Effect of hydrogen peroxide on human tendon allograft.

Bacterial contamination of tendon allografts at the completion of processing has historically been about 2 %, with tendons that are found to be culture positive being discarded. Treatment of tendon allograft with hydrogen peroxide at the beginning of tissue processing may reduce bacterial contamination, however, the potential side effects of hydrogen peroxide treatment include hydrolysis of the collagen and this may alter the mechanical properties of the graft. Pairs of human tendons were used. One was washed in 3 % hydrogen peroxide for 5 min and the untreated tendon was used as a control. The ultimate tensile strength of the tendons was determined using a material testing machine. A freeze clamp technique was used to hold the tendons securely at the high loads required to cause tendon failure. There was no statistical difference in the ultimate tensile strength between the treated and untreated tendons. Mean strength ranged from Extensor Hallucis Longus at 588 Newtons to Tibialis Posterior at 2,366 Newtons. Hydrogen peroxide washing may reduce bacterial contamination of tendon allograft and does not affect the strength of the tendon.

Airway management for infants with severe micrognathia having mandibular distraction osteogenesis.

We retrospectively reviewed the charts of seven neonates and infants with severe micrognathia and upper airway obstruction who underwent mandibular distraction osteogenesis as an alternative to long-term tracheostomy at the Children's Hospital at Westmead, Sydney, from 2004 to 2007. All patients required a variety of airway interventions at an early age and had poor feeding or failure to thrive. These children had other anomalies and required many investigations and procedures and presented repeated airway challenges for the anaesthetist. The mean number of anaesthetics per child in this series was 6.7 (range 4 to 13). Under anaesthesia, four patients had grade 4 laryngoscopy, one was grade 3 but two were only grade 2. Patients with a preoperative grade 4 laryngoscopy were very likely to need fibreoptic endotracheal intubation. Duration of intubation after surgery for mandibular distraction was a mean of 8.17 days (range 1 to 19). Three were extubated in the operating theatre and three in intensive care. Five patients had an improved laryngoscopy grade after completion of mandibular distraction and one remained grade four The remaining patient had a tracheostomy from birth.

High anion gap metabolic acidosis secondary to pyroglutamic aciduria (5-oxoprolinuria): association with prescription drugs and malnutrition.

Two cases of High Anion Gap Metabolic Acidosis (HAGMA) due to pyroglutamic acid (5-oxoproline) are described. In both cases the HAGMA developed during an episode of hospital treatment, in conjunction with paracetamol and antibiotic prescription, and the surviving patient made an uneventful recovery after the drugs were withdrawn. Clinicians need to be aware of this cause for metabolic acidosis because it may be a more common metabolic disturbance in compromised patients than would be expected, and the discontinuation of drugs implicated in the aetiology is therapeutic.

Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines.

The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.

Purification of a pluripotent neural stem cell from the adult mouse brain.

The adult mammalian central nervous system (CNS) contains a population of neural stem cells (NSCs) with properties said to include the generation of non-neural progeny. However, the precise identity, location and potential of the NSC in situ remain unclear. We purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties. Here we show that one type of NSC, which expresses the protein nestin but only low levels of PNA-binding and HSA proteins, is found in both ependymal and subventricular zones and accounts for about 63% of the total NSC activity. Furthermore, the selective depletion of the population of this stem cell in querkopf mutant mice (which are deficient in production of olfactory neurons) suggests that it acts as a major functional stem cell in vivo. Most freshly isolated NSCs, when co-cultured with a muscle cell line, rapidly differentiated in vitro into myocytes that contain myosin heavy chain (MyHC). This demonstrates that a predominant, functional type of stem cell exists in the periventricular region of the adult brain with the intrinsic ability to generate neural and non-neural cells.

Controlled hypertension, a transgenic toggle switch reveals differential mechanisms underlying vascular disease.

A novel inbred rat model with inducible hypertension has been generated using a renin transgene under the transcriptional control of the cytochrome P450, Cyp1a1 promoter. The degree and duration of hypertension are regulated tightly by administration of the natural xenobiotic indole-3 carbinol and can be readily reversed. Induction experiments reveal distinct temporal and mechanistic responses to hypertensive injury in different vascular beds, which is indicative of differential susceptibility of organs to a hypertensive stimulus. The mesentery and heart exhibited the greatest sensitivity to damage, and the kidney showed an adaptive response prior to the development of malignant hypertensive injury. Quantitative analysis of morphological changes induced in mesenteric resistance arteries suggest eutrophic remodeling of the vessels. Kinetic evidence suggests that locally activated plasma prorenin may play a critical role in mediating vascular injury. This model will facilitate studies of the cellular and genetic mechanisms underlying vascular injury and repair and provide a basis for the identification of novel therapeutic targets for vascular disease.

Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity.

Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.

Endogenous IGF-1 regulates the neuronal differentiation of adult stem cells.

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, 20-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like growth factor (IGF)-1 or heparin were added subsequent to FGF-2 removal. The spontaneous production of neurons after FGF-2 removal was shown to be due to endogenous IGF-1, since antibodies to IGF-1 and an IGF-1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron-promoting effects of heparin. Thus, it appears that endogenous IGF-1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF-1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated.

