Mesenteric lymphatic vessels adapt to mesenteric venous hypertension by becoming weaker pumps.

Lymphangions, the segments of lymphatic vessels between two adjacent lymphatic valves, actively pump lymph. Acute changes in transmural pressure and lymph flow have profound effects on lymphatic pump function in vitro. Chronic changes in pressure and flow in vivo have also been reported to lead to significant changes in lymphangion function. Because changes in pressure and flow are both cause and effect of adaptive processes, characterizing adaptation requires a more fundamental analysis of lymphatic muscle properties. Therefore, the purpose of the present work was to use an intact lymphangion isovolumetric preparation to evaluate changes in mesenteric lymphatic muscle mechanical properties and the intracellular Ca(2+) in response to sustained mesenteric venous hypertension. Bovine mesenteric veins were surgically occluded to create mesenteric venous hypertension. Postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 6) and sham surgery (Sham; n = 6) animals were isolated and evaluated 3 days after the surgery. Spontaneously contracting MVH vessels generated end-systolic active tension and end-diastolic active tension lower than the Sham vessels. Furthermore, steady-state active tension and intracellular Ca(2+) concentration levels in response to KCl stimulation were also significantly lower in MVH vessels compared with those of the Sham vessels. There was no significant difference in passive tension in lymphatic vessels from the two groups. Taken together, these results suggest that following 3 days of mesenteric venous hypertension, postnodal mesenteric lymphatic vessels adapt to become weaker pumps with decreased cytosolic Ca(2+) concentration.

Adaptation of mesenteric lymphatic vessels to prolonged changes in transmural pressure.

In vitro studies have revealed that acute increases in transmural pressure increase lymphatic vessel contractile function. However, adaptive responses to prolonged changes in transmural pressure in vivo have not been reported. Therefore, we developed a novel bovine mesenteric lymphatic partial constriction model to test the hypothesis that lymphatic vessels exposed to higher transmural pressures adapt functionally to become stronger pumps than vessels exposed to lower transmural pressures. Postnodal mesenteric lymphatic vessels were partially constricted for 3 days. On postoperative day 3, constricted vessels were isolated, and divided into upstream (UP) and downstream (DN) segment groups, and instrumented in an isolated bath. Although there were no differences between the passive diameters of the two groups, both diastolic diameter and systolic diameter were significantly larger in the UP group than in the DN group. The pump index of the UP group was also higher than that in the DN group. In conclusion, this is the first work to report how lymphatic vessels adapt to prolonged changes in transmural pressure in vivo. Our results suggest that vessel segments upstream of the constriction adapt to become both better fluid conduits and lymphatic pumps than downstream segments.

Decreased myosin phosphatase target subunit 1(MYPT1) phosphorylation via attenuated rho kinase and zipper-interacting kinase activities in edematous intestinal smooth muscle.

BACKGROUND: Intestinal edema development after trauma resuscitation inhibits intestinal motility which results in ileus, preventing enteral feeding and compromising patient outcome. We have shown previously that decreased intestinal motility is associated with decreased smooth muscle myosin light chain (MLC) phosphorylation. The purpose of the present study was to investigate the mechanism of edema-induced decreases in MLC in a rodent model of intestinal edema. METHODS: Intestinal edema was induced by a combination of resuscitation fluid administration and mesenteric venous hypertension. Sham operated animals served as controls. Contractile activity and alterations in the regulation of MLC including the regulation of MLC kinase (MLCK) and MLC phosphatase (MLCP) were measured. KEY RESULTS: Contraction amplitude and basal tone were significantly decreased in edematous intestinal smooth muscle compared with non-edematous tissue. Calcium sensitivity was also decreased in edematous tissue compared with non-edematous intestinal smooth muscle. Although inhibition of MLCK decreased contractile activity significantly less in edematous tissue compared with non-edematous tissue, MLCK activity in tissue lysates was not significantly different. Phosphorylation of MYPT was significantly lower in edematous tissue compared with non-edematous tissue. In addition, activities of both rho kinase and zipper-interacting kinase were significantly lower in edematous tissue. CONCLUSIONS & INFERENCES: We conclude from these data that interstitial intestinal edema inhibits MLC phosphorylation predominantly by decreasing inhibitory phosphorylation of the MLC targeting subunit (MYPT1) of MLC phosphatase via decreased ROCK and ZIPK activities, resulting in more MLC phosphatase activity.

