Quantifying Filopodia in Cultured Astrocytes by an Algorithm.

Astrocytes in vivo extend thin processes termed peripheral astrocyte processes (PAPs), in particular around synapses where they can mediate glia-neuronal communication. The relation of PAPs to synapses is not based on coincidence, but it is not clear which stimuli and mechanisms lead to their formation and are active during process extension/ retraction in response to neuronal activity. Also, the molecular basis of the extremely fine PAP morphology (often 50 to 100 nm) is not understood. These open questions can be best investigated under in vitro conditions studying glial filopodia. We have previously analyzed filopodial mechanisms (Lavialle et al. PNAS 108:12915) applying an automated method for filopodia morphometry, which is now described in greater detail. The Filopodia Specific Shape Factor (FSSF) developed integrates number and length of filopodia. It quantifies filopodia independent of overall astrocytic shape or size, which can be intricate in itself. The algorithm supplied here permits automated image processing and measurements using ImageJ. Cells have to be sampled in higher numbers to obtain significant results. We validate the FSSF, and characterize the systematic influence of thresholding and camera pixel grid on measurements. We provide exemplary results of substance-induced filopodia dynamics (glutamate, mGluR agonists, EGF), and show that filopodia formation is highly sensitive to medium pH (CO2) and duration of cell culture. Although the FSSF was developed to study astrocyte filopodia with focus on the perisynaptic glial sheath, we expect that this parameter can also be applied to neuronal growth cones, non-neural cell types, or cell lines.

Lymph node hypoplasia is associated with adverse outcomes in node-negative colon cancer using advanced lymph node dissection methods.

PURPOSE: Lymph node size as a prognostic parameter has not been investigated well in the past. Recent data, however, have indicated that this parameter could be even more important than the lymph node count. METHODS: Based on the results of earlier studies, we analyzed the lymph node size and number of node-negative colon cancer patients with regard to survival. Data from 115 node-negative cases of colon cancer were analyzed. Lymph nodes with diameters ≤5 mm were defined as small, and all other lymph nodes were classified as intermediate/large in size and labeled LN5. All of the cases were categorized according to the number of LN5s. The LN5 very low (LN5vl) group included cases with less than two LN5s. All of the other cases were assigned to the LN5 low/high (LN5l/h) group. RESULTS: The overall survival analysis revealed significantly worse outcomes for the LN5vl group, with a mean survival of 34 months compared to the LN5l/h group, with a mean survival of 40 months (P = 0.022). After adjusting for the pT1/2 and pT3/4 stages, we still found a significant outcome difference (P = 0.012). Multivariate analysis identified LN5vl and T-stage as being independently correlated with the outcome. The vast majority of LN5vl cases (91 %) were located in the left colon. The location itself, however, was not prognostic (P = 0.478). CONCLUSION: LN5 count, as a marker of immune response, could be shown as being prognostic in node-negative colon cancer. Patients with low LN5 counts showed poor outcomes. These patients could perhaps profit from adjuvant chemotherapy.

Lymph node size as a simple prognostic factor in node negative colon cancer and an alternative thesis to stage migration.

BACKGROUND: Stage migration is an accepted explanation for the association between lymph node (LN) yield and outcome in colon cancer. To investigate whether the alternative thesis of immune response is more likely, we performed a retrospective study. METHODS: We enrolled 239 cases of node negative cancers, which were categorized according to the number of LNs with diameters larger than 5 mm (LN5) into the groups LN5-very low (0 to 1 LN5), LN5-low (2 to 5 LN5), and LN5-high (≥6 LN5). RESULTS: Significant differences were found in pT3/4 cancers with median survival times of 40, 57, and 71 months (P = .022) in the LN5-very low, LN5-low, and LN5-high groups, respectively. Multivariable analysis revealed that LN5 number and infiltration type were independent prognostic factors. CONCLUSIONS: LN size is prognostic in node negative colon cancer. The correct explanation for outcome differences associated with LN harvest is probably the activation status of LNs.

Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors.

The peripheral astrocyte process (PAP) preferentially associates with the synapse. The PAP, which is not found around every synapse, extends to or withdraws from it in an activity-dependent manner. Although the pre- and postsynaptic elements have been described in great molecular detail, relatively little is known about the PAP because of its difficult access for electrophysiology or light microscopy, as they are smaller than microscopic resolution. We investigated possible stimuli and mechanisms of PAP plasticity. Immunocytochemistry on rat brain sections demonstrates that the actin-binding protein ezrin and the metabotropic glutamate receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but not to the GFAP-containing stem process. Further experiments applying ezrin siRNA or dominant-negative ezrin in primary astrocytes indicate that filopodia formation and motility require ezrin in the membrane/cytoskeleton bound (i.e., T567-phosphorylated) form. Glial processes around synapses in situ consistently display this ezrin form. Possible motility stimuli of perisynaptic glial processes were studied in culture, based on their similarity with filopodia. Glutamate and glutamate analogues reveal that rapid (5 min), glutamate-induced filopodia motility is mediated by mGluRs 3 and 5. Ultrastructurally, these mGluR subtypes were also localized in astrocytes in the rat hippocampus, preferentially in their fine PAPs. In vivo, changes in glutamatergic circadian activity in the hamster suprachiasmatic nucleus are accompanied by changes of ezrin immunoreactivity in the suprachiasmatic nucleus, in line with transmitter-induced perisynaptic glial motility. The data suggest that (i) ezrin is required for the structural plasticity of PAPs and (ii) mGluRs can stimulate PAP plasticity.

Ventral rectus fascia closure on top of mesh hernia repair in the sublay technique.

Sublay prosthetic herniorrhaphy has become a widely accepted procedure for incisional hernias. To evaluate the effect of fascia closure on top of mesh repair on infection, and the recurrence rate, the authors reviewed their data regarding herniorrhaphy in the sublay technique. This study was a retrospective analysis of 175 consecutive patients who underwent hernia repair by implantation of prostheses by means of the Stoppa-Rives technique from December of 1994 to December of 2001. All 175 patients had the mesh implanted in the subfascial plane, 130 received a light-weight or heavy-weight polypropylene mesh (Vypro or Prolene) (74 percent), eight had a polyester mesh (Mersilene) (5 percent), and 37 had an expanded polytetrafluoroethylene patch (Gore-Tex) (21 percent). After sublay mesh positioning, the mesh could not be covered by the fascia in 50 cases; in 31 of these cases, a second mesh was placed into the fascial defect. To evaluate the influence of the fascia closing procedure on top of the sublay mesh, three groups were differentiated: initial fascia closure (n = 125), no fascia closure and concomitant mesh interposition (n = 31), and no fascia closure without mesh interposition (n = 19). After a mean follow-up of 20 months, 11 deep prosthetic infections (8 percent) and 15 hernia recurrences (9 percent) were observed. There was an increased risk of mesh infection when the fascia could not be closed, but there was no influence of fascia closure on hernia recurrence. When the fascia was left open, the placement of a second mesh inlay technique reduced mesh infection. The authors' data give evidence that closing the ventral fascia after mesh repair in the sublay position is beneficial. When the edges of the hernia defect could not be approximated, the suturing of a second mesh into the fascia defect was a useful tool for reducing the prosthetic infection rate; however, no significant influence on hernia recurrence was observed.