Regulation of neural stem cell differentiation in the forebrain.

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR). The evidence suggests that in the absence of LIFR signalling, the stem cell population is present at approximately the same frequency and can generate neurons but is blocked from producing astrocytes that express glial fibrillary acidic protein (GFAP) or have trophic functions. The block can be overcome by other growth factors such as BMP-2/4 or interferon-gamma, providing further evidence that the inhibition to astrocyte development does not result from loss of a precursor population. Signalling through the LIFR, in addition to stimulating astrocyte differentiation, may also inhibit neuronal differentiation, which may explain why this receptor is expressed at the earliest stages of neurogenesis. Another signalling system which also exerts its influence on neurogenesis through active inhibition is Delta-Notch. We show in vitro that at high cell densities which impede neuronal production by FGF-1, lowering the levels of expression of the receptor Notch by antisense oligonucleotide results in a significant increase in neuronal production. Thus, stem cell differentiation appears to be dependent on the outcome of interactions between a number of signalling pathways, some which promote specific lineages and some which inhibit.

Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA receptor function in cerebellar granule cells.

Evidence has accumulated to suggest that the NMDA glutamate receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in culture are vulnerable to NMDA-induced neuronal excitotoxicity. In these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA. However, little is known about the molecular mechanisms underlying the protective properties of these trophic factors. Using cultured rat cerebellar granule cells, we investigated whether BDNF and FGF2 prevent NMDA toxicity by downregulating NMDA receptor function. Western blot and RNase protection analyses were used to determine the expression of the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent decrease in the expression of NR2A and NR2C subunits. Because NMDA receptor activation leads to increased intracellular Ca2+ concentration ([Ca2+]i), we studied the effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C synthesis on the NMDA-evoked Ca2+ responses by single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced the NMDA-mediated [Ca2+]i increase with a time dependency that correlates with their ability to decrease NR2A and NR2C subunit expression, suggesting that these trophic factors also induce a functional downregulation of the NMDA receptor. Because sustained [Ca2+]i is believed to be causally related to neuronal injury, we suggest that BDNF and FGF2 may protect cerebellar granule cells against excitotoxicity by altering the NMDA receptor-Ca2+ signaling via a downregulation of NMDA receptor subunit expression.

Neural stem cells.

This article is concerned with the idea that neural precursor cells in vertebrates can self-renew and give rise to all cell types within the nervous system. Supportive evidence for this notion of neural stem cells comes from clonal analyses undertaken both in vivo and in vitro. Neural stem cells also give rise to other cells in the body, including skin melanocytes and a range of mesenchymal cells in the head and neck. What determines the fate of these stem cells is their initial location within the developing neural tube and their final location post migration from the proliferative zone of the neural tube. A population of cells in the adult brain also have the characteristics of classical stem cells, a finding that opens the way for potential replacement therapy in nervous system-degenerative diseases. Much of the work in our laboratory has been concerned with the regulation of expansion and differentiation of these cells into their myriad progeny and the role of a series of various growth factors in this process. Different factors, such as members of the fibroblast growth factor family, act at different times to regulate stem cell proliferation and differentiation. Some factors, including members of the TGF beta superfamily, appear to be directly involved in the specification of cell fate. Finally, we are beginning to be able to determine the steps in the development of some lineages from multipotential stem cell to fully functional differentiated cell.

Fate of adult neural precursors grafted to adult cortex monitored with a Y-chromosome marker.

Freshly dissected and dissociated subventricular zone cells from adult male mice were transplanted into the right cerebral cortex of adult female mice. Host mice were perfused 3 or 7 weeks later. To detect donor male cells, paraffin sections (10 microm) were hybridized in situ with a Y-chromosome-specific probe. Some hybridized sections were also immunostained with antibodies against glial fibrillary acidic protein (GFAP), NeuN or beta-tubulin. Grafted cells were identified in 80% of host female brains; they were found in both grey and white matter and were located close to the initial injection site. Double-labelling studies showed that, of the surviving cells containing hybridized male nuclei, 5-10% expressed a glial (GFAP) marker and 6-7% expressed neuronal (NeuN or beta-tubulin) markers.

Targeted inactivation of the Ren-2 gene in mice.

Several recent studies have demonstrated that ablation of genes of the renin-angiotensin system can have wide-ranging and sometimes unexpected effects. Renin is directly involved in blood pressure regulation and is encoded by a single gene in most mammals. Wild mouse strains and some inbred laboratory strains have a duplicated renin gene (Ren-2), the physiological significance of which is unclear. Significant differences exist in the structure and expression of these renin genes, but as yet, no distinct biological function that distinguishes these genes has been defined. We have used gene targeting to discover the effects of inactivating the duplicated (Ren-2) gene in strain 129 mice, and we show that mice lacking the Ren-2 gene are viable and healthy. There appear to be no histopathological differences in renin-expressing tissues between Ren-2-null mice and their controls. Studies of our Ren-2-null mice allow, for the first time, a direct evaluation of the ability of the Ren-1d gene to regulate blood pressure in the absence of expression of the Ren-2 enzyme. We observed no alteration to blood pressure in adult mice homozygous for the mutated Ren-2 gene, even though the concentration of active renin is increased and of prorenin is decreased in plasma of these mice. Ren-1d is therefore capable of regulating normal blood pressure and despite a different tissue expression profile, is functionally equivalent to Ren-1c.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

TrkA mediates the nerve growth factor-induced intracellular calcium accumulation.