Evaluating the effects of immediate application of negative pressure therapy after decompression from abdominal compartment syndrome in an experimental porcine model.

PURPOSE: The purpose of this large-animal study was to assess the safety and effects of negative pressure therapy (NPT) when used as temporary abdominal closure in the immediate post-decompression period after abdominal compartment syndrome (ACS). METHODS: Using a hemorrhagic shock/resuscitation and mesenteric venous pressure elevation model, ACS was physiologically induced in 12 female Yorkshire swine. At decompression, animals were allocated to either NPT (n = 6) or Bogota bag (n = 6) as temporary abdominal closure and studied for a period of 48 h or until death. Outcomes measured included morbidity and mortality, as well as hemodynamic parameters, ventilator-related measurements, blood gases, coagulation factors, and organ (liver, kidney, lung, and intestinal) edema and histology at the time of death/sacrifice. RESULTS: All animals developed ACS. Early application of NPT was associated with decreases in mesenteric venous and central venous pressure, and significantly increased drainage of peritoneal fluid. In addition, there was no increase in the incidence of mortality, recurrent intra-abdominal hypertension/ACS, or any deleterious effects on markers of organ injury. CONCLUSIONS: Early application of NPT in this porcine ACS model is safe and does not appear to be associated with an increased risk of recurrent intra-abdominal hypertension. The results of this animal study suggest that the application of NPT following decompression from ACS results in greater peritoneal fluid removal and may translate into augmented intestinal edema resolution secondary to more favorable fluid flux profiles.

Evaluation of gravimetric techniques to estimate the microvascular filtration coefficient.

Microvascular permeability to water is characterized by the microvascular filtration coefficient (K(f)). Conventional gravimetric techniques to estimate K(f) rely on data obtained from either transient or steady-state increases in organ weight in response to increases in microvascular pressure. Both techniques result in considerably different estimates and neither account for interstitial fluid storage and lymphatic return. We therefore developed a theoretical framework to evaluate K(f) estimation techniques by 1) comparing conventional techniques to a novel technique that includes effects of interstitial fluid storage and lymphatic return, 2) evaluating the ability of conventional techniques to reproduce K(f) from simulated gravimetric data generated by a realistic interstitial fluid balance model, 3) analyzing new data collected from rat intestine, and 4) analyzing previously reported data. These approaches revealed that the steady-state gravimetric technique yields estimates that are not directly related to K(f) and are in some cases directly proportional to interstitial compliance. However, the transient gravimetric technique yields accurate estimates in some organs, because the typical experimental duration minimizes the effects of interstitial fluid storage and lymphatic return. Furthermore, our analytical framework reveals that the supposed requirement of tying off all draining lymphatic vessels for the transient technique is unnecessary. Finally, our numerical simulations indicate that our comprehensive technique accurately reproduces the value of K(f) in all organs, is not confounded by interstitial storage and lymphatic return, and provides corroboration of the estimate from the transient technique.

A murine model for the study of edema induced intestinal contractile dysfunction.

BACKGROUND: We have published extensively regarding the effects of edema on intestinal contractile function. However, we have found the need to expand our model to mice to take advantage of the much larger arsenal of research support, especially in terms of transgenic mouse availability and development. To that end, we have developed and validated a hydrostatic intestinal edema model in mice. METHODS: Male C57 Black 6 mice were subjected to a combination of high volume crystalloid resuscitation and mesenteric venous hypertension in an effort to induce hydrostatic intestinal edema. Wet to dry ratios, myeloperoxidase activity, mucosal injury scoring, STAT-3 nuclear activation, phosphorylated STAT-3 levels, NF-κB nuclear activation, myosin light chain phosphorylation, intestinal contractile activity, and intestinal transit were measured to evaluate the effects of the model. KEY RESULTS: High volume crystalloid resuscitation and mesenteric venous hypertension resulted in the development of significant intestinal edema without an increase in myeloperoxidase activity or mucosal injury. Edema development was associated with increases in STAT-3 and NF-κB nuclear activation as well as phosphorylated STAT-3. There was a decrease in myosin light chain phosphorylation, basal and maximally stimulated intestinal contractile activity, and intestinal transit. CONCLUSION & INFERENCES: Hydrostatic edema in mice results in activation of a signal transduction profile that culminates in intestinal contractile dysfunction. This novel model allows for advanced studies into the pathogenesis of hydrostatic edema induced intestinal contractile dysfunction.

