Congenital deafness is a major pediatric problem, affecting about 1.5–3 per 1000 newborns. The early treatment through cochlear implantation and auditory rehabilitation has been a historic milestone. Early diagnosis of congenital deafness is an essential requirement to obtain the best results, which is achieved through neonatal screening, a diagnostic practice that we began systematically at the Hospital Clínico in Valencia (Spain) 30 years ago. Neonatal hearing screening is successful in most developed countries. Its implementation has been slow due to the multiple difficulties that its universal application entails since it involves several health professionals and must be carried out, in a short time interval after birth. In addition, it must have a good performance that prevents the overload of other services and that requires experience and continuous adjustments in search of proper protocols. The aim of this review is to shed some light on some key points of neonatal hearing screening, highlighting our experience in the solutions to common problems. We will discuss about techniques, protocols and neonatal or nutritional factors that can influence the screening results. To a summary of our work, an update on the subject is provided with the intention of sharing experiences and facilitating the start-up of the new units. Congenital deafness is a major pediatric problem, affecting about 1–3 per 1000 newborns ,3,4 to tThe early treatment of these children through auditory rehabilitation programs for learning sound processing and cochlear implantation has been a historic milestone. This fact supports the need for the diagnosis of congenital deafness to be universal and, as soon as possible, that is, through neonatal screening. In this line, it is worth remembering that more than 90% of deaf newborns have healthy parents and that many of profoundly deaf require a cochlear implant ,6.Neonatal hearing screening has been successfully implemented in most Western countries. Its set-up has been slow due to the multiple difficulties that its universal application entails. The organization at a regional level is complex and the technical equipment, preparation of health personnel and administration in general makes it a hard and costly enterprise to run. It involves several care services and levels of health staff; it should be universal, and it should be carried out mainly in the short postpartum stay in maternity ward. A good performance is needed to avoid the overload of hospital services concerned. There is currently a strong discussion about the more appropriate technical equipment and protocols to be used, but all the programs manage to increase the early detection of congenital deafness in the neonatal period, which has made it possible to reduce the age of treatment and have better clinical prognosis and psychosocial adaptation ,8,9,10.We have systematically applied this practice at the Hospital Clínico in Valencia (Spain) during the last 30 years ,12,13.This review summarizes our experience over more than three decades and we will discuss the key points to achieve a satisfactory neonatal hearing screening, highlighting the solutions to the problems that usually appear in regard to equipment, techniques, protocols and neonatal or nutritional factors that can influence the results.Much time and effort has been devoted to finding the most efficient and accurate procedures, protocols, and equipment for screening, diagnosing, and treating children who are deaf or hard of hearing . Universal neonatal screening is the most useful procedure for early detection of congenital deafness. Neonatal hearing screening facilitates a confirmed diagnosis of neonatal deafness in the first 4 months of life while without this practice the diagnosis is usually delayed up to 35 m. In the same way, the treatment of children diagnosed from neonatal screening begins before 7 m, and in its absence, it is delayed on average up to 35 m ,16.Neonatal hearing screening (<1 month) should check both ears and be universal (>95% of newborns). It must be bilateral because unilateral congenital deafness affects neuropsychological development ,17,18. Under normal conditions if the technical application is adequate, the referral of patients to otorhinolaryngology (ENT) service will be less than 4%. Diagnostic confirmation must be conducted in good conditions before 3 m and audiologic study before 5–6 m in order to begin treatment at approximately 6 m  7,18]. ,18. 7,18A universal neonatal screening raises some problems of organization and distribution of tasks related to the responsible hospital service , health personnel in charge of carrying out the test , where and when the newborn examination must be performed, and finally, if the study should be carried out in the presence of the parents. In any case, regular check-ups should be made to ensure that all newborns are examined. Likewise, periodic meetings of the services and staff directly involved  should be scheduled. In any case, the verbal consent of those responsible for the child is required for legal purposes or a written disclaimer kept in neonatal history if family does not want to do the test.At the present there is controversy about the technical equipment and protocols to be used, but all the programs reduce the age of treatment achieving a better prognosis for hearing and psychosocial adaptation ,8,9,10. When defining the main characteristics of universal neonatal screening for deafness, it must be taken into account that the child will not cooperate and that a large population  will be included. Consequently, those procedures should be chosen that, similar to other analytical tests, are sensitive, specific and objective but, given the number of those studied, which are mostly healthy neonates, they must also be atraumatic, simple, repeatable, fast and cheap.It should be emphasized that screening does not seek a firm diagnosis of the disorder but rather the identification of suspected newborns in order to focus the subsequent effort to its diagnostic confirmation. Currently, most neonatal hearing screenings are performed with audiological techniques, mainly those based on acoustic otoemissions  and those based on auditory nerve potentials . Both techniques are sensitive and specific enough to be used in screening but have some differences, the most important of which is the fact that otoemissions only explore hearing up to the cochlear level (which constitutes the most frequent defect with more than 90% cases) but not the neural pathway, which may be affected in a small proportion of cases. On the other hand, otoemissions are faster and cheaper than AABR and require hardly any consumables. They are also less traumatic since they do not need to put electrodes on the skin and are easier to do. Regarding performance, AABR have fewer false positives than otoemissions, especially in the first days after birth ,19,20,21Given that TEOAE do not allow the identification of neural deafness, most authors advise the use of AABR in all children with auditory risk factors due to neural or syndromic pathology ,9,10,18.Discussion persists about the preferred technique in children without risk factors. The sequential use of both techniques in specific cases  is gaining followers, which is facilitated by the manageability of the available devices that can do TEOAE and AABR at the same time ,10,22,23Currently, more than half of the countries with neonatal hearing screening use TEOAE thanks to its low cost as well as its easy and fast technical implementation, although more programs are increasingly using a mixed protocol (TEOAE-AABR) or, less frequently, only AABR ,9,24,25.Given the differences in approach on the subject, the Joint Committee on Infant Hearing (JCIH) recommends that each country adopt the most appropriate norms to develop its own protocol in accordance with regional administrative possibilities, government decisions and budget . At the Concerning the place to carry out the first audiological examination, two alternatives are observed according to local availability of resources, one that conducts the examination at the maternity ward before discharge  and another that prefers to perform the test later in external office . The former is better performed by all nurses and the latter is better conducted in an external office by dedicated staff that filters the referral to ENT. This is recommended also for all retests in babies that fail the first test .It is necessary to monitor the patient in a longitudinal database, accessible to all specialists involved. It reduces the losses in follow-up that are the Achilles heel of the screening programs!There are a series of general and specific aspects that must be taken into account. 1. The test should be performed on all newborns in a quiet room by trained health personnel that also will fill the database with the results. 2. Although the study can be carried out by doctors or nurses, either from the pediatric as well as from the otolaryngology or obstetrics services, due to their location and numerical availability, nurses from obstetrics are the most suitable professionals. If possible, it is better that all obstetric nurses are trained in hearing screening because it permits to do the test in all shifts, all day long and covering holidays. Nevertheless, in large hospitals one can dedicate a special team of five health personnel to do it (it improves reliability but worsens continuity because of holidays and leaves).3. It is advisable to have two devices available in order not to stop testing if one malfunctions. For the same reason, two or three probes, ear tips and cables must be ready.4. Do not spend more than 5 min on a test. If it is impossible to carry out due to the child’s restlessness or other causes, it must be stopped and repeated later. Try to do it after feeding, in a quieter place or using breastfeeding or a pacifier. 5. Do not insist on testing, if a test is a “fail” two times, refer to next level after assuring that the ear tip and probe fit, were right and there were no technical problems  . On AABR6. In cases where it is required to repeat the test for failing to pass the first test/step it is important to discuss the inpatient versus outpatient retest issue because the former has more failures (because of middle ear status) but a lower lost to follow-ups; the later has opposing figures. The second test/step must be performed by an expert health personnel before 2–3 weeks of age , it must be bilateral, not testing just the ear that fail before and controlling the time you are spending, if it fails a pair of tries just send it to ENT (Joint Committee on Infant Hearing recommend just one try in good conditions) .7. Parents should be informed as soon as possible of the test results. It is very important to explain what results of the screening test mean: the pass means a normal auditory function but refer means only that technique cannot, at that moment, detect a normal function and that can be also for technical reasons or lack of aeration of middle ear, so another test must be performed in order to confirm the diagnosis some days later. This avoids the excess of anxiety in the family. It is basic to choose the best moment when to communicate results and explain clearly what the screening tests really mean showing the differences of screening versus diagnosis. On the other hand, it is important to remark also the possibility of a late onset hearing loss in spite of a normal screening test. Finally, it is necessary to give all information of this issue in a proper culturally competent way offering an appropriate support .In our experience, keeping these considerations in mind, an effective neonatal hearing screening program can be obtained without too many difficulties. With these tips you can do a sustainable local program.In any case, no matter how detailed and precise the screening is, the lack of follow-up is frequently observed in those newborns who did not pass the examination in the hospital. There is a high percentage of this babies that do not assist to controls or appointments in external office and also some documentation or contact problems. In most programs represent an 8–10% of the total of babies scheduled for retest ,9. AccorIn addition, high risk neonates are controlled in our external office and ENT services in order to detect late onset hearing loss till they have at least two years and usually after that age . Some additional problems in neonatal hearing screening deserve to be taken into consideration: infection by cytomegalovirus, the type of lactation and the increasing importance of genetic diagnosis.Cytomegalovirus infection (CMV) reaches a frequency of about 0.5% of all newborns in developed countries. Most of them are asymptomatic (more than 94%) and about 22% develop either neonatal or late-onset hearing loss.Approximately 6% are symptomatic and, of these, 33–60% develop hearing loss. The hearing loss is progressive in 11–50% and late-onset in 5–18% of these children. Early diagnosis is important for considering drug treatment with ganciclovir or valganciclovir, given that several studies have shown their usefulness for improving hearing loss or preventing its progression in these children. For a congenital diagnosis it is necessary to have a positive PCR test in urine before 21 postnatal days and that is the reason for doing retest in hearing screening before this date ,31,32. Breastfeeding is an important factor related to a normal response in TEOAEs test. The reasons are unclear but probably is due to a better middle ear aeration because a more powerful sucking. It may improve the final results of newborn hearing screening reducing the number of neonates who need to be rescheduled for a retest, as well as the associated anxiety and the possibility of losing patients during follow-up. This is another good reason to encourage newborn breastfeeding ,34.Finally, it is important to highlight the increasing importance of the genetic component in the origins of congenital deafness; recent research detects genetic abnormalities in up to 80% of cases including more than 400 known syndromes. Of the genetic cases, 70% are non-syndromic and 30% are syndromic. Still, among the non-syndromics, about 80% of the cases hearing loss is related to the autosomal recessive inheritance mechanism and 10–20% to the autosomal dominant one, being that genetic deafness presents all the Mendelian inheritance mechanisms, including the mitochondrial one. In the near future the best option for diagnosis could be a newborn hearing screening with the techniques discussed above or new ones , combined with CMV and genetic diagnosis in order to solve the limitations inherent to current neonatal programs because CMV and genetic treatment will be a real option ,37,38,39Neonatal hearing screening is a necessary, effective and efficient procedure. All techniques currently available are useful, specific and sensitive, although there are other techniques under development  and also complementary tests  that may improve the current performance of established programs.It is very important that, if possible, the programs are national in scope and involving primary care, in order to assure measures to control losses in the follow-ups and with post-neonatal audiological protocols for early diagnosis of delayed deafness. Furthermore, and of course, a good communication and implication between the specialists involved is mandatory, ruled by a general coordinator and with a good database to make easy knowing and controlling the quality of the programs, thus justifying the time and money invested.


