that the conformation of polymer chains obtained using the developed method of grafting for ldCS-NC hdCS-NC is effective for significantly improving the cytotoxicity of chitosan-based nanocapsules. Moreover, it should be noted that in the case of PEG-grafted nanocapsules the decrease in the cytotoxicity is still associated with a very high degree of internalization, indicating that the developed material can be considered a very effective carrier for drug delivery applications.Among all kinds of nanocarriers, nanocapsules are frequently the material of choice for biomedical applications since they offer the advantage of providing a good stability of encapsulated drugs and a favorable pharmacokinetic. The aim of the work was to develop nanocapsules to be used in the future as potential drug reservoir and whose composition could allow maximum flexibility of application together with great feasibility for the administration by different routes .In this work, chitosan—a biocompatible polymer—has been used to obtain a potentially smart nanocarrier whose surface properties could be properly tuned depending on the desired application. A nanoemulsion-based nanocapsule has been used as template to be coated with a chitosan shell.The process of chitosan hydrogel coating of a nanoemulsion template was studied in terms of exposure of amino groups on the surface depending on the interaction produced during the sonication process. It could be hypothesized that the sonication promoted interactions and produced stronger hydrophobic interaction between polymer chains, leading to a condensation of the structure that would lead to a higher exposure of amino groups and so to a higher availability for the interaction with the dye. On the other hand, it was also supposed that a different amount of polymer could be incorporated on the surface of the nanoemulsion template when sonication is used during the synthesis, leading to a different total amount of glucosamine units (and so amines) in the shell. The characterization of the material and the comparison between the sonicated and not sonicated one alloowed finally to confirm that the sonication process lead to a higher amount of amino groups exposed on nanocapsule surface.Chitosan shell offers the possibility of an easy functionalization through amino groups on its surface so chitosan-based nanocapsules have been further coated with PEG since this is known to be a very effective strategy to stabilize surfaces in biologically relevant media. The possibility to tune the surface properties by varying the PEG density coverage has been explored and results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their aminated terminal.3 linker on nanocapsule surface. Once they are linked, their tendency is to assume a mushroom-like conformation until the density of PEG molecules on the surface does not reach a value high enough to produce a change into a brush-like conformation [It is reported in literature that the attempt to produce high-density coverage of surfaces with PEG can result in a loss of efficiency of grafting. In fact, similarly to what reported for the grafting of other surfaces, it is reasonable to suppose that not all PEG molecules react immediately with BSormation . In the Despite the fact that the obtained grafting degree was slightly lower than the one expected, a highly improved stability in physiological medium  was observed with both low and high PEG-covered nanocapsules  indicating that the presence of PEG on the surface will allow the use of the grafted capsules in physiological media for biomedical applications.We also demonstrated that the presence of PEG on chitosan surface of nanocapsules was effective in avoiding the unspecific adsorption of proteins. This issue is of the utmost importance as, following intravenous injection, non-passivated nanoparticles tend to adsorb plasma proteins (protein corona), changing the charge and size of them. This binding of plasma components will be responsible of the final fate of the nanocapsules, and can greatly influence the biodistribution and therapeutic efficacy . To dateFollowing the encouraging results obtained in physiological media and protein-rich media, preliminary tests with cell cultures have been carried out showing very interesting results.The obtained coating demonstrated effectiveness in tuning the cytotoxicity of chitosan-coated nanocapsules. Moreover, the nanocapsules showed a high degree of internalization in Vero cells, a useful property for the potential application as drug reservoirs directed to cytoplasm release. Finally, the tunability of all the properties of the synthesized chitosan-based nanocapsules was obtained by a very easy and reproducible chemical grafting, indicating that the developed nanocarrier was very promising for further studies oriented toward drug encapsulation for biomedical application.® 20 , absolute ethanol, sodium sulfate anhydrous (99%–100.5%), and sodium chloride (99%), were purchased from Panreac. Span® 85 (sorbitanetrioleate) , oleic acid (90%), chitosan (medium molecular weight), Orange II sodium salt, and Bovine Serum Albumine (BSA) were obtained from Sigma-Aldrich. Bis(sulfosuccinimidyl) suberate (BS3) was purchased from Pierce Biotechnology and α-methoxy-ω-amino poly(ethylene glycol)  from IRIS Biotech GmbH. Water (double processed tissue culture) used in all nanocapsule synthesis was from Sigma. Millipore Biomax 300 kDa Ultrafiltration Discs were purchased from Merck Millipore.TweenVero cells  were purchased from the American Type Culture Collection . Dulbecco’s modified Eagle’s medium (DMEM), Phosphate-Buffered Saline (PBS), Dulbecco’s Phosphate-Buffered Saline (DPBS) were purchased from Lonza. MTT -2,5-diphenyltetrazolium bromide), penicillin, streptomycin, glutamine solutions, and 4′,6-diamidino-2-fenilindolphenylindol (DAPI) were purchased from Invitrogen .® 85 in 40 mL of absolute ethanol was added to the aqueous one, containing 136 mg Tween® 20 solved in 80 mL water, under magnetic stirring during 15 min for the formation of the nanoemulsion. Then 25 mg from a 5 mg/mL chitosan solution in acetic acid 1% (v/v) were added and again the mixture was left under stirring 15 min. Finally, the chitosan-coated nanoemulsion was added to 200 mL of 50 mM Na2SO4 under sonication (or stirring in the case of optimized nanocapsules). Capsules were separated from Na2SO4 through ultracentrifugation , washed with 100 mL of water, centrifuged again, and resuspended in water. The concentration of the nanocapsules in water suspension was obtained by measuring the weight of 1 mL of sample after freeze-drying.For the preparation of chitosan-based nanocapsules an organic solution containing 400 mg oleic acid and 86 mg Span2) was added and the mixture was kept under stirring for 2 h at 37 °C. Finally, 20 mL of Tris-HCl buffer 10 mM pH 8.0 was added to quench the linker that eventually did not react with PEG. Grafted nanocapsules were filtered using an Amicon Ultrafiltration unit using Millipore Biomax 300 kDa Ultrafiltration Discs to separate them from unreacted PEG. After a washing with fresh water nanocapsules, they were concentrated to a final volume of 2 mL.For the grafting of nanocapsule surface suspensions, 20 mg of nanocapsules at a concentration of 2 mg/mL in borate buffer 10 mM pH 8.3 were added with different amounts of the linker bis(sulfosuccinimidyl) suberate (BS3) (20–100 nmol/mgNC) and they were kept under stirring for 30 min. Then a double amount of α-methoxy-ω-amino poly (ethylene glycol)  technique. Nanoparticle hydrodynamic diameter and polydispersity index (PDI) have been measured in water at the concentration of 0.05 mg/mL.Electrophoretic mobility  of nanoparticles at different pH values has been determined by measuring the potential of a 0.05 mg/mL nanoparticle suspension in 10 mM KCl with a Plus Particle Size Analyzer (Brookhaven Instruments Corporation).−1 with a resolution of 2 cm−1 and a scanning number of 32.Nanocapsule composition was analyzed by Fourier Transform Infrared Spectroscopy analysis in a JASCO FT/IR—4100 Fourier transform infrared spectrometer in a frequency range of 600–4000 cmThermogravimetric analysis was performed in a TASTD 2960 thermogravimetric analyzer, by heating the sample at 10 °C/min under air atmosphere.Environmental Scanning Electron Microscopy (ESEM) images were obtained using a QUANTA-FEG 250 microscope in Scanning Transmission Electron Microscopy (STEM) mode. Bright Field Transmission Electron Microscopy (BF-TEM) analysis was carried out in a FEI Tecnai T20 microscope operating at 200 kV. Due to the sensitive nature of the sample, previous fixation, dehydration, and epoxy resin embedding were necessary for both techniques. This process can be briefly achieved as follows. A fresh sample was synthesized and it was fixed with glutaraldehyde 0.25% in phosphate buffer 10 mM at pH 7.4 for 2 h. It was washed three times with buffer and incubated with 1% osmium tetroxide in PBS for further fixation and staining. Finally, the sample was accurately washed with water and resuspended in 5% gelatin. The sample was centrifuged to obtain a pellet and was incubated overnight at 4 °C. The obtained solid sample was cut in very small pieces before undergoing the subsequent steps. The dehydration of the samples was carried out using the following steps: incubation in ethanol 30%, ethanol 50%, and incubation overnight in ethanol 70%; incubation in ethanol 90% for 1 h; and finally incubation three times in absolute ethanol. After that, samples were incubated overnight in a 1:1 mixture absolute ethanol/epoxy resin (r.t.). The mixture was then removed, changed with absolute epoxy resin and samples were left for impregnation for 8 h at room temperature. After another change of the medium, the final incubation in epoxy resin was carried out overnight at 60 °C to obtain the polymerization.®  and OriginLab®  softwares to measure the diameters of more than 100 nanocapsules and for the frequency count statistical analysis respectively.From different ESEM images , an estiNanocapsule amino content was measured by the Orange II spectrophotometric assay ,35. 