P < 0.001). The foveal avascular zone (FAZ) was significantly enlarged, and the central foveal thickness (CFT) and the inner nuclear layer (INL) thickness were significantly thinner postoperatively. The displacements were significantly correlated with the changes in the FAZ area , the CFT , and the INL thickness . In conclusion, the distance between the retinal bifurcations and the fovea was asymmetrically expanded after the surgery and was significantly correlated with the morphological changes. These results indicate that a horizontal macular contraction is correlated with vertical retinal contraction in the eyes with an ERM.The purpose of this cross-sectional retrospective study was to determine the relationship between the retinal displacements and the retinal thickness in eyes with epiretinal membrane (ERM) after vitrectomy with internal limiting membrane (ILM) peeling. To accomplish this, we measured the retinal thickness using optical coherence tomography (OCT) and the retinal displacement using OCT angiography to obtain 3 mm × 3 mm en face images before, and 2, 4, and 8 weeks following the surgery from 20 eyes of 20 patients. The distance between the retinal vessel bifurcations and the fovea was significantly displaced centrifugally and asymmetrically in the 4 quadrants postoperatively ( These alterations cause traction on the retina in the macular area which can lead to visual dysfunction and metamorphopsia. Vitrectomy and removal of the ERM with or without internal limiting membrane (ILM) peeling is used to release the traction and is effective in improving the visual dysfunction, metamorphopsia, and visual acuity6. Thus, it is important to know how the retina in the macular area is displaced in more detail after the surgery because the displacement will affect the visual function.An idiopathic epiretinal membrane (ERM) is a relatively common retinochoroidal disorder in older individuals9. The integrity of the outer retinal layers is important for vision in retinochoroidal disorders, and their alterations have been evaluated12. However, there have been only a few reports on the changes of the inner retinal layers in eyes with ERM after surgery13. This is important because the integrity of the inner retina is a major determinant of the visual dysfunctions in eyes with an ERM.Optical coherence tomography (OCT) enables clinicians to obtain information of the microstructures of the fovea, and earlier studies have reported that the retinal layer at the fovea is thickened and the outer retinal layers are disrupted in eyes with an ERM15. The displacements of the inner retina can be evaluated using the superficial OCTA en face images of the retina before and after surgery16. Thus, OCTA is a suitable method for evaluating whether there is a displacement of the inner retina postoperatively. Akahori et al. evaluated the displacement of the inner retina after surgery in eyes with a macular hole16. However, there has not been a study that investigated the displacement of the retina after vitrectomy that removed an ERM.OCT angiography (OCTA) is a noninvasive and safe technique that can obtain images of the microvasculature of the retina and the choroid. OCTA has enabled clinicians to investigate the retinal vasculature in situ repeatedly in short intervalsThus, the purpose of this study was to determine the relationship between the displacements of the inner retina and the retinal thicknesses in eyes with an ERM after vitrectomy with ILM peeling. To accomplish this, the superficial foveal avascular zone (FAZ) and the displacement of the retina were measured in the OCTA images, and the retinal thickness including the central foveal thickness (CFT) was measured in the spectral-domain OCT (SD-OCT) B scan images of eyes with an ERM before and after its removal. In addition, the relationships between the values determined by OCTA and SD-OCT were evaluated after vitrectomy with ILM peeling for the removal of an ERM.®, Heidelberg Engineering, Heidelberg, Germany) images were obtained before, and 2, 4, and 8 weeks following the surgery. The size and the location of the ERM and ILM peeling were determined from the videos recorded during the surgery. Eyes with severe cataract, macular diseases, history of other ocular diseases, and high myopia of > − 5 diopters (D) were excluded.We reviewed the medical records of all patients with a unilateral, idiopathic ERM who had undergone vitrectomy with ILM peeling at the Nagoya University Hospital from December 2016 to March 2019. All of the patients had undergone comprehensive ophthalmic examinations. Additionally, OCTA and SD-OCT  OCTA device16. The images of the superficial capillary network layer were used to measure the displacements of the retina and the area of the foveal avascular zone  and low quality images were excluded from the analyses. The two graders measured the vascular displacements independently and were masked of the other clinical findings.16. We adjusted the changes between the pre- and postoperative OCTA images by matching the retinal vasculature around the optic disc  three-port pars plana vitrectomy (PPV) was performed by one surgeon (TI) using the Constellation system . After core vitrectomy, the ERM and ILM were peeled circumferentially from the retina with ILM-peeling forceps in all cases. Finally, we performed a peripheral vitrectomy with shaving and carefully inspection of the periphery over 360 degrees.Cataract surgery and implanted a foldable acrylic IOL into the capsular bag were performed on all 19 phakic eyes.One-way analysis of variance (ANOVA) was used to determine the significance of the correlations between the displacements in the four quadrants and the time of the surgery. Pearson’s correlation coefficient tests were used to determine the significance of the associations between the changes in the displacement of each bifurcation and other variables. All statistical analyses were performed using the Statistical Package for Social Sciences for Windows 21.0 .The Ethics Committee of Nagoya University Hospital approved the procedures used in this cross-sectional retrospective study. The procedures conformed to the tenets of the Declaration of Helsinki. All of the patients signed an informed consent form before the surgery.Forty-one eyes of 41 patients with an ERM underwent vitrectomy with ILM peeling and SD-OCT and OCTA examinations between December 2017 and March 2019. Twenty-one eyes were excluded: 8 for a pseudo macular hole, 3 for macular degeneration, 1 for macular edema, 3 for severe cataract that prevented obtaining high quality OCTA images, and 6 moved to another hospital immediately after the surgery that did not have sufficient postoperative examinations. In the end, 20 eyes of 20 patients  were studied. The demographics of the patients and the surgical procedures are shown in Table P < 0.001). The mean size of the ILM peeled area was 4.78 ± 0.44 disc diameters (DDs).The mean postoperative best-corrected visual acuity (BCVA) was significantly improved  and temporal (P = 0.036) quadrants, and it was significantly greater in the superior quadrant than the inferior quadrant . The mean postoperative FAZ was 0.07 ± 0.03 mm2 at 2 weeks, 0.08 ± 0.03 mm2 at 4 weeks, and 0.09 ± 0.03 mm2 at 8 weeks after surgery. The increase in the area during the postoperative period was significant (P < 0.001). Of the 20 eyes, the area of FAZ in 18 eyes became larger after surgery.The mean preoperative FAZ area was 0.06 ± 0.03 mmP < 0.001). The mean postoperative CFT was significantly thinner than the baseline thickness during the entire follow-up period (P = 0.020: Table The mean preoperative CFT area was 406.3 ± 79.9 µm in eyes with an ERM which was significantly thicker than that of the fellow eye at 183.8 ± 16.7 µm (P < 0.001).The mean preoperative INL thickness was 70.9 ± 22.8 µm in the temporal, 91.8 ± 48.9 µm in the superior, 77.6 ± 27.5 in the nasal, and 69.5 ± 30.8 µm in the inferior quadrants. The mean postoperative INL thickness was significantly thinner only in the temporal quadrant during follow-up period (P < 0.001).The mean preoperative ORL thickness was 238.9 ± 36.3 µm in the temporal, 221.6 ± 25.5 µm in the superior, 226.6 ± 28.1 µm in the nasal, and 235.8 ± 33.2 µm in the inferior quadrant. The mean postoperative ORL thickness was significantly thinner only in the temporal quadrant during the follow-up period   Fig. , and the19. Our results showed that the distance between the retinal bifurcations and the fovea was significantly displaced centrifugally and asymmetrically in all 4 quadrants after the removal of the ERM by vitrectomy with ILM peeling. The FAZ area became significantly smaller and the CFT significantly thicker than that of the fellow eyes before surgery, and the FAZ area was enlarged and the CFT became thinner after the surgery. The changes in the displacement of the retinal bifurcations was significantly correlated with that in the area of the FAZ, the CFT, and the INL thickness.One of the advantages of using the OCTA images to evaluate the displacement is the ability to measure many points in a short time. We measured an average of 71.0 points for each eye in the OCTA images, which was a much larger number than previous reports20. Thus, we planted to perform ILM in addition to ERM peeling for eyes with ERM.Recent advances in vitrectomy surgery and ERM removal with ILM peeling have improved surgical safety and visual outcomes for ERM patients. Actually, there have been reports showing that vitrectomy with ILM peeling resulted in better visual improvement and lower ERM recurrence rates19.The retina was centrifugally displaced in all of the 4 quadrants, and the size of FAZ, which was much smaller than the fellow eye before surgery and was enlarged with time after the surgery. These results clearly show that the retina in the macular region was significantly displaced centrifugally after the surgery and which is consistent with previous reportsIn comparing the displacements between the inner and outer sectors of the superior and inferior quadrants, the distance in those sectors should be similar from the optic disc and the nasal displacement caused by the ILM peeling should not be different. The displacement of the bifurcation in the outer sector was greater than that in the inner sector in those quadrants. Accordingly, the farther the bifurcations were to the fovea, the greater were the displacements. These results corroborate the centrifugal displacements after the surgery.22 including eyes with a macular hole24. Consequently, the retina in the macula area was displaced nasally in addition to centrifugally in our cohort. Akahori et al. reported that there was greater displacements of the bifurcations farther from the optic disc14. The postoperative asymmetrical displacement in the temporal and nasal quadrants can be explained as a displacement of the nasal retina by the same directional forces, viz., centrifugally and nasally, but the temporal retina was displaced by different directional forces. This asymmetrical displacement might affect the recovery of the visual dysfunction and distortion postoperatively.On the other hand, the displacements in the inner sectors were greater than that in the outer sectors only in the nasal quadrant. In addition, the retina in the nasal quadrant was displaced the most among the four quadrants after the surgery. It has been reported that the retina is displaced nasally after ILM peeling in eyes with retinochoroidal diseases28.Our study showed that the INL thickness became thinner after surgery and the change in the INL thickness was significantly correlated with the degree of retinal displacement. These results imply that the INL is thickened by a centripetal traction of the ERM on the macula resulting in the development of aniseikonia and metamorphopsia. The release of the traction causes the retinal displacement which is correlated with the reduction of the INL thickness. These observations support the earlier findings30. In addition, the preoperative FAZ area and the CFT were correlated with the displacement of retina. Furthermore, the change in the FAZ area was correlated with the displacement of the bifurcations and the change in the CFT which is in good agreement with a previous report. These results suggest that the enlarged FAZ is related to the greater displacement of retina and reduction of the CFT. There have been several reports showing an association of the displacement of the retina with the changes in the retinal structures using OCT31 or retinal vasculature using OCTA33. Our results corroborate these earlier reports. Thus, a smaller preoperative FAZ and a thicker CFT may indicate greater macular contraction caused by the traction of the ERM and the progression of the disease process. Taken together, these findings indicate that the horizontal contraction in the macular area is correlated with the vertical contraction of the retina in eyes with an ERM.A contraction of the ERM exerts tangential traction on the inner retina and overlying vessels, causing a movement of the retinal vessels toward the center34. Second, there are several reports that the structure at the fovea is related to the vision in many retinochoroidal diseases which was not evaluated35.In our cohort, the BCVA was not significantly correlated with the retinal displacement. There are several explanations for this. First, we evaluated the inner retinal displacement because retinal vessels are located in the inner retina but the structure of the outer retina may be more important for vision than the retinal displacementsThis study has several limitations. First, we evaluated the displacement after a relatively short period of 8 weeks after the surgery. Second, the mean size of the ERM and ILM peeled was 4.78 ± 0.44 DD, and the size was not consistent among the eyes. Third, an imaging of the 6 × 6 mm area would be more helpful in terms of recognizing vasculature in a wider area. However, the resolution of the image is worse than that in smaller area of 3 × 3 mm, and this would make it more difficult to detect the bifurcations. Thus, we chose to use the 3 × 3 mm image. Fourth, we did not evaluate the eyes for aniseikonia and metamorphopsia, and it might have been better to investigate these parameters in addition to the BCVA to determine the functional changes after surgery. To resolve these limitations, prospective studies on a larger number of eyes with a longer follow-up periods are needed.In conclusion, the distance between the retinal bifurcations and the fovea was asymmetrically increased after removal of the ERM by vitrectomy with ILM peeling. The change in the distances was correlated with the changes in the FAZ area and the CFT. These results indicate that a horizontal macular contraction is correlated with vertical retinal contraction in the eyes with an ERM.


Intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury (ONI) or laser-induced ocular hypertension (OHT). In models of glaucoma, BDNF therapy can delay or halt RGCs loss, but this protection is time-limited. The decreased efficacy of BDNF supplementation has been in part attributed to BDNF TrkB receptor downregulation. However, whether BDNF overexpression causes TrkB downregulation, impairing long-term BDNF signaling in the retina, has not been conclusively proven. After ONI or OHT, when increased retinal BDNF was detected, a concomitant increase, no change or a decrease in TrkB was reported. We examined quantitatively the retinal concentrations of the TrkB protein in relation to BDNF, in a course of adeno-associated viral vector gene therapy (AAV2-BDNF), using a microbead trabecular occlusion model of glaucoma. We show that unilateral glaucoma, with intraocular pressure ( IOP) increased for five weeks, leads to a bilateral decrease of BDNF in the retina at six weeks, accompanied by up to four-fold TrkB upregulation, while a moderate BDNF overexpression in a glaucomatous eye triggers changes that restore normal TrkB concentrations, driving signaling towards long-term RGCs neuroprotection. We conclude that for glaucoma therapy, the careful selection of the appropriate BDNF concentration is the main factor securing the long-term responsiveness of RGCs and the maintenance of normal TrkB levels. Increased intraocular pressure (IOP) remains a major, though not the only, risk factor for developing glaucoma. Available therapies that reduce IOP do not always prevent the progressive death of retinal ganglion cells (RGCs) and the development of a pathology that leads to blindness. There is therefore a need to develop an effective neuroprotection method that can interact with cellular signaling and that promotes RGCs survival. The vast majority of RGCs contain both brain-derived neurotrophic factor (BDNF) and its high-affinity TrkB receptor, raising the possibility that locally produced BDNF may play an important role in the activation of RGC through TrkB receptors in addition to the retrogradely transported ligand ,2. ExogeTo secure therapeutic action, an effective intraocular concentration of BDNF requires that the expression of the BDNF high-affinity TrkB full-length receptor in retinal cells is maintained. Increased IOP was reported to change BDNF and TrkB availability owing to the impairment of retrograde transport in the optic nerve ,18,19. FWe have chosen a microbead trabecular occlusion model of glaucoma, with rapid elevation of IOP, which serves as the equivalent of a severe glaucoma attack . ContralWe will show that unilateral glaucoma leads to the bilateral reduction in BDNF concentration, accompanied by up to a four-fold TrkB upregulation. An increase in TrkB concentration did not compensate the reduced BDNF signals sufficiently to promote signaling towards the protection of the retina from significant cell loss after six weeks of pathology. Significantly, we found out that long-term, moderate BDNF overexpression in glaucomatous eye restores normal TrkB levels. In effect, increased BDNF signaling led to long-term RGCs neuroprotection both in the central and peripheral regions of the retina. We conclude that in severe glaucoma attacks, (1) a single administration of AAV2-BDNF is sufficient for significant RGCs neuroprotection, and (2) for glaucoma therapy, the careful control of BDNF concentration is the main factor securing the long-term responsiveness of RGCs and the maintenance of TrkB normal levels.A modification of the rat experimental model of glaucoma, incorporating rapid-onset elevation of intraocular pressure (IOP) , has beeIOP values significantly increased immediately after microbead injection. A rapid, over five-fold elevation of IOP with an initial “high-pressure injury” confirms that the model may be used as the equivalent of a severe glaucoma attack . During The effect of glaucoma and AAV-BDNF transgene administration on the density of retinal ganglion cells (RGCs) was examined by comparing glaucomatous and non-treated, contralateral retinas see . We comp2 amounted to 1686 ± 30 in the central retina, and to 1250 ± 133 in the peripheral zone .In naive animals, the number of RCGs per mmF  = 172.6, p < 0.001; 2 in the glaucomatous retina amounted to 1071 ± 137, while in the contralateral retina it was 1556 ± 145.After 6 weeks, the rats with glaucoma induction showed a 31% loss of RGCs in the central retina of the glaucomatous eye, as compared to the fellow eye  was markedly reduced (to approx. 15%) as compared to rats with no BDNF gene transfer .In rats which received BDNF transgene, the loss of RGCs in the glaucomatous retina  = 67.9, p < 0.001). The number of RGCs per mm2 in the glaucomatous retina amounted to 758 ± 124, while in the contralateral retina it was 1060 ± 124. In the glaucomatous retina of the rats which received the BDNF transgene, more RGCs survived than in the untreated rats . In that group, the density of RGCs in the glaucomatous retina amounted to 912 ± 37 and differed by 7% only from the non-treated retina (982 ± 82). Again, the importance of BDNF overexpression for the protection of RGCs in the peripheral zone has been confirmed by the significant interaction of glaucoma with BDNF overexpression (p = 0.0003).As shown on Next, we performed ELISA to answer the question of whether at six weeks after the induction of glaucoma BDNF protein concentrations in the retina are reduced, and if so, whether a deficit also develops in the contralateral eye. To examine whether AAV2-BDNF pretreatment effectively upregulates BDNF and whether the effect is unilateral, measurements were done in rats without and with intraocular administration of the AAV2 vector carrying the BDNF transgene to the Right eye. In parallel, to test whether altered retinal BDNF concentrations led to adaptive changes in BDNF TrkB receptor protein availability in the retina, we performed ELISA of TrkB in the same rats.n = 9)  A.F  = 14.71, p = 0.0001; Games–Howell post-hoc test; non-treated retina p = 0.003, glaucomatous retina p = 0.138, vs. intact rats) , indicating that the glaucomatous eye is not isolated and draws the line of changes in the contralateral eye.Six weeks after the induction of unilateral glaucoma, there was a bilateral decrease of BDNF concentration in the retina, in five of the six rats. The BDNF concentration reached, on average, 70% of control values  A. The BDn = 6); on the injected side it was over two-fold higher than in the intact rats  and three times higher than in the rats with glaucoma, BDNF-untreated , suggesting some BDNF transport from the injected to the contralateral eye, dependent on BDNF production in the transfected cells and on the neurotrophin content in their cellular milieu.AAV2-BDNF injection resulted in a marked, bilateral upregulation of BDNF concentration in the retina . n = 6), which was over four-fold higher (12.38 ± 5.84 ng/mg prot.) on the glaucoma side, and two-fold higher (6.70 ± 1.87 ng/mg prot.) on the contralateral side, than in the intact rats  = 8.19, p = 0.0018; Games–Howell post-hoc test: glaucomatous retina, p = 0.052; non-treated retina, p = 0.019; L/R retina p = 0.272). A weak correlation between the TrkB protein levels in the Left and Right retinas  indicated that the adaptive processes, reflected by the increased TrkB synthesis/maintenance in the glaucomatous and contralateral eyes, do not develop in parallel.In the intact rats, the retinal TrkB protein concentrations were in the picomolar range, and amounted to 2.17–3.58 ng/mg prot.  suggesting a common, strong response to conditions evoked by increased BDNF supply. We queried the dependence of TrkB expression on BDNF concentration, and examined the correlation between BDNF and TrkB levels in the AAV2-BDNF-untreated and -treated groups. The analysis revealed that within a subset of data from the AAV2-BDNF-treated group, in which BDNF exceeds control levels, the correlation is positive . That result indicated that in conditions of moderate BDNF overexpression in the retina, the TrkB protein does not undergo downregulation, but follows BDNF levels, which may be of significant functional importance.AAV2-BDNF injection led to the molecular changes restoring normal TrkB concentrations in the retina . The TrkB concentration values in the Left and Right retinas were highly correlated (Pearson correlation coefficient 13 GC/mL) of AAV2-BDNF, causing BDNF protein overexpression, which was maintained at moderate levels for at least 9 weeks from transgene administration.We have shown for the first time that in the microbead trabecular occlusion model of glaucoma characterized by an initial severe glaucoma attack , a singlWe decided to inject AAV-BDNF three weeks prior to glaucoma induction to ensure stable BDNF overexpression in the retina prior to glaucomatous changes development. This was based on the data of others  and on obdnf is moderately upregulated (2.5-fold), the expression of the trkb gene (coding for Ntrk2 receptor) is close to intact controls : WPRE) were produced and supplied by VectorBuilder Inc. . Rats were anesthetized with ketamine and medetomidine and given topical Alcon eye drops as described for microbead injection. The AAV2-BDNF vector  and received transcardial perfusion with 400 mL of ice-cold 0.1 M PBS with heparine (0.4 mL), followed by 150 mL ice-cold paraformaldehyde solution (4% PFA in 0.1M PBS pH 7.4). The eyeballs were removed with retro-bulbar stumps of optic nerves, and post-fixed in ice-cold 4% PFA for several hours. After triple PBS rinsing, the retinas were isolated and incised to facilitate their flat arrangement on the glass slides. The retinas were stored in PBS in 4 °C for 24 h.For the quenching of tissue autofluorescence, the retinas were incubated in 100 mM glycine solution . After PBS washing (3×) the retinas were incubated with 20% normal goat serum (NGS) in PBST for 45 min to eliminate non-specific binding. Subsequently, the retinas were incubated with a monoclonal primary antibody recognizing BRN3a antigen , in 4% NGS in PBST, for 24 h. The Brn3a protein is a strong nuclear marker for the rodent RGCs population; in the rat this antibody labels repetitively 96% of the RGCs population ,51,52. NImages of retinas were captured with a Zeiss Spinning Disc confocal microscope , using a 10× (for the whole retina image reconstruction) and 20× (for selected fields) objective lens. Z-stacks of 16-bit images consisted of 4–10 optical slices collected at 1 μm intervals and processed with a Max-projection tool.2), and their density was calculated per 1 mm² area.The quantification of the immunolabeled RGCs was performed using ImageJ open source image processing software in four quadrants of flattened retinas: in the peripheral regions located about 2.5 mm from the optic disc, and the central parts, centered at about 1 mm diameter away from the center of the optic disc . BecauseThe rats were deeply anesthetized with a lethal dose of pentobarbital and quickly perfused transcardially with 400 mL of ice-cold PBS. The brain and eyeballs were removed from the skull and placed on ice. Both the left and right visual cortex and the retinas were isolated and