Auditory diseases are disabling public health problems that afflict a significant number of people worldwide, and they remain largely incurable until now. Driven by continuous innovation in the fields of chemistry, physics, and materials science, novel materials that can be applied to hearing diseases are constantly emerging. In contrast to conventional materials, new materials are easily accessible, inexpensive, non-invasive, with better acoustic therapy effects and weaker immune rejection after implantation. When new materials are used to treat auditory diseases, the wound healing, infection prevention, disease recurrence, hair cell regeneration, functional recovery, and other aspects have been significantly improved. Despite these advances, clinical success has been limited, largely due to issues regarding a lack of effectiveness and safety. With ever-developing scientific research, more novel materials will be facilitated into clinical use in the future. According to the latest World Health Organization estimates, 466 million people around the world (over 5% of the world population) experience disabling hearing loss. By 2050, this number is projected to rise to approximately 900 million, and nearly 2.5 billion people are at risk of contracting auditory diseases . The earThe practical application of new materials is continuously undergoing considerable advancements, and substantial success has been achieved in some aspects. Whereas the new materials are being applied to treat auditory diseases, the abundant advances made in the fields of diabetes, cardiovascular disease, and neuromuscular disease using new materials are progressing. Recently, the Food and Drug Administration (FDA) approved the first RNA interference-based gene silencing technology drug—Patisiran—which regulates gene expression by the delivery of RNA to target cells, improving the prognosis of patients with rare cardiac and neurologic disease . For anoHearing loss is attributable to genetic factors, specific viral infections, chronic ear infections, birth complications, exposure to excessive noise, aging, and ototoxic drugs. CHL may occur as middle-ear effusion, tympanic membrane perforation, physical external trauma, infection, canal stenosis, cholesteatoma, otitis media, otosclerosis, ossicular erosions, and so on . At presDiseases that could contribute to SNHL are age-related hearing loss (ARHL), inherited hearing impairments, Meniere’s disease (endolymphatic hydrops), autoimmune inner-ear disease, ear infection, drug-induced deafness, ear trauma, and idiopathic SNHL . In addrvia γ-secretase inhibitors enables mice to regenerate hair cells in response to noise-induced damage, simultaneously gaining an improvement in their hearing level  application for cartilage regeneration, cartilage stem/progenitor cells (CSPCs) have also been used for the successful ear reconstruction and modeling the pathogenesis of auricular deformities  is a commonly seen disease in otorhinolaryngology. Although the preferred autograft materials, such as temporal muscle, cartilage, and fat, have a variety of applications, they have also been reported to be highly susceptible to infection and have pronounced dependence on nutrients from the surrounding tissue . Therefoneration . The intneration . A new cnsertion .Tympanoplasty exerts significant actions on the treatment of auditory diseases such as chronic otitis mastoidea, tympanosclerosis, cholesteatomatous chronic otitis media, and middle-ear sound transmission defect caused by trauma. Adhesive otitis patients often require surgical interventions when they present with concomitant middle-ear atelectasis, structural adhesion in the tympanic chamber, sound transmission disorder, recurrent infection, and persistent otorrhea . To avoiThe ear is one of the predilection sites for keloids, frequently following trauma, surgery, burns, and ear piercing. Due to the high rate of recurrence, the search for excellent postoperative coverage materials and preventing recurrence has become a particular research hotspot in the treatment of keloids . Park etin vitro experiments have newly demonstrated that the cGMP-dependent atrial natriuretic peptide (ANP) and the permissive environment created by novel silicon micro-pillar substrates (MPS) could facilitate the survival and growth of SGNs , gene therapy, and cell therapy from different perspectives  see Fig.via physical trapping, adsorption, or electrostatic interactions, then the sustained release can be obtained using diffusion of NGF and/or degradation of carriers, with the aim of local, sustained drug release . Hence, developing highly efficient and inexpensive materials is of substantial importance . Current release . One suc release . Moreove release . However release , but OW  release . Chen et release , but fur release  as well  release , anti-ap release , antioxi release , connexi release , cochlea release  genes. T release . Other v release . AAV2/An release . Tan et ng level . MicroRNtionally . Luo et tionally . A singltionally . Autoimmtionally . Therapitionally . Mesenchtionally . The apptionally . In regetionally . The pretionally , 3D printionally , tailoretionally , and eletionally . Recent ntiation . Other nntiation , Ti3C2Txembranes , microflembranes , and aniembranes . SuggestCis with dexamethasone during cochlear implantation may alleviate inflammation in the surgical sites (In terms of hearing ancillary equipment, the application of CI for bilateral severe-to-profound SNHL has also reached maturity. However, implantation surgery can potentially cause invasive damage. Recent studies have increasingly found that degradable cell coatings for CI electrodes can increase neuronal survival by persistently releasing BDNF from cell-coated surfaces . In addial sites .The control of tinnitus by external noise stimuli has become a therapy of particular interest. Tinnitus afflicts approximately 15% of the general population worldwide, and there is currently no cure for this disorder. Attempts have been made to ameliorate tinnitus with individually applied spectrally optimized near-threshold noise , with movia the diminishing hair-cell uptake of gentamicin, activating Nrf2-Kcap1-ARE, up-regulation of Nrf2, and the expression of related antioxidant genes without damaging mechanoelectrical transduction channels (via inhibiting the expression of TNF-α, IL-4, and Bax and up-regulating ACTH proteins, at the same time regulating auditory system homeostasis and improving the microvasculature circulation of the inner ear and the ischemia conditions of tissue cells in rats (P. aeruginosa (The discovery of novel pharmaceuticals is one of the important directions for future research. Today, drug candidates with potential therapeutic applications for hearing loss include antioxidants, regulators of mitochondrial function, ion channel modulators, hair-cell protectants, and anti-inflammatory drugs . Other pchannels . It has channels . Multiplchannels  and allechannels . Howeverchannels . Another in rats . Thus, Truginosa .This study has highlighted some bottlenecks in the application of therapeutic materials for auditory diseases, such as the tumorigenicity of repair-promoting substances, immune reaction to implanted materials, high cost, and a difficult fabrication process. More research combining materials science, basic medicine, and clinical medicine is required to address these bottlenecks, with the aim of obtaining more cost-effective and safer new materials for treating hearing loss.Presently, various deficiencies still exist regarding insufficient treatment options and their modest efficacy for auditory diseases, leading to CHL and SNHL. Nevertheless, the development and application of novel materials are gradually improving the condition. The novel materials are able to hasten perforated tympanic membrane healing, tympanoplasty of the middle ear, and repair of the microtia, as well as promote the regeneration of inner hair cells, spiral ganglion cells, and the synaptic ribbons, decrease inflammation level, and modify hearing level adjunctively. Although the efficacy and safety of novel materials in clinical application remain to be verified, with more progression of research and substantial technological innovation, these novel materials will be continuously improved, and more refined materials could be employed to treat hearing loss clinically.ML conceived and wrote the manuscript. YD modified the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mycoplasma hyopneumoniae, each administered in herd with subclinical PCV2d infection and enzootic pneumonia. One vaccine was a bivalent containing PCV2a and M. hyopneumoniae and the other was a trivalent vaccine containing PCV2a and 2b (PCV2a/b), and M. hyopneumoniae. The defining difference between these two vaccines was the inclusion or absence of PCV2b antigen. A total of 480, 21day-old pigs were randomly allocated to one of four treatment groups . These groups included; one-dose trivalent-vaccinated, two-dose trivalent-vaccinated, one-dose bivalent-vaccinated, and unvaccinated. The one- and two-dose trivalent vaccinated pigs exhibited significantly better growth performance when compared with those vaccinated with the bivalent vaccine. The one- and two-dose trivalent vaccinated pigs also reduced the amount of PCV2d loads in the blood and feces, and resulted in a lower M. hyopneumoniae load in the larynx when compared with one-dose bivalent vaccinated pigs. Statistical differences were not observed between the one- and two-dose trivalent-vaccinated groups in terms of growth performance, serology, amount of PCV2d loads in the blood and feces, amount of M. hyopneumoniae load in larynx, and pathological lesions. The results of the present study will provide swine practitioners and producer with comparative clinical field data to select the proper vaccine and vaccination regiment for herds suffering from subclinical PCV2d infection and enzootic pneumonia.The present field trial compared two combined vaccines of porcine circovirus type 2 (PCV2) and  Porcine circovirus type 2 (PCV2), a member of the family Circoviridae, is a common virus of pigs found throughout the world and is recognized as one of the most economically threatening pathogens to the global pork industry . PCV2 maMycoplasma hyopneumoniae is prevalent and highly contagious in the majority of swine herds throughout the global pig industry. M. hyopneumoniae causes mycoplasmal pneumonia, which is characterized by a chronic, non-productive cough with high morbidity and low mortality. Enzootic pneumonia that is caused by M. hyopneumoniae when combined with opportunistic bacteria, such as Pasteurella multocida, continues to be a significant chronic respiratory disease [ disease . Enzooti disease .M. hyopneumoniae are economically important pathogens and the primary agents involved in the PRDC found within global pig production systems. Vaccination for PCV2 and M. hyopneumoniae is one of the most effective strategies in the control of both pathogens, especially Asian pork industry [M. hyopneumoniae for more