0.2 Resistance of nanocapsules to aggregation has been determined by incubating nanocapsules (3 mL of a 0.15 mg/mL suspension) at different concentrations of albumin from bovine serum (BSA). The hydrodynamic diameter of the capsules under the incubation conditions was measured after 10 min using a Brookhaven 90Plus DLS instrument.2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and glutamine (2 mM). 7500 cells were seeded using a standard 96-well plate (five replicates per sample). After 24 h of incubation in a humidified atmosphere containing 5% CO2, the medium was replaced with new medium containing five different concentrations of nanocapsules and a negative control containing no capsules (non-treated cells). After 24 h of incubation, the medium was replaced with fresh medium containing MTT dye solution (0.5 mg/mL in DMEM). After 2 h of incubation at 37 °C and 5% CO2, the medium was removed and the formed crystals were dissolved in 200 μL of DMSO. The absorbance was read on a ThermoScientificMultiskan GO TM microplate reader at 570 nm. The relative cell viability (%) related to control cells without nanocapsules was calculated using the percentage ratio between absorbance of the sample and the absorbance of the control. Experiments were carried out in triplicate.In vitro cell viability test was carried out to determine the cytotoxicity of nanocapsules using 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Vero cells were grown at 37 °C in a 5% CO4 cells were seeded on glass coverslips in a 24-well plate at 37 °C. 24 h later, nanocapsules were added at 50 μg/mL in DMEM and incubated for 24 h at 37 °C. Non-internalized nanocapsules were removed, washing with DPBS twice. Cells were fixed with 4% paraformaldehyde for 20 min at 4 °C, washed twice with DPBS, and incubated for 10 min at room temperature with 4′,6-diamidino-2-fenilindolphenylindole (DAPI) for nucleus labeling. The coverslips were mounted on glass microscope slides using ProLong®  Gold Antifade. Optical microscopy was performed using an inverted microscope (Nikon Eclipse Ti-E), and images were analyzed using NIS-Elements Advanced Research software.To perform optical microscopy analysis, 3 × 10


Experiments 1 to 4 tested 16, 23, 9 and 6 subjects, 49/54 being unique. Experiment 1, Experiments 2 and 3, and Experiment 4 used three variants of the mfPOP method that provided increasingly improved signal quality. Experiments 1 to 3 examined centrally directed attention, and Experiment 4 compared effects of attention directed to different peripheral targets. Attention reduced the sensitivity of the peripheral locations in Experiment 1, but only for the white stimuli not yellow. Experiment 2 confirmed that result. Experiment 3 showed that blue stimuli behaved like white. Peripheral attention showed increased sensitivity around the attentional targets. The results are discussed in terms of the cortical inputs to the pupillary system. The results agree with those from multifocal and other fMRI and VEP studies. mfPOP may be a useful adjunct to those methods.Multifocal pupillographic objective perimetry (mfPOP) has recently been shown to be able to measure cortical function. Here we assessed 44 regions of the central 60 degrees of the visual fields of each eye concurrently in 7 minutes/test. We examined how foveally- and peripherally-directed attention changed response sensitivity and delay across the 44 visual field locations/eye. Four experiments were completed comparing  In seeming agreement early fMRI studies of attention reported reduced BOLD signals for unattended parts of the visual field9. Recent studies indicate that these changes are not due to blood stealing10, and that they are correlated with independent measures of reduced brain responsivity15.The study of visual attention has grown prodigiously over the past 25 years. Although there is some evidence of increased sensitivity to attended items, behavioural studies have generally indicated that there is suppression of sensitivity peripheral to the focus of attentionmultifocal methods, where responses are obtained to many independent stimuli that are concurrently presented across the visual field. For such stimuli, there is scope for attended and unattended stimuli amongst the concurrently presented ensemble, as can occur in nature. Attention has been reported to enhance multifocal VEP (mfVEP) responses at the attended location, and also to produce a mixture of milder enhancement and suppression at adjacent unattended locations16. Those patterns of sensitivity change are similar to those reported for fMRI methods that use multifocal-like methods18. One of us has extended multifocal methods to fMRI assessing retinotopic brain mapping20. That multifocal fMRI method has been applied to visual attention showing focal enhancement and peripheral suppression21. Here we describe a convenient and lower-cost multifocal method with which to explore attentional effects across the visual field: multifocal pupillographic objective perimetry (mfPOP).The suppressive effects are best seen when several peripherally placed distractors are presented together. A genre of experimental methods where this occurs as a matter of course are the 24, macular degeneration26, and early-stage diabetic retinopathy28. More recently the method has shown clinically useful results in multiple sclerosis29. The pupillary system is known to receive input from at least 7 extra-striate visual cortical areas30. Two of our recent studies confirm cortical input to mfPOP responses31. Furthermore, by using combined mfPOP and mfVEPs, we have recently verified extra-striate cortical drive of the irises in 42 subjects for stimuli like those used here33. A recent fMRI study reported that extra-striate cortical areas beyond V4 have been shown to have wider zones of peripheral suppression extending to 10 to 20 degrees away from the focus of attention18, well within the parts of the visual field tested by the mfPOP stimuli used here.Early versions of mfPOP have produced good diagnostic performance for glaucoma33. In our initial experiments on 16 subjects, the results for centrally directed attention were counter-intuitive: responses to peripherally presented white stimuli were suppressed, but this was not seen for the yellow stimuli. We therefore repeated the experiment with 23 different subjects, different experimenters, and mfPOP stimuli that had improved signal to noise ratios. Since white stimuli differ from yellow in containing blue we also examined blue vs. yellow stimuli in a further 9 subjects. Finally we compared the effects of attention directed to peripheral targets using the newest mfPOP stimulus methods26 on 6 subjects. We present the results of the four studies here and discuss them in terms of inputs to the pupillary system.Here we mainly examine whether a foveally-directed attentional task suppresses mfPOP responses to ensembles of 44 multifocal stimuli per eye, presented at locations spanning 1 to 30 degrees eccentricity. This study arose as part of an investigation of mfPOP responses to stimulus ensembles of yellow or white stimuli presented at a range of luminance levelsExperiment 1 examined 16 volunteer participants . Experiment 2 included 23 subjects . Experiment 3 examined 9 subjects . Experiment 4 tested 6 subjects . Subjects had normal or corrected visual acuity of 6/9 or better. Their visual fields were tested with the Humphrey FDT-C20 visual field test , and none showed visual deficits. The study conformed to the Declaration of Helsinki and subjects gave written informed consent under the Australian National University’s Human Experimentation Ethics protocols 04/238 and 10/061. Subjects were requested to refrain from caffeine or alcohol at for least one hour before each session.Multifocal visual stimuli were presented using a prototype of the FDA-cleared nuCoria Field Analyzer (nCFA) , which records pupillary responses to dichoptic stimuli presented to both eyes Fig. . Refract2, as was used in the original mfPOP methods27. The stimuli of Experiments 2 to 4 had slightly different luminances across the regions. These differences were introduced to balance out differences in visual field sensitivity23. The maximum luminance of any test stimulus was 150 cd/m2. Aside from yielding more similarly sized responses at each tested region this luminance balancing also improves the median signal to noise ratios obtained by about 15% in the least responsive regions of the visual field23. The stimuli of Experiment 4 used the clustered volleys method which improve signal to noise ratios by 40% relative to the presentation methods of Experiments 2 and 326. Thus the experiments of this paper illustrate the evolution of the mfPOP method to date.The stimulus ensemble for each eye contained 44 test regions extending from 2 to 30 degrees eccentricity. The stimuli are arranged in 5 concentric rings Fig. . In Expe31. All stimuli were presented against a background of 10 cd/m2. The temporal presentation of each of the stimulus regions was modulated by independent pseudo-random sequences. The transient onset stimuli were presented for a duration of 33 ms. In Experiment 1 the mean presentation interval was 1 s/region, generating 44 stimuli/s/eye; in Experiments 2 to 4 the mean interval was 4 s/region or 11 stimuli/s/eye. Those slower presentation rates have been shown to further improve signal to noise ratios23. In Experiment 1 each test was divided into eight segments of 30 seconds duration , whereas in Experiments 2 to 4 there were nine segments of 40 seconds (6 minutes). Rest periods of 7 seconds were given between segments. In the central Attention condition the centrally located fixation object changed transiently from a cross to a dot at random intervals during the course of each test sequence. This dot contained the same number of pixels as the cross. The fixation object remained a cross for the duration of the No-Attention condition. The fixation stimuli were red, were 1.6 deg square, and were presented within the central 2 deg of the stimulus ensembles. It is worth noting that in the No-Attention condition that the slow delivery of the transient onset stimuli meant that attention could not be distributed across a great number of stimuli at any one time. Experiment 4 employed a red 1.1 deg diameter circular attentional target displayed at either plus or minus10 deg eccentricity along the vertical midline. The mfPOP stimuli do not cross the