Some Alzheimer’s disease (AD) patients die without ever developing cognitively impaired basic activities of daily living (basic ADL), which may reflect slower disease progression or better compensatory mechanisms. Although impaired basic ADL is related to disease severity, it may exert an independent risk for death. This study examined the association between impaired basic ADL and survival of AD patients, and proposed a multistate approach for modeling the time to death for patients who demonstrate different patterns of progression of AD that do or do not include basic ADL impairment.1029 patients with probable AD at the Baylor College of Medicine Alzheimer’s Disease and Memory Disorders Center met the criteria for this study. Two complementary definitions were used to define development of basic ADL impairment using the Physical Self-Maintenance Scale score. A weighted Cox regression model, including a time-dependent covariate (development of basic ADL impairment), and a multistate survival model were applied to examine the effect of basic ADL impairment on survival.As expected decreased ability to perform basic ADL at baseline, age at initial visit, years of education, and sex were all associated with significantly higher mortality risk. In those unimpaired at baseline, the development of basic ADL impairment was also associated with a much greater risk of death (hazard ratios 1.77–4.06) over and above the risk conferred by loss of MMSE points. A multi-state Cox model, controlling for those other variables quantified the substantive increase in hazard ratios for death conferred by the development of basic ADL impairment by two definitions and can be applied to calculate the short term risk of mortality in individual patients.The current study demonstrates that the presence of basic ADL impairment or the development of such impairments are important predictors of death in AD patients, regardless of severity. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that impairs cognition and daily functioning. Affected patients all die with AD, although the factors that predict survival overlap with those that predict survival in non-demented individuals of similar age: especially age, education, and comorbid conditions –11. StroIf the development of basic ADL impairment exerts an effect on survival that is separate from disease severity, estimating the time to death for patients with and without such basic ADL impairment could be helpful in planning effectively for future demands on medical and social resources . SpecifiPatients came to the Baylor College of Medicine Alzheimer’s Disease and Memory Disorders Center (ADMDC) between January 1989 and February 2008 were eligible to participate in the ADMDC longitudinal follow-up database . This stOf 1484 probable AD patients , were added to the final Cox model to evaluate their corresponding effects on survival, adjusting for the effects of other selected variables. In addition, the inverse probability of censoring weights (IPCW) [First, descriptive statistics of the demographic and clinical characteristics were generated, and comparisons between the two groups of interest (patients with intact ability to perform basic ADL and those with impaired ability to perform basic ADL) were performed using either the s (IPCW)  was usedFinally, the effect of change in basic ADL status on the prediction of patient outcome was examined using a novel application of multistate survival analysis methods –22. In tBaseline characteristics of the 1029 patients in our sample are shown in In the weighted Cox regression model , age, edOf the 458 patients whose basic ADL were not impaired at baseline 50.9% (N = 233) developed such impairment by the first definition (PSMS ≥ 7) and 41.5% (N = 190) by the second definition (gained 2 or more points). Because the baseline model does not quantify the effect of developing basic ADL impairment on survival over time, we looked at a time-dependent model. The results of the Cox regression analysis including time-dependent variables (the development of basic ADL impairment) are also displayed in Finally, we used a novel multistate modeling approach to estimate the appropriate coefficients or contributions of each variable to the risk of death. We included all potential predictors from the baseline model . We set up a multi-state Cox model defining one state as pre-basic ADL impairment and one state as post ADL impairment. First we designated basic ADL impairment as the outcome event and, the best-fit model revealed that only age at first visit, baseline PSMS and MMSE scores were significant predictors of time to impairment, when controlling for other relevant factors . We thenIn order to demonstrate the value of the multi-state model, we generated survival curves for male and female example patients. ADMDC population information was used to determine the characteristics of an average patient: 75.1 years old at the initial clinic visit with 13.6 years of education and a baseline MMSE score of 19.0. We used the estimates from The current study is the first to demonstrate a strong relationship between either the presence or development of basic ADL impairment and the risk of death among AD patients, and the first to quantify this risk over a relevant clinical interval (6 years). The development of basic ADL impairment was a strong signal of increased likelihood of death in both men and women, surpassing the importance of the known risk factors of age, sex, and baseline cognitive scores. This increase in risk was significant and substantial even when disease severity was controlled for in the analysis. Our data can be used to provide quantitative estimates of how changes in an individual patient’s functional abilities will affect their predicted survival over a 6-year period.et al who investigated residents newly admitted to nursing homes with a diagnosis of probable Alzheimer’s disease found that the baseline basic ADL impairment was associated with increased mortality. However, Aguero-Torres et al found the impaired baseline functional status was associated with decreased survival in demented adults, but it became insignificant in patients with Alzheimer’s disease.Other results used in our model were consistent with previous studies showing that male sex, older age, less education, lower baseline MMSE score, and lower baseline basic ADL function significantly increase the risk of death in patients with AD –11,24–28Researchers have not reached a consensus regarding the best methods for modeling the progression of Alzheimer’s disease ,30. To bOne advantage of the current study is that it included a larger cohort of AD patients than many other studies. In fact, the participants in the Baylor ADMDC cohort represent one of the largest samples of individuals diagnosed with AD by standardized criteria and followed longitudinally in the United States. Another strength of our study was that only cases diagnosed as probable AD by NINCDS-ADRDA criteria  were useDespite the benefits of our large sample size, this particular cohort does have some limitations. The Baylor College of Medicine ADMDC is a specialty center associated with an academic institution. The sample used in this non-population-based study may contain a disproportionate number of well-informed participants who have better access to health care and fewer comorbid conditions  than the general population . HoweverThere is a relative lack of data in the literature regarding longitudinal aspects of AD. It is essential to determine which factors contribute to AD patients’ risk of death so that preventive and protective measures may be implemented and to improve planning for resources over the course of the disease. Such analyses have clear implications for health economic analysis and public policy. The current study demonstrates that both relative and absolute impairment in basic ADL are important predictors of death in patients diagnosed with AD even when controlling for other factors that influence survival. Our method can quantify this increased risk of death in individual patients when they develop such impairment. In the future, we hope to explore how AD treatment influences the likelihood of developing basic ADL impairment at any given time, as well as the survival of patients who have already developed such impairment.S1 File(XLSX)Click here for additional data file.


Diplodocus, Spinosaurus, Tyrannosaurus) have accessory intervertebral articulations that have been suggested to support these large body sizes. Some pseudosuchian archosaurs have been reported to have these articulations as well, but few have been characterized in these taxa because of a lower abundance of complete, three-dimensional pseudosuchian vertebral material in relation to dinosaurs. We describe the axial column of the large (∼4–5 m) poposauroid pseudosuchian Poposaurus langstoni from the Upper Triassic of Texas . P. langstoni was originally named from pelvic girdle elements and vertebrae; here we describe newly discovered and prepared presacral vertebrae and a presacral rib from the original excavation of the holotype in the 1940s. The well-preserved vertebrae have well-defined vertebral laminae and clear hyposphene–hypantrum intervertebral articulations, character states mentioned in pseudosuchians but rarely described. The new material demonstrates variation present in the hyposphene–hypantrum articulation through the vertebral column. We compared these morphologies to other pseudosuchians with and without the hyposphene–hypantrum articulation. Based on these careful comparisons, we provide an explicit definition for the hyposphene–hypantrum articulation applicable across Archosauria. Within Pseudosuchia, we find the hyposphene–hypantrum appeared independently in the clade at least twice, but we also see the loss of these structures in clades that had them ancestrally. Furthermore, we found the presence of large body sizes  and the presence of the hyposphene–hypantrum is correlated in most non-crocodylomorph pseudosuchian archosaurs with a few exceptions. This result suggests that the presence of the hyposphene–hypantrum is controlled by the increases and decreases in body size and not strictly inheritance.Dinosaurs and their close relatives grew to sizes larger than any other terrestrial animal in the history of life on Earth, and many enormous dinosaurs (e.g.,  The clade Archosauria, which includes extant birds and crocodylians, contains some of the largest land animals ever to exist  from the Dockum Group  in detail that were previously figured by P. langstoni we describe here is of interest because it includes a variety of presacral vertebrae  from throughout the column, and it is preserved in three dimensions so that articulation surfaces are easy to discern. These characteristics are not present in most Triassic pseudosuchian fossils , Dockum Group, Howard County, TX, USA. Biostratigraphic and lithostratigraphic correlations indicate the Otis Chalk localities are Upper Triassic (latest Carnian–early Norian) ( Norian) .Holotype: TMM 31025-12, right ilium.Previously Referred Specimens:TMM 31025-257, right ischium.TMM 31025-259, fused last cervical  and first trunk  vertebrae.TMM 31025-177, presacral vertebra .TMM 31025-257, presacral vertebra (anterior trunk).Newly Referred Specimens:TMM 31025-1261.5, presacral vertebra .TMM 31025-1262, presacral vertebra .TMM 31025-1261.3, presacral vertebra .TMM 31025-1261.4, presacral vertebra (anterior to mid-trunk).TMM 31025-1261.1, presacral vertebra (anterior to mid-trunk).TMM 31025-1261.2, presacral vertebra (mid- to posterior trunk).TMM 31025-2160, most