than 50% of their pigs (http://www.kahpha.or.kr (accessed on 29 April 2021)). Combined vaccination is consequently considered part of routine management practices.PCV2 and industry . Korean M. hyopneumoniae  was introduced into the global market (http://www.zoetisus.com (accessed on 29 April 2021)). The PCV2b antigen of the trivalent vaccine is of particular interest as it is genetically closely related to PCV2d (formerly referred to as mutant PCV2b), which is currently the predominant PCV2 genotype in Asian pig populations [M. hyopneumoniae and a trivalent vaccine containing PCV2a/b and M. hyopneumoniae has yet to be undertaken. The objective of this study was to compare a bivalent and trivalent vaccine, with an emphasis on the evaluation of growth performance in herds in the presence of subclinical PCV2d infection and enzootic pneumonia.Recently, a new trivalent vaccine containing PCV2a and 2b (PCV2a/b), and ulations ,5,6. Altulations ,11,12, tulations ,14,15. Iulations . Neverth®, Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO, USA) and M. hyopneumoniae  at 3 weeks of age. Submitted cases met the definition of subclinical PCV2 infection [M. hyopneumoniae infection was determined in all three, 68 day-old pigs by display of severe dry coughing, histopathological peribronchiolar lymphoid tissue hyperplasia, and the detection of M. hyopneumoniae in lung samples by real-time PCR [M. hyopneumoniae in the herd. Pre-trial investigations identified a PCV2 serological profile that presented an increase in antibody titers starting around 7 weeks of age. Pigs that were 7–15 weeks of age tested positive for PCV2 in their blood by PCR methodology. M. hyopneumoniae serology tested as partially positive in 7 week-old pigs, and completely positive in 10 week-old pigs. Together, these results show early and prolonged PCV2 and M. hyopneumoniae infections were circulating within the herd.The clinical field trial was conducted on a 1200-sow, farrow-to-finish swine farm that implemented an all-in-all-out production system. The farm was selected based on its history of subclinical PCV2d infection and enzootic pneumonia. The status of porcine reproductive and respiratory syndrome virus (PRRSV) at the farm was stable; with no active PRRSV circulation . No PRRS modified-live virus vaccine was administered for at least one year in sows and piglets. Piglets were vaccinated for PCV2  at 21 days of age. The pigs in the VacA2 group were intramuscularly vaccinated with a 1.0 mL dose of the trivalent vaccine (Fostera® Gold PCV MH) at 21 and 42 days of age, respectively. Pigs in the VacB group were intramuscularly vaccinated with a 2.0 mL dose of the bivalent vaccine  at 21 days of age. Pigs in the UnVac group were injected intramuscularly with 2.0 mL of phosphate buffered saline  at 21 days of age. Pigs were comingled and randomly assigned into 48 pens within the same building. Each pen contained 10 pigs with a similar proportion of each treatment per pen. Pens were identical in design and equipment which included free access to a feed and water trough.To minimize sow variation, eight, 21 day-old pigs were randomly selected using the random number generator function  from each sow and assigned evenly (two pigs per sow) to each of the four groups. A total of 480 pigs was randomly divided into 4 groups  using the same software and function . The pigWhole blood, and fecal and laryngeal swabs were collected at 0 (21 days old), 28 (49 days old), 49 (70 days old), 91 (112 days old) days post-vaccination (dpv). Pigs were snared and restrained with a mouth gag for laryngeal swab collection. Swabs were guided with a laryngoscope down into the larynx. The internal walls of the laryngeal cartilages were then swept with the swabs once the larynx was visualized and the epiglottis was in a low position .The pigs were monitored daily for abnormal clinical signs and scored weekly using scores ranging from 0  to 6 (death) . ObserveThe live weight of each pig was measured at 0 (21 days old), 49 (70 days old), and 154 (175 days old) days post-vaccination. The average daily weight gain  was analyzed over two time periods: (i) between 21 and 70 days old and (ii) between 70 and 175 days old. ADWG during the different production stages was calculated as the difference between the starting and final weight divided by the duration of the stage. Data for dead pigs were included in the calculation.PCV2d strain  was isolated in inguinal lymph node from 68 day-old pig in submitted diagnostic case. The relatedness between vaccine sequences and field strain was analyzed by T cell epitope contents comparison (EpiCC) analysis as previously described [DNA was extracted from serum and fecal samples using the commercial kit  to quantify PCV2d genomic DNA copy numbers by real-time PCR .M. hyopneumoniae genomic DNA copy numbers by real-time PCR [DNA was extracted from laryngeal swabs using the commercial kit  to quantify the time PCR .M. hyopneumoniae  and PCV2 . Serum samples were considered positive for M. hyopneumoniae antibody if the sample-to-positive (S/P) ratio was ≥0.4, and positive for anti-PCV2 antibodies if the reciprocal ELISA titer was >350, in accordance with the manufacturer’s instructions for each kit.The serum samples were tested using the commercially available enzyme-linked immunosorbent assay (ELISA) kits for The severity of macroscopic lung lesions was scored to estimate the percentage of the lung affected by pneumonia. The scoring was done by two pathologists (Chae and one graduate student) at the Seoul National University . For the entire lung  . Two bli10 values. Statistical analyses were performed IBM SPSS Statistics for Windows version 23.0 . The Shapiro–Wilk test will be utilized to test the collected data for a normal distribution. One-way analysis of variance (ANOVA) was used to examine whether there are statistically significant differences at each time point within different groups. A one-way ANOVA test result with such a statistical significance was be further evaluated by conduction a post-hoc test for a pairwise comparison with Tukey’s adjustment. If the normality assumption was not met, the Kruskal–Wallis test was be performed. Results from Kruskal–Wallis test which showed statistical significance were further evaluated with the Mann–Whitney test to include Tukey’s adjustment to compare the differences among the groups. Results were reported in p-value where a value of p < 0.05 was considered to be significant.Prior to statistical analysis, real-time PCR data were transformed to logp < 0.05) in vaccinated animals  than those in unvaccinated animals (UnVac group) at 21 to 126 dpv. A comparison between vaccinated groups determined that respiratory signs, such as dyspnea and tachypnea in the VacA1 group were significantly lower (p < 0.05) than those in the VacB group at 63 and 98 dpv.Respiratory signs, such as dyspnea and tachypnea, were significantly lower (p < 0.05) than that of unvaccinated animals (UnVac group) during the fattening period (70 to 175 days of age) and overall period (21 to 175 days). In a comparison of vaccinated groups, the ADWG of the VacA1 group was significantly higher (p < 0.05) than that of the VacB group during the fattening period (70 to 175 days of age). The ADWG of the VacA1 and VacA2 groups was significantly higher (p < 0.05) than that of the VacB group during the overall period (21 to 175 days of age)  and unvaccinated (UnVac group) animals at the time the study began (21 days of age). The ADWG of vaccinated animals  was significantly higher ( of age) .M. hyopneumoniae in unvaccinated animals. In the VacA1 group, one pig died of unknown hemorrhagic diarrhea at 63 days of age. Two additional pigs from the VacA1 group died of bronchopneumonia as determined by a combination of M. hyopneumoniae as detected with PCR, and P. multocida that was isolated from the lungs at 72 and 75 days of age. In the VacA2 group, three pigs died of bronchopneumonia, as determined by a combination of PCV2d that was detected with PCR, and Glaesseralla parasuis that was isolated from the lungs at 52, 70, and 78 days of age. Three pigs in the VacB group died of bronchopneumonia, as determined by a combination of M. hyopneumoniae that was detected with PCR, and Trueperella pyogenes that was isolated from the lungs at 60, 80, and 82 days of age. Two additional VacB pigs died of bronchopneumonia, as determined by a combination of PCV2d that was detected with PCR and P. multocida that was isolated from the lungs at 72 days of age. In the UnVac group, one pigs died of salmonellosis, as determined by Salmonella typhimurium that was isolated from the large intestine at 52 days of age. Four UnVac group pigs died of bronchopneumonia, as determined by a combination of PCV2d and M. hyopneumoniae that were detected with PCR, and G. parasuis that was isolated from the lungs at 64, 75, 88, and 110 days of age. Three additional UnVac group pigs died of bronchopneumonia from a combination of M. hyopneumoniae that was detected with PCR, and P. multocida and T. pyogenes that were isolated from the lungs at 72 (2 pigs) and 80 days of age.Diagnostic results indicated that mortality was primarily related to co-infection with PCV2 and Shared EpiCC score was higher in the trivalent vaccine compared to the bivalent vaccine. T cell epitope coverage of bivalent vaccine against field PCV2 strain (SNUVR202002) was 62% and of trivalent vaccine against the same field PCV2d strain was 83%. This represented 33% improvement of an epitope coverage .p < 0.05) than that of unvaccinated animals (UnVac group) at 28, 49, and 91 dpv. PCV2d DNA loads in blood from the VacA1 and VacA2 groups were significantly lower (p < 0.05) than that of the VacB group at 49 dpv (p < 0.05) than that of unvaccinated animals (UnVac group) at 28, 49, and 91 dpv  were significantly lower  were significantly lower (p < 0.05) than that of unvaccinated animals (UnVac group) at 28, 49, and 91 dpv. In comparison of vaccinated groups, the amount of M. hyopneumoniae DNA loads in laryngeal swabs from the VacA1 group was significantly lower (p < 0.05) than that of the VacB group at 49 dpv (The amount of t 49 dpv .p < 0.05) PCV2 ELISA titers at 28, 49, and 91 dpv than that of unvaccinated animals from the UnVac group. In comparison of vaccinated groups, the PCV2 ELISA titers of the VacA1 and VacA2 groups was significantly higher (p < 0.05) than that of the VacB group at 49 and 91 dpv .Vaccinated animals from VacA1, VacA2, and VacB groups produced significantly higher (p < 0.05) M. hyopneumoniae ELISA S/P ratios at 28, 49, and 91 dpv than that of unvaccinated animal from the UnVac group. In comparison of vaccinated groups, the M. hyopneumoniae ELISA S/P ratios of the VacA1 and VacA2 groups was significantly higher (p < 0.05) than that of the VacB group at 91 dpv  macroscopic and microscopic lung lesion scores when compared to unvaccinated (UnVac) group at 154 dpv  groups had significantly lower  body weight by 1.245 kg/pig , leading to an increase of revenue by 2.98 US dollars  per pig.The present field trial is used to compare two different