Despite the considerable progress made towards understanding ALS pathophysiology, several key features of ALS remain unexplained, from its aetiology to its epidemiological aspects. The glymphatic system, which has recently been recognised as a major clearance pathway for the brain, has received considerable attention in several neurological conditions, particularly Alzheimer’s disease. Its significance in ALS has, however, been little addressed. This perspective article therefore aims to assess the possibility of CSF contribution in ALS by considering various lines of evidence, including the abnormal composition of ALS-CSF, its toxicity and the evidence for impaired CSF dynamics in ALS patients. We also describe a potential role for CSF circulation in determining disease spread as well as the importance of CSF dynamics in ALS neurotherapeutics. We propose that a CSF model could potentially offer additional avenues to explore currently unexplained features of ALS, ultimately leading to new treatment options for people with ALS. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive fatal neurodegenerative disorder characterised by the selective death of motor neurons. Although the underlying cause of ALS is unknown, recent discoveries in the genetics and molecular pathology of ALS have provided important new insights. These include the finding that monogenetic causes of ALS—accounting for approximately 10% of cases—are phenotypically and pathologically largely indistinguishable from sporadic ALS. Furthermore, over 97% of ALS cases and half of frontotemporal dementia (FTD) are pathologically defined by cytoplasmic mis-accumulation of insoluble TDP-43, leading to these disorders being classified as TDP-43 proteinopathies .Although much remains to be established about ALS pathophysiology, multiple mechanisms are implicated including proteostasis, glutamate excitotoxicity, dysregulation of RNA metabolism, nuclear-cytoplasmic transport and autophagy , 36, 73.The glymphatic system has recently been recognised as an important clearance pathway for the brain, playing a major role in the regulation of brain metabolites, including glucose and lipids . NotablyAgainst this background we describe the various potential roles of cerebrospinal fluid (CSF) in ALS pathophysiology, including as a possible driver for the disease process. We start by providing a brief overview of CSF circulation, followed by a primer on the glymphatic system, including the different factors affecting its function (Box 1), both of which have been comprehensively described by excellent reviews , 86. We Box 1: Factors affecting glymphatic functionCurrent evidence suggests that exchange between the cerebrospinal and interstitial fluid (CSF-ISF) is mainly driven by arterial pulsation. Reducing arterial pulsation through internal carotid artery ligation resulted in significantly reduced exchange between CSF and ISF . AccordiNeuroimaging results have also revealed both rostrally- and caudally-directed CSF flow produced by inspiration and expiration respectively , with foSleep has been shown to play a major role in waste clearance from the brain. Injected radiolabelled amyloid-beta was found to be cleared much more rapidly in sleeping mice than in awake mice . Sleep iEarly suggestions for the impact of ageing on CSF dynamics arose when several studies observed changes in resistance to CSF outflow, secretion and turnover in older individuals , 154. AgSurrounding most of the brain and spinal cord, CSF is believed to originate primarily from the choroid plexus, which is an extension of the ependymal lining of the brain ventricles. The majority of CSF is secreted into the two lateral ventricles, from where it converges into the third ventricle through the foramen of Monro, and subsequently flows into the fourth ventricle through the aqueduct of Sylvius. CSF then leaves the fourth ventricle and reaches the subarachnoid space via the foramen of Magendie and the two foramina of Luschka, with an indeterminate fraction of CSF also thought to flow into the central canal of the spinal cord.CSF drainage however remains a topic of debate, with recent studies challenging the traditional view of reabsorption into dural venous sinuses by arachnoid granulations, which are outgrowths of the arachnoid mater , 163. AlThe total volume of CSF in an adult is widely estimated to be about 150 mL, with the larger majority of this volume distributed in the subarachnoid spaces and about 25 mL present in the brain ventricles. Renewal of CSF takes place three to four times in a single day. Whilst the primary role of CSF has long been considered to be the provision of buoyant support to the brain and protection against mechanical damage, another important function of CSF, which will be the focus of this review, is the clearance of metabolic waste products.The earliest descriptions of perivascular spaces, or Virchow-Robin spaces, arose as early as the mid-1800s, when investigators, including Rudolf Virchow and Charles Robin, observed spaces surrounding blood vessels penetrating the brain parenchyma . HoweverAnatomically, these perivascular spaces are formed by pial arteries that perforate the brain parenchyma after traversing the subarachnoid space. As these pial arteries transition into penetrating arterioles, CSF from the subarachnoid space also extends into the brain parenchyma, bordering the blood vessels and creating CSF-filled spaces that are able to interact with the extracellular space , 217. ThAQP4-knockout mice support the importance of AQP4 channels in glymphatic transport and amyloid-beta clearance [Whilst evidence for the role of perivascular spaces in solute transport from the brain interstitium had already emerged in the 1980s , the implearance . FollowiDespite past studies usually focussing on either the glymphatic system or the meningeal lymphatic system, increasing evidence supports the view that these two systems could be interdependent, with recent findings demonstrating a decrease in glymphatic influx and efflux of interstitial solutes following disruption of the meningeal lymphatic vasculature . SimilarAlthough CSF from ALS patients is now known to be constitutionally abnormal , 27, witThe in vitro effect of ALS-CSF also extends to both astrocytes and microglia, demonstrating a non-cell autonomous component to CSF toxicity. Upon exposure to ALS-CSF, astrocytes undergo a change in morphology, accompanied by increased GFAP reactivity, further acquiring a neuroinflammatory profile . ReleaseSupporting these in vitro findings, several studies have also reported various changes caused by ALS-CSF in animal models. Neurofilament phosphorylation was observed in rat motor neurons following injection of ALS-CSF into the spinal subarachnoid space . MoreoveAccepting inherent limitations in patient studies, including sample heterogeneity, and that CSF concentration may not reflect cellular levels, these in vitro and in vivo studies nonetheless show that ALS-CSF is toxic to neurons and possesses pro-inflammatory properties . The in Evidence for the disruption of CSF flow in ALS includes findings from a phase-contrast electrocardiography-triggered MRI study, which revealed different CSF dynamics in ALS patients, with a delay in CSF flow upon systole and a higher maximum velocity . AbnormaAdditionally, with human neuroimaging studies revealing changes in CSF dynamics with age , 170 andThe finding of increased norepinephrine concentration in CSF from ALS patients is also intriguing , 35. NorVascular function, which is intimately connected to the glymphatic system, is known to be affected in ALS. Multiple studies employing single-photon emission computed tomography (SPECT) have demonstrated hypoperfusion in the brain of both ALS and FTD patients, with greater involvement of the frontal and temporal lobes , 82, 199ANG and VEGF, provide further indirect evidence of the role of vascular factors in ALS [VEGF has been heavily implicated in ALS pathophysiology, with several studies demonstrating the protective effect of VEGF therapy on neuronal death, both in vitro and in vivo [VEGF hypoxia-response element in mice also results in motor neuron degeneration [VEGF-associated neurodegeneration has been suggested to arise from ischaemia following reduced vascular perfusion, although this has yet to be established.Genetic association studies linking ALS risk with angiogenic genes, s in ALS , 195. VE in vivo , 198. Laneration . VEGF-asThe link between hypertension and ALS is currently less clear. Multiple associations have been made between hypertension and ALS onset and survival , 129, buOverexpression of AQP4 in ALS is well documented in rodent models , 48, 137SOD1 mice triggered earlier disease onset and reduced survival [SOD1 ALS.Another intriguing finding is that reduced AQP4 expression in survival . One expTaken together, there are multiple and converging lines of evidence that suggest that the glymphatic system plays a role in ALS pathophysiology. We therefore outline several key processes in ALS that might be impacted by a dysregulated glymphatic system Fig. .C9ORF72. TDP-43 proteinopathy is known to be the most prevalent and has been implicated in many pathways, including mitochondrial dysfunction, autophagy dysregulation and impaired endocytosis [Accumulation of various proteins that may directly or indirectly be toxic in ALS is well-established (Box 2). These include mis-accumulated phosphorylated TDP-43, SOD1 aggregates and dipeptide repeats (DPRs) found in the most common inherited form of ALS, due to a repeat expansion mutation in ocytosis .VCP, OPTN and UBQLN2, argues for an important role for dysregulated clearance in ALS. Clear evidence supporting the overproduction of TDP-43 in ALS, however, remains to be established.The reasons for accumulation could reflect excess production and/or impaired clearance through autophagy and the ubiquitin-proteasome system (UPS). These are both involved in TDP-43 and SOD1 clearance , 212, wiGiven the established role of the glymphatic system in protein clearance, impairment of glymphatic function could also contribute to the accumulation of the different toxic proteins in ALS. The presence of TDP-43, SOD1 and DPRs in ALS-CSF is well-evidenced and points to a potential role for CSF in their regulation. It is therefore possible that ageing, coupled to the different possible sources of glymphatic disturbance already discussed, could result in an imbalance between production and clearance of these pathogenic proteins. Notably, ageing has further been argued to promote the deterioration of processes such as autophagy and the UPS , which cNotwithstanding this, protein inclusions associated with ALS, including those enriched in TDP-43, SOD1 and DPRs, are predominantly intracellular, as opposed to amyloid-beta aggregates, found in Alzheimer’s disease, which also reside extracellularly. Thus, glymphatic clearance in the context of ALS could potentially follow from processes such as exosome secretion , 78 or nWhilst considerable progress has been made towards understanding ALS pathophysiology, its primary cause has remained a matter of debate, with genetic mutations accounting for only a minority of ALS cases. Hence, the significance of ageing in the impairment of clearance mechanisms could provide a promising avenue to explore in ALS.Box 2: Key ALS proteinsTARDBP gene, possesses a wide range of functions, most notably in RNA regulation. About 5% of familial ALS cases can be attributed to mutations in the TARDBP gene [TDP-43 inclusions are now widely considered as the pathological hallmark of ALS, with more than 95% of ALS patients known to exhibit ubiquinated TDP-43 deposits . The TDPDBP gene . TDP-43 DBP gene .SOD1 was the first ALS-linked gene to be discovered and mutations in the SOD1 gene are now known to account for about 15% of familial ALS cases and 1% of sporadic cases [SOD1 gene codes for the Cu-Zn superoxide dismutase enzyme whose cellular function is to convert superoxide radicals into hydrogen peroxide and oxygen. Although increasing evidence indicates a gain-of-function mechanism for SOD1 pathogenicity [ic cases . The SODgenicity , its neugenicity . Considegenicity .FUS gene are responsible for a relatively small fraction of familial ALS cases (about 3%), and an even smaller proportion of sporadic ALS cases [FUS mutations promote neurodegeneration is, however, unclear.Another ALS protein known to form cytoplasmic inclusions is the FUS protein. Mutations in the LS cases . The FUSLS cases . The exaC9ORF72 locus, accounting for about 40% of familial ALS and about 10% of sporadic ALS [C9ORF72-mediated ALS is the non-canonical translation of repeated RNA sequences, giving rise to five classes of DPR proteins, namely, poly-GA, poly-GP, poly-GR, poly-PA and poly-PR [The commonest known cause of ALS is a repeat expansion in the adic ALS , 114. On poly-PR . Althoug poly-PR , 213.Glutamate excitotoxicity has long been viewed as an important pathophysiological mechanism in ALS and FTD. Riluzole, which inhibits glutamate release, is currently the only globally approved drug for ALS . One reaSOD1 mice [Glutamate excitotoxicity could also be caused by direct overstimulation of glutamate receptors, due to an increased level of extracellular glutamate . AlthougOD1 mice , 203. InOD1 mice , 71, 103OD1 mice .Whilst the cause of increased glutamate levels could result from several processes, the role of CSF in glutamate regulation  could inDysregulated energy metabolism is now a widely known feature of ALS, with patients generally suffering from weight loss, hypermetabolism and hyperlipidaemia . AlthougHence, given the recently characterised role of the glymphatic system in lipid transport , an impaTo date, several studies have helped to demonstrate the inflammatory profile of ALS-CSF, revealing raised levels of various immune components, including C3c, albumin and IgG in ALS-CSF , 67, 102Given their high metabolic rate as well as their inability to divide, motor neurons could be particularly vulnerable to accumulation of toxic metabolites, including lactate, 4-hydroxynonenal (HNE) and peroxynitrite . High laThe variable sites of disease onset and subsequent disease evolution have raised the question as to what determines ALS disease spread. This is unknown and debate has hitherto focussed on questions of anterograde or retrograde spread , 57. SomMore recently, a growing body of literature suggests that ALS spread could be explained by a prion-like mechanism. Notably, the mutant SOD1 protein, which has been demonstrated to possess high fibril-forming propensity under certain conditions, can promote fibrillation in wild-type SOD1 . TDP-43 Although the prion-like properties of the different ALS proteins are starting to gain acceptance within the scientific community, less is known about how intercellular spread could take place in vivo. Trans-synaptic transmission has been suggested as one possible mechanism , 32. AnoA further pathway already described by others is that spread of the disease could occur via the CSF circulation . The preHence, whilst we do not propose that spread of the disease occurs solely through CSF pathways, such a mechanism of spread could clearly complement other proposed models. It would also help to explain the non-contiguous pattern of spread and the multifocal pattern of initiation observed in some ALS cases , and eveOne major caveat in considering glymphatic involvement in ALS is that its function has so far only been demonstrated in the brain, with its contribution in the spinal cord still unclear. Indeed, spinal cord and brain anatomy differ considerably both in terms of parenchymal and leptomeningeal organisation, suggesting possible mechanistic differences in waste clearance. Whether clearance from the brain and clearance from the spinal cord are influenced by similar factors and could therefore be modulated via similar mechanisms are also not known. These distinctions may therefore need to be further explored, given their possible importance with regard to spinal cord involvement in ALS. Another point to consider is that most studies on the glymphatic system have been carried out in rodents, with definitive evidence for its existence in humans yet to be established. However, results have indicated a high possibility of this being the case, with some studies reporting a number of inter-species similarities, not only in terms of the associated anatomical structures, but also the factors affecting clearance, such as sleep , 216.Hence, the glymphatic system could play an important role in reconciling disease mechanism with epidemiology and other currently unexplained aspects of ALS. It would firstly support the existence of a threshold effect suggested by the significance of ageing as a risk factor. Secondly, it could help to explain the intriguing gender disparity in ALS, with the established protective influence of oestrogen in preserving vascular, and therefore glymphatic, function possibly accounting for the lower female incidence. This could further underlie the postmenopausal increase in female:male ratio observed by some studies , 117. AdC9ORF72 mouse models that recapitulate the disease phenotype bears a connection to glymphatic function also deserves mention. Both CSF production and turnover rate are much greater in mice than in humans [C9ORF72 mouse models have failed to exhibit motor dysfunction. Interestingly, however, cognitive defects have been observed both in mutant TDP-43 and C9ORF72 mouse models [Whether the intriguing difficulty in generating n humans , and when humans . This coe models , althouge models .Understandably, many areas need to be addressed before acknowledging glymphatic dysfunction as a contributor to ALS pathophysiology. Despite the involvement of certain angiogenic genes, evidence for a clear genetic link between ALS and genes implicated in glymphatic function is still missing. Furthermore, definitive evidence for the significance of sleep  and hypeWhilst the glymphatic system has more often than not been associated with waste clearance from the brain, regulating glymphatic influx could also significantly impact ALS neurotherapeutics. With BBB impermeability acting as a major hurdle to drug delivery in ALS and other neurological disorders, intrathecal therapy has emerged as a promising treatment modality in recent years. Indeed, administering the drug directly into the cerebrospinal circulation has multiple advantages, such as allowing for more accurate monitoring of drug levels, thereby ensuring better pharmacokinetic results . The obvSeveral intrathecal therapies are currently being trialled in ALS, including anti-sense oligonucleotides (ASOs), mesenchymal stem cells (MSCs) and growth factors, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and vascular endothelial growth factor (VEGF) . PromisiTo further emphasise the significance of achieving more efficient drug delivery, different variables, such as diffusion rate and anatomical constraints, have been suggested to limit drug access to deeper parts of the brain and the spinal cord . AdditioThe glymphatic system has promise in potentially unifying many different aspects of ALS, and may therefore be viewed as a novel paradigm when considering ALS pathophysiology. We suggest that further avenues be explored to specifically examine features of disease onset and spread. Several lines of enquiry from pre-clinical to epidemiological studies are thus required to shed light on the extent of CSF contribution in ALS, with the hope of ultimately identifying new treatment options for people with ALS.


TIR3B thyroid nodules are considered to be at risk of malignancy (15–30%) but guidelines recommend conservative surgery with lobectomy with primary diagnostic porpoise. Risk stratification mainly based on ultrasound, elastography and genetic mutations usually may influences the surgical approach.P values less than 0.05 as significant.We retrospectively analyzed 52 cases of TIR3B underwent between 2015 and 2017 total thyroidectomy (TT) and lobectomy (L), focusing mainly on the observed rate of malignancy. Chi-squared test and Fisher’s exact probability test were used for analysis, considering a Out of 52 patients 49 underwent TT and 3 L. In TT group a multinodular goiter was associated in 67.3% of patients. Malignancy rate was 81.6 and 33.3% respectively after TT and L (P 0.003). Multicentric and contralateral tumors were detected respectively in 36.7% and in 32.6% of patients underwent TT. No main post-operative complications were registered.Ultrasound and elastography are useful to define within the TIR3B group those lesions at higher risk and therefore requiring a more radical approach. TT seems an appropriate approach to TIR3B lesions, especially in multinodular goiter, considering the incidence of malignancy with probably higher rate than previously reported. In 2014, the Italian Society for Anatomic Pathology and Cytology (SIAPEC) together with the Italian Thyroid Association (AIT) modified the previous thyroid cytology classification of 2007, by replacing the TIR3 class with two new subclasses, TIR3A and TIR3B comparable respectively to the Bethesda category AUS/FLUS and FN/SFN . Recent S. Maria University Hospital, Terni, University of Perugia, Italy, referral centre of Umbria region , by the same surgical team, with standard surgery for both total thyroidectomy (TT) and lobectomy (L). The diagnostic work out adopted standard technique as US, RTE and cytology. Molecular analysis was available in a limited number of patients (5 cases) and therefore not considered for the present study. Risk stratification was done in all cases. US features of the nodule were evaluated in terms of cancer risk according to ATA guidelines [We retrospectively analyzed 52 cases of follicular proliferation classified as TIR3B according to SIAPEC 2014, treated between January 2015 and December 2017 in the Unit of Endocrine Surgery, idelines . Size laidelines , 11 was P values less than 0.05 were considered as statistically significant.The statistical comparison between groups was performed by using chi-squared test and Fisher’s exact probability test. n = 40) and in 33.3% (n = 1) respectively after TT and L (P 0.003), with a cumulative malignancy rate of 78.8% (41 out of 52 patients). Only 6 patients out of 49 undergone TT, presented a an isolated low risk, T2 (< 4 cm) carcinoma. The patient with carcinoma after L was treated with contralateral surgery after 2 months. Main size of the TIR3B nodules was 1.7 cm. Size larger than 2 cm was not significantly associated to presence of carcinoma (P 0.057) in the TT group. Among malignant TIR3B a microcarcinoma, less than 1 cm in main diameter, was detected in 15 (36.5%) patients. Out of 49 patients, TT allowed detection of multicentric tumor in 18 (36.7%) cases and in 16 (32.6%) a contralateral carcinoma was detected. The principal histology detected was follicular variant of papillary carcinoma in 37 cases out of 41 (92.6%). High preoperative risk of the TIR3B lesions stratified on US feature, RTE and clinical behaviour fitted the diagnosis of carcinoma in 36/41 patients (87.8%). No main post-operative complications were registered. No recurrent laryngeal nerve lesions were observed after both TT and L. Only 3 cases of transient hypoparathyroidism were observed in the TT group.Out of 52 patients, 34 females and 28 males, presenting at admission a TIR3B lesion, 49 underwent TT and only 3 thyroid L. In TT group a multinodular bilateral goiter was associated in 33 (67.3%) patients, all patients of the L group presented uninodular goiter. Malignancy was detected in 81.6% (The new SIAPEC classification of 2014, assign to TIR3B lesions, referable to the Bethesda FN/SFN group, an higher risk of malignancy compared to TIR3A, based on the evidence of mild/focal nuclear atypia , based oThe decision on what is the best treatment to propose to our TIR3B patients, although affected by several variables and sometimes weak risk stratification criteria, represents both for patient and surgeon a decisional conflict . This coOur data and recent publications show an increased rate of TIR3B lesions with associated higher malignancy rate than expected. This evidence might recommend a radical approach to TIR3B nodules by TT differently to what stated by ATA guidelines which considers conservative surgery with L with diagnostic primary porpoise. Risk stratification based on US pattern, size, clinical behaviour, genetic mutations supports, in clinical practice, the choice of the more appropriate surgery. The presence of concomitant multinodular goiter and risk for bilateral and multicentric tumors are the main factors supporting the choice for a TT in TIR3B. The above considerations must be otherwise analyzed in the general scenario of the recent approach to thyroid cancer for which after a risk stratification, less invasive forms and microcarcinoma are referred to observation or L rather than to TT as in the past.