Balance point characterization of interstitial fluid volume regulation.

The individual processes involved in interstitial fluid volume and protein regulation (microvascular filtration, lymphatic return, and interstitial storage) are relatively simple, yet their interaction is exceedingly complex. There is a notable lack of a first-order, algebraic formula that relates interstitial fluid pressure and protein to critical parameters commonly used to characterize the movement of interstitial fluid and protein. Therefore, the purpose of the present study is to develop a simple, transparent, and general algebraic approach that predicts interstitial fluid pressure (P(i)) and protein concentrations (C(i)) that takes into consideration all three processes. Eight standard equations characterizing fluid and protein flux were solved simultaneously to yield algebraic equations for P(i) and C(i) as functions of parameters characterizing microvascular, interstitial, and lymphatic function. Equilibrium values of P(i) and C(i) arise as balance points from the graphical intersection of transmicrovascular and lymph flows (analogous to Guyton's classical cardiac output-venous return curves). This approach goes beyond describing interstitial fluid balance in terms of conservation of mass by introducing the concept of inflow and outflow resistances. Algebraic solutions demonstrate that P(i) and C(i) result from a ratio of the microvascular filtration coefficient (1/inflow resistance) and effective lymphatic resistance (outflow resistance), and P(i) is unaffected by interstitial compliance. These simple algebraic solutions predict P(i) and C(i) that are consistent with reported measurements. The present work therefore presents a simple, transparent, and general balance point characterization of interstitial fluid balance resulting from the interaction of microvascular, interstitial, and lymphatic function.

Edemagenic gain and interstitial fluid volume regulation.

Under physiological conditions, interstitial fluid volume is tightly regulated by balancing microvascular filtration and lymphatic return to the central venous circulation. Even though microvascular filtration and lymphatic return are governed by conservation of mass, their interaction can result in exceedingly complex behavior. Without making simplifying assumptions, investigators must solve the fluid balance equations numerically, which limits the generality of the results. We thus made critical simplifying assumptions to develop a simple solution to the standard fluid balance equations that is expressed as an algebraic formula. Using a classical approach to describe systems with negative feedback, we formulated our solution as a "gain" relating the change in interstitial fluid volume to a change in effective microvascular driving pressure. The resulting "edemagenic gain" is a function of microvascular filtration coefficient (K(f)), effective lymphatic resistance (R(L)), and interstitial compliance (C). This formulation suggests two types of gain: "multivariate" dependent on C, R(L), and K(f), and "compliance-dominated" approximately equal to C. The latter forms a basis of a novel method to estimate C without measuring interstitial fluid pressure. Data from ovine experiments illustrate how edemagenic gain is altered with pulmonary edema induced by venous hypertension, histamine, and endotoxin. Reformulation of the classical equations governing fluid balance in terms of edemagenic gain thus yields new insight into the factors affecting an organ's susceptibility to edema.

Application of local heat induces capillary recruitment in the Pallid bat wing.

Skin blood flow increases in response to local heat due to sensorineural and nitric oxide (NO)-mediated dilation. It has been previously demonstrated that arteriolar dilation is inhibited with NO synthase (NOS) blockade. Flow, nonetheless, increases with local heat. This implies that the previously unexamined nonarteriolar responses play a significant role in modulating flow. We thus hypothesized that local heating induces capillary recruitment. We heated a portion (3 cm2) of the Pallid bat wing from 25 degrees C to 37 degrees C for 20 min, and measured changes in terminal feed arteriole (approximately 25 microm) diameter and blood velocity to calculate blood flow (n = 8). Arteriolar dilation was reduced with NOS and sensorineural blockade using a 1% (wt/vol) NG-nitro-L-arginine methyl ester (L-NAME) and 2% (wt/vol) lidocaine solution (n = 8). We also measured changes in the number of perfused capillaries, and the time precapillary sphincters were open with (n = 8) and without (n = 8) NOS plus sensorineural blockade. With heat, the total number of perfused capillaries increased 92.7 +/- 17.9% (P = 0.011), and a similar increase occurred despite NOS plus sensorineural blockade 114.4 +/- 30.0% (P = 0.014). Blockade eliminated arteriolar dilation (-4.5 +/- 2.1%). With heat, the percent time precapillary sphincters remained open increased 32.3 +/- 6.0% (P = 0.0006), and this increase occurred despite NOS plus sensorineural blockade (34.1 +/- 5.8%, P = 0.0004). With heat, arteriolar blood flow increased (187.2 +/- 28.5%, P = 0.00003), which was significantly attenuated with NOS plus sensorineural blockade (88.6 +/- 37.2%, P = 0.04). Thus, capillary recruitment is a fundamental microvascular response to local heat, independent of arteriolar dilation and the well-documented sensorineural and NOS mechanisms mediating the response to local heat.

