PKCalpha mediates CCL18-stimulated collagen production in pulmonary fibroblasts

A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha G?6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.     

Femoral-to-port through-and-through wire access to reestablish subcutaneous port function

OBJECTIVE: Central venous catheter malfunction often results from fibrin sheath formation and is routinely addressed with thrombolytic therapy or mechanical stripping. Mechanical stripping from a distant access site such as a femoral vein is the only option for a subcutaneous port that has failed thrombolytic therapy. When a fibrin sheath has rendered the catheter tip inaccessible to snaring, catheter salvage cannot be achieved, requiring port exchange. We report two cases in which an inaccessible catheter tip was mobilized via advancing a wire through the port and through the catheter, allowing for successful snaring, mechanical stripping, and return of normal port function. CONCLUSION: Passage of a hydrophilic wire through a subcutaneous port and beyond the catheter tip is technically possible. The wire can be snared from a femoral access to achieve successful catheter stripping when direct catheter snaring is not possible.     

Physiological evidence that pyramidal neurons lack functional water channels

The physiological conditions that swell mammalian neurons are clinically important but contentious. Distinguishing the neuronal component of brain swelling requires viewing intact neuronal cell bodies, dendrites, and axons and measuring their changing volume in real time. Cultured or dissociated neuronal somata swell within minutes under acutely overhydrated conditions and shrink when strongly dehydrated. But paradoxically, most central nervous system (CNS) neurons do not express aquaporins, the membrane channels that conduct osmotically driven water. Using 2-photon laser scanning microscopy (2PLSM), we monitored neuronal volume under osmotic stress in real time. Specifically, the volume of pyramidal neurons in cerebral cortex and axon terminals comprising cerebellar mossy fibers was measured deep within live brain slices. The expected swelling or shrinking of the gray matter was confirmed by recording altered light transmittance and by indirectly measuring extracellular resistance over a wide osmotic range of -80 to +80 milliOsmoles (mOsm). Neurons expressing green fluorescent protein were then imaged with 2PLSM between -40 and +80 mOsm over 20 min. Surprisingly, pyramidal somata, dendrites, and spines steadfastly maintained their volume, as did the cerebellar axon terminals. This precluded a need for the neurons to acutely regulate volume, preserved their intrinsic electrophysiological stability, and confirmed that these CNS nerve cells lack functional aquaporins. Thus, whereas water easily permeates the aquaporin-rich endothelia and glia driving osmotic brain swelling, neurons tenatiously maintain their volume. However, these same neurons then swell dramatically upon oxygen/glucose deprivation or [K+]0 elevation, so prolonged depolarization (as during stroke or seizure) apparently swells neurons by opening nonaquaporin channels to water.     

Developing a multimedia environment for customized teaching of an adrenalectomy

We have developed a computer based simulation process which allows a surgical expert to create a customized operative environment. This virtual environment, the Toolkit for Illustration of Procedures in Surgery (3D TIPS), is deployed on a low-cost computer system and requires minimal training for the programmer. The learner can be engaged in training immediately and the educator can modify the system and annotate the procedure to highlight specific points using video clips, operative images, and the like. A laparoscopic adrenalectomy is presented as a proof of concept in the accompanying article.     

Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus

Astroglia are integral components of synapse formation and maturation during development. Less is known about how astroglia might influence synaptogenesis in the mature brain. Preparation of mature hippocampal slices results in synapse loss followed by recuperative synaptogenesis during subsequent maintenance in vitro. Hence, this model system was used to discern whether perisynaptic astroglial processes are similarly plastic, associating more or less with recently formed synapses in mature brain slices. Perisynaptic astroglia was quantified through serial section electron microscopy in perfusion-fixed or sliced hippocampus from adult male Long-Evans rats that were 65-75 days old. Fewer synapses had perisynaptic astroglia in the recovered hippocampal slices (42.4% +/- 3.4%) than in the intact hippocampus (62.2% +/- 2.6%), yet synapses were larger when perisynaptic astroglia was present (0.055 +/- 0.003 microm2) than when it was absent (0.036 +/- 0.004 microm2) in both conditions. Importantly, the length of the synaptic perimeter surrounded by perisynaptic astroglia and the distance between neighboring synapses was not proportional to synapse size. Instead, larger synapses had longer astroglia-free perimeters where substances could escape from or enter into the synaptic clefts. Thus, smaller presumably newer synapses as well as established larger synapses have equal access to extracellular glutamate and secreted astroglial factors, which may facilitate recuperative synaptogenesis. These findings suggest that as synapses enlarge and release more neurotransmitter, they attract astroglial processes to a discrete portion of their perimeters, further enhancing synaptic efficacy without limiting the potential for cross talk with neighboring synapses in the mature rat hippocampus.     

Whole isolated neocortical and hippocampal preparations and their use in imaging studies

This study shows that two whole isolated preparations from the young mouse, the neocortical 'slab' and the hippocampal formation, are useful for imaging studies requiring both global monitoring using light transmittance (LT) imaging and high resolution cellular monitoring using 2-photon laser scanning microscopy (2PLSM). These preparations share advantages with brain slices such as maintaining intrinsic neuronal properties and avoiding cardiac or respiratory movement. Important additional advantages include the maintenance of all local input and output pathways, the absence of surfaces injured by slicing and the preservation of three-dimensional tissue structure. Using evoked extracellular field recording, we demonstrate long-term (hours) viability of both whole preparations. We then show that propagating cortical events such as anoxic depolarization (AD) and spreading depression (SD) can be imaged in both preparations, yielding results comparable to those in brain slices but retaining the tissue's three-dimensional structure. Using transgenic mice expressing green fluorescent protein (GFP) in pyramidal and granule cell neurons, 2PLSM confirms that these preparations are free of the surface damage observed in sliced brain tissue. Moreover the neurons undergo swelling with accompanying dendritic beading following AD induced by simulated ischemia, similar to cortical damage described in vivo.