Background: Midazolam is recommended by health guidelines for sedation and hypnosis in children. Oral solution is a suitable dosage form for children. But there is no conclusive evidence for sedative-hypnosis and antianxiety effects by midazolam oral solution in children.Methods: Relevant studies were identified through searching PubMed, Embase, Cochrane Library, CINAHL, International Pharmaceuticals, four Chinese electronic databases, and relevant lists. Two reviewers independently selected trials, assessed trial quality, and extracted the data.Results: Eighty-nine randomized controlled trials (RCTs) comparing midazolam oral solution with placebo or blank (n = 33), dexmedetomidine (n = 15), ketamine (n = 11), different midazolam doses (n = 10), midazolam injection (n = 8), chloral hydrate (n = 7), diazepam (n = 5), N2O (n = 5), triclofos (n = 4), butorphanol (n = 2), fentanyl (n = 2), hydroxyzine (n = 1), and thiopental (n = 1) were identified. Meta-analysis showed no significant difference in the success rate and duration of sedation and hypnosis between midazolam oral and injectable solution (P > 0.05). The success rate of sedation and hypnosis of midazolam was higher than that of ketamine . No significant difference was found in the success rate of sedation and hypnosis, mask acceptance, and parental separation between midazolam oral solution and dexmedetomidine (P > 0.05), and the result of one cohort study was consistent. The results of RCTs and a prospective cohort study showed that the incidence of adverse drug reactions (ADR) was 19.57% (189/966). Incidence of adverse reactions between dose groups of  = 0.10  and Pf (95% CI) = 0.09 , respectively], higher than that of the dose group of  = 0.01 ].Conclusions: Available evidence suggests that midazolam oral solution is as good as midazolam injection and dexmedetomidine and is better than ketamine. Based on efficacy and safety results, an oral midazolam solution dose of 0.5–1 mg/kg is recommended for children. Data from World Bank showed that children (aged 14 years and younger) accounted for 25.79% of the world's total population in 2018. The use of sedative-hypnotic drugs in assisting children to complete medical examinations and surgery has become more and more extensive  and (“child” or “newborn” or “infant” or “neonate” or “toddler” or “teenager” or “adolescent” or “pediatric”). We systematically searched the official website of the National Drug Administration and the Center for Adverse Reaction Monitoring for reports of midazolam adverse reactions in countries and regions around the world.PubMed, Embase, Cochrane Library, CINAHL, International Pharmaceuticals, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), Wanfang Database, VIP Database for Chinese Technical Periodicals (VIP), the WHO Clinical Trials Registry Platform, Cochrane Central Registry of Controlled Trials, and A report was selected for inclusion if (a) participants were children aged 0–18 years; (b) the intervention group only used midazolam oral solution, and the route of administration was oral; (c) for comparisons, the control group was blank control, placebo, midazolam injection , or other sedative-hypnotic drugs, and the dose and course of treatment were not limited; (d) studies focused on the efficacy and safety outcome of midazolam; (e) studies were randomized controlled trials (RCTs), cohort studies, case–control studies, case series studies, case reports, and cross-sectional survey studies.Studies were excluded if (a) they were repeated published studies; (b) they were non-Chinese and non-English studies; (c) their full text is not available; (d) they were comparative studies with different routes of administration of midazolam .The primary outcome was the success rate of sedation and hypnosis . Secondary outcomes were depth of sedation (sedative hypnosis depth scores), anxiety scores, duration of sedation and hypnosis (the time when children fell asleep to the time responding to command), the time of falling asleep , and the type and incidence of adverse reactions.Two reviewers (Cheng and Xu) independently screened the titles and abstracts of every record. Full articles were obtained when either information conformed to satisfy the selection criteria outlined previously or not enough to ascertain because of limited information. Data were independently extracted by each reviewer and entered into a standardized form. The data extraction form included general characteristics and outcome measurements. Discrepancies were resolved by a third researcher, Chen.P ≤ 0.05 was considered statistically significant. Final outcomes of treatment vs. placebo or other medicines were used for the analysis, as recommended by the Cochrane Handbook for Systematic Review of Interventions, except where large pretreatment differences were identified; for these studies, the change from baseline was compared instead to prevent skewing of results. Where mean and/or standard deviation values were not reported, these were calculated based on reported CIs or P-values.Results for dichotomous outcomes were expressed as risk ratios (RR) with 95% confidence intervals (CIs), and for continuous outcomes, the mean difference (MD) with 95% CIs was accounted for. 2 tests. Heterogeneity was quantified, where 25% = small, 50% = moderate, and 75% = high heterogeneity  software. Descriptive analysis was employed for data that cannot be meta-analyzed. The incidence of adverse reactions was analyzed by meta-analysis of uncontrolled dichotomous data. Pf[95% CI] referred to the effect volume after correcting the relevant factors. Pf was equal to odds ratio (OR) divided by (1 + OR). With reference to MedDRA 20.1, the types of adverse reactions were divided into cardiovascular system, digestive system, nervous system, and so on. In combining the studies, the conservative random effects model was employed, since the underlying effects can differ across studies and populations that are not necessarily homogeneous  sequence generation, (2) allocation concealment, (3) blinding, (4) incomplete outcome data, (5) selective outcome, and (6) other biases. The Newcastle–Ottawa Scale (NOS) was used to assess the quality of cohort studies and case–control studies. Critical appraisal checklists of the Joanna Briggs Institute (JBI) were used to assess the bias of case reports and case series. Two researchers (Cheng and Xu) independently completed the quality evaluation, and discrepancies were resolved by the third researcher Chen.The search yielded a total of 15,865 references . We excluded 11,547 references after reviewing the title and abstract. A further 241 references were excluded after full-text reviews, because 87 involved combination therapy or did not concern midazolam oral solution, 79 did not concern children, and 75 were review or conference papers. In total, we identified 89 RCTs, eight cohort studies, 12 case series, and one case report that met our inclusion criteria .n = 33), dexmedetomidine (n = 15), ketamine (n = 11), midazolam injection solution (n = 8), chloral hydrate (n = 7), diazepam (n = 5), N2O (n = 5), triclofos (n = 4), butorphanol (n = 2), hydroxyzine (n = 1), fentanyl (n = 2), thiopental (n = 1), and different midazolam oral solution doses (n = 10) were identified. Eight cohort studies evaluating the effect and safety of midazolam oral solution with placebo or blank (n = 4), chloral hydrate (n = 1), dexmedetomidine (n = 1), and different midazolam oral solution doses (n = 3) were identified. Of these 89 RCTs, the sample size ranged from 10 to 442 (median 60). The location of the first author had the following distribution: India , United States , China , Iran , UK , Japan , Canada , Turkey , Israel , Australia , Brazil , the United Arab Emirates , the Sultanate of Oman , Ireland , Germany , Netherlands , the State of Kuwait , Mexico , the Republic of South Africa , Nepal , Thailand , Uruguay , and Italy . Only one trial was a multicenter RCT. A total of 7,457 children were included in the 89 RCTs for effectiveness and safety analysis. Twenty-two RCTs were included in the current meta-analysis. Other characteristics of the studies were summarized in Eighty-nine RCTs evaluating the effect and safety of midazolam oral solution with placebo or blank  of studies used an adequate method of random sequence generation. Thirteen percent (12/89) of studies implemented adequate allocation concealment. Fifty-two percent (46/89) used the methods of blinding to patients and researchers. Thirty-nine percent (35/89) used the methods of blinding to the outcome measurer. The mean score of risk of bias of eight cohort studies was 5.75. Results of quality assessment of 12 case series and one case report were in I2 = 78%, P < 0.01, and for cohort studies, RR = 1.41, 95% CI , P < 0.01.Four RCTs , P > 0.05].Four RCTs , I2 = 0%, P > 0.05] , P < 0.01].Five RCTs , P < 0.05].Two RCTs , P < 0.01].Two RCTs , P < 0.01]. Another showed that the depth of sedation of midazolam oral solution was deeper .Four RCTs , P > 0.05].One RCT , P > 0.05] , P > 0.05].Two RCTs , P > 0.05] , the dose of midazolam oral solution was divided into three dose groups (0–0.25), mg/kg (0.25–0.5), mg/kg and (0.5–1.0) mg/kg.P > 0.05]. One RCT showed that the depth of sedation of (0.25–0.5) mg/kg midazolam oral solution was lighter , and the cohort study proved the same outcome .Three RCTs , P < 0.01].One RCT , , and  mg/kg were Pf (95% CI) = 0.01 , 0.10 , and 0.09 , respectively.I2 = 29%, P = 0.69; RR = 6.63, 95% CI , P = 0.18]. Two RCTs , P = 0.87]. Four RCTs , I2 = 58%, P = 0.03] , I2 = 0%, P < 0.05] , I2 = 58%, P = 0.66]. But the incidence of mental system adverse effects in midazolam oral solution was higher   receptor , I2 = 34%, P = 0.02]. One cohort study showed no statistically significant difference in each system, but the ratio of experimental groups compared with the control group was approximately 1:20, which could lead to false-negative results , Our evidence suggests that clinical midazolam oral solutions are used in children for sedative hypnosis at doses ranging from 0.25 to 1.0 mg/kg. Our review showed that the time for children to fall asleep was longer in the dose of (0.25–0.5) mg/kg compared to (0.5–1.0) mg/kg, and the two groups were equivalent in relieving anxiety and parental success rate. And the incidence of adverse reactions was similar [10% for (0.25–0.5) mg/kg and 9% (0.5–1.0) mg/kg]. These results suggest that the oral solution of 0.25–1.0 mg/kg of midazolam is effective and safe, and the safe and effective dose of midazolam in the pediatric surgery outdoor anesthesia/sedation expert consensus (2017) is 0.50–0.75 mg/kg, so they are consistent to some extent , (0.25–0.5), and (0.5–1.0) mg/kg. The results did not differ from those before grouping. This may be the result of the insufficient numbers of included studies.This systematic review had some limitations. First, the literature included in this study is limited to Chinese and English, and thus, there may be language bias. Second, 77 of the 89 RCTs did not clearly describe the allocation concealment. If researchers and authors document their experimental methods in future clinical trials and