Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

Dichotomous effects of in vivo and in vitro ionizing radiation exposure on lymphatic function.

On the one hand, lymphatic dysfunction induces interstitial edema and inflammation. On the other hand, the formation of edema and inflammation induce lymphatic dysfunction. However, informed by the earlier reports of undetected apoptosis of irradiated lymphatic endothelial cells (LECs) in vivo, lymphatic vessels are commonly considered inconsequential to ionizing radiation (IR)-induced inflammatory injury to normal tissues. Primarily because of the lack of understanding of the acute effects of IR exposure on lymphatic function, acute edema and inflammation, common sequelae of IR exposure, have been ascribed solely to blood vessel damage. Therefore, in the present study, the lymphatic acute responses to IR exposure were quantified to evaluate the hypothesis that IR exposure impairs lymphatic pumping. Rat mesenteric lymphatic vessels were irradiated in vivo or in vitro, and changes in pumping were quantified in isolated vessels in vitro. Compared with sham-treated vessels, pumping was lowered in lymphatic vessels irradiated in vivo but increased in vessels irradiated in vitro. Furthermore, unlike in blood vessels, the acute effects of IR exposure in lymphatic vessels were not mediated by nitric oxide-dependent pathways in either in vivo or in vitro irradiated vessels. After cyclooxygenase blockade, pumping was partially restored in lymphatic vessels irradiated in vitro but not in vessels irradiated in vivo. Taken together, these findings demonstrated that lymphatic vessels are radiosensitive and LEC apoptosis alone may not account for all the effects of IR exposure on the lymphatic system.NEW & NOTEWORTHY Earlier studies leading to the common belief that lymphatic vessels are radioresistant either did not characterize lymphatic pumping, deemed necessary for the resolution of edema and inflammation, or did it in vivo. By characterizing pumping in vitro, the present study, for the first time, demonstrated that lymphatic pumping was impaired in vessels irradiated in vivo and enhanced in vessels irradiated in vitro. Furthermore, the pathways implicated in ionizing radiation-induced blood vessel damage did not mediate lymphatic responses.

Algebraic formulas characterizing an alternative to Guyton's graphical analysis relevant for heart failure.

Although Guyton's graphical analysis of cardiac output-venous return has become a ubiquitous tool for explaining how circulatory equilibrium emerges from heart-vascular interactions, this classical model relies on a formula for venous return that contains unphysiological assumptions. Furthermore, Guyton's graphical analysis does not predict pulmonary venous pressure, which is a critical variable for evaluating heart failure patients' risk of pulmonary edema. Therefore, the purpose of the present work was to use a minimal closed-loop mathematical model to develop an alternative to Guyton's analysis. Limitations inherent in Guyton's model were addressed by 1) partitioning the cardiovascular system differently to isolate left ventricular function and lump all blood volumes together, 2) linearizing end-diastolic pressure-volume relationships to obtain algebraic solutions, and 3) treating arterial pressures as constants. This approach yielded three advances. First, variables related to morbidities associated with left ventricular failure were predicted. Second, an algebraic formula predicting left ventricular function was derived in terms of ventricular properties. Third, an algebraic formula predicting flow through the portion of the system isolated from the left ventricle was derived in terms of mechanical properties without neglecting redistribution of blood between systemic and pulmonary circulations. Although complexities were neglected, approximations necessary to obtain algebraic formulas resulted in minimal error, and predicted variables were consistent with reported values.

A Modern View of the Interstitial Space in Health and Disease.

Increases in the volume of the interstitial space are readily recognized clinically as interstitial edema formation in the loose connective tissue of skin, mucosa, and lung. However, the contents and the hydrostatic pressure of this interstitial fluid can be very difficult to determine even in experimental settings. These difficulties have long obscured what we are beginning to appreciate is a dynamic milieu that is subject to both intrinsic and extrinsic regulation. This review examines current concepts regarding regulation of interstitial volume, pressure, and flow and utilizes that background to address three major topics of interest that impact IV fluid administration. The first of these started with the discovery that excess dietary salt can be stored non-osmotically in the interstitial space with minimal impact on vascular volume and pressures. This led to the hypothesis that, along with the kidney, the interstitial space plays an active role in the long-term regulation of blood pressure. Second, it now appears that hypovolemic shock leads to systemic inflammatory response syndrome principally through the entry of digestive enzymes into the intestinal interstitial space and the subsequent progression of enzymes and inflammatory agents through the mesenteric lymphatic system to the general circulation. Lastly, current evidence strongly supports the non-intuitive view that the primary factor leading to inflammatory edema formation is a decrease in interstitial hydrostatic pressure that dramatically increases microvascular filtration.

