Thoracolumbar myelopathies associated with spinal cord and vertebral column lesions, with a similar clinical phenotype, but different underlying etiologies, occur in pugs.To further characterize the clinical and neuropathological characteristics of pugs with longstanding thoracolumbar myelopathy.Thirty client‐owned pure‐bred pugs with a history of more than a month of ataxia and paresis of the pelvic limbs, suggesting a myelopathy localized to the thoracolumbar spinal cord, were included in the study.Prospective clinicopathological study. Included pugs underwent a complete neurological examination and gross and histopathologic postmortem studies with focus on the spinal cord. Computed tomography (n = 18), magnetic resonance imaging (n = 17), and cerebrospinal fluid analysis (n = 27) were performed before or immediately after death.Twenty male and 10 female pugs had a median age at clinical onset of 84 months . Affected pugs presented with a progressive clinical course and 80% were incontinent. There was circumferential meningeal fibrosis with concomitant focal, malacic, destruction of the neuroparenchyma in the thoracolumbar spinal cord in 24/30 pugs. Vertebral lesions accompanied the focal spinal cord lesion, and there was lympho‐histiocytic inflammation associated or not to the parenchymal lesion in 43% of the pugs.Meningeal fibrosis with associated focal spinal cord destruction and neighboring vertebral column lesions were common findings in pugs with long‐standing thoracolumbar myelopathy. CAPcaudal articulate processCNScentral nervous systemCSFcerebrospinal fluidCTcomputed tomographyCVMcongenital vertebral malformationGFAPglial fibrillary acidic proteinIVDHintervertebral disc herniationMRImagnetic resonance imagingSADsubarachnoid diverticula1Myelopathy involving the thoracolumbar spinal cord is well recognized in dogs and is clinically characterized by ataxia, paresis, or paralysis of the pelvic limbs, whereas the thoracic limbs are spared.The high prevalence of neurological gait abnormalities in pugs2The study was approved by the local Ethical Committee . Client‐owned pure‐bred Swedish pugs were included in the study between 2011 and 2018 at Albano Animal Hospital, Danderyd, and at the University Animal Teaching Hospital, Uppsala.2.1Pugs were included in the study provided that they fulfilled the following criteria: a history of more than 1 month of a bilateral pelvic limb gait abnormality described by the owner as incoordination and weakness , and a neurological examination confirming pelvic limb ataxia and paraparesis suggestive of a myelopathy localized to the thoracolumbar spinal cord. For the dog to be included, the owners also agreed to donate their dog for postmortem studies after naturally occurring death or euthanasia.2.2Dogs were excluded if histopathology of the thoracolumbar spinal cord was not performed at the time the pug died or was euthanized.2.3Signalment and clinical variables were included in the data set. A thorough case history, including onset and course of clinical signs, presence of incontinence , signs indicating the dog was in pain, and result of any given treatment, was obtained at the time of inclusion. All pugs underwent a complete neurological examination performed the same day they were euthanized, by a board‐certified neurologist (C. Rohdin). After euthanasia, the included pugs underwent a full postmortem examination with focus on the spinal cord and vertebral column. If practically possible, the included pugs underwent advanced diagnostic imaging, including magnetic resonance imaging (MRI) and computed tomography (CT), as well as cerebrospinal fluid (CSF) sampling and analysis, and repeat neurological examinations (by C. Rohdin). Advanced imaging was carried out before or immediately after death in order to associate any potential spinal cord lesion with the presence of vertebral column lesions, as well as to guide the histopathologic investigation of the spinal cord. Some of the recruited dogs were also included in another study investigating the presence of CVMs in pugs with and without neurological deficits.2.4Computed tomography was performed with 2 mm slice thickness, using a dual slice CT scanner , or a 64 slice CT scanner . The studies were reconstructed with a soft tissue and a bone algorithm using medium and high‐frequency reconstruction algorithms. For CT studies, the examination included the entire thoracic and lumbar vertebral column and the presence of vertebral malformations, such as hemivertebra, associated kyphosis, transitional vertebrae, and hypo‐ or aplasia of the CAPs was evaluated.The MRI studies were acquired using either a magnet operating at 0.2 Tesla , or at 1.5 Tesla . At a minimum, the MRI study included sagittal T1‐ and T2‐weighted images of the entire thoracolumbar spine and transverse T1‐ and T2‐weighted images of the area of interest. Intervertebral discs were described as compressing , or not compressing the spinal cord.All CT and MRI studies were submitted to and evaluated by a single board‐certified imager at a diagnostic imaging company .2.5The CSF samples were taken at the University Animal Teaching Hospital, Uppsala, Sweden, or at Albano Animal Hospital, Danderyd, Sweden, and analyzed within 30 minutes of collection. The analyses included total leucocyte‐ and erythrocyte cell counts, and differential leucocyte counts, and measurement of protein concentration (turbidimetry). Protein concentration was considered to be increased if >0.25 g/L at the cerebellomedullary cistern, or >0.40 g/L at the lumbar cistern.2.6Gross pathology was performed at the Section of Pathology, Swedish University of Agricultural Sciences (site 1) or at the National Veterinary Institute, Uppsala, Sweden (site 2).The entire central nervous system (CNS) were carefully sampled and fixed in 10% neutral buffered formalin for transport to a board‐certified pathologist. After the spinal cord had been evacuated, the vertebral column was carefully examined and cut sagittally. Midsagittal sections were performed of thoracolumbar vertebrae and intervertebral discs with macroscopically degenerative changes, and overt disc herniations on macromorphology, or where compression of the spinal cord had been demonstrated on MRI. Discs adjacent to disc herniations were also included. The sections were placed in 10% neutral buffered formalin and decalcified using 20% formic acid (Kristensens solution).Histopathological examination of the CNS was performed at site 2, at the Department of Pathology, University of Veterinary Medicine, Hannover (site 3), or at the Section of Clinical and Comparative Neuropathology, Ludwig‐Maximilians‐Universität, Munich (site 4), Germany. After fixation, specimens of various anatomical sites of the brain and spinal cord were processed for routine histopathology, embedded in paraffin wax and sectioned at 3‐4 μm. Tissues were stained using routine hematoxylin and eosin (HE), and examined by light microscopy.Histopathology of the vertebral column was performed at site 1. Decalcified tissue was embedded using paraffin wax and processed to 4‐5 μm sections. Tissues were routinely stained with HE. The overall morphology and integrity of the intervertebral discs and adjacent vertebrae, in addition to other apparent features, were assessed and recorded.2.7P < .05.Descriptive statistical analyses were performed using a commercially available statistical software program . Continuous variables are presented as means and SDs and as medians and interquartile range (IQR). Associations between clinical variables and pathological findings were investigated using the Chi‐square, Fishers exact test. Level of statistical significance was set at 33.1Thirty pugs fulfilled the inclusion criteria with pathology being performed by the time the pug was euthanized. Information about dog signalment, clinical variables, CSF findings, and whether or not the dog was receiving medical treatment is presented in Table All 30 pug owners described an insidious, progressive course of their pug's pelvic limb gait abnormality. Mean duration of clinical signs before veterinary consultation was 7.1 months . None of the owners perceived their dogs to be in pain, but a reluctance to go for walks was reported by the owners of 13 pugs.At the physical examination, all 30 pugs presented with moderate to severe pelvic limb muscle atrophy. Spinal pain was not recognized in any of the pugs upon examination. In 13 pugs, a repeated neurological examination had been performed. In 3 of them, the pelvic limb flexor reflexes gradually became weaker, with loss of muscle tone in the pelvic limbs, at reexaminations performed within 9.5, 11, and 12 months after the initial examination. These 3 pugs were examined neurologically on 7, 4, and 3 occasions respectively. In 2 out of these 3 pugs, with a normal perineal reflex upon initial examination, loss of the perineal reflex and anal tone developed over time.All 30 pugs were still ambulatory at the day of euthanasia. Twenty‐eight pugs were euthanized because of their gait abnormality or because of their associated incontinence. Other causes for euthanasia (n = 2) were pyometra (n = 1) and osteosarcoma of the mandibula (n = 1). The pug with pyometra had a neurological examination performed, confirming pelvic limb ataxia and paraparesis, 1 month before acute onset of clinical signs of the pyometra. The pug with osteosarcoma had a neurological examination, confirming pelvic limb ataxia and paraparesis, and a CT of the entire thoracolumbar vertebral column, with no signs of vertebral involvement, performed 2 months before euthanasia.3.2Fifteen pugs underwent both CT and MRI imaging. All CT studies n = 18) were performed immediately after death. Magnetic resonance imaging was performed under general anesthesia (n = 7) or immediately after death (n = 10). Mean time between MRI and euthanasia in the 7 pugs examined under anesthesia was 6.3 months SD, 4.2 months). In the 18 pugs examined by CT, 64 CVMs were identified, mean 3.6 , in the thoracolumbar vertebral column. In CT‐examined pugs with