frozen on dry ice. Dissected tissues were stored at −80 °C.g for 30 min at 4 °C. ELISA was performed on supernates diluted 50×. All samples were run in duplicate.A ChemiKineTM BDNF Sandwich ELISA Kit  detecting both human and rat BDNF protein was used, according to the manufacturer’s instructions. Crude tissue homogenates (20% w/v) were prepared in 100 mM Tris buffer (pH 7.0), containing 2% BSA, 1M NaCl, 2% Triton X-100 and Complete Protease Inhibitor Cocktail, 200 μm phenylmethyl-sulphonyl fluoride  and 157 μg/mL benzamidine hydrochloride . An IKA Ultraturrax  tissue grinder was used to disrupt the tissue. The homogenates were incubated on ice for 30–60 min and centrifuged at 15,000× g for 10 min at 4 °C. ELISA was performed on supernates diluted 10× following the manufacturer’s instructions. All samples were tested in duplicate.For TrkB ELISA , tissue homogenates prepared for BDNF ELISA and stored at −18 °C were thawed, re-homogenized and centrifuged at 2000× 20, sampled and measured as above. A standard curve was prepared and a regression coefficient was calculated to determine the linearity of the curve. The absorbances of the experimental samples were compared with the values from the standard curve, and the total protein concentration in each sample was calculated.Protein concentration was determined by the modified Bradford method in cuvettes  with bovt-test for dependent variables was used. STATISTICA 13.1  and R software were used for the data analysis.The Shapiro–Wilk test was used to verify the normality of data distribution. The homogeneity of variance in the groups was analyzed with the Leven and Bartlet’s tests. Two-way ANOVA with repeated measurements followed by a Sidak’s multiple comparisons test was used to compare the density of RCG between groups. Because the homogeneity of variance was violated in sets of data on BDNF and TrkB levels in some groups, one-way ANOVA with Welch’s correction followed by Games–Howell pos-hoc tests were used. For the comparison of IOP levels in time, a


The employed TiO2 nanotube scaffolds allow for organotypic long-term preservation of adult tissues ex vivo and support strong tissue adhesion without the application of glues, a prerequisite for tissue investigations under tension. In combination with finite element calculations we found that the deformation behavior is highly dependent on the displacement rate which results in Young’s moduli of (760–1270) Pa. Image analysis revealed that the elastic regime is characterized by a reversible shear deformation of retinal layers. For larger deformations, tissue destruction and sliding of retinal layers occurred with an equilibration between slip and stick at the interface of ruptured layers, resulting in a constant force during stretching. Since our study demonstrates how porcine eyes collected from slaughterhouses can be employed for ex vivo experiments, our study also offers new perspectives to investigate tissue biomechanics without excessive animal experiments. Numerous eye diseases are linked to biomechanical dysfunction of the retina. However, the underlying forces are almost impossible to quantify experimentally. Here, we show how biomechanical properties of adult neuronal tissues such as porcine retinae can be investigated under tension in a home-built tissue stretcher composed of nanostructured TiO Mechanical properties of cells and tissues play a crucial role in many biological processes, ranging from embryologic development to cancer progression ,3,4,5,6.Besides medical treatment, surgical intervention of the retina is the therapy of choice. However, also here retinal mechanical properties can strongly intervene positive outcome: The retina is located in the spherical eyecup and always under mechanical tension. Thus, it tends to detach and enrolls once the surgeon cuts into the tissue. Difficulties with reattachment and local tissue degradation can be a severe negative side effect ,25,26. ATo quantify the local mechanical properties of retinae ex vivo, scanning force microscopy has been employed to map tissue elasticity on the retinal surface , while a2 nanotube scaffolds are ideal scaffolds for long-term organotypic culture of adult neuronal tissues including the mammalian retinae for at least 14 days [The mammalian retina of pigs is similarly composed as in humans ,32. It i 14 days ,35. HereMany diseases are characterized by retinal detachment in which the retina on its photoreceptor side disrupts from the underlying retinal pigment epithelium. To mimic the situation of force exertion on the retina’s photoreceptor layer, we attached the retina on the photoreceptor side to the nanotube scaffolds and applied a loading force on it which results in a displacement propagating from the photoreceptor side through the retina to the opposite surface where the ganglion cell layer is located. In combination with finite element calculations, we obtained a displacement rate dependent mechanical response dominated by the time scales of viscoelasticity as well as the relaxation of internal structural degrees of freedom. To this end, our setup and the very sensitive force sensor in combination with fluorescence microscopy images of retina structures can shed more light on the ability of adult retinal tissue to withstand external forces, while providing quantitative data pertaining to forces the retina can withstand without irreversible damage, and if damage occurs, which retinal layer seems to be the “weak link” during rupture.2 nanotube scaffolds which were initially located next to each other without a gap in between with the retina positioned at the interface of the two scaffolds , forces acting during deformation can be derived from Hooke’s law. After positioning the fresh pig retina on the tissue stretcher at the interface of the two adjacent nanotube scaffolds E, the moThe FD curves displace four different regimes: (1) For small displacements below 0.05 mm, the force increases linearly, characteristic for elastic deformation. From the slope the elastic constant  of the retina can be determined. This linear regime is preceded by a non-linear force increase for displacements below 0.01 mm  N/m was found for the first linear regime, while for a slower velocity of 0.1 µm/s, the elastic constant was higher with a value of (33.5 ± 4.7) N/m. Also, the yielding forces increased from (1.5 ± 0.1) mN for 2.0 µm/s to (2.2 ± 0.1) mN for a velocity of 0.1 µm/s. To this end, the maximum forces the retina can withstand without irreversible damage are velocity dependent, as well as the corresponding displacements at which yielding occurred . SimilarIn case of a pure elastic tensile extension of the retina during stretching, a conversion of the force into stress and the displacement into a dimensionless strain would result in a linear stress-strain dependency from which the slope describes the Young’s modulus of the retina. However, the initial sample length (viz. the area of the tissue where stretching takes place) is immeasurably small because the two nanotube scaffolds with the retina on top are initially touching each other. Thus, it is not possible to normalize the displacement to calculate the strain. Moreover, since the retina is stretched by pulling onto the bottom of the retina perpendicular to the cross-sectional area along which the displacement is expected, the deformation is not pure uniaxial strain within the substrate plane, but contains additional shear components. Thus, we performed finite element simulations to gain more details on the displacement field and the material properties of the retina. Within the simulation, the same experimental conditions were employed as obtained in the experiments. To gain the effective Young’s modulus of the retina from our experiments, we varied the Young’s modulus within the simulation iteratively until we got the same displacement for a certain loading force found experimentally within the first linear (viz. elastic) regime in the FD curves. As an example, In summary, we found an effective Young’s modulus for the first linear increase (regime 1) of around 1270 Pa for stretching velocities of 0.1 µm/s, approximately 1000 Pa for 0.5 µm/s and 760 Pa for 2.0 µm/s, respectively. Thus, the retina behaves softer for faster loading rates. Moreover, even for small displacement rate variations of less than one order of magnitude, a remarkable change in material properties become visible. In order to get a first impression how the retina deforms during stretching, we performed immunostaining to visualize retinal layers during a 0.1 µm/s deformation after a stretch distance of 30 µm (elastic regime: reversible deformation), 60 µm (yielding: the retina begins to rupture), and 600 µm, respectively. Here, the retina was fixed and removed from the stretcher device. Subsequently, we employed different fluorescently labelled antibodies to image Müller radial glial cells and cone photoreceptor cells compared to the un-stretched retina (control) . The con5 Pa was reported, while for an increased strain rate, the Young’s modulus increased as well. However, the retinal strips dried during stretching which was expected to stiffen the material compared to fresh tissue. Furthermore, Chen and coworkers used uniaxial tension tests for porcine retinae and determined an elastic modulus around 5 × 104 Pa [Even though it has been known for decades that changes in the mechanical properties of the retina are often related to eye diseases which can result in loss of vision ,39,40,41× 104 Pa . They al× 104 Pa . Worthin× 104 Pa , similar× 104 Pa . For oth× 104 Pa ,28,50.Studies on retinal layer elasticity by Qu et al. showed that the ganglion cell layer (GCL) of porcine eyes is softest with a Young’s modulus of (1.33 ± 0.37) kPa – similar to the Young’s moduli observed in our experiment for regime 1 – while the photoreceptor layer (PRL) and ONL are hardest compared to other retinal layers with a Young’s modulus of (25.9 ± 7.36) kPa . In addiIn our present study, we developed a tissue stretcher with a self-designed force sensor to quantify the forces acting during uniaxial retina deformation under tension. Additionally, we show how fresh adult porcine eyes collected from slaughterhouses can be employed to investigate eye mechanics ex vivo. After validating the working principle of our stretcher device, we studied the force–distance behavior and displacement rate dependence of porcine retinae deformation. Even though biological tissues are never the same for different animals since they exhibit small variations in size, cellular and molecular structure, we found that the results in terms of the measured force–distance (FD) curves are remarkably reproducible with small standard deviations of elastic constants and yielding forces. In every stretching experiment, we observed four different deformation regimes : (1) a lBy using finite element calculations for a more detailed data analysis, we determined the Young’s modulus of the retina during elastic deformation (regime 1). We found values in the order of (760–1270) Pa depending on the deformation velocity. Interestingly, for an increasing displacement rate from 0.5 µm/s to 2.0 µm/s, we observed a decrease of the determined Young’s