horizontal of vertical meridians . We also fitted effects for sex and consensual pupil response (response of a pupil to stimuli presented to the opposite eye). To examine the effect of eccentricity and deal with issues of multiple comparisons/subject, we fitted linear mixed-effects models. For Experiments 1 to 3 these were based on the mean response for each of the five rings of the stimuli . Thus, subjects fixated the central cross while attending to the superiorly- or inferiorly-placed target. This was repeated for white and yellow versions of the stimuli in six subjects. Experiments 1 to 3 all compared an Attention and No-Attention cases. The No-Attention case could be thought of as a distributed attention case, although the transient onset stimuli meant that only a few stimuli were presented at any one time . Nevertheless, it was useful to compare conditions for similarly directed attention. To quantify the effect we computed the difference between the results for superiorly and inferiorly directed attention. These data used the newer Clustered Volleys mfPOP method and the signal quality of these data was higher than the previous experiments providing a median (±SD) t-statistic for response amplitude of 5.53 ± 2.17 for yellow, and 5.58 ± 2.18 for white cf. Fig. .Figure 21. A point of difference was that stimuli containing blue (white stimuli) seemed to show stronger effects than yellow stimuli. To our knowledge, no such colour differences have been explored using fMRI. Conversely, attention to yellow stimuli accelerated pupil responses, while those to blue or white stimuli had little to no effect on delay. We are unsure as to why these delay effects occur but their presence strengthens the case that something different is occurring for stimuli that do, or do not contain blue. When the effects of peripheral attention were examined 40, and the superior colliculus43. Thus, our results need to be discussed in terms of the blue sensing system and thalamo-cortical connectivity to the pupillary system of its visual inputs.Attentional effects are thought to be mediated by higher cortical areas exerting top-down influences30. Perhaps the best-known cortical input to the pupils is the luminance-based accommodative triad, which converges the eyes, accommodates the lenses and constricts the pupils when objects draw near44. This cortically derived, stereopsis-driven, system also appears to make the pupils sensitive to equiluminant temporal modulation of very high spatial frequency luminance gratings45. Pupil responses to red-green equiluminant stimuli, can be reversibly modulated from macaque extrastriate cortex46, or blocked by related cortical lesions in humans47. Those patients display red-green achromatopsia, thus the cortically mediated visual sensation and the pupil responses are concordant. As we have confirmed with 44-region multifocal stimuli31, these cortically driven pupil responses to red-green equiluminant stimuli are delayed compared to responses to luminance stimuli48. Pupil responses to 13 Hz modulation of blue-yellow contrast are similarly delayed49.The pretectal olivary nuclei (PON) drive the pupils and receive about half their input directly from retinal ganglion cells (RGCs), and the remainder from brain areas including the striate and extra-striate cortex50 due to the different blue-blocking capacity of brunescent lenses in older persons. The subjects in the present study were too young for this to be a major factor. At the level of the retinal ganglion cells (RGCs) the blue system is quite heterodox compared to other RGC types. In primates, the luminance and red/green RGCs come in anatomically similar pairs of parasol and midget RGCs, with the inputs to their receptive field centres exchanged to provide ON or OFF luminance centres, and red-ON or green-ON centres. The anatomically similar members of each pair of RGC types differ principally in which strata of the inner plexiform layer (IPL) their dendrites reside51. By contrast, the blue-ON and blue-OFF signals we experience are gathered by anatomically very different sub-populations of RGCs. The small-bistratified RGCS carry the cone-driven Blue-ON/Yellow-OFF signal, while the cone-driven blue-OFF/Yellow-ON signal is transmitted by the giant-monostratified RGCs52. These cells are much larger than the bistratified RGCs and have sub-populations defined by their stratification in the IPL and whether their axons project to the LGN or the PON.The blue content of white stimuli has been reported to be a major source of between-subject variability in multifocal ERGsgiant-monostratified RGCs contain melanopsin, making them intrinsically photo-receptive to blue light52, and so they are sometimes referred to as ipRGCs. The melanopsin has no light adaptation mechanism, which gives the ipRGCs a Blue-ON response that tracks absolute luminance in bright conditions53. The melanopsin response is very sluggish and is the main determinant of the steady state pupil diameter in bright light55. The cone derived Blue-OFF and melanopsin derived Blue-ON responses are thus temporally duplexed. Thus, post-synaptic cells could largely select which blue signal they are attending to (ON or OFF) by either integrating with a time constant of about 1 second, or by differentiating the input to monitor frequencies above 1 Hz.All the 30. That multifocal stimuli such as those used here influence the pupils via this route has been recently demonstrated by several of our studies. We have compared yellow and red-green stimuli in healthy subjects31 and in glaucoma patients23. As shown by other studies the cortically derived pupil responses to red-green stimuli were delayed compared to luminance stimuli. Both stimulus types picked up visual field losses in glaucoma suggesting the pupillary responses to these transiently presented stimuli are concordant with visual perception. By contrast, when extremely slow, long duration, blue stimuli that drove melanopsin were used the ability to detect glaucomatous visual field loss was poor compared to our standard transiently presented yellow stimuli24. That may be because of the very large dendritic trees of the ipRGCs or the branching of ipRGC axons within the retina56. Overall, it seems we can at least partially choose cortical versus retinal sources by altering the temporal properties of the stimuli. This idea is supported by our demonstration that a visual field marker for early-stage macular degeneration and diabetic eye disease is seen both for yellow mfPOP and mfVEP testing on the same 47 patients when the mfVEP electrodes are positioned to pick up extrastriate cortical activity32. In summary, several sources of evidence indicate that mfPOP responses to transiently presented stimuli are substantially driven by the extrastriate cortex, as PON connectivity would suggest30.The key factor for the present study is that both types of blue sensitive RGCs appear to contribute to visual perception via their projections to the LGN. As summarised above these signals then return to the pupillary system via the extra-striate cortical input to the PON58. Similarly, constriction and dilation effects have been shown with respect to modulations of attention and the apparent brightness of stimuli60. Attention, but not day-dreaming, modulates pupil dilation mediated by the slow sympathetic nervous system61. Here we have concentrated on the higher-bandwidth pupil constriction system driven by the parasympathetic system. That being said we have demonstrated that mfPOP can quantify the temporal evolution of the sympathetic-driven re-dilation phase out to several seconds24. Those parts of the pupillary response have been shown to quantify very interesting aspects of attentional modulation61. Thus, these effects could also be assessed in a multifocal setting, and differences between contraction and dilation effects quantified in one test.Reports of the ability of the pupils to track aspects of attention and cognition have been growing, but so far not with a detailed spatial mapping of the attentional affects as shown here. For example, pupil constrictions to dim photographs of the sun have been reported21, but with a cheaper and more user-friendly apparatus. One could also combine pupillometry and fMRI, or pupillometry, fMRI, and Near Infrared Spectroscopy. As we have recently demonstrated mfVEPs measured using EEG electrodes that largely sample extra-striate visual cortex verify some aspects of mfPOP responses33, and there would be no reason not to do both simultaneously to better identify the sources of pupil responses to attention. Certainly, the set-up for mfPOP is shorter than for EEG, and the system is potentially very portable. The dichoptic set-up also allows for a variety of between-eye stimulus manipulations. An aspect we have not addressed is that by stimulating both eyes concurrently and recording both pupils both direct and consensual responses are recorded. This allows for differential examination of afferent versus efferent effects of attention. Some of our results indicate that efferent effects were large but confirmation will await the use of newer more reliable stimuli26.The multifocal methods used here open up the possibility of examining retinotopic mechanisms of attention as has begun to be explored with fMRIPerhaps the rate of delivery of multifocal stimuli needs to be considered more. Differences between Experiment 1 and the other three were observed, and the stimulus delivery rate in Experiment 1 was 4 times higher. Aside from the stimuli being different, an idea suggested by a reviewer is that with the more rapid stimuli attention would possibly be more divided between them, when there was no central task.21. Experiment 4 examined differences between different direct attention conditions and revealed that attention in the inferior field may be more variable.A problem with the first three experiments was the possibility that the participants either guessed the purpose of the experiment, or invented some attentional task of their own during the experiments, when they were not directed to attend to the central changes. It was therefore useful to compare results obtained for different foci of continuously directed attention as in Experiment 4 Fig. . Neverth


