Mycoplasma hyopneumoniae (M. hyopneumoniae) swine experimental model objectives is to reproduce mycoplasmal pneumonia (MP). Unfortunately, experimental validated protocols to maximize the chance to successfully achieve lung lesions induced by M. hyopneumoniae are not available at the moment. Thus, the objective of this work was to identify those factors that might have a major influence on the effective development of MP, measured as macroscopic lung lesions, under experimental conditions. Data from 85 studies describing M. hyopneumoniae inoculation experiments were compiled by means of a systematic review and analyzed thereafter. Several variables were considered in the analyses such as the number of pigs in the experiment, serological status against M. hyopneumoniae, source of the animals, age at inoculation, type of inoculum, strain of M. hyopneumoniae, route, dose and times of inoculation, study duration and co-infection with other swine pathogens. Descriptive statistics were used to depict M. hyopneumoniae experimental model main characteristics whereas a recursive partitioning approach, using regression trees, assessed the importance of the abovementioned experimental variables as MP triggering factors. A strong link between the time period between challenge and necropsies and lung lesion severity was observed. Results indicated that the most important factors to explain the observed lung lesion score variability were: (1) study duration, (2) M. hyopneumoniae strain, (3) age at inoculation, (4) co-infection with other swine pathogens and (5) animal source. All other studied variables were not relevant to explain the variability on M. hyopneumoniae lung lesions. The results provided in the present work may serve as a basis for debate in the search for a universally accepted M. hyopneumoniae challenge model.One of the main  Mycoplasma hyopneumoniae (M. hyopneumoniae) has been recognized as the etiological agent of enzootic pneumonia (EP) and a relevant infectious agent of the porcine respiratory disease complex (PRDC). Both are chronic respiratory diseases affecting mainly grow-finishing pigs, causing major economic losses in the pig industry worldwide. Th. Th22]. M. hyopneumoniae-inoculated pigs (with or without co-infection with other swine pathogens). Therefore, those experimental units in which pigs were mock inoculated, immune stimulated (through vaccination or adjuvantation) or contact-exposed to M. hyopneumoniae were not considered. On the contrary, experimental units within a study meeting the two abovementioned inclusion criteria were systematically introduced into a database .Eligible studies were those providing: 1) information in regards of MP prevalence and/or macroscopic score ; and 2) at least one experimental unit including M. hyopneumoniae strain (Strain) variable, the term “pool” was applied in those experimental units in which more than one strain were used simultaneously or subsequently to inoculate the animals. In addition, for the additional challenge with other swine pathogens (CoIn), it was considered either when M. hyopneumoniae was co-inoculated before, at the same time or after another bacterial or viral microorganism. Lastly, a major setback of the systematic review process was the fact that different lung lesion scoring systems were used along the different compiled studies. In consequence, recently reported formulae of equivalence were applied to homogenize mean lung lesion scores between experimental units [Several variables were annotated from each experimental unit accomplishing the aforesaid inclusion criteria . For theal units . Thus, aDescriptive statistics were used to analyze variables and summarize their main characteristics . Distribution, central tendency and dispersion parameters were calculated for continuous variables whereas frequency distribution bar charts were created for categorical variables.rpart 4.1–10 package [mgcv 1.8–12 package [The contribution of the previously mentioned variables to the experimental induction of MP was assessed through a recursive partitioning analysis via decision tree models construction. Thus, tree-based models were grown and pruned by using the  package  in the R package . The pru package , 18. In  package , which d package  and the  package .®/PubMed® database produced an initial list of 643 references until 1990 (last entry in May 2016). An additional article written in Chinese was identified in other sources [M. hyopneumoniae experimental inoculations reported), which, after removal of duplicities, were reduced to 136. After examination of full text, 85 studies, including a total of 261 experimental units, complied with the inclusion criteria and therefore, they were included in the analyses.Study selection flow diagram is shown in  sources . ExaminaM. hyopneumoniae strains together with an alphanumerical reference and the countries where they were used.Since different variables were specified in each experimental unit, the number of experimental units considered for the analyses differed between variables. Main descriptive statistics calculated for continuous variables and the number of experimental units considered for each variable is summarized in M. hyopneumoniae antibody animals were principally chosen. In regards of the inoculation procedure, pigs were often challenged at 6 weeks of age through the IT inoculation route in one time. Concerning the inoculum, M. hyopneumoniae broth culture (CT) was applied in the majority of cases. The dosage given per pig was greatly variable, reaching a maximum of 1.50 x 1015 CCU [8 CCU, being 107CCU/pig the most prevalent inoculum dose. Nearly one quarter of the experimental units used co-infection with other viral and bacterial swine respiratory pathogens. In decreasing order of importance, the pathogens co-inoculated were Porcine reproductive and respiratory syndrome virus, Swine influenza virus, Porcine circovirus type 2 and bacterial pathogens such as Actinobacillus pleuropneumoniae or Pasteurella multocida. The mean study duration was found to be about 6 weeks, although in most of the cases the animals were necropsied after 4 weeks post-inoculation (wpi). As outcomes, the mean percentage of lungs affected by MP within an experimental unit was 72% and the mean lung lesion score (Ph. Eur.) was around 10% of lung weight damaged. Importantly, from the 261 included experimental units, 30 (11.5%) did not manage to reproduce MP. The use of a given strain was found to be closely related with the laboratory or research group. M. hyopneumoniae 232 was the most largely used strain, mainly by the United States research groups. In Europe, a higher variability is seen, but French M. hyopneumoniae BQ14 and 116 strains were the most prevalent through experimental units from this continent. Notably, the use of a pool of M. hyopneumoniae strains, meaning simultaneous or subsequent inoculations of at least two different strains, has been reported in experimental inoculation models only in Europe. In Asia, the Japanese E-1 strain was found to be the most commonly used one. Lastly, two different strains were handled in Australia, named Beaufort and Hillcrest.The mean sample size within experimental units was approximately of 8 inoculated pigs. Nonetheless, experimental units including only one pig were the most prevalent ones. The latter is probably explained because of the impact of a unique study with numerous experimental units of 1 pig each [1015 CCU . Such a M. hyopneumoniae seropositive pigs [Since the Llscore variable was more commonly specified within experimental units (n = 227) than the Llprop one (n = 160), the former was chosen as response variable in the multivariate analysis (74 studies considered). The explanatory variables were: InAge, Asource, InRoute, Strain, InType, Dose, InDays, CoIn and Nweek. Giving the fact that only 3 experimental units from 2 studies used ive pigs , 25, Serad hoc ANOVA test . Using GAM, the strong connection between Nweek and Llscore variables was reinforced . Indeed, Llscore peaked when necropsies were performed at 4 wpi, dropping later at 8 wpi .Nonetheless, sole inoculation with 75/2002 or JS strains resulted in similar Llscore values  to those reached in co-infection. All the remaining strains resulted in lower Llscore outcomes, particularly when the age of pigs at inoculation was lower than 3.5 weeks .When above 3.5 weeks of age, inoculated specific pathogen-free (SPF) pigs showed higher Llscore values  than their C or cesarean-derived, colostrum-deprived (CDCD) counterparts .All the other analyzed variables, namely InRoute, InDays, InType and Dose,werenot found relevant to explain Llscore.In a first obtained tree-based model, in which all explanatory variables were fitted to get the full picture of the model, Nweek was the most important variable by far as shown by an at 8 wpi . Thus, tat 8 wpi  and, in M. hyopneumoniae experimental models, though highlighting the existing inconstancy between protocols. Works studying how a unique variable may influence MP under experimental settings have been already made [M. hyopneumoniae challenge model pursuing this goal.Numerous attempts to induce MP at experimental level have been reported and, as evidenced by the present results, variation in both the number of animals affected by MP as well as in severity of such lung lesions do occur. The latter is accompanied by wide differences between ady made , 10–13. et al. (2008) identified factors with a relevant influence on the expression of clinical postweaning multisystemic wasting syndrome (PMWS) under experimental conditions, albeit different statistical techniques were applied [In order to accomplish the abovementioned objective, data from a series of studies were summarized through a systematic review and analyzed by means of recursive partitioning. To authors’ knowledge, this is the first time that such statistical method has been applied to evaluate the performance of experimental infections in pigs. Along the same line, Tomás  applied . Recursi applied . In the  applied . MissingM. hyopneumoniae transmission and persistence instead of lung pathology [M. hyopneumoniae strain (Strain) followed by the age of animals at inoculation (InAge), co-infection with other swine pathogens (CoIn) and, finally, the source of the animals (Asource).In the first fitted regression tree containing all explanatory variables, the most determinant factor associated to the presence of lung lesions in experimental trials was found to be the study duration (Nweek). Henceforth, and to further examine such relationship, a complementary analysis was performed by means of GAM. In this way, GAM analysis revealed that lung lesion scores peaked when the Llscore was assessed at 4 wpi, being congruent with those studies indicating that macroscopic lung lesions reach their maximum level around that moment , 8. Hencathology –30.TakinM. hyopneumoniae strains have been described [M. hyopneumoniae strains assessed were split into two groups, one leading to higher lung lesion score outcomes than the other. Thus, the obtained results support the already reported high virulence of some M. hyopneumoniae strains such as Hillcrest [M. hyopneumoniae commercial vaccines are based [M. hyopneumoniae strains. As a distinctive condition, simultaneous or subsequent inoculations with more than one strain did not result in more severe lung lesions. Although some studies have suggested an enhanced effect of multiple infections with different M. hyopneumoniae strains [et al. (2014), in which no link was found between lungs harboring more than one strain and lung lesion severity. Notwithstanding, given that data from single M. hyopneumoniae strain inoculations