histopathologically confirmed focal spinal cord lesions (n = 17), at least 1 CVM presented immediately adjacent to the focal lesion in 16 pugs (94%)  or from the lumbar area n = 1) when the dogs were under general anesthesia (n = 1) or in association with euthanasia (n = 26). The median number of mononuclear cells in the CSF was 6/μL  ; of protein 0.3 g/L  ; and of erythrocytes 5/μL   (Table  when the3.4Gross pathology was performed at site 1 (n = 29), and at site 2 (n = 1).Histopathology of the CNS was performed at site 3 (n = 19), at site 4 (n = 10), and at site 2 (n = 1).Fifteen dogs had intervertebral discs evaluated histopathologically (site 1).3.4.1Vertebral column pathology included hemivertebra (n = 6), with (n = 4) or without (n = 2) kyphosis, and herniating intervertebral discs (n = 15). The morphology of the CAPs could not be assessed, as they were destroyed by the processing procedure. There was occasional focal spinal cord atrophy of the thoracolumbar spinal cord.3.4.2Lesions were confirmed in the CNS of all examined pugs including meninges, spinal cord, and nerve roots. Meningeal fibrosis and concomitant spinal cord parenchymal lesions were a consistent finding in 29/30 pugs.Meningeal fibrosis was confirmed in the thoracolumbar spinal cord of 29 pugs. Focal, circumferential, meningeal fibrosis, limited in length along the spinal cord, in many cases involving only a few millimeters Figure , was preMild to moderate meningeal proliferation was also recognized throughout the CNS unrelated to the focal spinal cord pathology in 14 pugs.A single, thoracolumbar, focal spinal cord lesion was repeatedly found accompanying the meningeal fibrosis in 28 pugs. In 1 other pug, the lesion was more extensive, presenting between T10 and L1. The parenchymal spinal cord lesions were characterized by malacia (n = 24), by syringomyelia (n = 4), or by hydromyelia (n = 1). The level of these focal lesions corresponded to the intramedullary T2W hyperintensity on MRI. The focal malacic lesions within the spinal cord were delineated by microgliosis, astrogliosis, gemistocytes, and astrocytosis, forming astroglial scars Figure . The malWallerian‐like degeneration could be traced further cranial from the malacic spinal cord lesions, mainly through the fasciculus gracilis in the cervical spinal cord Figure . CompareNerve root entrapment and vascular encasement was found with meningeal vessels and nerve roots embedded in the circumferential meningeal fibrosis. Loss of nerve fiber density, seen involving both dorsal and ventral nerve roots Figure , hypertrVarying degrees of lympho‐histiocytic CNS inflammation, including encephalitis (n = 9) Figure , myelitiSevere gray matter destruction was recognized in the cranial cervical spine in 3 pugs. One of these pugs had had surgery to remove a SAD in the dorsal C2‐C3 spinal cord area 4 years before presenting with signs of a T3‐L3 myelopathy. These lesions displayed the same features as the lesions in the thoracolumbar spinal cord, with (n = 2) or without (n = 1) Figure  associatMeningeal fibrosis with a concomitant spinal cord lesion was absent in 1 pug. At 10 months before euthanasia, this pug was diagnosed by MRI (1.5 Tesla) with a focal intramedullary T2W hyperintensity and a probable SAD at T6/7. Histopathology displayed neurodegenerative features with mild‐severe dilated myelin sheaths, swollen axons, and myelinophagia in the cervical, thoracic, and lumbar spinal cord, however, no focal lesions was identified.3.4.3Fifteen out of 30 pugs had a total of 39 thoracolumbar discs evaluated histopathologically. Herniating intervertebral discs were found at the identical site as the focal lesion in 4 out of 15 pugs. On MRI, these 4 herniations were mildly moderately compressing the spinal cord. In 1 pug with evidence of moderate spinal cord compression from a disc diagnosed on MRI, IVDH could not be confirmed by histopathology 8 months later.In 6 of the 15 examined pugs, protrusions of chondroid tissue, presumably originating from the nucleus pulposus with chondroid metaplasia, into the adjacent endplate and subsequently into the subchondral bone, so called Schmorl's nodes, were found Figure . Schmorl4Apart from sharing a uniform clinical presentation and course of the disease, pugs in this study also exhibited similar neuropathological features, including thoracolumbar meningeal fibrosis and concomitant spinal cord destruction. Although the homogenous clinical and pathological findings could represent a true disease, the additional finding of meningeal fibrosis unrelated to the thoracolumbar spinal cord lesion more likely suggest a predisposition, possibly breed related, to meningeal proliferation. The profound meningeal fibrosis could interfere with vascular supply to the spinal cord as well as cause obstruction of CSF flow with subsequent parenchymal spinal cord destruction.Although the finding of concomitant CNS inflammation and neighboring vertebral column pathology might be unrelated findings, their high, and for CNS inflammation unexpected, prevalence suggest they are part of the underlying etiology.Histopathological evidence of ongoing mild to severe lympho‐histiocytic CNS inflammation, presenting distant to or at the focal spinal cord lesion, was found in 43% of the examined pugs. In addition, 52% pugs presented with a cell composition in the CSF indicating, in general mild, intrathecal mononuclear inflammation. Although an infectious etiology was considered unlikely because of the stereotype and longstanding history, this cannot be entirely excluded. On the other hand, glial fibrillary acidic protein (GFAP), the hallmark intermediate filament protein in astrocytes,without (1.5‐30 months) and those with inflammatory infiltration of the arachnoidea. Mild inflammatory meningeal changes have previously been described in pugs with SAD.The spinal cord lesions from the pugs with thoracolumbar myelopathy in this study shared many features with the spinal cord of laboratory dogs, used in research about adhesive, also called constrictive, arachnoiditis.hout 1.5‐0 months Pugs with intraparenchymal malacia presented with a longer duration of clinical signs before euthanasia and less often with lympho‐histiocytic inflammation in the CNS on histopathology compared to pugs with syringo/hydromyelia. Although not statistically significant, this may indicate that there is an early inflammatory phase and a later chronic proliferative state in which fibrosis and adhesions become permanent in pugs with a longstanding thoracolumbar myelopathy, as suggested in human literature related to adhesive arachnoiditis.Vertebral column lesions, including CVMs and IVDHs, accompanied the focal meningeal fibrosis and spinal cord lesions in pugs diagnosed by CT. Unfortunately, all pugs in the present study were not examined by advanced imaging, and some CVMs, in particular articular process dysplasias, are difficult to document without CT.An interesting finding was the prolapse of the nucleus pulposus through the cartilaginous endplate into the body of the vertebral body, consistent with a Schmorl's node. Schmorl's nodes are uncommonly described endplate abnormality in canines.Histopathology showed intradural tethering and hypertrophy of the dentate ligaments in pugs of this study. The spinal cord, meninges, and nerve roots move within the vertebral canal upon posturing of the head and spine.Although all pugs included in this study presented with the similar, predictive, clinical phenotype, thoracolumbar meningeal fibrosis and parenchymal spinal cord pathology were not confirmed in 1 dog, which indeed had a T2W hyperintensity in the thoracolumbar spinal cord on MRI. As the extent of the parenchymal spinal cord pathology in general was limited in length along the spinal cord, it might therefore easily have been missed on trimming procedures.The majority of pugs presented with incontinence, and fecal incontinence was more likely to develop before urinary incontinence. Reports on fecal and urinary incontinence in dogs, including pugs, with upper motor neuron spinal cord lesions have been sparse and mostly associated with SAD.Spinal pain was not described by any of the owners nor recognized upon examination in any of the pugs. However, reluctance to go for walks was described by more than a third of the owners. Although vascular compromise to the spinal cord in dogs, except initially, is described as non‐painful,We acknowledged that this descriptive study, having more than 1 pathologist examining the pugs, was limited by the lack of possibility for objective comparison and statistical analysis. However, the pathologists independently confirmed the major pathological findings of this study, that is, meningeal fibrosis, and concomitant spinal cord lesions, and also the presence of lympho‐histiocytic CNS inflammation. Furthermore, each of them provided valuable input related to additional pathology and potential pathogenesis of pugs with longstanding thoracolumbar meningeal fibrosis. Owner attitude and financial limitations early in the project might have resulted in exclusion of some dogs undergoing the same diagnostic protocol and may have limited the opportunity to evaluate the importance of neighboring vertebral lesions.5Pugs with a longstanding thoracolumbar myelopathy presented with a progressive clinical course and a high prevalence of fecal incontinence. The pathology, characterized by meningeal fibrosis with associated malacic spinal cord lesions, was accompanied by neighboring vertebral column lesions. In addition, lympho‐histiocytic CNS inflammation was recognized in the CNS related and unrelated to the focal spinal cord lesion. Increased knowledge of the clinical and pathological characteristics is important for our understanding and treatment of this debilitating condition in pugs. Future studies should focus on the mechanisms behind the development of meningeal fibrosis in pugs.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Animal Ethics Committee of Sweden  C202/2014.Authors declare human ethics approval was not needed for this study.


