Spatiotemporal microRNA profile in peripheral nerve regeneration: miR-138 targets vimentin and inhibits Schwann cell migration and proliferation.

While the peripheral nervous system has regenerative ability, restoration of sufficient function remains a challenge. Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration, including myelination, neuroplasticity, kinase signaling in nerve axoplasm, and cell migration; however, the mechanisms regulating its expression within Schwann cell (SC) remain unexplored. The aim of this study was to profile the spatial and temporal expression profile of microRNA (miRNA) in a regenerating rat sciatic nerve after transection, and explore the potential role of miR-138-5p targeting vimentin in SC proliferation and migration. A rat sciatic nerve transection model, utilizing a polyethylene nerve guide, was used to investigate miRNA expression at 7, 14, 30, 60, and 90 days during nerve regeneration. Relative levels of miRNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction. In vitro assays were conducted with cultured Schwann cells transfected with miRNA mimics and assessed for migratory and proliferative potential. The top seven dysregulated miRNAs reported in this study have been implicated in cell migration elsewhere, and GO and KEGG analyses predicted activities essential to wound healing. Transfection of one of these, miRNA-138-5p, into SCs reduced cell migration and proliferation. miR-138-5p has been shown to directly target vimentin in cancer cells, and the luciferase assay performed here in rat Schwann cells confirmed it. These results detail a role of miR-138-5p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.

Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays.

BACKGROUND: Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. RESULTS: The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and ß-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. CONCLUSIONS: In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite spatiotemporal expression profile of peripheral nerve regeneration.

Role of sagittal reformatted computed tomographic images in the evaluation of orbital floor fractures.

BACKGROUND: The sagittal plane computed tomographic (CT) scan has been proposed as the most important radiologic view in the diagnosis of orbital floor fractures. Before the advent of high-resolution CT, several articles were published in the radiology literature debating the need for direct sagittal views, which required cumbersome patient positioning. Today, the ability to easily create high-quality sagittal reformations requires reassessment of the importance of this view in the evaluation of orbital floor fractures. MATERIALS AND METHODS: Computed tomographic studies of 24 orbital floor fractures for which coronal and sagittal images were available were included. Five independent reviewers evaluated all image sets and recorded maximum fracture width, depth, posterior shelf length (PSL), and presence of medial wall fracture and of trap door deformity. Direct fracture width measurements were obtained via coronal images, whereas depth and PSL were measured directly in the sagittal plane. Indirect measurements in a given plane were obtained by counting the number of slices in which the fracture was present and multiplying by the slice thickness. Cronbach alpha analysis was used to provide an intraclass correlation coefficient, where greater values signify less interreviewer variability. RESULTS: The Cronbach alpha values for width, depth, and PSL were 64%, 59%, and 85% in the coronal view and 76%, 55%, and 51% in the sagittal view, respectively. The alpha values for presence/absence of medial wall fracture and trap door deformity were greater in the coronal view at 73% and 55%, respectively. The intrareviewer variability was not significantly correlated with the degree of patient rotation or CT scan slice thickness. The interreviewer variability was significantly less among attending reviewer then resident reviewers. CONCLUSIONS: There was less interreviewer variability when the measured variables were assessed indirectly (width was best assessed in the sagittal plane, and depth and PSL were best assessed in the coronal view). These findings support the idea that additional views aid the surgeon's ability to further define fracture anatomy than might be possible with any single view, thereby improving preoperative planning and reconstructive strategies.

Duration of smoking cessation and its impact on skin flap survival.