predominate over data from multiple strain co-inoculations, the obtained results should be corroborated. Additionally, differences between M. hyopneumoniae strains might be explained, among others, by the number of in vitro passages, which has been discussed to result in pathogenicity differences [Differences in virulence between escribed , 10. Desescribed , 32, it escribed , 34. By illcrest  or even illcrest , 36, frore based . On the re based , 38, wer strains , 34, 39,M. hyopneumoniae infection [M. hyopneumoniae between 3 and 12 weeks of age. Furthermore, in almost all the analyzed studies, inoculated pigs were tested to be seronegative against M. hyopneumoniae, excluding conferred protection of younger pigs by passively acquired maternal antibodies. While M. hyopneumoniae is considered a primary respiratory pathogen, thus, able to induce disease by itself, the use of co-infections have been also proven effective to induce M. hyopneumoniae-like lung lesions in several experiments. However, whether co-infection with other swine pathogens influences the severity of M. hyopneumoniae infection (macroscopic lung lesions) is still under debate. Whereas some studies found no potentiation of pneumonia by other microorganisms [M. hyopneumoniae [M. hyopneumoniae alone. Regrettably, the lack of more experimental units using co-infection models hindered the evaluation of the co-inoculated pathogens separately, being all of them pooled and evaluated together. The latter might be taken into account since the lung lesion outcome may vary depending on the co-inoculated microorganism. Lastly, the use of SPF pigs resulted to be an important feature to effectively reproduce MP. Unfortunately, criteria applied to define SPF pigs by the authors reporting their use were unknown, becoming difficult to precisely explain the importance of such factor on lung lesion outcome. Nevertheless, and independently of the source, pigs used in M. hyopneumoniae experimental models were, in general terms, M. hyopneumoniae-free by means of serology and/or polymerase chain reaction (PCR) techniques. Hence, pigs naive to M. hyopneumoniae are probably required to increase the chance to achieve severe MP.Immunopathological events are considered to play an imperative role in the pathogenesis of nfection , 40. Connfection . Indeed,nfection , no diffrganisms , 25, 42,eumoniae , 44. DesM. hyopneumoniae challenge studies, in which very high doses are necessary to achieve clinical disease [All the other evaluated variables  were found not relevant to explain MP variability. Contrarily, higher percentage of animals affected by MP as well as mean lung lesion score have been reported in pigs inoculated endotracheally in comparison with pigs inoculated intranasally or by aerosolization . Inocula disease . In agreM. hyopneumoniae experimental inoculation protocols could be overestimated in the statistical analysis.Importantly, care must be taken in drawing conclusions from the obtained results. The statistical approach performed in the present study was probably hampered by the broad differences in the applied conditions between experimental units, which make it difficult to assess the effect of a particular condition in the lung lesion outcome. Moreover, inherent limitations of the included publications and the systematic review process must be also taken into account. In this respect, the trend towards publishing only positive results (e.g. experiments in which MP was successfully achieved) and the exclusion of those studies that did not fulfil all the systematic review inclusion criteria (e.g. experiments in which MP was induced but no information in regards of lung lesion score was provided), probably lead to biases that could have influenced the obtained results. Another important reason for bias is linked to the high prevalence of some particular conditions within a variable, such as the lung lesion assessment at 4 wpi as striking example. Thus, the importance of such widely extended conditions within M. hyopneumoniae experimental swine models and constitutes the first insight into conditions needed to induce lung lesions suggestive of MP under experimental settings. Thus, obtained results might serve as a basis for debate in the search for a M. hyopneumoniae experimental model seeking to induce severe MP. While the M. hyopneumoniae strain used may depend basically on the strain availability, other easily modifiable conditions might be taken into account in experimental models. Therefore, the highest likelihood to achieve severe MP would require lung lesion assessment within a period below 8wpi and include inoculation of SPF (M. hyopneumoniae free) pigs older than3.5 weeks of age and, preferably, in co-infection with another swine respiratory pathogen as a triggering factor.The present study broadens the current understanding in regards to S1 PRISMA ChecklistProtocol followed in accordance with the PRISMA guidelines.(DOC)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Background. We describe the unusual case of a recently transplanted cadaveric renal transplant recipient who presented with recurrent pulmonary mucormycosis. Case Report. An 18-year-old man with end stage renal disease secondary to congenital renal agenesis status after cadaveric kidney transplant 4 months before presented with acute onset of fever, hemoptysis, and back pain. The patient underwent an emergent left lower lobectomy due to the critical nature of his illness. He was also treated with amphotericin with resolution of his symptoms. One week later, he had evidence of recurrent disease on imaging with a surgical site infection. He underwent reexploration with evacuation of an empyema and debridement of a surgical site infection. He was continued on IV antifungal therapy with isavuconazonium and amphotericin. Radiographic clearance of disease with three months of treatment was apparent with no evidence of recurrence at seven-month follow-up. Discussion. Opportunistic infections in solid organ transplant patients represent a significant source of morbidity and mortality. Most patients are treated with prophylactic anti-infective agents. However, rare infections such as pulmonary mucormycosis remain a risk. The transplant physician must be aware of these uncommon infections and their treatment strategies, including the management of uncommon recurrent disease.  Rhizopus and Mucor, represents a rare but aggressive infection of immunosuppressed patients. Less than one percent of solid organ transplant patients are infected; however mortality can exceed greater than 80% [Mucormycosis, which includes fungi in the Mucorales order such asthan 80% –5. Rhinothan 80% .Most infections occur within the first year of solid organ transplant . The vasMucorales have known resistance to caspofungin and voriconazole. Accordingly, amphotericin B is regarded as the first-line therapeutic agent for mucormycosis . HoweverSurgical debridement is essential for effective treatment of mucormycosis. Due to the aggressive behavior of this fungus in immunocompromised patients, extensive surgical debridement has appeared to be necessary in successful clearance of disease reported in various case series , 14, 15.We report the unusual case of a renal transplant recipient with refractory pulmonary mucormycosis. The objective of our study is to describe the management of recurrent lung mucormycosis in a recent cadaveric renal transplant recipient. This case report adds to the growing body of literature surrounding this phenomenon and to elucidating effective treatment algorithms.3 and elevated creatinine. His initial chest X-ray demonstrated a consolidation of the left lower lobe  and isavuconazonium (372 mg daily) after discharge. His follow-up chest X-ray 7 months after treatment showed no evidence of residual disease. His medications were discontinued at this time. To our knowledge, he has had no further issues.Mucormycosis is a rare infection with a high likelihood of devastating outcomes in immunocompromised patients. Currently, there is a paucity of data on optimal treatment strategies. In particular, there are no clinical studies that demonstrate successful treatment of patients with recurrent mucormycosis. Our case report demonstrates a strategy to combat recurrent mucormycosis using salvage therapy with isavuconazonium. An initial strategy of IV liposomal amphotericin B combined with a newer medication, isavuconazonium, was employed along with aggressive surgical debridement. Despite initial efforts, the patient had evidence of recurrent pulmonary mucormycosis with dissemination of the infection to his surgical incision.The current gold standard for mucormycosis is IV liposomal amphotericin B, surgical debridement, and, if possible, modification of risk factors , 10, 11.There are several important limitations to our study. First, we presented evidence from a single patient. High-quality randomized control trials are needed to further elucidate the use of isavuconazonium. Additionally, it is possible to consider that the first pneumonectomy was inadequate and contributed to the spread of the initial disease to the patient's skin.In conclusion, we present a rare case of recurrent pulmonary mucormycosis with skin dissemination in an immunocompromised patient that was successfully managed with a combination of surgical treatment and aggressive pharmacologic therapy, including amphotericin B and isavuconazonium. It is also important to be aware of this possible diagnosis in all immunosuppressed patients and to be well-versed on potential treatment options for recurrent or refractory disease.


Sudden hearing loss in the only hearing ear cases are rarely published in the English literature; most of the cases are idiopathic. It is an otologic emergency needing urgent treatment. Delayed diagnosis can interfere with patient's social life with interrupting the verbal communication. In this case report we presented a 33-year-old female patient having sudden sensorineural hearing loss in the only hearing ear diagnosed as bilateral large vestibular aqueduct syndrome.  Sudden sensorineural hearing loss (SSHL) can be described as at least 30 dB sensorineural hearing loss (SNHL) in at least three consecutive frequencies within a three-day period . It is aA 33-year-old woman was admitted with complaints of sudden hearing loss in the right ear for one day. She had no vertigo or dizziness complaint. On her medical history, she had total hearing loss in the left ear from the childhood. She had no additional illnesses. Otoscopic examination was normal. In the pure tone audiometry she had 52 dB sensorineural hearing loss in the right ear and a profound sensorineural hearing loss in the left ear . She hadThe vestibular aqueduct (VA) is a bony canal extending from medial wall of the vestibule to the posterior fossa dura at the level of anterior part of the sigmoid sinus. Ductus endolymphaticus is the main structure that courses in the VA. Normal VA diameter is reported to be between 0.4 and 1 mm, and large VA is mostly accepted to be a VA greater than 1.5 mm at its anteroposterior diameter or greater than 2.0 mm as measured at the midway the common crus and external aperture . LVAS isSudden hearing loss in the only hearing ear is an audiological emergency interrupting the patient's verbal communication as well as psychological and social health status . Due to In conclusion, for cases with sudden hearing loss in the only hearing ear, LVAS must also be considered in differential diagnosis. Early addition of intratympanic steroid and hyperbaric oxygen treatment modalities to oral steroids may have supportive effect on hearing, rescuing the patient from cochlear implantation surgery.