combination vaccines; a bivalent vaccine containing PCV2a and M. hyopneumoniae vaccine strains provide better protection against field PCV2d strain compared to bivalent vaccine containing PCV2a vaccine strain only. These results agree with previous EpiCC analysis, in which combination of PCV2a and PCV2b vaccine shared on average more T cell epitope content with strains from all the different genotypes than monovalent PCV2a vaccines [The improved growth performance of the trivalent-vaccinated groups over the bivalent-vaccinated group may be attributed to the different epitope determinant between vaccine and field PCV2 strain; T cell epitope coverage of bivalent vaccine against field PCV2d strain was 62% and of trivalent vaccine against the same field PCV2d strain is 83%. Therefore, trivalent PCV2a/b and vaccines . PCV2a-bvaccines ,10,11,12vaccines ,14,15. Ivaccines ,26,27. TM. hyopneumoniae antigen and adjuvant formulation differed between the two combination vaccines. In particular, adjuvant formulation is known to affect the immunogenicity and protective effect of inactivated whole-cell M. hyopneumoniae bacterins [M. hyopneumoniae load in larynx when compared to the bivalent vaccine. Although correlation between the reduction of M. hyopneumoniae in the larynx and vaccine protection is not well known, reducing the amount of M. hyopneumoniae loads in the larynx are more likely to reduce horizontal transmission to neighboring pigs.The strains of the acterins . TrivaleM. hyopneumoniae reduces pneumonic lung lesions in field trials [Regardless of vaccine type, vaccinated animals had a significantly greater reduction in mycoplasmal lung lesions compared to unvaccinated animals. These results are consistent with previous studies, where vaccination of pigs with d trials ,30,31. Ad trials .M. hyopneumoniae vaccine and trivalent PCV2a/b and M. hyopneumoniae vaccine. Broader coverage resulting from the PCV2 vaccine’s two genotypes provides additional insurance against the evolving PCV2 virus in the field. It is clinically meaningful to conduct comparative field clinical trial on farm with subclinical PCV2d infection and enzootic pneumonia.This is the first comparative field trial that evaluated the differences between a bivalent PCV2a and M. hyopneumoniae and a trivalent vaccine containing PCV2a/b and M. hyopneumoniae. Pigs vaccinated with the trivalent vaccine exhibited significantly better growth performance when compared with the bivalent vaccine. No statistical differences in growth performance were observed between one-dose and two-dose trivalent-vaccinated groups. The improved growth performance of the trivalent-vaccinated groups over the bivalent-vaccinated group may be attributed to the different epitope determinant between vaccine and field PCV2 strain; T cell epitope coverage of bivalent vaccine against field PCV2d strain is 62% and of trivalent vaccine against the same field PCV2d strain is 83%. It is clinically meaningful to conduct comparative field clinical trial on farm with PCV2d subclinical infection and enzootic pneumonia.This is the first comparative field trial that evaluated the differences between a bivalent and trivalent vaccine containing PCV2a and


Blast exposure can induce various types of hearing impairment, including permanent hearing loss, tinnitus, and hyperacusis. Herein, we conducted a detailed investigation of the cochlear pathophysiology in blast-induced hearing loss in mice using two blasts with different characteristics: a low-frequency dominant blast generated by a shock tube and a high-frequency dominant shock wave generated by laser irradiation (laser-induced shock wave). The pattern of sensorineural hearing loss (SNHL) was low-frequency- and high-frequency-dominant in response to the low- and high-frequency blasts, respectively. Pathological examination revealed that cochlear synaptopathy was the most frequent cochlear pathology after blast exposure, which involved synapse loss in the inner hair cells without hair cell loss, depending on the power spectrum of the blast. This pathological change completely reflected the physiological analysis of wave I amplitude using auditory brainstem responses. Stereociliary bundle disruption in the outer hair cells was also dependent on the blast’s power spectrum. Therefore, we demonstrated that the dominant frequency of the blast power spectrum was the principal factor determining the region of cochlear damage. We believe that the presenting models would be valuable both in blast research and the investigation of various types of hearing loss whose pathogenesis involves cochlear synaptopathy. Blast exposure causes various types of pathologies in the external, middle, and inner ear6. The most common feature of blast exposure is tympanic membrane perforation (TMP)8. Temporary and permanent hearing loss, tinnitus, and hyperacusis are also among the common signs of blast exposure10. Although permanent sensorineural hearing loss (SNHL) is currently untreatable, middle ear pathologies, including TMP, can be treated even though they require surgery. Moreover, permanent SNHL caused by blast exposure is often associated with a decline in the quality of life12. Therefore, blast-induced SNHL animal models have been established by various studies to investigate the detailed mechanisms of this pathology17. Conventionally, blast injury models generated using a blast tube, which consists of an air-compressed tube structure, have been used to study traumatic brain injury (TBI)14. However, since the blast intensity required to induce a permanent threshold shift (PTS) is relatively high, adjusting the intensity to induce a PTS without fatal TBI using the conventional chamber is a difficult task. Moreover, the blast intensity required to generate a PTS is usually strong enough to induce conductive hearing loss, such as TMP, which makes it impossible to accurately evaluate the inner ear functions using auditory brainstem responses (ABRs) or distortion-product otoacoustic emissions (DPOAEs). Multiple exposures to low-intensity blasts were also used in an attempt to generate pure SNHL without TMP17. However, a single exposure would be ideal to investigate the relationship between the property of the shock wave and the degree of inner ear damage.The necessity for research on blast-induced hearing impairment has escalated, as the ear is the most vulnerable organ in the event of blast exposure18. This model was originally used to create blast-induced mild traumatic injury (bmTBI) using single shock-wave irradiation; therefore, the intensity of the irradiated shock wave is relatively mild. We induced mild SNHL without TMP in our preliminary experiment. The other model of unilateral hearing loss and tinnitus was generated via irradiation with a laser-induced shock wave (LISW)19, which is our unique blast injury model of pure unilateral hearing loss and tinnitus following unilateral cochlear damage20. Furthermore, this model was reported to have good reproducibility for generating mild SNHL without TMP19. The advantage of these two models for hearing-related research is that they do not cause damage to the middle ear (including TMP), making it possible to investigate pure SNHL.A mouse model is useful for detailed pathological analyses because of its small size, cost-effectiveness, and availability of transgenic animals. Hence, we developed two different types of blast-induced SNHL mouse models. The first was created using a conventional shock tube, which consists of the driver (high-pressure) and driven sections separated by a diaphragm22, to facilitate the comparison of the effect of the two different blasts on the inner ear. We investigated the relationship between the site of inner ear dysfunction physiologically and anatomically and the peak pressure of the exposed shock wave.Using these two blast-induced SNHL mouse models, this study aimed to analyze the relationship between the property of the blast shock wave and the pathophysiological effect on the inner ear after blast exposure. Originally, we utilized the LISW model as blast exposure with ear protection, which is a common situation for soldiers on the battlefield, as LISW propagates into the inner ear via bone conduction, a condition similar to blast exposure with ear protection. The shock tube approach models a mix of air- and bone-conduction exposure, similar to human blast exposures without hearing protection. The intensities of shock wave were adjusted to induce almost the same severity of damage as that at 16 kHz, a low-hearing-threshold frequency in miceWe used two different types of blast-induced hearing loss mouse models in the present study. The waveform of the shock wave generated by the shock-tube system used in this study showed a steep rise in the positive peak with 25.0 kPa Fig. e. The duMoreover, we calculated the frequency spectra from the observed pressure waveforms using fast Fourier transform. The frequency of the median root mean squared (RMS) amplitude of the shock tube-induced shock wave was 976.5 Hz, whereas the frequency of the median RMS amplitude of the LISW was 448 kHz (500 times higher than that of the shock tube induced shock wave). Moreover, the LISW consisted of a substantially higher range of frequencies compared to the shock tube induced shock wave.We observed the tympanic membrane in all cases immediately after blast exposure before conducting the hearing assessment. We did not observe TMP in the mice after blast exposure in any group , which serves as a biological motor for amplifying the motion of the sensory epithelium24. The shock-tube group exhibited a remarkable elevation in the ABR threshold one day after the shock wave exposure at all tested frequencies, but the ABR threshold was not significantly elevated 28 days after exposure compared to that at pre-exposure, although the thresholds 28 days after exposure at all frequencies were larger than the pre-exposure thresholds   represents the summed activity of the cochlear nerve4, 120 = 14.86, p < 0.001; Bonferroni multiple comparison significance at 22.65 kHz [p = 0.025], at 32.00 kHz [p = 0.040], respectively)    , and fewer in the LISW group at a higher frequency (Bonferroni multiple comparison significance at 32.0 kHz [p = 0.042]): these trends were similar to those of the ABR wave I amplitude. We also counted the number of orphan ribbons, i.e., synapse structures lacking apposed glutamate receptor patches (green: anti-GluA2), in the blast-exposed ears .Subsequently, we focused on the synapses between the IHCs and cochlear nerve fibers. Synaptic ribbons are a characteristic structure of hair cell afferent synapses that are involved in vesicle delivery to the active zoneWe further analyzed the SGCs 28 days after blast exposure using frozen cross-sections. The number of SGCs in the blast-exposed ear did not change in any cochlear region in any group , and in the entire cochlear region in response to the shock tube (low-frequency dominant shock wave). The peak forces potentially affect the high-frequency dominant cochlear damage especially in the LISW