publications in detail, readers and reviewers can better understand the true content of the study. Third, of these 89 RCTs, the smallest sample size is 10 (median 60). A small sample size results in a less authentic result. We look forward to a larger population and long-term data to fully assess the efficacy and risk.Limited evidence suggests that midazolam is effective and safe prior to a diagnosis or treatment procedure for sedation and hypnosis in children. Available evidence suggests that midazolam oral solution is as good as midazolam injection and dexmedetomidine and is better than ketamine. The success rate of sedation and hypnosis of midazolam oral solution was lower than that of chloral hydrate. Neuropsychiatric adverse reactions of midazolam are higher than those of chloral hydrate and dexmedetomidine. Digestive system adverse reactions of midazolam are lower than those of chloral hydrate.LZh, ZC, LZe, LH, DY, XC, and PX conceived and designed the review. XC, PX, FQ, XJ, YW, and ML reviewed the literature and extracted data. XC and ZC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: To explore cerebrospinal fluid (CSF) levels of soluble triggering receptor expressed on myeloid cells 2 (sTREM2) and neurofilament light proteins (NFL) in patients with neurosyphilis (NS).Methods: We enrolled 71 NS patients (41 early-NS and 30 late-NS patients) and 20 syphilis but non-NS patients whose CSF samples were collected. The CSF levels of the microglial activation biomarker sTREM2 and neuronal injury biomarker NFL were measured using ELISA.Results: CSF sTREM2 levels were significantly higher in NS patients compared to those in syphilis/non-NS patients (p < 0.001). In a subgroup analysis, the CSF sTREM2 levels elevated significantly in late-NS patients than those in early-NS patients (p < 0.001). The CSF sTREM2 levels in early-NS group were also significantly higher than those in syphilis/non-NS group (p = 0.024). Like CSF sTREM2, similar differences between groups were also found in CSF NFL. There was a moderate correlation between CSF sTREM2 and CSF NFL  in NS group.Conclusions: CSF sTREM2 levels elevated in NS and peaked at the late stage, suggesting that CSF sTREM2 may be a useful marker to quantify microglia activation in NS and may play a role in the progression of NS. The positive correlation between CSF sTREM2 and CSF NFL indicates a linkage between microglial activation and neuronal injury in NS. Treponema pallidum invading the central nervous system (CNS), which mainly damages the meninges, blood vessels, and brain, and spinal cord parenchyma  is a cell surface receptor protein, which is mainly expressed in myelocytes . Most rein vitro –8. Increin vitro , 10, andin vitro , 12. Howin vitro , 4. Neurin vitro . The levThe aim of this study was to determine the levels of CSF sTREM2 in different stages of NS patients and to explore the relationship between CSF sTREM2 and CSF NFL, so as to better understand the neuropathogenesis and progression of NS.Treponema pallidum particle agglutination (TPPA) and fluorescent treponemal antibody absorption (FTA-ABS), with increased CSF protein (>45 mg/dl) or white blood cells (WBC) (> 5/μl) in absence of other known causes of these abnormalities. All of the enrolled patients were HIV negative. The exclusion criteria were as follows: treatment with antibiotics within the last 1-month, other infectious diseases , neurodegenerative disease  and autoimmune diseases . The patients enrolled in the control group were a serofast status without neurological symptoms and signs and underwent lumber puncture to rule out neurosyphilis.Between May 2018 and June 2019, 71 NS patients and 20 syphilis but non-NS controls hospitalized in the neurology department of Beijing Ditan Hospital and Beijing Tiantan Hospital were analyzed in this study. We recorded the medical history, neurological symptoms and signs, and serum and CSF laboratory testing results. According to the guidelines of NS in the USA, Europe and related literatures –18, the This study was approved by the Ethics Committee of Beijing Ditan Hospital Affiliated to Capital Medical University, Beijing, People's Republic of China and written informed consent was obtained from all participants.The enrolled NS patients were divided into early-NS that occurred during the primary stage or secondary stage, including asymptomatic, meningeal and meningovascular NS and late-NS that occurred years to decades after the primary infection, including general paresis and tabes dorsalis , 4.CSF samples were immediately centrifuged and the supernatants were collected and stored at −80°C until the time of the biomarker assays. ELISA, to quantify CSF sTREM2  and NFL , was performed blinded to clinical information. All testing was performed according to the manufacturer's protocols and by the same technique.10 transformed where appropriate to reduce skewness. Continuous data were compared with the non-parametric Mann-Whitney U-test when the data were non-parametric or independent two-sample t-test when the data were parametric. Gender differences were assessed by χ2-test. Pearson' test (for parametric data) and Spearman's test (for non-parametric data) were used to ascertain the associations for analysis of correlations. The significance level was established at a two-sided p < 0.05.Data were analyzed by SPSS software (IBM SPSS 25.0 version) or Prism (GraphPad software 7.0 version). CSF levels of sTREM2 and NFL were logn = 34), meningeal NS (n = 1) and meningovascular NS (n = 6). In addition, 30 patients with late-NS were enrolled, including general paresis (n = 26) and tabes dorsalis (n = 4). The vast majority of patients received anti-syphilis treatment, and only 2 patients with paralytic dementia had no treatment history. The 30 cases of late-NS showed a variety of neurological symptoms and signs, including mental and behavior disorders (24/30), cognitive changes (23/30), sensory impairment  (8/30), parkinsonism (4/30), headache (3/30) and seizures (3/30). The symptoms of 41 cases of early-NS were asymptomatic (34/41), headache (1/41), stroke (6/41) and seizures (1/41).The clinical characteristics and concentrations of different CSF biomarkers were summarized in p = 0.65 and p = 0.29). The serum RRP titer was significantly higher in the NS group than in the syphilis/non-NS group (p < 0.001). As for CSF laboratory findings between the late-NS group and early-NS group, we found that the incidence of positive CSF RPR rate was significantly higher in the late-NS group than in the early-NS group (p < 0.001), but no significant difference of serum RPR titer, CSF protein concentration and CSF WBC was found between the two groups .There was no significant difference in age and gender between the NS group and the syphilis/non-NS group (10) values in the NS group were significantly higher than those in the syphilis/non-NS group  values between the two NS groups  values in the early-NS group were also significantly higher than those in the syphilis/non-NS group  pg/ml in NS group and 14783.12 (10567.25-17894.95) pg/ml in non-NS group. CSF sTREM2  values and CSF NFL (log10)  and CSF NFL (log10) in the syphilis/non-NS group  levels were weak correlated with age in the NS group  and age in the NS group  and CSF NFL (log10) were better correlated with age in syphilis/non-NS group  in the two groups  of our NS patients had CSF RPR positive, and the higher CSF sTREM2 levels were found in CSF RPR positive group. There were significant differences between CSF sTREM2 . The patients/participants provided their written informed consent to participate in this study.WL, HC, WW, LY and XZ conceived and designed the study. WL, MJ, JG, and YX carried out the laboratory analyses. LY, WL, DX, and YH collected and organized the data and drafted the manuscript. WL, HC, and XZ performed the statistical analysis. All authors read, reviewed, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1−/− donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM. In addition, clinical studies show that organ transplant recipients receiving calcineurin inhibitor therapy have impairment of Foxp3 + T-regulatory (Treg) cells, whereas Treg function is preserved under rapamycin (RPM) therapy5. Experimentally, peri-transplant costimulation blockade (COB) alone, using CD154 monoclonal antibody (mAb) or CTLA4-Ig, or COB plus a brief course of RPM, can induce long-term, Treg-dependent survival of organ transplants9. Critically, allograft recipients of COB-based protocols can develop donor-specific tolerance and remain free from the long-term development of chronic allograft rejection without maintenance immunosuppression9. Hence, understanding whether peri-transplant therapies can also benefit VCA recipients and avoid maintenance calcineurin inhibitor-based immunosuppression is an important consideration in efforts to promote the broader application of VCA procedures and to improve the subsequent quality of life in VCA recipients.The widespread application of vascularized composite allotransplantation (VCA) is currently limited by various factors, including the perceived risks of maintenance immunosuppression with calcineurin inhibitors that involve increased risks of the development of infection, neurotoxicity, nephrotoxicity and carcinomaWe hypothesized that a clinically relevant protocol such as peri-transplant delivery of some form of COB plus RPM might promote long-term VCA survival by inhibiting the allo-activation of conventional T cells and allowing the development of Treg-dependent mechanisms that maintain allograft survival. Corollaries of this over-arching hypothesis are that potentially both donor and recipient Tregs might contribute to graft survival; that Tregs must prevent potent host responses to allogeneic skin that is typically considered highly immunogenic; and that therapeutic agents that negatively or positively affect Treg function might impair or further promote outcomes, respectively. We sought to test these concepts in murine limb transplant models that are ideally suited to mechanistic investigations.6 cells, i.v.) is very effective at inducing tolerance of various kinds of allografts, including that of hearts, kidneys or islets transplanted across fully MHC-disparate combinations such as BALB/c->C57BL/6 mice (H-2d->H-2b)7. However, this protocol, while able to prolong VCA survival to >30 days, was unable to induce long-term (>100 days) VCA survival  plus donor splenocyte transfusion  indicated that CXCR4i altered the proportions of multiple cell types including T and B cells from donor bone-marrow13. Allografts derived from CXCR4i-treated mice  underwent acute rejection despite therapy with CD154/DST/RPM , a CXCR4 inhibitor (CXCR4i) that is used clinically to mobilize bone-marrow cells for subsequent hemopoietic stem cell transplantationRPM Fig. . Collectb-negative) CD4 and CD8 cells in untreated controls, in contrast to their essentially normal numbers in large numbers in recipients receiving CD154/DST/RPM  plus DST (5 × 106 on day 0) extended 50% survival to about 3 weeks (p < 0.05)  and CTLA4Ig , that led to long-term survival of skin and cardiac allografts in the BALB/c-> C57BL/6 