Michael C. McCormack

Intestinal alkaline phosphatase is a gut mucosal defense factor maintained by enteral nutrition.

Under conditions of starvation and disease, the gut barrier becomes impaired, and trophic feeding to prevent gut mucosal atrophy has become a standard treatment of critically ill patients. However, the mechanisms responsible for the beneficial effects of enteral nutrition have remained a mystery. Using in vitro and in vivo models, we demonstrate that the brush–border enzyme, intestinal alkaline phosphatase (IAP), has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier. IAP expression and function are lost with starvation and maintained by enteral feeding. It is likely that the IAP silencing that occurs during starvation is a key component of the gut mucosal barrier dysfunction seen in critically ill patients.

The Differing Roles of the Classical and Mannose-Binding Lectin Complement Pathways in the Events following Skeletal Muscle Ischemia-Reperfusion

Complement is an important mediator of the injuries observed after skeletal muscle ischemia and subsequent reperfusion. Although the classical pathway had been assumed to be the major pathway of activation leading to injury, the mannose-binding lectin (MBL) pathway might also play a contributing role. In this study, we found that MBL-deficient mice had significant protection after skeletal muscle reperfusion injury compared with wild-type, classical pathway-specific C1q-deficient mice, or MBL-deficient mice reconstituted with recombinant human MBL. MBL-deficient mice, however, were not protected from permeability edema or secondary lung injury after ischemia-reperfusion. These data indicate that blockade of the classical pathway alone (C1q) is protective against permeability edema and remote pulmonary injury but not protective against histologic muscle injury. In contrast, blocking the MBL pathway alone protects against histological injury but is not protective against permeability edema or lung injury. Thus, the activation of both pathways is likely responsible for the full spectrum of injuries observed after skeletal muscle reperfusion injury.

The effect of pressure and shear on autologous fat grafting.

1. Aygit AC, Basaran K, Mercan ES. Transaxillary totally subfascial breast augmentation with anatomical breast implants: Review of 27 cases. Plast Reconstr Surg. 2013;131:1149–1156. 2. Hwang K, Kim DJ. Anatomy of pectoral fascia in relation to subfascial mammary augmentation. Ann Plast Surg. 2005;55:576–579. 3. Jinde L, Xiaoping C, Wanquan Z, Xia G, Ligang X. Can the pectoral fascia integrity be preserved during subfascial breast augmentation through the axillary approach? Aesthetic Plast Surg. 2010;34:29–32. 4. Salgarello M, Visconti G, Barone-Adesi L, et al. Inverted-T skin-reducing mastectomy with immediate implant reconstruction using the submuscular-subfascial pocket. Plast Reconstr Surg. 2012;130:31–41. 5. Salgarello M, Visconti G, Barone-Adesi L. One-stage immediate breast reconstruction with implants in conservative mastectomies. In: Salgarello M, ed. Breast Reconstruction: Current Techniques. Rijeka, Croatia: In-Tech; 2012:49–82. Available at: www.intechopen.com.

The impact of liposuction cannula size on adipocyte viability.

PurposeAutologous fat transfer (“fat grafting”) is widely used in cosmetic and reconstructive surgery, but long-term outcomes remain inconsistent. Each step in the transfer process can cause mechanical damage to the graft tissue. In particular, liposuction breaks aspirated adipose tissue into distinct globules and subjects it to shear forces, both of which can impact subsequent fat graft viability. The optimal size of the liposuction cannula for use in fat grafting is not known. Methods and TechniquesControlled lipoaspirate samples were collected from adult female patients undergoing elective liposuction of the abdomen and flanks with uniform aspiration pressure (−25 in Hg) and either a 3- or 5-mm standard blunt-tip liposuction cannula. Individual grafts of 1.00 ± (0.01) gram were prepared and injected into the bilateral flanks of nude mice with a 14-gauge catheter. After six weeks, these grafts were explanted and analyzed by weight and histology. ResultsAt six weeks, fat lobules in the 5-mm group retained 25% more weight than those in the 3-mm group [mean (SD), 0.70 (0.07) vs 0.56 (0.09) g, n = 24/group, P < 0.01). Histologic analysis revealed more intact, nucleated adipocytes in the 5-mm group than in the 3-mm group [4.42 (0.92) vs 3.10 (0.56) on a 1–5 rating scale]. The 5-mm group exhibited both less infiltrate [1.58 (0.17) vs 3.13 (0.70)] and less fibrosis [1.67 (0.45) vs 3.13 (0.89)] than the 3-mm group. ConclusionsIn this controlled model of fat grafting with either a 5- or 3-mm aspiration cannula, the use of a larger aspiration cannula led to improved graft retention and quality. This finding has important implications for clinical applications of fat grafting.