A plumber's guide to the cardiovascular system.

Blood flow through the cardiovascular system is governed by the same physical rules that govern the flow of water through domestic plumbing. Using this analogy in a teaching laboratory, a model of the cardiovascular system constructed of pumps and pipes was used to demonstrate the basic interactions of pressure, flow, and resistance in a regulated system, with student volunteers providing the operational actions and regulatory components. The model was used to validate predictions and explore solutions prompted by student discussion. This interactive teaching laboratory provides an engaging experiential exercise that demonstrates regulation of flow and pressure in an intact cardiovascular system with apposite changes in heart rate and resistance. In addition, the system provides strong clinical correlates and illustrates how that regulated system responds to challenges such as heart failure, inappropriate vasodilation, and hemorrhage. The results demonstrate that, with limited practice, the instructor can effectively guide the students to reliably reproduce physiologically appropriate results.

Adaptation of the hepatic transudation barrier to sinusoidal hypertension.

The role of the hepatic transudation barrier in determining ascites volume and protein content in chronic liver disease is poorly understood. Therefore, the purpose of the present study was to characterize how chronic sinusoidal hypertension impacts hepatic transudation barrier properties and the transudation rate. The suprahepatic inferior vena cava was surgically constricted, and animals were exposed to either short-term (SVH; 2-3 wk) or long-term venous hypertension (LVH; 5-6 wk). Compared with SVH, LVH resulted in lower peritoneal fluid pressure, ascites volume, and ascites protein concentration. The transudation barrier protein reflection coefficient was significantly higher, and the transudation barrier hydraulic conductivity, transudation rate, and transudate-to-lymph protein concentration ratio were significantly lower in LVH animals compared with SVH animals. The sensitivity of transudation rates to acute changes in interstitial fluid pressures was also significantly lower in LVH animals compared with SVH animals. In contrast, there was no detectable difference in hepatic lymph flow rate or sensitivity of lymph flow to acute changes in interstitial fluid pressures between SVH and LVH animals. Taken together, these data suggest that decreased hepatic transudation barrier permeability to fluid and protein and increased reflection coefficient led to a decrease in the hepatic contribution to ascites volume. The present work, to the best of our knowledge, is the first to quantify an anti-ascites adaptation of the hepatic transudation barrier in response to chronic hepatic sinusoidal hypertension.

Hepatic transudation barrier properties.

OBJECTIVE: Fluid and protein continuously transude from the surface of the liver. Despite a common understanding that transudation plays a critical role in hepatic interstitial and peritoneal fluid balance, transudation from the entire liver has not been studied. Therefore, the goal of the present work was to provide the first direct measurement of the hepatic transudation rate and transudation barrier properties. METHODS: Transudation rates were determined by collecting transudate from the entire liver. Hydraulic conductivity, and fluid transudation and protein reflection coefficients of the transudation barrier (formed by the subscapular interstitial matrix, capsule, and peritoneum) were determined from changes in fluid and protein transudation rates in response to hepatic venous pressure elevation. RESULTS: Following hepatic venous pressure elevation from 6.1 ± 0.9 to 11.1 ± 0.6 mm Hg, transudation rate increased from 0.13 ± 0.03 to 0.37 ± 0.03 mL/min·100 g. Transudation barrier hydraulic conductivity, fluid transudation and protein reflection coefficients (3.9 × 10-4  ± 5.7 × 10-5  mL/min·mm Hg·cm2 , 0.36 ± 0.04 mL/min·mm Hg, and 0.09 ± 0.03, respectively) were comparable to those reported for hepatic sinusoids. CONCLUSIONS: Taken together, these findings suggest that the hepatic transudation barrier is highly permeable at elevated sinusoidal pressures. These fundamental studies provide a better understanding of the hepatic transudation barrier properties and transudation under conditions that are physiologically and clinically relevant to ascites formation.