modulus, as well as a decrease in yielding force succeeding the elastic deformation. Also, for the second linear force increase (regime 3), the slope decreases for higher stretching velocities, as well as the maximum force plateau following the force increase. The observation that yielding occurs faster, viz. for smaller displacements or strains when a higher deformation rate is applied, is usually found in many biological and classical (hard) materials ,52,56,57Considering tissue as a liquid-like material which can also exhibit glass-like behavior, we surmise that the observed high displacement rate dependence can be attributed to non-Newtonian flow during viscoelastic deformation within regime 1. Here, athermal effects contribute, viz. the employed displacement rates ulations . Additioulations . Even thAt the microscale, we expect that local heterogeneities and the layered structure of the retina determine the observed FD behavior. Thus, we employed immunostaining to visualize retinal layers before and during stretching. During elastic deformation after a displacement of 30 µm, we observed that the retinal structure was preserved compared to control, while the entire thickness of the retina was reduced during tissue expansion , we still see an intact ONL and no rupture, while the other layers of the retina were not detectable anymore and the structure was fully disrupted  placed on ice bags in an insulated transportation box. After about 1-h transportation time, eyes were removed from the tubes, placed in a plastic dish with ice cold PBS and all irrelevant tissues around the eyeballs were cut off . Subsequently, isolation of the retina was performed in a sterile room dedicated to primary culture experiments. First, the eyeball was shortly dipped in 70% ethanol and washed twice with sterilized PBS before transferring to the Petri dish with PBS. An incision was made behind the ora serrata to release the pressure of the posterior cavity allowing to hold the eyeball easily. Equatorial sectioning of an eyeball was started from about 2 mm behind the cornea by using scalpel, eye scissors and forceps. After removal of the lens, the retina remained attached to the vitreous body. By using forceps, the retina was removed carefully from the underlying pigment epithelium. Subsequently, the remaining vitreous body was gently removed as well.2. All tissue samples were stamped horizontally at the same position at the center of the retina close to the macula. Subsequently, the retina explants were placed on the tissue stretcher as described below. Supply with nutrients was ensured with retina culture medium composed of AMES medium  with 20% horse serum  and 0.1% gentamicine .To perform all experiments with reproducible conditions, the tissue was stamped with a self-designed rectangular tissue stamper to a size of 10 × 6 mm2 nanotube scaffolds located next to each other with the tissue placed on top above the interface of the scaffolds . A rectangular dot on white background sticker was glued on top of the stainless-steel plate to track the alteration of glass fibers position during the experiment  mm2 nanotube scaffold attached to the force sensor as employed in our stretching experiment. Subsequently, extra masses were added to the force sensor to determine the spring constant. The result displayed almost no effect with an increase in the spring constant value of less than 1.5%.To determine the spring constant, we added extra masses of brass wires with known values (150‒2200 mg) to the force sensor gadget and measured the elongation of the glass fibers with a camera as a function of gravitational force. After calculating the acting gravitational forces from Newton’s law, we obtained a linear force increase as function of glass fiber extension). From the slope, the spring constant can directly by derived and we obtained a value of (54.96 ± 0.52) N/m. For control, the experiment was repeated with the TiO2 nanotube scaffolds with the photoreceptor-side down  up to 1 mm. After stretching, the tissue stretcher was removed from the incubator and the retina was fixed on the nanotube scaffolds with 4% paraformaldehyde  in PBS for 48 h. Subsequently, the retina was gently removed with forceps. For each experiment new nanotube scaffolds were used. Prior to retina experiments, nanotube scaffolds were cleaned with 70% alcohol while the entire tissue stretcher was deconstructed, autoclaved, and subsequently reconstructed inside the sterile room.Immunohistochemical staining of the porcine retina was applied to visualize retinal structures inside the retina after mechanical testing. The change in cell layer structure inside the retina was revealed by tracking Müller radial glial cells, and cone photoreceptor. After removing the fixed retina from the nanotube scaffolds with forceps, it was embedded in 3% agarose gel in PBS and subsequently segmented by a vibration microtome with slice thickness of 30 µm. Then, 3–5 sliced samples were transferred into 24-well plates and filled with 2 mL of PBS. Next, the samples were incubated with 1 mL of washing buffer (1% DMSO + 0.3% Triton X-100 in PBS) for 10 min at room temperature. After removing the washing buffer, 1 mL of blocking solution (5% of donkey serum in washing buffer) was added and subsequently incubated for 1 h. In the meantime, primary antibodies were diluted to 1:100 in blocking solution. In this study, anti-glutamine synthetase produced in mouse  was used to track Müller radial glial cells, and peanut agglutinin conjugated biotin  for cone photoreceptor ,46. After overnight incubation, samples were washed three times with washing buffer for a total of 3 h. For fluorescence detection of all tracking cell types, staining kits with cyanine conjugated dyes (Cy2 and Cy3) with 1:200 in washing buffer were utilized. Cy2 produced in donkey anti mouse  served to visualize Müller cells incubated with anti-glutamine synthetase. Cy2 conjugated with streptavidin  was employed for cone photoreceptor visualization. Hoechst 33342  was used as a nuclear counterstaining (1:1000 in washing buffer). Negative controls were carried out without primary antibodies in order to ensure specific binding of primary and secondary antibodies. After 2 h incubation in dark room, sections were washed with PBS several times. Finally, slides were mounted on glass coverslip with glycerol and visualized with a laser scanning confocal microscope .For a displacement rate (viz. motor velocity) of 0.5 µm/s and 2.0 µm/s, three stretching experiments were performed, while for 0.1 µm/s five stretching experiments were conducted. Raw data obtained from tissue stretcher were used to generate force-distance curves. After calculating the slope of linear regimes and determining yielding forces, we determined the mean values and standard deviation (SD) of these numbers for the three different velocities. ® 5.3 were employed to determine the effective Young’s moduli of the retina during deformation from the experimentally determined forces for various displacements and displacement rates. First, the “model builder” was used to build up two scaffolds onto which the retina is placed. Since both pure titanium and TiO2 nanotube scaffolds exhibit Young’s moduli decades larger than the soft retinal tissue [3 [Finite element calculations using COMSOL Multiphysicsl tissue , we can l tissue ,31,36. Fissue [3 , and a Pissue [3 .Free tetrahedral structural solid finite elements were defined before generating a fine mesh where the scaffold including the retina was discretized into 93373 elements. Subsequently, we used the “solid mechanics” module with position constrains on one titanium scaffold to keep it fix in all directions and position constrains on the second scaffold in z-direction see . Additio2 nanotube scaffolds which support adhesion and long-term culture of adult neuronal tissues ex vivo, we showed how these scaffolds integrated into a tissue stretcher with a self-designed force sensor can be employed to study tissue mechanics under tension. In fact, numerous eye diseases are linked to biomechanical dysfunction such as hole formation in the macular or rupture of retinal layers. Here, forces acting on the photoreceptor side of the retina can result in retinal detachment during many eye diseases. To address these issues, we studied the influence of tension acting onto the photoreceptor side which then propagated through the retina towards the opposite surface. We observed a high displacement rate dependence of elastic behavior which can be attributed to the heterogeneous structure of the vascularized retina. For different displacements and displacement rates, we showed that a viscoelastic material such as the retina can behave elastically, or even liquid-like, while fast displacements resulted in decreased stiffening due to reduced friction between structural components. Based on TiOEspecially in older patients, the vitreous body begins to shrink and pulls away from the retina. When the vitreous body sticks to the retina, macular stretching and irreversible hole formation can follow. For a better understanding of the underlying biomechanical properties, our setup offers the possibility to culture the retina with the ganglion cell layer attached to the nanotube scaffolds to quantify the resulting displacement field and acting forces during tension. Additionally, eye surgeons also inevitably have to apply a force to the retina which can cause severe negative side effects such as rupture. To reduce possible complication during surgery, modern surgery is assisted by computer and robots ,64,65. N


