Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells

Long-term propagation of inner ear-derived progenitor/stem cells beyond the third generation and differentiation into inner ear cell types has been shown to be feasible, but challenging. We investigated whether the known neuroprotective guanidine compound creatine (Cr) promotes propagation of inner ear progenitor/stem cells as mitogen-expanded neurosphere cultures judged from the formation of spheres over passages. In addition, we studied whether Cr alone or in combination with brain-derived neurotrophic factor (BDNF) promotes neuronal differentiation of inner ear progenitors. For this purpose, early postnatal rat spiral ganglia, utricle, and organ of Corti-derived progenitors were grown as floating spheres in the absence (controls) or presence of Cr (5 mM) from passage 3 onward. Similarly, dissociated sphere-derived cultures were differentiated for 14 days in the presence or absence of Cr (5 mM) and spiral ganglia sphere-derived cultures in a combination of Cr with the neurotrophin BDNF (50 ng/ml). We found that the cumulative total number of spheres over all passages was significantly higher after Cr supplementation as compared with controls in all the three inner ear cultures. In contrast, sphere sizes were not affected by the administration of Cr. Administration of Cr during differentiation of spiral ganglia cells resulted in a significantly higher density of β-III-tubulin-positive cells compared with controls, whereas densities of myosin VIIa-positive cells in cultures of utricle and organ of Corti were not affected by the treatment. Importantly, a combination of Cr with the neurotrophin BDNF resulted in further significantly increased densities of β-III-tubulin-positive cells in cultures of spiral ganglia cells as compared with single treatments. In sum, Cr promoted continuing propagation of rat inner ear-derived progenitor cells and supported specifically in combination with BDNF the differentiation of neuronal cell types from spiral ganglion-derived spheres.

Type II DeBakey dissection with complete aortic rupture visualized by unenhanced postmortem imaging

We present postmortem computed tomography (pmCT) as well as postmortem magnetic resonance (pmMR) imaging findings in a case of type II DeBakey aortic dissection with a complete rupture of the ascending aorta compared to the findings obtained at forensic autopsy. PmCT only allowed a presumptive diagnosis of aortic dissection based on an anterior mediastinal enlargement. However, at pmMR the dissection including the aortic rupture was clearly visible. Visualization was realized in an unenhanced manner without the need for postmortem angiography.

Subcritical Propagation and Coalescence of Oil-Filled Cracks: Getting the Oil Out of Low-Permeability Source Rocks

We use a fracture mechanics model to study subcritical propagation and coalescence of single and collinear oil-filled cracks during conversion of kerogen to oil. The subcritical propagation distance, propagation duration, crack coalescence and excess oil pressure in the crack are determined using the fracture mechanics model together with the kinetics of kerogen-oil transformation. The propagation duration for the single crack is governed by the transformation kinetics whereas the propagation duration for the multiple collinear cracks may vary by two orders of magnitude depending on initial crack spacing. A large amount of kerogen (>90%) remains unconverted when the collinear cracks coalesce and the new, larger cracks resulting from coalescence will continue to propagate with continued kerogen-oil conversion. The excess oil pressure on the crack surfaces drops precipitously when the collinear cracks are about to coalesce, and crack propagation duration and oil pressure on the crack surfaces are strongly dependent on temperature. Citation: Jin, Z.-H., S. E. Johnson, and Z. Q. Fan (2010), Subcritical propagation and coalescence of oil-filled cracks: Getting the oil out of low-permeability source rocks, Geophys. Res. Lett., 37, L01305, doi:10.1029/2009GL041576.

Magma-Driven Multiple Dike Propagation and Fracture Toughness of Crustal Rocks

Dike swarms consisting of tens to thousands of subparallel dikes are commonly observed at Earth's surface, raising the possibility of simultaneous propagation of two or more dikes at various stages of a swarm's development. The behavior of multiple propagating dikes differs from that of a single dike owing to the interacting stress fields associated with each dike. We analyze an array of parallel, periodically spaced dikes that grow simultaneously from an overpressured source into a semi-infinite, linear elastic host rock. To simplify the analysis, we assume steady state (constant velocity) magma flow and dike propagation. We use a perturbation method to analyze the coupled, nonlinear problem of multiple dike propagation and magma transport. The stress intensity factor at the dike tips and the opening displacements of the dike surfaces are calculated. The numerical results show that dike spacing has a profound effect on the behavior of dike propagation. The stress intensity factors at the tips of parallel dikes decrease with a decrease in dike spacing and are significantly smaller than that for a single dike with the same length. The reduced stress intensity factor indicates that, compared to a single dike, propagation of parallel dikes is more likely to be arrested under otherwise the same conditions. It also implies that fracture toughness of the host rock in a high confining pressure environment may not be as high as inferred from the propagation of a single dike. Our numerical results suggest fracture toughness values on the order of 100 MPa root m. The opening displacements for parallel dikes are smaller than that for a single dike, which results in higher magma pressure gradients in parallel dikes and lower flux of magma transport.