In humans and other mammals, the neural retina does not spontaneously regenerate, and damage to the retina that kills retinal neurons results in permanent blindness. In contrast to embryonic stem cells, induced pluripotent stem cells, and embryonic/fetal retinal stem cells, Müller glia offer an intrinsic cellular source for regenerative strategies in the retina. Müller glia are radial glial cells within the retina that maintain retinal homeostasis, buffer ion flux associated with phototransduction, and form the blood/retinal barrier within the retina proper. In injured or degenerating retinas, Müller glia contribute to gliotic responses and scar formation but also show regenerative capabilities that vary across species. In the mammalian retina, regenerative responses achieved to date remain insufficient for potential clinical applications. Activation of JAK/STAT and MAPK signaling by CNTF, EGF, and FGFs can promote proliferation and modulate the glial/neurogenic switch. However, to achieve clinical relevance, additional intrinsic and extrinsic factors that restrict or promote regenerative responses of Müller glia in the mammalian retina must be identified. This review focuses on Müller glia and Müller glial-derived stem cells in the retina and phylogenetic differences among model vertebrate species and highlights some of the current progress towards understanding the cellular mechanisms regulating their regenerative response. In humans and other mammals, the retina, like most other regions of the central nervous system (CNS), does not spontaneously regenerate; and damage to the retina or neurodegenerative disease that kills retinal neurons results in permanent blindness. Worldwide, more than 12% of people over the age of 40 have visual impairment or blindness caused by age related macular degeneration and glaucoma, two of the neurodegenerative diseases affecting the retina [A variety of stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells, and fetal-derived neural and retinal stem cells, are currently under investigation for regeneration and subsequent transplantation of retinal neurons (see reviews in –10). WitMüller glia are intriguing candidates for intrinsic retinal stem cells. Müller glia are radial glial cells within the retina and are generated from the same lineage as retinal neurons. In the mature retina, Müller glia maintain retinal homeostasis, buffer ion flux associated with phototransduction, and form the blood/retinal barrier within the retina proper. Although they contribute to gliotic responses and scar formation following retinal injury, Müller glia also show regenerative capabilities that vary across species. This review focuses on Müller glia and Müller glial-derived stem cells in the retina and the phylogenetic differences among model vertebrate species and highlights current progress towards understanding and harnessing their regenerative response.The retina is a thin layer of neural tissue located at the posterior pole of the eye. It consists of (a) photoreceptors (rods and cones) that convert light stimuli into neurochemical signals, (b) three major classes of interneurons  that perform initial information processing, (c) Müller glia that perform a multitude of support functions, and (d) projection neurons  that extend axons through the optic nerve and optic tract to convey the visual image information to higher processing centers within the brain –18. The The initial evidence for the stem cell characteristics of Müller glia came from research to discover the cellular source of ongoing neurogenesis and regenerative responses in the retinas of fish. The capacity for neurogenesis in the mature retinas of fish appears to be teleologically related to their overall pattern of indeterminate growth and the associated continuous growth of their eyes. Eye growth in fish results in large part from a general expansion/stretching of the retina, leading to decreasing retinal density for most types of retinal neurons within the central retina . There iRetinal regeneration in fish has been studied for over 50 years –34. A vaIn warm blooded species, spontaneous regeneration by Müller glia does not occur to any appreciable extent in vivo, leading to questions about the designation of Müller glia as retinal stem cells in these species, particularly in mammals . In the  Ascl1a [ Atoh7 [Evidence for neurogenesis by Müller glia in vivo following acute retinal injury has been demonstrated in posthatch chick, rat, and mouse , 73–77 b Ascl1a , 79 and  [ Atoh7 , 81. Int [ Atoh7 , 82. The [ Atoh7  and in r [ Atoh7 , 83, 84. [ Atoh7 . A betteα) [β) [Promoting clinically relevant levels of regeneration from Müller glia in the mammalian or human retina will require suppression of gliosis and enhancement of their proliferative and neurogenic responses. Multiple signaling molecules and their downstream signal transduction cascades have been implicated in regulating the injury and regenerative responses in the retina, including notch –88, tumoα) , transfoα) [β) , 90–92, α) [β) , 73, midα) [β) –95, ciliα) [β) –99, epidα) [β) , 84, 100α) [β) , 102. Maα) [β) –106. Howα) [β) , 107. AlIntracellular signaling through the JAK/STAT pathway is activated by receptor binding of a variety of ligands, including cytokines . BAmong the factors that can activate JAK/STAT and MAPK signaling, CNTF plays multiple roles in the retinal injury response and particularly in activating the gliotic responses of Müller glia. CNTF expression is upregulated in Müller glia following a variety of retinal injuries in the zebrafish , posthatIn addition to activating JAK/STAT signaling, injury activates MAPK signaling in Müller glia in zebrafish and mice , 126. VaDespite similarities between fish, birds, and mammals in the Müller glial response to exogenous growth factors, modulation of JAK/STAT and MAPK signaling in the mammalian retina remains insufficient to stimulate clinically relevant regenerative responses. There is evidence that both intrinsic and extrinsic mechanisms contribute to inhibition of the neurogenic/regenerative response of Müller glia in the retinas of higher vertebrates. Although regeneration in the fish retina can occur throughout life, there is an age-associated decline in the proliferative response of Müller glia to retinal injury and exogenous mitogens in rodents , 131. Th Sox2), as well as nestin, an intermediate filament typically associated with neural stem cells. Only rMC-1 has robust GFAP expression, consistent with its origin from light-injured retinas [Several Müller glial cell lines have been described: ImM10, conditionally immortalized Müller glia from P10 mouse retinas , 133; MI retinas , althoug retinas . There aThere is a temporal change in cell cycle kinetics of primary Müller glia, which initially proliferate slowly, even in the presence of serum containing medium . After c Pax6 and nestin [Immortalized Müller glia can be induced to generate cells expressing neuronal genes , 139–142d nestin , 139. Usd nestin . Transpld nestin . TransplThere have been few systematic studies of the activity of specific signal transduction cascades in Müller glia in vitro. Since activation of JAK/STAT or MAPK signaling in Müller glia can regulate gliosis and proliferation, we analyzed activation of STAT3 and MAPK pathways in ImM10 cells by western blot. There were no changes in total STAT3 or pSTAT3 in neurosphere or differentiation cultures; and, unexpectedly, despite the presence of both EGF and FGF2 in sphere forming media, there was no change in pERK1/2 in neurospheres . In contDuring neonatal retinal development, CNTF has been proposed to modulate a “molecular switch” that promotes either a glial or neuronal cell fate via concentration dependent activation of STAT3 versus MAPK signaling, respectively , 145. In , retinal ganglion cells , and bipolar cells (Prkca). However, addition of CNTF did not alter the overall patterns or levels of gene expression [To test if low levels of CNTF could promote neurogenesis in differentiation cultures of ImM10 cells, we analyzed gene expression following addition of low concentrations of CNTF (20 ng/ml) . Consistpression . In addipression .The JAK/STAT and MAPK signaling pathways are implicated in retinal cell fate choice, proliferation, and gliotic hypertrophy after retinal injury and can be activated by a variety of growth factors including EGF, HB-EGF, FGF2, insulin, and CNTF. The JAK/STAT pathway appears to mediate both beneficial (proliferation) and detrimental (hypertrophy/gliotic) aspects of the injury response in vivo . Our finα [β [ ccnd1 and p27(kip1) [ Ascl1a [ Atoh7 [Although activation of JAK/STAT and MAPK signaling by CNTF, EGF, and FGFs can promote proliferation and the glial/neurogenic switch, these signal transduction pathways do not act in isolation. Rather, they function within the context of a wide variety of other cellular mechanisms that contribute to the retinal injury response and regeneration in various contexts. Extensive discussion of all the cellular mechanisms regulating the injury and regenerative responses of Müller glia is beyond the scope of this article; several recent reviews can provide more information , 149–154α , 89, TGFα [β , and BMPα [β , 91, 16427(kip1) , 85, 16527(kip1) , 166–168[ Ascl1a , 162 and [ Atoh7 , 81, 141ξ), to modulate downstream signal transduction cascades [ξ blocks its phosphatase activity, resulting in increased phosphorylation of a variety of tyrosine-kinase receptors and their substrates and potentiating downstream signal transduction cascades [ Mdkn mRNA is upregulated in ImM10 cells under differentiation condition in vitro [Analysis of retinal regeneration in fish has identified a variety of signaling molecules and their downstream signal transduction cascades that have shown promise for enhancing regenerative responses of Müller glia and warrant continued study. One intriguing, yet understudied, molecule that plays a role in retinal neurogenesis and regeneration in zebrafish is midkine (MDKN) . MDKN iscascades , 169–174cascades . In zebrcascades , 95. Morcascades . Much lecascades  and ratscascades . We founin vitro . Given tMüller glia are particularly appealing as a cellular source for retinal regeneration because they are intrinsic to the retina and offer the potential to regenerate neurons in situ, without transplantation. Despite a growing body of research showing the neurogenic potential of Müller glia in the mammalian retina, a level of regenerative response sufficient for potential clinical applications has yet to be achieved. Given the overall modest outcomes to date, cultured Müller glia seem unlikely to provide a clinically relevant path for generating sufficiently large numbers of retinal progenitors for transplantation. Successful regenerative strategies using transplantation are more likely to build on the ongoing progress in generating retinal progenitors and neurons from other sources, such as induced pluripotent stem cells , 177–184


This study at first demonstrated that the transplantation of TCs could improve allergen‐induced asthma by obviously inhibiting airway inflammation and airway hyper‐responsiveness preclinically, with the down‐regulation of Th2‐related cytokine IL‐4, transcription factor GATA‐3 and Th2 cell differentiation, while up‐regulation of Th1‐related cytokine IFN‐γ, transcription factor T‐bet and Th1 cells proliferation in asthma, just like MSCs. Co‐transplantation of TCs with MSCs showed better therapeutic effects on experimental asthma, even though the therapeutic effects of TCs alone were similar to those of MSCs alone. TCs and the combination of TCs with MSCs could improve the airway inflammation and airway hyper‐responsiveness and can be a new alternative for asthma therapy.Telocytes (TCs) newly discovered as the mesenchyme‐derived interstitial cells were found to have supportive effects on mesenchymal stem cells (MSCs). The present study aimed at investigating effects of TCs or TCs gathered with MSCs on experimental airway inflammation and hyper‐responsiveness. The TCs were isolated from the lung tissue of the female BALB/c mice. The ovalbumin (OVA)‐induced asthma model was established. TCs (1 × 10 Telocytes (TCs) are newly discovered as the mesenchyme‐derived interstitial cells in recently years Pathophysiological changes of TCs quantity or function were noticed in a large number of diseases, such as myocardial infarction, heart failure, inflammatory bowel disease, liver fibrosis and systemic sclerosis Female BALB/c mice, weighing 18–22 g (6–8 weeks), were provided by Animal Facility in Biomedical Research Center of Zhongshan Hospital, Fudan University. This study was approved by the Fudan University Ethical Committee for animal experiments.2). The supernatant was moved into new culture bottles 30 min. after cell adhesion on the plate. Cells were selected, purified and further amplified to meet the need for the experiment. The TCs were identified according to the morphology and immunofluorescent staining using mouse anti‐cKit antibody, rat anti‐vimentin antibody and rabbit anti‐CD34 antibody . After incubated overnight at 4°C with the first antibodies diluted in 1% bovine serum albumin (BSA) in PBS, the slides were washing in PBS for three times. Then, sections were incubated with PE conjugated antimouse secondary antibodies, PE conjugated anti‐rat secondary antibodies or FITC conjugated anti‐rabbit secondary antibodies according to the manufacturer . DAPI was used to mark nuclear according to the manufacture (Abcam).The isolation and identification of lung TCs were described by previous study 2), after red blood cells were lysed. The medium was changed every 48 hrs, and MSCs were identified according to the morphology and flow cytometry for cell markers such as CD90‐PE, CD105‐PE and CD34‐PE .The femur and tibia of mice were isolated under sterile condition. The osteoepiphysis of femur and tibia were cut off, and the marrow cavities were washed with DMEM/F12 medium. The lavage fluid was centrifuged at 100 g for 10 min., and cells were collected and cultured in incubator  at 1, 8 and 15 days (d). At 25–28 days, the mice were per‐nasally instilled with 50 μl 0.4% OVA. The airway reactivity was measured, and specimens were collected 24 hrs after the last provocation. At 22–24 days, the mice were intravenously injected with the suspension of TCs alone, MSC alone, or the