Subjective age refers to how young or old people experience themselves to be. It is associated with health status, physical function, and mental processes that influence the occurrence of fear of falling, which is a severe problem in older adults. The present study aims to contribute to existing knowledge by examining the relationships between subjective age and fear of falling. Participants were community-dwelling adults aged 65 years and older drawn from the 2011 (T1) - 2015 (T5) waves of the National Health and Aging Trends Study. Subjective age was measured by asking, “What age do you feel most of the time?” Fear of falling was measured with the question: “In the last month, did you worry about falling down?” The analyses included 1,984 participants without fear of falling at baseline, on average, 74.9±6.6 years old, female (53%), and non-Hispanic whites (76%). Eighty percent of the participants felt younger, and 4% felt older than their chronological age, but 21% of them had a fear of falling. Participants who experienced fear of falling at T5 tended to be female, non-Hispanic whites, and live alone. Generalized estimating equations revealed that an “older” subjective age independently predicted fear of falling  controlling for demographics and health conditions. Fear of falling predicted an “older” subjective age  after adjustment. These findings indicated bidirectional relationships between subjective age and fear of falling. Subjective age may help identify individuals who are at risk for fear of falling in older adults.


The predictive power for NIV failure of Model III  was better than that of Model I . Considering the improvement in parameters, patients with successful NIV exhibited significantly improved heart rates, arterial pH and PaCO2 levels, and patients with NIV failure exhibited a significantly improved PaCO2 level only. In conclusion, NIV is reasonable to apply to elderly patients with pneumonia, but should be done with caution. For the early identification of NIV failure, the heart rate and arterial blood gas parameters should be monitored within 2 h after NIV commencement.Noninvasive ventilation (NIV) is useful when managing critically ill patients. However, it is not easy to apply to elderly patients, particularly those with pneumonia, due to the possibility of NIV failure and the increased mortality caused by delayed intubation. In this prospective observational study, we explored whether NIV was appropriate for elderly patients with pneumonia, defined factors that independently predicted NIV failure, and built an optimal model for prediction of such failure. We evaluated 78 patients with a median age of 77 years. A low PaCO Noninvasive ventilation (NIV) refers to ventilatory support delivered via a nasal, full- face, or helmet mask, without bypassing the upper airway, which is different from tracheal intubation or tracheostomy ,2,3,4. NThe proportion of elderly patients in intensive care units (ICUs) is rapidly growing, because global populations are aging . If eldeThe aim of this study was to investigate whether NIV was appropriate for elderly patients with pneumonia, to define factors that independently predicted NIV failure, and to make an optimal model for prediction of NIV failure.This was a prospective observational study conducted at 21 university-affiliated hospitals of South Korea from June 2017 to February 2018. Among ICU patients prescribed NIV in accordance with international guidelines, we selected a subset by applying inclusion and exclusion criteria. The NIV machines, interfaces, and circuits were selected by respiratory intensivists from among the equipment available; the intensivists also chose initial NIV settings and durations by reference to patient conditions and the international guidelines. Patients who deteriorated after NIV underwent tracheal intubation at the discretion of respiratory intensivists. The study was approved by the Institutional Review Boards (IRBs) of all participating hospitals and the Korea Medical Center IRB . Written informed consent was obtained from all enrolled patients or their legal surrogates.The inclusion criteria were age ≥ 65 years and NIV prescribed at admission or during the hospital stay because of acute hypoxic/hypercapnic RF or acute on chronic RF. The exclusion criteria were age <65 years; a lack of written informed consent; NIV use despite contraindications, such as myocardial infarction or post cardiac arrest; NIV use because of acute postoperative RF; NIV weaning; and NIV palliative therapy.2)/percentage of inspired oxygen (FiO2) ≤300 and a normal or low partial pressure of arterial carbon dioxide (PaCO2). Acute hypercapnic RF was defined as a PaCO2 >50 mmHg [2/FiO2 > 300 or PaCO2 < 50 mmHg) when on NIV and resolution of any acute exacerbation. NIV failure was defined as a transition to mechanical ventilation , a “hopeless” discharge on NIV, or death while on NIV.Acute RF was divided into acute hypoxic and acute hypercapnic RF. Acute hypoxic RF was defined by a partial pressure of arterial oxygen  with 95% confidence intervals (CIs). We sought a model that optimally predicted NIV failure. The discriminatory power of each tested model was assessed using the Harrell C-index and the area under the curve (AUC); the models were compared using a bootstrap method. A two-tailed p-value < 0.05 was taken to indicate significance. All statistical analyses were performed using SAS ver. 9.4 software .Descriptive statistics are presented as medians (the 25th to 75th percentiles) or as numbers (percentages). The Fisher’s exact test was used to analyze categorical data, and the Mann-Whitney n = 36; age <65 years, n = 37; NIV weaning, n = 40; postoperative NIV, n = 3; NIV after myocardial infarction, n = 1; NIV after cardiac arrest, n = 3; and palliative NIV, n = 2); thus, 78 patients were included, of whom 46 patients experienced NIV success and 32 patients experienced NIV failure . We used logistic regression to identify factors associated with failure. Univariate analyses showed that the heart and respiratory rates, the PaO2/FiO2 ratio, PaCO2, use of the assist control mode, and pneumonia were significantly associated with NIV failure. Multivariate logistic regression (using backward elimination) revealed that a low PaCO2 , a high heart rate , and pneumonia  were significant independent predictors of NIV failure . Models II and III were slightly better . The differences in the AUCs of Models II and III (compared to that of Model I) were significant   .2) between 30 min and 2 h after NIV commencement, should be considered when predicting NIV failure.Therefore, not only pneumonia status, but also a vital sign (heart rate) and arterial blood gas status . Therefore, NIV is not contraindicated by pneumonia alone. Improvement of heart rate and PaCOp = 0.535).Our study makes several important contributions to the literature. When elderly patients are admitted to ICUs, intensivists find it difficult to decide whether aggressive treatment is appropriate, because advanced age is associated with poorer outcomes after mechanical ventilation, and survival does not always ensure a satisfactory quality of life ,20,23,24Early identification of NIV failure is important; delayed identification increases mortality. In a previous study, the timing of NIV failure was categorized as follows: (1) immediate failure (within minutes to < 1 h), (2) early failure (1–48 h), and (3) late failure (after 48 h) . The maj2 level independently predicted NIV failure. Although the PaO2/FiO2 ratio has been associated with NIV failure in patients with hypoxic respiratory failure, the arterial pH and PaCO2 values of patients with hypercapnic respiratory failure were more closely associated with NIV failure than the PaO2/FiO2 ratio [2 value more meaningfully predicted NIV failure than the PaO2/FiO2 ratio.We found that heart rate and PaCOO2 ratio . In our 2 level after NIV commencement was associated with NIV failure. A low PaCO2 level was associated with severe disease and agitation, because the increased tidal volume and tachypnea caused by labored breathing led to reduction of the PaCO2 level [2 level after NIV commencement was related to a PaCO2 level before NIV commencement. Therefore, patients with a low PaCO2 level after NIV commencement initially had more severe disease, compared to patients with a high PaCO2 level after NIV commencement. Disease severity was associated with NIV failure, so NIV failure was more likely in patients with a low PaCO2 level than in patients with a high PaCO2 level. A more important finding related to the evaluation of NIV failure was the variation in PaCO2 level after NIV commencement. As shown in 2 level in the NIV failure group was associated with a less-improved arterial pH than that of the NIV success group. In other words, although the absolute median variation was similar in both groups, PaCO2 improvement was less clinically effective in the NIV failure group than in the NIV success group. This finding is probably because the number of patients with hypoxic respiratory failure was higher in NIV failure group than in NIV success group. However, a PaCO2 level reflects the clinical condition. Consequently, when predicting NIV failure, we should consider whether a PaCO2 level has improved properly to the point where pH reaches to the normal range. The results of a subgroup analysis of patients with only hypercapnic RF were similar to the findings in the present study . Therefore, pneumonia was severe. We found that the C index of pneumonia alone was 0.639; thus, pneumonia was only weakly associated with NIV failure. Therefore, NIV is reasonable to apply in elderly patients with pneumonia, if we could carefully observe patients after NIV commencement.We also explored the utility of NIV in patients with pneumonia. This has been controversial; prior studies have reported conflicting results, probably because of differences in clinical characteristics  ,35,36,37n = 78). However, the number was sufficient to reveal the factors associated with NIV failure in elderly patients with acute RF, because a post hoc power analysis revealed that the power of each variable was appropriate. Second, we did not include clinical data, such as chest X-ray or laboratory findings, when determining disease severity in patients with pneumonia. However, given the SOFA scores and PaO2/FiO2 ratios, most pneumonia patients suffered from sepsis. Third, we did not set detailed indications for weaning or intubation, relying rather on respiratory intensivist discretion. However, all intensivists had extensive experience with NIV, and most decisions were based on international guidelines.Our study had certain limitations. First, the patient number was relatively small (NIV is reasonable to apply in elderly patients with pneumonia, if we could carefully observe patients after NIV commencement. For the early identification of NIV failure triggering immediate intubation, the heart rate and arterial blood gas parameters should be closely monitored between 30 min and 2 h after NIV commencement. Additional studies with larger patient cohorts are warranted to validate our findings.