Comparative analysis of processing methods in fat grafting.

Background: Centrifugation is a popular processing method, with an unclear mechanism of action. Hypotheses include fat concentration, reduced inflammatory response by removal of blood, and concentration of adipose-derived stem cells. The authors performed multiple experiments to determine the role of centrifugation and compared it with a different processing method (mesh/gauze technique). Methods: Lipoaspirate components were quantified after centrifugation at increasing speed to determine concentration efficacy. For comparison, the authors quantified the concentration efficacy of mesh/gauze. They also compared the number of adipose-derived stem cells isolated by either method. To determine the effects of each component, they compared fat alone to fat mixed with various spinoff components in a mouse model. They also compared centrifugation to mesh/gauze. Results: The adipocyte fraction remains constant above 5000 g, whereas 1200 g results in 91 percent concentrated fat. Mesh/gauze also results in 90 percent concentrated fat. The number of adipose-derived stem cells in 1 g of fat was 1603 ± 2020 and 1857 ± 1832 in the centrifuge and mesh/gauze groups, respectively (p = 0.86). Five “add-back” groups were created: fat plus oil, fat plus surgical tumescence, fat plus fresh tumescence, fat plus cell pellets and fresh tumescence, and fat plus cell pellets. The fat-only group had better retention than the groups mixed with tumescence, regardless of whether it was surgical, fresh, or had cell pellets. Oil did not affect grafts. Centrifugation at 1200 g was equivalent to mesh/gauze (0.73 ± 0.12 g and 0.72 ± 0.13 g, respectively). Conclusions: Centrifugation improves graft retention by concentration of the adipocyte fraction. The concentration efficacy of mesh/gauze is equivalent to centrifugation at 1200 g, with equivalent in vivo outcomes.

Ischemic Preconditioning of Skeletal Muscle Mitigates Remote Injury and Mortality

BACKGROUND Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality. METHODS Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Students t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio. RESULTS Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59). CONCLUSIONS We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.

Poloxamer 188 protects against ischemia-reperfusion injury in a murine hind-limb model.

Background: Ischemia-reperfusion injury can activate pathways generating reactive oxygen species, which can injure cells by creating holes in the cell membranes. Copolymer surfactants such as poloxamer 188 are capable of sealing defects in cell membranes. The authors postulated that a single-dose administration of poloxamer 188 would decrease skeletal myocyte injury and mortality following ischemia-reperfusion injury. Methods: Mice underwent normothermic hind-limb ischemia for 2 hours. Animals were treated with 150 &mgr;l of poloxamer 188 or dextran at three time points: (1) 10 minutes before ischemia; (2) 10 minutes before reperfusion; and (3) 2 or 4 hours after reperfusion. After 24 hours of reperfusion, tissues were analyzed for myocyte injury (histology) and metabolic dysfunction (muscle adenosine 5′-triphosphate). Additional groups of mice were followed for 7 days to assess mortality. Results: When poloxamer 188 treatment was administered 10 minutes before ischemia, injury was reduced by 84 percent, from 50 percent injury in the dextran group to 8 percent injury in the poloxamer 188 group (p < 0.001). When administered 10 minutes before reperfusion, poloxamer 188 animals demonstrated a 60 percent reduction in injury compared with dextran controls (12 percent versus 29 percent). Treatment at 2 hours, but not at 4 hours, postinjury prevented substantial myocyte injury. Preservation of muscle adenosine 5′-triphosphate paralleled the decrease in myocyte injury in poloxamer 188–treated animals. Poloxamer 188 treatment significantly reduced mortality following injury (10 minutes before, 75 percent versus 25 percent survival, p = 0.0077; 2 hours after, 50 percent versus 8 percent survival, p = 0.032). Conclusion: Poloxamer 188 administered to animals decreased myocyte injury, preserved tissue adenosine 5′-triphosphate levels, and improved survival following hind-limb ischemia-reperfusion injury.

Autologous Fat Processing Via the Revolve System: Quality and Quantity of Fat Retention Evaluated in an Animal Model