In this study, four novel variants in POU4F3, c.696G>T (p.Glu232Asp), c.325C>T (p.His109Tyr), c.635T>C (p.Leu212Pro), and c.183delG (p.Ala62Argfs∗22), were identified in four different Chinese families with ADNSHL by targeted next-generation sequencing and Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, c.183delG (p.Ala62Argfs∗22) is classified as a pathogenic variant, c.696G>T (p.Glu232Asp) and c.635T>C (p.Leu212Pro) are classified as likely pathogenic variants, and c.325C>T (p.His109Tyr) is classified as a variant of uncertain significance. Based on previous reports and the results of this study, we speculated that POU4F3 pathogenic variants are significant contributors to ADNSHL in the East Asian population. Therefore, screening of POU4F3 should be a routine examination for the diagnosis of hereditary hearing loss.Hereditary hearing loss is one of the most common sensory disabilities worldwide. Mutation of POU domain class 4 transcription factor 3  POU4F3) and approximately 70 other loci, have been reported (http://hereditaryhearingloss.org/).Hearing loss is one of the most common hereditary sensory disabilities worldwide . Hair cePOU4F3 gene encodes POU4F3, a POU-domain class IV protein, has two exons, and encodes a protein of 338 amino acids that belongs to the POU-domain family of transcription factors, which are expressed specifically in inner ear hair cells and play a critical role in the maturation, differentiation, and maintenance of inner ear hair cells . Gfi1 not only plays a late role in the differentiation and maintenance of hair cells but also promotes the formation of hair cells in cooperation with atonal BHLH transcription factor 1 (Atoh1) [POU4F3 inhibits the expression of myosin VI, which plays a large role in the maintenance of stereocilia of hair cells that are responsible for auditory transduction [POU4F3.Although mice require only one copy of the functional POU4F3 to retain hearing , 46, sev variant , 23, 24.h ADNSHL . Anotherop decay . Also, top decay , 20. ExA (Atoh1) . In addi (Atoh1) . Thus, r (Atoh1) . Anothersduction . Tornarisduction . AlthougPOU4F3 (three missense variants and one frameshift variant) involved in hearing loss. The missense variant c.696G>T (p.Glu232Asp), detected in family A, is located in the POU-specific domain, and a different missense variant at the same locus, c.694G>A (p.Glu232Lys), has been reported previously [POU4F3 leads to substitution of the glutamate at position 232 with an aspartic acid, which probably alters the structure of the α-helix of the POU-specific domain. The structural changes in the helix might affect the DNA-binding ability, what was probably responsible for the hearing loss in this family. The missense variant found in family C, c.635C>T (p.Leu212Pro), is also localized in the POU-specific domain, and it is possible that the mechanism of action is likely the same as described above. The missense variant observed in family B, c.325C>T (p.His109Tyr), is located in the transcriptional activation domain, which is not a functional domain, and this is likely why the hearing impairment in this family was mild. This variant is heterozygous in 0.016%  of East Asians, according to the gnomAD database. We speculate that its detection in the public database is due to the mild hearing loss associated with this variant. The frameshift variant, c.183delG (p.Ala62Argfs∗22), identified in family D results in a truncated protein with loss of both functional domains crucial for high-affinity binding to DNA.In this study, we identified four novel variants in eviously . The misPOU4F3 were identified in four different families. These consisted of three missense variants, c.696G>T (p.Glu232Asp), c.325C>T (p.His109Tyr), and c.635C>T (p.Leu212Pro), and one frameshift variant, c.183delG (p.Ala62Argfs∗22). These variants of POU4F3 are considered to be responsible for ADNSHL, designated as DFNA15. POU4F3 variants are not rare, and therefore, screening of POU4F3 should be included in routine examinations for diagnosis of ADNSHL. Further studies are required to determine the specific mechanisms underlying hearing loss.In summary, four novel variants in


The past 5 years have seen incredible advances in approaching hearing loss as a major public health issue. National efforts include the 2015 President’ Council of Advisors on Science and Technology and the National Academies of Science, Engineering, & Medicine’s 2016 Commission on Hearing Health Care for Adults, which led to the 2017 OTC hearing aid legislation and the expected debut of OTC hearing aids in 2020-2021. The World Report on Hearing amplifies these efforts. This presentation will cover the role of the Report in the context of the rapidly evolving hearing care landscape in the US and how the Report’s call for affordable, accessible hearing care fit within current national efforts focused on older adults. Finally, the WHO recognized 2020-2030 as the Decade of Healthy Aging. We will discuss how the World Report on Hearing integrates with broader efforts to support healthy aging locally and globally.


Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms.Immunization of pigs with an inactivated M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA.In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti- Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (PEP). PEP is a widespread chronic respiratory disease of swine that is characterized by coughing, reduced weight gain, and decreased feed conversion ) between runs were evaluated as previously described . BrieflyM. hyorhinis (Mhr), A. pleuropneumoniae (App), S. suis serotype 2 (SS2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and pseudorabies virus gB protein (gB-PRV) positive sera that were detected using an established ELISA, and 2 M. hyopneumoniae negative and 2 convalescent sera that served as negative and positive controls, respectively, were used to determine specificity.M. hyopneumoniae convalescent sera diluted at 1:100, 1:500, 1:1,000, 1:2,000, 1:4,000, 1:8,000, 1:16,000, and 1:32,000 in blocking buffer were tested using the other optimized working conditions of the ELISA. The sensitivity of the ELISA was evaluated based on the cut-off.Five M. hyopneumoniae IgG in sera from vaccinated pigs at Farm A and unvaccinated pigs at Farm C was detected with the commercial IDEXX IgG-ELISA kit and the M. hyopneumoniae convalescent serum IgG-ELISA. SIgA in nasal swabs collected from Farm C was detected with the sIgA-ELISA kit, kindly provided by Dr. Zhixin Feng. M. hyopneumoniae DNA in laryngeal swabs was detected using nested PCR [Anti-sted PCR . Each saPretreated bacteria or purified protein were solubilized and loaded onto SDS polyacrylamide gels. After electrophoresis, samples were stained with Coomassie brilliant blue, or transferred (2 h at 100 V) to a polyvinylidene difluoride membrane  using a transblotting apparatus . The membrane was blocked in 5 % skimmed milk-TBST at 4℃ overnight. The membrane was incubated with His-tag (4C2) monoclonal antibody  at RT for 1 h followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  at RT for 1 h, and visualized with enhanced chemiluminescence .


Lawsonia intracellularis is widespread in pigs and associated with severe enteropathy, mainly in the ileum, commonly resulting in substantial reduction in weight gain. The impact of three in-feed probiotics and a commercial live L. intracellularis vaccine was compared in a pig challenge model. Probiotic treatment was associated with reduced L. intracellularis fecal shedding and reduced gut lesions. Here, the bacterial microbiota of the ileum of these pigs was characterized with 16S rRNA gene sequencing and was subsequently analyzed with bioinformatics tools.Clinical intervention during bacterial infections in farm animals such as pigs commonly includes the use of antimicrobials. With the rise of antimicrobial resistance and the attempts to reduce the use of antibiotics in food animals, effective alternatives are urgently needed to reduce or even remove pathogens and disease risks. Improving clinical outcomes and overall pig health by using probiotics appears attractive. However, reliable data sets on the efficacy of probiotics are scarce. The obligate intracellular bacterium Bacillus pumilus; the microbiota of these pigs had the greatest diversity and highest richness. We also observed depleted and enriched core microbiota amongst the groups; however, there was no correlation with clinical characteristics. The results suggest that an increased diversity of the ileal microbiota is associated with a reduction in shedding, i.e. a unit increase in Shannon diversity index resulted in 2.8 log reduction in shedding.The greatest microbial richness was observed in the probiotic treated group T03-LAW, which accounted for 87% of richness observed in the study. Treatment had a significant impact on both the microbiota structure and taxonomic profile in the ileum, explaining between 26 and 36% of the structural variation, with the strongest association in the T03-LAW group. Overall, the largest changes were observed for the pigs treated with in-feed L. intracellularis challenge. An even and diverse microbiota community benefits pigs infected with L. intracellularis, however, investigations are needed to determine if this is also true for other pathogens. The study unambiguously demonstrates the usefulness of probiotic supplementation in reducing the impact of enteric pathogens and pathogen shedding rates in food animals without the use of antimicrobials.Probiotic supplementation of a base feed ration increased ileum microbiota diversity leading to a mitigation of the effects of a pathogenic The online version contains supplementary material available at 10.1186/s42523-021-00084-6. Firmicutes-to-Bacteroidetes ratio in weaned pigs [Lactobacillaceae numbers were increased after administration of the prebiotic lactulose [Escherichia coli, suggesting that the gut microbiome of piglets may have shifted functionally and mechanistically [Probiotics are increasingly used in commercial food animal production and are also slowly being implemented in pork production. Interactions of diet, gut microbiota, and host appear to impact animal health and disease . Common ned pigs . At the actulose . Probiotstically . Antimicstically . FeasiblLawsonia intracellularis (L. intracellularis), an obligate intracellular bacterium with a tropism for intestinal epithelial cells, especially in the ileum [L. intracellularis is associated with ileitis, also known as proliferative enteritis, which manifests as an acute or chronic form [L. intracellularis among pigs occurs by the oral fecal route [L. intracellularis infection after weaning has been associated with microbial dysbiosis resulting from the change in feeding regiments [L. intracellularis infection is also known to affect the gut microbiome [he ileum , is of ghe ileum . In pigsnic form . In addinic form . Transmial route , most pial route  and the al route –14. The egiments . Similarcrobiome , 15, 16.L. intracellularis infection is often accomplished by either vaccination or antimicrobial treatment. High doses of antibiotics such as tiamulin, tylosin, chlortetracycline, lincomycin and olaquindox have been shown to be effective [L. intracellularis [L. intracellularis shedding is not completely eliminated, regardless of vaccine type or vaccination route [L. intracellularis and the amount of prescribed antimicrobials [L. intracellularis infection. In addition, there is limited knowledge on the effect of live attenuated L. intracellularis vaccine on the microbiota of the intestine [Control of ffective , 18. Orallularis , 20. In on route , 22. A rcrobials . Moreoventestine .L. intracellularis challenge study we demonstrated  changes in a) time to onset of clinical pathology, b) levels of shedding and c) severity of shedding associated with a commercial live L. intracellularis vaccine supplemented via feed with one of three Bacillus strains and control (non-supplemented/non-vaccinated) compared to non-treated infected pigs [In a previous controlled ted pigs . Here weL. intracellularis infection characteristics including pathology and shedding results unrelated to this investigation have been published [2 each) of 10 pigs each equipped with a nipple waterer and a self-feeder. Specifically, the T01-LAW, T02-LAW and T03-LAW pigs were housed in a single room but in three separate pens approximately 2 m apart from each other. Similarly, the POS-CONTROL group was also housed in a single room in two separate pens. The NEG-CONTROL and VAC-LAW pigs were housed in separate single rooms with a single pen each [L. intracellularis , were supplied feed supplemented with one of the three Bacillus probiotics , or remained non-treated . At 7 weeks of age all groups, excluding the NEG-CONTROL pigs, were challenged with gut homogenate