Regulation of the cytosolic free Ca2+ concentration by nerve growth factor was investigated in C6-2B glioma cells newly expressing the high affinity nerve growth factor receptor trkA, using Fura-2 fluorescence ratio imaging. In these cells, nerve growth factor (50 ng/ml) evoked a novel approximately 3-fold increase in cytosolic free Ca2+ concentration, while no measurable Ca2+ response was observed in wild type or mock-transfected cells lacking a functional trkA receptor. K-252a, a tyrosine kinase inhibitor which prevents nerve growth factor-mediated responses in C6-2B cells expressing trkA, also blocked the rise in cytosolic free Ca2+ concentration by nerve growth factor. Moreover, basic fibroblast growth factor, which in these cells elicits biochemical changes similar to nerve growth factor, failed to affect cytosolic free Ca2+ concentration, further supporting the specificity of nerve growth factor/trkA receptor in mediating a Ca2+ response. While insensitive to chelation of extracellular Ca2+, the response was abolished following depletion of Ca2+ stores or blockade of intracellular Ca2+ release, providing strong evidence that intracellular Ca2+ is the main source for nerve growth factor-evoked cytosolic free Ca2+ concentration increase. Nerve growth factor increased the cytosolic free Ca2+ concentration also in NIH3T3 cells overexpressing trkA but devoid of p75 nerve growth factor receptor. Our data suggest that trkA but not p75 is required for nerve growth factor-evoked Ca2+ signaling.

Rat Sertoli cell calcium response to basement membrane and follicle-stimulating hormone.

Sertoli cells cultured on basement membrane substrates differentiate morphologically into polarized cells and exhibit an enhanced responsiveness to FSH. The signal transduction mechanisms by which the extracellular matrix induces changes in the morphology and function of Sertoli cells are not known. Since calcium has been implicated in mediating changes in cytoskeletal assembly and organization, we investigated to see if basement membrane can modulate cytosolic free calcium concentrations during the process of adhesion and spreading of Sertoli cells. A direct quantification of the intracellular free cytosolic calcium concentration [Ca2+]i in freshly isolated immature rat Sertoli cells plated on laminin was performed by digital imaging microscopy using the fluorescent probe Fura-2 AM. [Ca2+]i levels rose by 1.5-2-fold within 1 h after plating on laminin, suggesting that calcium may be involved in adhesion and spreading of the cells on basement membrane. Furthermore, the possibility that matrix influences [Ca2+]i levels upon stimulation with FSH was examined by adding FSH directly to the cells spreading on laminin. A dramatic decrease in [Ca2+]i was observed compared to the level in untreated cells. Similarly, a significant decrease in [Ca2+]i in response to FSH was observed in cells already spread on laminin or Matrigel. Addition of dibutyryl cAMP did not significantly alter the basal calcium levels. Long-term exposure of Sertoli cells cultured on either laminin or Matrigel to FSH was studied by incubating the cells with 45CaCl2 in the presence or absence of FSH for 24 h. FSH induced a decrease or no change in 45Ca concentration in cells cultured on basement membrane. Addition of dibutyryl cAMP, instead of FSH, did not alter the basal 45Ca concentrations. In cells cultured on the peptides derived from laminin (RGD and SIKVAV), FSH increased the uptake of 45Ca significantly, whereas on YIGSR, also a laminin-derived peptide, it did not have any effect. Thus, basement membrane induces an early increase in [Ca2+]i in cultured Sertoli cells during spreading, and FSH appears to significantly decrease [Ca2+]i levels.

Regulation of neural precursor differentiation in the embryonic and adult forebrain.

1. Precursors form the neuroepithelium of the developing cortex and also from the adult sub-ventricular zone, can be cloned in vitro after stimulation with fibroblast growth factor (FGF)-2 and have the potential to give rise to both neurons and glia. The generation of neurons from these clones can be stimulated by either a factor derived from an astrocyteprecursor line, Ast-1, or FGF-1. 2. Neuronal differentiation stimulated by FGF-1 can be inhibited by diacylglycerol-lipase inhibitor and mimicked by arachidonic acid, suggesting that the neuronal differentiation is signalled through the PCL gamma pathway. 3. The sequential expression of FGF-2 and FGF-1 within the developing forebrain neuroepithelium fits with the different functions the two FGF play in precursor regulation. 4. We have shown that the precursor response to FGF-1 is regulated by a heparan sulphate proteoglycan (HSPG) expressed within the developing neuroepithelium. Precursors restricted to the astrocyte cell lineage can be stimulated by epidermal growth factor or FGF-2; however, the differentiation into GFAP positive astrocytes appears to require a cytokine acting through the leukaemia inhibitory factor beta receptor.