Skeletal muscle extracellular matrix structure under applied deformation observed using second harmonic generation microscopy.

The mechanical and structural properties of passive skeletal muscle are important for musculoskeletal models in impact biomechanics, rehabilitation engineering and surgical simulation. Passive properties of skeletal muscle depend strongly on the architecture of the extracellular matrix (ECM), but the structure of ECM and its realignment under applied deformation remain poorly understood. We apply second harmonic generation (SHG) microscopy to study muscle ECM in intact muscle samples both under deformation and in the undeformed state. A method for regional relocation was developed, so that the same ECM segment could be viewed before and after applying deformations. Skeletal muscle ECM was viewed at multiple scales and in three states: undeformed, under compression and under tension. Results show that second harmonic generation microscopy provides substantial detail of skeletal muscle ECM over a wide range of length scales, especially the perimysium structure. We present images of individual portions of skeletal muscle ECM both undeformed and subjected to tensile/compressive deformation. We also present data showing the response of the perimysium to a partial thickness cut applied to a section under tensile deformation. STATEMENT OF SIGNIFICANCE: Second Harmonic Generation (SHG) microscopy is an imaging technique which takes advantage of a non-linear and coherent frequency doubling optical effect that is present in a small number of biological molecules, primarily collagen Type I, II and myosin. Collagen I is the most abundant collagen type in skeletal muscle, making SHG a promising option for visualisation of the skeletal muscle extracellular matrix (ECM). SHG microscopy does not require fixing or staining. This short communication presents the application of SHG microscopy to skeletal muscle ECM to improve our understanding of how collagen fibres reorganise under applied tensile and compression, including microscopic observations of collagen fibre reorganisation for intact samples by using a method to re-identify specific regions in repeated deformation tests.

Subcapsular haematoma following laparoscopic cholecystectomy.

Laparoscopic cholecystectomy (LC) is now considered the gold standard treatment for symptomatic gallbladder disease. Over the last two decades, a reduction in postoperative morbidity, mortality and hospital stay have seen a complete shift from open surgery to a laparoscopic approach. Intrahepatic subcapsular haematoma (ISH) is a rare and potentially life-threatening complication of LC. A 34-year-old female underwent LC for uncomplicated cholelithiasis. No complications were observed intra-operatively. 2 h postoperatively, the patient developed severe abdominal pain and tachycardia. Ultrasonography demonstrated an echogenic collection adjacent to the gallbladder fossa. Laparoscopy showed an ISH involving the right and left lobes of the liver, and no evidence of any intra-abdominal haemorrhage. Subsequent urgent triphasic CT identified a large ISH and a hypervascular lesion on the right lobe of the liver. This lesion demonstrated delayed enhancement with contrast filling suggestive of a hepatic haemangioma. This case report demonstrates the impact of imaging on postoperative management and the importance of postoperative patient monitoring in patients who have undergone laparoscopic surgery. Imaging explorations have a decisive role in the detection and characterization of haematomas.

Thermosensitive hydrogel for prolonged delivery of lentiviral vector expressing neurotrophin-3 in vitro.

BACKGROUND: The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/ß-glycerol phosphate (ß-GP) as a carrier. METHODS: Three chitosan/ß-GP scaffolds were prepared with varying concentrations of chitosan and ß-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/ß-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. RESULTS: Collagen displayed its most significant elution of virus on day 1 and chitosan/ß-GP (with a final concentration of 2.17% chitosan) on day 3. CONCLUSIONS: The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system.