containing L. intracellularis by gastric gavage. The gut homogenate, obtained from a commercial vendor, was produced by collecting intestines from a field pig suffering from ileitis. The affected mucosa was collected and processed (i.e. grinded and diluted), and the resulting homogenate was passaged in experimental specific pathogen free pigs to obtain mucosal scrapings with a high L. intracellularis load free of other common pig pathogens. A homogenized stock was used for inoculation of all pigs to guarantee that each pigs was equally exposed to microbial loads that potentially could have been present in the inoculum. All pigs were euthanized 16 days post L. intracellularis challenge, at approximately 9 weeks old [Details on the experimental design Table  and clinublished . In briepen each . At 3 weeeks old .Table 1L. intracellularis shedding was evaluated by measuring the bacterial load in rectal swabs. Rectal swabs were collected at dpc 0, 2, 4, 6, 8, 10, 12 and 15 using polyester swabs and stored in 5 mL plastic tubes containing 1 mL of sterile saline solution at − 80 °C until testing. Testing was done using a quantitative real-time PCR specific for L. intracellularis as described [10 transformed to normalize them. These values were used to calculate the total area under the curve (AUC) for each group, one of the outcomes utilized in this analysis. L. intracellularis associated histopathology was described using a combined ileum lesion score based on microscopic lesions and amount of L. intracellularis antigen within lesions. In brief, individual scores of crypt enterocyte hyperplasia , inflammation  and amount of L. intracellularis antigen as determined by immunohistochemistry  were combined for a maximal score of 9 [escribed . Prior tore of 9 .At necropsy, luminal ileum content was collected from each pig approximately 10 cm anterior to the ileocecal junction and stored at − 80 °C until use. In addition, the inoculum stock, used to infect the pigs, was also processed and sequenced. Approximately 1 g (wet weight) of the luminal ileum content from each pig or 1 g of the challenge material was used to extract genomic DNA using PowerMag DNA Isolation Kit  following the manufacturer’s instructions. The quality of the DNA was assessed by Qubit 4 Fluorometer . The 16S rRNA amplicon library was prepared as described  with minThe paired end reads from the sequencing step above were processed using the Quantitative Insights Into Microbial Ecology-2™ (QIIME-2) software . The rawphyloseq object [p-value set at < 0.05 [The analysis of the microbiota community structure was based on characteristics of the alpha and beta diversities. In brief, data from QIIME-2 was exported into the R statistical package (v3.5.1). Four files, including the taxonomic map, the OTU biome table, the experimental metadata and the rooted phylogenetic tree, were combined to generate a q object  which waq object . Then thq object . Pairwist < 0.05 . We alsot < 0.05 –34. EachTo assess multivariate associations of experimental factors and microbial structural changes, a permutation multivariate analysis of variance (PerMANOVA) model with the Adonis function (9999 permutations) was used in the “microbiome” package in R. Here a model was run for each of the beta diversity indices with the aim to assess the individual and cumulative variation explained by the factors  used in this study.The “metacoder” package (v0.3.2) in R  was usedL. intracellularis [” package (v0.4.0) in R. First, we assessed the correlation between shedding and histopathology, then examined how each is associated with primary microbiota parameters such as abundance, diversity and richness as well as secondary parameters such as clustering. Parameters and characteristics were defined as directly or inversely correlated and a statistical significance of p < 0.05 determined the significance of the association. This allowed us to identify treatment-mediated parameters associated with reduced shedding and histopathology.To identify any relationship between the microbiota characteristics and the clinical signs of infection, i.e. histopathology and fecal shedding of llularis , we usedL. intracellularis, were included. Each group contained 10 animals, excluding the POS-CONTROL group which contained 20 animals. The pigs were purchased from a commercial farm in the Midwest of the US and were cross-bred pigs. Overall, 54.3% (38/70) of the pigs were male and 45.7% (32/70) were female. Samples used for this study were collected from 9-week-old pigs of which the majority had been experimentally challenged with L. intracellularis 16 days earlier. They spent 44 days on their specific diet with or without probiotic supplementation.A total of 70 pigs, sero-negative for L. intracellularis antibodies and DNA as determined by ELISA on serum and PCR on fecal samples [L. intracellularis vaccination, all VAC-LAW pigs had seroconverted and, at necropsy, 10–65% of the pigs in all other challenged groups were also seropositive. Successful L. intracellularis challenge was confirmed by a high prevalence of pigs shedding L. intracellularis with the highest bacterial DNA levels detected in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs  in all three alpha diversity indices as shown in Fig. The mean species richness, Simpson index, and Shannon index across all groups were 93, 0.53 and 1.9, respectively. A comparison between groups showed significant differences  correlation for shedding and the histopathology score. The unit increase in abundance of Streptoccocaeae and Erysipelotrichacea was associated with a 0.25–0.35 and 0.24–0.32 log reduction in shedding and histopathology respectively , Coriobacteriaceae  and Peptostreptococaceae   but the trends were similar compared to shedding.A direct correlation between histopathology in the ileum and fecal shedding of eae Fig. d and f. L. intracellularis infected pigs under different experimental treatment regiments were defined and characterized. We specifically compared possible prophylactic interventions to prevent L. intracellularis associated disease as opposed to traditional therapeutic methods such as antibiotic treatment. In future it may be worthwhile to repeat this study and to add a traditional antimicrobial treatment as an additional group. The ileum, as the primary site of L. intracellularis infection in pigs [L. intracellularis and 44 days post commercial L. intracellularis vaccination (VAC-LAW) or the start of three different probiotics  which were added to the regular base diet given to pigs in all groups. This differs from other L. intracellularis studies focusing on the enteric microbiome which investigated fecal [L. intracellularis infection dynamics at the site of colonization. We specifically examined the bacterial community as earlier L. intracellularis experiments in gnotobiotic pigs suggested that a certain microbial background is required for L. intracellularis infection to establish [L. intracellularis infection including fecal shedding and histopathology in the ileum. Overall, we observed a strong association between treatment and changes in ileum microbiota structure based on alpha diversity indices. These changes were significant and not limited to richness (i.e. the number of unique OTUs) but also to evenness and relative abundance. For example, richness was highest in T03-LAW and lowest in VAC-LAW  with either L. intracellularis, the established microbiome, or both. Alternatively, other microorganisms, induced by the probiotic bacteria or their metabolites, could have a major impact. Also, the probiotic bacteria could directly affect the protective biofilm formation on the enteric mucosal surface, such as changing the redox potential, resulting in an unfavorable environment for L. intracellularis. It may also be beneficial to increase the microbiota richness for the robustness of a niche in warding off a pathogen invasion. It is recognized that enteric pathogens, including L. intracellularis, have the potential to alter the bile acid metabolism [L. intracellularis infection, the disruption of the physiological microbiome (dysbiosis) at that site brought on by the infection or both [L. intracellularis infection by impacting the bile acid metabolism and other similar physiological processes. In this particular context further research is required to identify the microbial combination of probiotics and mechanistic basis for these effects.Our approach assumed that clustering by richness and abundance corresponding to treatment is indicative of a strong taxonomic effect for the experiment. The extent to which treatment impacted the ileal microbial structure differed for each group. These differences potentially define the exploitable usefulness of these treatments, specifically in augmenting clinical outcomes to control tabolism . Physioltabolism . Hence,  or both . The favPrevotella, Clostridium, Alloprevotella, and Ruminococcus were identified [Prevotellaceae, Ruminococcaceae, and Lactobacillaceae [Shifts in the ileal core genera were associated with changes in the threshold for shedding level, i.e. the clusters with the most depleted core (cluster 5) and the most enriched cores (clusters 1 and 3). This suggests that depleting the ileal core genera results in a higher threshold for shedding. It is difficult to compare the core genera across studies. Besides age, commonly studies are done in pigs around weaning , 46, houentified . A more entified . Besidesillaceae . The anaClostridiaceae, Streptoccaceae or Erysipelatricheae was associated with 3.9, 3.1 and 3.4 log reduction in copies of L. intracellularis shedding and pathology rank, respectively. In particular, reduction in shedding and pathology in the VAC-LAW group appeared to be influenced by the increase in abundance of Clostridiaceae. A similar but weaker relationship was noted for T01-LAW and T02-LAW. In a previous study, analysis of the microbiota of L. intracellular is vaccinated pigs showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum [In this study it was established that a direct relationship exists between pathology and shedding and identifying microbial factors that disrupt it provides a foundation for limiting the impact of this infection. The precise mechanism of this microbial interaction is unknown. The data obtained here will be useful to design future experiments in an attempt to further reduce bacterial shedding and pathology. This perhaps could advance probiotic supplementation to limit the impact of infectious diseases in food production. Limiting pathology arguably reduces not only the adverse effects on an animal’s productivity but also shedding and thus limits transmission to other pigs. In this regard, T03-LAW (cluster 1) appears to be the only treatment group in which the shedding dynamics were reduced. At the family level, a log increase in abundance of utyricum  which isL. intracellularis. In addition to T03-LAW, a reduction in shedding associated with increased diversity was also evident for T01-LAW and T02-LAW. An increase in diversity was however associated with increased shedding among VAC-LAW pigs and this still requires an explanation. Perhaps this could reflect the effect of a longer term dysbiosis as