Merging Clinical Cases, Client Communication, and Physiology to Enhance Student Engagement, Learning, and Skills.

Understanding disease processes, making diagnoses, and guiding clinical therapy are predicated on an understanding of normal physiologic function. However, we have observed that many first-year students fail to appreciate the important role that a clear understanding of normal function plays in becoming well-prepared, practicing veterinarians. Students also struggle with application of basic knowledge to the diagnosis and treatment of disease, as evidenced by poor performance on exam questions requiring application. The purpose of this project was to help students link the physiologic concepts in the classroom with clinical application, as well as to improve their ability to explain those concepts to a client. We found that, as a result of this assignment, students developed a deeper understanding of physiologic processes and their clinical relevance and, subsequently, felt more confident conveying this knowledge to simulated clients. Implementation of this case project has been very well received by the students. Students improved their grasp of the material, and they indicated that the project contributed positively to their motivation to study and learn physiology.

Functional adaptation of bovine mesenteric lymphatic vessels to mesenteric venous hypertension.

Lymph flow is the primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to determine the functional responses of postnodal mesenteric lymphatic vessels. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension to elevate mesenteric lymph flow. Three days after surgery, postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 7) and sham surgery (Sham; n = 6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98 ± 0.75 min(-1); Sham: 5.42 ± 0.81 min(-1)) and fractional pump flow (MVH: 1.14 ± 0.30 min(-1); Sham: 2.39 ± 0.32 min(-1)) were significantly lower in the venous occlusion group. These results indicate that postnodal mesenteric lymphatic vessels adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.

Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

Award article: Microcirculatory Society Award for Excellence in Lymphatic Research: time course of myocardial interstitial edema resolution and associated left ventricular dysfunction.

OBJECTIVE: Although the causal relationship between acute myocardial edema and cardiac dysfunction has been established, resolution of myocardial edema and subsequent recovery of cardiac function have not been established. The time to resolve myocardial edema and the degree that cardiac function is depressed after edema resolves are not known. We therefore characterized temporal changes in cardiac function as acute myocardial edema formed and resolved. METHODS: Acute myocardial edema was induced in the canine model by elevating coronary sinus pressure for three hours. Myocardial water content and cardiac function were determined before and during coronary sinus pressure elevation, and after coronary sinus pressure restoration. RESULTS: Although no change in systolic properties was detected, accumulation of water in myocardial interstitium was associated with increased diastolic stiffness. When coronary sinus pressure was relieved, myocardial edema resolved within 180 minutes. Diastolic stiffness, however, remained significantly elevated compared with baseline values, and cardiac function remained compromised. CONCLUSIONS: The present work suggests that the cardiac dysfunction caused by the formation of myocardial edema may persist after myocardial edema resolves. With the advent of new imaging techniques to quantify myocardial edema, this insight provides a new avenue for research to detect and treat a significant cause of cardiac dysfunction.

Blood flow augmentation by intrinsic venular contraction in vivo.

Venomotion, spontaneous cyclic contractions of venules, was first observed in the bat wing 160 years ago. Of all the functional roles proposed since then, propulsion of blood by venomotion remains the most controversial. Common animal models that require anesthesia and surgery have failed to provide evidence for venular pumping of blood. To determine whether venomotion actively pumps blood in a minimally invasive, unanesthetized animal model, we reintroduced the batwing model. We evaluated the temporal and functional relationship between the venous contraction cycle and blood flow and luminal pressure. Furthermore, we determined the effect of inhibiting venomotion on blood flow. We found that the active venous contractions produced an increase in the blood flow and exhibited temporal vessel diameter-blood velocity and pressure relationships characteristic of a peristaltic pump. The presence of valves, a characteristic of reciprocating pumps, enhances the efficiency of the venular peristaltic pump by preventing retrograde flow. Instead of increasing blood flow by decreasing passive resistance, venular dilation with locally applied sodium nitroprusside decreased blood flow. Taken together, these observations provide evidence for active venular pumping of blood. Although strong venomotion may be unique to bats, venomotion has also been inferred from venous pressure oscillations in other animal models. The conventional paradigm of microvascular pressure and flow regulation assumes venules only act as passive resistors, a proposition that must be reevaluated in the presence of significant venomotion.