combinationval Fig. . Likewis05) Fig. . Additio05) Fig. . With an05) Fig. . Hence, We sought to compare the contributions of donor cells in the peritransplant CTLA4Ig/RPM protocol with that seen in our studies with CD154/DST/RPM, described above. As summarized in Fig. 17. In addition, bone-marrow reticular and endothelial cells strongly express functional stromal-derived factor (CXCL12), the ligand for CXCR418, and murine and human Tregs traffic to, and are retained in, BM through CXCR4/CXCL12 signals17. There is also evidence from hemopoietic stem cell transplant models that the BM may be an immunologically privileged site, whereby donor Tregs can protect allogeneic cells from immune destruction19. Hence, we investigated whether Foxp3+ Treg cells present pre-transplant in donor limb bone-marrow are central to the efficacy of COB-based protocols.Our finding that the same factors blocked the efficacy of 2 protocols that are highly effective in prolonging orthotopic VCA survival, CD154/DST/RPM and CTLA4Ig/RPM, led us to focus on which T or B cell population might be required. In murine bone-marrow, as with human bone-marrow, there is a higher frequency of CD4+ CD25+ FoxP3+ Treg cells than in any other secondary lymphoid organsUsing flow cytometry, we found that use of the CXCR4i in donors pre-transplant led to a > 50% reduction in donor Foxp3+ Tregs within the bone-marrow by the day of transplantation whereas the splenic population of Tregs was unchanged Fig. . In cont20. Administration of diphtheria toxin markedly reduced Foxp3+ Tregs in donor bone-marrow pre-transplant  within fully functional Foxp3 + CD4+ Treg cellsant Fig. , and useant Fig. . These d17. Treatment of Tregs with the CXCR4i, AMD3100, led to mildly impaired Treg suppressive function in vitro , which suppresses phosphoinositide 3-kinase (PI3K) and Akt signalingR4i Fig. , and simR4i Fig. . ConsistR4i Fig. . These d5. While the many reports of the efficacy of costimulation blockade in experimental transplant models have not led to corresponding success clinically, COB using a modified form of CTLA4Ig (Belatacept) has utility in curtailing alloresponses and avoiding calcineurin inhibitor use in renal transplant recipients, thereby helping preserve glomerular filtration rates long-term27. Whether ongoing Belatacept therapy impairs Treg function long-term by blocking the inhibitory actions of CTLA4 remains unclear29, though its use clinically does require monthly infusion. A critical role for chemokine-dependent recruitment of Tregs to allografts in mice undergoing COB has been reported8, but there are no data on donor Tregs being required for successful engraftment of solid organs or VCA tissues. Hence, the current studies are significant in showing how the efficacy of 2 different peri-transplant COB protocols both depended upon the presence of Foxp3+ Treg cells present within donor bone-marrow.Given that well over a decade of clinical trials with Tregs as adoptive cell therapy in transplant recipients has not produced any compelling successes, it may be time to reconsider how best to exploit Treg biology in the context of transplantation. In particular, physiologic levels of Foxp3+ Tregs may be sufficient to maintain allograft survival following various therapeutic interventions, involving “Treg-friendly” COB and/or RPM, and the avoidance of calcineurin inhibitor-based protocols so as to allow optimal Treg functioning30, the cognate ligand for the G protein-coupled receptor, CXCR4, which is expressed by mononuclear leukocytes and bone-marrow progenitor cells. This ongoing production of CXL12 in the BM leads to Treg recruitment and immobilization; there are greater than 2-fold more Tregs in the human BM than in the thymus, blood or lymph nodes, and these bone-marrow Treg cells express more Foxp3 and have greater suppressive activity than non-bone-marrow Treg cells17. Foxp3+ Treg cells are thought to maintain hemopoietic stem cell (HSC) quiescence by their generation of adenosine, and this quiescent state hence promotes HSC longevity31. Components of this large Treg reservoir within the bone-marrow are thereby present during limb VCA procedures. We found that donor bone-marrow cells are destroyed by alloreactive host cells in the first week post-transplant but are preserved in mice receiving COB, and we observed accumulation of recipient Tregs within the donor bone-marrow. The spatial and contextual details of the interactions of donor and recipient cells within the bone-marrow compartments of limb allografts remain to be dissected, just as the details of the concept that this site is immunologically privileged remains to be elucidated19. However, it might provide a site at which alloreactive host Teff cells undergo immunomodulation by resident Foxp3+ Treg cells.Osteoblasts, endothelial cells and reticular cells within the BM are the major sources of CXCL12 production32, and with the utility of CXCR4 targeting in disrupting the functions of tumor associated Treg cells35. Foxo1 is a transcription factor essential for control of the expression of Foxp3 and its target genes; phosphorylation of Foxo1 leads to its nuclear export and Treg dysfunction. Hence, the local production of CXCL12 by cells within bone-marrow niches promotes Treg accumulation and also stabilizes their function25.Our studies also suggest that beyond promoting the bone-marrow accumulation of Tregs, CXCL12 signaling through the CXCR4 receptor may have important effects in terms of Treg homeostasis and stability, since targeting of the receptor impaired Treg function, decreased PTEN expression and increased phosphorylation of Foxo1. These findings are consistent with studies of signaling within cancer cellsOn a practical level, further studies are required to image serial events within the donor bone-marrow post-transplant, as well as to test aspects of host acceptance of the grafts. We still need insights into whether host alloreactive T cells are being suppressed by donor Tregs within the donor bone-marrow or as a result of donor Treg migration beyond their bone-marrow niche. Of particular interest will be the assessment of whether donor-specific tolerance can be achieved, the relative contribution of donor vs. recipient bone-marrow cells (including Tregs) in long-surviving donor long-bones as well as in recipient bone-marrow, and the potential for ongoing freedom from the development of chronic rejection at periods considerably later than 100 days post-transplant. However, the current studies already suggest optimism in seeking to develop therapeutic protocols for VCA procedures that involve donor BM, given the ability to administer clinically approved agents, such a CTLA4Ig and RPM, in the peri-transplant period and achieve sustained engraftment without maintenance immunosuppression. Hence, there may be better and less toxic ways to immunosuppress patients after VCA than the current calcineurin inhibitor-based protocols, and the data from experimental models may be especially relevant given that the small numbers of patients currently undergoing VCA procedures do not lend themselves to extensive clinical experimentation.10; DEREG/B6 mice engineered to express the diphtheria toxin receptor plus a green fluorescent protein (DTR-eGFP) within fully functional Foxp3 + CD4 + Treg cells20; Foxp3YFP-Cre mice36 and CXCR4fl/fl mice37 (both strains on the B6 background) from The Jackson Laboratory. All mice were used at 8–12 weeks of age. Animal study protocols were approved and undertaken in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee of The Children’s Hospital of Philadelphia (19-001052). Experiments were performed with age‐ and sex‐matched mice and using animals that were littermates or were maintained in the same room and/or were co‐housed within the same cages for >2 weeks to limit potential effects of microbiome differences.We purchased WT BALB/c and WT C57BL/6 mice; B6/Rag−/− mice lacking mature T and B cells38, except that the anastomoses of donor and recipient blood vessels were performed by direct suturing rather than utilizing a cuff technique. Nerve sheaths were anastomosed by direct suturing. Mice were examined daily for signs of vascular compromise and rejection; the latter was apparent as hair loss, swelling and erythema of the skin that rapidly progressed to skin necrosis and was confirmed by histology38.Heterotopic and orthotopic hindlimb transplants were performed using BALB/c donors and C57BL/6 recipients, as describedWe purchased CXCR4i ; CD8 mAb (clone YTS 169.4), CD154 mAb (clone MR1) and murine CTLA4-Ig (BioXcell); diphtheria toxin (Sigma-Aldrich) and RPM (Sigma-Aldrich).In some experiments, WT BALB/c mice were subjected to pre-Tx whole-body irradiation (800 cGy), on the same day as their use as limb transplant donors. DEREG mice were injected daily for 6 days with diphtheria toxin  to deplete Tregs prior to their use as limb transplant donors. Additional donor mice were treated with AMD3100, a CXCR4 inhibitor (CXCR4i), on days −4, −2 and on the day of transplantation .6 cells), i.v., at transplantation, and in some series, mice also received RPM (2 mg/kg/d) delivered by a 4-week Alzet osmotic pumps inserted at transplantation. In additional studies, recipients were treated with murine CTLA4‐Ig , i.p., at days 0, 2 and 4 post-Tx, alone or in conjunction with RPM (2 mg/kg/d) delivered by a 4-week Alzet pumps inserted at transplantation. Recipient CD8 T cells were depleted by weekly i.p. injection of CD8a mAb  until rejection39.Recipients were treated with CD154 mAb  with or without donor splenocyte transfusion , H-2Kd , CD34  and Lineage Antibody Cocktail , and performed flow cytometry using a CyAn flow cytometer . We also purchased unconjugated CD3  and CD28  mAbs from BD Bioscience.We purchased mAbs directed against murine CD4 , CD8 , Foxp3 , CD25 , H-2K41.Limb specimens were fixed in 10% neutral buffered formalin, decalcified in Formical-2000, and embedded in paraffin. Histologic sections (6 μM) were stained with hematoxylin and eosin, reviewed by a pathologist (W.W.H) blinded to conditions, and graded using Banff criteria9; qPCR was performed using Taqman primer and probe sets, with data normalized to 18 s rRNA and relative expression determined by the formula 2−ΔCT.RNA was isolated using RNeasy kits (Qiagen), with RNA integrity and quantity analysis using a NanoDrop ND-1000 and a Nanochip 2100 Bioanalyzer (Agilent Technologies)9.Western blots were performed using Abs directed against Foxo1, phospho-Foxo1, PTEN (eBioscience) and β-actin We used GraphPad Prism to analyze data. Differences between two groups were assessed with a 2-tailed Student’s t-test if data were normally distributed, or Mann-Whitney U unpaired test if not normally distributed. Groups of three or more were analyzed by 1-way ANOVA, with Tukey’s multiple comparison test if normally distributed, or the Kruskal-Wallis with Dunn’s multiple comparison test if not normally distributed. Graft survival was evaluated with Kaplan-Meier followed by log-rank test. P < 0.05 was considered significant.Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Wayne W. Hancock (whancock@pennmedicine.upenn.edu).Supplementary information.