Sports-related concussions are an increasing public health issue with much concern about the possible long-term decrements in cognitive function and quality of life that may occur in athletes. The measurement of cognitive function is a common component of concussion management protocols due to cognitive impairments that occur after sustaining a concussion; however, the tools that are often used may not be sensitive enough to expose long term problems with cognitive function. The current paper is a brief review, which suggests that measuring cognitive processing through the use of event related potentials (ERPs) may provide a more sensitive assessment of cognitive function, as shown through recent research showing concussion history to influence ERPs components. The potential influence of genetics on cognitive function and ERPs components will also be discussed in relation to future concussion management. The Centers for Disease Control and Prevention estimates that as many as 3.8 million sport and recreation-related concussions occur each year in the United States . Sports-Although concussion has been considered a transient injury, a number of recent studies link multiple concussions with long-term neurodegenerative issues ,4,5. OneThe prevalence and potential lifelong effects of concussion demand that more attention must be directed towards continually improving concussion management in an effort to minimize short and long-term injury. Proper concussion management allows for safe and effective return-to-play decisions to be made. This reduces the chances of exacerbating a current concussion, which, as shown by CTE research, may lead to serious long-term brain dysfunction. Current clinical concussion management protocol for athletic trainers includes neurocognitive evaluation . The ImmSome studies suggest that cognitive ERPs, which can be used to assess a variety of cognitive processes ranging from visual attention  to spatial analysis (N2) and visual perception (P3), can be influenced by genetics ,14,15,16The major ERP components include N1, Mismatch Negativity (MMN), the N2 family, the P3 family, and Error Related Negativity (ERN) . The N1 The MMN component appears when an individual has been attending to a repetition of identical stimuli and then is presented with an out-of-the ordinary stimulus. The MMN peak component is indicative of a process that compares the most recent stimuli to the sensory memory trace of previous stimuli. MMN is observed as a negative wave that is maximum size at central midline scalp sites and peaks between 160–220 ms.N2 is the second negative wave peak of an ERP and occurs between 200 ms and 350 ms following the presentation of a stimulus . An N2 dThe P3 component corresponds to the devotion of attention to significant environmental events, and records the constant refreshing of working memory ,21,22,23Two ERP components that are stimulus-locked are the ERN and the Error Related Positivity (Pe). The ERN is a peak component characterized by a negative-going deflection at the frontal and central electrode sides following the time of response. It is generally thought that the ERN corresponds to the system that monitors responses or is sensitive to conflicts between intended and actual responses ,26. The For more comprehensive reviews of the literature regarding ERPs and their use in concussion, please see the reviews by Alderman et al. and Broglio et al. ,29. A stGaetz et al. studied the effects of multiple concussions in groups of hockey players using N2/P3 and CNV (Contingent Negative Variation) paradigms . The resLedwidge and Molfese found larger N2/P3 amplitudes in athletes with multiple concussions and longer P3b latencies . A recenThese waveforms have also been connected to post-concussion symptoms. In a study done by Lavoie et al., P3b amplitude and symptomatology were inversely related, as symptomatic athletes had smaller P3b amplitudes relative to asymptomatic athletes . In thisThe studies detailed above demonstrate the ability of event-related potentials, specifically that of the P3b and N2 waveforms, to detect cognitive dysfunction in those with a history of concussion. Because P3b amplitude is associated with the allocation of attentional resources to a stimulus, it is possible that the amplitude is a function of the amount of resources being employed at a specific point in time following stimulus presentation. It then follows that a decrease in amplitude post-concussion indicates that the ability to recruit neural networks needed to attend to a stimulus has been impacted. Overall, these findings suggest that concussion history may affect the amplitude or latency of ERP components, and thus EEG may prove a reliable technology for detecting and measuring underlying cognitive deficits. Impairments in cognition are often seen acutely following a concussion, and long-term deficits in cognitive function as a result of concussion are a major concern. The use of commonly used neurocognitive assessments, such as ImPACT, may not be sensitive enough to detect these changes . TherefoOne of the most widely used computerized neurocognitive tests in concussion diagnosis and management is the ImPACT . ImPACT In 2001, the first International Conference on Concussion in Sport held in Austria deemed neurocognitive testing the paramount concussion diagnostic tool and placed much promise in computerized neurocognitive test batteries regarding detection of concussions . HoweverThe 2001 International Symposium on Concussion in Sport first expressed interest in the manifestations of neurocognitive deficits in relation to genetic phenotype . This coOne genetic factor of particular interest is the allelic variation of the Apolipoprotein E (ApoE) gene. The ApoE gene on chromosome 19 is essential in plasma lipid metabolism. The ApoE gene has three major isoforms: e2–e4 . The vasSpecifically, the e4 allele has been associated with an increased risk for developing Alzheimer’s Disease (AD), and, when coupled with a history of head injury, the risk of Alzheimer’s Disease may increase ten-fold . ScientiThe e4 allele of the ApoE gene is also associated with decreased performance on tests of global cognitive function and episodic memory ,57,58. NCrawford et al. (2002) linked the e4 allele to deficient memory performance and rehab outcome following a TBI. Athletes who have suffered an mTBI (mild traumatic brain injury) and who carry the e4 allele are more likely to experience post-concussion headache and report more severe post-concussion headache symptoms than concussed athletes who lack the e4 allele . SubcommIn addition to cognitive function, the e4 allele has been associated with lower N2 amplitude and may be indicative of a gene-dose relationship, with e4 homozygotes exhibiting lower amplitudes than e4 heterozygotes . A studyA specific single-nucleotide polymorphism (SNP) in the ApoE gene (rs405509) is commonly known as the APOE Promoter. This SNP is characterized by a T allele in lieu of a G allele, and is linked to increases concussion susceptibility ,66). Ind. Ind66])COMT) gene is responsible for the breakdown of dopamine and serotonin in the brain. The presence of the COMT Met allele may increase performance on tasks of executive functioning [COMT gene have extended P300 latencies [The catechol-O-methyltransferase  be examined in relation to concussions, because it has been hypothesized to effect executive function and possibly be related to Alzheimer’s disease . A studyMore research must be done on the effects of ApoE, COMT, and DRD2 on ERP before EEG technology can be used in conjunction with genetics as a concussion evaluation and management tool. Additionally, research is needed to demonstrate that there is a link between concussion history, genetics and ERP components, and until this link is made, any recommendations should be made with caution. However, if these relationships can be confirmed, these genes could serve as useful biomarkers in concussion management for their potential relation to cognitive function.In order to confirm the relationships hypothesized in this paper, future studies could focus on incorporating possibly more sensitive measures of cognitive function such as those provided by ERPs and genetics into the diagnosis and management of sports-related concussions, but also may be important in determining long-term implications of concussions in athletes. Studies could be conducted that examine ERP components during recovery and while returning-to-play. Additionally, a more thorough understanding of the influence of type of task and the ERP components elicited need to be undertaken. McDevitt & Krynetskiy have suggested genotyping of athletes as a potential method for clinicians to improve concussion management and return-to-play decisions . Specifi