of a presacral rib.Justification for Newly Referred Material:The holotype and the newly referred material were collected from the same quarry (TMM 31025 Quarry 1) during the 1940 and 1941 excavations . The preservation and color of the holotype, referred, and newly referred material are identical. The relative size is also consistent between this new material and the previously referred material and holotype, but appears to represent two individuals (see below) .P. langstoni based on general morphology of their centra and neural arches, well-defined vertebral laminae, and an accessory rib articulation in the posterior cervical vertebrae .We assign six presacral vertebrae to ertebrae  and 3. Tangstoni . The posangstoni  because fragilis  and otheiquensis . The sha ischium , we can P. langstoni based on a bimodal distribution of the prezygapophyseal height to centrum height, and it appears at least one of the vertebral positions is duplicated. We refer two of these vertebrae  to the same individual  because they most closely resemble that material in size. These vertebrae  were both figured but not fully described by Lythrosuchus langstoni, because these presacral vertebrae were more compressed and taller than previously described material of P. gracilis and possessed an accessory rib articulation. Poposaurus, because a specimen of P. gracilis (TTU P-10419) also possessed the accessory rib articulation in a posterior cervical vertebra. The presacral specimens TMM 31025-1261.5, TMM 31025-1262, TMM 31025-1261.3, TMM 31025-1261.4, TMM 31025-1261.1, and TMM 31025-1261.2 we refer to as belonging to individual “B”  as the dual “B”  because Poposaurus langstoni is only known from a single locality and other archosauromorphs are known from the same locality. The stem archosaur Trilophosaurus buettneri and a phytosaur are also known from TMM 31025, Quarry 1. The vertebral morphologies of the three taxa known from the locality are disparate and can easily be distinguished from one another. For example, the centra of T. buettneri are much longer anteroposteriorly than they are dorsoventrally tall, whereas the centra of P. langstoni are dorsoventrally taller than they are anteroposteriorly long. The vertebrae of T. buettneri  are also much smaller in overall size than the known material of P. langstoni , Parringtonia gracilis (NMT RB426), and a number of specimens of Alligator mississippiensis .The axial column of slightly  and 3. EP. langstoni, cervical vertebrae were identified by the presence of the parapophyses on the anterior rim of the centrum located anywhere from the base of the centrum to the base of the neural arch just ventral to the location of the neurocentral suture, whereas the parapophyses of trunk vertebrae are only present on the neural arch. The parapophyses migrate dorsally from the base of the centrum posteriorly along the tetrapod vertebral column (such as in P. gracilis and A. mississippiensis). Because the diapophyses are located on the neural arch in vertebrae posterior to presacral 7 in other paracrocodylomophs  we can constrain the position of the anteriormost vertebra of P. langstoni (TMM 31025-1261.5) at presacral ∼7 or 8. This vertebra also has a very slight ventral keel, which further constrains its position to presacral ∼7 or 8 because this is characteristic of the most anteriorly located cervical vertebrae in other paracrocodylomorphs .In P. langstoni include four cervical vertebrae and four trunk vertebrae  and the three cervical vertebrae of individual “B”   are amphangstoni  and the P. kirkpatricki , and A. babbitti (MSM 4590). Each of the accessory rib articulations are dorsoventrally long and thin, projecting laterally from their respective centra. The accessory rib articulation of TMM 31025-1262 is broken at its base on the left lateral side, but it is intact on the right lateral side and protrudes 0.5 cm from where it attaches to the rest of the centrum. It is located 1 cm dorsal to the parapophyses. The accessory rib articulations of TMM 31025-1261.3 are intact on both sides, protrude 1.5 cm laterally, are located 3.2 cm from the diapophyses, and are located just dorsal to and touching the parapophyses. On the right lateral side of TMM 31025-177, the accessory rib articulation is just dorsal to and separated by 1 cm from the parapophyses. The left lateral side does not preserve this articulation; however, the paradiapophyseal lamina is clearly broken in the place where the articulation would be present. The accessory rib articulations on each cervical vertebra connect to the diapophyses through the paradiapophyseal laminae. This accessory rib articulation is also present in the posterior cervical vertebrae of P. gracilis (TTU P-10419) and A. babbitti (MSM 4590), and it is a synapomorphy of the clade Poposauroidea  and the posterior cervical vertebra of individual “A” (TMM 31025-177) possess a well-preserved accessory rib articulation between the diapophysis and the parapophysis. The presence of these accessory articulations provides evidence that these vertebrae had double-headed ribs associated with them, an uncommon occurrence in pseudosuchians, reported only in this specimen, auroidea .TMM 31025-177 is a posterior cervical vertebra. The positions of the parapophyses and diapophyses place it as likely between presacral 7 and 9, and the positions of these articulations are very similar to those in TMM 31025-1262. The parapophyses are low on the centrum and well separated from the diapophyses, which are located on the neural arch  and 2H.This specimen is mostly intact but is missing the neural spine, which is broken at the base, and the left prezygapophysis is broken. Both postzygapophyses are broken slightly at the edges and the diapophyses are both broken off where they connect to the neural arch and laminae  and 2H. TMM 30125-177 has distinct laminae forming thin, pronounced ridges on its neural arch. These laminae are thinner than those on the cervical vertebrae attributed to individual “A.” On its lateral sides there are clear posterior and anterior centrodiapophyseal laminae, which connect the diapophyses to the posterior and anterior portions of the neurocentral junction, respectively. There is a clear paradiapophyseal lamina connecting the parapophyses and diapophyses, and the accessory rib articulation is located on this lamina. This paradiapophyseal lamina extends nearly straight ventrally from the diapophysis, less than 5° from vertical. The postzygapophyseal laminae are thin and well defined and connect the parapophyses and the lateral aspect of the postzygapophyses at about a 10° angle dorsal to the anteroposterior horizontal. The prezygadiapophyseal laminae are laterally thick and short with the prezygapophyses and diapophyses less than 0.5 cm apart. These laminae are at a ∼45° angle dorsal to the anteroposterior horizontal.TMM 30125-1261.5 is the anteriormost vertebra of this newly referred material and can be identified as a posterior cervical vertebra . It is nearly complete, with only slight breakage and very minor post-depositional compression in some areas including on and just ventral to the prezygapophyses. It also has a very slight ridge along the midline of the ventral surface of the centrum. The parapophyses on the lateral sides of this vertebra are located on the base of the anterior rim of the centrum and are lowest on the centrum relative to the other vertebrae and well separated from the diapophyses. The neural arch is fused to the centrum, and the neurocentral suture is nearly obliterated, but there is a slightly raised area with a distinctive texture on the lateral surface where the suture was originally present . In anteNundasuchus songeaensis (NMT RB48), the anterior and posterior trunk vertebrae of F. tenax , the trunk vertebrae of P. chiniquensis (UFRGS-PV-0156-T), and anterior trunk vertebrae of B. kupferzellensis (SMNS 80294). From lateral view, the neural spine of TMM 31025-1261.5 remains relatively consistent in anterior–posterior length at about 2.5 cm.The neural spine is tall and laterally compressed in TMM 31025-1261.5. A long depression extends dorsoventrally along the posterior surfaces of the neural spine. The neural spine is virtually identical in preserved morphology to that of TMM 31025-1262, but it is broken off at the dorsal end. In posterior view, the spinopostzygapophyseal laminae are paired and form distinct ridges from the base of the neural spine to nearly the distal end of the neural spine. The neural spine is flat in anterior view, and no spinoprezygapophyseal laminae are present on that side. The neural spine is much wider at its base, at 2.5 cm just dorsal to the postzygapophyses in posterior view, and tapers to about 0.5 cm thick in anterior and posterior views at its most dorsal point. It does not expand laterally into a “spine table” as in the mid-trunk vertebrae of Revueltosaurus callenderi, PEFO 34561) and are found outside Archosauria among stem members  and are more similar in terms of lateral expansion to the other cervical vertebrae of individual “A,” TMM 31025-1261.3 and TMM 31025-1262. On the lateral sides of TMM 31025-1261.5, there are clear anterior and posterior centrodiapophyseal laminae, which connect the diapophyses to their respective portions of the neurocentral junction. The paradiapophyseal lamina connects the parapophysis with the diapophysis, and is angled ∼5° from vertical in lateral view. The postzygapophyseal laminae are well defined and connect the parapophyses and the lateral aspect of the postzygapophyses at about a 45° angle dorsal to the anteroposterior horizontal. This specimen is crushed so that the left postzygapophysis is pushed toward the diapophysis and this has compressed the lateral side of the left postzygapophyseal lamina  and 2C.  members . There aTMM 31025-1262 is a nearly complete posterior cervical vertebra, with slight breakage and minor post-depositional compression in some areas, including the prezygapophyses being pushed toward the neural spine  and 2D. N. songeaensis (NMT RB48), the anterior and posterior trunk vertebrae of F. tenax , the trunk vertebrae of P. chiniquensis (UFRGS-PV-0156-T), and anterior trunk vertebrae of B. kupferzellensis (SMNS 80294). From lateral views, the neural spine remains relatively consistent in thickness mediolaterally at about 2.7 cm. The distal end of the neural spine is also rounded slightly in lateral view and is slightly taller posteriorly.The neural spine is tall and mediolaterally compressed. A long depression extends dorsoventrally along the posterior surfaces of the neural spine. The neural spine is nearly identical to that of TMM 31025-1265.5. In posterior view, the spinopostzygapophyseal laminae are paired and form distinct ridges from the base to nearly the distal end of the neural spine. There are no spinoprezygapophyseal laminae present on the neural spine in anterior view. The neural spine is much wider at its base, at 3 cm dorsal to the postzygapophyses in posterior view, and tapers to about 0.5 cm mediolaterally in anterior and posterior views at its most dorsal point. It does not expand into a spine table as in the mid-trunk vertebrae of TMM 30125-1262 has distinct laminae forming thin, pronounced ridges on its neural arch; however, these laminae are more laterally expanded than the laminae of the posterior cervical of individual “B” (TMM 31025-177) and more similar to the anterior cervical vertebrae of individual “A” (TMM 31025-1261.3 and TMM 31025-1261.5). On the lateral sides of the vertebra, there are clear posterior and anterior centrodiapophyseal laminae, which connect the diapophyses to the posterior