BACKGROUND Currently, fat graft viability and retention cannot be reliably predicted. The reasons for this variability are not fully understood, although fat processing has been implicated. OBJECTIVES The authors compare the in vitro quantity and in vivo fat retention from lipoaspirate processed by the Revolve system (LifeCell, Bridgewater, New Jersey) compared with centrifugation and decantation. METHODS Ten patients were enrolled in this prospective study. Lipoaspirate from each patient was processed by each of 3 methods: decantation, centrifugation, and the Revolve system. Biochemical characteristics and free oil, adipose, and aqueous phases of the processed fats were determined. Fat grafts were implanted in nude mice; volume retention and quality of the fat grafts were evaluated after 28 days. Viability of retained fat was demonstrated by intact adipocytes and neovascularization on histology. RESULTS Of the 10 patients, 9 were women and 1 was a man. Mean patient age was 40.7 ± 8.9 years (range, 30-55 years). Fat tissue obtained from all methods had good physiological properties with neutral pH and isotonic salt concentrations. The Revolve system yielded significantly less blood cell debris, a higher percentage of adipose tissue, and a lower percentage of free oil compared with the other 2 methods. Fat tissue retention from Revolve samples was significantly higher (73.2%) than that from decanted samples (37.5%) and similar to that from centrifuged samples (67.7%). CONCLUSIONS The Revolve system produced physiologically compatible, preinjection fat with reduced contaminants and free oil in conjunction with high fat content. In an animal model, volume retention of Revolve-processed fat grafts was significantly greater than decanted samples. The Revolve system presents a fat-processing option that was less time-consuming, easier to use, and more efficient in this study than standard centrifugation or decantation.

A Novel Murine Island Skin Flap for Ischemic Preconditioning

BACKGROUND Ischemia reperfusion injury is a well-known phenomenon affecting skin flap viability. One method to improve flap viability is ischemic preconditioning. Previous murine flap models used random flaps. We developed a single pedicle island skin flap which allows us to create true ischemia by clamping the single pedicle. Our first aim was to describe a novel murine skin flap model with a definable, reproducible injury. Our second aim was to test the usefulness of this model by demonstrating mitigation of injury via ischemic preconditioning. MATERIALS AND METHODS Dorsal lateral thoracic artery pedicle island skin flaps (3.5 x 1.5 cm) were elevated in 39 male C57/BL6 mice: a Control group (n = 7), 10 h of ischemia (n = 21), and Preconditioning (2 cycles of 20 min ischemia: 20 minutes reperfusion) + 10-h ischemia (n = 11). After flap elevation, a silicon sheet barrier was placed. The axial pedicles were occluded, and the flaps were inset with 6-0 prolene. In all mice, ischemia was followed by 1 wk of reperfusion. At 1 wk, percent necrosis was measured and an analysis of variance was performed. RESULTS The percent of flap necrosis was 1.1% +/- 1.11% in controls. Animals that were subjected to 10 h of ischemia developed 33.14% +/- 7.23% necrosis. Preconditioned animals that underwent 10 h of ischemia demonstrated a 43% reduction in necrosis (18.82% +/- 5.68%). There was a statistically significant difference among all groups (P < or = 0.001). CONCLUSION Rat models have been the standard for skin flap experiments. We have developed a novel murine single pedicle island skin flap model with reproducible injury. This model has numerous advantages, including ease of handling, low cost, appropriateness for biomedical studies, and the availability of genetically altered animals. We also confirmed this models usefulness in a study of mitigation of ischemia reperfusion injury through ischemic preconditioning.

Sequential limb ischemia demonstrates remote postconditioning protection of murine skeletal muscle.

Background: Ischemic postconditioning, the process of exposing tissues to brief cycles of ischemia-reperfusion after critical ischemia, can mitigate local ischemia-reperfusion injury. Remote protection of skeletal muscle has never been demonstrated in postconditioning models of ischemia-reperfusion injury. Methods: Mice were subjected to 2 hours of ipsilateral hind limb ischemia followed by reperfusion. Contralateral limb ischemia was subsequently induced for 2 hours after either 0 (n = 6), 20 (n = 6), or 120 (n = 5) minutes of ipsilateral limb reperfusion. These groups were compared with animals subjected to bilateral simultaneous injury (n = 8) and sham animals that did not undergo ischemia (n = 6). The gastrocnemius muscles were harvested for histologic evaluation, and injury was recorded as the percentage of injured fibers. Results: The first limbs undergoing injury in the 20-minute interval group had a 59 percent injury reduction compared with contralateral limbs (16.0 ± 2.4 percent versus 39.5 ± 6.5 percent) after 24 hours of reperfusion and 62 percent reduction after 48 hours (24.4 ± 3.0 percent versus 63.6 ± 5.5 percent). In animals with no interval or a 120-minute interval between the onset of limb ischemia, there was no significant difference in injury between hind limbs. The injury in these groups was similar to that in hind limbs subjected to simultaneous bilateral ischemia. Conclusions: A 20-minute reperfusion interval between hind limb ischemia significantly protects against injury in the initially ischemic limb, while similar injury is observed with simultaneous ischemia or an interval of 120 minutes. This study demonstrates remote postconditioning of skeletal muscle and may lead to the development of post hoc therapies.