combination for 3 consecutive days.The mice were intraperitoneally injected with 100 μl 0.02% ovalbumin (OVA) and 100 μl Al(OH)n = 10/group): (1) animals were intraperitoneally sensitized with OVA, intratracheally provoked with vehicle and intravenously treated with vehicle as negative controls (PBS); (2) animals sensitized with OVA, provoked with vehicle and treated with TCs at 106 per day (PBS + TCs); (3) animals sensitized and provoked with OVA, and treated with vehicle as positive controls (OVA); (4) animals sensitized and provoked with OVA, and treated with TCs at 106 per day (L‐TCs); (5) animals sensitized and provoked with OVA, and treated with TC at 2 × 106 per day (H‐TCs); (6) animals sensitized and provoked with OVA, and treated with MSCs at 106 per day (MSC); and (7) animals sensitized and provoked with OVA, and treated with the combination of TCs and MSCs at 106 per day, respectively (TCs + MSC) .BALB/c mice were randomly divided into seven groups . Frozen sections of lungs were prepared to observe the distribution of TCs and MSCs using Leica TCS SP5 confocal microscope .An additional experiment was designed to confirm the migration of TCs and MSCs into the lung after the intravenous injection of the living TCs labelled with PKH26 (Red) and MSC with 5(6)‐(N‐succinimidyloxycarbonyl)‐3′,6′,O,O’‐diacetylfluorescein (CFSE) (Green) (Sigma‐Aldrich)  as lung resistance (RL). The concentrations of methacholine for provocation were 6.25, 12.5 and 25 mg/ml, respectively. The percentage of RL value and basal RL value were used to reflect the airway responsiveness after the provocation of methacholine.Pathological changes of lung injury were evaluated according to Underwood's standard of lung histopathological scoring Leucocytes in the bronchoalveolar lavage fluid (BALF) were harvested, stained with Wright‐Giemsa dye and counted after centrifugation at 200 g for 15 min. (4°C). Levels of inflammatory mediators, for example interleukin (IL)‐4, interferon (IFN)‐γ, transforming growth factor‐beta (TGF‐β) (Sigma‐Aldrich) and OVA‐specific IgE  were measured with ELISA kits as suggested by the manufactory.An additional experiment was designed and performed to evaluate CD4+ T‐cell phenotypes. The mouse spleen was acquired under sterile condition, cut into small pieces and filtered with 70‐μm strainer to remove the capsule and connective tissue. The cell supernatant was collected and centrifuged at 1500 rpm for 10 min., with thrice washes atTris‐NH4 cl. FACSAria II flow cytometry  was used to test spleen CD4+ T‐cell subgroups in animals mentioned above. The CD4+ T cells were isolated by flow cytometry and then were labelled with IFN‐γ‐PE, IL‐4‐PE and Foxp3‐PE antibodies (BD). The proportion of CD4 + IFN‐γ+ T cells, CD4 + IL‐4+ T cells and CD4 + Foxp3+ T cells was accounted. The mRNA expression of T‐bet, GATA‐3 and Foxp3 in lung tissues harvested from various groups was measured on basis of gene probes as listed in Table t‐test, and statistical differences among more than two groups were determined using anova. P < 0.05 was considered significant.Data were analysed using IBM SPSS Statistics 20 . Each point corresponds to the mean ± S.E.M. Statistical differences between two groups were determined by The lung‐origin TCs were identified with the morphological features Fig. A combineP = 0.14, Fig. P < 0.05 or less, respectively). When stimulated with the concentration of methacholine is 12.5 mg/ml, the airway hyper‐responsiveness of OVA animals treated with L‐TCs, H‐TCs, MSCs or TCs + MSC was significantly lower than those treated with vehicle  after the exposure of middle concentration of methacholine at 12.5 or 25 mg/ml, respectively. At exposure to high concentration of 25 mg/ml, the level of airway hyper‐responsiveness in OVA animals treated with TCs + MSCs was lower than those treated with L‐TCs alone (P = 0.004).The similar levels of airway hyper‐responsiveness were found in OVA animals treated with vehicle and L‐TCs after the exposure to the low concentration of methacholine at 6.25 mg/ml . Figure P < 0.05 or less, respectively). The percentage of airway goblet cells in OVA animals treated with TCs + MSC was significantly lower than in those treated with either TCs or MSCs alone, respectively , similar to the levels of animals sensitized with OVA and provoked with vehicle (P = 0.149). The percentage of airway goblet cells in OVA animals with MSC group was significantly lower than in those treated with TCs at low and high concentrations .The score of pathological changes in OVA animals treated with L‐TCs, H‐TCs, MSC or TCs + MSC was significantly lower than those treated with vehicle, while still higher than animals provoked and treated with vehicle (P < 0.05), which was prevented by treatments with L‐TCs, H‐TCs, MSCs or TCs + MSC , although the levels are still higher than in those provoked and treated with vehicle. The number of total leucocytes in BALF of OVA animals treated with TCs + MSC was significantly lower than those treated with either TCs or MSCs alone . The number of eosinophils in animals with TCs + MSC was not different from those provoked and treated with vehicle (P = 0.155), while the number of total leucocytes was still higher (P = 0.049).The total leucocytes Fig. A and eosP < 0.05 or less, respectively).Ovalbumin provocation significantly increased levels of IL‐4 and OVA‐specific IgE and decreased IFN‐γ proteins in BALF . The proportion of spleen CD4 + IL4+ T cells in OVA animals treated with TCs + MSCs was significantly lower, as compared with those with L‐TCs or H‐TCs alone .The proportion of spleen CD4 + IL4+ T cells was significantly increased in OVA animals treated with vehicle, while CD4 + IFN‐γ+ or CD4 + Foxp3+ T cells decreased, as shown in Figure P = 0.011, P = 0.042, respectively).mRNA expression of GATA‐3 Fig. E up‐reguPreclinical studies demonstrated that MSC could alleviate airway inflammation and airway hyper‐responsiveness in allergic models Th1/Th2 imbalance was considered as the pathogenesis of asthma, and the decreased quality and quantity of Treg cells were suggested to play the critical roles in the development of asthma Inflammatory mediators play regulatory effects on the differentiation of CD4+ T‐cell subgroups, for example the promotion of IFN‐γ in the differentiation of naive CD4+ T cells into Th1 cells. Moreover, TGF‐β plays a key role in Treg differentiation. Our results showed that TCs could increase the TGF‐β and IFN‐γ in asthmatic animals. TCs were found to release extracellular vesicles with mediators to transfer signalling to neighbouring cells by paracrine The previous studies found that there were many TCs distributed around the stem cell niches in organ tissue In conclusion, the present study at first demonstrated that the transplantation of TCs could improve allergen‐induced airway inflammation and hyper‐responsiveness preclinically, with the down‐regulation of Th2‐related cytokine IL‐4, transcription factor GATA‐3 and Th2 cell differentiation, while up‐regulation of Th1‐related cytokine IFN‐γ, transcription factor T‐bet and Th1 cells proliferation. Co‐transplantation of TCs with MSCs showed better therapeutic effects on experimental asthma, even though the therapeutic effects of TCs alone were similar to those of MSCs alone. Thus, our data indicate that the combination of TCs and MSCs can be a new alternative for asthma therapy.The authors declare that they have no conflict of interest to disclose.Fig. S1 The flow chart of establishment of acute asthma model.Click here for additional data file.


Age-related hearing loss (ARHL), presbycusis, is a chronic health condition that affects approximately one-third of the world's population. The peripheral and central hearing alterations associated with age-related hearing loss have a profound impact on perception of verbal and non-verbal auditory stimuli. The high prevalence of hearing loss in the older adults corresponds to the increased frequency of dementia in this population. Therefore, researchers have focused their attention on age-related central effects that occur independent of the peripheral hearing loss as well as central effects of peripheral hearing loss and its association with cognitive decline and dementia. Here we review the current evidence for the age-related changes of the peripheral and central auditory system and the relationship between hearing loss and pathological cognitive decline and dementia. Furthermore, there is a paucity of evidence on the relationship between ARHL and established biomarkers of Alzheimer's disease, as the most common cause of dementia. Such studies are critical to be able to consider any causal relationship between dementia and ARHL. While this narrative review will examine the pathophysiological alterations in both the peripheral and central auditory system and its clinical implications, the question remains unanswered whether hearing loss causes cognitive impairment or vice versa. Age-related hearing loss (ARHL) or Presbycusis, used interchangeably in this paper, is a common chronic health problem affecting individuals above 65 years old WHO, . ARHL isMost of the ARHL epidemiological studies have reported hearing loss prevalence data in the 500 Hz−4 kHz , 2005-2006 data on participants aged > 70 years old revealed that on average, speech frequency (0.5–4 kHz), and high-frequency (3–8 kHz) PTA thresholds of the black participants were better than white participants by −5.8 (95% CI: −8.6 to −3.1), and −11.1 (95% CI: −13.9 to −8.2), respectively  located in GRM7, a gene encoding metabotropic glutamate receptor type 7 protein (mGluR7)  SNPs in 13-kb region in the middle of the KCNQ4, a gene which encodes a voltage-gated K-channel found in both outer and inner hair cells of cochlea , an isoform of N-acetyltransferases-encodes metabolism of reactive oxygen species  ε4 allele  and increase in action potential (CAP) of the auditory nerve  decline in overall volume, total number, and neuron size of octopus cells in PVCN  are available. The SOC comprises three main nuclei, namely the medial and lateral superior olive  and medial nucleus of the trapezoid body (MNTB),  is considered one of the major convergence centers for both mono-and-binaural auditory information Figure . It simuAuditory thalamus/medial geniculate body (MGB) neurons transmit auditory stimuli to cortex and other subcortical structures by filtering and enhancing acoustic features of the auditory signal Figure  Bartlet. Gamma-aA number of animal experiments have reported age-related changes of A1. These include, (i) decline in the number of calcium binding positive neurons in rat A1  to measure brain volumes and MRI spectroscopy for delineating neurochemical and metabolic changes  difficulties rather than peripheral hearing deficits (Panza et al., Numerous attempts have been made to provide plausible explanations for the association between ARHL and cognitive decline. Cognitive load on perception hypothesis argues that the decline in cognitive capacity increases the cognitive load resulting in a sensory loss CHABA, . SensoryCommon cause hypothesis argues that a common mechanism underlies age-associated changes observed in cognition, hearing, and other sensory modalities CHABA, . It has Wayne and Johnsrude  proposedRecently, a cognitive reserve hypothesis has been proposed to explain how individuals with similar neuropathological conditions differ substantially in their ability to make efficient use of brain reserve during tasks Stern, . IntelliEven though, ARHL is still not considered as a major risk factor for AD, it is a modifiable age-associated condition linked to dementia and late-life cognitive decline (Livingston et al., ARHL is an important marker in frailty (Panza et al., p = 0.003]. The association was more pronounced in females than in males (Ramage-Morin, Social isolation and loneliness is considered one of the risk factors for ARHL. Mick and Lin  found a Based on the above reported findings, there seems to be a bidirectional association between ARHL and cognitive impairment, depression and social isolation, depression, and frailty Figure . As showA number of limitations can be identified in the existing body of literature on neuro- patho-physiological changes in the peripheral and central auditory systems due to aging. As mentioned in the previous sections, ARHL is influenced by different factors including aging, lifestyle choices, genetics, and environmental factors (Van Eyken et al., In conclusion, aging results in pathological and physiological changes in both peripheral and central auditory systems. A number of genetic, environmental, and health co-morbid factors increase the risk of ARHL. Peripheral hearing loss further exacerbates the changes in the central auditory pathway. In this review, we have described the evidence for the association between ARHL and cognitive impairment, AD, depression, social isolation, and frailty. There is no substaintial evidence of how ARHL and AD-related biomarkers are associated prior to the clinical manifestation of the dementia syndrome. This is a necessary yet expensive research question that will inform our understanding of the statistical associations reported between the two. However, it is clear that ARHL represents a modifiable condition and a useful target for secondary prevention of cognitive impairment, frailty, social isolation, and depression in older adults. Further research is required to comprehensively address underlying mechanisms and determine whether hearing loss treatment could delay or arrest late life cognitive decline or dementia. Specifically, longitudinal studies including randomized clinical trials with representative samples of hearing aid, and hearing implant users and rehabilitation programs could further elucidate the association between ARHL and other components of our proposed model in Figure All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.DJ and PF have no conflicts of interest to declare. RM is the founder and Chief Scientific Officer of the Alzhyme and KaRa Minds Institute. HS has, and continues to receive remuneration from activities with Pfizer and Takeda Pharmaceuticals. Although grants and funds for our research were provided by public and private organizations, they in no way influenced any part of this review, its conclusions or the decision to submit this manuscript for publication to this journal.