and anterior portions of the neurocentral junction, respectively  and 4C. TMM 31025-1261.3 is a posterior cervical vertebra that can be attributed to presacral 9 or 10. The diapophyses are located on the neural arch and are well separated from the parapophyses. The parapophyses are also more dorsal on the centrum than in both TMM 31025-1261.5 and TMM 31025-1262. Although this specimen’s neural arch is detached from the centrum, the two portions were fused and then broken post-depositionally. There is evidence of where a neurocentral suture was located, because there are some slightly raised areas on the lateral sides of the centrum ventral to the break . There iThe detached neural arch of this specimen is broken through the center of the neural canal, and is not a sufficiently close fit with the centrum to be reattached because small bits of bone are missing. The neural canal is deeply excavated; it is circular in anterior view and elliptical with the long axis oriented dorsoventrally in posterior view. The diapophyses are broken off at the point where they begin to extend out from the neural arch. A few laminae are broken and the specimen is slightly post-depositionally compressed in some areas, but otherwise it is intact.TMM 31025-1261.3 has distinct laminae forming thin, pronounced ridges on its neural arch, and these laminae are more laterally expanded than the laminae of the cervical vertebra of individual “B” (TMM 31025-177) and more similar to the other cervical vertebrae of individual “A” (TMM 31025-1262 and TMM 31025-1261.5). On the left lateral side there is a clear posterior centrodiapophyseal laminae, which connects the diapophysis to the posterior portion of the neurocentral junction, but this lamina is broken off on the right lateral side. There is a clear paradiapophyseal lamina, which connects the parapophyses and diapophyses, but the lamina on this vertebra connects the diapophyses to the accessory rib articulation instead of the parapophyses because the articulation is located just dorsal to the parapophyses and nearly touching. The paradiapophyseal lamina extends nearly straight down from the diapophysis, ∼5° from vertical. The postzygapophyseal laminae are well defined and connect the parapophyses and the lateral aspect of the postzygapophyses at about a 45° angle dorsal to the anteroposterior horizontal. The prezygadiapophyseal laminae are thick in lateral view and the prezygapophyses and diapophyses are ∼1.5 cm apart. These laminae extend dorsally from the diapophyses at a right angle to the anteroposterior horizontal with the prezygapophyses being dorsal to the diapophyses. The diapophyses are broken off about where they connect to the neural arch, but the left one extends about 0.5 cm laterally before its break. The neural spine is broken off at its base, so spinoprezygapophyseal laminae are unable to be seen.B. kupferzellensis  and all three trunk vertebrae of individual “B”   are waisAll four of the trunk vertebrae preserve one or both of the accessory articulation structures  that form the hyposphene–hypantrum articulation. TMM 31025-1261.4  and 3C iTMM 31025-257 is an anterior mid-trunk vertebra  and 3H. P. kirkpatricki , and the posterior trunk vertebra of F. tenax  and the anterior and posterior trunk vertebrae of F. tenax (PVL 3850). The neural spine of TMM 31025-257 is angled on the posterior edge and extends further dorsally on the anterior edge, causing it to be slightly wider at the distal end than at the base in lateral view, unlike the neural spines of the posterior trunk vertebrae of P. kirkpatricki (TTU P-9002) and B. kupferzellensis (SMNS 52970), which are both more rectangular in lateral view.The neural spine is laterally compressed, and roughly the same height as the height of the rest of the vertebra to the base of the neural spine. Grooves extend dorsoventrally along both the posterior and anterior surfaces of the neural spine. The spinoprezygapophyseal laminae are paired and cease to form distinct ridges about halfway up dorsoventrally from the base of the neural spine. The dorsal edge of the neural spine is rounded and the top centimeter of the neural spine is slightly laterally expanded, although it is not sufficiently wide to form a “spine table” as in the mid-trunk vertebrae of TMM 31025-257 has distinct laminae between the prezygapophyses, postzygapophyses, parapophyses, and diapophyses. On the lateral sides, the specimen has paradiapophyseal laminae, which connect the diapophyses with the parapophyses. There are anterior centroparapophyseal laminae, which connect the parapophyses with the anterior portion of the neurocentral junction, and there are also posterior centrodiapophyseal laminae, which connect the diapophyses with the posterior portion of the neurocentral junction, and all of these laminae are at a 45° angle ventral to the anteroposterior horizontal  and 4D. TMM 30125-1261.4 is an anterior to mid-trunk vertebra. It is weathered post-depositionally, and the neural arch is broken horizontally through the mediolateral middle and separate completely from the centrum  and 3C. On the left lateral side, TMM 30125-1261.4 has distinct laminae forming pronounced ridges, the posterior and anterior centrodiapophyseal laminae, which connect the diapophyses to the posterior and anterior portions of the neurocentral junction, respectively. The anterior centrodiapophyseal lamina is recognizable but not clearly defined on the right lateral side. On both lateral sides the prezygapophyseal laminae are recognizable and connect the parapophyses and the lateral aspect of the prezygapophyses at a steep angle (>80°) to the anteroposterior horizontal.The posterior facet of the neural arch is sufficiently weathered so that no hyposphene structure is recognizable, however there is a well-defined hypantrum visible on the anterior facet of the neural arch  and 3C. TMM 31025-1261.1 is an anterior to mid-trunk vertebra that is nearly complete, but is well weathered post-depositionally, more so on the anterior and right lateral portions. The specimen is attributed to presacral 12–14, as the parapophyses are well separated from diapophyses, and both are located on the neural arch. The neural arch is completely fused to the centrum with only a slight textural trace of where the neurocentral suture was located .The parapophyses are slightly higher on the neural arch than those on the trunk vertebra we attribute to individual “B” (TMM 31025-257). Both of the parapophyses are broken off at their bases. They are circular in lateral view with a diameter of about 1 cm. The left parapophysis is in the correct anatomical position, but the right parapophysis has broken from its originally position and has shifted about 3–4 cm posteriorly. The right diapophysis is broken off at its base, and the left diapophysis is broken off about 1.5 cm from its base. This left diapophysis is dorsoventrally compressed, shaped as a narrow ellipse in lateral view with the long axis along the anteroposterior horizontal. There is a clear paradiapophyseal lamina connecting the parapophysis and the diapophysis on the left side. There is also a posterior centrodiapophyseal lamina connecting the diapophysis with the posterior section of the neurocentral suture junction. The right side is too weathered and broken to see any laminae. No zygapophyses are preserved. The neural canal is filled with sediment but appears to be only slightly laterally compressed  and 3D.The neural spine of TMM 31025-1261.1 is complete but is broken and offset dorsally by ∼25°. The neural spine is tall relative to the rest of the neural arch and laterally compressed and the dorsal edge of the neural spine is sloped at 45°, increasing in height posteriorly, in lateral view. The anteroposterior width of the neural spine is nearly uniform throughout in lateral view. The distal end of the neural spine is slightly rounded. The neural spine is posteriorly shifted in relation to the anteroposterior center of the centrum. The anterior surface of the neural arch is well weathered post-depositionally so that a hypantrum is not recognizable. The posterior surface of the neural arch preserves a clearly defined hyposphene. The shape of the hyposphene is triangular in posterior view, with a point directed dorsally along the dorsoventral midline, and the ventral side is slightly concave, forming the dorsal border of the neural canal  and 3D. TMM 31025-1261.2 is a posterior trunk vertebra and we attribute it to presacral position somewhere among 14–17, as the parapophyses are only separated from the diapophyses by about 1 cm, and both are located on the neural arch. It is well weathered post-depositionally. The neural arch is completely fused to the centrum with slight bowing where the neurocentral suture was located, but no suture is present within the bone. The neural canal is deeply excavated and laterally compressed in anterior view, but appears round and less laterally compressed in posterior view  and 3G.On the lateral sides of this specimen, the diapophyses are broken about 2 cm from where they connect to the neural arch. Both are dorsoventrally compressed and thin out posteriorly from a circle with a diameter ∼1 cm across to a flat surface with a thickness of 0.5 cm. Both diapophyses are intact and both are dorsoventrally compressed so that they are shaped as a narrow elliptical with the long axis along the anteroposterior horizontal. There are clearly defined paradiapophyseal lamina connecting the parapophysis and the diapophysis on both lateral sides. There are also posterior centrodiapophyseal laminae connecting the diapophyses with the posterior sections of the neurocentral suture junction on both lateral sides.The left postzygapophysis is broken off at its base, and the right postzygapophysis is broken off about 1.5 cm from its base and there is a subtle but recognizable postzygadiapophyseal lamina connecting the postzygapophysis to the diapophysis on the right lateral side. The right prezygapophysis is broken off at its base, and the left prezygapophysis is only broken slightly at the tip with no more than half a centimeter of missing material. There are clear prezygadiapophyseal laminae connecting the prezygapophyses with the diapophyses on both sides. There are also clear prezygaparapophyseal laminae connecting the prezygapophyses with the parapophyses on both sides.The neural spine is broken off immediately dorsal to the prezygapophyses, so spinoprezygapophyseal laminae are obstructed from view. However, the break is not horizontal and there is more of the neural spine preserved in posterior view with the break on the posterior aspect of the neural spine located ∼3 cm dorsal to the postzygapophyses  and 3G. P. gracilis  and presacral 12 of individual “B” (TMM 31025-257). The articulation is also present in presacral 12 in individual “A” (TMM 31025-1261.2). All three of the preserved hyposphenes are triangular in posterior view, and the ventralmost edges of the triangular hyposphenes are oriented horizontally with their apices pointed dorsally along the midline. The ventrolateral corners of the triangular hyposphenes curve slightly ventrally around the neural canal. The hyposphenes  appear to be more dorsoventrally compressed  in individual “B” than in individual “A.” This material described herein includes the twelfth presacral from both individuals , so it is likely that this variation is not based on location in the vertebral column. The hypantra  reflect a complementary shape of the hyposphene structures on their respective vertebrae. The horizontal distance between the prezygapophyses is narrower across the structure