Agrostis stolonifera grasses growing on toxic waste from an abandoned lead mine  and identified them using DNA sequencing (nrITS barcoding). The endophytes were then tested as a consortium of eight strains in a growth chamber experiment in association with the grass Festuca arundinacea at increasing concentrations of lead in the soil to investigate how they influenced several growth parameters. As a general trend, plants treated with endophytes performed better compared to the controls at each concentration of heavy metal, with significant improvements in growth recorded at the highest concentration of lead . Indeed, this set of plants germinated and tillered significantly earlier compared to the control, with greater production of foliar fresh and dry biomass. Compared with the control, endophyte treated plants germinated more than 1-day earlier and produced 35.91% more plant tillers at 35 days-after-sowing. Our results demonstrate the potential of these fungal endophytes used in a consortium for establishing grassy plant species on lead contaminated soils, which may result in practical applications for heavy metal bioremediation.Bioremediation is an ecologically-friendly approach for the restoration of heavy metal-contaminated sites and can exploit environmental microorganisms such as bacteria and fungi. These microorganisms are capable of removing and/or deactivating pollutants from contaminated substrates through biological and chemical reactions. Moreover, they interact with the natural flora, protecting and stimulating plant growth in these harsh conditions. In this study, we isolated a group of endophytic fungi from  Some metals are essential to plant growth such as zinc and manganese, but lead does not have any known biological function and is toxic to plants even at low concentration [Lead (Pb) is classified as a “heavy metal” due to its high relative atomic weight  in the cells (non-specific defense mechanism) [Due to the high toxicity of lead, plants activate several responses in the presence of the metal including (1) immobilization of the metal in the rhizosphere through the release of exudates or biosorption in the roots walls (exclusion); 2) assimilation of the metal into the root and activation of detoxification reactions involving the sequestration of the metal through the production of metal ligands such as phytochelatin and metallothioneins, and sometimes accumulation in cellular vacuoles (detoxification); and (3) activation of the NO-dependent stress response and other metabolic pathways to minimize the amount of bioavailable lead (Pbchanism) ,5. Moreo assimilachanism) . Howeverchanism) . Lead causes huge environmental and human health problems ,8. ThereEndophytes offer bioremediation potential . EndophyNeotyphodium are foliar endophytes that have been studied for their beneficial effect on grasses such as Festuca spp. and Lolium spp. in soil polluted with aluminum, zinc, copper, and cadmium [Festuca sp. treated with N. coenophialum and N. uncinatum produced a greater foliar and root biomass compared with untreated plants at a high concentration of cadmium in a hydroponic system. Endophytes are also able to support plant growth in the presence of toxic heavy metals positively influencing the plants’ absorption, degradation, and dislocation of the contaminant . For exa cadmium ,17,18,19 cadmium  demonstrVerbascum lychnitis grown in post-mining waste when co-inoculated with Rhizopagus irregularis, an arbuscular mycorrhizal fungus (AMF), Cochliobolus sativus, Diaporthe sp., and Phoma exigua var. exigua compared with non-inoculated and AMF plants. When the plants were inoculated only with Diaporthe sp., a negative effect was measured in the plant, an effect that was offset with a positive response when the plant was also inoculated with the AMF [Agrostis capillaris when co-inoculated with an Epichloë species. They hypothesized that excessive production of nitrogen by the DSE was used for the production of alkaloids for plant defense by the Epichloë species, balancing the costs for the plant in hosting the DSE [Recent research has emphasized the importance of using endophytes as a consortium rather than as a single strain. Indeed, Wężowicz et al.  found an the AMF . Vandegr the AMF , in a si the DSE . Agrostis stolonifera) from soil highly contaminated with lead. The aim of the research was first to establish the identity of the endophytes recovered from the grass and to investigate the literature for any bioremediation effects reported for these microorganisms. Once this was established, and considering their coexistence in the same sampled plant, the microorganisms selected from the recovered endophytes were tested as a consortium in a laboratory experiment in association with Festuca arundinacea Schreb.  Darbysh). The grass was grown in the presence of increasing concentrations of lead in the soil to determine the influence of the microbial consortium on plant metal toxicity.However, relatively little is known about fungal endophytes and their potential application in bioremediation . TherefoPlant samples were collected from an abandoned mine site in Ballycorus, County Dublin, Ireland. The mine was manually excavated to recover lead and silver in the 20th century up until the 1920s. Previous research revealed an average lead concentration at the site of 1.5%  in spoil and slag waste, which exceeds the UK guideline limit of lead for soils  on industrial sites (> 750 mg/kg Pb). The site  includes three solid waste heaps  and an additional waste heap on the banks of the Loughlinstown River (SLAG01) . 2 and its altitude ranges between 205 to 225 m along a northwest 10–30° slope. The soil is dry and loose, and vegetation is limited to sparsely distributed grasses such as Agrostis stolonifera and the site is surrounded by birch trees (Betula sp.) [2 and altitude between 188 to 194 m and features a northwest facing 10–20° slope. It is populated by Agrostis stolonifera, Festuca ovina, and Galium verum. Gorse (Ulex europaeus), pine (Pinus sylvestris), birch (Betula sp.), and bracken (Pteridium aquifolium) are also found in the surrounding area. The soil is equally as dry and rocky as SP02, but with no humic or mossy layer [2. The heap is between 177 and 183 meters above sea level and has a northwest slope of between 10–30°. The area is surrounded by various plant species including Rubus fruticosus and Ulex europaeus, with a surface layer of moss and loose, rocky soil [SP02 is the largest and highest of the three heaps closest to the top of the mine, which constitutes the main volume of solid waste. The total area is 1505 mula sp.) . Concentula sp.) . A recensy layer . Lead insy layer . SP04 iscky soil . Lead coOther minerals such as silver, zinc, nickel, and barium were found throughout the mine site at varying concentrations (see ).Agrostis stolonifera plants were sampled at each of the three spoil heaps  using a small spade without distressing the plant and root system. Isolated grass clumps were chosen away from the main grass carpet to avoid areas where the soil was restored and was therefore less rich in heavy metals. Plants for endophyte isolation were collected from the three waste heaps described above. Two Once in the laboratory, five root samples were collected from each plant and surface sterilized by first washing each root sample in 70% ethanol and then submerging in 5% sodium hypochlorite for 3–5 min. Then, the roots were washed three times in deionized sterile water to remove any chemical residues. Each of the roots was cut into five non-contiguous segments of 1 cm length, which were then laid on ½ strength potato dextrose agar  in Petri dishes. The plates were labelled, sealed with parafilm and stored in the dark at 20 °C in an incubator. The Petri dishes were observed daily until the endophytes began to grow out from either end of the root about a week after the culturing. Each emergent endophyte was isolated on a separate Petri dish and a minimum of three subcultures were established on separate plates.g for 10 min and the upper CTAB phase of approximately 900 µL was transferred to a new sterile 1.5 mL tube. An equal volume of 500 µL ice-cold isopropanol was added to each tube, which were mixed a few times by inversion and stored at −20 °C for an hour. The tubes were centrifuged at 8000× g for 5 min to spin down the DNA. The supernatant was removed, and the pellet was washed with 800 µL of 70% ethanol and the samples were centrifuged at 8000× g for 3 min. The supernatant was removed, and the samples were left to dry at room temperature under a chemical hood for between 30 minutes to one hour. The pellets were resuspended in 100 µL TE buffer and the samples were kept at 37 °C in a heat box for 30 min to allow the DNA to fully dissolve. The final concentrations of DNA (ng·µL−1) in the extracted samples was measured with a Nanodrop UV spectrophotometer . For the identification of the endophytes, total genomic DNA was isolated using a CTAB (Hexadecyltrimethylammonium bromide) extraction in 2 mL microcentrifuge tubes as follows: the extraction buffer was prepared by adding 1 mL of 2× CTAB solution and 0.2% β-mercaptoethanol into a sterile 2 mL microcentrifuge tube and was preheated in a heat block at 65 °C for 15 min. Fungal material of 200 mg average weight for each sample was added to the preheated buffer with a metal bead and the contents were homogenized using a Mixer Mill MM 300 (Retsch) for 1.5 minutes at 30 Hz each side. The samples were then incubated