Noninvasive Follow-up Imaging of Ruptured Pediatric Brain AVMs Using Arterial Spin-Labeling.

BACKGROUND AND PURPOSE: Brain AVMs represent the main etiology of pediatric intracranial hemorrhage. Noninvasive imaging techniques to monitor the treatment effect of brain AVMs remain an unmet need. In a large cohort of pediatric ruptured brain AVMs, we aimed to investigate the role of arterial spin-labeling for the longitudinal follow-up during treatment and after complete obliteration by analyzing CBF variations across treatment sessions. MATERIALS AND METHODS: Consecutive patients with ruptured brain AVMs referred to a pediatric quaternary care center were prospectively included in a registry that was retrospectively queried for children treated between 2011 and 2019 with unimodal or multimodal treatment (surgery, radiosurgery, embolization). We included children who underwent an arterial spin-labeling sequence before and after treatment and a follow-up DSA. CBF variations were analyzed in univariable analyses. RESULTS: Fifty-nine children with 105 distinct treatment sessions were included. The median CBF variation after treatment was -43 mL/100 mg/min (interquartile range, -102-5.5), significantly lower after complete nidal surgical resection. Following radiosurgery, patients who were healed on the last DSA follow-up demonstrated a greater CBF decrease on intercurrent MR imaging, compared with patients with a persisting shunt at last follow-up (mean, -62 [SD, 61] mL/100 mg/min versus -17 [SD, 40.1] mL/100 mg/min; P = .02). In children with obliterated AVMs, recurrences occurred in 12% and resulted in a constant increase in CBF (mean, +89 [SD, 77] mL/100 mg/min). CONCLUSIONS: Our results contribute data on the role of noninvasive arterial spin-labeling monitoring of the response to treatment or follow-up after obliteration of pediatric AVMs. Future research may help to better delineate how arterial spin-labeling can assist in decisions regarding the optimal timing for DSA.

Pre-activated antiviral innate immunity in the upper airways controls early SARS-CoV-2 infection in children.

Children have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates and a substantially lower risk for developing severe coronavirus disease 2019 compared with adults. However, the molecular mechanisms underlying protection in younger age groups remain unknown. Here we characterize the single-cell transcriptional landscape in the upper airways of SARS-CoV-2-negative (n = 18) and age-matched SARS-CoV-2-positive (n = 24) children and corresponding samples from adults (n = 44), covering an age range of 4 weeks to 77 years. Children displayed higher basal expression of relevant pattern recognition receptors such as MDA5 (IFIH1) and RIG-I (DDX58) in upper airway epithelial cells, macrophages and dendritic cells, resulting in stronger innate antiviral responses upon SARS-CoV-2 infection than in adults. We further detected distinct immune cell subpopulations including KLRC1 (NKG2A)+ cytotoxic T cells and a CD8+ T cell population with a memory phenotype occurring predominantly in children. Our study provides evidence that the airway immune cells of children are primed for virus sensing, resulting in a stronger early innate antiviral response to SARS-CoV-2 infection than in adults.

TROPHY registry - status report.

INTRODUCTION: The TROPHY registry has been established to conduct an international multicenter prospective data collection on the surgical management of neonatal intraventricular hemorrhage (IVH)-related hydrocephalus to possibly contribute to future guidelines. The registry allows comparing the techniques established to treat hydrocephalus, such as external ventricular drainage (EVD), ventricular access device (VAD), ventricular subgaleal shunt (VSGS), and neuroendoscopic lavage (NEL). This first status report of the registry presents the results of the standard of care survey of participating centers assessed upon online registration. METHODS: On the standard of treatment forms, each center indicated the institutional protocol of interventions performed for neonatal post-hemorrhagic hydrocephalus (nPHH) for a time period of 2 years (Y1 and Y2) before starting the active participation in the registry. In addition, the amount of patients enrolled so far and allocated to a treatment approach are reported. RESULTS: According to the standard of treatment forms completed by 56 registered centers, fewer EVDs (Y1 55% Y2 46%) were used while more centers have implemented NEL (Y1 39%; Y2 52%) to treat nPHH. VAD (Y1 66%; Y2 66%) and VSGS (Y1 42%; Y2 41%) were used at a consistent rate during the 2 years. The majority of the centers used at least two different techniques to treat nPHH (43%), while 27% used only one technique, 21% used three, and 7% used even four different techniques. Patient data of 110 infants treated surgically between 9/2018 and 2/2021 (13% EVD, 15% VAD, 30% VSGS, and 43% NEL) were contributed by 29 centers. CONCLUSIONS: Our results emphasize the varying strategies used for the treatment of nPHH. The international TROPHY registry has entered into a phase of growing patient recruitment. Further evaluation will be performed and published according to the registry protocol.

Risk Factors for Early Brain AVM Rupture: Cohort Study of Pediatric and Adult Patients.