at the dorsal most edge than at the ventralmost extent, creating a triangular space for articulation. In summary, the hyposphene–hypantrum articulation appears triangular in all vertebrae described herein and therefore does not vary in general shape along the vertebral column of one individual of P. langstoni, but the two recognized individuals of P. langstoni have hyposphene structures that are markedly different in terms of height to width ratio   . As longR. callenderi (PEFO 34561) (Erythrosuchus africanus (SAM 905) (P. gracilis (NMT RB426), or N. songeaensis (NMT RB48), or they may not converge at all and appear well separated, as in Deinosuchus riograndensis (TMM 43632-1)  , ErythroSAM 905) , P. grac43632-1)  or A. miR. callenderi, PEFO 34561; P. gracilis, NMT RB426; E. okeeffeae, AMNH FR 30587; D. riograndensis, TMM 43632-1; A. mississippiensis, The lateral surfaces of the hyposphene articulate with the medial surfaces of the hypantrum, which is located between and ventral to the prezygapophyses and dorsal to the neural canal . The artP. langstoni allows for comparisons with the presacral vertebrae of other pseudosuchian archosaurs. Other closely related members of Poposauroidea, P. gracilis—the sister taxon to P. langstoni—and the smaller E. okeeffeae, have also been reported to have a hyposphene–hypantrum between trunk vertebrae , the current sister taxon of E. okeeffeae (S. longicervix (PVSJ 85), is poorly preserved  has not been reported to have the hyposphene–hypantrum articulation.The presacral vertebrae of the poposauroid keeffeae , 2011, treserved , and theArizonasaurus babbitti is one of the oldest members of Poposauroidea, outside of L. adentus + Shuvosauridae, and it has been reported to have the hyposphene–hypantrum articulation  is rectangular with the long axis oriented dorsoventrally in posterior view and located ventral to the postzygapophyses and dorsal to the neural canal , and it has been cited as having the hyposphene–hypantrum as well . The trunk vertebra of B. kupferzellensis figured in lateral view by P. chiniquensis that was figured in posterior view by B. kupferzellensis (SMNS 80296) (P. kirkpatricki (TTU P-9002). S. galilei (PVSJ 32) has a hyposphene–hypantrum articulation present on at least one of its posterior trunk vertebrae   and P. kertebrae , but it P. kirkpatricki has been reported to have the hyposphene–hypantrum articulation present on its middle and posterior trunk vertebrae  also appear triangular in anterior view with a shape and size complementary to the hyposphene of the same vertebra. The hyposphene structure of the mid-trunk vertebra of P. kirkpatricki  has clear hyposphene and hypantrum structures (P. kirkpatricki (TTU P-9002), with the midline apex directed dorsally and the ventrolateral corners curved slightly around the dorsal edge of the neural arch . The posterior trunk vertebrae of D. spurensis have hyposphenes that have a width and height nearly equal to each other. These hyposphenes also curve around the neural canal and appear triangular in posterior view , and Aetobarbakinoides brasiliensis  and posterior cervical (E.M. 30R) vertebrae  may have a hyposphene–hypantrum articulation in its trunk vertebrae, but this is ambiguous because of the poor preservation of known material. The trunk vertebra of L. meadei, TMM 31100-448 appears to have a hyposphene that is square in posterior view; however, the postzygapophyses and surrounding portions of the neural arch are broken and compressed, making it difficult to definitively discern a hyposphene structure according to our definition  (P. gracilis (NMT RB426), the sister taxon of R. callenderi (PEFO 34561) + Aetosauria  do converge; however, they do not have a bony and dorsoventrally elongated process that could definitively be a hyposphene. The cervical vertebrae (PEFO 34561-CV) of this specimen definitively do not have the hyposphene–hypantrum articulation. Therefore, both R. callenderi and P. gracilis are small pseudosuchian archosaurs that lack the hyposphene–hypantrum.Although it is present in a few aetosaurs, the hyposphene–hypantrum articulation is not present in the sister taxon to Aetosauria, O 34561) . It is atosauria . The posSphenosuchus acutus, UCMP 129740; D. riograndensis, TMM 43632-1; A. mississippiensis, TMM M-12606)  , which iM-12606)  than theM-12606) , such asSmilosuchus gregorii, 545 mm; Machaeroprosopus pristinus, 444 mm)  . The abs 444 mm) , which iIn summary, our observations of pseudosuchian archosaurs both with and without the hyposphene–hypantrum articulation showed a clear relationship between larger body sizes and the presence of the hyposphene–hypantrum articulation in trunk vertebrae across pseudosuchian archosaurs. Because of this close fit, this feature may be controlled by increases or decreases in body size and not strictly by inheritance. If so, the presence or absence of hyposphene–hypantrum would have limited use as a character in phylogenetic analyses of archosaurs  and the


The incidence of sensorineural hearing loss is between 1 and 3 per 1000 in healthy neonates and 2–4 per 100 in high-risk infants. The national universal neonatal hearing screening carried out in Poland since 2002 enables selection of infants with suspicion and/or risk factors of hearing loss. In this study, we assessed the incidence and risk factors of hearing impairment in infants ≤33 weeks’ gestational age (wga).We analyzed the database of the Polish Universal Newborns Hearing Screening Program from 2010 to 2013. The study group involved 11438 infants born before 33 wga, the control group—1487730 infants. Screening was performed by means of transient evoked otoacoustic emissions. The risk factors of hearing loss were recorded. Infants who failed the screening test and/or had risk factors were referred for further audiological evaluation.Hearing deficit was diagnosed in 11% of infants ≤25 wga, 5% at 26–27 wga, 3.46% at 28 wga and 2–3% at 29–32 wga. In the control group the incidence of hearing deficit was 0.2% (2.87% with risk factors). The most important risk factors were craniofacial malformations, very low birth weight, low Apgar score and mechanical ventilation. Hearing screening was positive in 22.42% newborns ≤28 wga and 10% at 29–32 wga and in the control group.Hearing impairment is a severe consequence of prematurity. Its prevalence is inversely related to the maturity of the baby. Premature infants have many concomitant risk factors which influence the occurrence of hearing deficit. Childhood hearing impairment is the result of the overlapping factors of genetic predisposition and environmental impact. Worldwide reporting of hearing loss finds that the prevalence of moderate and severe bilateral hearing deficit (>50 dB) is 2–3 per 1000 live births in well baby nursery population and 2–4 in 100 infants in an intensive care population [For effective treatment, congenital or perinatal hearing loss should be recognized within three months of birth so that early intervention can begin prior to 6 months of age. If not detected and hearing amplification provided, significant hearing impairment negatively impacts speech development and results in disorders in psychological and mental behavior .The Joint Committee on Infant Hearing in 2000 listed 10 factors that identify infants at greatest risk for hearing impairment . This liThe implementation of hearing screening programs for newborns continues to grow. Some institutions used to perform screening based on risk factors only –13. HoweInfant hearing screening is considered to be an effective procedure in early detection of hearing impairment in infants and therefore it should be one of the priorities in neonatal care , 4. The The techniques most often employed and successfully used in the universal neonatal hearing screens are:Automated auditory brainstem response (ABR)Otoacoustic emissions (OAE).Both OAE and automated ABR technologies provide noninvasive recordings of physiologic activity underlying normal auditory function and both are easily performed in neonates and infants .Otoacoustic emissions recording usually takes less than 1 min and can be achieved without audiological knowledge . The priAuditory brainstem response is an auditory evoked potential that originates from the auditory nerve. It can detect injury on the level of cochlea, auditory nerve and auditory pathway in the brainstem. It is now recommended that infants admitted to the neonatal intensive care units (NICU) for more than 5 days have ABR included as part of their screening so that neural hearing loss will not be missed [st level of the hearing screening program). Infants in whom the result of OAE screen is positive, i.e. that receive the result ‘refer’ for at least one ear and/or those who have at least one risk factor of hearing impairment, are referred for further evaluation by means of ABR to the audiological centers responsible for hearing evaluation and appropriate intervention (2nd level of the hearing screening program).In Poland, the group of Polish neonatologists and otorhinolaryngologists, together with The Great Orchestra of Christmas Charity Foundation initiated the program of universal newborn hearing screening in 2002. This program is based on otoacoustic emissions testing and a questionnaire aimed at identifying risk factors defined by the Joint Committee on Infant Hearing published in 2000 , in all It is also now recommended that in all infants readmitted to the hospital in the first month of life, when there are conditions associated with potential hearing loss , a repeat hearing screening is performed before discharge .The group of infants that is particularly at risk of hearing deficit are premature newborns, born < 33 weeks of gestational age (wga), especially those treated in NICUs. Apart from congenital sensory or conductive hearing loss, these infants may develop sensory-neural hearing loss, known also as auditory neuropathy/auditory dyssynchrony, due to the treatment received in the NICU .The aim of this study was to assess the incidence of hearing impairment in preterm infants (≤33 wga) and to analyze the risk factors of hearing loss in this population.st level of the hearing screening program). Screening was performed with OtoRead screener (Interacoustics). The outcomes of screening were presented as either ‘refer’ (i.e. hearing problem detected in at least one ear) or ‘pass’ (i.e. hearing problem not detected). The list of risk factors used in the questionnaire was adopted from the JCIH statement of 2000  and the risk factors of hearing loss were recorded in the questionnaire, filled in by medical staff based on the medical records and family interview . Infants who did not have OAE screen performed on the 1st level were also referred to the 2nd level.Infants who failed the screening test and/or had risk factors of hearing impairment were referred to the outpatient audiology clinic for further evaluation within 3 months of life or immediately after hospital discharge  and finally examined by means of auditory brainstem response method (Racia Alvar Centor C.). Absolute thresholds of hearing were defined using a click as stimulus and a specific stimulus for frequency 500 and 1000 Hz. Latencies of waves I, III and V were measured. Basing on the results, the diagnosed hearing deficit was classified as mild (21–40 dB HL), moderate (41–70 dB), severe (71–90 dB HL) and profound (>90 dB HL).At the 2The hearing screening program covered more than 98% of infants born in Poland in this period of time (n = 1 525 094). These infants had at least one OAE screen performed and the questionnaire of risk factors filled in. The missing infants (n = 25 926) were not screened due to various logistic and medical situations.The population was divided into two groups. The study group consisted of 11 438 infants born < 33 wga and was further divided into two subgroups:≤28 wga–2884 neonates29–32 wga–8554 neonates.The control group consisted of 1 487 730 newborns born ≥33 wga.nd level of screening program. In the control group (≥33 wga) only infants with risk factors of hearing impairment or with positive result of the screening OAE screen, were referred for further examination at the 2nd level. Therefore, some of the analysis are limited only to those infants who were screened at 1st level and examined at the 2nd level of the program (n = 31348). Other infants of this group were treated as infants with normal hearing . Flowchart of children registered in the central database of PUNHSP between 1 January 2013 and 31 December 2013 with diagnostic tests performed is presented in All infants in the study group were treated as infants with risk factors of hearing impairment and independently of the screening result they were directed to the audiologists for further examination by means of ABR. Only about 55% of infants referred for further assessment showed up in the outpatient audiology clinics taking part in the neonatal hearing screening program. Among infants born before 33 wga it was 58.9%, thus a portion of our results is limited to the group of 6742 infants of the study group examined at the 2By terms hearing impairment and hearing deficit in PUNHSP we understand any permanent hearing problem either unilateral or bilateral of the level ≥20 dB, irrespectively of its etiology and time of diagnosis.The STATISTICA 10 package was used for the analysis. Pearson Chi square, Odds Ratio (OR) and 95% confidence intervals (CI) were calculated. Multivariate logistic regression analysis was performed for the investigated risk factors and Cohen’s kappa coefficient for test agreement was calculated.The final diagnosis of permanent hearing impairment was given predominantly in most premature infants, born ≤ 25 weeks of gestation– 11% (32 / 291). In infants born between 26 and 28 wga hearing deficit was diagnosed in 4.2% of patients (53 / 1257) and in those between 29 and 32 wga in 2.3% (122 / 5194)–Pearson Chi-square p<0.0001. The exact results for each week of gestation are shown on We analyzed the incidence of different types and intensity of hearing deficits in relation to gestational age at birth. The results are presented in th day of life.The mean time of the final diagnosis in all groups of infants was 89Among infants with permanent profound bilateral sensorineural hearing loss 349 received cochlear implants (94.6%). 897 children with other types of hearing deficit received hearing aids. Most children received also audiological, logopedical and psychological care.Risk factors of hearing deficit were found in 64 580 (4.3%) infants. All infants born < 33 wga were treated as infants with risk factors due to their prematurity. At least one other risk factor was identified in 84.4% (n = 9651) of them. Most frequent risk factor was exposure to “ototoxic medications”, accounting for 63.0% in this population. The second and the third most frequent risk factors were “low birth weight <2500 g”– 53.3% and “treatment in the intensive care unit”– 43.9%. The use of ototoxic medications was also the most frequent risk factor in the control group, however it was only recorded in 1.72% of infants born ≥ 33 wga. The second and third most frequent risk factors in the control group were congenital infections (0.83%) and family history of hearing loss (0.61%), however their frequency was comparable in the study group and was not significant.st minute of life and mechanical ventilation. In infants born between 29 and 32 wga the most significant were craniofacial anomalies, complex congenital anomalies, extremely low birth weight and low Apgar scores. In the near term and term newborns (≥ 33 wga) the most important role played the family history of hearing loss, craniofacial anomalies and complex congenital anomalies, as well as very low birth weight and low Apgar scores. The exact data are shown in Hearing evaluation in patients with specific risk factors revealed that in the group of infants born ≤ 28 wga in whom hearing impairment was diagnosed, the most important risk factors were craniofacial anomalies, low Apgar score at 1Lastly, we analyzed the association between a positive result of the hearing screening and the final diagnosis of the hearing impairment. There was also highly significant difference in the occurrence of hearing loss between those three groups .Hearing impairment is an important and severe consequence of preterm birth and its prevalence is inversely related to the maturity of the infant. In our study, 4.2% of preemies born between 26 and 28 weeks of gestation and 2.3% of infants born between 29 and 32 wga were diagnosed with hearing impairment, similarly to the findings of other authors , 8, 17. The pathophysiology of hearing loss in preterm infants is very complex and although prematurity alone may not have a severe impact on hearing, it is commonly associated with multiple other risk factors that can influence hearing in a synergistic fashion. Therefore, the risk of hearing loss in preemies is substantially higher than in the general newborn population.There are many different known causes of neonatal hearing loss. It is generally accepted that genetic and environmental factors are each responsible for half of the cases , 19. In Other causes particularly important for hearing loss among premature infants include ototoxic drugs: aminoglycosides and loop diuretics, as well as noise exposure, hyperbilirubinemia and hypoxia . AminoglExposure to the constant background noise generated by contemporary life-support equipment in the NICU is another risk factor of hearing loss . Recent Hyperbilirubinemia which can produce selective injury of the brainstem auditory nuclei and may damage the auditory nerve and ganglion cells , 23 doesHypoxia is strongly associated with hearing loss, as adequate oxygenation and perfusion are crucial for normal cochlear function , 25. It According to our analysis, an important risk factor for hearing deficit is very low birth weight (VLBW < 1500 g). We could see this clearly in infants born between 29 and 32 wga and in those born ≥33 wga, where its significance was confirmed by the statistical analysis, however we must realize that in most premature newborns it was almost a constant feature (92.23%). Some studies also show this association while others do not , 26, whiIt is known from many studies that some infants treated in NICU may begin to develop hearing impairment at the age of 2–4 years . The patnd level of hearing screening and were explained about the importance of further audiological tests and rehabilitation. We think that some of these children were eventually diagnosed and treated in institutions that do not take part in the national program of hearing screening and that is why they are missing but we also suppose that some parents simply ignored the risk of hearing deficit and did not keep the scheduled appointments for their baby’s further assessment.In our study, we tried to analyze the prevalence of hearing deficit and the risk factors influencing hearing impairment in premature infants. Although involving large number of patients, the study has some limitations. First one is the referral rate of infants to the second level of hearing screening program which is about 60% and thus does not allow to perform the complete analysis. When discharged from the hospital parents were given written referral note to the 2st level of hearing screening). We know from our other study that in some cases there might be mistakes in entries to the database, which in consequence cause underestimation of the data on the percentage of follow-up visits at the 2nd level of PUNHSP [The study was based on the database of the national program and data was collected from 405 neonatal centers  are referred to the second level of screening where ABR and other exams are performed, while term infants are screened only once before discharge from the hospital and if they have no risk factors, they are treated as infants with normal hearing. Therefore, in our statistical analysis we could include only 31348 infants born ≥33 wga. These were infants who were examined at the second level of the hearing screening program and not the whole population of near term and term infants. We realize however that this limitation might change the picture of the whole population.As stated before, the questionnaires of risk factors were filled in by medical staff before infants were discharged from the hospital after birth, based on the medical records of the patients. We admit however, that there might be some discrepancies between hospitals especially when reporting such risk factors as “the use of ototoxic drugs” or “hyperbilirubinemia”, which are not precisely defined in terms of duration or peak level.nd level of screening. These children, independently of the results of OAE screen are referred to the 2nd level center where they have all necessary diagnostic tests performed and the final diagnosis is given. Additionally, when we checked the agreement between the screening test and the final diagnosis of normal or impaired hearing, the results were very good–above 78% in infants ≤28wga and almost 90% in those born between 29 and 32 wga. Anyway, it is planned in the nearest future to introduce ABR in some NICU departments and perform the pilot study using these both techniques of screening.Another limitation of our study might be the fact that we used only otoacoustic emissions as screening test, while it is suggested by the experts that all infants treated in NICU should be tested by means of auditory brainstem response . HoweverThe important result of the PUNHSP is that nearly all infants with permanent severe bilateral sensorineural hearing deficit obtained cochlear implants and most other infants received other hearing aids. All children were also included in audiological, logopedical and psychological care program.th day of life. This is appropriate time according to the standards of care and it enables an early intervention within 6 months of age [The mean time of the final diagnosis and intervention in all groups of infants was 89s of age , 31.Following extreme prematurity, hearing impairment is a major adverse outcome that is commonly associated with other severe disabilities. What is also important this pathology may be postponed in time as delayed-onset and progressive hearing loss is not uncommon in this group of patients.The extent to which prematurity alone is responsible for high prevalence of hearing impairment remains unclear. However, these patients are commonly exposed to other risk factors for hearing loss such as low Apgar score, intensive care treatment with mechanical ventilation, hypoxia, ototoxic drugs and hyperbilirubinemia. Therefore, long-term careful monitoring and the appropriate audiological management of hearing loss is essential among very premature infants.Hearing impairment is a severe consequence of prematurity and its prevalence is inversely related to the maturity of the baby.Premature infants have many concomitant risk factors which influence the occurrence of hearing deficit. The most important of them seem to be low Apgar scores, mechanical ventilation, very low birth weight and craniofacial anomalies.Some risk factors, e.g. “ototoxic drugs”, although frequently recorded, does not seem to be very significant.