group. However, by using a stronger LISW than that in the present study, broader damage toward the cochlear apex was introduced as reported previously29. This previous report also indicates that the blast delivery method would be less involved in this frequency distribution. Therefore, we strongly suggest that the frequency-distribution pattern of the exposed blast would be the biggest factor to determine the distribution of cochlear damage.Our analysis of the longitudinal pattern of cochlear damage after blast exposure clearly showed that the degree of the inner ear dysfunction measured using the ratio of ABR changes was directly associated with the frequency distribution of the power spectrum of the exposed shockwave, irrespective of the shock wave generator. The primary site of cochlear damage is determined by the frequency spectrum of the exposed noise in noise-induced hearing loss24. However, loss of OHC was not observed in both groups in this research, whereas only a disruption of the OHC stereociliary bundle was observed, which could be the reason why the differences in DPOAE elevation could not be detected.Conversely, although a clear relationship was observed between the physiological (obtained with the ABR) and histological findings, the trend in the DPOAE after blast exposure was not clearly commensurate with the histological damage in the OHC at lower frequencies. The DPOAE can directly measure OHC function, which acts as a biological motor for amplifying the motion of the sensory epithelium30. This type of hearing dysfunction is described as cochlear synaptopathy31, which is revealed to be closely related to the pathogenesis of tinnitus32 and hyperacusis33. Conventionally, animal models of acute cochlear synaptopathy are created by noise exposure34; however, the damaged area is generally limited to the cochlear basal turn. By using different shock waves in the current study, we could easily induce synaptopathy in the whole cochlear area. Cochlear synaptopathy has recently been discovered in various types of SNHL, including age-related hearing loss and congenital/genetic hearing loss36. Therefore, we believe that our blast-induced hearing loss models would be valuable both in blast research and for the investigation of various types of hearing loss, whose pathogenesis involves cochlear synaptopathy. However, it needs to be mentioned that our presenting models did not purely constitute cochlear synaptopathy, as the relationship between the ABR amplitude and number of synapses is not correlated when threshold shift or HC damage occurs.We confirmed the degeneration of the synapses in the IHCs and disruption of the stereociliary bundle in the OHCs, whereas there was no reduction in the number of the hair cells in the spiral ganglion after the blast exposure used in this research. The reduction in the synapses without hair cell loss is compatible with the reduction in the ABR wave I amplitude after LISW exposure because synaptic ribbon counts in normal ears provide an accurate metric of the afferent innervation of the IHCs19. We used confocal microscopic analysis using immunohistochemistry for the stereociliary bundle in addition to the conventional SEM in this study, as it is difficult to eliminate the artifacts in the bundle structures arising from their deformation by the vapor deposition process38. Therefore, we obtained the same result with both confocal scanning and SEM, and we believe that we were able to count the exact ratio of the disrupted bundles. We strongly suggest that the stereociliary bundle disruption was the main pathology, which caused an ABR threshold shift without any hair cell loss. However, various ratios of the disrupted stereociliary bundle were observed in the cochlear apical part even in the control group. The OHC hair bundle at the apical part is structurally wider and larger than that at the basal turn39; therefore, the stereociliary bundle at the apical turn is more fragile. This is the most plausible reason for the lack of a significant difference between the ratios of the disrupted bundles of the shock-tube and LISW groups, especially in the lower-frequency regions.The stereociliary disturbance without hair cell loss observed in this study is a unique characteristic of blast-induced hearing dysfunction, which was also observed in our previous study using LISW29 and SGCs19 could be induced by using more intense shock waves generated by the shock tube or LISW. We could easily control the intensity of the shock wave (shock tube or LISW) using our shock-wave generator system. This adjustability of the shock-wave generator is an advantage of our system.In this study, we did not observe the loss of the IHCs, OHCs, and SGCs in any group. The hair and ganglion cells may be more resilient to shock waves compared to the synapses or stereociliary bundles. Indeed, the loss of both types of hair cells40. However, the mouse is the most widely used experimental animal due to its size, cost-effectiveness, and availability of transgenic variants. The physiological findings from LISW seem to be similar to hearing impairment involving a high-frequency hearing loss in humans caused by blast exposure that is encountered in clinical situations (based on the comparisons of the two shock-wave groups)10. The shock wave generated by our shock-tube chamber is low frequency-dominant for mice owing to the size of the cochlea (whereas it lies in the middle audible range for humans or chinchillas). From this perspective, LISW would be a suitable stimulation to generate mouse models of high-frequency blast-induced hearing loss, akin to the pathological state in humans.The chinchilla model of hearing loss has been widely used for a long time, partially due to its human-like hearing range, low-impedance tympanic membrane, and large ear canal41. Recent research has revealed that tympanic membrane rupture could limit the damage to the inner ear by absorbing a part of the shock wave’s energy15. Our LISW model could not induce TMP, because the shock wave (even the high-intensity ones) was irradiated directly to the inner ear29. A high-intensity blast generated using a shock tube would be the ideal shock-wave generator to analyze mixed hearing loss with TMP and SNHL. It is possible to choose the type of blast depending on the use or aim of the research. One of the limitations of this research is that the total energy of exposed blasts induced by different generators was not standardized. In this research, we prioritized the degree of damage at 16 kHz, a low-hearing-threshold frequency in mice, to facilitate the comparison of the effect of the two different blasts on the inner ear. To compare these two different blast delivery settings, standardizing the total energy of blast pressure would be needed.In this study, we created a blast-exposed mouse model without TMP because we aimed to analyze the pure sensorineural component of hearing loss. However, TMP is one of the most frequently observed symptoms in patients exposed to real explosions42. Recent studies have revealed that neurotrophin-343 and Rho-associated coiled-coil containing protein kinase inhibitor44 confer protective or regenerative effects against the loss of ribbon synapses after inner ear damage. Moreover, the disruption of the stereociliary bundle is also an important area of focus for SNHL, including noise-induced hearing loss. Forced expression of the Atoh1 gene reportedly has a regenerative function after the loss of stereociliary bundles produced by noise-induced hearing loss45. Our multiple blast-induced hearing loss models would be ideal platforms for research on various types of SNHL.The development of therapeutic strategies for blast-induced hearing loss is an urgent issue. The treatment modalities for blast-induced hearing loss involving cochlear synaptopathy or stereociliary bundle disruption can be applied to other types of SNHL with the same underlying pathology. As mentioned above, our blast-induced hearing loss models also represented cochlear synaptopathy, which is a good target for SNHL treatmenthttp://www.nc3rs.org.uk/page.asp?id=1357). All efforts were made to minimize the number and suffering of the animals.Thirty CBA/J mice  weighing 17–20 g were purchased from Charles River Laboratories . They were given free access to water, a regular diet, and were individually housed and maintained at 23–25 °C. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical College and conducted per the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science and Technology of Japan . All experiments were performed per the ARRIVE guidelines , divided by a polyester diaphragm into high-and-low pressure parts  Fig. . a, b. C19: a laser target was irradiated with a 532-nm Q-switched neodymium-doped yttrium aluminum garnet (Nd: YAG) laser   to examine the TMP on both ears.https://www.masseyeandear.org/research/otolaryngology/eaton-peabody-laboratories/engineering-core). The sound level was raised in increments of 5 dB from ≥ 10 dB below the threshold to < 80 dB sound pressure level (SPL). At each sound level, 1024 responses were averaged with alternated stimulus polarity. The “ABR threshold” was defined as the lowest SPL level at which any wave could be detected on visual inspection of the tacked waveforms, usually corresponding to the level just below that at which the peak-to-peak response amplitude rose significantly above the noise floor. When no response was observed at the highest available sound level, the threshold was designated as being 5 dB greater, to allow statistical tests. For amplitude versus level functions, the wave I peak was identified by visual inspection at each sound level and the peak-to-peak amplitude was computed.Cochlear function tests were conducted for each animal at 6 log-spaced frequencies  before and 1, 7, and 28 days after blast exposure. The mice were anesthetized with ketamine  and medetomidine hydrochloride . Needle electrodes were inserted at the vertex and pinna to detect the ABRs, with a ground electrode placed near the tail. The ABRs were evoked using 5 ms tone pips . The response was amplified, filtered, and averaged using a LabVIEW-driven data-acquisition system  was 1.2 and the f2 level was 10 dB below the f1 level. For each f2/f1 primary pair, the levels were swept in increments of 5 dB from 20 to 80 dB SPL (for f2). Waveform and spectral averaging were used at each level to increase the signal-to-noise ratio of the recorded ear-canal sound pressure, and the amplitude of the DPOAE at 2f1–f2 was extracted from the averaged spectra along with the noise floor at the spectrum’s adjacent points. The threshold was defined as the f1 level required to produce a DPOAE at 0 dB SPL.All histological examinations were conducted after the final ABR assessment (28 days after blast exposure). For whole-mount preparation, the mice were transcardially perfused with heparinized phosphate buffer saline (PBS) followed by 50 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) at room temperature (20 °C–25 °C) under general anesthesia. After decapitation, the