at 65 °C for one hour, mixing the tubes by inversion 3–4 times. A volume of 500 µL of chloroform:isoamylalcohol (24:1) was added to each tube, which were shaken vigorously for 15 seconds and laid on a shaking plate at 220 rpm for 10 min to mix well. The samples were centrifuged at 8000× The fungal isolates were grouped based on their colony morphology as observed on the ½ strength potato dextrose agar (PDA). Twelve different morphologies were observed, out of which eight were chosen to use for further analysis based on their rapid growth at room temperature and their early sporulation.https://unite.ut.ee). Similarity criteria for assigning taxonomic rank to the endophyte strains was allocated based on an initial survey of existing fungal taxa in UNITE and GenBank, as follows: >97% similarity was assigned to the same species, 90–96% to the same genus, 85–90% to the same order, and <85% to no significant match.Amplification of the nrITS barcode intergenic regions were carried out using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′)  and ITS4Furthermore, β–tubulin (Bta2) and translation elongation factor 1-α (tef) sequences were amplified following the cycle described by Raja et al. (2017) to confirm the identification of some samples .6 spores/mL. Each isolate spore solution  were mixed in equal quantities to reach a final inoculum volume of 12 mL.For the inoculum, single endophyte isolate spore solutions were prepared by washing the fungal colony with sterile water and delicately shaking the plate to dislodge the spores into the solution that was then collected. The concentration of spores was measured using a hemocytometer and the solution modified to give a concentration of 10Lead is naturally bound with other elements and is commonly found as galena. Galena (mainly PbS), containing on average 86% of Pb, 13% of S, and 1% Ag is the main ore of lead . This mi0 = 0, Pb400 = 400, Pb600 = 600, Pb800 = 800 galena mg/kg) were mixed separately with 5 kg of John Innes No. 2 compost  and then, divided between 20 pots of 1 L volume for a total of 80 pots. Then, two seeds of Festuca arundinacea  were sown in each pot at a 1 cm depth. The 20 pots for each concentration of galena were equally split between the control and endophytic set by inoculating the seeds with either 200 µL of sterile water for the control or 200 µL of the microbial inoculate for the endophytes set, for a total of 10 pots for each treatment set  was programmed to produce a 16 h photoperiod at a compost surface illumination of 230 µmol mThe plants were given no supplementary nutrients during the experimental period and were harvested after 50 days. Germination and tillering time were recorded for each plant during the course of the experiment and fresh and dry weight (g) of above ground tissue was measured post-harvest. Foliar biomass from plants in the same pot were collected and measured as a unique sample to assess the fresh foliar weight per pot, which were then dried in the oven at 65 °C for three days before measuring the dry weight. The germination index (GI), described by formula (1), was designed to qualitatively compare the seed germination rate and the timing of germination between each of the treatment sets. A higher GI index means a higher number of seeds germinated at earlier times versus lower index, which described a set with a lower number of seed germinated. Indeed, for each seed that did not germinate, the index value was heavily penalized.(1) GI = (N seed germinated ≤ 15d) × 4 + (N seed germinated > 15d) × 2 − (N seed NOT germinated) × 5 The GI was calculated by summing two factors: the number of seeds that germinated before 15 days-after-sowing (das) were multiplied by 4 and this figure was added to the number of seeds that germinated after 15 days multiplied by 2. Indeed, 92.50% of seeds germinated before 15 das (days-after-sowing). The number of seeds that did not germinate was multiplied by 5 and subtracted from the total. Boxplots were drawn using Microsoft Excel 365© to statistically assess germination and tillering, and for comparing post-harvest plant biomass. Boxplots were chosen as they visually show the distribution of numerical data and skewness through displaying the data quartiles (or percentiles) and averages. Data analysis was further carried out using single factor and two-way analysis of variance (ANOVA) in Microsoft Excel 365© and IBM SPSS Statistics .Agrostis stolonifera roots collected from the three waste heaps at the Ballycorus mine site  (p < 0.01). However, overall, the two-way ANOVA analysis also demonstrated that Festuca arundinacea seeds treated with endophytes (E) germinated significantly earlier than the control set (p < 0.05) (E Pb800) was significantly earlier compared with the control (C Pb800) (p < 0.05).A higher concentration of lead in the soils negatively influenced the germination of  < 0.05) . In part0 Pb400, Pb600, and Pb800) were analyzed. This result confirmed that the effects induced by the endophytes were not influenced by increasing concentration of lead in the soil.Moreover, no significant results were obtained when the interaction between endophyte and control treatments (E vs. C) and concentration of lead in the soil (PbE Pb600) germinated compared with the control set (C Pb600) (p < 0.05). Furthermore, a greater germination index (GI) was measured for each set of endophyte treatments at different concentrations of lead in the soil, which means that a greater number of seeds sprouted compared with the untreated sets . Indeed, more than 90% of endophyte treated plants were tillering at 35 days-after-sowing with an average of 13.08% more tillered plants compared with the control. Moreover, plants at treatment E Pb800 had higher percentage tillering than untreated plants at 35 days-after-sowing (p < 0.01)  .Festuca arundinacea as revealed by two-way ANOVA. Increasing heavy metal concentration in the soil induced a significant reduction of the fresh and dry biomass for each set  (p < 0.01).A higher concentration of lead in the soils negatively influenced the biomass production by C) and plants treated with endophytes (E) (E Pb800). This set of plants produced a greater fresh and dry foliar biomass compared with the control set. However, no statistical significance was observed due the high variability within the dataset, here represented by the box size and the bars in the box plots  a,b with ox plots a,b.800).The production of tillers was also generally negatively affected by an increasing concentration of lead in the soil . HoweverFestuca arundinacea in a laboratory experiment to assess the impact of the endophyte consortium on this host at increasing concentrations of lead. Results demonstrated a better performance of the plants treated with the endophyte consortium for each of the growth parameters recorded, hence reducing the detrimental effect of the heavy metal. Little is still known about the role of fungal endophytes in supporting the growth of plants in soil contaminated with toxic heavy metals such as lead. Therefore, in this study, root endophytes were isolated from grasses that were growing in an abandoned lead mine. The endophytes were inoculated onto the grass Agrostis spp. and Festuca spp. generally grow relatively well in heavy metal contaminated soils, even in the absence of any symbiotic microbiota, due to a local evolutionary adaptation leading to heavy metal tolerant ecotypes [Grasses such as ecotypes ,31. HoweAgrostis stolonifera (creeping bent grass); eight of which were selected and further identified. Other research has demonstrated that the abundance and diversity of the microbial community is negatively correlated with a high concentration of toxic heavy metal in the soil, although populated by more resistance strains [A total of 62 fungal cultures were isolated in the laboratory from the root tissue of  strains ,33. ThusAcremonium, Sarocladium and Tolypocladium; Eurotiomycetes: Penicillium; Leotiomycetes: Phialocephala). Four isolates were Penicillium species and this was the dominant genus isolated (as identified by nrITS and colony morphology under microscope), which was not unexpected considering their high tolerance and efficiency in heavy metal detoxification [Tolypocladium spp. and Phialocephala spp. have been isolated in previous studies in association with toxic heavy metals [Acremonium spp. and Sarocladium spp. were for the first time connected with this topic. All the different genera isolated belonged to different fungal orders and three different classes of fungi were represented . Therefore, a beneficial effect was predicted in using these fungi as a unique inoculum rather than separately as single strains. Indeed, many previous studies observed a better performance of the endophytes when used as a consortium rather than single endophyte species [b when Verbascum lychnitis was co-inoculated with Xylaria sp. and Diaporthe sp.; the effects were not visible if the plants were inoculated with a single species. Indeed, the biosynthetic pathway of this chlorophyll is particularly negatively influenced by Pb; therefore, the authors declared that the inoculation of a endophytic consortium positively impacts the composition of the photosynthesis system, which was consequently associated with an increase in photosynthesis efficiency for plants grown in heavy metal conditions [The eight endophytes described above were then used as a consortium in a laboratory experiment in association with  species ,22,35,36 species  