Influenza H9N2 subtype viruses and their reassortants (such as H7N9) are posing increasing threats to birds and humans in China. During 2009–2013, multiple novel subtype viruses with H9N2 original genes emerged in China. Yet, the genetic evolution of H9N2 viruses in various host organisms in China has not been systematically investigated since 2009. In the present study, we performed large-scale sequence analysis of H9N2 viral genomes from public databases, representing the spectrum of viruses isolated from birds, mammals and humans in China from 1994 to 2013, and updated the clade classification for each segment. We identified 117 distinct genotypes in 730 H9N2 viruses. We analyzed the sequences of all eight segments in each virus and found three important time points: the years 2000, 2006 and 2010. In the periods divided by these years, genotypic diversity, geographic distribution and host range changed considerably. Genotypic diversity fluctuated greatly in 2000 and 2006. Since 2010, a single genotype became predominant in poultry throughout China, and the eastern coastal region became the newly identified epidemic center. Throughout their 20-year prevalence in China, H9N2 influenza viruses have emerged and adapted from aquatic birds to chickens. The minor avian species and wild birds exacerbated H9N2 genotypes by providing diversified genes, and chickens were the most prevalent vector in which the viruses evolved and expanded their prevalence. It is the necessity for surveillance and disease control on live-bird markets, poultry farms and wild-bird habitats in China. The increased prevalence of H9N2 viruses in chickens after 2010 enhanced infectivity and antigenic drift.5 Furthermore, H9N2 viruses preferentially bind to the human-type sialic acid receptor and spread between ferrets by respiratory droplets.4 Between 2013 and 2016, an increasing number of human cases of H9N2 virus infection (n=18) were laboratory confirmed in China . From this evidence, it is apparent that H9N2 viruses have undergone considerable changes in their biologic characteristics, but the underlying genetic evolution driving these changes is largely unknown.H9N2 subtype influenza viruses have become endemic in various types of terrestrial poultry in Eurasian and African countries and have caused sporadic infections in humans and mammals.8 In the 1980s, the viruses were isolated from quail, which was the first evidence of H9N2 viruses in land-based poultry.9 Subsequently, H9N2 viruses spread to chickens in the Guangdong province of Southern China in 1994.11 The viruses then adapted to chickens and other land-based birds, such as pheasants, chukars and other minor domestic poultry.10 Therefore, quail populations in China most likely contributed to the interspecies transmission of H9N2 viruses from ducks to chickens.2 After 1998, the viruses have sporadically crossed to humans, pigs and other mammals in China, causing mild respiratory disease.10In China, H9N2 viruses were initially isolated from domestic ducks during an influenza virus surveillance in Hong Kong from 1976 to 1985.12 The Eurasian lineage comprises three distinct sublineages: A/chicken/Beijing/1/94-like (BJ/94-like), A/quail/Hong Kong/G1/97-like (G1-like) and A/duck/Hong Kong/Y439/97 (Y439-like or Korean-like). The BJ/94-like and G1-like viruses mainly circulated in chickens and quails, respectively, in China since the mid-1990s.13 However, G1-like viruses also caused outbreaks in chickens in the Middle East and Germany.2 Korean-like viruses were first isolated from domestic ducks in Hong Kong in 1997, but similar viruses have been identified in disease outbreaks in chickens in South Korea since 1996.2Epidemiologic and genetic studies revealed that the hemagglutinin (HA) gene of H9N2 viruses bifurcates into the Eurasian and American lineages.14 A previous investigation of 330 H9N2 strains isolated from birds, pigs and humans in China from 1994 to 2008 identified 54 genotypes, which were further categorized into five distinct series , according to the sources of their genes.14 However, the genotype evolution of H9N2 from all hosts since 2009 is not well known.The internal genes of H9N2 viruses are more diversified than are the surface genes, which is the result of extensive reassortment of H9N2 viruses among chickens, ducks, minor poultry and wild-bird species.5 The continuous evolution of H9N2 viral genes resulted in additional phylogenetic groups that exceeded the previously designated clade boundaries in chickens. However, the genetic evolution in other hosts is relatively unknown. Therefore, it is necessary to update the divergent phylogenic clade classification of H9N2 viruses prevailing in China and to perform long-term, systematic genotypic analyses to elucidate the spatiotemporal evolution of H9N2 viruses in various hosts.We previously analyzed the sequence similarities of H9N2 viruses isolated specifically from chickens in China from 1994 to 2013 and found that the genes  underwent extensive variation, forming 10 different clades and generating 69 genotypes.In this study, we performed a large-scale genomic sequence analysis of the sequences of 730 H9N2 viruses with all eight gene segments and the sequences of 560 viruses with partial segments. These viruses were isolated in China from 1994 to 2013. We identified 117 genotypes from 730 H9N2 viruses isolated from avian, mammalian and human hosts. In addition, we determined the spatiotemporal changes in genotypic diversity and viral host range.www.FluDB.org), Global Initiative on Sharing Avian Influenza Data (www.gisaid.org) and the Influenza Virus Resource at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/genomes/FLU). The accession numbers of the previously published sequences were summarized in the 15 manually adjusted to correct frameshift errors, and subsequently translated. Downstream phylogenetic analyses were performed on regions of the alignments containing few gaps across the sequences. These regions included the following intervals: HA g.190_1566, neuraminidase (NA) g.91_1272, nucleoprotein (NP) g.67_1014, nonstructural protein (NS) g.79_807, matrix protein (M) g.88_948, polymerase acidic protein (PA) g.796_2103, polymerase basic protein 1 (PB1) g.61_1437 and polymerase basic protein 2 (PB2) g.1168_2199.All previously published sequences of H9N2 viruses isolated in China from 1994 to 2013 and related sequences were downloaded from the Influenza Research Database . We used 100-bootstrap maximum-likelihood trees to construct a majority-rule consensus tree from each bootstrap sample with the SumTrees (version 4.0.0) program.17 Bayesian phylogenetic trees were constructed for each segment by using the Bayesian evolutionary analysis sampling trees (BEAST version 1.6.1) software.18 The model of substitution and rate variation in each tree was duplicated from the corresponding RAxML tree, with the additional assumption of a lognormal distribution molecular clock. The BEAST trees were initiated with the RAxML majority-rule consensus tree to reduce the computation time required for the Markov chain Monte Carlo sampler to reach stationary distribution. Maximum clade credibility trees were constructed by using 2000 equidistant samples from the final 30% of the posterior sample of trees (n=25 × 106 samples) with a Python script and tree annotator.18 The resulting trees were partitioned into clades with a median branch length distance threshold of 0.20.19 Clades with median root-to-tip distances equal to or less than 20% of the median root-to-tip distance of all clades in the tree were selected as clusters. Clades were then manually merged, if necessary, by their branching posterior and reported classification.20 Each clade was assigned a unique clade identification number.The randomized accelerated maximum-likelihood (RAxML7.2.8 Alpha) program was used to construct maximum-likelihood phylogenies for each segment.5 The preceding 69 genotypes (G01–G69) stuck to the already reported genotypes and the additional new genotypes were added subsequently.5Viral genotypes were analyzed for H9N2 viruses isolated from all hosts in China from 1994 to 2013. Isolates were genotyped if the sequences and clade assignments were available for all eight segments. The genotypes of such isolates  were determined by the combination of clade assignments of each of the eight segments. Five hundred sixty isolates with partial segments were not included in the genotype analysis. Genotype identification numbers were assigned according to the previous study.P value <0.05 was considered to indicate statistical significance.Analysis of variance based on general linear model, and three multiple comparison tests  were performed using SAS (version 9.4). A 19 We then manually merged the clades, if necessary, by their branching posterior and reported classification.20 We found the HA sequences clustered into 16 clades .We constructed phylogenetic trees for all eight segments of the H9N2 viruses isolated from various hosts in China from 1994 to 2013. We partitioned the phylogenetic trees into clusters by using a top 20th-percentile cutoff.6 clades . Accordi14 contained 31 strains isolated from quail and other minor avian species from 2000 to 2004. Thus, the HA gene of H9N2 viruses has undergone extensive variation, especially from the HA gene of the BJ/94-original viruses. Recently, clade 15 has replaced other clades, making it the unique predominant lineage.In our analyses, we observed that 12 independent clades evolved from the ancestral A/chicken/Beijing/1/94  virus, indicative of the extensive variation of the BJ94-like H9 segment in China. Among the BJ/94-original viruses, the majority (1523 of 1607) was distributed in clades 5, 8, 12 and 15; whereas the most recent isolates were consolidated in clade 15. Unlike the BJ/94-original clade, other previously identified clades were relatively stable. We observed that 32 strains with HA genes corresponding with the G1-like clade were clustered in clade 4. Twenty-seven aquatic bird isolates of Korean-like lineage formed the present clade 2. Clade 3, which was the previously identified as an unknown avian clade,14 Here we found that all NA genes clustered within four clades (clade 0–3)  . Among t22 Here we used numbers to name each clade according to the phylogeny of the internal genes, which we also did for the surface genes. All six internal segments were divided into 42 independent clades , representative strains , geographic locations (Korean lineage) or viral subtypes .t clades .PB2 genes of H9N2 viruses were previously classified into seven clades.14 However, we found that the PB2 genes formed nine distinct lineages (clade 0–8) , which contained the Korean-like PB2 gene. The strains from clades 3 and 4 possessed H5N1-like PB2 genes.22The ade 0–8) . Before 14 Clades 0 and 5 were the two largest groups, which were primarily restricted to chicken isolates. Clade 0 was previously characterized by the BJ/94 lineage, and clade 5 evolved from the A/Chicken/Shanghai/F/1998 virus, becoming the most prevalent clade in China after 2010. Clade 1 was also predominant