Elastic stable intramedullary nailing (ESIN) has became a well-accepted method of osteosynthesis of diaphyseal fractures in the skeletally immature patient for many advantages, the purpose of this study is to evaluate the preliminary results of this minimally invasive treatment for severely displaced distal tibial diaphyseal metaphyseal junction (DTDMJ) fractures.This study was carried out over a 6-year period. Twenty-one severely displaced DTDMJ fractures treated using ESIN were evaluated clinically and radiographically. Complications were assessed: the patients were evaluated with regard to nonunion, malunion, infection, growth arrest, leg length discrepancy, implant irritation, and joint function.Mean age at the time of surgery was 7.8 years (range between 5.3 and 14.8 years), mean body weight 34.1 kg, all fractures were transverse or mild oblique type, including 3 open fractures, 5 multifragmented fractures, and 4 fractures associated with polytrauma; 6 cases were treated with antegrade ESIN of tibia while 15 cases need combined retrograde fibula and antegrade tibia fixation treatments. Follow-ups were ranging from 11 to 36 months, 19 fractures showed both clinical and radiographic evidence of healing within 5 months; all cases had full range motion of knee and ankle with symmetrical foot progress angle. Nail removal was at a mean 7.1 months, at final follow-up, no growth arrest or disturbances occurred. Five patients had complications; leg length discrepancy had decreased yet affected 2 patients, 2 cases showed delayed union, and 1 case developed restricted dorsal extension at the metatarsophalangeal joint of the hallux.ESIN is the treatment of choice for pediatric severely displaced DTDMJ fractures that cannot be reduced by closed reduction or ones that cannot be casted. The advantages include faster fracture healing, excellent functional and cosmetic results, safe and reliable surgical technique, and lower severe complication rate. Surgical treatment should be considered for those cases where closed treatment cannot achieve and maintain acceptable alignment, rotation, and length. As severely displaced distal tibial diaphyseal metaphyseal junction (DTDMJ) fractures, the treatment is more challenging, and often requires intervention, operative treatment is needed in polytrauma patients who have most unstable fractures, open fractures, neurovascular injuries, and compartment syndrome.–4 ESIN can be considered the “gold standard” for the treatment of midshaft fractures, but there are concerns about their use in distal tibial diaphyseal and proximal metaphyseal fractures, its proximity to the ankle makes the surgical technique more complicated than the treatment of the midshaft fractures, and the stability supplied by the nails cannot be achieved optimally.Tibial diaphyseal displaced fractures in children are frequently treated with the elastic stable intramedullary nailing (ESIN) method. The advantages are minimally invasive surgery with a short hospitalization duration, primary bone union and early weight bearing.Due to the paucity of the relevant literature to investigate the outcomes of surgical treatment of severely displaced DTDMJ fractures using ESIN in children, we report our initial experience and retrospectively evaluate the clinical and radiographic outcomes.2,6 the metaphysis area is marked by a rectangle containing the base of the growth plate of both distal tibia and fibula , another small square is made at the side of tibial epiphysis that the height was equal to the widest portion of the tibial epiphysis. The segment of distal tibia between 2 squares is defined as the DTDMJ  with 2.5 to 4.0 mm diameters were employed on the basis of one-third of the medullary canal of the tibial shaft at its narrowest point. The tips of the nails  may need to be greater precontoured approximately 1.3 times as it does in shaft fracture, for more divergence can be achieved with more stabilization of the fracture when nails are advanced into the distal fragment. At the beginning, 2 nails were introduced through symmetrical skin incisions (1 cm in length) which were made at the proximal medial and lateral metaphyseal cortices 2 cm distal to the tibial tubercle, and both nails were advanced proximally to the fracture site; then close reduction of the fracture was performed using C-arm fluoroscopy, distal traction was continued until length has been reestablished; after that, distal fragment was manipulated to contact with proximal one. Once a satisfactory reduction has been achieved, the first nail was advanced distally into the tibal metaphysis . The position of distal fragment could be corrected indirectly by turning the first nail, and then the second nail was advanced into the proper position. Make sure that the tips of nails were in divergent positions and the reduction and stability of the fracture were satisfactory , and 4 cases of polytrauma included 3 multiple fractures and 1 had craniocerebral injury. In the series, all cases were associated with distal fibular fractures, from which 18 fibular fractures were completely displaced.There were 11 boys and 10 girls. And the mean age at the time of their surgeries was 7.8  years, mean body weight 34.1  kg, in which 9 cases involved the left side. The main cause of injury was traffic accidents in 10 cases, by tumbling in 6 cases, and by falling from height in 5 cases. The most common was the transverse type of fracture (13 cases) followed by the mild oblique type (8 cases); 4 multifragmented fractures were classified as comminuted type “wedge” while 1 multifragmented fracture was “complex” based on AO's definition of fracture severity code and proposed simplification;In tibial fractures, 19 cases were fixed with 2 nails Figs. , while 2Mean duration of surgery was 46.7 ± 20.0  minutes. Mean hospitalization duration was 3.9 (range 2–10) days; it did not exceed 5 days except in the case of the patient with craniocerebral injury. Follow-up ranged from 11 to 36 months, averaged 24.6 ± 8.5 months, 19 fractures showed both clinical and radiographic evidence of healing within 21 weeks  and 2 cases (9.5%) showed delayed healing at 10 months  months. At the final follow-up, all cases had full range motion on knee and ankle with symmetrical foot progress angle, no limping and they were able to have unrestricted physical activity at the finial visit. There were no infections or nail end irritation, as no growth arrest or disturbances by radiological and clinical assessment.Five patients had complications. Leg length discrepancy affected 2 cases, initially, lengthening was observed in 2 patients  at 12 months after surgery. At the final visit (24 months and 36 months after surgery), the length discrepancy had decreased, and mean tibia overgrowth was10.5 mm  . However, the optimal treatment of unstable distal tibia metaphyseal fractures remains controversial. Crossed Kirschner wires fixed with an acute angle or entry point are very close to the fracture line, and this would lead to unstable fracture reduction. Plates may sometimes result in extensive soft tissue dissection and may be associated with nonunion, wound infections despite that they could bring complete anatomical reduction. As external fixation provides a stable immobilization, which allows direct surveillance of the limb and wound status. It is particularly useful in open fractures, some studies reported that the use of external fixator was associated with 50% of superficial pin tract infections, a high rate of refracture and joint stiffness. The use of ESIN in the proximal and distal tibia is technically demanding and not free of complications. Only few reports about its application in the distal tibial metaphyseal fracture were found. In 2014, Cravino et al showed good results in the pediatric population who had sustained closed traumatic, displaced fracture of the distal tibial metaphysis treated by ESIN. We separated distal tibial metaphysis into 2 parts, proximal part and distal part. DTDMJ is the proximal part of the distal tibial metaphysis. All the 21 cases in this study were located at this junction, and satisfied results have been achieved. If the fracture is located at the distal part, which is more adjacent to the epiphyseal plate, in our opinion, it will be too difficult to fix the fracture and keep its stabilization by the nails unless the tips of the nails are perforated at the growth plate. The reason is that the fracture line is very close to the growth plate and nails are insufficient for keeping the reduction while the nails stop at the growth plate. Cravino et al reported in some distal tibia fractures, for example, stable fixation could only be achieved by anchoring the tip of the nails in the epiphysis; however, these cases just had short follow-up and it is not possible to exclude growth plate disturbances. Due to the high risk of growth disturbances, in this study, all fractures were located at DTDMJ and none of the nails advanced across the distal epiphyseal plate.In spite of the internal fixation of pediatric diaphyseal tibial fractures with ESIN has been a rapid, well-codified, and effective method.Treatment of the DTDMJ fractures with ESIN involves a totally different concept from diaphyseal fracture management. Although the Me’taizeau technique is minimally invasive and easy to learn, distal tibia metaphysis fractures can be challenging due to the relatively short length of the distal fragment and the proximity of the growth plate. First, the tips of the nails may need to be greater precontoured approximately 1.3 times as it does in shaft fracture to get much divergency with stable osteosynthesis. Secondly, if conventional 2 nails cannot achieve sufficient stabilization of reduction, an extra nail could be used to get more stabilization of the fracture, and all the 3 tips of the nails should be in divergent positions. In this study, 2 of 21 cases fixed with 3 nails in tibial fracture, and 1 case showed in Fig.  reported 5 patients developed clawing of the hallux following a fracture of the tibia, and relieved by performing a tenolysis proximal to the medial malleolus, the operative findings demonstrated that the etiology was associated with a localized subclinical compartment syndrome. Our patient refused to accept the further treatment because he did not have any limitation during physical activity.In this series, 19 fractures achieved bony union within 21 weeks and 2 cases showed delayed healing at 10 months after surgery, no angular malalignment or malrotation was identified. This technique is to preserve the blood supply and soft tissues at the fracture site while providing adequate stabilization of the fracture. It included 2 cases with 3 nails fixation of tibia and 15 cases with retrograde fixation of the fibula. Other complications were 2 cases of overgrowth  and 1 case developed restricted dorsal extension at the metatarsophalangeal joint of the hallux, 1 studyLimitations of this study include a retrospective evaluation of surgical treatment with a small number of patients, the absence of long-term follow-up to evaluate the outcome of growth plate disturbances, and lack of comparison group to carry out randomized controlled trials.5This study showed good functional and radiological results in the pediatric population who had severely displaced DTDMJ fractures that cannot be reduced by closed reduction or be casted, and we believe that ESIN is the treatment of choice for the transverse and mild oblique fractures, “wedge” multifragmented fractures, and mild open fractures. The advantages include fracture healing faster, excellent functional and cosmetic results, safe and reliable surgical technique, and lower severe complication rate. However, further prospective studies are required to compare ESIN with other treatment modalities.


