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.A mong the most critical functions of the mammalian gut mucosa is to provide a barrier to luminal microbes and toxins while simultaneously allowing for the necessary digestion and absorption of dietary nutrients. The molecular mechanisms that govern barrier function are incompletely understood, but it is clear that under conditions of starvation and disease, the gut barrier becomes impaired, leading to significant morbidity and mortality (1-8). Trophic enteral feeding to prevent gut mucosal atrophy and resultant barrier dysfunction has become part of the standard treatment of intensive care unit patients (9-14). The mechanism(s) responsible for the beneficial effects of trophic feeding are not understood.Intestinal alkaline phosphatase (IAP), a brush-border protein that hydrolyzes monophosphate esters, is expressed exclusively in villus-associated enterocytes and is considered an excellent marker for crypt-villus differentiation (15-18). Narisawa et al. (19) reported that compared with their wild-type (WT) littermates, mice lacking IAP gained more weight under conditions of a high-fat diet. In addition, several studies have shown that the IAP enzyme is capable of detoxifying LPS, likely through dephosphorylation of the lipid A moiety, the primary source of its endotoxic effects (20). Despite these few reports, the physiological role of IAP within the gut has not been elucidated. Results and DiscussionTo examine the functional role of IAP, we developed in vitro model systems using intestinal cell lines (T84, HT-29, and IEC-6) that express little or no IAP under basal conditions. Stable cell lines were created that overexpress IAP (Fig. 1A), and enzyme assays were used to determine the cellular localization of the ectopically expressed IAP protein. The results in Fig. 1B show that parent cells make little, if any, endogenous IAP. In contrast, large amounts of IAP enzyme are seen in the stably transfected cells. Importantly, the vast majority of the IAP activity is in the membrane fraction as opposed to the cytosol. Fig. 1C shows the results of the control experiment used to validate our separation of membranous and cytosolic fractions, confirming that the majority of the MAPK enzyme activity exists within the cytosol rather than the membrane. This membrane ...

Higher aspiration pressures up to -0.83 atm did not affect fat graft viability in vivo. Positive pressure up to 6 atm also did not affect fat graft viability. The degree of shear stress, which is a function of flow rate, did significantly affect fat graft viability. Fat grafts injected slowly with low shear stress significantly outperformed fat injected with high shear stress. These data suggest that shear stress is a more important variable regarding fat graft viability than pressure.

The McGivney hemorrhoidal ligator (MHL), a band designed to cause tissue necrosis, is the preferred experimental tool to create hindlimb ischemia-reperfusion (I/R) injury in rodents. This report defines and compares the ex vivo band tension exerted by MHL and orthodontic rubber bands (ORBs) along with select in vivo characteristics of I/R. As to method, ex vivo band tension was measured over relevant diameters using a tensiometer. In vivo assessment of murine limb perfusion during ischemia with ORB and MHL was compared using laser Doppler imaging and measurement of wet weight-to-dry weight ratio. Neuromuscular scoring and histological extent of muscle fiber injury after I/R with MHL and ORB were also compared. A dose-response curve, between the duration of ORB-induced I/R with both mitochondrial activity (methyl-thiazol-tetrazolium) or tail perfusion [laser Doppler imaging (LDI)], was generated. As a results, ex vivo measurements showed that ORB exerted significantly less force than the MHL. Despite less tension in ORB, in vivo testing of the ORB confirmed complete ischemia by both LDI and wet weight-to-dry weight ratio. After I/R, caused by ORB, there was significantly less neuromuscular dysfunction. Histological assessment confirmed similar degrees of muscle fiber injury after I/R with either the MHL or ORB. Increasing durations of ischemia created by the ORB followed by reperfusion significantly decreased mitochondrial activity and tail perfusion after 24 h of ischemia. In conclusions, ORB produced similar levels of tissue ischemia in murine models of limb I/R with fewer levels of nonspecific injury. ORB may be the preferred model for selected studies of limb I/R.

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