cochleae were quickly dissected and small openings were created at the round and oval windows, and the cochlea apex, which was then bathed overnight in 4% PFA in 0.1 M PB at 4 °C. After decalcification with 0.5 M ethylenediaminetetraacetic acid (EDTA) in PBS  for 4 days at 4 °C with shaking, each cochlea was microdissected into six pieces for whole-mount preparation.15, initiated by blocking the tissues for 1 h with 0.1% Triton X-100 in PBS supplemented with 5% normal goat serum to detect the hair cells and synapses. The fixed and permeabilized pieces were incubated overnight at 37 °C with a rabbit polyclonal antibody to mouse (IgG1) anti-CtBP2 at a 1:200 titer , mouse (IgG2a) anti-GluR2 antibody at 1:2000  and rabbit anti-Myosin 7a at 1:200 (#25-6790 Proteus Biosciences). The cochlear pieces were washed, and the following secondary antibodies were incubated for 2 h at 37 °C: Alexa Fluor 488-conjugated goat anti-mouse (IgG2a) at 1:1000 , Alexa Fluor 568-conjugated goat anti-mouse (IgG1) at 1:1000 , and Alexa Fluor 647-conjugated chicken anti-rabbit at 1:200 . All primary and secondary antibodies were diluted in 1% normal horse serum with 0.3% Triton X-100. The immunostaining protocol for stereocilia analysis was also based on a previous study15. The tissue was permeabilized by freezing on dry ice, incubated for 1 h at room temperature in a blocking solution with 5% normal donkey serum in PBS and 0.3% Triton X-100 for 2 h at room temperature. The primary antibody, rabbit anti-espin 1:100 , was incubated overnight at 37℃, and the secondary antibody, i.e., biotinylated donkey anti-rabbit FAB fragment 1:400 (Jackson ImmunoResearch #711-067-003), was incubated for 2 h at 37 °C. Following tertiary labeling, streptavidin-conjugated Alexa Flour 568 1:200 (Jackson ImmunoResearch #S-11226) was incubated for 1 h at room temperature. All antibodies were diluted in 1% normal donkey serum with 0.3% Triton X-100.The immunostaining protocol was performed according to the previous studieshttp://www.masseyeandear.org/research/otolaryngology/investigators/laboratories/eaton-peabody-laboratories/epl-histology-resources/imagej-plugin-for-cochlear-frequency-mapping-in-whole-mounts) were used to measure the total length of the cochlear whole mounts and the length of the individual segments.The specimens were washed thrice with PBS and mounted on slides containing an antifade medium  and viewed under a fluorescence microscope  or confocal fluorescence microscope . The cochlear lengths were measured for each animal, and a cochlear frequency map was computed to precisely localize the IHCs in the 5.6, 8, 11.3, 16, 22.6, and 32 kHz regions, respectively. PhotoShop CC , ImageJ software , and ImageJ Plugin  and embedded in an optimal cutting temperature compound for cryosectioning. Serial frozen sections of 10 µm were cut along a horizontal plane parallel to the cochlear modiolus, stained with hematoxylin and eosin, and viewed under a microscope (BZ-X700).4 at 4 °C for 30 min, dehydrated in ethanol, critical point dried with liquid CO2, sputter-coated with osmium, and examined under an electron microscope  operated at 5.0 kV.The specimens for scanning electron microscopy (SEM) were obtained thusly: the mice were transcardially perfused with 0.01 M PB (pH = 7.4) containing 8.6% sucrose under general anesthesia, followed by fixation with freshly depolymerized 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB (pH = 7.4) containing 5% sucrose. After decapitation, the cochleae were quickly removed, creating small openings in the round and oval windows, and apex of the cochlea, which were bathed overnight in the same fixative at 4 °C. After decalcification with 0.5 M EDTA in PBS [Decalcifying Solution B (EDTA method); Wako Pure Chemical Industries Ltd.) for 4 days at 4 °C with shaking, each cochlea was microdissected into six pieces for whole-mount preparation. The tissues were subsequently fixed with 1% OsOOHCs and IHCs were counted using confocal microscopy conducted at 5.6, 8, 11.3, 16, 22.6, and 32 kHz, while focusing on the presynaptic ribbons in the basolateral portion of the IHCs; an oil-immersion 100 × or water-immersion 60 × objective and a 0.2-μm z-step were used. Z-stacks were acquired at three adjacent areas for each frequency region in each cochlea: each stack contained approximately 10 IHCs per row. The number of OHCs and IHCs per 200 µm was counted at each point described above. The densities of the OHCs and IHCs per 200 µm were calculated and compared at each site.The numbers of synaptic ribbons in the IHCs (CtBP2-positive puncta) and glutamate-receptor patches (GluA2 puncta) per 200 µm were counted at 5.6, 8, 11.3, 16, 22.6, and 32 kHz. The number of synaptic ribbons and glutamate-receptor patches per IHC was calculated and compared at each site. The counts were performed by three different blinded individuals.The number of spiral ganglion cells (SGCs) in the basal, middle, and apical-middle turns of the cochleae was counted. We calculated the average SGC density obtained from the following three sections: the section considered as the center of each frequency site and 20 µm before and after the center section.Quantitative analysis of stereociliary bundle disruption in the OHCs was conducted by calculating the ratio of the disrupted stereocilia  from each energy group (n = 5 for each group) using the confocal stereocilial immunohistochemistry images. The number of stereocilia per 100 μm was counted at the center of the 5.6, 8, 11.3, 16, 22.6, and 32-kHz areas. One or more rows of OHC bundles that were bent toward the lateral side, tangled, or lacking their base were designated as “disrupted.” The counts were performed by three different individuals who were blinded to the experimental groups to minimize bias.t-test. p-values of < 0.05 were considered statistically significant. The standard error (SE) of the mean was represented with error bars.Statistical analyses were performed using Prism 5 . All data values for the thresholds and amplitude of hearing function tests, and histological analysis were compared using the two-way ANOVA, followed by Bonferroni correction for multiple comparisons. The data values for the threshold shift and amplitude ratio were compared using a 2-tailed, unpaired Student’s Supplementary Information.


Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein,  Retinal ganglion cells (RGCs) are the only neurons in the retina that convey visual information to the brain, doing so via the optic nerve. Major causes of optic nerve damage and subsequent RGC death are ocular trauma and optic neuropathies. At the end-stage pathology in the retina, remaining RGCs have high neuroprotective capacity, and active resilience mechanisms exist. In contrast, in this study, we demonstrate neurodegenerative mechanisms based on the early susceptibility of peripheral ventrotemporal (VT) RGCs to degeneration after optic nerve injury. We identified the serotonin transporter (SERT) as the factor mediating VT RGC vulnerability and axon regeneration. Loss of SERT affected activation of integrin β3 and gene expression in the peripheral VT retina, leading to peripheral VT RGC protection and axon regeneration. Interestingly, GPNMB, a newly identified molecule mediating neuroprotection and axon regeneration in this study, is expressed in all retinal regions. However, GPNMB expression is acutely downregulated by the presence of SERT in the peripheral VT retina, leading to regional differential vulnerability of RGCs. These findings provide new molecular mechanisms underlying selective RGC vulnerability and clues that could inform further neuroprotective and regenerative strategies. Mature retinal ganglion cell (RGC) axons that comprise the optic nerve degenerate, RGCs die and blindness ensues after ocular trauma or during optic neuropathies such as glaucoma. Ocular trauma, such as facial fractures, leads to 67% of patients who have ocular injuries, of which 4% present with optic nerve abnormalities . RetinalA number of studies have described intrinsic and extrinsic molecules and signaling pathways that mediate RGC death or survival pathways as well as axon regeneration after optic nerve crush (ONC) –9. TargeSlc6a4), is highly expressed in VT RGCs during retinal development and at early postnatal stages [Axonal connections from RGCs in specific retinal regions are required for the formation of visual maps . In micel stages –28. SERTl stages . HoweverGpnmb, which results in reduced VT RGC death and enhanced axonal regeneration. Taken together, this study identifies a novel molecular axis underlying selective regional vulnerability of RGCs after ocular trauma.Utilizing a well-established optic nerve crush (ONC) mouse model, first we characterized regional differential RGC vulnerability at the early time points of axon degeneration. We demonstrate that the peripheral VT retina in mouse, the region where ipsilaterally-projecting RGCs are located, is more susceptible to degeneration after ONC compared to other retinal regions. To understand the molecular mechanisms underlying this susceptibility, we focused on SERT, which is upregulated on VT axons after ONC, and demonstrate that it functions in peripheral VT RGC death and impaired axon regeneration. Furthermore, loss of SERT in the peripheral VT retina attenuates the activation of integrin β3 and affects molecular signatures in the peripheral VT retina including + cells in the VT RGC layer was detected three days after ONC when compared to the DT retina  . Thus, wT retina . Next, wl retina . Thus, pSlc6a4::Cre;R26RZsGreen mouse (serotonin transporter (SERT), encoded by Slc6a4), and visualized ipsilateral RGCs showing ZsGreen-SERT expression as we previously utilized [Slc6a4::Cre;R26RZsGreen mice (+/βIII-tubulin+) from the peripheral edge (0 μm) to the central region (600 μm) in the uninjured adult VT retina  began to die three days after ONC and only 39.6% of those RGCs survived five days after ONC (+/βIII-tubulin+) and contralaterally-projecting RGCs (ZsGreen-SERT-negative/βIII-tubulin+) within the peripheral VT retina four days after ONC  expression was upregulated on VT RGC axons within one day after ONC, and its expression remained high three days after ONC Slc6a4::CSlc6a4-/- constitutive mutant mice [Slc6a4-/- mice was similar to that in WT  and two weeks (late RGC degeneration) after ONC in ant mice  Fig 3A)Slc6a4-/-at in WT . Five dasurvived . In othee extent . Two weesurvived . In contal rates . Next, wfter ONC . In the o injury . Loss ofwo weeks . We founwo weeks . Howeverne cells . Thus, STo explore the molecular mechanisms of SERT-mediated VT RGC death after ONC, we focused on integrin β3, which binds to SERT and regulates SERT-induced functions in platelets and the nervous system ,34. FirsItgb3 in most RGCs of the peripheral VT retina, AAV2-GFP-Cre virus was intravitreally injected into the peripheral ventral retina of WT or of Itgb3flox/flox mice (Itgb3flox/flox retina expressing AAV2-GFP-Cre (referred to as Itgb3-/- retina) was compared to WT retina expressing AAV2-GFP-Cre (referred to as WT retina). First, we