Development of techniques for time-lapse imaging of the dynamics of glial-axonal interactions in the central nervous system

Background: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualise the various stages of myelination. Methodology/Principal findings: Two approaches have been taken to follow myelination over time i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. The data demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, evidence from time-lapse fluorescence microscopy and confocal microscopy suggest that the oligodendrocytes’ cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs which then extend longitudinally along the axons. From this model it is predicted that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion: These experiments show the feasibility of visualising the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualisation of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

Astroglial-axonal interactions during early stages of myelination in mixed cultures using in vitro and ex vivo imaging techniques

<b>Background</b><p></p> Myelination is a very complex process that requires the cross talk between various neural cell types. Previously, using cytosolic or membrane associated GFP tagged neurospheres, we followed the interaction of oligodendrocytes with axons using time-lapse imaging in vitro and ex vivo and demonstrated dynamic changes in cell morphology. In this study we focus on GFP tagged astrocytes differentiated from neurospheres and their interactions with axons.<p></p> <b>Results</b><p></p> We show the close interaction of astrocyte processes with axons and with oligodendrocytes in mixed mouse spinal cord cultures with formation of membrane blebs as previously seen for oligodendrocytes in the same cultures. When GFP-tagged neurospheres were transplanted into the spinal cord of the dysmyelinated shiverer mouse, confirmation of dynamic changes in cell morphology was provided and a prevalence for astrocyte differentiation compared with oligodendroglial differentiation around the injection site. Furthermore, we were able to image GFP tagged neural cells in vivo after transplantation and the cells exhibited similar membrane changes as cells visualised in vitro and ex vivo.<p></p> <b>Conclusion</b><p></p> These data show that astrocytes exhibit dynamic cell process movement and changes in their membrane topography as they interact with axons and oligodendrocytes during the process of myelination, with the first demonstration of bleb formation in astrocytes

Acral Lentiginous Melanoma: A Case Control Study and Guidelines Update

Background. Malignant melanoma incidence is increasing dramatically. We report herein a case of the rarest acral lentiginous type. Case Report. A 58-year-old man presented with a melanoma resembling lesion over the sole of his right foot, measuring 15–20 mm in diameter. An excisional biopsy with a narrow (2 mm) margin of surrounding skin was obtained. Histological findings were consistent with a diagnosis of acral lentiginous melanoma. Sentinel lymph node biopsy was also performed and micrometastases were not identified in frozen-section examination. According to the AJCC system, the tumor stage was IB (T2aN0M0). A wide local excision of the biopsy scar with a margin of 2 cm was performed. A split-thickness thick skin graft was used to reconstruct the excisional defect. During an 18-month followup, no local or distant recurrence has been observed. This paper aims to extract an updated rational approach to the management of this disease out of an enormous body of knowledge

Transperitoneal laparoscopic right radical nephrectomy for renal cell carcinoma and end-stage renal disease: a case report

Nephron-sparing surgery (partial nephrectomy) results are similar to those of radical nephrectomy for small (<4 cm) renal tumors. However, in patients with end-stage renal disease, radical nephrectomy emerges as a more efficient treatment for localized renal cell cancer. Laparoscopic radical nephrectomy (LRN) increasingly is being performed. The objective of the present study was to present a case of a patient under hemodialysis who was submitted to LRN for a small renal mass and discuss the current issues concerning this approach. It appears that radical nephrectomy should be the standard treatment in dialysis patients even for small tumors. The laparoscopic technique is associated with acceptable cancer-specific survival and recurrence rate along with shorter hospital stay, less postoperative pain and earlier return to normal activities

Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

Time-lapse imaging of glial-axonal interactions

In the central nervous system (CNS), myelin is formed by oligodendrocytes that are derived from precursor cells, known as oligodendrocyte precursor cells (OPCs). Successive stages of OPC interactions with the axons can be visualized in vitro and ex vivo using mixed neural cell cultures and pieces of intact spinal cord, respectively. OPCs and their differentiation can be imaged using cell-type-specific markers or green fluorescent protein (GFP) tags. This protocol describes methodology for generating these two systems for time-lapse imaging of dynamic cell interactions using fluorescent and 2-photon microscopy

Time-lapse imaging of fluorescently labelled cells in association with neurites <i>in vitro</i>.

<p><b>A–E</b>) Myelinating cultures generated from a mix of wild type/beta-actin mice were visualised using time-lapse microscopy (Nikon TE2000 (60×, 0.75NA) over 16 hr in 5 min intervals on DIV 27 after the addition of wild type neurospheres previously infected with lentivirus carrying dsRed/GFP gene and addition of cyto-GFP cells. Two cell types were followed over time, one that expressed DS red/cyto-GFP and the other cyto-GFP. <b>A–E</b>) Strongly positive green cells typical of cyto-GFP morphologically resembled oligodendrocytes in contact with neurite bundles. The membrane appears to ruffle and form flaps/bubbles (arrow). In addition, the soma changes its location with respect to the neurite processes, over time, by moving closer to the neurite bundle. <b>C–E</b>) Dynamic imaging over 7.5 hours of a dsred/GFP labelled cell (asterisk) which was engulfed by a cell resembling a microglial cell (yellow arrow). This fluorescence was very much weaker than the cells generated from the beta-actin cyto-GFP mouse. Time frames obtained with 40× magnification (long distance working lens) and without perfect focus. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030775#pone.0030775.s002" target="_blank">Video S2</a>. <b>F</b>) Immunostaining of a similar culture around the same time confirmed MBP expression. Representative video from 1 experiment, seen in duplicate.</p

Confocal images of transplanted cyto-GFP expressing cells in the s<i>hiverer</i> spinal cord.

<p>Four weeks after transplantation of cyto-GFP expressing neurospheres, fixed sections of the <i>shiverer</i> spinal cord were immunolabelled for MBP (red) and GFP (green). <b>A</b>) A cyto-GFP labelled cell appears to extend spirals of cytoplasm around an MBP-positive myelin-like sheath (yellow arrows). Below the cell body, cyto-GFP is seen at the lateral edges (in relation to the long axis of the sheaths, white arrow) of adjacent sheaths and probably represents the cytoplasm filled paranodal loops on either side of the node of Ranvier (asterisks). <b>B</b>) Schematic of visualisation of the sections in C and D. <b>Ci–ii and Di–ii</b>) Spiral of GFP cytoplasm was followed by focussing up and down through the plane of view where they crossed up, traversed the axonal surface, then crossed down again representing the looping as shown in the schematic in B. <b>E</b>–<b>G</b>) 3D reconstruction of cyto-GFP structures (<b>E</b>), illustrates cyto-GFP either side of a space typical of a node of Ranvier (white arrows). <b>F</b>) is a tilted perspective of E) and shows the cyto-GFP form complete rings (white arrows representing the same position in E), consistent with the morphology of paranodal loops. <b>I</b>) Asymmetric caspr positive structures in association with cyto-GFP, at either side of a heminode. On the left, caspr forms a single vertical line and co-localises with cyto-GFP from the myelinating cell. On the right, caspr appears like a loose coil, consistent with its pattern of expression in non-myelinated axons. All images were acquired using an Olympus FV1000 confocal microscope (×60, 1.35NA). Representative images from at least 10 separate experiments.</p