The in vitro release study of the film-coated NPRBO tablets was performed in three-phase simulated gastrointestinal media. The cultivar KGLP was superior to the other samples in terms of the ORY, tocotrienol and tocopherol contents and anti-inflammatory activity. Aerosil® was the most suitable absorbent for transforming NPRBO into a free-flowing powder and was used to prepare the NPRBO core tablets. The in vitro KGLP-NPRBO film-coated tablet release profile showed that no ORY was released at gastric pH while 85% of ORY was released at pH 7.4 after 6 h; this would be expected to occur in the colorectal area. Therefore, this study demonstrates the potential of KGLP-NPRBO to prevent colorectal cancer via a specific colorectal dietary supplement delivery system.Colorectal cancer occurs due to various factors. The important risks are dietary lifestyle and inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis. It has been found that the inhibitory enzyme cyclooxygenase-2 (COX-2) in the colorectal region can potentially reduce the risk of colorectal cancer. The present study investigated rice bran oil from natural purple rice bran, which exhibits antioxidant and anti-inflammatory activity. This study aimed to evaluate the bioactive compound content of natural purple rice bran oil (NPRBO) derived from native Thai purple rice and the anti-inflammatory activity of NPRBO in colorectal cancer cells, and to develop a colorectal delivery platform in the form of film-coated tablets. NPRBO from the rice bran of five different Thai purple rice cultivars, namely Khao’ Gam Leum-Phua (KGLP), Khao’ Gam Boung (KGB), Khao’ Gam Thor (KGT), Khao’ Gam Pah E-Kaw (KGPEK), and Khao’ Niaw Dam (KND), were extracted using the supercritical carbon dioxide extraction technique. The amount of γ-oryzanol (ORY), tocotrienols, and tocopherols present in NPRBOs and the in vitro anti-inflammatory activity of NPRBO were investigated. The highest anti-inflammatory NPRBO was transformed into a dry and free-flowing powder by liquisolid techniques. Then, it was compressed into core tablets and coated with Eudragit Colorectal cancer is a type of cancer that develops in the colon or rectum. Symptomatically, patients suffer from bloody stools, weight loss, bowel movement changes , and fatigue. Colorectal cancer occurs due to various factors. Inadequate fiber intake and inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, significantly increase the risk of colorectal cancer ,2 The ocRice is an important plant for the Thai economy. It is one of the most famous Thai agricultural products and is exported worldwide. Rice bran is a valuable byproduct of the rice milling process. Particularly, it is rich in dietary fibers, γ-oryzanol, essential vitamin E complex, tocotrienols, and β-sitosterol . SeveralThai purple rice is a native plant that is cultivated in the Northern part of Thailand, particularly in the highlands and mountainous areas. The natural purple rice bran oil (NPRBO) obtained from the Khao’ Gam Pah E-Kaw, Khao’ Gam Thor, and Khao’ Gam Boung varieties with an γ-oryzanol content greater than 5% has demonstrated excellent in vitro antioxidant and inhibitory effects on nitric oxide production in mouse macrophages . To the ®L100, as the outer coating layer and a time-controlled release polymer, Eudragit® NE30D, as the inner coating layer of the NPRBO core tablets. The outer coating layer protects the core tablets from dissolving in the stomach, duodenum and early part of the ileum, and the inner coating layer delays NPRBO release until the colorectal area.Colorectal-targeted delivery is an approach that allows the active ingredient to be released in the specific region from the colon to the rectum. This can be achieved by using a polymer with pH-dependent solubility (soluble at pH 6.0–7.0) combined with a time-dependent controlled release polymer to prevent premature release in the gastrointestinal region. This can be achieved with a polymeric matrix containing active ingredient or polymer-coated tablets. This study selected the double coating layer approach, i.e., a pH-dependent soluble polymer, EudragitOur study aimed to evaluate the bioactive compounds in NPRBO, including γ-oryzanol, tocotrienols, and tocopherols and to investigate the anti-inflammatory activities of NPRBO by inhibiting inducible nitric oxide (iNOS) and COX-2 production in a cell-based study. Additionally, this study provided a proof of concept in vitro dietary supplement delivery system for NPRBO film-coated tablets, with the aim of releasing NPRBO in the colorectal region where it can act as a colorectal cancer chemopreventive agent.® NE30D and Eudragit® L100 were supplied by Evonik industries AG . Colloidal silicon dioxide (Aerosil®) was purchased from Bio-Chemical Technology Co., Ltd. . Microcrystalline cellulose Avicel® was purchased from Sigma-Aldrich .γ-Oryzanol was purchased from Wako Pure Chemical Industries (Japan). δ-, β-, γ-, and α-tocotrienol, and δ-, β-, γ-, and α-tocopherol were purchased from Merck Co., Ltd. . All solvents and chemicals used were either HPLC grade or analytical grade and were purchased commercially from Sigma Chemical Co. , Fluka Chemie GmBH, Merck , Invitrogen , and Roche . A murine macrophage cell line (RAW 264.7), a human adenocarcinoma cell line (HT-29), and a colorectal carcinoma cell (HCT 116) were purchased from American Cell Culture Collection . Talcum was purchased from Union Science Co., Ltd. . EudragitOryza sativa L.) were collected from Northern Thailand in December 2010. Khao’ Gam Leum-Phua (KGLP), Khao’ Gam Thor and Khao’ Gam Boung were collected from the Phob-Pra Agricultural Extension Office, Tak Province. Khao’ Gam Pah E-Kaw and Khao’ Niaw Dam were collected from the Mae Hong Son Rice Research Center, Mae Hong Son Province. All samples were dried in a hot air oven at 60 °C for 48 h. Then, fresh rice bran samples were obtained from a rice milling process  and sieved through a 60-mesh sieve, then subjected to a heating–cooling process to inactivate endogenous lipase [Five local glutinous purple rice cultivars  ,15. The ®PFP column was obtained from Phenomenex Co., Ltd. . The mobile phase consisted of methanol and de-ionized water in a ratio of 9:1. The flow rate was set at 0.6 mL/min. All samples were analyzed in triplicate.The δ-, β-, γ-, and α-tocotrienol and δ-, β-, γ-, and α-tocopherol contents in the NPRBOs were optimized according to the improved method of Pengkumsri et al.  with sliThe anti-inflammatory activities of NPRBOs were investigated through inhibitory effects on nitric oxide and iNOS production in RAW 264.7 cells. Moreover, the inhibitory effect of COX-2 production in HCT 116 and HT-29 cells was also investigated. The passage number of RAW 264.7 mouse macrophage and colorectal HCT116 and HT-29 for anti-inflammatory activity determination was between 5–10.TM M Cell Lysis Buffer  to perform the assay for iNOS. The quantity of nitrite in the culture medium was measured using Griess reagent as an indicator of nitric oxide production. Finally, the absorbance was measured at 540 nm against a standard curve of potassium nitrite [The inhibition of nitric oxide and iNOS production was assayed using the improved methods of Hong et al. , Hu et a nitrite . Fresh c nitrite . Concurr nitrite  and Saen nitrite  with sli5 cells/well) were pre-incubated in a black 96-well plate with a clear bottom and incubated for 24 h. Then, cells were given fresh medium containing various concentrations of the tested samples . After 12 h of incubation, LPS and IFN-γ were added. After 72 h of incubation, cell supernatants, which were extracted with 10 mM Tris, pH 8.0, 1% NP-40, 0.15 M NaCl, and 1 mM EDTA were collected to analyze total COX-2 levels. The procedure to determine the level of production followed the manufacturer’s protocol. Curcumin and γ-oryzanol were used as the positive controls. Concurrently, the viability of HT-29 and HCT116 cells were assayed using the cell proliferation reagent, WST-1. The amount of DNA was quantified by the Quant-iT PicoGreen Assay , in accordance with the manufacturer’s protocols, while that of the protein produced by HT-29 and HCT116 cells was analyzed using Bradford reagent prepared in-house [The inhibition of COX-2 production was assayed using a human total COX-2 immunoassay  following the methods of Hong et al.  and Coluin-house .® pH 101) and colloidal silicon dioxide (Aerosil®). Then, the binding solution, 10% PVP-K 90 in isopropanol, was added while continuously mixing until a wet mass was obtained. The damp mass was sieved through No. 8 mesh sieve, and the NPRBO granules were dried at room temperature overnight, followed by another sieving through No. 12 mesh sieve. Then, they were mixed with tableting excipients, including talcum (1%), Ac-Di-Sol® (0.5%), and magnesium stearate (0.5%), by tumbling the mixture in a polypropylene bag for 2 min. A B3B rotary tablet compressor , equipped with 8.2 mm dies, was used to prepare tablets, with a compression force between 12 and 15 kN. Some tablets were selected to perform tablet quality control tests, including assessing the γ-oryzanol content, weight variation, hardness, and friability. The hardness of the tablet was tested using a Monsanto Tablet Hardness Tester , and the average values were obtained. Tablet friability was measured using a friability tester .NPRBO was converted into a dry powder using absorbents, including microcrystalline cellulose (Avicel® NE30D (inner coating) and Eudragit® L100 (enteric coating), were coated onto the NPRBO tablets for the purpose of colorectal-targeted delivery.Two coating layers, including Eudragitw/v Eudragit® NE30D and 25% talcum as the coating material. The outer layer coating solution contained Eudragit® L-100 D and 25% talcum as the coating material. A Thai coater  was used to coat the NPRBO core tablets. The rotating rate of the coating pan was 9–11 rpm, the spraying rate of the coating solution was 2.83–3.11 g/min, the air pressure was 2.5–3.5 bar, the temperature in the coating pan was 97–103 °C, and the coating duration was 1.5–6 h, at 17–18% relative humidity. After coating, the tablets were cured at 40 °C for 2 h. Then, some of the coated tablets were used to perform tablet quality control tests as described in the previous section.Two types of coating solution (inner coating and outer coating solution) were used to coat the NPRBO core tablets. The inner layer coating solution contained 10% A dissolution apparatus II  was used to perform the dissolution study at 37 ± 0.5 °C with a paddle speed of 100 rpm. The NPRBO coated tablets were studied regarding the release of γ-oryzanol in a series of media, including 1 N hydrochloric acid, pH 1.2, for 2 h; phosphate buffer, pH 6.8, for 3 h; and phosphate buffer, pH 7.4, for 2 h, to simulate the human gastrointestinal environment. The media were sampled at time points of 120, 300, 310,320, 330, 360, 390 and 420 min. The γ-oryzanol in the media was evaluated by HPLC (Agilent 1200).The tablets were crushed, extracted with hexane, and then the solvent was evaporated. The extracts were analyzed for their γ-oryzanol content by HPLC, as stated previously.p-values ≤ 0.05 were considered significant.All of the results are expressed as the mean of three replicates ± standard deviation (SD). All statistical analyses were conducted using SPSS (version 16). In this study, it was found that the NRPBOs