Blood removal from cerebrospinal fluid (CSF) in post-subarachnoid hemorrhage patients may reduce the risk of related secondary brain injury. We formulated a computational fluid dynamics (CFD) model to investigate the impact of a dual-lumen catheter-based CSF filtration system, called Neurapheresis™ therapy, on blood removal from CSF compared to lumbar drain.A subject-specific multiphase CFD model of CSF system-wide solute transport was constructed based on MRI measurements. The Neurapheresis catheter geometry was added to the model within the spinal subarachnoid space (SAS). Neurapheresis flow aspiration and return rate was 2.0 and 1.8 mL/min, versus 0.2 mL/min drainage for lumbar drain. Blood was modeled as a bulk fluid phase within CSF with a 10% initial tracer concentration and identical viscosity and density as CSF. Subject-specific oscillatory CSF flow was applied at the model inlet. The dura and spinal cord geometry were considered to be stationary. Spatial–temporal tracer concentration was quantified based on time-average steady-streaming velocities throughout the domain under Neurapheresis therapy and lumbar drain. To help verify CFD results, an optically clear in vitro CSF model was constructed with fluorescein used as a blood surrogate. Quantitative comparison of numerical and in vitro results was performed by linear regression of spatial–temporal tracer concentration over 24-h.2 = 0.89 with + 2.13% and − 1.93% tracer concentration confidence interval).After 24-h, tracer concentration was reduced to 4.9% under Neurapheresis therapy compared to 6.5% under lumbar drain. Tracer clearance was most rapid between the catheter aspiration and return ports. Neurapheresis therapy was found to have a greater impact on steady-streaming compared to lumbar drain. Steady-streaming in the cranial SAS was ~ 50× smaller than in the spinal SAS for both cases. CFD results were strongly correlated with the in vitro spatial–temporal tracer concentration under Neurapheresis therapy (RA subject-specific CFD model of CSF system-wide solute transport was used to investigate the impact of Neurapheresis therapy on tracer removal from CSF compared to lumbar drain over a 24-h period. Neurapheresis therapy was found to substantially increase tracer clearance compared to lumbar drain. The multiphase CFD results were verified by in vitro fluorescein tracer experiments. A detailed understanding of cerebrospinal fluid (CSF) physiologic function may help improve treatment of CSF-related central nervous system (CNS) diseases and debilitating neurological conditions. CSF is a clear, colorless fluid that occupies the subarachnoid space (SAS) and the ventricular system within the brain . CSF is The importance of CSF dynamics has been investigated in several CNS diseases that include neuroinflammatory conditions such as multiple sclerosis , 8 and nNeurapheresis™ therapy  is being investigated as a means to potentially rapidly remove red blood cells from the SAS and consequently decrease the incidence or severity of secondary complications. In brief, Neurapheresis therapy involves aspiration of CSF from the lumbar spinal SAS, filtration of CSF and removal of red blood cells and detritus to a waste bag, and then return of filtered CSF to the SAS at the thoracic spine. The different locations of the aspiration and return ports may facilitate advective bulk movement of CSF between the ports within the SAS. Additional details on Neurapheresis therapy are provided by Khani et al. .Computational and in vitro modeling constitute potential methods to study complex transport phenomena occurring during blood clearance from CSF spaces and, subsequently, to improve devices and protocols to treat neural disorders. Clinical trials to develop guidelines for more effective clearance are difficult due to limited availability of subjects and associated study costs. Empirical models limit our ability to investigate CSF filtration technologies, highlighting the need for a computational tool. Human clinical trials of Neurapheresis therapy in SAH patients are being conducted , butSeveral in vivo, in vitro, and in silico studies have been conducted to better understand Neurapheresis therapy. A rabbit model was used to investigate Neurapheresis therapy in the context of cryptococcal meningitis. This study showed a 5-log reduction in yeast concentration and 1-log reduction in its polysaccharide antigen over 24-h. A drawback of the study was that the rabbit model has an extremely small SAS, thus it is unclear how rabbit CSF dynamics compare to humans . A studyWhile the previous studies provided insight into Neurapheresis therapy, they had the following important limitations: (a) they lacked realistic system-wide CSF geometry, (b) they did not consider the multi-phase solute transport within the CSF, (c) they were conducted over relatively short time periods, (d) they utilized animal models that have different CSF dynamics than humans, and/or (e) they lacked numerical model solution verification. This study seeks to address these limitations by formulating a multi-phase numerical model of CSF system-wide solute transport within an anatomically realistic human geometry and by verifying the numerical solution versus in vitro measurements.The overall approach was to build a subject-specific multiphase computational fluid dynamics (CFD) model and to verify model results using a corresponding bench-top model. After verification, the models were used to quantitatively assess tracer removal by Neurapheresis therapy compared to lumbar drain. Fluorescein solution was used as a surrogate tracer and modeled as a bulk fluid phase within the CSF –32 to trA high-resolution T2-weighted magnetic resonance imaging (MRI) sequence was used to acquire a subject-specific geometry of the CSF system of a 23-year-old healthy female subject Fig. a. This mA detailed intracranial CSF space geometry obtained with high-resolution MRI was added to the spinal SAS. The CSF space schematic in this region is shown in Fig. The dual-lumen Neurapheresis catheter geometry was identical with our previous publication . In brieAn unstructured tetrahedral computational mesh was generated using ANSYS ICEM 19.2 . Details on the prism layer and Mesh parameters are included in Table Clearance of tracer after SAH with Neurapheresis therapy and lumbar drain was studied under the following boundary conditions Fig. a. The mo−1. A zero-pressure outlet boundary condition was defined at the cranial opening. No-slip boundary conditions were imposed at the model walls  with the walls modeled as stationary. CSF was modeled as incompressible with a density of 998.3 kg/m3 and viscosity of 0.89 mpa s. Tracer density and viscosity was assumed to be identical to CSF.To represent CSF pulsation around the brain and spinal cord, an oscillatory velocity inlet boundary condition was defined at the model caudal opening using a User Defined Function. The exact waveform was derived from the C2–C3 CSF flow rate waveform  by solving the continuity and Navier–Stokes equationsk and the bulk fluid to be zero. Thus, the drift velocity was considered to be zero.The 3D CFD computations used the ANSYS multi-phase fluid model to track the dispersion of a tracer within the CSF, with tracer volume fraction given byBased on clinical SAH observations and previous research, we assumed a baseline tracer concentration of 10%  is much smaller than steady-streaming and oscillatory CSF velocities . HoweverTo help understand the relative importance of diffusive versus advective mass transport, the Sherwood number (2/s), to molecular diffusion coefficient , \docum−10 m2/s , 44 and −11 m2/s .Simulations were carried out using the PISO Scheme (pressure-implicit with splitting of operators) to solve the flow equations with second-order upwind for momentum discretization, PRESTO! (PREssure STaggering Options) for pressure discretization, and first-order upwind for volume fraction discretization. Default values were used for under relaxation factors. The implicit formulation was used for volume fraction parameters and a dispersed model was used for phase interface modeling. The convergence criteria for velocity, continuity, momentum, and phase volume fraction were set to 1E−06 with time step = 0.1 s and maximum of 100 iterations per time-step. Total simulation time was 14 days to compute 24-h of real-world time in parallel mode with 126 GB RAM and 38 processors. Time required to solve the fixed flow field (11 flow cycles) was 36 h.Axial distribution of average tracer concentration at 1 h was verified by numerical sensitivity studies for time-step size and mesh resolution. Results were computed for a “coarse”, “medium”, and “fine” mesh with wall prism layers. For the medium mesh, time-step sensitivity was then checked with time-step resolution given by fractions of the cardiac cycle, T = 0.9 (s), for T/18, T/9, T/5. Based on these results, a “Medium” mesh with time step = T/9 was carried forward for computation of final results.To help verify the numerical model results, an in vitro CSF model was constructed with a fluid domain geometry identical to the numerical model. For the in vitro model, the fluid domain was encased in a 2 mm thick transparent shell. The shell was divided into cranial Fig. b, upper To mimic SAH blood distribution within the CSF, an aqueous solution of fluorescein sodium (25 mg/1000 mL) was used to fill the entire model representing a uniform distribution of blood. A uniform distribution was assumed because previous research from Tangen et al.  showed tA continuous flow syringe pump  was used to aspirate fluid via the Neurapheresis catheter at 2.0 mL/min. This same pump was also configured in parallel to return fresh deionized water at a rate of 2.0 mL/min. This clean return flow was split and 0.2 mL/min was diverted by a second continuous flow syringe pump for CSF production originating from the ventricles. Thus a net flow of 1.8 mL/min was delivered to the return port of the Neurapheresis catheter. Another syringe pump was used to generate the subject-specific CSF flow wave form ) light emitting diodes triggered to a camera flash. For model calibration, a baseline image was obtained without any tracer introduced to the model. To establish baseline decay of the fluorescein tracer, time-lapse imaging was obtained at 5-min intervals over a total of 24-h with the tracer solution at a known concentration 15 μM. To eliminate aliasing artifacts due to CSF oscillations, images were collected with a trigger set to the cardiac systole. An identical series of images was then collected for the same experiment under Neurapheresis therapy and also with lumbar drain.Spatial–temporal distribution of tracer concentration for each case was computed as 12-bit images were collected with 12 MP resolution using a digital camera  with a 50 mm focal length prime lens with f/10 aperture (Sony E 50 mm f/1.8 OSS). Imaging settings were 2000 ISO, white balance 5500 K, and 1/100 s shutter speed. A 525 nm bandpass filter  was used in front of the lens to improve image signal-to-noise ratio.DH is hydraulic diameter, Acs is the cross-sectional area and Qpeak defined as the maximum flow rate at peak systole and diastole at each slice.Based on the 3D reconstruction and meshing, the following geometric and hydrodynamic parameters were calculated along the spine at 1 mm intervals using our previously described methods . ReynoldTo quantify steady-streaming, the cyclic mean velocity in the z-direction, lication . \documeA detailed comparison of numerical and in vitro results was performed by the following correlation analysis similar to that previously conducted by our group . In