confirmed that GFP-Cre expression was detected in ~95% of RGCs in the peripheral VT retina  VT RGC death after ONC, Slc6a4::Cre;Itgb3flox/flox mice were generated  survived. In fact, integrin β3 was also upregulated in dorsal RGCs after ONC where SERT is not expressed  compared to the intact retina (3.58%)  . Howeveregrin β3 . Thus, SSlc6a4-/- mutant mice and Itgb3flox/flox mice with AAV2-GFP-Cre virus infected into the peripheral VT retina, regenerated axons were labeled with the anterograde axonal tracer Alexa Fluor 555-conjugated cholera toxin subunit B (CTB) and fixed two days later as we previously described [ClearT and ScaleS [+ axons within the optic nerve were visualized by confocal microscopy. In Itgb3flox/flox retina expressing AAV2-GFP-Cre in the VT retina (referred to as Itgb3-/-) and Slc6a4-/- retina two weeks after ONC, robust axon regeneration was detected when compared to WT  from the VT retina of WT and Slc6a4-/- mice one day after ONC  expression is completely eliminated in the injured VT retina of Slc6a4-/- mice  [Gpnmb was chosen as a top candidate, and we analyzed whether GPNMB is a regulator of RGC protection and axon regeneration after ONC. However, expression and functions of GPNMB in the retina, especially RGCs remain unclear. First, we investigated expression of GPNMB in RGCs and found that GPNMB is expressed in >95% of RGCs in the VT retina and DN retina  , Parkinsis (ALS)  and afteis (ALS) . Moreoveis (ALS) ,56. GPNMis (ALS) . NeurotoN retina . In the fter ONC . Thus, lSlc6a4 (SERT) via antisense Slc6a4 sequences under CMV promoter (referred to as AAV2-shSlc6a4) and control sequences (referred to as AAV2-shCtr) were generated, and we utilized DBA/2J mouse carrying mutations in Gpnmb that result in a truncated and dysfunctional GPNMB protein, or the DBA/2J-Gpnmb+ mouse, carrying wild-type Gpnmb, as a control [Gpnmb+ mice  was generated, and intravitreally injected into the peripheral ventral retina  RGCs to degeneration after ONC. We found that neurodegenerative and regeneration-inhibitory signals conveyed from the optic nerve to VT RGCs after ONC are mediated by SERT-integrin β3 molecular axis, which acutely reduces GPNMB expression. Taken together, our study identifies a novel molecular mechanism mediating the region-specific neuronal vulnerability of RGCs to acute injury.Slc6a4-/- mice has shown that SERT mediates RGC vulnerability and axon regeneration in the VT retina. Several studies have shown that local translation of specific molecules on axons after nerve injury and in neurodegenerative diseases leads to regulation of neuronal survival/death and axon regeneration [Peripheral VT RGCs possess anatomically and molecularly distinct features compared to RGCs in other regions of the mouse retina . In mousneration ,61. Thusitgb3  in the injured RGCs after optic nerve injury when compared to intact RGCs [itgb3 after injury [Slc6a4-/- mice. It is intriguing that integrin β3 effects seem to be specific to VT RGCs. It is known that activated integrin β3 enhances SERT-dependent platelet functions [Intriguingly, integrin β3, a molecule mediating SERT-induced functions in other systems ,34, is aact RGCs . Howeverr injury ,63, whilr injury –66. Sincunctions . Thus, iSlc6a4-/- VT retina after ONC could be triggered by (i) reduced serotonin reuptake and/or (ii) binding of excess extracellular free 5HT to serotonin receptors expressed in VT RGCs. The dose  of fluoxetine was utilized in this study since it has been reported to show robust effects on stress-related behavior [Slc6a4-/- mice showed these effects  loss of SERT and integrin β3 led to VT RGC protection and axon regeneration, (iii) a downstream molecule, GPNMB is normally downregulated in WT VT RGCs after ONC, but not in Itgb3-/- or Slc6a4-/- RGCs, and (iv) GPNMB promotes neuroprotection and axon regeneration in the VT retina as observed in Itgb3-/- or Slc6a4-/- VT RGCs. Taken together, SERT and integrin β3 could play a critical role in mediating degenerative and regenerative-inhibitory signal transduction after ONC. Further studies are needed to identify ligands mediating downstream signals via integrin β3-SERT after ONC, leading to development of new therapeutic strategies after ocular trauma.How does SERT mediate degenerative and regeneration-inhibitory signals? A precedent has shown that SERT binds to platelet-derived growth factor receptor (PDGFR), mediates phosphorylation of PDGFR and serves as a modulator that facilitates transmission of signals from PDGFR to its associated signaling molecules upon the presence of its ligand, PDGF . This fuDynlt1b , which was upregulated in the Slc6a4-/- VT retina, has been reported to enhance axon outgrowth by modulating Rho signaling pathways and actin/microtubule dynamics [Nell1 and Srp54, which were downregulated in the Slc6a4-/- VT retina, are involved in cell death in a variety of cellular contexts [Chac1 [JunD [Socs3 [Rab3c [Slc6a4-/- mice compared to WT mice  is normally expressed in the pigment cells such as the RPE, and the melanosomes in Pmel−/− RPE showed abnormal melanin aggregates and poorly preserved membranes, suggesting PMEL maintains normal pigment production in the RPE [Slc6a4-/- VT retina after optic nerve injury could facilitate pigment production and maintain RPE integrity, resulting in altered expression of signaling molecules that may affect neuroprotection and axon regeneration via specific molecular interactions between the RPE and VT RGCs.Our RNA-seq data show that  the RPE . Intrigu the RPE . Moreove the RPE . Thus, eThe magnitude of RGC vulnerability after optic nerve injury and in ocular diseases varies depending on the RGC subtypes –14,16. ASlc6a4-/- mice after ONC compared to WT retina such as Col25a1 (1.16-fold increase) a marker of D- and V-ooDSGCs [Ebf3 (1.17-fold decrease), a marker of F-RGCs [Tac1 (1.38-fold increase), Crhbp (1.96-fold increase) and Gabra2 (1.95-fold increase) are also affected in the VT retina of Slc6a4-/- mice after ONC .Slc6a4-/- mice , Itgb3flox/flox mice , B6.FVB(Cg)-Tg(Slc6a4-cre)ET33Gsat/Mmucd , B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J mice , DBA/2J mice  and DBA/2J-Gpnmb+/sjJ mice  were purchased and used for experiments. In particular, Slc6a4::Cre;Itgb3flox/flox mice and Slc6a4::Cre;Itgb3+/+ mice as control were generated by intercrossing Slc6a4::Cre;Itgb3flox/+ mice. Slc6a4::Cre;R26RZsGreen mice were generated by crossing Slc6a4::Cre mice with R26RZsGreen mice. All mice were housed in a pathogen-free barrier facility. Mice were maintained in a 12 h light/dark cycle with access to standard laboratory chow and water ad libitum. Both sexes were used for all experiments.Animals used are described as follows: C57BL/6J mice , 8-10-week-old animals were anesthetized with ketamine/xylazine. To expose the optic nerve, the dural sheath surrounding the optic nerve in the left eye was carefully incised. The optic nerve in the left eye was then crushed using #5 Dumont forceps, applying pressure for 5s ~1 mm behind the optic disc. The right eye was intact (uninjured) retina. Following various time points after optic nerve crush, animals were humanely euthanized, perfused with 4% paraformaldehyde (PFA) following PBS, and the head was cut and post-fixed in 4% PFA overnight at 4°C and washed with PBS for further analysis. For analysis of effects of fluoxetine, fluoxetine hydrochloride was purchased from Sigma-Aldrich (#1279804), prepared fresh daily and given by intraperitoneal injection (10mg/kg) one day before ONC and daily after ONC for two weeks as previously utilized for analysis of retinal axon projection ,76. TimeCre virus , AAV2-CMV-GFP-shSlc6a4 virus (target sequences: GCCCTCTGTTTCTCCTGTTCA) [Gpnmb-IRES-eGFP  was slowly intravitreally injected into the peripheral ventral or dorsal retina of 6-week-old WT or transgenic mice.For intravitreal injection of all materials described below, glass micropipettes made by a micropipette puller was used. For axon regeneration assay, 2 μl of 2 μg/μl of Alexa Fluor 555 conjugated cholera toxin β subunit (CTB)  were injected into the central retina 2 days before euthanasia. 1 μl of AAV2-CMV-GFP-CTGTTCA)   in the whole mount retina, the retina was incubated in cold methanol for 10 mins, and PBS was used for washing and during further immunostaining processes. Immunolabeling was performed with the following primary antibodies: mouse βIII-tubulin , guinea pig RBPMS , chick GFP , goat osteopontin (OPN) , rabbit SERT , mouse phosphorylated-integrin β3 (pY759.7A) , mouse integrin β3 , rabbit neurofilament L , goat GPNMB  and mouse Iba1. Secondary antibodies were used at 1:500 and purchased from Lifetechnology: donkey anti-Rabbit IgG 647 , goat anti-Mouse IgG 488 , goat anti-Rabbit IgG 594 , donkey anti-Mouse IgG 647 , goat anti-Guinea Pig IgG 647 , goat anti-Guinea Pig IgG 488 , donkey anti-Goat IgG 488 , goat anti-Chick IgG 488 , donkey anti-Mouse IgG 594 . DAPI was used at 1:500 and purchased from ThermoFisher . ZsGreen+ VT RGCs and axons were detected with intrinsic fluorescence of ZsGreen protein without the antibody.For whole mount immunofluorescent staining of the injured and intact retina, after removal of the cornea, lens and retinal pigment epithelium, the ventral retina was cut with microscissors, creating a small incision as a marker for orienting the retina. The injured and intact retina were dissected out from the left and right retina, respectively and incubated in different tubes for 1 hour at 4°C in PBS containing 0.4% Triton X-100 (PBST). Each retina was incubated with primary antibodies in PBST overnight at 4°C and washed with PBST three times. The retina was incubated with secondary antibodies in PBST overnight at 4°C and washed with PBST three times. The dorsal, nasal and temporal regions in the intact and injured retina were also cut with micro scalpels on the microscope slide and mounted with Fluoro-Gel mounting medium . For cryosectioning, the retina or optic nerve was incubated in 30% sucrose PBS for 48 h at 4°C, and then embedded with Tissue-Tek O.C.T. and 14 μm retinal or optic nerve cryosections were prepared and immunostained described above. For immunostaining with integrin β3 and SERT  antibodies, PBS was used instead of PBST during the immunostaining process and sections were incubated with the primary antibodies overnight at room temperature and with the secondary antibodies for 4 hours at room temperature. We also tested goat SERT  antibody in Whole optic nerve labeled with CTB, or whole retina or cryosections after immunostaining were imaged on Olympus Fluoview 1000 laser scanning microscope with Fluoview software, a 20× objective lens , a 40× objective lens (UPlanFL N NA = 1.3) or a 60x objective lens (PlanApo NA = 1.42).ClearT [leS [ClearT/ScaleS clearing solutions are composed of formamide , methyl-β-cyclodextrin , γ-cyclodextrin , D-(-) Sorbitol , and Triton X-100 . Stock solution I (10 mM methyl-β-cyclodextrin and 10 mM γ-cyclodextrin in ddH2O) and stock solution II (20% D-(-) Sorbitol and 0.2% Triton X-100 in ddH2O) were prepared and stored at room temperature. Fixed CTB 555-labeled optic nerve was incubated in the 1.5ml tube containing 50 μl of the stock solution I, 250 μl of the stock solution II and 200 μl of PBS at room temperature overnight. The optic nerve was then incubated in the tube containing 200 μl of formamide and 300 μl of PBS at room temperature for two hours, and in the tube containing 200 μl of formamide and 300 μl of ddH2O at room temperature for two hours. Finally, the optic nerve was incubated in 500 μl of formamide for 30 mins—one hour at 42°C, and mounted on the microscope slide with the cover glass and ~100 μl of formamide. 