were comprised of δ-, γ-, and α-tocotrienol, δ-, γ-, and α-tocopherol, and γ-oryzanol, which corresponds to a previous report by Pengkumsri et al., who reported that Chiang Mai black rice bran oil prepared by supercritical carbon dioxide extraction is composed of δ-, γ-, and α-tocotrienol, δ-, γ-, and α-tocopherol, and γ-oryzanol, while Supanburi rice bran oil (white rice) is composed of δ- and γ-tocotrienol, δ- and γ-tocopherol, and γ-oryzanol . The amoThe anti-inflammatory- activity of the NPRBOs was determined through their inhibitory effects on nitric oxide and iNOS production in RAW 264.7 cells induced with LPS and IFN-γ as well as the inhibition of COX-2 production by HT-29 and HCT116 cells induced with LPS and IFN-γ. NPRBOs were effective in inhibiting the LPS/IFN-γ-mediated induction of nitric oxide, iNOS, and COX-2 production, which was comparable to the positive controls—curcumin and γ-oryzanol—as shown in 2, COX-2, and phospho-Akt (active) levels in the highly malignant (+SA) mammary epithelial cell line. Moreover, combined treatment with γ-tocotrienol and celecoxib also downregulated NF-κB and induced apoptosis in mammary epithelial cells [The results from this study demonstrate that NPRBOs exhibited potent anti-inflammatory activity, especially regarding an inhibitory effect on COX-2 production in both colorectal cell lines (HT-29 and HCT116 cells). COX-2 plays a vital role in colorectal carcinogenesis and nonsteroidal anti-inflammatory drugs (NSAIDs), especially COX-2 selective inhibitors, have potential chemopreventive properties in human colorectal cancer . Indeed,al cells . The pre® PH 101 and Aerosil®. The ratios of NRPBO extract to the absorbents that provided an appropriate damp mass were 1:4 and 1:1 for Avicel ® PH 101 and Aerosil®, respectively. Polyvinylpyrrolidone (PVP) was added to enhance the compression property of the KGLP-NRPBO tablets, and tableting excipients  were added to NRPBO granules to enable the tableting process. Both KGLP-NRPBO: Avicel® PH 101 1:4 and KGLP-NRPBO: Aerosil® 1:1 tablets showed less than one percent weight loss in the friability test, which is suitable for the film coating process [The KGLP-NRPBO extract was in liquid form; thus, to formulate it in tablets, the NRPBO was absorbed using the absorbents Avicel process .® PH 101 as the absorbent. This phenomenon contributes to the poor absorption properties of Avicel® PH 101 at the coating temperature. Consequently, the KGLP-NRPBO extract diffused from the core tablets to the films, and caused them to swell and peel off from the tablets. On the other hand, the core tablets that used Aerosil® as the absorbent were smoothly coated film tablets. Therefore, they were used in the in vitro release assay in simulated gastrointestinal pH media.In the coating process, the peeled film-coated tablets were observed in the batch that used Avicel® L-100 D was used as the outer coating of the KGLP-NPRBO tablets. Approximately 15% of γ-oryzanol was released in the small intestinal phase. This was a consequence of the Eudragit® L-100 D outer coat that dissolved at pH 6.8, so that the Eudragit® NE30D inner coating layer swelled but did not dissolve since it is a time-dependent controlled-release polymer. Thus, some γ-oryzanol diffused through the swelling layer of the Eudragit® NE30D in a zero-order fashion, according to the γ-oryzanol gradient between the core tablet and the media. Premature release is a common problem that occurs with time-dependent controlled-release polymers [® NE30D film was not thick enough, so, as a consequence, some γ-oryzanol was released earlier in the small intestinal phase. However, up to 85% of the γ-oryzanol was released in the colorectal phase, as the Eudragit® NE30D film dissolved entirely after 120 min. The lag-time of 5 h in the gastric and small intestinal phases was sufficient to deliver the KGLP-NPRBO tablets to the colorectal area, since the gastro-intestinal transit time is approximately 3–5 h, after which the bolus stays in the colon for the next 36–72 h [The simulated gastrointestinal study was divided into three phases: the gastric phase at pH 1, the small intestinal phase at pH 6.8, and the colorectal phase at pH 7.4. γ-Oryzanol was used as a marker for the in vitro release study. polymers . In this 36–72 h ,35. BaseIt was found that the NRPBOs were comprised of δ-, γ-, and α-tocotrienol, δ-, γ-, and α-tocopherol, and γ-oryzanol. Khao’ Gam Leum-Phua exhibited the highest δ- and γ-tocotrienol and δ-, γ-, and α-tocopherol contents, while the NPRBO prepared from Khao’ Gam Pah E-Kaw showed the highest α-tocotrienol and γ-oryzanol contents.The present results indicate that the NPRBOs exhibited potent anti-inflammatory activity in a cell-based study, comparable to the positive controls, curcumin and γ-oryzanol. The NPRBO prepared from Khao’ Gam Leum-Phua showed the highest inhibitory activity on nitric oxide and iNOS production in LPS/IFN-γ-stimulated RAW 264.7 cells and also exerted the highest inhibitory activity on COX-2 production in both colorectal cell lines, i.e., HT-29 adenocarcinoma cells and HCT116 carcinoma cells. It is suggested that there is a potential use for NPRBO containing tocotrienols, tocopherols, and γ-oryzanol as a colorectal chemopreventive agent.® as an absorbent with KGLP-NPRBO to produce KGLP-NPRBO core tablets. Eudragit® NE30D and Eudragit® L-100 D were successfully used as coating materials for delivering NPRBO in simulated gastrointestinal pH media. The film-coated NPRBO tablets did not show premature release at gastric and duodenum pH. Eighty-five percent of γ-oryzanol was released after 6 h at pH 7.4; thus, the film-coated NPRBO tablets are expected to reach the colorectal area. This study demonstrates the potential anti-inflammatory activity (COX-2 inhibition) of KGLP-NPRBO which is one of the pathways that causes colorectal cancer and provides proof of concept for a colorectal-specific dietary supplement delivery system. However, further in vivo and clinical studies are required to explore the pharmacological significance and pharmacokinetics of NPRBO in colon cancer and local diseases.Moreover, this study demonstrated that the NPRBO colorectal dietary supplement delivery concept is feasible by using Aerosil


Gamma Knife radiosurgery (GKRS) is an important part of the neurosurgical armamentarium in the treatment of acoustic neuromas. However, the treatment outcome related to the morphology of the tumor has not been rigorously studied. In this cohort, we evaluated the morphological features of the tumor in the tumor response and neurological outcomes after GKRS.From July 2003 to December 2008, there were 93 cases of acoustic neuromas treated upfront with GKRS with 64 cases with serviceable hearing and 29 cases without serviceable hearing to fulfill the margin dose of 12Gy with at least follow up 5 years.p < 0.001) and associated no-hearing symptom was 70% and 35%, respectively (p < 0.001). There was 81.2% of hearing preservation after GKRS in serviceable hearing group including 27 cases of pear type (84%), 14 of linear type (70%), and 9 cases of sphere type (90%) (p < 0.01); however, there was no case of hearing improvement in the no-serviceable hearing group (0 of 29). There were 85% of patients with decreased tinnitus in serviceable hearing groups as compared to 61.5% of patients in no serviceable hearing group (p < 0.05). In multivariate analysis, the tumor morphology was highly correlated to hearing preservation rate (p < 0.01).The duration of symptom before GKRS in serviceable /no serviceable hearing was 7.9 ± 1.2 and 15.3 ± 3.1 months (In the limited case of this cohort, we found that the tumor morphology and timing of treatment was highly correlated to the rate of hearing preservation. The sphere type of tumor morphology was associated with the best chance of hearing preservation. Gamma Knife Radiosurgery (GKRS) serves as a powerful tool in the neurosurgical armamentarium for the treatment of small to moderately sized acoustic neuromas , 2. In l. In general, a margin dosage less than 13 Gy were more likely to preserve hearing compared to earlier series that used higher margin doses [. With dose less than 12.5Gy, further reduction in cranial neuropathy was observed [. Either volume of tumor or tumor growth rate remain controversial in predicting the preservation of hearing after GKRS and some have even implicated that a large tumor did not increase the risk of hearing loss as compared to the smaller tumor [. Those with pre radiosurgical hearing function of Gardner-Robertson Class I showed better preservation than those GRC of II-IV [. The cochlear dosage limited within 4 to 5.3 Gy showed the better hearing preservation and further reduction to a dose less than 2.7 Gy exerted still better hearing preservation [. In addition, the subset of tumor morphology with large intracanaicular volume exerted worse hearing preservation [There were several factors contributing to high hearing preservation rate including the peripheral dosage of tumor, tumor size, cochlear dose, volume of intracanalicular portion of the tumor, and pre-radiosurgical hearing capacity –20. In gin doses . With doobserved . Either er tumor , 22. Thoof II-IV , 14. Theervation , 23. In ervation , 24, 25.. In addition, the scope of brain stem compression related to hearing preservation was still debated [. The shape and morphologic alteration of acoustic neuromas subjected to gamma knife treatment and their implications in terms of hearing preservation have not been rigorously investigated. In this study, we retrieved the data from our radiosurgical patient registry and investigated the relationship of the shape of tumor, grade of brainstem compression, pre-radiosurgical hearing function and associated symptoms, and radiosurgical parameters to radiological tumor response and hearing preservation after GKRS.As known, the tumor volume alteration responded to gamma knife treatment was different with smaller volume change in the intracranial portion and larger tumor response outside the intracranial portion . In addi debated , 27. TheFrom July 2003 to December 2008, there were 150 cases of acoustic schwannoma treated with GKRS. There were 32 cases with previous operation, 10 with neurofibromatosis type II, and 10 cases without regular follow up which were excluded in this study. Seven cases were excluded from this study due to not be categorized into either of three types of tumor morphology. Finally, there were only 93 cases entered into this study. The data analysis has been approved by Taichung Veterans General Hospital Institute Review Board (CG12319B).Stereotactic radiosurgery was performed using a Leksell Gamma Knife model 4C (Elekta AB). Treatment planning was performed using Leksell GammaPlan software . A dose of 12 Gy was prescribed to the 50% isodose line, and it covered more than 95% of the tumor in all treatments.Patients were followed up at our otolaryngology and neurosurgical outpatient clinics 3 months after radiosurgery and then 6 months periodically until the last follow up. MRI follow up was conducted 6 months after GKRS and then yearly until last follow up. The clinical data obtained at follow-up included the audiogram and a detailed neurological function assessment.Serviceable hearing was defined as a speech repetition threshold or pure tone audiogram value less than 50 dB and a speech discrimination