and contained a G1-like PB1 segment, which was mainly isolated from quail and other minor avian species. Unlike most other clades, clade 2 consisted of multiple lineages (Dk1 and Dk2) and was isolated from diverse sources, including chicken, duck, swine and minor avian species. A single isolate of duck origin (A/duck/Shantou/7488/2004) with a Dk3-like PB1 segment constituted clade 3. Seventeen H9N2 strains from different sources, containing H5N1-like PB1 genes,22 formed clade 4.In the PB1 phylogenetic tree, H9N2 viruses formed six lineages (clade 0–5) , unlike 14 formed clades 0 through 7  and 23 wild waterfowl isolates constituted clade 3. Clade 4, whose strains were mainly isolated between 2000 and 2005 in China, was previously characterized by the Dk1 lineage. Only two strains had Dk2-like PA segments and formed clade 5.We observed that eight different lineages of the PA segment, rather than the nine previously described,hrough 7 . Most of14 forming clades 0 through 6. It should be noted that over half of the NP genes in the analyzed strains clustered into clade 6, which was previously characterized by the F/98 lineage. The NP genes of the clade 5 isolates were apparently derived from the A/goose/Hong Kong/W217/97 (H6N9) strain, which was previously deemed the Aq lineage.2 Both clades 0 and 1 were previously characterized by the BJ/94 lineage. Clade 3 consisted of H9N2 isolates from minor poultry and chickens with the G1-like NP gene. Only 27 H9N2 strains were of duck and waterfowl origin and contained a Dk-like NP segment and clustered in clade 4.The NP segment segregated into seven lineages, similar to those previously identified,M gene showed much less diversity than the other segments. Unlike the three previously identified clades,14 we categorized the M genes into four clades (clades 0–3)  . The M sNS genes of H9N2 viruses in China. The number of lineages increased from three, as previously classified,14 to eight clades (clades 0–7) (We found considerable diversity in the des 0–7) . Clades P<0.05). In the first stage (1994–1999), fewer genotypes (n=19) were prevalent, indicating limited gene reassortment  were notably associated with the geographic distribution of the viruses . We calcFrom 2006 to 2009, H9N2 viral genetic diversity sharply decreased, and the prevalence of isolates was diffusely distributed among the 16 provinces. However, the proportions of isolates and genotypes in Shandong province and Hong Kong were relatively higher. After 2010, the Pearl River Delta and eastern coastal regions became the concomitant epicenters of H9N2 viral outbreaks . We defi5 Together, our findings demonstrate that H9N2 viruses originating from South China spread throughout the country over 20 years, and the Pearl River Delta and the newly identified Eastern Coastal Regions are the two epidemic centers. Additional surveillance is necessary to fully appreciate the geographic distribution of H9N2 viruses in China.In 2010, the number of H9N2 genotypes decreased and the virus was only isolated in seven provinces. After 2010, however, the number of affected provinces sharply rose to 19, and the eastern coastal region was the site of more frequent viral circulation . This ph22 In contrast, the remaining series were distributed in multiple hosts  to other hosts (n≥2), indicative of frequent viral transmission or wider viral host tropism of these genotypes  in the evolution of these viruses in China. In the periods divided by these time points, the genotypic diversity, geographic distribution and host range of H9N2 viruses underwent considerable changes.5 To understand the ecology of H9N2 generation and transmission, we employed the same strategy to analyze the genotypic evolution of the H9N2 viruses from all hosts in China from 1994 to 2013. Compared with the traditionally classified clades, the clade classification advanced here revealed great changes in the HA and NS genes of these viruses. The BJ/94-original HA and NS genes further evolved into 13 and 5 clades, respectively. Among these newly identified clades, clade 15 HA and clade 7 NS were components of the predominant G57 genotype, which contributed six internal genes to the novel H7N9 viruses.5 Moreover, the reassortment of the clade 15 HA gene seems to increase the viral host range. Therefore, it is necessary to continually update H9N2 clade classification to follow their genetic evolution. Such continual updating of clade classification is well established in the surveillance of H5 viruses.23We previously performed a phylogenetic study of H9N2 viruses isolated specifically from chickens in China from 1994 to 2013.Based on the clade classification, we analyzed the genotypic evolution of H9N2 viruses in China during 1994–2013. The period from 1994 to 1999 represents an initial era of adaptation and transmission of H9N2 viruses from aquatic birds to terrestrial poultry in China. H9N2 viruses originally circulated in the Pearl River Delta region in southern China, and gradually spread to 15 provinces. At this time, the G1, BJ/94, BG and F/98 genotypic series were restricted to quail, chickens and other minor avian species, with few gene exchanges among these species.14 The increase in genotypic diversity of H9N2 viruses in China may be explained by two hypotheses. The first hypothesis suggests that chickens, which are the largest avian population, increased the concentration of H9N2 viruses in one region, which greatly contributed to viral transmission between chickens and other hosts. The second hypothesis suggests that immune pressure within the chicken population contributed to the diversification of H9N2 genotypes. Since the late 1990s, chicken farmers in China have administered commercially inactivated vaccines raised against the BJ/94 and F/98 series to control H9N2 viral infections. This immune pressure may drive H9N2 viruses to become more adaptive through reassortment with G1-like and the Korean-like genes from quail and wild birds, respectively. During the second period of our analysis, the extent of the geographic distribution of H9N2 viruses was not considerably changed, and the Pearl River Delta region remained the epidemic center.In the following period between 2000 and 2005, the genotypic diversity of H9N2 viruses greatly increased, which resulted from frequent gene exchanges within different hosts. This was particularly evident in the G1-like genes and the Korean-like genes in quail and other minor avian species, which reassorted with the BJ/94-like and F/98-like series to enhance the diversity of their genotypes. This phenomenon was also observed in other studies.5 The G57 genotype was found in 2007 and initially prevalent in the eastern coastal region. In the 2010–2013 period, the eastern coastal region became the more important epicenter of H9N2 infections. Like Pearl River Delta, the environmental factors, such as warm climate, bird migration, the developed poultry industries and trade, may facilitate the distribution of H9N2 viruses in the eastern coastal region. In the third period between 2006 and 2013, H9N2 evolution has been largely characterized by G57 genotypic selection and expansion.After 2006, the diversified genotypes of the F/98 series generated in chickens were evolutionarily selected and become predominant, while the genotypes of the other series gradually decreased. This suggests that H9N2 viruses adapted by selecting the most advanced genotype. Subsequently, in 2010, the G57 genotype was further selected from the F/98 series and became the only predominant genotype. This is the first such event to occur during the 20-year prevalence of H9N2 viruses in China. We previously revealed that this G57 genotype contributed to the country-wide outbreak of H9N2 viruses in chickens and the subsequent emergence of the novel H7N9 reassortant with six H9N2 internal genes.H9N2 genes by generating and transmitting novel genotypes. Since 2010, the chicken-origin H9N2 viruses account for the majority H9N2 strains. Besides, the genotypes generated in chickens have shown changed biologic properties. Particularly, the recent predominant G57 genotype was generated in chickens and transmitted to other birds and mammals. Despite no human-origin G57 H9N2 strains were found in our data, it was reported that this genotype was isolated from a human patient in Hong Kong at the year of 2014.5 These recent H9N2 strains exhibited increased infectivity and transmission in chickens and ferrets,5 which creates serious concerns for public health. In addition, the G57 H9N2 viruses have provided genes to generate 11 novel reassortants of subtypes other than H7N9 . These novel reassortants were isolated in birds, swine and humans primarily after 2010.5 From 2013 to 2016, an increasing number of human H9N2 cases (n=18) were confirmed in China . Therefore, chickens have profoundly affected the evolution of H9N2 viruses.Since H9N2 viruses were isolated in farmed chickens and became endemic, this host has played an increasingly important role in the evolution of P<0.05). The important time points of 2000,14 200614 and 20105 were also, respectively, indicated in different studies. The changes of genotypes in poultry species that we found have good correspondences with the isolation rate  in these hosts demonstrated by previous epidemiological surveys.26 Recent phylogeography study also confirmed our results about the geographic distribution of H9N2 viruses in China.27 Our study presents a comprehensive summary of H9N2 evolution in the recent decades. However, more systematic epidemiological surveillance and analysis are urgently needed to better understand the circulation of H9N2 influenza virus in China.In this analysis, we included all the available 8162 sequences of H9N2 influenza viruses isolated in China from 1994 to 2013 uploaded in the NCBI, FluDB and GISAID, however the sample bias or the limitation of sequences on hosts and geographical coverage cannot be avoided, which may not be possible to adequately profile the emergence and evolution of H9N2 viruses. But, our findings of the H9N2 genetic evolution had the match with those of previous studies on understanding the viral behaviors. Statistical analysis showed that the genotypic diversity of H9N2 viruses between any two consecutive time periods was changed significantly (In summary, our findings indicate that throughout the 20-year prevalence of H9N2 viruses in China, minor avian species and wild birds exacerbate the genotypes by providing diversified genes, and chickens act as a vector, permitting genetic selection of potentially virulent genotypes and expanding prevalence. In this ecologic environment, H9N2 viruses appear to have evolved into an adaptive pathogen with a wide host range. Not surprisingly, the infectivity of H9N2 viruses has increased rapidly, especially in mammals. H9N2 viruses are predicted to enter into another evolutionary phase with increased genotypic diversity and accelerated changes in biologic characteristics. In addition to live-bird markets, poultry farms and wild-bird habitats are important sites for surveillance and disease control.