reported species . Wężowic species  measurednditions . Toxic effects of lead on the plants were observed during this experiment in each parameter measured, from the delay in germination to the decrease in harvested foliar biomass. Shama and Dubey  reportedFestuca arundinacea treated with endophytes overall performed better in the first stage of development of the plant with statistical significance compared with the control set, with particular improvements for plants grown at the highest concentration of lead (E Pb800). However, it was observed in this study that Penicillium-produced gibberellins not only positively stimulated the growth of the plant in conditions of high salinity, but also altered the abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) levels in the host plant and reduced ROS in the intracellular plant environment, alleviating the negative effect of the salt [Festuca arundinacea, but further experiments are required to confirm that.Endophytes have been found to positively influence seed germination and stimulate plant growth though the production of plant-like hormones under stress conditions . Leitão the salt . SimilarF. arundinacea plants was demonstrated here, with major effects at the highest concentration of lead (E Pb800 vs C Pb800). The mechanisms by which endophytes positively influenced the fitness of the plants were not the focus of this study. However, some assumptions can be made. Overall, a positive influence by the endophytes in mitigating the effect of the heavy metal in Lactuca sativa grown in cadmium contaminated soil and treated with an endophytic consortium had greater foliar and root biomass compared with the untreated set. They also demonstrated that the application of the endophytes not only improved plant fitness in stressful conditions, but also reduced the accumulation of cadmium in the edible part of the lettuce, reducing the risk for human health [Bibi et al.  demonstrn health . Exophiala pisciphila H93 in the presence of toxic heavy metals including lead, zinc, and cadmium, and they observed higher crop biomass production compared with the untreated set [Similar results were found by Li et al. , who inoated set . 2+ ions in the plant’s cells through mechanisms of metal exclusion and stabilization in the soil (phytostabilization) or metal extraction from the soil and compartmentalization in specific organs such as microbial membranes and plant vacuoles (phytoextraction) [Both the above experiments highlighted a correlation between a reduction in the concentration of metal in the shoot and a better performance of the crops in the presence of the endophytes, which, they deduced, locked the metal in soils or roots, reducing its bioavailability in the plant system. Endophytes generally reduce the bioavailability of Pbraction) ,9,11,13.Penicillium were identified among the endophytes isolated in this study. Penicillium spp. have been extensively studied for their ability of biosorption and bioaccumulation of toxic heavy metals [P. canescens (TCDAs18R2A2 and TCDAs18R2A7), was shown to strongly biosorb into its membranes toxic heavy metals, with largely higher affinity for lead (Pb2+) compared to cadmium (Cd2+), mercury (Hg2+), and arsenic (As3+) [P. sanguifluum (TCDAs18R2A6) was capable of producing siderophores, which, in this particular condition, can be released in the soil and bind the heavy metal, thus reducing the absorption by the plant roots [Three species of y metals ,13,42. Ty metals . In anotc (As3+) . Furtherc (As3+)  found thnt roots .Phialocephala spp. (TCDAs18R1B3) are sometimes classified as dark septate endophytes (DSEs) [s (DSEs) , which as (DSEs) ,48,49,50s (DSEs) .Tolypocladium cylindrosporum (TCDAs18R1A9) has not previously been isolated on soil contaminated with heavy metal. However, a closely related species, T. inflatum, is commonly found in Norwegian soil with elevated concentrations of lead originating from galena deposits and was found to be capable of growing in high metal concentrations in media experiments (10 mM Pb) [Tolypocladium inflatum, Tolypocladin, which is capable of binding di- and trivalent metals. Unfortunately, lead was not included during this experiment; however, they demonstrated that Tolypocladin was capable of binding other heavy metals such as Zi2+, Cu2+, Co2+ and Ni2+, and causing their precipitation as complex salts in weak acid conditions [0 mM Pb) . When th0 mM Pb)  declared0 mM Pb)  isolatednditions . Acremonium spp. (TCDAs18R2A9) are frequently associated with grasses such as tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) and can beneficially interact with the host improving tolerance against biotic stresses such as herbivorous or insect attack, or abiotic stresses such as drought [Acremonium spp. have not been isolated often from heavy metal contaminated soil except for isolates from the genus Neotyphodium, which were formerly part of the Acremonium section [Acremonium pinkertoniae was isolated from iron smelter wastes in Villerupt (France) by Zapotoczny et al. [A. pinkertoniae was capable of growing at toxic levels of heavy metal using two mechanisms of detoxification: (1) avoidance, which involved the thickening of the membrane to limit the exposure of cells’ interiors, and (2) an adaptive tolerance, which involved increasing the chitin–glucan content that bound the metal in the wall membrane [A. persicinum from contaminated soil in a lead–zinc mine in Iran. The microorganism was able to grow in the presence of heavy metals and better biosorb Zn, Cd, and Pb compared with other fungal isolates [ drought ,54. Howe section ,56,57 any et al. , who obsmembrane . Mohammamembrane  recurrenisolates .Other than these detoxification mechanisms, fungal isolates are known to produce other kinds of compounds that could stimulate plant growth in stress conditions such as antioxidant enzymes, organic molecules , plant-like hormones , and antimicrobial compounds ,62,63,64F. arundinacea in the presence of lead in our experiment. Regardless of the lack of knowledge on the actual mitigation mechanisms used by these microorganisms, fungal endophytes are very promising for the bioremediation and restoration of contaminated soils, not only for their detoxification effects, as demonstrated here, but also for their higher resistance to heavy metals than bacteria [To summarize, a combination of these detoxification microbial mechanisms may be involved in the improved performance of bacteria .Epichloë and AMF; this would allow us to identify the effect that different classes of microorganisms have in the plant. Indeed, Wężowicz et al. [Future research should focus further on elucidating the mechanisms of heavy metal stress alleviation in the plant and on gaining new insights into the complex relationship between these fungi and their hosts. Thus, isolation of other endo-organisms from different plant tissue could be used to obtain a different variety of microorganisms, which could include, for example, z et al.  and Vandz et al.  observedAgrostis spp., but also showing a flexibility to interact with other species such as Festuca. Indeed, the quicker establishment of the plants in these conditions, induced by the endophytes, would accelerate the restoration of deteriorated areas, thus reducing the presence of bioactive metals and improving the ecology, variety of soil microflora, and soil fertility, and, where possible, reestablishing healthy crop production.Overall, these results confirmed the vital role of the endophytes for the survival and the quicker establishment of new vegetation in such extreme conditions of heavy metal stress, not only selectively interacting with just one plant such as


Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors. Gliomas are the most common type of pediatric brain tumors3. Among these, pediatric high grade gliomas are the most aggressive, with an average 5-year overall survival of only 20%. The high rate of mortality poses a need for improved understanding of tumor biology, so this information can be used to develop new targeted therapies and design effective clinical trials.Brain tumors are the most common childhood malignancy, and are the leading cause of cancer-related mortality in children4. Tumor driver mutations found in pediatric glioblastoma (GBM) include BRAFV600E point mutations, alterations in NF1, TP53, EGFR, and PDGFRA with or without concurrent CDKN2A/B deletions, and histone 3.3 G34R/V mutations10. Tyrosine receptor kinase (TRK) fusions  are also seen in up to 50% of infant GBM12.Discoveries of epigenetic and molecular subgroups of pediatric high grade glioma are primarily based on genomic studies conducted on tumors collected at the time of primary diagnosis, recurrence, or post-mortem. Hemispheric, non-midline GBMs are typically surgically resected at diagnosis, and exhibit intratumor heterogeneity and plasticity, where glioma stem cells resemble neural progenitor-, oligodendrocyte precursor-, astrocyte and mesenchymal-like states13. Previously, the lack of surgical specimens, coupled with the rarity of this disease, led to a paucity of tissue specimens available for molecular studies and the generation of preclinical models. However, over the past 8 years, there has been a surge in the molecular understanding of DMG tumors including the identification of novel oncohistones, the putative cell of origin, and mutational landscape20. Our improved understanding of DMG tumor biology occurred, in large part, due to the increased availability of