The treatment options for high-risk prostate cancer are either radical prostatectomy or radiotherapy/brachytherapy depending on the patients’ prognosis. In older men with multiple comorbidities, radiotherapy with androgen deprivation therapy is an attractive option. Common side effects of androgen deprivation therapy include hot flushes, tiredness, increased risk of fractures, increased risk of metabolic disorders, coronary heart disease, and psychological effects. This case highlights the potential side effect of lipodystrophy secondary to leuprolide acetate injections. To the best of our knowledge, this is the first reported case of such an instance.In this case report, we describe a 70-year-old white man with prostate-specific antigen of 1.8 ng/mL, clinical stage T2bN0M0, Gleason 4+5=9 prostate cancer who developed an unusual side effect from leuprolide acetate as part of his androgen deprivation therapy. Approximately 2 months after the initial 3-monthly injection of leuprolide acetate (Eligard 22.5 mg) our patient developed abnormal lipid deposition particularly in his deltoid and abdominal region. His upper limb mobility gradually became compromised due to the size of these abnormal fat depositions. He had liposuction to correct this lipodystrophy and had a good functional outcome and cosmesis from the procedure.To the best of our knowledge, this is the first reported case of lipodystrophy secondary to leuprolide acetate injections. Leuprolide acetate in commonly used as one of the gonadotrophin-releasing hormone agonists and thus we should be mindful of the potential effect of producing lipodystrophy, especially in patients with cirrhosis, and to watch for any signs and symptoms as appropriate. The implication of this potential side effect poses difficult management strategies for such patients, and second-line alternatives such as chemotherapy may need to be considered. The treatment of prostate cancer depends on the risk stratification and staging of the patient. Initial evaluation of a patient with suspected prostate cancer includes a detailed history, digital rectal examination, pre-treatment serum prostate-specific antigen (PSA), and Gleason score from the initial prostate biopsy. Imaging studies that are used include radionuclide bone scan and computed tomography (CT)/magnetic resonance imaging (MRI) of the abdomen and pelvis to look for nodal and metastatic involvement. With those findings patients are staged according to American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) classifications , 2.Based on their staging workup we can stratify patients into various classifications as suggested by the National Comprehensive Cancer Network (NCCN) . PatientEligard  is generally well tolerated, with the most common adverse event being flushing and injection site erythema . It has A 70-year-old white man presented with a history of lower urinary tract symptoms suggestive of prostatic enlargement. His background history included appendicectomy, diverticulitis, hypertension, and past heavy alcohol intake. He denied any significant family history. On examination, his prostate was found to be smooth and mildly enlarged. At this stage his PSA was measured at 1.8. Within 2 months it nearly doubled to 3.2 and a hard nodule was now felt on the right side of his prostate. He had a prostate biopsy under ultrasound guidance 3 months later and was found to have adenocarcinoma with Gleason score of 9 (5+4). Of the cores examined, 11 out of 17 were positive for adenocarcinoma. His prostate volume was 27 cc.He underwent staging with a bone scan and CT of his chest, abdomen, and pelvis. There was no evidence of metastatic disease but there was evidence suggestive of liver cirrhosis on CT. After lengthy discussion with his urologist and radiation oncologist he proceeded with external beam radiotherapy with neoadjuvant and adjuvant androgen deprivation therapy. He received 76 Gy in 38 fractions using a conformal technique within 8 months of the prostate biopsy. He was also commenced on 3-monthly Eligard  subcutaneous injections (22.5 mg) over his lower abdomen for androgen deprivation.He started to note the development of abnormal swelling over his deltoids to the extent that it was interfering with his activities of daily living. The swelling was so large that he was unable to wear some shirts and he was unable to abduct and forward flex his shoulder joint which made reaching over his head to retrieve items from cupboards very difficult. He noticed these swellings over the 2 months post his first Eligard  injection Fig. ; howeverOn his most recent follow up, he unfortunately showed signs of clinical relapse with a rising PSA from 0.75 ng/mL and testosterone of 26.8 nmol/L (January 2014) to a PSA of 24 ng/mL and testosterone level of 34.1 nmol/L (September 2014) as well as evidence of bone metastases on a repeat bone scan. He was then recommenced on goserelin acetate 10.8 mg 3-monthly subcutaneous injection as a single agent for further treatment which has so far seemed successful.The use of luteinizing hormone-releasing hormone (LHRH) agonists is not only limited to advance prostate cancer. They are commonly used in fertility treatments, endometriosis, precocious puberty, uterine fibroids, and hirsutism . For ferIt is interesting to note that lipodystrophy can also be seen in patients with HIV. The mechanism for this has not yet been proven but one theory suggests that it is related to HIV type 1 protease inhibitors and nucleoside reverse transcriptase inhibitors such as stavudine and zidovudine , 14.Although the mechanism for the lipodystrophy in our patient is not clear, we postulate that due to the androgen surge that is associated with LHRH agonists such as leuprorelin , it creaIn this case report we discuss an uncommon side effect from leuprolide acetate injections for the treatment of prostate cancer. To the best of our knowledge this is the first reported case of lipodystrophy secondary to leuprolide acetate injections. Leuprolide acetate is commonly used as one of the gonadotrophin-releasing hormone agonists and thus we should be mindful of the potential effect of producing lipodystrophy, especially in patients with cirrhosis, and to watch for any signs and symptoms as appropriate. The implication of this potential side effect poses difficult management strategies for such patients, and second-line alternatives such as chemotherapy may need to be considered.