Strategies for modulating the inflammatory response after decompression from abdominal compartment syndrome.

BACKGROUND: Management of the open abdomen is an increasingly common part of surgical practice. The purpose of this review is to examine the scientific background for the use of temporary abdominal closure (TAC) in the open abdomen as a way to modulate the local and systemic inflammatory response, with an emphasis on decompression after abdominal compartment syndrome (ACS). METHODS: A review of the relevant English language literature was conducted. Priority was placed on articles published within the last 5 years. RESULTS/CONCLUSION: Recent data from our group and others have begun to lay the foundation for the concept of TAC as a method to modulate the local and/or systemic inflammatory response in patients with an open abdomen resulting from ACS.

Peritoneal fluid: a potential mechanism of systemic neutrophil priming in experimental intra-abdominal sepsis.

BACKGROUND: Recent studies suggest that peritoneal fluid (PF) may be an important mediator of inflammation. The aim of this study was to test the hypothesis that PF may drive systemic inflammation in intra-abdominal sepsis by representing a priming agent for neutrophils. METHODS: PF was collected 12 hours after the initiation of intra-abdominal sepsis in swine. Naive human neutrophils were primed with PF before treatment with N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate to elucidate receptor-dependent and receptor-independent mechanisms of neutrophil activation. Flow cytometry was used to quantify neutrophil surface adhesion marker expression of integrins and selectins and superoxide anion production. Additionally, proinflammatory cytokines were quantified in PF. RESULTS: PF primed neutrophils via receptor-dependent and receptor-independent mechanisms. There were significant increases in the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α in PF correlating with the development of intra-abdominal sepsis. CONCLUSIONS: PF represents a priming agent for naive polymorphonuclear cells in intra-abdominal sepsis. This may be secondary to increased levels of proinflammatory cytokines. Strategies to reduce the amount of PF may decrease the systemic inflammatory response by reducing a priming agent for neutrophils.

Acute renal response to rapid onset respiratory acidosis.

Renal strong ion compensation to chronic respiratory acidosis has been established, but the nature of the response to acute respiratory acidosis is not well defined. We hypothesized that the response to acute respiratory acidosis in sheep is a rapid increase in the difference in renal fractional excretions of chloride and sodium (Fe(Cl) - Fe(Na)). Inspired CO(2) concentrations were increased for 1 h to significantly alter P(a)CO(2) and pH(a) from 32 ± 1 mm Hg and 7.52 ± 0.02 to 74 ± 2 mm Hg and 7.22 ± 0.02, respectively. Fe(Cl) - Fe(Na) increased significantly from 0.372 ± 0.206 to 1.240 ± 0.217% and returned to baseline at 2 h when P(a)CO(2) and pH(a) were 37 ± 0.6 mm Hg and 7.49 ± 0.01, respectively. Arterial pH and Fe(Cl) - Fe(Na) were significantly correlated. We conclude that the kidney responds rapidly to acute respiratory acidosis, within 30 min of onset, by differential reabsorption of sodium and chloride.

Resuscitation-induced intestinal edema and related dysfunction: state of the science.

High volume resuscitation and damage control surgical methods, while responsible for significantly decreasing morbidity and mortality from traumatic injuries, are associated with pathophysiologic derangements that lead to subsequent end organ edema and dysfunction. Alterations in hydrostatic and oncotic pressures frequently result in intestinal edema and subsequent dysfunction. The purpose of this review is to examine the principles involved in the development of intestinal edema, current and historical models for the study of edema, effects of edema on intestinal function (particularly ileus), molecular mediators governing edema-induced dysfunction, potential role of mechanotransduction , and therapeutic effects of hypertonic saline. We review the current state of the science as it relates to resuscitation induced intestinal edema and resultant dysfunction.

Sodium hydrogen exchanger as a mediator of hydrostatic edema-induced intestinal contractile dysfunction.