score of 50% or greater.The HB scale was used to approximate the quantity of facial nerve function that the patient had at presentation as well as at each follow-up interval. The HB scale has 6 grades and each grade is reported as a fraction . In the HB scale, Grade I indicates perfectly normal, Grade II indicates slight or mild weakness, Grade III indicates moderate weakness with good  eye closure, Grade IV indicates moderate weakness with no volitional eye closure, Grade V indicates severe weakness, and Grade VI indicates total facial paralysis.The MR imaging slice thickness was 1-2 mm for T1-weighted images or time of flight with and without Gd contrast administration for tumor morphologic assessment. The MR imaging slice thickness was 1-2 mm for T2-weighted images and FLAIR without contrast administration for the assessment of surrounding tissue reaction. The tumor volumes were determined on MR imaging images using a picture archiving and communications system (PACS) and GKRS planning for further assessment.The calculation of brainstem compression was assessed including the brainstem compression and fourth ventricle compression ratio. For this calculation, the A line was determined from the midpoint between bilateral internal carotid artery and junction of bilateral transverse sinus. B line was drawn perpendicular to the A line at the maximum site of tumor compression. The distance of the tumor to the A line divided by the distance from the brainstem surface to the A line was determined as brain stem compression. C line was determined by the line perpendicular to A line at the maximum diameter of 4th ventricle. The distance of border of the fourth ventricle to A line divided by the distance from the fourth ventricle surface to A line at the opposite side was determined as the degree of fourth ventricle compression Fig. .Fig. 1IlA line to determine the intra/extracanalicular tumor /nerve was made along the ridge of petrous bone to posterior surface of the petrous bone. The tumor lateral to this line was considered to intracanalicular part and medial to this line was considered to extracanalicular part. The total length of facial-vestibular bundle was determined from the surface of brainstem to cochlea. The nerve width was determined by the maximum width of the nerve bundle in the consecutive MRI. The tumor morphology was categorized into three types. The linear type was defined as the morphology of tumor when it was linear shaped either with the internal canal, outside the canal, or combined both Fig. . The peaThe descriptive statics were computed using the standard methods to calculate median and mean values. Univariate and multivariate analysis were performed to assess for variable predictive of preservation of serviceable hearing after GKRS. The following variables were assessed: the tumor morphology, length of tumor in IAC, width of tumor in IAC, percentage of brain stem compression, percentage of fourth ventricle compression, cochlear dosage, dosage to the lateral semicircular canal, dosage to the median semicircular canal, dosage to the inferior semicircular canal, maximum brainstem (BS) dosage, and 10 Gy volume in brainstem. The unpaired student t test or analysis of variance was used for continuous variables. Nominal or ordinal data were compared using the two-tailed Fisher exact test. All statistical analyses were conducted with the aid of commercially available software (SPSS 16.0). Statistical significance was defined as a probability of value less than or equal to 0.05.3. The length of opposite side of vestibular-facial nerve bundle was 19.9 ± 0.2 mm. The mean diameter of opposite side of ICA was 4.3 ± 0.6 mm. The morphology analysis showed 60 cases with pear type, 22 cases of linear type, and 11 cases of sphere type.There were 93 cases in which GKRS was used as the upfront treatment. The male to female ratio was 60 to 33. The right and left lateralization was 60 to 33. The median age was 58(57.7 ± 15.1) years old. The follow up period was 76.3 ± 3.6 months. There were 64 cases with serviceable hearing and 29 cases without serviceable hearing at the time of presentation for GKRS. There were 5 cases that presented with hemifacial spasm. One case presented with grade I facial palsy. There were 46 cases with tinnitus and 7 cases of facial numbness. One case had trigeminal neuralgia. In imaging analysis, there were 20 cases with tumor invasion to trigeminal nerve. There were 11 cases of brainstem dysfunction. There were 43 cases of tumor involved the whole vestibular-facial nerve bundles and 50 cases with only part of the tumor located within the IAC. The mean tumor volume was 3.14 ± 0.05 cmP < 0.05)The demography of patient without operation before GK was sub-categorized into serviceable hearing and no serviceable hearing showed in Table p < 0.05), length  and width of tumor  in ICA and 10 Gy volume in brain stem .The anatomic structure and associated radiation dosage are depicted in Table P < 0.01). There were 85% of patients with decreased tinnitus after GKRS, and 61.5% of patients in non-serviceable hearing group had decreased tinnitus (p < 0.05). One of 64 cases in the serviceable hearing presented with severe dizziness and underwent a tumor resection with improvement of dizziness but resulting hearing loss  as compared to pear type (84% hearing preservation) (27 of 32 patients) and linear type (70%) (14 of 20 patients). In the non-serviceable hearing group, there was 48% of linear group (11 of 23 patients) and 40% of linear group (2 of 5 patients) retained stable hearing. However, there was no patient who obtained serviceable hearing after GKRS in the non-serviceable hearing group.P < 0.01). The length of tumor in the IAC were significant higher in the linear type than the pear and sphere type (p < 0.01). The width of tumor in the IAC was significant higher in the linear and pear type than in sphere type (p < 0.01). The percentage of brainstem compression was higher in the sphere and pear types than in linear type (p < 0.05). The percentage of 4th ventricle compression was significant higher in sphere type than the pear and linear types (p < 0.01).There was significant higher cochlea dose in the linear and pear type than the sphere type (p < 0.01). The dose to the semicircular canal was higher in the linear and pear types than in sphere type . There were significant higher dosage to the brainstem in the sphere and pear type than linear type (p < 0.01). The associated brainstem 10 Gy volume was also higher in the sphere and pear type than the linear type.The distributions of patients by tumor morphology is illustrated in Table The uni-variate and multi-variate analyses are shown in Table . In this study, the tumor volume seemed to be larger in the sphere type, but this same morphological type predicted improved high hearing preservation despite the overall larger tumor volume. In contrast, the morphology of the tumor rather than tumor volume better predicted the probability of hearing preservation.Beyond local tumor control and maintenance of facial nerve function, hearing preservation is a paramount outcome to assess after GKRS. The initial tumor volume was a crucial factor to determine the radiologic response after GKRS. However, Gamma Knife treatment parameters can be correlated to tumor volume including the radiation dosage to the cochlea, brainstem, and semicircular canal , 22. In . In this study, we found that the morphology of the sphere type demonstrated better hearing preservation rate as compared to the pear and linear type of tumors. The ratio of brain stem compression in sphere type was highly less than the pear type but there was no significant difference between the pear types. This data suggests that brainstem compression was not a key factor to predicting the outcome of hearing preservation. On the contrary, the initial tumor growth pattern was predictive of hearing preservation at last follow up.The location of tumor and degree of brainstem compression were reasonable predictors of hearing outcome after GKRS. There was higher hearing preservation in tumor without the involvement of the whole internal acoustic canal, a greater distance away from the cochlea, and less brainstem compression , 22, 23.. In this study, we found that a high proportion of serviceable hearing was noted in those patients with shorter interval of symptom as compared to those longer symptoms. In addition, there was no patient without serviceable hearing that regained serviceable hearing after GKRS. These data support early treatment as a crucial factor for better hearing preservation.The timing of treatment for acoustic neuromas especially in those patients with small-medium size of tumor is hotly debated. There were some reports concerning the treatment of acoustic tumor with deterioration of hearing after GKRS and considered to be an adverse effect from the radiation. “Wait and see” treatment option is advocated in some studies. But there were also researchers that encourage the treatment as soon as possible and against the “wait and see” policy , 28, 29.. This study found that no hearing associated symptom was significantly higher in serviceable hearing than in the serviceable hearing group, but the higher regression of no hearing associated symptom was noted in the serviceable hearing. It can be explained to be that the no hearing associated symptom appeared earlier than hearing impairment symptom and it forward the patient to receive examination and entered into the treatment program.The response of no hearing associated factors after GKRS seemed to be a facilitated factor for assessment of hearing outcome. In some reports, the regression of no hearing associated factor exerted the higher hearing preservation rate than those without regression , 30. ThiIn this study, most of the small to medium sized acoustic neuromas can be morphologically categorized into the sphere, pear and linear types. Based on the classification, the sphere type of tumor had the original site of tumor growth outside the internal acoustic canal; the pear type of tumor had the growth pattern either from the originality outside the IAC with some part of tumor invasive to proximal part of IAC or from the distal part of IAC with most of tumor invasive outside the IAC; the pear type had the originality of tumor in the ICA canal. There was a high degree of hearing preservation in the sphere type followed by the pear and to a lesser extent the linear type. This suggests that of the tumor growth and morphology seem to be a crucial determinant in predicting the result of hearing outcome after GKRS.. The number of cases in this series is small, which attenuated the statistic power in prediction.There were several limitations in this study. First, some of acoustic neuroma could not be subcatergozied into either of linear, pear and sphere type and it reduced the power in prediction of hearing preservation. The numbers of slices obtained from MRI for the calculation of values in anatomic structure were various in different sizes of tumor , which may be a confounding factors in measurement . The numThose with fewer symptoms and favorable hearing status at GKRS were more likely to have hearing function at the last follow up. The tumor morphology influenced the outcome of hearing and those with a spherical tumor were likely to have high hearing preservation rate.


