Myocutaneous flap ischemia: flow dynamics following venous and arterial obstruction.

To further clarify the pathogenesis of the poorer prognosis in skin flaps exposed to venous stasis compared with arterial insufficiency, a microsphere study was conducted in bilateral rectus abdominis island flaps in seven pigs. The relationship between capillary blood flow and arteriovenous (A-V) shunting was studied during progressive 1-hour intervals of arterial insufficiency and venous stasis and during 3 hours of reperfusion. Under controlled conditions, total blood flow was reduced from 100 percent to both 50 and 25 percent by application of an adjustable clamp on the artery supplying one flap and on the vein draining the contralateral flap. The relative distribution between A-V shunt flow and capillary blood flow was different in arterial insufficiency when compared with venous stasis at both the 50 percent and the 25 percent blood flow levels. In the arterial insufficiency flaps, the A-V shunt flow and capillary blood flow shared the total blood flow in the following percentages: 64/36 (at 100 percent total blood flow), 44/56 (at 50 percent total blood flow level), and 22/78 (at 25 percent total blood flow level). In the venous stasis flaps, the A-V shunt flow and the capillary blood flow shared the total blood flow in percentages of 70/30, 66/34, and 55/45, respectively. Hence, in arterial insufficiency flaps, capillary blood flow was spared by a relatively greater decline in A-V shunting compared with venous stasis flaps. Redistribution of capillary blood flow from subcutaneous tissue to muscle was observed, whereas blood flow was equally distributed throughout the length of the flaps at all flow levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine bladder wall biomechanics following partial bladder obstruction

Evaluation of bladder wall mechanical behavior is important in understanding the functional changes that occur in response to pathologic processes such as partial bladder outlet obstruction (pBOO). In the murine model, the traditional approach of cystometry to describe bladder compliance can prove difficult secondary to small bladder capacity and surgical exposure of the bladder. Here, we explore an alternative technique to characterize murine mechanical properties by applying biaxial mechanical stretch to murine bladders that had undergone pBOO. 5–6 week old female C57/Bl6 mice were ovariectomized and subjected to pBOO via an open surgical urethral ligation and sacrificed after 4 weeks (n=12). Age matched controls (n=6) were also analyzed. Bladders were separated based on phenotype of fibrotic (n=6) or distended (n=6) at the time of harvest. Biaxial testing was performed in modified Kreb's solution at 37 °C. Tissue was preconditioned to 10 cycles and mechanical response was evaluated by comparing axial strain at 50 kPa. The normal murine bladders exhibited anisotropy and were stiffer in the longitudinal direction. All mice showed a loss of anisotropy after 4 weeks of pBOO. The two phenotypes observed after pBOO, fibrotic and distended, exhibited less and more extensibility, respectively. These proof-of-principle data demonstrate that pBOO creates quantifiable changes in the mechanics of the murine bladder that can be effectively quantified with biaxial testing.     

Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction)

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP.Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue.In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase.The following is a summary of the results: 1) The bladder mass increased from 2.2 ± 0.2 gm to 11.5 ±1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a V_max = 1120 nmoles/mg protein/4 min and K_m = 147 µM CP. 2) The creatine kinase activity of obstructed bladders also displayed linear kinetics with a V_max = 1125 nmoles/mg protein/4 min tissue, and K_m = 276 µM CP.These studies demonstrate that whereas both control and obstructed bladders have virtually identical maximum creatine kinase activities, the K_m for the obstructed tissue is significantly higher than the K_m for the control tissue. This may indicate that under cellular conditions (at sub-maximum substrate concentrations), the creatine kinase activity of the obstructed bladders may be significantly lower than the activity of the control bladders. In addition, the reduced tissue concentrations of ATP and CP would certainly be consistent with the reduced functional response to bethanechol and field stimulation.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni²⁺-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litreE. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search forras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidateras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identifyras binding proteins present in cellular extracts.Abbreviations his-ras histidine-tagged vHa-ras - Ni-NTA Ni²⁺ nitriloacetic acid agarose - IPTG isopropyl thio--D-galactoside     

A free arterialized venous loop flap.

An arterialized venous loop flap measuring 6 x 16 cm was transferred from the left forearm to the right lower leg for reconstruction of an unstable skin graft on the tibia. The transferred flap became softer and thinner, as intended, after ligation of the afferent artery, which was performed 50 days after the first operation. The operative results were satisfactory.     

Reliable assessment of skin flap viability using orthogonal polarization imaging

Intraoperative evaluation of skin flap viability has primarily been dependent on clinical judgment. The purpose of this study was to determine whether an orthogonal polarization spectral imaging device could be used to accurately predict viability of random-pattern skin flaps. Orthogonal polarization spectral imaging is a newly developed technique that visualizes the microcirculation using reflected light without the use of fluorescent dyes and allows for noninvasive real-time observation of functional microvascular networks. In Sprague-Dawley rats (n = 24), three types of random skin flaps were designed with unknown zones of viability (n = 8 per group). After flap elevation, the skin flaps were evaluated by both clinical examination and orthogonal polarization spectral imaging. Areas of the flap determined to be nonviable by clinical examination were measured and marked. Orthogonal polarization spectral imaging was subsequently performed, and areas of the skin flap with stasis (i.e., cessation of red blood cell movement) in the dermal microcirculation on orthogonal polarization spectral imaging were measured and marked. The skin flaps were then secured in place. Flaps were evaluated on a daily basis for clinical signs of ischemia and necrosis. On postoperative day 7, the total amount of random skin flap necrosis was measured and recorded. Clinical examination of the random skin flaps significantly underestimated the actual amount of eventual flap necrosis, and as result was a very poor predictor of flap necrosis. By contrast, assessment of microcirculatory stasis using the orthogonal polarization spectral imaging device correlated well with the subsequent development of necrosis in all groups. In the three groups, the average amount of flap necrosis predicted by clinical examination deviated from actual necrosis by approximately 2 to 4 cm. However, the amount that orthogonal polarization spectral imaging differed from actual necrosis was 0.1 to 0.3 cm. Therefore, orthogonal polarization spectral imaging was an excellent predictor of eventual flap necrosis and much more accurate than clinical observation (p < 0.001). Intraoperative evaluation of axial and random pattern flap viability has traditionally been based on clinical examination as no other reliable, convenient test currently exists. The authors demonstrated that an orthogonal polarization spectral imaging device accurately predicts zones of necrosis in random pattern flaps by directly visualizing cessation of microcirculatory flow. Intraoperative stasis in the dermal microcirculation correlated precisely with subsequent flap necrosis. Orthogonal polarization spectral imaging was significantly more accurate than clinical examination, which consistently underestimated flap necrosis. The orthogonal polarization spectral imaging technique may have value in the intraoperative assessment of skin flap perfusion such as that required after skin-sparing mastectomy.     

Free transplantation of venous network pattern skin flap

We report a patient treated by transplantation of a venous network pattern skin flap containing two superficial cutaneous veins. Through one of the veins, arterial blood passed into the capillaries, restoring circulation and ensuring survival of the flap.