tumor tissue through rare diagnostic biopsies22 and more frequently from the donation of postmortem specimens23.In contrast to hemispheric gliomas, diffuse midline gliomas (DMGs) are rapidly growing tumors that arise in midline region of the brain and are typically not amenable to surgical resection. DMGs affect over 1,000 children every year in the United States and Europe with a 5-year overall survival of only 2%24. The predominant presence of heterozygous K27M mutations in genes encoding the histone 3 variants 3.1, 3.2, and 3.3  in midline gliomas led to the revised classification of “H3K27M diffuse midline glioma” by the World Health Organization in 201625. Diffuse intrinsic pontine glioma (DIPG), which originates in the pons of the brainstem, belongs to the H3K27M DMG classification of tumors.Oncohistones are genetic driver events found in over 60% of pediatric high grade gliomas, where histone 3.3 G34R/V mutations primarily occur in hemispheric gliomas and histone 3 K27M (H3K27M) mutations occur in midline  brain anatomical location28. The putative cell of origin of H3K27M DMG is an oligodendrocyte precursor-like cell, which is highly proliferative, capable of self-renewing, and exhibits high expression of PDGFRA14. Among H3K27M DMGs, H3.3 K27M mutated tumors occur in all midline locations, co-occur most frequently with TP53 pathway alterations, are resistant to radiotherapy, and harbor the worst overall survival (median 11 months) compared to H3.1 K27M, and H3 wild type (WT) tumors. DMG tumors harboring H3.1 K27M are mainly restricted to the brainstem, and occur in younger children (median age of 5 years), and are commonly comprised of ACVR1 mutations31. Recent findings have shown rare H3 WT DMG tumors overexpress EZHIP, which is an oncohistone-mimic of H3K27M, and these patients have an overall survival similar to H3.1 K27M tumors (median 15 months)32.We and others have shown that H3K27M is thought to arise as an initial oncogenic driver event in midline gliomas, followed by secondary genomic alterations in cell cycle regulatory and growth factor signaling pathways27. Recently, upfront biopsies matched with postmortem tissue showed longitudinal molecular changes across the tumor genome33, allowing for a better understanding of tumor evolution at progression. Analysis of postmortem specimens has also expanded our understanding of DMG tumor heterogeneity, highlighting histologic and molecular heterogeneity within the tumor, and the implications this can have on responses to treatment34. Finally, postmortem specimens have been a major source of existing preclinical disease models as outlined previously by us23 and others37.Unlike diagnostic biopsies, postmortem brain tissue can be used to analyze tumor dissemination within brain parenchyma, in order to identify spatial genomic signatures and clonal evolution across tumor cell migration39. However, a comprehensive protocol outlining the harmonized effort of coordinating a successful postmortem donation, and details of whole brain and spinal cord specimen processing that allows for maximized utility of tumor and healthy brain regions for molecular analyses and generation of preclinical models, is still needed.Existing postmortem tissue donation literature describes the consenting process, socioeconomic/religious factors affecting tissue donation, generation of preclinical models, and sample integrity of postmortem tissueThe postmortem tissue donation program at Children’s National Hospital (CNH) was established in 2010, with the aim of acquiring DMG tumor specimens to allow for further understanding of disease biology. Over time, the CNH postmortem donation program has evolved to include other brain tumor specimens upon family’s request. In this study, we first describe our experience in coordinating postmortem donations for pediatric central nervous system (CNS) cancers and outline a comprehensive protocol that provides detailed information on logistics, organization and execution of the donation process. We further outline examples of how donated specimens were utilized for downstream biological studies of the disease. Our comprehensive protocol allows for the compilation and annotation of relevant clinical information, the collection and processing of whole brain, spinal cord, biofluids ), and germline controls for molecular characterization, and the generation of preclinical models using post-mortem specimens.Since its establishment in 2010, the CNH tissue donation protocol has evolved to include the following cascade of events, which allow for maximum efficiency of sample procurement and processing while minimizing burden on the patient’s family in the immediate postmortem period: (1) identification of a patient/family interested in donation in partnership with other institutions, (2) consent process, (3) patient enrollment, (4) assignment of a point of contact who can provide updates on the patient’s status to the clinical coordinator, (5) collection of patient’s clinical data such as drug treatment, surgical procedures and MR images, (6) coordination with the funeral home, (7) identification of a neuropathologist or diener for specimen acquisition, (8) coordination with laboratory personnel responsible for the collection and biobanking of tissue and biofluid specimens, and (9) follow up with the family , supratentorial glioma (n = 4), and other brain cancers n = 10)  models were attempted and twenty-four models were generated (58%) utilizing postmortem tumor samples Fig. b. GeneraPDX model generation was performed using either brainstem, thalamus, or cortex as the primary site of injection Fig. b. The si40, three of the four H3K27M DMG patients (patients 1–3) had disseminated tumor cells located in other midline locations such as the thalamus, basal ganglia, the periventricular regions, and cerebellum   on multiple regions of tumor and healthy brain of these four patients diagnosed with H3K27M DMG was performed. WGS was also performed on blood when available to serve as germline controls. The primary mutation, es) Fig. c. All foFRA Fig. c. IntereVAT1, NDST3), and one clonal mutation (PRPF4B) in common with the pontine tumor. Similarly, disseminated tumors in patients 1, 2, and 3 harbored subclonal mutations that were not detected in the pons. Strikingly, the cerebellar disseminated tumor in patient 1 contained seven subclonal mutations that were absent in the pons. Among these were mutations in genes encoding a cell adhesion gene (CTNNA2), and the chromatin regulators KMT2A and SMARCB1, known to be altered in other types of pediatric cancers43.Remarkably, disseminated tumors in the cerebellum, cervico medullary junction (CMJ) and occipital lobe of patient 4 did not harbor the H3K27M or other partner mutations found in the primary pontine tumor  of 8H9. In order to assess the effects of this specialized treatment on the pontine tumor and surrounding tissue, a mid-sagittal slice of the whole brain was obtained during post-mortem processing  and the primary and extended tumor (n = 3) from DMG cases. CD45 (leukocyte common antigen) immunohistochemistry (IHC) staining revealed infiltrating immune cells intermixed with tumor cells (which were identified through H&E and H3K27M IHC staining) in both the primary and extended tumors , neuropathologist, funeral home, and the laboratory team in charge of processing tissue.Despite careful planning, there are occasional challenges in the coordination and execution of postmortem donation. In some cases, particularly during off-hours and holidays, the pathologist and/or laboratory personnel are not available for immediate tissue processing, limiting the center’s ability to accept a post-mortem donation. A diener is a trained autopsy technician who assists a pathologist with dissection during an autopsy. To address this challenge, dieners can be hired for tissue procurement when a neuropathologist is not available. In order to execute the comprehensive whole brain and spine processing protocol, it typically requires four to six lab personnel for efficient freezing and fixing of tissue, as well as processing of fresh tissue for DMSO and the generation of preclinical models. During times when there are insufficient staff members available for processing, a modified SOP protocol, that allows for rapid processing of samples with fewer personnel, can be used instead  proved to be challenging as tumor was not visible by gross examination of postmortem tissue. While prior MRI reports were obtained, the most recent MR images were not available at the time of postmortem, precluding maximally efficient tumor sampling. In contrast, availability of MR images (patient 65) helped to determine tumor dissemination to the cerebellum and spinal cord. Therefore, the donation process was modified to allow for collection of both primary and metastatic sites which were preserved in DMSO to be used for future pre-clinical models. Subsequently, gross and histological examination of the samples by a neuropathologist confirmed tumor infiltration of the cerebellum, hippocampus and spinal cord. Therefore, it is critical to use an SOP that includes the use of the latest MR images in order to prioritize tumor locations for collection.44. However, this observation needs to be validated in a larger cohort of samples.The accuracy of genetically and histologically faithful preclinical models is important for translation of novel agents into the clinic. Preclinical models were derived from tumors representing various