vitrOverall, numerical and in vitro results showed agreement in terms of spatial–temporal concentration of tracer over the 24-h simulation period. Neurapheresis therapy was found to clear tracer from the CSF more rapidly than lumbar drain with most of the clearance occurring within the thoracic SAS after 1-h of treatment.3 [2, respectively. As expected, maximum area and perimeter was located at the cranium  Fig. c. \documThe square of the Womersley number and oscillatory Peclet numbers were calculated to estimate the potential enhancement of dispersion by shear. For both tracer and hemoglobin 2/s in the cortical SAS and 3.0677 E−04 m2/s in the spinal SAS. Baseline tracer concentration was set to 10% throughout the model. After 24-h, tracer concentration was reduced to 4.9% under Neurapheresis therapy compared to 6.5% under lumbar drain. Tracer clearance in the thoracic region occurred more rapidly after 1 h under Neurapheresis therapy compared to lumbar drain  under both Neurapheresis therapy Fig. a and lumOverall, the distributions and clearance rates of tracer concentration in Neurapheresis therapy and lumbar drain match the bench-top patterns. Numerical simulations predicted slightly faster clearance rates under Neurapheresis therapy and lumbar drain than in vitro Fig. .Differences between spatial–temporal cross-sectional average tracer concentration over 24-h obtained from in vitro and CFD were quantified using Bland–Altman plots Fig. . A relatots Fig. a2, b2 shots Fig. a2, b2.FiThe CFD and in vitro model provided a systematic comparison of lumbar drain to Neurapheresis therapy after SAH. These models allow detailed comparison of results without the confounding impact of many variables that would be difficult to control for in vivo animal models or SAH patients. Computer simulations provided the theoretical basis to interpret bench top in vitro results by better elucidating the complex tracer clearance patterns in pulsatile CSF after SAH.Studies show that wide variability exists in CFD modeling techniques and the choice of numerical solvers and settings are complex and can yield disparate results for biofluids simulations . Thus, i2 = 0.89, Fig. 2 = 0.65, Fig. Comparison of spatial–temporal cross-sectional average tracer concentration profiles revealed similar clearance trends for both CFD and in vitro under Neurapheresis therapy and lumbar drain conditions Fig. . A stronOur model results are difficult to directly compare with previous research as no study has been conducted previously with an anatomically realistic model and with Neurapheresis therapy applied with an exact catheter geometry. However, Tangen et al.  used an After 24 h, results from Neurapheresis therapy showed that 4.9% of tracer remained in the model Fig. a while 6The Neurapheresis therapy flow rate applied in our study was 2.0 mL/min with a 1.8 mL/min return flow rate. A flow rate of 2.0 mL/min is not possible to apply using a lumbar drain because it would remove CSF more rapidly than it is being produced at the choroid plexus (~ 500 mL/day) . To helpTransient simulations of oscillating fluids are computationally intensive, in particular when conducted over long time periods with small time-step size. For example, in the present case representing CSF oscillations, computation of a single CSF flow cycle requires ~ 3.6 h using 38 processors (Intel(R) Xeon(R) Gold 6148 CPU @ 2.40 GHz) and 126 (GB) Memory. Simultaneously solving the passive transport equation requires additional time. Neurapheresis therapy is conducted over a period of more than 24-h. As such, we applied a two-part CFD method that neglected diffusion to obtain a computationally tractable solution over the 24-h timeframe. First, a transient Navier–Stokes solution of 11 flow cycles was performed to obtain the steady-streaming velocity field. Steady-streaming is postulated to be responsible for the time-average bulk movement of CSF in the SAS that results from nonlinear cumulative effects of convective acceleration . Steady The In this study, we did not include the potential impact of microscopic anatomy within the domain such as arachnoid trabeculae or blood vessels, nor hydrodynamic affects on finite-size particles . Other numerical studies have investigated the potential impact of microscopic structures  within tmax is large compared to unity. Therefore, the chosen tracer provides good similitude for blood. The in vitro experiments were performed at 19 °C. We did not create a thermostatic environment due to size limitations, because the density ratio between CSF and fluorescein tracer is not different whether the experiment is conducted at 19 °C or at body temperature of 37 °C.In this study, blood dispersion was modeled by fluorescein tracer mixed in a single continuum CSF phase at room temperature. Physiologically, blood cells and debris create a suspension when mixed into CSF. The biochemistry of blood coagulation within the CSF was not reproduced. Additionally, once exposed to the SAS environment, blood cells can rupture releasing oxyhemoglobin which is further enzymatically converted to bilirubin , 64. WhiIn the comparison between simulation and experiments, the highest deviations occurred in the cranial SAS. It is likely these differences were larger in the cranial SAS due to the 2D imaging technique that used a picture obtained for a single angle relative to the model, whereas, the CFD concentrations were precisely averaged across each 3 mm thick slice, including fluid located within the ventricles of the brain. Future work could potentially improve agreement of in vitro and numerical results by utilizing tomographic projection imaging  of the iThe numerical simulations in this study were based on MRI measurements for a single subject-specific CSF system geometry and CSF flow waveform. These parameters should be investigated in a larger cohort to determine the potential impact of age, sex, and disease states on CSF solute transport. However, the consistency of CSF dynamics across humans in the healthy state and with ALS has been studied by our group and we found relatively small differences across subjects . TherefoOur modeling approach did not include flow oscillations within the ventricles , 70 or aOur model only had one single site of CSF production in the lateral ventricles because the focus of our study was on CSF solute transport within the subarachnoid space, external to the ventricles, we simplified CSF production to occur at a single site within the lateral ventricle. CSF production was assumed to flow out into the cisterna magna where mixing occurs with CSF in the subarachnoid space. Also, the in vitro system did not allow imaging of tracer concentration within the ventricles, and therefore we were not able to compare in vitro to computational results within the ventricles. Future studies should investigate the impact of CSF production location by adding the choroid plexus in the third and fourth ventricles.No attempt was made in this study to optimize catheter design or positioning for Neurapheresis therapy. The effect of Neurapheresis therapy on CSF steady-streaming velocities in the spinal SAS were investigated in our previous study . The preA subject-specific CFD model of the CSF system was formulated and applied to compare the impact of Neurapheresis therapy on tracer removal from CSF compared to lumbar drain over a 24-h period. Results were verified with an in vitro model built identical to the CFD model. The numerical modeling approach using a frozen flow field to represent solute transport resulted in similar solute transport dynamics as that seen in vitro. Using the verified computational model with in vitro system, the results predict that Neurapheresis therapy significantly increases tracer clearance compared to a lumbar drain. The overall tracer concentration after a 24-h period for Neurapheresis therapy was 4.9% compared to 6.5% with lumbar drain. This effect was maximized within the region between the return and the aspiration ports in Neurapheresis therapy.


The aim of this study was to evaluate the therapeutic effects of coronary granulocyte colony-stimulating factor (G-CSF) on rats with chronic ischemic heart disease (CIHD).n = 10) after the CIHD model was established. Left ventricular ejection fraction (LVEF), myocardial injury area, myocardial perfusion area and viable myocardium were observed by coronary angiography, dual-isotopic myocardial imaging and first-pass delayed myocardial perfusion magnetic resonance imaging (MRI) before modeling as well as 2 and 4 weeks after surgery.Thirty healthy rats were randomly divided into control, subcutaneous and intracoronary G-CSF injection groups (P < 0.05). The distal stenosis degrees of target lesions in subcutaneous and intracoronary G-CSF injection groups were significantly lower than that of the control group (P < 0.05). Compared with the situation before mobilization, LVEF was significantly improved after 2 weeks in intracoronary and subcutaneous G-CSF injection groups (P < 0.01). Their infarcted myocardial areas were reduced, the left ventricular remodeling was relieved, the percentage of viable myocardium was increased, angiogenesis was promoted and cardiomyocyte apoptosis was inhibited.The peak times of peripheral blood and subcutaneous G-CSF levels were 3 and 5 days after mobilization, respectively. The peripheral blood CD34+/CD133+ cell ratio of subcutaneous or intracoronary G-CSF injection group significantly exceeded that of the control group (Intracoronary G-CSF injection is safe and effective as subcutaneous injection, improving the cardiac function of CIHD rats. In receThe aim of this study was to compare the effects of intracoronary injection and subcutaneous injection of G-CSF on the cardiac function and the left ventricular remodeling in rats with CIHD and to evaluate its safety. The results provide valuable experimental evidence for clinical application.22.1Thirty healthy Sprague Dawley rats weighing 200–220 g of either gender were purchased from SLAC Laboratory Animals Co., Ltd .Recombinant human G-CSF was purchased from Harbin Pharmaceutical Group Co., Ltd . G-CSF ELISA kit and TUNEL DeadEnd kit were bought from Santa Cruz (USA). Fluorescein isothiocyanate (FITC)-labeled monoclonal mouse antihuman CD34 antibody and APC-labeled monoclonal mouse antihuman CD133 antibody were obtained from Amresco (USA). TNF-α, cTnT and BNP ELISA kits, reverse transcription system and Access RT-PCR kits as well as ECL Plus kit were provided by Sigma (USA). Goat antimouse Akt1 and vWF polyclonal antibodies were purchased from Shanghai Baoman Biotechnology Co., Ltd (China). Magnevist (Gd-DTPA) injection was bought from Schering AG . Omnipaque (iohexol) injection was obtained from GE Healthcare . 