Cre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. Using this extensive database, we have established the Mouse Model of Pancreatic Cancer Atlas (MMPCA) to serve as a platform for meaningful and interactive discussion among researchers and pathologists who study pancreatic disease. We hope that the MMPCA will be an effective tool for promoting a more consistent and accurate consensus of PanIN classifications in the future.Pancreatic ductal adenocarcinoma (PDAC) is one of the leading forms of cancer related deaths in the United States. With limited treatment options and unreliable diagnostic methods, long-term survival rates following a diagnosis of pancreatic cancer remain poor. Pancreatic intraepithelial neoplasia (PanIN) are precancerous lesions that precede progression towards PDAC. PanIN occur in increasing complexity as the disease progresses and the description of PanIN plays a critical role in describing, staging and diagnosing PDAC. Inconsistencies in PanIN classifications exist even amongst leading pathologists. This has led to debate and confusion among researchers and pathologists involved in pancreatic cancer research, diagnosis and treatment. We have sought to initiate a discussion with leading pathologists with a goal of increasing consensus in the interpretation of PanIN and associated structures within the precancerous pancreas. Toward achieving this goal, we are in the process of conducting an extensive study of over 1000 male and female pancreata in varying stages of PanIN progression isolated from the Ptf1a The high mortality rate of PDAC is due in part due to the typical inability to detect the disease before reaches an advanced stage. A major challenge to early detection is that the size of key precancerous lesions, known as pancreatic intraepithelial neoplasia (PanIN), are below the detection limit of current imaging capabilities [Pancreatic cancer is one of the most lethal cancers and is currently estimated as the third leading cause of cancer related deaths in the United States –4. Pancrbilities , and parbilities .The description of PanIN plays a central role in the diagnosis and staging of pancreatic disease. PanIN have historically been described assuming a model that PanIN emerge from metaplasia of pancreatic ductal epithelia. PanIN are currently classified into three main groups, PanIN-1, PanIN-2 and PanIN-3, with PanIN-1 being further broken down into PanIN-1A and PanIN-1B. However, often a histological feature in a diseased pancreas does not fit neatly into one of the three PanIN classes, and this is where consensus begins to break down. Although the existing PanIN classification schemes are descriptive, they may not accurately reflect PanIN etiology since an alternative model for PanIN emergence from acinar-to-ductal metaplasia (ADM) is gaining acceptance , 11. As In 1999, the Pancreatic Cancer Think Tank, sponsored by the National Cancer Institute, was formed and established the nomenclature and classification scheme for PanIN. Members were composed of pathologists from the United States and Europe . The groAnother group of international experts was established to further discuss the classification system. This meeting was held at Johns Hopkins Hospital in 2003 . At thisNormal ductal epithelium cells are cuboidal in appearance with no signs of mucinous cytoplasm or nuclear crowding. Squamous  metaplasia refers to transitioning of normal cuboidal epithelial cells into another distinct type of cells, as occurs during elongation of the cuboidal cells seen in PanIN-1A. PanIN-1A is distinguishable by flat, basally located nuclei within columnar cells and with mucin present. PanIN-1B is similar to PanIN-1A, but display a papillary or basally located pseudostratified structure. PanIN-2 may be present as flat or papillary and display abnormal nuclear characteristics without cribriforming luminal necrosis. PanIN-3 are typically papillary with cribriforming of the lumen and loss of nuclear polarity –15. TablAn alternative classification system has been introduced  based onCre/+ model [Cre/+;LSL-KrasG12D/+ mouse strain was used as a model of human pancreatic cancer. It should also be noted that in the adult mouse, the islet cells contain Pdx-1 expression, while the acinar cells contain Ptf1a expression [Here, we have used a mouse model to investigate progression of pancreatic cancer and PanIN initiation. Mouse models of pancreatic cancer have been developed that appear to recapitulate all recognized features observed in human pancreatic cancer progression, from the precancerous PanIN precursor state to invasive carcinoma . The mos/+ model , and we /+ model , and we pression .Cre/+;LSL-KrasG12D/+ mouse strain as a model for human pancreatic cancer development, it should be noted that Kras mutations have been reported to be present in >90% of human pancreatic adenocarcinomas and found in more than 95% of the earliest pre-cancerous PanIN-1 features [Cre/+;LSL-KrasG12D/+ strain used in this study has been shown by others, and observed in our lab, to progress from a PanIN pre-cancerous stage all the way through tumor formation, which seems to closely mimic pancreatic cancer progression in humans [Regarding the relevance of the Ptf1afeatures . In thatfeatures . Given tfeatures . Consequn humans ,20–22.st release of the MMPCA were obtained from over 500 male and female mice at ages ranging from 5- to 16-months from the Ptf1aCre/+;LSL-KrasG12D/+ mouse model. The MMPCA provides multiple images from different mice at multiple magnifications and in the form of an open and continuous discussion. By providing an online hub with one of the most extensive publicly accessible and annotatable histology databases available for a mouse model of pancreatic cancer, experts will be able to discuss their opinions on classification patterns and interactively address classification discrepancies in specific histology images. After this interaction takes hold, the online atlas will allow for researchers in the field to collaborate on refining future guidelines for classification of PanIN and other features observed in pancreatic cancer tissues. The MMPCA will also enable comparison with what is known about pancreatic cancer progression in humans, and as more data becomes available, allow further assessment of the Ptf1aCre/+;LSL-KrasG12D/+ mouse strain as a model for human pancreatic cancer.The goal in creating the Mouse Model of Pancreatic Cancer Atlas (MMPCA) is to utilize our extensive histology database to establish an extensive resource in which researchers and pathologists in the field of pancreatic cancer can engage in interactive conversations about the PanIN classification and broader interpretation of pancreas tissue features involved in pancreas pathology. Images for the 1The Institutional Animal Care and Use Committee (IACUC) at Miami University approved the procedures performed in this study as documented by IACUC project numbers 854 and 855. The protocols were approved by both the ethics committee and the IACUC of Miami University . All surgeries were performed under isoflurane anesthesia, and all efforts were made to minimize animal suffering. All authors and personnel involved were required to complete online and in class animal training prior to any work involving animals. All dissections were conducted as previously described . The proCre/+;LSL-KrasG12D/+ mouse model of pancreatic cancer was used in this study. Control mice were represented by Ptf1aCre/-;LSL-KrasG12D/- mice and study mice were represented by Ptf1aCre/+;LSL-KrasG12D/+ mice. Mice were acquired from The Jackson Laboratory and then bred in-house at Miami University to obtain the desired number needed for the study. The KrasG12D mice, scientific strain name B6.129-Kras<tm4Tyj>, contains a mutant Kras that is silenced by a Lox-stop-Lox cassette. The mutation is unsilenced through the crossbreeding with Ptf1a mice, specific strain name B6.Ptf1a(tm1.1(cre)Cvw), that express cre-recombinase under the control of the Cre knockin at the Ptf1a-p48 locus, which is primarily expressed in the pancreas. Genotyping for the Kras mutation and Cre-recombinase was conducted to determine the classification of each mouse. DNA from each mouse was obtained through an ear punch for later extraction and also for identification purposes throughout the study.The Ptf1a2 (Fisher Scientific), water, and either the Kras, Cre, or control primers (IDT). For the Kras program, the mixture was denatured at 94°C for 30 seconds, then annealed at 69°C for 1 minute, and elongated at 72°C for 1 minute using the Gene Amp PCR System 9700 thermocycler for 35 cycles, then held at 4°C when complete. For the Cre program, the mixture was denatured at 94°C for 1 minute, then annealed at 63°C for 20 seconds, and elongated at 72°C for 45 seconds using the thermocycler for 30 cycles, then held at 4°C when complete.DNA was extracted through the use of a thermocycler, Gene Amp PCR System 9700, after submerging the ear clipping in 25 uL of 25 mM NaOH/0.2 mM EDTA at a pH of 12. After an hour at 98°C, the sample was neutralized with 25 uL of 40 mM Tris-HCl at a pH of 5. The sample is stored at -20°C until PCR amplification of the Kras, Cre, and control primers are conducted. The extracted DNA was added to a mixture containing Dreamtaq Green Master Mix (Fisher Scientific), MgClAn agarose gel stained with ethidium bromide was used to separate the amplified gene product through gel electrophoresis. A UV light on the UVP ChemiDoc-It 2 815 Imager was used to show the presence or absence of the amplified DNA. When present, the Kras mutation is shown at 550 bp and the Cre at 250 bp. Classification as a study mouse means both the Kras and Cre were present, and classification as a control mouse means both the Kras and Cre were absent. In this atlas, cohorts of nine study mice and one control mouse per age category were generated for age groups 5-, 11-, and 15- months. The Institutional Animal Care and Use Committee (IACUC) at Miami University approved all procedures conducted in this research.Cre/+;LSL-KrasG12D/+) and control  mice were used in this study. Starting at age 5-months, 24 mice of each gender for both control and study mice were sacrificed and pancreata harvested for histological analysis extending out to 15-months of age. The complete distribution of mice utilized in the study, and therefore representing the MMPC Atlas, is summarized in Collectively, over 1000 study  were generated from groups of 5-, 11-, and 15-month old mice. Histology images from these mice were used to determine PanIN grades and frequencies in the study mice in comparison to normal pancreatic tissue. It has been previously reported that more than 80% of the ducts are expected to be normal at approximately 2.25 months, 68% of the ducts are expected to be normal from age 4–5 months, and 18% of the ducts are expected to be normal for mice in the age range from 7–10 months . BrieflySlides were stained with hematoxylin and eosin. First, the slides were placed in xylene substitute for 4 minutes. Then, the slides were transferred to absolute ethanol for 4 minutes, 95% ethanol for 2 minutes, and 70% for 2 minutes. They were then rinsed with tap water for 4 minutes