BACKGROUND: Resuscitation-induced intestinal edema is associated with early and profound mechanical changes in intestinal tissue. We hypothesize that the sodium hydrogen exchanger (NHE), a mechanoresponsive ion channel, is a mediator of edema-induced intestinal contractile dysfunction. METHODS: An animal model of hydrostatic intestinal edema was used for all experiments. NHE isoforms 1-3 mRNA and protein were evaluated. Subsequently, the effects of NHE inhibition (with 5-(N-ethyl-N-isopropyl) amiloride [EIPA]) on wet-to-dry ratios, signal transduction and activator of transcription (STAT)-3, intestinal smooth muscle myosin light chain (MLC) phosphorylation, intestinal contractile activity, and intestinal transit were measured. RESULTS: NHE1-3 mRNA and protein levels were increased significantly in the small intestinal mucosa with the induction of intestinal edema. The administration of EIPA, an NHE inhibitor, attenuated validated markers of intestinal contractile dysfunction induced by edema as measured by decreased STAT-3 activation, increased MLC phosphorylation, improved intestinal contractile activity, and enhanced intestinal transit. CONCLUSION: The mechanoresponsive ion channel NHE may mediate edema-induced intestinal contractile dysfunction, possibly via a STAT-3 related mechanism.

Nonlinear lymphangion pressure-volume relationship minimizes edema.

Lymphangions, the segments of lymphatic vessel between two valves, contract cyclically and actively pump, analogous to cardiac ventricles. Besides having a discernable systole and diastole, lymphangions have a relatively linear end-systolic pressure-volume relationship (with slope E(max)) and a nonlinear end-diastolic pressure-volume relationship (with slope E(min)). To counter increased microvascular filtration (causing increased lymphatic inlet pressure), lymphangions must respond to modest increases in transmural pressure by increasing pumping. To counter venous hypertension (causing increased lymphatic inlet and outlet pressures), lymphangions must respond to potentially large increases in transmural pressure by maintaining lymph flow. We therefore hypothesized that the nonlinear lymphangion pressure-volume relationship allows transition from a transmural pressure-dependent stroke volume to a transmural pressure-independent stroke volume as transmural pressure increases. To test this hypothesis, we applied a mathematical model based on the time-varying elastance concept typically applied to ventricles (the ratio of pressure to volume cycles periodically from a minimum, E(min), to a maximum, E(max)). This model predicted that lymphangions increase stroke volume and stroke work with transmural pressure if E(min) < E(max) at low transmural pressures, but maintain stroke volume and stroke work if E(min)= E(max) at higher transmural pressures. Furthermore, at higher transmural pressures, stroke work is evenly distributed among a chain of lymphangions. Model predictions were tested by comparison to previously reported data. Model predictions were consistent with reported lymphangion properties and pressure-flow relationships of entire lymphatic systems. The nonlinear lymphangion pressure-volume relationship therefore minimizes edema resulting from both increased microvascular filtration and venous hypertension.

Evaluating the potential role of nitric oxide as a mediator of hydrostatic edema mediated intestinal contractile dysfunction.

BACKGROUND: Administration of L-nil, a selective inhibitor of inducible nitric oxide synthase (iNOS), improves ileus in an animal model of resuscitation induced intestinal edema. The purpose of this study was to elucidate the iNOS/nitric oxide (NO) signal transduction pathway in intestinal edema. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into two groups; CONTROL and RESUS+VH (edema, 80 cc/kg normal saline (resuscitation) with mesenteric venous hypertension). iNOS mRNA and protein, iNOS activity, NO tissue levels, soluble guanylyl cyclase (sGC) expression, and cyclic guanosine monophosphate (cGMP) levels were measured. As a functional endpoint, we evaluated intestinal contractile strength and frequency in L-nil treated animals. RESULTS: Edema was associated with increased iNOS mRNA and protein expression without subsequent increases in iNOS activity or tissue NO levels. There was no significant change in sGC expression or increase in cGMP induced by edema. Administration of L-nil did not decrease edema development or preserve contractile strength, but increased contractile frequency. CONCLUSION: Hydrostatic intestinal edema is not associated with increased iNOS activity or tissue NO levels. Administration of L-nil in edema increases intestinal contractile frequency. This may represent a potential mechanism for the amelioration of ileus seen with the administration of L-nil.