The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the embryonic retina by inducing chemokinesis in these cells.Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7 Microglia are resident macrophages of the central nervous system (CNS) that derive from myeloid hematopoietic progenitors –3. They th day of incubation (E7). Then, amoeboid microglia colonize the entire retina by tangential migration in a central-to-peripheral direction. Subsequent radial migration in a vitreal-to-scleral direction allows amoeboid microglia to reach the plexiform layers, where they differentiate into ramified microglia. Other studies in our lab showed that in vitro cultures of quail embryo retina explants mimic the migration and differentiation of microglial precursors in the in situ developing retina [Our previous studies showed the developmental program of microglia in the quail embryo retina –18. Thusg retina , 20. TheNumerous studies have highlighted the temporal and spatial coincidence between microglial immigration and programmed cell death (PCD) during the normal development of the vertebrate CNS , 21–27, Abundant research has shown that ATP/ADP and UTP/UDP are “find-me” signals that can stimulate both chemotaxis and chemokinesis in microglial cells, attracting them to sites of neuronal damage in the injured adult brain –39. Micrin vitro model system based on organotypic cultures of quail embryo retina explants to show that purinergic signaling by extracellular ATP and UDP, presumably released from dying retinal cells, is involved in the entry and chemokinetic migration of microglial cells into the retina, similar to observations in the developing zebrafish brain. Hence, this mechanism for microglial colonization of the CNS appears to be a conserved process across vertebrate species.We demonstrate here a chronological coincidence between the onset of microglial cell entry into the quail embryo retina and a striking increase in the death of retinal cells in regions dorsal to the BP/ONH area. We also used our Coturnix coturnix japonica) embryos at E6 and E7 were used to study the relationship between the first entry of microglial cells into the in situ retina and the apoptotic cell death taking place during these stages of retinal development. In addition, organotypic cultures of explants from E6.5 quail retinas were used to examine molecular mechanisms that control the first entry of microglial cells into the retina.Eyes from quail .Explants from E6.5 retinas were cultured i et al.  with somi et al. . Retinasin vitro incubation. In each experiment, reagent treatment was carried out on the retina explant obtained from the right eye of each quail embryo, whereas the retina explant from the left eye of the same embryo was cultured in reagent-free medium and served as a control. Apyrase , a potato enzyme that catalyzes the hydrolysis of extracellular tri- and di-phosphate nucleotides, was used at 10U/ml. Suramin sodium salt , a broad spectrum antagonist of the P2X and P2Y purinergic receptors, was used at 100 μM. Adenosine 5′-triphosphate disodium salt  was added to the culture medium at 10 μM, 100 μM, and 1mM. Adenosine 5′-(3-thiotriphosphate) tetralithium salt , a non-hydrolyzable ATP analogue, was used at 100 μM. Uridine 5´-diphosphate disodium salt hydrate  was added at 100 μM and 1mM. Q-VD-OPh , a broad spectrum caspase inhibitor, was used at 100 μM.E6.5 retina explants were subjected to experimental treatments with different reagents (as described below), which were added to the culture medium from the beginning of Cell viability in reagent-treated and control E6.5+24hiv retina explants was determined by flow cytometry of cell suspensions obtained by dissociation of retinal explants, using fluorescein diacetate (FDA) and propidium iodide (PI).After their culture, retinal explants were rinsed in 0.01M phosphate buffered saline (PBS) and dissociated in 1 ml PBS using a Dounce homogenizer . Then, the resulting suspensions were incubated for 15 minutes with 10 μg/ml PI (Sigma) and 15 μg/ml FDA (Sigma) in the dark. Samples were analyzed by flow cytometry using FACS Canto II flow cytometer . Cell viability was determined in each sample as the percentage of FDA-positive cells with respect to the total cell number (FDA-positive plus IP-positive cells). The mean cell viability was determined in at least six reagent-treated E6.5+24hiv retina explants and their corresponding controls for each treatment.Microglial cells were identified in cultured retina explants and non-cultured retinas by immunolabeling with the monoclonal antibody QH1 , which recognizes all quail hemangioblastic cells except for mature erythrocytes , includiDouble QH1/anti-active caspase-3 immunolabeling was carried out on whole-mounted non-cultured retinas, which were fixed in paraformaldehyde-lysine-periodate (PLP)  for 1 hoCross cryosections of non-cultured retinas were also used for double QH1/anti-active caspase-3 immunolabeling. Specimens were fixed for 1 hour at 4°C in PLP, thoroughly rinsed in PBS-Tr, cryoprotected overnight at 4°C in 20% sucrose in PBS-Tr, and embedded into 7.5% gelatin and 20% sucrose in PBS-Tr. Solidified blocks containing the specimens were then embedded in OCT compound, frozen in isopentane cooled with liquid nitrogen, and stored at -40°C before sectioning on a Leica CM1850 cryostat. Twenty micrometer-thick cryosections were obtained on Superfrost slides , hydrated in PBS, and treated for double QH1/anti-active caspase-3 immunolabeling following a similar schedule to that described for whole-mounts, with minor modifications . In addition, cell nuclei were stained in immunolabeled cryosections with the nuclear dye Hoechst 33342 (Sigma).Whole-mounted E6.5+24hiv retina explants underwent single QH1 immunolabeling or double QH1/anti-active caspase-3 antibody immunolabeling, following a similar protocol to that described for whole-mounted non-cultured retinas. Cross cryosections of control and reagent-treated E6.5+24hiv retina explants were also used for single anti-active caspase-3 (1:100), H5 (1:40) and 39.4D5 (1:100) immunolabeling to test whether experimental treatments did or did not produce structural alterations in the explants.The localization of apoptotic cells observed in E6 and E7 non-cultured retinas by anti-active caspase-3 immunofluorescence was compared with that demonstrated by TUNEL staining. Hydrated and permeabilized cross cryosections were treated for 1 hour at 37°C with a solution containing 0.01 units/μl of terminal deoxynucleotidyl transferase enzyme  in TdT buffer (Promega) and 4 nmol/ml biotin-16-dUTP . After incubation, sections were washed with PBS and incubated in Alexa Fluor 488-conjugated streptavidin (Molecular Probes) for 1 hour at room temperature. Following a blocking step in 10% NGS-PBS-Tr, sections underwent single anti-active caspase-3 (1:100) immunolabeling, using the protocol described above. Cell nuclei were stained with the nuclear dye Hoechst 33342.The proportion of active caspase-3-positive cells colabeled with TUNEL was determined by counts (with 40X objective) in specific microscopic fields in the dorsal part of double anti-active caspase-3/TUNEL-labeled histological sections from E6 and E7 non-cultured retinas (three retinas per age). These microscopic fields spanned 300 μm in width and the entire retinal thickness in height and were selected from 10 histological sections per retina, collected at intervals of 200 μm along the anterior-posterior axis of the eye. Results were expressed as the percentage of caspase-3-positive cells colabeled with TUNEL with respect to the total number of caspase-3-positive cells.Active caspase-3 positive cells were quantified by flow cytometry of cell suspensions obtained from reagent-treated and control E6.5+24hiv retina explants labeled with anti-active caspase-3 antibody. Explants were fixed in 4% paraformaldehyde for 15 minutes, rinsed in PBS, and incubated overnight at 4°C in anti-active caspase-3 antibody (1:100). After rinsing in PBS, retina explants were incubated for 1 hour at room temperature in the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG and subsequently dissociated in 1 ml PBS. After obtaining a cell suspension, the percentage of caspase-3 positive cells was determined by flow cytometry with respect to the total cell number  analyzed in at least six reagent-treated E6.5+24hiv retina explants and their corresponding controls for each treatment.In order to test the correlation between cell death and the entry of microglial cells into the retina, caspase-3-positive cells and microglial cells in the retina were simultaneously quantified in E6.5+24hiv retina explants treated with the caspase inhibitor Q-VD-OPh and in their respective controls. In brief, after fixation, each explant was incubated for 1 hour at room temperature in Alexa Fluor 594-conjugated QH1 antibody (1:500) and extended on a slide to obtain micrographs for subsequent microglial cell quantification. Then, the explant was rinsed in PBS, and the above protocol was followed for the quantification of caspase-3-positive cells by flow cytometry.in vitro culture, given that microglial cells enter the retina from E7 onward. Retina explants were then fixed in PLP for 1 hour and rinsed in PBS, followed by DNA denaturation with 2N HCl for 1 hour at 37°C and neutralization with 0.1 M Na borate (pH 8.5) for 25 minutes at room temperature. They were blocked for 15 minutes in PBS-Tr containing 5% normal goat serum (NGS) at room temperature and incubated overnight at 4°C in a mix of monoclonal anti-BrdU (1:800) (Sigma) and polyclonal anti-iNOS (1:500)  antibodies, both diluted in BSA-PBS-Tr. Polyclonal anti-iNOS antibody was used as microglial marker, because amoeboid microglia express iNOS during the normal embryonic development of quail retina [Microglial proliferation in ATP- and UDP-treated E6.5+24hiv retina explants and their respective controls was detected by adding 5-bromo-2’-deoxyuridine (BrdU) (Sigma) to the medium at a final concentration of 10 μM for the last 12 hours of l retina . The expThe percentage of proliferating cells was evaluated by counting BrdU-positive microglial cell nuclei and calculating their percentage with respect to the total number of microglial cells in at least eight reagent-treated explants and their corresponding controls for each treatment.Observations of fluorescent specimens were made with a Leica TCS-SP5 confocal microscope . Stacks of confocal optical sections of selected microscopic fields were collected at 0.5–1 μm intervals and projection images were obtained, stored in TIFF format, and digitally prepared with Adobe Photoshop .QH1-labeled microglial cells that had entered the retina were counted on microscopic fields of 2 mm x 2 mm in each whole-mounted E6.5+24hiv retina explant. Mean values of microglial cell numbers were determined from counts made in at least 10 reagent-treated explants and their corresponding controls.2 (500 μm x 500 μm) in each specimen.Indicators of morphological features of QH1-labeled microglial cells were determined in reagent-treated and control E6.5+24hiv retina explants by morphometric analysis using Image Tool 2.0 software . Cell profile area and cell elongation index were used as indicators to assess changes in the migratory phenotype of microglial cells after treatment with different reagents. The elongation index of a cell was defined as the ratio of its major axis length to its minor axis length. The mean values of these cell parameters were determined in at least 10 reagent-treated E6.5+24hiv retina explants and their corresponding controls, based on data obtained in three different square microscopic fields of 0.25 mmData are reported as means ± standard error of the mean (SEM). Statistical differences between reagent-treated retina explants and their controls were determined with the Student´s t-test. All analyses were performed using IBM SPSS statistics software version 20.0.0 for Windows . Differences were considered significant at P<0.05.Previous studies of our group showed that amoeboid microglial cells enter the retina of quail embryos from the BP/ONH area between E7 and hatching (E16) and then migrate tangentially in a central-to-peripheral direction on the vitreal part of the embryonic retina –17. In tin vitro system to reproduce the physiological-like behavior of microglial cells, which follow a similar chronotopographical pattern of migration and ramification to that observed in the in situ developing retina [in vitro study covered a period from 12 hours before to 12 hours after the first entry of microglial cells into the retina (at E7). In each quail embryo, the retina explant obtained from the right eye was used for experimental treatment, whereas the retina explant from the left eye served as non-treated control. This procedure was selected because the elongated phenotype of tangentially migrating microglial cells and their rate of entry from the BP/ONH area were similar between non-treated retina explants obtained from the two eyes in each quail embryo  or UDP (1 mM) had a significant effect on the entry of microglial precursors into the retina, probably because of the rapid hydrolysis of these nucleotides into nucleosides, including adenosine, by ectonucleotidases , 58, 94,In conclusion, this study offers experimental evidence of a relationship between retinal cell death and the entry of microglial cells into the developing quail retina. In addition, the findings indicate a major role for purinergic signaling in the early entry and subsequent migration of microglial precursors in the retina. Our results demonstrate that extracellular ATP and UDP exert a chemokinetic effect on microglial cells entering the retina through purinergic receptors, probably of P2Y subtype. Nucleotide signaling also participates in the recruitment of microglial progenitors into the developing zebrafish brain , suggest