Local heat produces a shear-mediated biphasic response in the thermoregulatory microcirculation of the Pallid bat wing.

Investigators report that local heat causes an increase in skin blood flow consisting of two phases. The first is solely sensory neural, and the second is nitric oxide mediated. We hypothesize that mechanisms behind these two phases are causally linked by shear stress. Because microvascular blood flow, endothelial shear stress, and vessel diameters cannot be measured in humans, bat wing arterioles (26.6 +/- 0.3, 42.0 +/- 0.4, and 58.7 +/- 2.2 microm) were visualized noninvasively on a transparent heat plate via intravital microscopy. Increasing plate temperature from 25 to 37 degrees C increased flow in all three arterial sizes (137.1 +/- 0.3, 251.9 +/- 0.5, and 184.3 +/- 0.6%) in a biphasic manner. With heat, diameter increased in large arterioles (n = 6) by 8.7 +/- 0.03% within 6 min, medium arterioles (n = 8) by 19.7 +/- 0.5% within 4 min, and small arterioles (n = 8) by 31.6 +/- 2.2% in the first minute. Lidocaine (0.2 ml, 2% wt/vol) and NG-nitro-L-arginine methyl ester (0.2 ml, 1% wt/vol) were applied topically to arterioles (approximately 40 microm) to block sensory nerves, modulate shear stress, and block nitric oxide generation. Local heat caused only a 10.4 +/- 5.5% increase in diameter with neural blockade (n = 8) and only a 7.5 +/- 4.1% increase in diameter when flow was reduced (n = 8), both significantly lower than control (P < 0.001). Diameter and flow increases were significantly reduced with NG-nitro-L-arginine methyl ester application (P < 0.05). Our novel thermoregulatory animal model illustrates 1) regulation of shear stress, 2) a nonneural component of the first phase, and 3) a shear-mediated second phase. The time course of dilation suggests that early dilation of small arterioles increases flow and enhances second-phase dilation of the large arterioles.

Inhibition of intestinal transit by resuscitation-induced gut edema is reversed by L-NIL.

BACKGROUND: Post-resuscitation gut edema and associated gut dysfunction is a common and significant clinical problem that occurs after traumatic injury and shock. We have shown previously that gut edema without ischemia/reperfusion injury delays intestinal transit [1]. We hypothesized that gut edema increases expression of inducible nitric oxide synthase (iNOS) protein, and that selective iNOS inhibition using L-NIL reverses the delayed intestinal transit associated with gut edema. MATERIALS AND METHODS: One hour prior to laparotomy, rats were pretreated with 10 mg/kg body weight of intraperitoneal L-NIL or saline vehicle and underwent 80 ml/kg body weight of 0.9% saline + superior mesenteric venous pressure elevation (Edema) or sham surgery (Sham). A duodenal catheter was placed to allow injection of a fluorescent dye for the measurement of intestinal transit. At 6 h, the small bowel was divided and the mean geometric center (MGC) of fluorescent dye was measured to determine transit. Ileum was harvested for histological assessment of mucosal injury, evaluation of iNOS protein expression by Western blotting, and MPO activity. Tissue water was determined using the wet-to-dry weight ratio to assess gut edema. Data are expressed as mean +/- SEM, n = 3-6 and * = P <0.05 using ANOVA. RESULTS: Gut edema, expressed as increased wet-to-dry ratio, was associated with decreased intestinal transit and elevated iNOS protein expression. Pretreatment with l-NIL improved intestinal transit and decreased expression of iNOS protein without decreasing intestinal tissue water compared to edema animals. There was no difference in mucosal injury or MPO activity among groups. CONCLUSION: Gut edema delays intestinal transit via an iNOS-mediated mechanism.