Primary Oil Migration Through Buoyancy-Driven Multiple Fracture Propagation: Oil Velocity and Flux

We present a fracture-mechanics-based formulation to investigate primary oil migration through the propagation of an array of periodic, parallel fractures in a sedimentary rock with elevated pore fluid pressure. The rock is assumed to be a linearly elastic medium. The fracture propagation and hence oil migration velocity are determined using a fracture mechanics criterion together with the lubrication theory of fluid mechanics. We find that fracture interactions have profound effects on the primary oil migration behavior. For a given fracture length, the mass flux of oil migration decreases dramatically with an increase in fracture density. The reduced oil flux is due to the decreased fracture propagation velocity as well as the narrowed fracture opening that result from the fracture interactions.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.

The effects of doxycycline on nitric oxide and stromelysin production in dogs with cranial cruciate ligament rupture

OBJECTIVE To investigate the potential of doxycycline to reduce stromelysin and inducible nitric oxide synthase (iNOS) activity in dogs with osteoarthritis (OA) secondary to spontaneous cranial cruciate ligament (CCL) rupture. STUDY DESIGN Prospective, clinical study. ANIMALS Eighty-one dogs with OA secondary to CCL rupture and 54 normal dogs. METHODS Dogs with OA secondary to CCL rupture were divided into 2 groups before surgery. The Doxy-CCl group received 3 to 4 mg/kg doxycycline orally every 24 hours for 7 to 10 days (n = 35). The CCL group received no treatment (n = 46). Synovial fluid, articular cartilage, synovial membrane, and CCL samples were collected during surgery (Doxy-CCL group and CCL group) or immediately after euthanasia from healthy dogs (control group). Synovial fluid samples were examined cytologically. Total nitric oxide (NOt) concentrations were measured in the supernatant of explant cultures of all tissue samples, and stromelysin activity was measured in the supernatant of explant cultures of cartilage. RESULTS NOt concentrations measured in cartilage were significantly lower in the Doxy-CCL group than in the CCL group, but were not different from those measured in the control group. Doxycycline treatment did not have a significant effect on cartilage stromelysin levels. CONCLUSION The findings in this study indicate that doxycycline inhibits NO production in cartilage in dogs with CCL rupture. CLINICAL RELEVANCE Doxycycline may have a role in the treatment of canine OA by inhibiting NO production.

Apoptosis of ligamentous cells of the cranial cruciate ligament from stable stifle joints of dogs with partial cranial cruciate ligament rupture

OBJECTIVE To describe the presence and amount of apoptotic ligamentous cells in different areas of partially ruptured canine cranial cruciate ligaments (prCCLs) and to compare these findings with apoptosis of ligamentous cells in totally ruptured cranial cruciate ligaments (trCCLs). ANIMALS 20 dogs with prCCLs and 14 dogs with trCCLs. PROCEDURES Dogs with prCCLs or trCCLs were admitted to the veterinary hospital for stifle joint treatment. Biopsy specimens of the intact area of prCCLs (group A) and the ruptured area of prCCLs (group B) as well as specimens from trCCLs (group C) were harvested during arthroscopy. Caspase-3 and poly (ADP-ribose) polymerase (PARP) detection were used to detect apoptotic ligamentous cells by immunohistochemistry. RESULTS No difference was found in the degree of synovitis or osteophytosis between prCCLs and trCCLs. No difference was found in degenerative changes in ligaments between groups A and B. A substantial amount of apoptotic cells could be found in > 90% of all stained slides. A correlation (r(s) = 0.71) was found between the number of caspase-3-and PARP-positive cells. No significant difference was found in the amount of apoptotic cells among the 3 groups. No significant correlation could be detected between the degree of synovitis and apoptotic cells or osteophyte production and apoptotic cells. CONCLUSIONS AND CLINICAL RELEVANCE The lack of difference between the 3 groups indicates that apoptosis could be a factor in the internal disease process leading to CCL rupture and is not primarily a consequence of the acute rupture of the ligament.

Tibial Plateau angles with and without cranial cruciate ligament rupture: comparison between different dog populations and a wolf population.

The tibial plateau angles (TPA) of dogs with and without cranial cruciate ligament (CrCL) injuries were evaluated and further compared to the TPAs of dogs from a previous population and of wolves. Similar TPA measurements were found in all groups, suggesting that any possible changes in breeding practises, breed preferences, nutrition or other factors have not significantly influenced the TPA over time. Moreover, a difference was not found in the TPA between any group and the group affected with CrCL injury, suggesting that the TPA is not a clinically relevant predisposing factor in the development of canine CrCL rupture.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Perioperative and Anesthetic Management of Complete Tracheal Rupture in One Dog and One Cat.

ABSTRACT The authors describe two animals (one dog and one cat) that were presented with severe respiratory distress after trauma. Computerized tomographic imaging under general anesthesia revealed, in both cases, complete tracheal transection. Hypoxic episodes during anesthesia were relieved by keeping the endotracheal tube (ETT) positioned in the cranial part of the transected trachea and by allowing spontaneous breathing. Surgical preparation was performed quickly, and patients were kept in a sternal position to improve ventilation and oxygenation, and were only turned into dorsal recumbency shortly before surgical incision. A sterile ETT was guided into the distal part of the transected trachea by the surgeon, at which point mechanical ventilation was started. Both animals were successfully discharged from hospital a few days after surgery. Rapid and well-coordinated teamwork seemed to contribute to the good outcome. Precise planning and communication between anesthetists, surgeons, and technicians, as well as a quick course of action prior to correct ETT positioning helped to overcome critical phases.