1 hour after mounting, CTB-labeled regenerated retinal axons were imaged on the confocal microscope with a 20x objective lens and the whole optic nerve imaging was taken in ~80 images, 2 μm steps. Each image was stitched and the whole optic nerve image was acquired.Tissue clearing reagents based on the combinations of previously described clearing methods, ClearT  and ScalarT [leS  were pre+ or RBPMS+ RGCs in a 400 μm x 600 μm area in four quadrants, ventrotemporal (VT), ventronasal (VN), dorsotemporal (DT) and dorsonasal (DN) of the peripheral retina, the closest region to the peripheral edge as described in RGC survival analysis in the whole mount retina was performed by counting the number of βIII-tubulin+ (OPN+) RGCs (%) shown in +/βIII-tubulin+ number by the βIII-tubulin+ number in a 400 μm x 600 μm area of each quadrant of the intact naïve peripheral retina each animal.OsteopontinSlc6a4-Cre-derived ZsGreen expression in intraretinal axons in the VT retina, the pixel intensity of ZsGreen+ axons in a 400 μm x 400 μm area in the VT retina adjacent to the optic disc as shown in For quantification of + optic nerve in a 200 μm-optic nerve column adjacent to the lesion site in injured optic nerve or at the same location in the intact optic nerve was measured with ImageJ software. Fold change of integrin β3 expression in the optic nerve after injury was obtained by dividing the pixel intensity in the injured optic nerve by that in the intact nerve from the same animal. The analysis was performed on two non-consecutive cryosections per animal and the average of fold change was obtained per animal. For counting integrin β3+ RGCs in the VT retina, RBPMS+ RGCs showing bright integrin β3+ fluorescent signal intensity were counted after evaluating the threshold level compared to the background. The analysis of integrin β3+ RGCs was performed on two non-consecutive cryosections per animal and the average of the number of those RGCs was obtained per animal. Phosphorylated-integrin β3+ cells in the RGC layer of the whole mount retina were counted in a 200 μm x 200 μm area in the VT intact or injured retina of WT and Slc6a4-/- mice after evaluating the threshold level compared to the background, and the total number of phosphorylated-integrin β3+ cells was obtained per animal.For integrin β3 expression in the retinal and/or optic nerve cryosections, 14 μm retinal or optic nerve cryosections were prepared and immunostained with anti-integrin β3 antibody and the secondary antibody and images of injured and intact retina and/or optic nerve were taken with the same setting of the inverted confocal microscope. For quantification of integrin β3 expression in the injured or intact optic nerve cryosections, the pixel intensity of integrin β3+ cells in the whole mount retina were counted in a 300 μm x 300 μm area in the VT or DN intact or injured retina and GPNMB+ cell detection rate two days after ONC (%) was calculated by dividing the GPNMB+ cell number in the injured retina by the number in the intact retina.GPNMB+ microglia/macrophages accumulated in the optic nerve five days after ONC was measured by Fiji and normalized to WT mice treated with PBS.Area where activated Iba1For quantification of regenerative capacity in mutants and WT mice, merged stack of ~80 images of CTB-labeled regenerated axons in the optic nerve was prepared with Fiji software. For quantification of the average length of the five longest regenerated axons, the distance of the five longest axons from the lesion site was measured with ImageJ and the average of the longest axons was obtained each animal. In case of some long-regenerated axons turning back to the retinal side, the distance between the lesion site and the turning site was measured. For counting axon number from the lesion site, a line transecting the merged stack image at 400, 800 or 1200 μm from the lesion site was drawn, and the number of the CTB-labeled axons crossing the transection line was counted per animal.In situ Cell Death Detection kit  was utilized according to the manufacture’s protocol. Four sections including VT and DT regions per mouse were used, and TUNEL+ cells in the RGC layer within 300 μm from the peripheral edge of VT and DT retina were counted. The average of TUNEL+ cells in the RGC layer per section was assessed and evaluated depending on locations and time postinjury.RGC death two and three days after ONC was confirmed with the TUNEL assay on 14 μm cryosections from snap-frozen eyeballs. Fluorescein Gapdh gene. The results of qRT-PCR in the injured retina were normalized to the mean value of the intact retina for each experiment, and experiments were repeated three times with specific primers described in PrimerBank: Slc6a4  and Gapdh .The whole injured and intact retina were dissected out in cold RNase-free PBS. The peripheral VT region of the injured and intact retina in Slc6a4-/- mutants, three peripheral VT retinae from WT mice or Slc6a4-/- mutants were pooled in the same tube after dissection, and total RNA was extracted. This sample preparation was conducted in triplicates for both WT and Slc6a4-/- mutants. RNA was isolated using TRIzol RNA isolation reagent (ThermoFisher) and RNA quality was verified by the University of Pittsburgh Health Genomics Core using Agilent Tapestation RNA and Qubit Fluorometric Quantification. RNA samples with the RNA integrity number of (RIN) >7 were utilized. Briefly, cDNA was synthesized with SuperScript IV and strands underwent Adenylation of 3’ ends followed by adapter ligation and library amplification with indexing. Library preparation was conducted with TruSeq Total RNA library kit (Illumina) using at least 1 μg of total RNA following the manufacture’s instruction. Modifications during the library preparation include rRNA reduced RNA cleaned up was done with AMPureXP beads and library amplified with 13 cycles of PCR and cleaned up with 37μl AMPureXP beads. Libraries were sequenced using NextSeq500 (Illumina) with 75 base paired end reads resulting in 40M reads per sample. FASTQ files were analyzed using CLC Genomics Grid Workbench v20.0. For RNA sequencing analysis, sequenced reads were aligned to the mouse genome (GRCm38). Read counts were generated and differentially expressed genes were obtained using CLC Genomic Workbench v20.0. Transcripts with at least 10 read events in two samples of either WT or Slc6a4-/- mutants and with a p-value lower than 0.05 were collected so that 701 transcripts out of 25,676 transcripts were utilized for further analysis. For further expression shown in heatmaps and volcano plots and functional analysis, 29 transcripts which show the false discovery rate (FDR) p-value (<0.05) and a fold change higher than 1.5 were selected. Heatmaps and volcano plots were generated using CLC Genomic Workbench v20.0.One day after optic nerve crush in three WT mice and three Sample sizes were based on past experience with studies in mouse models of retinal axon crush and other CNS diseases where phenotypes are obvious, and this range of the number per experiment can be also found in our previous published paper  and otheS1 Fig+ RGCs in four quadrants of the peripheral intact retina. Osteopontin (OPN)+/βIII-tubulin+ RGCs are detected in both VT and DN retina. (B) Quantitative analysis of osteopontin (OPN)+ RGCs (%) in four quadrants of the intact peripheral retina . Data presented as mean ± SD. N.S., not significant; Scale bar represents 20 μm.(A) Distribution of osteopontin (OPN)(TIF)Click here for additional data file.S2 Fig+ RGCs in four quadrants of the intact peripheral retina in Slc6a4-/- and WT mice . Data presented as mean ± SD; N.S., not significant.Quantification of βIII-tubulin(TIF)Click here for additional data file.S3 Fig+ activated microglia/macrophages at the lesion site in the optic nerve compared to WT or Slc6a4-/- mice with saline . Data presented as mean ± SD. Scale bar represents 100 μm.(A-B) Five days after ONC, WT mice treated with fluoxetine show a reduced area of Iba1(TIF)Click here for additional data file.S4 FigItgb3 in the dorsal retina of Itgb3flox/flox mice after intravitreal injection of AAV2-GFP-Cre virus and RBPMS+ RGC survival analysis in the peripheral DN WT or Itgb3flox/flox + AAV2-GFP-Cre retina two weeks after ONC. (C-D) DN RGCs in WT and Itgb3flox/flox + AAV2-GFP-Cre retina are similarly vulnerable to injury . Data presented as mean ± SD. N.S., not significant; Scale bars represent 20 μm.(A) One day after ONC, integrin β3 is upregulated in DN RGCs (arrows). (B) Schema of deletion of (JPG)Click here for additional data file.S5 FigSlc6a4-/- compared to WT mice one day after ONC, the significant genes (P < 0.05) which are known to regulate RGC death/survival and axon regeneration after ONC, are listed. (B) Enriched cellular processes and pathways in the VT Slc6a4-/- retina compared to WT retina one day after ONC are shown. Bars indicate the degree of -log10 P value.(A) In the VT retina of (TIF)Click here for additional data file.S6 FigGpnmb+ mice, but not DBA/2J mice. Scale bar represents 20 μm.GPNMB expression in RGCs are detected in DBA/2J-(TIF)Click here for additional data file.S7 FigGpnmb virus infection in the dorsal retina of DBA/2J mice and RBPMS+ RGC survival analysis in the peripheral DN and DT retina two weeks after ONC. (B-C) In in DBA/2J mice, peripheral DN and DT RGCs are protective by AAV2-GFP-Gpnmb virus compared to AAV2-Ctr virus two weeks after ONC . (D) CTB-labeled regenerated axons beyond the injury site (*) are observed in DBA/2J mice after infection of AAV2-GFP-Gpnmb virus, but not AAV2-Ctr into the dorsal retina. (E-F) Quantitative analysis of axon regeneration in DBA/2J mice having AAV2-Ctr or AAV2-GFP-Gpnmb virus in the dorsal retina two weeks after ONC. The average of the distance of the CTB-labeled five longest axons from the lesion site (E) and the number of regenerated axons at 400 and 800 μm from the lesion site are shown (F). , two-way ANOVA (F)). Data presented as mean ± SD. N.S., not significant; Scale bars represent 20 μm (B) and 100 μm (D).(A) Schema of AAV2-Ctr or AAV2-GFP-(TIF)Click here for additional data file.S1 TableSlc6a4-/- compared to WT mice one day after ONC are listed. Gene expression levels in Slc6a4-/- mutant are shown as Fold change of their expression over that in WT. Genes (in red) have been identified as genes differentially expressed in specific RGC subtypes after optic nerve injury [Significant genes (P < 0.05) in the VT retina of e injury .(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.