99mTc-MIBI injection was provided by Guangzhou Medical Isotope Service Center . All other reagents were analytically pure.Ameroid constrictor rings were purchased from Labnet (USA). PCR system and FACSCalibur flow cytometer were bought from Thermo Fisher Scientific (USA). Inverted fluorescent microscope and image analysis system were obtained from Tohnichi (Japan). Fluorescent imaging system was provided by Medica (USA). Innova 2000 Digital Cardiac Cath Lab System was purchased from GE Healthcare .2.2T wave changes (from elevation to inversion or inversion to elevation) in leads I, aVL and (or) II, III, aVF and/or V4 to V6 found on ECG; (2) coronary angiography showing subtotal or complete occlusion of left circumflex (LCX) branch (stenosis > 95%); (3) cardiac MRI showing perfusion defects and/or dyskinesia of left ventricular lateral wall; (4) myocardial perfusion imaging of single-photon emission computed tomography (SPECT) suggesting perfusion defects and/or metabolic/perfusion mismatch of the left ventricular lateral wall  and cardiac magnetic resonance imaging (MRI) were performed. Coronary angiography data were analyzed by a specialist who did not know the grouping, and computerized quantitative analysis (QCA) was made according to the standard method on the minimum lumen diameter of the vessel proximal to the constrictor ring, reference lumen diameter and diameter stenosis, while the collateral branches of coronary arteries were classified according to the Rentrop collateral circulation classification method. The data of myocardial radionuclide scans were analyzed by ECTb 3.0 software to determine the defect area of the left ventricular myocardial perfusion and viable myocardium. The percentage of myocardial perfusion defect area of the left ventricle (%) = , lactic dehydrogenase (LDH) , TNF-α , cardiac troponin T (cTnT)  and type B natriuretic peptide (BNP)  were determined by ELISA.Briefly, capture antibody was 1:200 diluted, added to a 96-well plate at 100 µL/well and coated overnight at 4°C. After capture antibody was shaken off, the plate was washed with 1× washing buffer (100 µL/well) four times, 1 min each time and dried on absorbent paper the last time. After blocking with 100 µL/well of blocking solution for 2 h at room temperature, the plate was washed four times with 1× washing buffer (100 µL/well), 1 min each time and dried on absorbent paper the last time. The standard was diluted into six concentrations, and each concentration had one replicate. Then, the blocking solution was shaken off, and the plate was washed twice. Blank well was added 100 µL of diluent, standard wells were added standards of each concentration, and 100 μL of supernatant was added to sample wells. Afterward, 100 µL/well of detection antibody was added to each well and incubated at room temperature for 2 h. Then, the liquid was shaken off, and the plate was washed four times. Subsequently, 100 µL/well of reaction solution was added, incubated at room temperature for 10 min and sealed with parafilm. Then, the liquid was shaken off, and the plate was washed four times. The optical density of each group was detected by a microplate reader 40 min after the color development reagent at 100 µL/well was added. Standard curves were thereafter plotted to calculate the corresponding concentrations.2.5.3After 2 weeks of G-CSF treatment, all rats were executed to separate the left anterior descending branch, LCX branch and their corresponding area of donor myocardium. HE staining was performed for the myocardium, liver, lung, kidney, spleen and lymph nodes.Paraffin sections were baked in a 60°C oven for 1 h, deparaffinized with xylene I and II solutions for 10 min each, hydrated with series concentrations of ethanol solutions until water and washed with PBS for 3 min. Afterward, they were stained with hematoxylin for 10 min, rinsed with tap water for 10 min, differentiated with 1% HCl-ethanol solution for 10 s, rinsed with tap water for 10 min, stained with eosin for 3 min and rinsed with tap water and distilled water for 30 s each. Subsequently, the sections were dehydrated with series concentrations of ethanol solutions, transparentized with xylene I and II solutions for 10 min each, mounted with neutral resin, observed under a microscope and analyzed with Image-Pro Plus 6.0 software.2.5.4TUNEL assay was used to detect the apoptosis of myocardial cells in the infarcted marginal area, local neovascularization and expression levels of factors related to apoptosis and angiogenesis.The sections were baked in a 60°C oven for 10 min, deparaffinized by using xylene twice, hydrated with series concentrations of ethanol solutions until water and washed with PBS three times, 1 min each time. After antigen retrieval by using 200 mL of 0.1 M citrate buffer (pH 6.0), the sections were blocked at room temperature for 10 min and washed with PBS three times, 1 min each time. Then, the TUNEL solution comprising 45 μL of label solution and 5 μL of enzyme solution was dropped onto the sections (50 μL each) for incubation at 37°C for 60 min. Subsequently, the sections were washed with PBS twice, 2 min each time, and the cell nuclei were stained with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution at room temperature. The liquid was shaken off, and the sections were washed with PBS, dropped antiquenching reagent, covered with coverslips and observed under a fluorescence microscope.2.5.5n = 3). Proteins were separated with 10% SDS-PAGE, and the products were transferred to a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% skim milk overnight at 4°C, incubated with primary antibody against vWF (1:500) overnight at 4°C and then with horseradish peroxidase (HRP)-conjugated secondary antibody  at room temperature for 2 h and exposed to a gel imaging system. Finally, the grayscale values of protein bands were analyzed.The expression level of vWF protein at the marginal area of infarcted myocardium was detected by Western blotting as follows . A small2.6t test was carried out for the same continuous variable at the beginning and after follow-up. Categorical variables were compared with the χ2 test, and Fisher’s exact test was performed when the sample size was small. P < 0.05 was considered statistically significant.All data were statistically analyzed by SPSS 13.0 software. Continuous variables were expressed as (mean ± standard deviation). The comparisons of numerical variables between groups were conducted by analysis of variance. For pairwise comparisons, the Bonferroni method was used in the case of homogeneity of variance. If there was no homogeneity of variance, Dunnett’s method was employed. The paired 33.1No death occurred during the establishment of the CIHD model. After randomization, no adverse events such as death, myocardial infarction, infection and heart failure occurred.3.2P > 0.05]  pg/mL vs (392.4 ± 80.4) pg/mL,  > 0.05] .3.3P > 0.05) %, (18.2 ± 3.1)% and (21.6 ± 3.3)%, respectively. The CD34+/CD133+ cell ratios were (1.3 ± 0.3)%, (17.8 ± 2.9)% and (20.9 ± 2.8)%, respectively, suggesting that different routes of G-CSF injection increased such ratios after 2 weeks. However, there was no significant difference between the two treatment groups ( > 0.05) .3.4P > 0.05]. In the subcutaneous G-CSF injection group and intracoronary G-CSF injection group, the stenosis degree of LCX artery significantly decreased . However, the difference between the two treatment groups was not statistically significant (P > 0.05). Compared with the postoperative 2nd week, the degree of proximal vascular stenosis decreased in the subcutaneous G-CSF injection group and intracoronary G-CSF injection group in the postoperative 4th week (P < 0.05). Also, there was no significant difference between the two treatment groups. Eight weeks after surgery, no collateral vessel was formed in the control group, and one animal in the subcutaneous G-CSF injection group and intracoronary G-CSF injection group each had the vessels % vs (25.4 ± 2.3)%,  vessels .3.5P < 0.01) . Besides, although the difference in LVEDV between the intracoronary G-CSF injection group and the subcutaneous G-CSF injection group was not statistically significant 4 weeks after surgery, the changes exceeded that of the control group (P < 0.05). The LVESV reduction of the intracoronary G-CSF injection group and the subcutaneous G-CSF injection group exceeded that of the control group, but there was no significant difference between the former two groups, suggesting that both administration routes can improve LVEF of CIHD rats and inhibit left ventricular remodeling. Additionally, the myocardial perfusion defect area and myocardial infarct size of the intracoronary G-CSF injection group and subcutaneous G-CSF injection group were also smaller than those of the control group 8 weeks after surgery (P < 0.05), but the difference between the former two groups was not statistically significant (P < 0.05)  . The impnificant . The per < 0.05) .3.6P < 0.05), but the level of the control group hardly changed. Compared with 2 weeks after surgery, the level of LDH in the control group increased significantly 4 weeks after surgery whereas decreased significantly after G-CSF treatment. The level of LDH in the G-CSF treatment group 4 weeks after surgery was significantly lower than that of the control group. After mobilization of G-CSF, the levels of TNF-α were significantly higher than those in the postoperative 2nd week, but the three groups had similar levels 4 weeks after surgery. In the postoperative 2nd week, the levels of cTnT in the peripheral blood of rats treated with G-CSF were lower than those before treatment . The TNF-α levels in 30 rats were significantly positively correlated with the degree of myocardial damage . There was no significant difference between the former two groups. Myocardial HE staining showed that intracoronary injection of G-CSF also relieved myocardial necrosis , suggesting that the number of new vessels in the infarct border area increased after G-CSF mobilization, but the two treatment groups had similar vWF expression levels .The expression levels of vWF protein and the corresponding mRNA in the infarcted area were determined by Western blotting and RT-PCR, respectively. The levels of vWF protein and the corresponding mRNA in the control group were very low 4 weeks after surgery, whereas those of the subcutaneous G-CSF injection group and the intracoronary G-CSF injection group significantly increased (n levels . The vWF3.9P < 0.05), but the numbers of the former two groups were similar. DNA ladder marker was used to identify the amplified bands at 223, 327 and 597 bp, respectively. The band positions were consistent with the theoretical ones of Bax, Bcl-2 and GAPDH. In the postoperative 8th week, the expression levels of Bcl-2 mRNA in the subcutaneous G-CSF injection group and intracoronary G-CSF injection group were higher than that of the control group, whereas the Bax mRNA expression levels of the former two groups were lower (P < 0.05). The two G-CSF treatment groups had similar Bax and Bcl-2 mRNA expression levels (The TUNEL assay showed that the numbers of apoptotic cardiomyocytes in the infarct border zone of subcutaneous G-CSF injection group and intracoronary G-CSF injection group were lower than that of the control group 4 weeks after surgery (n levels .4Death from ischemic heart disease accounts for 50% of all deaths from heart disease and 40% of all deaths from heart failure . At presG-CSF is a powerful mobilizing agent for bone marrow HSCs, which can simultaneously mobilize bone marrow HSCs, mesenchymal stem cells and endothelial stem cells to release them into the peripheral blood . G-CSF cG-CSF can cause the mobilization of bone marrow stem cells including EPC to the peripheral blood . This st5Coronary G-CSF injection did not promote the proliferation of distant organ cells or the increase of neovascularization, and no adverse reactions such as death, myocardial infarction and heart failure caused by coronary injection were found, which preliminarily proved that the method was safe and feasible. Meanwhile, MRI and SPECT confirmed that the effect of G-CSF injection via the coronary artery was similar to that of subcutaneous injection in improving the cardiac function. In addition, the dose of G-CSF was reduced, which suggests that, it is possible to mitigate the adverse reactions caused by the considerable use of G-CSF. Regardless, the number of animals in this study is not large, so the findings should be further confirmed by experiments using more animals and clinical studies.