molecular subtypes of DMG  and the fidelity of these preclinical models was shown by mutation analysis and histological characterization. Importantly, postmortem collection time did not seem to influence successful generation of a PDX model, as PDX models were generated with samples collected at a range of 5–40 h. In our experience, xenograft model generation was more successful than primary cell cultures. This could be due to tumor cell microenvironment requirements that are naturally present in a mouse. Our study also shows that utilizing fresh tissue is more favorable than cryopreserved tissue for the generation of PDX models in concordance with previous studiesATM only in the primary pontine tumor. For this patient, the key genomic aberration in the primary pontine tumor, which was absent in the disseminated tumors, may either be important for tumor initiation and maintenance but not tumor migration, or was a passenger mutation that was no longer present in the disseminated tumor. Three patients also harbored subclonal mutations that were absent from the primary pontine tumor, but present in disseminated tumors. The acquisition of these subclonal mutations may have been driven by specific microenvironment changes that promote adaptations to aid in the process of tumor migration. Future mechanistic studies are required to understand the role of clonal and subclonal mutations found within specific tumor sites (primary and disseminated).Postmortem tumor tissue is also useful for the histologic and molecular analysis of regions of tumor dissemination within the brain parenchyma. In our analysis of four patients with disseminated H3K27M DMGs, WGS sequencing revealed clonal and subclonal mutations within the primary and disseminated tumors. One patient harbored a mutation in Improved knowledge of the long- and short-term effects of treatment is necessary to determine why therapy is not successful. Molecular and histologic analyses of postmortem tissue can provide essential information about the combined effects of radiation, chemotherapy, and other molecularly targeted or immune based therapies on both tumor and healthy brain cells. This information can especially be helpful for patients with DMGs who undergo a variety of therapies on clinical trials. In this study, we used sagittal whole brain sections to analyze the effect of CED therapy on a H3K27M DMG tumor. This analysis revealed decreased cellularity in the region where CED therapy was delivered, indicating that the treatment was successful in this area. However, other areas of the brain that had tumor disseminated did not seem to be affected by therapy. Additional studies using a similar approach can help inform clinical trials for CED or other therapeutic agents, to optimize treatment delivery and effect. We also showed that whole brain postmortem samples can also be used to identify immune cells within the tumor and disseminated tumor sites. Additional studies, to understand the immune response following different treatment modalities, with a focus on immunotherapy, are still needed.47. Furthermore, upon consent, all whole genome- and RNA-sequencing data generated from our postmortem program will continually be deposited and shared through the platform OPEN DIPG via the Gabriella Miller Kids First Research Act, an international collaboration led by Children’s Brain Tumor Tissue Consortium (CBTTC). This platform allows for a large collection of sequencing data that can be mined by the scientific community to improve the understanding of disease biology and identify novel targets of therapy. In addition to sharing information with the medical community, it is important to engage with families in discussion about the impact of their donation, particularly when families express interest in receiving this information at the time of the donation. In the CNH postmortem program, unless they request not to receive updates, families are provided with information regarding publications or other mechanisms by which their donation contributed to the scientific field during specific time intervals , protocol #Pro00001339 (NCT01106794). Consent to the postmortem tissue donation program was obtained by the patient’s primary physician as per guidelines and regulations provided by our institutional IRB. The topic was discussed with the patient’s family either when patient’s health declined or earlier if the family broached the subject. Post-mortem donation consenting was primarily conducted during hospital visits; although consent could also be obtained via phone call should a patient’s sudden decline or distance inhibit face-to-face discussion. Informed consent/assent was obtained from the patient and family prior to the patient’s passing, and a guardian later authorized autopsy on behalf of the deceased. During the consenting process, particular emphasis was placed on the issues of confidentiality, samples to be collected, and access to medical records. After a careful reading of the consent, families were asked to voice any questions or concerns regarding the autopsy and potential risks. They then signed the consent and a copy was provided to the family.The protocol was restricted to children age 2 month—25 years with a cancer diagnosis. Patients consented to a limited autopsy allowing for collection of specimens including whole brain, spinal cord, blood, CSF, and a 5 mm skin biopsy punch. Families were assured that any incisions made would not be visible in the case of an open-casket funeral and that only the samples named in the consent would be collected. Families were given the opportunity to limit the extent of autopsy, retention of samples, long-term access to clinical data, and sharing of clinical data to outside institutions. Medical records review was conducted at the time of the postmortem to document diagnosis, treatments, outcomes, and time points of progression.23. Briefly, single cell suspension was prepared by digestion of fresh or cryopreserved tissue with DNase I and Collagenase followed by RBC lysis. The cell pellet was then filtered through 70 micron cell strainer to remove non-dissociated tissue and debris. Live cells were counted by trypan blue staining and plated for cell culture or orthotropic injection into NOD-SCID mice. NOD SCID gamma mice used for orthotopic injections ranged from 2 days-3 weeks old. Briefly, mice were anesthetized by hypothermia and the following coordinates were used for the injections into specific locations in the brain . Injected mice were monitored daily for a week for signs of distress and all procedures were conducted in accordance with CNHS IACUC protocol #30435. Mice were consequently monitored for 12 months post tumor cell injections for symptoms of brain tumors such as lethargy, enlarged head, and abnormal movements. Mice indicating signs of brain tumor development were euthanized, and whole brains were collected and analyzed by immunohistochemistry and molecular analyses for tumor confirmation.All experiments were carried out following guidelines given by Children’s National Institutional Animal Care and Use Committee (IACUC), approved protocol # 30425. Tissue obtained at postmortem was utilized to generate pre-clinical models as described previously45. Briefly, slides were deparafinnized at 60 °C for 1 h followed by xylene and ethanol solutions. Slides were then blocked and incubated with primary antibody for 1 h followed by HRP labeled secondary antibody. Cell grown on coated chamber slides were washed in PBC followed by fixation in 10%formalin for 5 min, and permeabilized in 0.1% Triton X-100 for 10 min. Blocking was performed in 1%BSA for 1 h, followed by primary and fluorescent tagged secondary antibodies incubation. Cells were stained with 4′,6-Diamidino-2-phenylindole (DAPI) and imaged.Formalin fixed paraffin embedded tissue samples were used for immunohistochemistry as previously describedDNA/RNA was isolated from frozen tissue using Qiagen nucleic acid isolation kits as per manufacturer’s instructions. All DNA samples were quantified using Qubit fluorometer, and RNA integrity was assessed using bioanalyzer, as per manufacturer’s instructions.48. Droplet digital PCR  was performed for H3F3A p.K27M mutant and wildtype copies in DNA isolated from cells and PDX models derived from H3K27M postmortem tumors.Genomic DNA was isolated as described above, and subjected to dropletization and PCR amplification, followed by detection of fluorescence signal as described previously33. DNA was isolated as described above and was sequenced using HiSeq X 150 bp read length. Samples were sequenced with 90X coverage and somatic variant calling was performed using workflow that has been setup in common workflow language on CAVATICA. The pipeline uses BWA-MEM, SAMBLASTER and finally Strelka was applied for identification of point mutations. Somatic mutations were compared to the Genome Aggregation Database (gnomAD) to control for population genetics, Catalogue of Somatic Mutations in Cancer (COSMIC) for somatic mutations reported in other cancers to call an alteration in our samples as mutation. All calls were made with an mutation allelic frequency of 25% or more.Whole genome sequencing was performed at Children’s Brain Tumor Tissue Consortium (CBTTC) in collaboration with Nant Health. All samples were processed as described previouslySupplementary file1 (DOCX 30 kb)Supplementary Figure S1Supplementary Figure S2Supplementary file2 (DOCX 23 kb)Supplementary file3 (DOCX 22 kb)Supplementary Figure S3