BACKGROUND AND PURPOSE: Whether architectural characteristics of ruptured brain AVMs vary across the life span is unknown. We aimed to identify angioarchitectural features associated with brain AVMs ruptured early in life. MATERIALS AND METHODS: Patients with ruptured brain AVMs referred to 2 distinct academic centers between 2000 and 2018 were pooled and retrospectively analyzed. Imaging was retrospectively reviewed for angioarchitectural characteristics, including nidus size, location, Spetzler-Martin grade, venous drainage, and arterial or nidal aneurysm. Angioarchitecture variations across age groups were analyzed using uni- and multivariable models; then cohorts were pooled and analyzed using Kaplan-Meier and Cox models to determine factors associated with earlier rupture. RESULTS: Among 320 included patients, 122 children (mean age, 9.8 ± 3.8 years) and 198 adults (mean age, 43.3 ± 15.7 years) were analyzed. Pediatric brain AVMs were more frequently deeply located (56.3% versus 21.2%, P < .001), with a larger nidus (24.2 versus 18.9 mm, P = .002), were less frequently nidal (15.9% versus 23.5%, P = .03) and arterial aneurysms (2.7% versus 17.9%, P < .001), and had similar drainage patterns or Spetzler-Martin grades. In the fully adjusted Cox model, supratentorial, deep brain AVM locations (adjusted relative risk, 1.19; 95% CI, 1.01-1.41; P = .03 and adjusted relative risk, 1.43; 95% CI, 1.22-1.67; P < .001, respectively) and exclusively deep venous drainage (adjusted relative risk, 1.46, 95% CI, 1.21-1.76; P < .001) were associated with earlier rupture, whereas arterial or nidal aneurysms were associated with rupture later in life. CONCLUSIONS: The angioarchitecture of ruptured brain AVMs significantly varies across the life span. These distinct features may help to guide treatment decisions for patients with unruptured AVMs.

Impairment of Sox9 expression in limb buds of rats homozygous for hypodactyly mutation.

Rat hypodactyly (hd) is an autosomal recessive mutation manifesting in homozygotes as reduction or loss of digits II and III. We mapped the hd allele to a short segment of chromosome 10, containing 16 genes. None of these genes has been shown to influence limb development yet. In situ hybridization showed no changes in several important patterning genes (Shh, Fgf8, Bmp2, 4, 7). However, we found that expression of cartilage condensation marker Sox9, and Bmp receptor Bmpr1b (acting as an upstream activator of Sox9 expression) is absent from the subepithelial mesenchyme of the digit condensations II and III. The failure of the chondrogenic condensations to extend towards the subepithelial mesenchyme may reduce the size of digit primordia and underlie the subsequent loss of phalanges and reduction of metacarpals/metatarsals in hd rats.

Regulation of long-term depression by increases in [guanosine 3',5'-cyclic monophosphate] in the hippocampal CA1 region of freely behaving rats.

A role for guanosine 3',5'-cyclic monophosphate (cGMP) and the protein kinase G (PKG) pathway in synaptic long-term depression (LTD) in the hippocampal CA1 region has been proposed, based on observations in vitro, where, for example, increases of [cGMP] result in short-term depression (STD) coupled with a reduction in presynaptic glutamate release. To date, no evidence exists to support that LTD in the intact, freely behaving animal involves these mechanisms. We examined the effect of increases of [cGMP] on basal transmission and electrically-induced STD at hippocampal CA1 synapses in vivo. We found that elevating [cGMP] dose-dependently caused a chemically-induced STD which occluded electrically-induced STD. Repeated administration of Zaprinast, an inhibitor of cGMP-degrading phosphodiesterase, resulted in persistent LTD (>24 h). Paired-pulse analysis supported a presynaptic mechanism of action. Application of an inhibitor of soluble guanylate cyclase prevented LTD induced by low-frequency stimulation (LFS), and impaired LFS-STD elicited in the presence of Zaprinast. These data suggest the involvement of cGMP in LTD in the CA1 region of freely behaving adult rats.

Triangular tibia with fibular aplasia associated with a microdeletion on 2q11.2 encompassing LAF4.

Nievergelt syndrome (NS) is an autosomal dominant mesomelic dysplasia characterized by specific deformities of the radius, ulna, fibula and a rhomboid shape of the tibia. Phenotypically overlapping conditions such as mesomelic dysplasia, Savarirayan-type (MIM 605274), have been described, but their pathogenesis also remains unknown. We report on a girl with fibular agenesis, severely abnormal, triangular tibiae, urogenital tract malformations, failure to thrive, convulsions and recurrent apnoeas leading to respiratory arrest at the age of 4 months. Her skeletal findings correspond to those of the mesomelic dysplasia, Savarirayan-type recently described in two patients. In addition to the skeletal findings, our patient had central nervous system manifestations and developmental anomalies of the urogenital tract. In the patient described in this study, array comparative genomic hybridization (CGH) analysis revealed a de novo interstitial microdeletion of 500 kb on chromosome 2q11.1 containing the LAF4/AFF3 (lymphoid-nuclear-protein-related AF4) gene. In situ hybridization analysis of Laf4 in mouse embryos revealed expression in the developing brain, in the limb buds and in the zeugopod corresponding to the limb phenotype. Haploinsufficiency for LAF4/AFF3 is associated with limb, brain and urogenital malformations and specific changes of the tibia that are part of the NS spectrum.