BACKGROUND: Empirical and experimental evidence abounds as to the negative effects of smoking on skin flaps. The ideal duration of preoperative smoking cessation is unclear. The present study evaluates the effect of various durations of smoking cessation on skin flap survival in a rat model. METHODS: Forty smoke-exposed and 10 non-smoke-exposed Sprague-Dawley rats were divided into five groups: controls and 0 days, 2 weeks, 4 weeks, and 8 weeks of smoking cessation. All animals had a dorsally based random pattern flap created and either bilateral pure axial superficial inferior epigastric (superficial inferior epigastric) flaps or a unilaterally superficial inferior epigastric axial with random component flap. The percentage of flap necrosis was assessed 2 weeks postoperatively. RESULTS: The mean amount of random skin flap necrosis was 16.6 +/- 13.0 percent, 30.3 +/- 8.4 percent, 27.6 +/- 7.9 percent, 27.1 +/- 6.1 percent, and 29.7 +/- 10.3 percent, respectively. There was significantly less flap necrosis in the controls than in all other groups (p < 0.03). There was no necrosis of any of the pure axial superficial inferior epigastric flaps. The mean amount of superficial inferior epigastric axial with random component skin flap necrosis was 11.1 +/- 6.2 percent, 31.1 +/- 6.0 percent, 36.0 +/- 8.4 percent, 21.7 +/- 4.0 percent, and 19.1 +/- 6.3 percent, respectively. All smoke-exposed groups had significantly greater flap necrosis than controls, with the exception of the 8-week group (p < 0.02). CONCLUSIONS: Smoking irreversibly increases the risk of flap necrosis in a random pattern flap out to 2 months of preoperative cessation. Preoperative smoking does not result in any necrosis of pure axial flaps. In axial with random component flaps, significant decreases in skin flap necrosis are not seen until 4 weeks of preoperative cessation.

Evaluation of a novel reverse thermosensitive polymer for use in microvascular surgery.

Microvascular clamps have several potential shortcomings, including the risk of vessel injury. LeGoo, a novel reverse thermosensitive polymer (Pluromed Inc., Woburn, MA), is investigated as a substitute to vascular clamping in a microsurgical model and the technical details are described. Femoral vessels of Sprague Dawley rats were used to evaluate the usefulness of this polymer for performing end-to-end arterioarterial (AA), venovenous (VV), and end-to-side arteriovenous (AV) microvascular anastomoses. The ability to obtain and maintain hemostasis was assessed. Secondary endpoints, including polymer volume, concentration, temperature, infusion technique, ability to reinfuse, blood vessel stenting effect, polymer dissolution characteristics, and reestablishment of flow, were also noted. Initial hemostasis occurred in every case. Mean duration of efficacy (hemostasis) after initial injection was 17.8 minutes (4 minutes to 44.5 minutes) for AA anastomoses and 31.8 minutes (13 minutes to 46 minutes) for VV anastomoses. Mean volume of polymer initially injected was 0.11 mL (0.01 mL to 0.20 mL) and 0.07 mL (0.06 mL to 0.10 mL) for AA and AV arteries, respectively, and 0.14 mL (0.10 mL to 0.20 mL) and 0.20 mL (0.15 mL to 0.27 mL) for VV and AV veins. Use of LeGoo in veins was clearly superior to arterial use with regard to the technical aspects of injecting LeGoo and reestablishing hemostasis, as well as greater vessel stenting effect and less vessel retraction. This novel polymer showed promise for its ability to allow for hemostasis while performing microvascular anastomoses. Improvements were made with regard to injection techniques, appropriate volumes, ability to reliably determine gel plug dissolution, and final vessel patency. Preliminary results demonstrate that this polymer may be a viable substitute for microvascular clamps.

Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel.

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

Enhanced peripheral nerve regeneration elicited by cell-mediated events delivered via a bioresorbable PLGA guide.

Using an established rat peripheral-nerve regeneration model, the authors have demonstrated enhancement of regeneration following subcutaneous priming of bioresorbable poly(lactic-co-glycolic)acid (PLGA) guides in vivo. Four weeks after nerve reconstruction, regeneration of the peripheral nerve through the cell-infiltrated guides displayed a significant increase in the total axon number and myelination status recorded in primed over unprimed guides, demonstrating the importance of cell-mediated events in the regeneration process. To define the different components enhancing nerve regeneration in this model, they have focused on identifying factors capable of eliciting Schwann-cell migration, since this has been identified as an early and necessary event in nerve regeneration. Using an in vitro migration assay, screening of a limited number of cellular and extracellular factors has demonstrated differential promotion of Schwann-cell migration. Of interest, combining fibronectin and bFGF resulted in a two-fold enhancement in Schwann-cell migration over that recorded with either alone. These results describe a rapid screening process for identifying various molecules and combinations thereof, with potential involvement in Schwann-cell migration. Coupling these findings to the use of the PLGA guide as an in vivo delivery system provides a rationale for the selection of exogenous factors to test for the enhancement of peripheral-nerve regeneration.