Marcus J. D. Wagstaff

Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CA1 neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constitutive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

Perineal and posterior vaginal wall reconstruction with superior and inferior gluteal artery perforator flaps

Perineal and posterior vaginal wall reconstruction following abdominoperineal and local cancer resection entails replacement of volume between the perineum and sacrum and restoration of a functional vagina. Ideal local reconstructive options include those which avoid functional muscle sacrifice, do not interfere with colostomy formation, and avoid the use of irradiated tissue. In avoiding the donor site morbidity of other options, we describe a fasciocutaneous option for the reconstruction of the perineum and posterior vaginal wall. We present our technique of superior and inferior gluteal artery perforator (SGAP or IGAP) flaps to reconstruct such defects. Fourteen patients between 2004 and 2008 underwent 11 SGAP and three IGAP flaps. There were no flap failures or partial flap losses and no postoperative hernias. All female patients reported resumption of sexual intercourse following this procedure. Our experience in both the immediate and delayed setting is that this technique produces a good functional outcome with low donor‐site morbidity.

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types – quiescent human CD34+ hematopoietic progenitor cells and dendritic cells – which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a sin-gle virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

Delivery of a constitutively active form of the heat shock factor using a virus vector protects neuronal cells from thermal or ischaemic stress but not from apoptosis

The heat shock proteins (HSPs) are induced by stressful stimuli and have a protective effect. Different HSPs protect with different efficiencies against different stresses indicating that optimal protection would be obtained with a non‐stressful agent which induced a range of HSPs. We have prepared a herpesvirus vector expressing a constitutively active mutant form of heat shock factor 1 (HSF1) which, unlike the wild‐type form of this transcription factor, does not require stress for its activation. Upon infection of neuronal cells, this virus induced a more restricted range of HSPs than in non‐neuronal cells. Infection with the virus protected neuronal cells against subsequent thermal or ischaemic stress in accordance with its ability to induce HSP70 expression but did not protect them against apoptotic stimuli. The mechanisms of these effects and their significance for the use of HSF to manipulate HSP gene expression is discussed.

Gene transfer using a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo.

The design of recombinant HSV-1 vectors for delivery of transgenes to the central nervous system is undergoing constant development. Problems associated with the construction and use of such vectors include the requirement for detection of recombinant versus nonrecombinant virus in vitro and also the identification of transduced cells in vivo. This could be overcome by the insertion of reporter genes such as lacZ or green fluorescent protein (GFP) under a separate promoter to the transgene to be expressed. In this case, however, reporter gene expression does not necessarily confirm transgene expression as a separate RNA must be produced. This study reports the use of an encephalomyocarditis virus internal ribosome entry site (IRES) to enable the translation of two reporter genes from a single mRNA transcript driven by the same promoter within a disabled HSV vector, and discusses the potential advantages of this approach.

Performing two DIEP flaps in a working day: an achievable and reproducible practice

BACKGROUND While the deep inferior epigastric artery perforator (DIEP) flap is a reliable technique for autologous breast reconstruction, the meticulous dissection of perforators may require lengthy operative times. In our unit, we have performed 600 free flaps for breast reconstruction over 8 years and have reduced operative times with a combination of preoperative computed tomographic angiography (CTA), various anastomotic techniques and the Cook-Swartz implantable Doppler probe for perfusion monitoring. We sought to assess the feasibility of performing two DIEP flaps within the working hours of a single day. METHODS A review of 101 consecutive patients undergoing DIEP flap breast reconstruction in a 12-month period was performed, comparing one DIEP flap per day (n=43) to two DIEP flaps per day (n=58). Complications, outcomes and techniques used were critically analysed. For cases of two DIEP flaps per day, a comparison was made between the use of two separate operating theatres (n=44) and a single consecutive theatre (n=14). RESULTS Complications did not increase when two DIEP flaps were performed in a single working day. The use of vascular closure staple (VCS) sutures and ring couplers resulted in statistically significant reductions in anastomotic times. The use of two separate theatres for performing two DIEP flaps resulted in a reduction of 59min in operative time per case (p=0.004). CONCLUSION Two DIEP flaps can be safely and routinely performed within the hours of a single working day. By minimising operative times, these techniques can improve productivity and substantially decrease surgeon fatigue.

The financial implications of computed tomographic angiography in DIEP flap surgery: A cost analysis

THE FINANCIAL IMPLICATIONS OF COMPUTED TOMOGRAPHIC ANGIOGRAPHY IN DIEP FLAP SURGERY : A COST ANALYSIS

A Reliable and Aesthetic Technique for Cephalic Vein Harvest in DIEP Flap Surgery

The need for a secondary source of venous drainage in deep inferior epigastric artery perforator flaps is common, with the cephalic vein commonly utilized as an alternative venous recipient vessel. In using the cephalic vein, previous studies have described the deltopectoral groove or the infraclavicular fossa as the site for harvest. We describe the use of an anterior axillary skin crease, which can improve aesthetic outcome, reduce the time needed for harvest, enable a greater length of vein to be transposed, and minimize surgical insult to the upper breast/chest wall. An anterior axillary-line skin fold can be used as the site for cephalic vein harvest, and using the methodology described, the technique can be fast and highly reliable and result in a final scar that is barely visible.

The course of anterolateral thigh perforators does not correlate between sides of the body: the role for preoperative imaging.

[Extract] The anterolateral thigh flap is well established as a versatile reconstructive option for a range of soft tissue defects, with excellent results reported. However, controversy exists regarding the reliability of the vascular anatomy of the flap. Some authors describe a consistent relationship of perforators to anatomical landmarks and between sides of the body, whereas others have described uncertainty in predicting individual perforator anatomy and advocated the use of preoperative imaging. A major advantage of preoperative imaging is the selection of the side of the body with better vascular anatomy. Computed tomographic angiography has been shown to be accurate in detecting the optimal perforator and limb for flap harvest. To confirm this benefit, the current study aimed to compare the course of anterolateral thigh perforators between body sides.

Buried Free Flaps for Breast Reconstruction: A New Technique Using the Cook-Swartz Implantable Doppler Probe for Postoperative Monitoring

Buried Free Flaps for Breast Reconstruction : A New Technique Using the Cook-Swartz Implantable Doppler Probe for Postoperative Monitoring