Early hearing deprivation could affect the development of auditory, language, and vision ability. Insufficient or no stimulation of the auditory cortex during the sensitive periods of plasticity could affect the function of hearing, language, and vision development. Twenty-three infants with congenital severe sensorineural hearing loss (CSSHL) and 17 age and sex matched normal hearing subjects were recruited. The amplitude of low frequency fluctuations (ALFF) and regional homogeneity (ReHo) of the auditory, language, and vision related brain areas were compared between deaf infants and normal subjects. Compared with normal hearing subjects, decreased ALFF and ReHo were observed in auditory and language-related cortex. Increased ALFF and ReHo were observed in vision related cortex, which suggest that hearing and language function were impaired and vision function was enhanced due to the loss of hearing. ALFF of left Brodmann area 45 (BA45) was negatively correlated with deaf duration in infants with CSSHL. ALFF of right BA39 was positively correlated with deaf duration in infants with CSSHL. In conclusion, ALFF and ReHo can reflect the abnormal brain function in language, auditory, and visual information processing in infants with CSSHL. This demonstrates that the development of auditory, language, and vision processing function has been affected by congenital severe sensorineural hearing loss before 4 years of age. Individuals with congenital sensorineural hearing loss usually have no hearing experiences after birth. Approximately 1‰ to 6‰ newborns suffered from severe to profound sensorineural hearing loss . Early hThere is the cross-modal reorganization of auditory-related brain area after long-term hearing deprivation , 15. FunBrain development is a gradual process of unfolding of a self-organizing and highly synchronous network from complex interactions between internal and external environment. The studies from both animal models and human children have demonstrated that the maturation of auditory cortex is critical at the first few years of life , 23. LotThe advances of fMRI had made it possible to study the intrinsic functional organization of the brain. For example, resting-state fMRI could be used to investigate the spatial-temporal correlations within the functional brain regions during rest, especially in the deaf children who could not cooperate the other kinds of task performance. The amplitude of low frequency fluctuations (ALFF) is one of the parameters to measure the total power in the range of 0.01 and 0.1 Hz which could be an index to reflect the neurophysiological changes in different brain diseases –30. RegiThis study aimed to use resting-state functional MRI to study the intrinsic functional changes of brain area due to auditory deprivation in infants with congenital severe sensorineural hearing loss.A total of 23 infants with congenital severe sensorineural hearing loss (CSSHL) who did not pass hearing screening using auditory brain stem response (ABR) test at 3 days and 42 days after birth were retrospectively included in our study. The ABR results of all infants showed greater than 90 dB which indicates severe or profound sensorineural hearing loss. The age of deaf infants at MRI examination was from 6 months to 48 months (mean age 24.18 ± 14.00 months). Seventeen age and sex matched normal hearing subjects were recruited. The age of the control group at MRI examination was from 11.2 months to 49 months (mean age 26.35 ± 12.67 months). Excluding criteria include a variety of central nervous system diseases, such as white matter hypoplasia, abnormal neuronal migration, and neuronal skin syndrome, tumor, trauma, infection, epilepsy, and so on. Informed consent was signed by the parents of the infants and all the examinations were approved by the hospital ethics committee.All the subjects were scanned with MRI at the 3.0T MR scanner  and 16-channel standard quadrature head coil. Before MRI examination, all the infants were given the oral administration of 10% chloral hydrate with an amount of 0.6 mL per kg and the maximum amount of no more than 80 mL.The detailed MRI sequences include the following: firstly, spin echo  and fast spin echo  and whole brain T1WI and T2WI were acquired to exclude central nervous system abnormalities. The parameters were as follows: T1WI: TR/TE = 300/2.5 ms, slice thickness = 4 mm, the interlayer spacing = 1.2 mm, matrix = 320 × 320, FOV = 220 × 220 mm, flip angle = 70°, NEX = 1,25 transverse slices covering the whole brain, T2WI: TR/TE = 6000/93 ms, slice thickness = 4 mm, the interlayer spacing = 1.2 mm, matrix = 320 × 320, FOV = 220 × 220 mm, and flip angle = 120°. Secondly, gradient-echo echo planar imaging (GRE-EPI) was scanned and the detailed parameters are TR/TE = 3000 ms/30 ms, FOV = 220 × 220 mm, flip angle = 90°, matrix = 64 × 64, slice thickness = 3 mm, 210 frames, and 38 transverse slices without gap covering the whole brain. Finally, magnetization prepared rapid gradient echo imaging (MP-RAGE) sequences. The parameters were set as follows: TR/TE = 1900/2.53 ms, FOV = 250 × 250 mm, flip angle = 9°, matrix = 256 × 256, slice thickness = 1 mm, and 160 sagittal slices without gap covering the whole brain.http://www.fil.ion.ucl.ac.uk/spm) and a pipeline analysis toolbox, REST, and DPARSF (http://www.restfmri.net/) [http://resting-fmri.sourceforge.net). Finally, the images were smoothed with a Gaussian filter of 6 mm full width at half maximum (FWHM). Template selected was infant template (using 9–15-month-old infant template) provided by the Imaging Research Center (https://irc.cchmc.org/software/infant.php).The resting-state fMRI data were preprocessed using SPM8 (ri.net/) –34. The  t-test was conducted based on the ALFF and ReHo maps by REST software. Age and gender were selected as covariates. AlphaSim corrected p < 0.05 (cluster size > 228 voxels). Correlation analysis was performed to calculate the relationship between ALFF and age in two groups by using SPSS version 19.0 . Significant difference was set at p < 0.05. The value of Cohen d was used to describe the effect size (ES).Two-samplep = 0.0059, ES = 0.94), left superior temporal gyrus (BA22) , left inferior frontal gyrus (BA44) , left inferior frontal gyrus (BA45) , left inferior prefrontal gyrus (BA47) , and left dorsolateral prefrontal cortex (BA46)  in infants with CSSHL (deaf < hearing), which are responsible for auditory, language, and executive function.ALFF differed significantly between the deaf and hearing group in left Heschl's gyri (BA41) , right BA19 , right BA17 , and right BA39  (deaf > hearing), which are responsible for processing visual information , left inferior frontal gyrus (BA45) , left inferior prefrontal gyrus (BA47) , left dorsolateral prefrontal cortex (BA46) , left medial frontal gyrus (BA32) , and left temporal polar gyrus (BA38)  (deaf < hearing), which are responsible for auditory processing, language perception, executive function, and behavior and decision-making.ReHo differed significantly between the deaf and hearing group in left superior temporal gyrus (BA22) , BA19 , and BA17  (deaf > hearing), which are responsible for visual information processing , but it showed an increased trend in the control group, though there is no significant correlation  . However, there was no significant correlation in control group   in both groups, which included left BAs 22, 41, 44, 45, 46, and 47 and right BAs 18, 19, and 39. The correlation between ALFF of these ROIs and age was calculated. ALFF of left BA45 was negatively correlated with age and showed a decreasing trend in the deaf group along with age increased (= 0.512) . ALFF of= 0.378) .r = 0.530, p = 0.029), but it showed no significant correlation in the deaf group  (p > 0.05). All above results indicated that the function of auditory, language, and vision was changed in infants with CSSHL.ALFF value of left BA47 was positively correlated with age and showed an increasing trend in the control group (= 0.989) . There wChanges of intrinsic brain organization are essential for exploring infants with the congenital severe sensorineural hearing loss (CSSHL). This is the first study using resting-state fMRI to evaluate the function of auditory and language-related brain areas in infants with CSSHL before four years of age. There were some important findings. ALFF and ReHo values in the deaf infants decreased in auditory, language processing, and executive function related brain areas. ALFF and ReHo values in the deaf infants increased in occipital lobe, which is responsible for visual information processing. Another important finding was that the changes of ALFF of left BA45 were positively correlated with deaf during, and ALFF of right BA39 was negatively correlated with deaf duration in infants with CSSHL. ALFF of left BA47 was also found positively correlated with age in normal subjects again, but the correlation disappeared in infants with CSSHL.ALFF indirectly reflects the spontaneous neural activity and indicates the functional changes of brain activity , 36. In Executive function includes lots of organizational and self-regulatory skills, such as personal and social behavior, decision-making, and emotional and cognitive processing , 45 In tIn deaf infants, not all the executive functions were impaired; some of them are preserved and even enhanced , 46, 47.The right occipital gyri  are responsible for visual information processing. In the present study, both ALFF and ReHo values of BA18, BA19, and BA17 increased. It suggests that the vision function had been enhanced due to the hearing loss. Another study also suggested the compensatory enhanced visual process after auditory deprivation, and meanwhile vision cortex is much more sensitive to reorganization or neuroplasticity without auditory input , 48. BehInterestingly, the significant correlation was found between ALFF of left BA45, right BA39, and deafness duration in deaf infants. ALFF of left BA45 was found to be negatively correlated with the deprivation duration in deaf infants. In contrast, no significant correlation was found between ALFF of left BA45 and age in normal controls although there was an increasing trend along with aging. This demonstrated that the function of left BA45 which is related to language was impaired more seriously along with deaf duration. ALFF of right BA39 was found to be positively correlated with the deprivation duration in deaf infants but with no significant correlation with age in hearing infants. The cortex is responsible for spatial orientation and semantic sensation. This suggests that the function of spatial orientation and semantic sensation was enhanced along prolonged deafness duration. Significant positive correlation was found between ALFF of left BA47 and age in normal infants, but no correlation was found in deaf infants. It may conclude that the function of left BA47 is enhanced along with age increasement in normal infants, but this increased trend disappeared in deaf infants, which suggests that the executive function is the cross-modal plasticity caused by the deprivation of auditory experience.The primary limitation of this study was the small sample size of infants (hearing loss and hearing group). Additionally, clinical information about the function of auditory and language recovery in deaf infants with cochlear implant was not followed up. The correlation between the recovery and abnormal function of auditory and language-related brain areas in deaf infants' precochlear implant needs to be investigated in the future study.In conclusion, ALFF and ReHo reflect the abnormal brain function in language, auditory, and visual information processing in infants with CSSHL before 4 years of age. Congenital severe sensorineural hearing loss affected the development of auditory and language processing function.