the VAC-LAW group was first exposed to attenuated L. intracellularis before being exposed to pathogenic L. intracellularis challenge. In addition, the VAC-LAW group did manifest associated changes in the ileal microbiota after L. intracellularis challenge  outside the realm of controlled experimentation. Ultimately such scalable innovation means livestock will be raised without adverse environmental effects while also minimizing the contribution to the evolution of antibiotic resistance. In this regard, our findings indicate the potential usefulness of microbial families such as Clostridiaceae, Streptoccaceae, Erysipelatricheae, Rummincococeae, Coriobacteriaceae and Peptostreptococaceae in augmenting clinical outcomes during L. intracellularis infections.A potential application of the study findings includes microbiota augmentation to modify pathology, shedding and, by extension, L. intracellularis, either with prophylactic treatment in the form of a commercial live L. intracellularis vaccine or in-feed probiotics. These changes exhibited as taxonomic shifts and included, but were not limited to, core and accessory genera depletion or enrichment. We observed that microbial changes such as diversity and richness had an inverse correlation with clinical characteristics such as ileal histopathology and fecal shedding. The extent to which this relation manifested varied depending on treatment, which raises the potential for using microbial manipulation to decrease pathology and shedding. Modulating these clinical parameters appears to be the foundation to limiting disease transmission as well as promoting individual animal resilience to disease. Further studies need to be done to explore the causal relationship and underlying mechanism(s) of probiotic feed supplementation on L. intracellularis induced ileal lesions. To unravel the complex associations between microbial diversity and L. intracellularis infection/pathogenesis, a longitudinal pig study with suitable design, such as sampling at different time points by staggered sacrifice of pigs within a group, introduction of the probiotic feed intervention at different time points before or after L. intracellularis infection, or ileal content transfer in addition to other tools would also be required.In this study, significant effects on the ileal microbiota structure were observed during experimental infection of pigs with Additional file 1 Number of positive pigs/total pigs per group (group mean ± SEM) for log10 L. intracellularis DNA in rectal swabs at different days post L. intracellularis challenge [A,B,C) indicate significantly different group means on a certain day.hallenge . DiffereAdditional file 2. Association of histopathology and shedding for each of the treatment groups.Additional file 3. Taxa and percentage of bacteria present the inoculum used to challenge the pigs.Additional file 4. Taxonomic characteristics of ileal microbiota. OTUs clustered according to their phylogenetic relationship and colored by abundance, also called a heat-tree. The core microbes can be identified by the dark green backbone while the transient/accessory microbes compose the rest of the tree branches and change by treatment groups.Additional file 5. Taxonomic association with shedding. The relationship between shedding and the abundance of individual families colored by treatment group is demonstrated.Additional file 6. Taxonomic association with pathology (microscopic ileum score). The relationship between pathology and the abundance of individual families colored by treatment group is demonstrated.


Sanfilippo syndrome is an untreatable form of childhood-onset dementia. Whilst several therapeutic strategies are being evaluated in human clinical trials including i.v. delivery of AAV9-based gene therapy, an urgent unmet need is the availability of non-invasive, quantitative measures of neurodegeneration. We hypothesise that as part of the central nervous system, the retina may provide a window through which to ‘visualise’ degenerative lesions in brain and amelioration of them following treatment. This is reliant on the age of onset and the rate of disease progression being equivalent in retina and brain.For the first time we have assessed in parallel, the nature, age of onset and rate of retinal and brain degeneration in a mouse model of Sanfilippo syndrome. Significant accumulation of heparan sulphate and expansion of the endo/lysosomal system was observed in both retina and brain pre-symptomatically (by 3 weeks of age). Robust and early activation of micro- and macroglia was also observed in both tissues. There was substantial thinning of retina and loss of rod and cone photoreceptors by ~ 12 weeks of age, a time at which cognitive symptoms are noted. Intravenous delivery of a clinically relevant AAV9-human sulphamidase vector to neonatal mice prevented disease lesion appearance in retina and most areas of brain when assessed 6 weeks later. Collectively, the findings highlight the previously unrecognised early and significant involvement of retina in the Sanfilippo disease process, lesions that are preventable by neonatal treatment with AAV9-sulphamidase. Critically, our data demonstrate for the first time that the advancement of retinal disease parallels that occurring in brain in Sanfilippo syndrome, thus retina may provide an easily accessible neural tissue via which brain disease development and its amelioration with treatment can be monitored.The online version of this article (10.1186/s40478-020-01070-w) contains supplementary material, which is available to authorized users. The temporal course of neurodegeneration in Sanfilippo syndrome or mucopolysaccharidosis type IIIA (MPS IIIA) is manifest by a normal infancy followed by progressive intellectual disability in early childhood and death at a median age of 18 years . The mosSanfilippo syndrome is presently untreatable, however several therapeutic approaches are under clinical evaluation in patients including but not limited to gene replacement , 19, 40.Significant retinal pathology occurs in utero in MPS III  with incA significant loss of retinal function has been reported in MPS III mice as they age , 21, 42.Embryologically, the retina and optic nerve are outpouchings of brain and are part of CNS. Importantly, the retina is the only part of the CNS that can be visualised non-invasively. Given that many other neurological disorders exhibit retinal/optic nerve pathology prior to, or in conjunction with the onset of brain pathology , 24, we The research protocol was approved by the Institutional Animal Ethics Committee  and the Institutional Biosafety Committee (#B144/12/20 and #B149-12-21) prior to study commencement.mps3a) were obtained from a breeding colony established and maintained at the Women’s and Children’s Hospital Network, North Adelaide, South Australia, Australia. Congenic MPS IIIA mice were also intercrossed with a reporter line , purchaAll mice were group-housed in a constant temperature/humidity-controlled facility with a 14-h light:10-h dark cycle, with food and water ad libitum. Mice were provided with toilet rolls and plastic cups plus nesting material for environmental enrichment. All breeding, housing and experimental procedures complied with the Australian code for the care and use of animals for scientific purposes  and the Association for Research in Vision and Ophthalmology (ARVO) statement (2016).SGSH mutation was undertaken on ear-notch tissue from which DNA was extracted (ThermoFisher Scientific), with subsequent PCR and restriction digest analysis performed according to methods in [C TGA CCT ACG 3′) were utilised. Reactions containing 2 × TaqMan® Genotyping Mastermix and 20 × GFP-FAM primer were aliquoted into MicroAmp® fast optical 384-well reaction plates and 1 µL DNA was added to each well . Negative, no DNA template controls were included in each assay. The plates were sealed with MicroAmp® optical adhesive film and amplification performed on a QuantStudio 7 Flex Real-Time PCR system (Thermofisher Scientifc).Genotyping for the thods in . The gen-fill CO2. Intra-cardiac perfusion with ice-cold saline was undertaken then brain, eyes and optic nerves were removed. Some brain tissue was dissected into five 2 mm hemi-coronal slices, with slice 1 containing olfactory bulb and the rostral aspect of the cerebral cortex and slice 5 containing cerebellum and brainstem. For mass spectrometry evaluation, the retina was carefully dissected from the eyes and both brain and retina were frozen at − 80 °C until used.No differences have previously been found in the time of onset or rate of disease lesion progression in the brains of male or female mice with MPS IIIA, therefore, mice of mixed genders were humanely killed at 3, 6, 12, 15, 20, 22 or 25 weeks of age using asphyxiation with slowBrain tissue destined for immunohistochemistry was immersion-fixed in 4% paraformaldehyde in PBS for up to 1 week. Eyes and optic nerves to be used for immunohistochemistry were immersion-fixed in Davidson’s solution  for 24 h and then held in 70% alcohol for processing. Davidson’s solution is the preferred fixative for whole eyes as it provides optimal tissue morphology while avoiding retinal detachment. A separate cohort of eyes was collected for retinal whole-mount preparation and was fixed in 4% PFA in PBS for 24 h before being placed in 30% sucrose at 4 °C until dissected as previously described .11 vg; 10 µL) via the superficial temporal vein. A repeat injection was made via the tail vein on day 5 of life. Control mice of both genotypes received AAV-CMV-mCherry at this time. This sequential injection strategy increases the number of (newly emerging) retinal neurons exposed to viral vector [To determine the impact of sulphamidase gene replacement on retinal and brain disease lesions, on the day of birth, cohorts of MPS IIIA and unaffected mice n = 5/group) received an intravenous injection of AAV9-CMV-hSGSH or AAV9-CMV-GFP  and mounted on glass slides . Retinae were sectioned in a consistent manner to the level of the optic nerve head and brain sections were taken at approximately 0.48 mm lateral to the midline (according to the Mouse Brain Atlas ). Oven-dDetails of tissue type, antigen retrieval pre-treatment, primary antibody/lectin reagents used are outlined in Table Immunohistochemical staining of brain and ocular tissues was performed according to established methods , 7. BrieFor lectin histochemistry, sections underwent antigen retrieval and then were incubated in hydrogen peroxide followed by peroxidase-conjugated isolectin-B4 overnight. Colour detection was achieved using DAB. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was carried out according to published methods , 33. AllH&E-stained sections were evaluated to determine the total thickness of the retina and that of individual retinal layers. To ensure inter-animal consistency, measurements were taken in two locations: 500 µm from the ciliary body  and 500 µm from the optic nerve head . Measurements were made on each side of the optic nerve head and the mean thickness of the peripheral and central retina was calculated. For accuracy, retinal thickness measurements were only taken of sections in a perpendicular orientation. Total thickness was measured from the retinal ganglion cell layer (RGC) to, but not including, the retinal pigmented epithelium (RPE). Other measurements taken were the thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), total photoreceptor segment layer (PS), inner segment layer (IS) and outer segment layer (OS). All measurements were carried out blind to genotype/age.The number of positively stained cells in a specified length of retina or in a given area of brain were either counted manually or quantified using threshold analysis. The relatively low number of TUNEL-positive nuclei were counted along the entire length and thickness of retinae. Peanut agglutinin-positive cone inner segment number were counted in a field of view using a 40X objective on each side of the central and peripheral retina. Calretinin positive amacrine cells were counted along 450 µm of retina starting 150 µm from each side of the optic nerve head. PKCα-positive bipolar cells were counted in the central and peripheral retina, for a total length of ~ 225 µm on both sides of the optic nerve head. Due to their relatively low number, calbindin-positive horizontal cells were counted along almost the entire length of retina, starting one field of view from the ciliary body to within one field of view from the optic nerve head using a 40X objective. Cell counts were expressed as number of immuno-positive cells/mm. Threshold analysis of LIMP2 and GFAP staining, based on the optical density of positive immunostaining, is reported as % immunoreactivity. Rhodopsin staining was undertaken for demonstration of rod outer segment changes however no quantification was performed.-X and then incubated in Alexafluor 488-labelled anti-rabbit secondary antibody for 1 day at 4 °C. Following further washing in PBS/triton-X, retinae were mounted on glass slides using Vectashield anti-fade mounting medium with DAPI  and stored in the dark. Flat mounts were evaluated on a confocal microscope  with excitation provided by a 488 nm laser with emission captured with 493–569 nm filters for the green channel.Immunolabelling with RBPMS was carried out on free-floating retinae, which were incubated in primary antibody for 3 days at room temperature, washed in PBS with 1% tritonCell counter available in AnalySIS Lifescience software  and are presented as the number of cells/mm2 of retina.The density of RGCs was quantified in retinas taken from n = 6 mice/genotype at each of 3, 6, 12 and 20 weeks of age. A further n = 3 mice/genotype were aged to 25 weeks. Using a × 40 objective, confocal z-stacks were taken through the RGC layer in three consistent regions in both central and peripheral retina. RGCs were counted manually using + RGCs per mouse  were obtained using a 20 × objective to capture the entire dendritic tree. Images were imported into Fiji open-source image processing software [NeuronJ available in Fiji. and the total number of branches/nodes was determined using Cell counter. All analyses were performed by a user blind to mouse genotype and age.To evaluate RGC dendritic morphology, z-stack images of three GFPsoftware  and dendsoftware . DendritUnfixed brain slices, optic nerve and retina were homogenised in 0.02 M Tris/0.5 M NaCl, pH 7.4, with HS disaccharides quantified using a previously published method . Peak art-test with a Welch’s correction was utilised. (GraphPad Prism version 8). Regression models with continuous scale outcome variables and time-dependant interaction effects were fitted to the data, and residuals analysed to validate the models’ distributional assumptions. Multiple comparisons were handled with ANOVA and post-hoc Bonferroni correction. Data were regarded as statistically significant when p < 0.05.Data are expressed as mean ± SEM or as individual data points. Graphical and analytical exploratory data analyses were performed, and patterns assessed. To compare differences between unaffected and MPS IIIA mice at each time point, an un-paired Student’s Figure Examining the ONL more closely Fig. a, b we nIn contrast, there was no significant change in the number of RBPMS-positive retinal ganglion cells (RGCs) in MPS IIIA retina to 25 weeks of age Fig. a–c. The We next quantified HS levels in retina, optic nerve and brain Fig. a–c, and Amoeboid, isolectin-B4-positive and therefore presumptively activated microglia were noted in MPS IIIA mouse retina and optic nerve, as well as visual cortex and superior colliculus from 3 weeks of age, the earliest time point evaluated Fig. a–l. A cuBrain tissue from MPS IIIA mice exhibits increasing numbers of ubiquitin-positive axonal inclusions with age . Whilst For retinal imaging to provide a non-invasive, quantitative method for visualising neurodegenerative changes in Sanfilippo syndrome, retinal disease must parallel brain disease. When we compared the rate of accumulation of HS Fig. a, the raFinally, given there is currently no effective therapy for patients with Sanfilippo syndrome, and any treatment strategy developed needs to be capable of preventing development of both brain and retinal disease lesions to afford patients maximum quality of life, we examined the short-term efficacy of i.v. delivery of AAV9-CMV-hSGSH to MPS IIIA mice at day 0 and 5 of life. One unaffected control mouse was cannibalised between day 0 and day 5 of life, however all other mice remained healthy post-injection. Mouse retina Fig. a and braOur goal was to establish the age at which disease lesions first become apparent in Sanfilippo mouse retina and brain and compare the rate of disease progression in each tissue in order to test the hypothesis that examination of the retina will provide insight into brain disease stage. In short-term studies, we then determined the ability of a clinically-relevant gene therapy approach to mediate improvements in disease sequelae in both neural tissues. Our objective is to ultimately provide a therapeutic monitoring strategy in addition to a prognostic tool, for children diagnosed with this devastating disorder. Whilst a few researchers have evaluated disease lesions in MPS III mouse retina, those studies have either started later in the disease course  or For the first time, our study reveals that the age of onset and rate of progression of retinal disease in mice with MPS IIIA are remarkably similar to those in the brain. Both tissues exhibit substantial accumulation of heparan sulphate by 3 weeks of age, as determined by sensitive mass spectrometry-based measurements. HS levels in optic nerve were also elevated at this pre-symptomatic age. Primary storage of HS led to massive expansion of the late endosomal/lysosomal system in retina, optic nerve and brain as visualised using LIMP2-staining. Similarly, the presence of presumptively activated isolectin-B4-positive microglia with an amoeboid morphology was noted in all three tissues in 3-week-old MPS IIIA mice. These cells remained present to the end of the study (mice aged 25 weeks) and were only rarely observed in unaffected mouse retina and optic nerve. Macrogliosis was observed from ~ 6 weeks of age in MPS IIIA mouse retina and brain although curiously, not in optic nerve. This may be explained by the fact that the optic nerve inherently expresses a high level of GFAP—astrocytic processes extend in all directions and through all planes of the optic nerve, rendering astrogliosis more challenging to quantify. Staining of axons in optic nerve with antibodies to ubiquitin revealed the appearance of axonal spheroids between 3 and 6 weeks of age, an observation that has also been made in brain . Thus, twww.clinicaltrials.gov; NCT04018755), could ostensibly be visualised using non-invasive retinal imaging.Disease lesion progression also occurs in parallel in the two structures. This suggests that methods that visualise and quantitate any of these lesions in MPS IIIA retina could inform clinicians about brain disease progression. Similarly, the efficacy of any treatment that is able to access retina as well as brain, for example i.v. AAV9-sulphamidase as examined in the present study, or small molecule therapeutics such as anti-inflammatory agents e.g. Anakinra; IL-1R inhibitor, which is currently under clinical evaluation in Sanfilippo patients . Koronyo-Hamaoui et al.  were firMore recent studies demonstrate that hyperspectral imaging enables brain-relevant disease lesions to be evaluated in retina . Changes+-cone photoreceptor cells are impacted. Tools capable of visualising these very early apoptotic events and/or detecting initial loss of outer segments may provide prognostic indicators of the early-onset form of Sanfilippo c.f. later-onset disease that manifests in adulthood [The second novel outcome of our study is the characterisation of retinal lesions present very early in the MPS III disease course, i.e. pre-symptomatically. Other studies have only evaluated Sanfilippo retina from a symptomatic stage onwards . Wedulthood .Other than photoreceptors, all other neuronal classes in the retina, including RGCs, bipolar cells, horizontal cells and amacrine cells, appear largely unaffected by the disease process. These parallels observations made in MPS IIIA mouse brain tissue: neither we , nor othLoss of rod photoreceptors presumptively leads to the impairments previously noted in the dark-adapted ERG in MPS III mice , 21, 42.In conclusion, this is the first study to evaluate the relationship between retinal and brain disease lesions in an animal model of a lysosomal storage disorder. We demonstrate that both retina and brain in Sanfilippo mice exhibit disease lesions at a very early disease stage  and that the rate of disease progression is comparable between the two structures. This bodes well for utilising non-invasive imaging of retina to inform our understanding of the state of brain disease. The loss of photoreceptor neurons during early disease progression and the possibility of visualising and monitoring this using non-invasive techniques may provide both a prognostic and therapeutic monitoring tool for MPS IIIA. At the very least, the early age at which retina displays disease lesions in Sanfilippo syndrome highlights the absolute necessity of directing therapeutics at both the brain and the retina, to ensure patient quality of life is maximised.Additional file 1: Figure S1. A-type retinal ganglion cells in unaffected Thy1-GFP (A) and MPS IIIA Thy1-GFP mouse retina (B). Scale bar = 50 μm. The complexity of the dendritic tree was determined in GFP-positive RGCs in unaffected and MPS IIIA mice aged three, six, 12, and 20 weeks of age. We observed no significant difference in dendritic tree area (C), total dendritic tree cable length (D), or the total number of branch points or nodes (E). Negligible change was observed in soma diameter over the 20 week time course (F). Data represent mean ± SEM. *p<0.05.Additional file 2: Figure S2. Representative images of ubiquitin immunolabelling in 25 week old unaffected and MPS IIIA optic nerve are shown . Arrows indicate ubiquitin-positive spheroids. Scale bar = 20 µm. (C) Ubiquitin-positive axonal spheroids >5 µm diameter were quantified in optic nerve obtained from unaffected and MPS IIIA mice aged 3- to 25-weeks of age. ****p<0.0001.