A new paradigm for graduate research and training in the biomedical sciences and engineering.

98Emphasis on the individual investigator has fostered discovery for centuries, yet it is now recognized that the complexity of problems in the biomedical sciences and engineering requires collaborative efforts from individuals having diverse training and expertise. Various approaches can facilitate interdisciplinary interactions, but we submit that there is a critical need for a new educational paradigm for the way that we train biomedical engineers, life scientists, and mathematicians. We cannot continue to train graduate students in isolation within single disciplines, nor can we ask any one individual to learn all the essentials of biology, engineering, and mathematics. We must transform how students are trained and incorporate how real-world research and development are done-in diverse, interdisciplinary teams. Our fundamental vision is to create an innovative paradigm for graduate research and training that yields a new generation of biomedical engineers, life scientists, and mathematicians that is more diverse and that embraces and actively pursues a truly interdisciplinary, team-based approach to research based on a known benefit and mutual respect. In this paper, we describe our attempt to accomplish this via focused training in biomechanics, biomedical optics, mathematics, mechanobiology, and physiology. The overall approach is applicable, however, to most areas of biomedical research.

Optimal lymphatic vessel structure.

Lymphatic vessels transport excess interstitial fluid from the low-pressure tissues to the higher pressure veins. The basic structural unit of lymphatic vessels is the lymphangion, a segment of the vessel separated by two unidirectional valves. Lymphangions cyclically contract like ventricles and can actively pump lymph. Lymphangions, as conduit vessels, also can act as arteries, and resist lymph flow. Functional parameters such as pressures, flow, and efficiency are determined by structural parameters like length, radius, and wall thickness. Since these structural parameters are unalterable experimentally, we developed a computational model to study the effect of a particular structural parameter, lymphangion length, to a particular functional variable, lymph flow. The model predicts that flow is a bimodal function of length, exhibiting an optimal length in the same order of magnitude as that observed experimentally. In essence, when the length to radius ratio is small, lymphangions act more like ventricles, where longer lengths yield greater chamber volume and thus lymph pumped. When the length to radius ratio is large, lymphangions act more like arteries, where longer lengths yield greater resistances to flow. This approach provides the means to explore how lymphatic vessel structure is optimized in a variety of conditions.

Myocardial fluid balance.

Fluid accumulation in the cardiac interstitium or myocardial edema is a common manifestation of many clinical states. Specifically, cardiac surgery includes various interventions and pathophysiological conditions that cause or worsen myocardial edema including cardiopulmonary bypass and cardioplegic arrest. Myocardial edema should be a concern for clinicians as it has been demonstrated to produce cardiac dysfunction. This article will briefly discuss the factors governing myocardial fluid balance and review the evidence of myocardial edema in various pathological conditions. In particular, myocardial microvascular, interstitial, and lymphatic interactions relevant to the field of cardiac surgery will be emphasized.

Effect of sialyl Lewis(x) selectin blockade on myocardial protection during cardioplegic arrest and reperfusion.

PURPOSE: Selectins play a crucial role in the neutrophil-mediated myocardial injury associated with ischemia/reperfusion. We investigated the effect of selectin inhibition on neutrophil-endothelial cell adhesion, myocardial water content, and left ventricular (LV) recovery after cardiopulmonary bypass (CPB) and cardioplegia. METHODS: Dogs were subjected to CPB and 60 minutes of hypothermic cardioplegia. A selectin inhibitor (SI) (25 mg/kg) was given five minutes prior to CPB and as a continuous infusion (5 mg/kg/h) throughout CPB (n = 6). Saline-treated controls (n = 6) received identical volumes. Preload recruitable stroke work (PRSW) was calculated by sonomicrometry and micromanometry. Myocardial water content was determined by microgravimetry. Myeloperoxidase (MPO) activity was measured to quantify polymorphonuclear neutrophil (PMN) infiltration. RESULTS: SI did not attenuate PRSW as well as post-CPB MPO tissue activity. While we found no difference in myocardial water gain between groups 120 minutes post-CPB, there was better edema resolution with SI. CONCLUSIONS: Selectin antagonism does not reduce CPB-associated myocardial injury, and contractile recovery is not enhanced.