and kept between 37–40°C to help distinguish the blues of the hematoxylin. They were placed in the hematoxylin  for 1 minute and were placed back into the tap water for 4 minutes. Then the slides were dipped for a second in the 1% acid alcohol solution and were rinsed again in the tap water for 4 minutes. After being placed for 2 minutes in “Scott’s Tap” water, a combination of distilled water, sodium bicarbonate, and magnesium sulfate, they were rinsed in tap water for 4 minutes. They were then placed in eosin for 30 seconds and put into 95% ethanol for 6 minutes. The slides were then placed into absolute ethanol for 6 minutes, then xylene substitute for 4 minutes. The undersides of the slides were wiped free from solution and 2 drops of permount was added with a coverslip. Images were then taken through the use of an Olympus AX70 Light Microscope in the Center for Advanced Microscopy and Imaging at Miami University.https://mousepancreaticcancer.wordpress.com/ and is under the control and maintenance of the Kennedy Research Group at Miami University. Currently, images from 5-, 11-, and 15-month-old mice are available on the database, and with the continued maintenance under the Kennedy group, additional age categories will be added and a new MMPCA version released each year.Once all of images were captured with the Olympus AX70, they were then uploaded into “slide decks” on a WordPress page established for the online atlas. The homepage for the MMPCA is shown in Cre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. A screen-shot of the home welcome page of the MMPCA is shown in The MMPCA is an online freely accessible atlas intended to facilitate broad participation of researchers and pathologists who are actively involved in pancreatic disease research in an online interactive conversation regarding classification of features that arise in the histological analysis of the pancreas in the Ptf1aCurrently, there are websites that offer in depth examples of the various classifications and subsequent stages, but none of these websites function as interactive databases that provide multiple images from multiple subjects at varying magnifications. By allowing pathologists to make comments on varying features within an image, discussion is generated, and thus conflict resolution is initiated. Multiple magnifications are also displayed to show varying features throughout the pancreatic tissue.The initial MMPCA has been established using images from mice ages 5, 11, and 15 months to ensure that early, intermediate, and late PanIN stages were represented. Control pancreas tissue is present from each age category to establish a basis for comparison with normal tissue within this mouse model. By providing multiple images from different mice, the database provides a comprehensive model of PanIN development that seeks to resolve ambiguity in the field. From this, we hope to use our ample amount of histology from the ages 5, 11, and 15 months to help set a standard classification system of the PanIN to be used in both research studies and clinical practices.Cre;LSL-KrasG12D mice . The MMPCA provides a large database of images at multiple magnifications, making vast amounts of information available to researchers that can be investigated and shared among experts around the world. The MMPCA will provide an opportunity for investigators and pathologists to refine current PanIN and pancreatic cancer classification schemes and the possibility for increasing the consensus regarding PanIN classifications and descriptions of pancreatic cancer pathology. The online and interactive nature of the MMPCA will make it possible for pathologists and researchers from around the world to participate in the dialogue and to contribute their expertise to the project by simply accessing the MMPCA web page, adding comments and interacting with other investigators.We introduce the MMPCA, an online and interactive resource for researchers and pathologists active in the investigation and classification of pancreatic cancer. The MMPCA is being populated from histology images prepared from pancreata collected from over 1000 age-matched and gender-matched Ptf1a


Valid HER2 testing is essential for optimal therapy of patients with HER2-positive gastric cancer and the correct use of first-line chemotherapy. While testing for HER2 status in breast cancer is routinely performed, this is not the case for HER2 testing in gastric cancer and it is usually only performed on clinician request. An interdisciplinary German expert group (pathologists and clinicians) took the challenges of HER2 testing in gastric cancer as an opportunity to address essential aspects and questions for the practical use of HER2 testing in this indication. The recommendations made in this manuscript reflect the consensus of all participants and reflect their opinions and long-term experience in this field. Valid human epidermal growth factor receptor 2 (HER2) testing is essential for the optimal care of patients with advanced gastric cancer and the correct use of first-line drug therapy. Prerequisites for the accurate diagnosis of HER2 status are the availability and quality of samples, quality-assured histopathological diagnosis and close communication between pathologists and clinicians in the interpretation of the findings with regard to their therapeutic consequences. While today all breast cancers are routinely tested for the HER2 status, this is not yet the case for HER2 testing in gastric cancer and it is often only performed when requested by the clinician. One underlying reason for this is due to the fact that trastuzumab is only approved for the treatment of patients with advanced or metastatic gastric cancer, while the breast cancer label also includes patients with early HER2-positive breast cancer , however, the cost implications and therapeutic relevance of this approach are controversial.HER2 gene amplification status for further clarification with ISH testing is required  study, which showed a significant survival benefit for patients treated with trastuzumab plus chemotherapy (capecitabine or 5-fluorouracil and cisplatin) as compared with chemotherapy alone   and negative ratio (<2.0) the tumor should be considered as HER2-positive in accordance with the updated recommendation for breast cancer  whose tumors have HER2 overexpression as defined by IHC2 + and a confirmatory positive ISH result, or by an IHC 3 + result. To ensure accurate and reproducible results, validated and standardized assay methods should be used  in all cases of adenocarcinoma of the stomach or gastroesophageal junction. Should this not be warranted, HER2 testing can also be performed upon request of the clinician who submitted the sample for analysis .HER2 amplification status should be assessed with ISH according to the currently recommended test algorithm.When up-front testing is performed, the pathologist should initially carry out an IHC-based assessment. In inconclusive cases (IHC2+), The minimum number of biopsies and tumor cells required for robust HER2 testing was discussed.Five tumor-containing biopsies from different areas of the tumor should be aimed for. If the number of biopsies for reliable HER2 testing is insufficient and/or in inconclusive cases , a rebiopsy is required. This also applies where sample quality does not allow valid assessment of HER2 status. In inconclusive cases, other tumor-containing tissue should also be assessed, e.g. metastatic tissue in addition to the primary tumor, if applicable. For validation of IHC2+ cases with ISH at least 50 tumor cells should be available. If further drug therapy is required in patients with HER2-negative primary tumors, additional tumor samples should be tested to check current tumor status; metastatic tissue is also suitable for this purpose.According to the German S3-guideline, when malignancy of the stomach or gastroesophageal junction is suspected, a minimum of eight biopsies should be taken from all suspicious lesions to ensure a reliable diagnosis  and in liver metastases (17.2%)  . Accordingly, any pathologist is obliged to provide tumor blocks upon a clinician’s request where determining HER2 status is relevant to treatment decision-making. Some countries have specific payment rules for shipping  in collaboration with the Reference Institute for Bioanalytics (RfB) in Germany (consensus).HER2-testing should be quality-assured. To support this, samples should be sent to pathology laboratories which are regularly and successfully participating in interlaboratory (proficiency or ‘round robin’) quality assurance schemes for HER2 testing of stomach cancer, such as that offered by the Quality-Assurance Initiative Pathology  (consensus).If the metastasis is accessible, rebiopsy should be performed provided this can be tolerated by the patient (consensus).If the metastasis is not accessible for biopsy, any additional archived material  should also be accessed if available, and the primary finding should be confirmed by testing of this additional material (consensus).In case of a HER2-positive primary finding and a HER2-negative metastasis, the positive finding remains valid, and there is the option for treatment with trastuzumab.The problem of tumor heterogeneity and dealing with discordant findings between biopsy and surgical specimens were discussed.In the case of discordant findings between biopsy and surgical specimens, if one of the specimens is HER2-positive, then the tumor should be evaluated as positive.Evaluation of the HER2 status can be performed on the surgical specimen and the endoscopic biopsy. The scoring system for the assessment of IHC staining pattern in gastric cancer includes both sample types, but gives different criteria for categorization of HER2 status between biopsy and surgical specimen  of the German Society of Pathology and the Federal Association of German Pathologists is available as an established tool to address the basic requirements of HER2 testing and recommendations of the guidelines. QuiP® offers pathology laboratories in Germany the opportunity to participate in interlaboratory comparisons as part of external quality assurance system for tumor diagnostics  and should preferably submit samples for the determination of HER2 to such facilities.The experts recommend that pathology facilities should regularly (at least every 2 years) participate in interlaboratory comparisons for HER2 testing of gastric cancer. Clinicians should ascertain which pathology facilities are appropriately certified via successful participation in interlaboratory comparison quality assurance for HER2 gastric cancer (the certified institutes are listed on the home page of the German Society of Pathology, see The expert meeting was held in Frankfurt a.M. on September 21st, 2015 at the invitation of the Roche Pharma AG. The recommendations referred to in this manuscript are the consensus of all participants and reflect their opinions and long-term experience in this field.Clinicians Lordick M, Al-Batran S, Hofheinz R, Lorenzen S, Möhler M, Thuss-Patience P.Pathologists Baretton G.B., Dietel M, Gaiser T, Kreipe H.H., Kirchner T, Quaas A, Röcken C, Rüschoff J, Tannapfel A.