Ataxia with oculomotor apraxia type 2: novel mutations in six patients with juvenile age of onset and elevated serum alpha-fetoprotein.

Ataxia with oculomotor apraxia type 2 (AOA2), a neurodegenerative disorder with juvenile to adolescent onset is caused by mutations within the SENATAXIN gene ( SETX). We performed molecular analyses in six patients showing clinically an AOA2 phenotype and moderate to significant elevated serum alpha-fetoprotein levels. Sequencing the 24 coding exons and flanking intronic sequences revealed 11 novel DNA variations, including seven unknown missense mutations, a dinucleotide deletion, a four-nucleotide deletion affecting the 5' splice site of exon 22 and two sequence variations, which are considered to be polymorphisms. By molecular testing the clinical diagnosis has been confirmed in all patients.

Detection of novel skeletogenesis target genes by comprehensive analysis of a Runx2(-/-) mouse model.

Runx2 is an essential factor for skeletogenesis and heterozygous loss causes cleidocranial dysplasia in humans and a corresponding phenotype in the mouse. Homozygous Runx2-deficient mice lack hypertrophic cartilage and bone. We compared the expression profiles of E14.5 wildtype and Runx2(-/-) murine embryonal humeri to identify new transcripts potentially involved in cartilage and bone development. Seventy-one differentially expressed genes were identified by two independent oligonucleotide-microarray hybridizations and quantitative RT-PCR experiments. Gene Ontology analysis demonstrated an enrichment of the differentially regulated genes in annotations to terms such as extracellular, skeletal development, and ossification. In situ hybridization on E15.5 limb sections was performed for all 71 differentially regulated genes. For 54 genes conclusive in situ hybridization results were obtained and all of them showed skeletal expression. Co-expression with Runx2 was demonstrated for 44 genes. While 41 of the 71 differentially expressed genes have a known role in bone and cartilage, we identified 21 known genes that have not yet been implicated in skeletal development and 9 entirely new transcripts. Expression in the developing skeleton was demonstrated for 21 of these genes.

Mutations in WNT7A cause a range of limb malformations, including Fuhrmann syndrome and Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome.

Fuhrmann syndrome and the Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome are considered to be distinct limb-malformation disorders characterized by various degrees of limb aplasia/hypoplasia and joint dysplasia in humans. In families with these syndromes, we found homozygous missense mutations in the dorsoventral-patterning gene WNT7A and confirmed their functional significance in retroviral-mediated transfection of chicken mesenchyme cell cultures and developing limbs. The results suggest that a partial loss of WNT7A function causes Fuhrmann syndrome (and a phenotype similar to mouse Wnt7a knockout), whereas the more-severe limb truncation phenotypes observed in Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome result from null mutations (and cause a phenotype similar to mouse Shh knockout). These findings illustrate the specific and conserved importance of WNT7A in multiple aspects of vertebrate limb development.

Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms.

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.

A single amphioxus and sea urchin runt-gene suggests that runt-gene duplications occurred in early chordate evolution.

Runt-homologous molecules are characterized by their DNA binding runt-domain which is highly conserved within bilaterians. The three mammalian runt-genes are master regulators in cartilage/bone formation and hematopoiesis. Historically these features evolved in Craniota and might have been promoted by runt-gene duplication events. The purpose of this study was therefore to investigate how many runt-genes exist in the stem species of chordates, by analyzing the number of runt-genes in what is likely to be the closest living relative of Craniota-amphioxus. To acquire further insight into the possible role of runt-genes in early chordate evolution we have determined the number of runt-genes in sea urchins and have analyzed the runt-expression pattern in this species. Our findings demonstrate the presence of a single runt-gene in amphioxus and sea urchin, which makes it highly likely that the stem species of chordates harbored only a single runt-gene. This suggests that runt-gene duplications occurred later in chordate phylogeny, and are possibly also associated with the evolution of features such as hematopoiesis, cartilage and bone development. In sea urchin embryos runt-expression involves cells of endodermal, mesodermal and ectodermal origin. This complex pattern of expression might reflect the multiple roles played by runt-genes in mammals. A strong runt-signal in the gastrointestinal tract of the sea urchin is in line with runt-expression in the intestine of nematodes and in the murine gastrointestinal tract, and seems to be one of the phylogenetically ancient runt-expression domains.

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms.

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

Injections of porcine sperm extracts trigger fertilization-like calcium oscillations in oocytes of a marine worm.

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.

Confocal laser scanning microscopy of calcium dynamics in living cells.

Confocal laser scanning microscopy (CLSM) is widely used to monitor intracellular calcium levels in living cells loaded with calcium-sensitive fluorophores. This review examines the basic advantages and limitations of CLSM in in vivo imaging analyses of calcium dynamics. The benefits of utilizing ratioed images and dextran-conjugated fluorophores are addressed, and practical aspects of handling confocal datasets are outlined. After considering some relatively new microscopical methods that can be used in conjunction with conventional CLSM, possible future applications of confocal techniques in analyses of intracellular calcium dynamics are discussed.