Flow in lymphatic networks: interaction between hepatic and intestinal lymph vessels.

OBJECTIVE: Lymph from both the liver and intestine flows into the cisterna chyli. We hypothesized that increasing liver lymph flow would increase cisterna chyli pressure and, thereby, decrease intestinal lymph flow, potentiating intestinal edema formation. METHODS: Anesthetized dogs were instrumented to measure and manipulate portal vein pressure and cisterna chyli pressure. The effects of directly increasing portal pressure with and without directly increasing cisterna chyli pressure on intestinal wet-to-dry ratio and intestinal ascites formation rate were determined. Target values for portal and cisterna chyli pressures were determined following elevation of inferior vena caval pressure to levels seen in patients with obstructive caval disease. RESULTS: Direct elevation of portal pressure (P(port)) alone to 17.5 mm Hg caused a significant increase in intestinal wet-to-dry ratio (3.98 +/- 0.24 vs. 3.40 +/- 0.43) and the rate of ascites formation (0.36 +/- 0.12 vs. 0.05 +/- 0.03 mL/g dry wt/h). Simultaneous direct elevation of cisterna chyli pressure to 6.0 mm Hg and P(port) to 17.5 mm Hg caused further increases in intestinal wet-to-dry ratio (5.52 +/- 1.20) and ascites formation (0.57 +/- 0.11 mL/g dry wt./h). CONCLUSIONS: Inferior vena caval hypertension increases liver lymph flow that elevates cisterna chyli pressure, which inhibits intestinal lymph flow and augments intestinal edema formation.

Esmolol and cardiopulmonary bypass during reperfusion reduce myocardial infarct size in dogs.

BACKGROUND: Infarct size can be reduced by beta-blockade in acute myocardial ischemia. However it is unknown whether myocardial salvage is still effective when beta-blockade is limited to reperfusion. METHODS: After initiation of cardiopulmonary bypass, 20 dogs were submitted to 2 hours of regional left ventricular ischemia, followed by 2 hours of reperfusion. In 11 dogs beta-blockade was started with the onset of reperfusion (esmolol group). The remaining dogs received no treatment (control, n = 9). Infarct size was determined by tetrazolium chloride staining. Myocardial water content (MWC) and ultrastructural damage (electronmicroscopy) were determined from transmural biopsies. RESULTS: Infarct size was significantly smaller in the esmolol group compared with control (49% versus 68%, p < 0.05). After 2 hours ischemia there was no difference in MWC between groups, whereas after 2 hours reperfusion MWC of ischemic myocardium was significantly lower in the esmolol group than in the control (p < 0.05). Ultrastructural changes were typical for ischemia-reperfusion injury in both groups. CONCLUSIONS: Beta-blockade may be cardioprotective during reperfusion through various mechanisms and may enhance myocardial salvage, even when treatment is initiated as late as with the onset of reperfusion.

Protein washdown as a defense mechanism against myocardial edema.

Myocardial edema occurs in many pathological conditions. We hypothesized that protein washdown at the myocardial microvascular exchange barrier would change the distribution of interstitial proteins from large to small molecules and diminish the effect of washdown on the colloid osmotic pressure (COP) of interstitial fluid and lymph. Dogs were instrumented with coronary sinus balloon-tipped catheters and myocardial lymphatic cannulas to manipulate myocardial lymph flow and to collect lymph. Myocardial venous pressure was elevated by balloon inflation to increase transmicrovascular fluid flux and myocardial lymph flow. COP of lymph was measured directly and was also calculated from protein concentration. Decreases occurred in both protein concentration and COP of lymph. The proportion of lymph protein accounted for by albumin increased significantly, whereas that accounted for by beta-lipoprotein decreased significantly. The change in the calculated plasma-to-lymph COP gradient was significantly greater than the change in the measured COP gradient. We conclude that the change in the distribution of interstitial fluid protein species decreases the effect of protein washdown on interstitial fluid COP and limits its effectiveness as a defense mechanism against myocardial edema formation.