Mesenteric Lymph Drainage Improves Cardiac Papillary Contractility and Calcium Sensitivity in Rats with Hemorrhagic Shock.

BACKGROUND: Myocardial dysfunction is an important adverse factor of hemorrhagic shock that induces refractory hypotension, and post-hemorrhagic shock mesenteric lymph (PHSML) return is involved in this adverse effect. This study investigated whether mesenteric lymph drainage (MLD) improves PHSML return-induced cardiac contractile dysfunction via the restoration of cardiomyocyte calcium sensitivity. MATERIALS AND METHODS: A hemorrhage shock model was established by using a controlled hemorrhage through the femoral artery that maintained a mean arterial pressure of 40 ± 2 mmHg for 3 h. MLD and mesenteric lymph duct ligation (MLDL) were performed from 1 to 3 h during hypotension. The papillary muscles of the heart were collected for measurement of calmodulin expression and for determining contractile responses to either isoprenaline or calcium. RESULTS: The results showed that either MLD or MLDL reversed the hemorrhagic shock-induced downregulation of calmodulin expression, a marker protein of cardiomyocyte calcium sensitization, in papillary muscles. MLD also improved the decreased contractile response and ±df/dt of the papillary muscle strip to gradient isoprenaline or calcium caused by hemorrhagic shock. CONCLUSION: These findings indicate that increased cardiac contractibility may be associated with the restoration of calcium sensitivity produced by PHSML drainage.

[ATP-sensitive potassium channel involved in modulation of nitride oxide regulating contractile activity of isolated lymphatics from hemorrhagic shock rats].

OBJECTIVE: To investigate the mechanism of ATP-sensitive potassium channel (K(ATP)) on nitride oxide (NO) regulating contractile activity of isolated lymphatics from hemorrhagic shock (HS) rats. METHODS: Eighty - four Wistar rats were randomly divided into control group (n=6), HS 0.5-hour group (n=36), HS 2-hour group (n=42). A segment of thoracic duct of rats was adopted for isolated lymphatics after HS, then the HS 0.5-hour and 2-hour lymphatics were incubated combined or respectively with K(ATP) inhibitor glibenclamide (Gli), opener of K(ATP) pinacidil (Pin), NO donor L-arginine (L-Arg), protein kinase A (PKA) inhibitor H-89, PKA donor 8-bromine-cyclic adenosine monophosphate (8-Br-cAMP), nitricoxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME), soluble guanylyl cyclase (sGC) inhibitor 1 h-[1,2,4]-oxadiazole-[4,3-a]- quinoxalin-1-one (ODQ), protein kinase G (PKG) inhibitor KT-5823 (named as HS 0.5 h, HS 0.5 h + L-Arg, HS 0.5 h + L-Arg + H-89, HS 0.5 h + L-Arg + Gli, HS 0.5 h + 8-Br-cAMP, HS 0.5 h + 8-Br-cAMP + Gli and HS 2 h, HS 2 h + L-NAME, HS 2 h + L-NAME + Pin, HS 2 h + ODQ, HS 2 h + ODQ + Pin, HS 2 h + KT-5823, HS 2 h + KT-5823 + Pin, n=6). By lymphatic perfusion in vitro, contractile frequency (CF) was recorded, and contractile amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated. RESULTS: The results suggested that the CF, TI, FPF of HS 0.5-hour lymphatics were significantly increased compared with control group, and the CF, TI, FPF decreased significantly when incubated with L-Arg. H-89 could deteriorate the decreased effect of L-Arg on CF and FPF, and Gli could deteriorate the decreased effect of L-Arg on FPF. When the HS 0.5-hour lymphatics incubated with 8-Br-cAMP, the CF and FPF were all decreased significantly, and when the HS 0.5-hour lymphatics incubated with 8-Br-cAMP and Gli, the CF was significantly higher than HS 0.5 h + 8-Br-cAMP group, and the TI and FPF decreased significantly compared with HS 0.5-hour group. The CF, FPF, TI of HS 2-hour lymphatics were significantly decreased compared with control group. L-NAME could increase the CF, TI, FPF; ODQ could increase the CF, TI; KT-5823 could increase the CF and FPF; when incubated with Pin respectively, the CF and FPF when incubated with L-NAME were decreased, the CF, TI and FPF when incubated with ODQ were decreased, in addition, the CA was recovered as level of control group, the CF and FPF when incubated with KT-5823 were decreased. CONCLUSIONS: K(ATP) involved in NO modulating pumping function of isolated lymphatics of HS rats, and the effect may be relative to the signal pathway of NO-cAMP-PKA and NO-cGMP-PKG.

[Role of mesenteric lymph drainage improving the metabolism of red blood cell in hemorrhagic shock rats following fluid resuscitation].

OBJECTIVE: To observe the effects of mesenteric lymph drainage on the metabolism of red blood cell (RBC) in hemorrhagic shock (HS) rats following fluid resuscitation. METHODS: Eighteen male Wistar rats were randomly divided into sham group (n=6), HS group (n=6), HS + drainage group (n=6). After 1.5 hours of HS model prepared, the animals were given fluid resuscitation by lost blood plus equal volume of Ringer solution within 30 minutes in HS and HS + drainage groups, and mesenteric lymph drainage was performed after 1 hour of hypotension in HS + drainage group. At 3 hours after resuscitation or corresponding time, blood samples were obtained from abdominal aorta. Membrane suspensions of RBC prepared from part of whole blood samples were used to measure the activities of adenosine triphosphate ase (ATPase) and contents of ATP and lactic acid (LA), the intracellular fluid of RBC prepared from part of whole blood samples was used to determine the concentration of 2,3-diphosphoglyceric acid (2,3-DPG), Na(+) and K(+), plasma samples isolated from blood by centrifugation were used to determine the concentration of Na(+), K(+), Cl(-) and total Ca. RESULTS: Compared with sham group, the content of ATP (µmol/g), activity of Na(+)-K(+)-ATPase (µmol×mg(-1)×h(-1)) and Ca(2+)-ATPase (µmol×mg(-1)×h(-1)) in RBC membrane and total Ca (mmol/L) in plasma were decreased markedly (ATP: 6.698±0.938 vs. 10.670±1.466, Na(+)-K(+)-ATPase: 0.042±0.010 vs. 0.066±0.019, Ca(2+)-ATPase: 0.054±0.015 vs. 0.081±0.017, total Ca: 2.27±0.18 vs. 2.66±0.21, P<0.05 or P<0.01) in HS group, and the content of LA (mmol/g) in RBC and K(+) (mmol/L), Cl(-) (mmol/L) in plasma were increased observably in HS group (LA: 3.472±0.853 vs. 1.743±0.395, K(+): 5.83±0.34 vs. 5.23±0.37, Cl(-): 113.37±3.63 vs. 106.35±4.99, P<0.05 or P<0.01), there was no significant difference in term of the contents of 2,3-DPG (mmol/L: 2.196±0.609 vs. 2.590±0.574, P>0.05). Compared with HS group, the contents of 2,3-DPG (4.459±0.900) and ATP (8.859±1.189), the activities of Na(+)-K(+)-ATPase (0.089±0.022), Ca(2+)-ATPase (0.082±0.020) of RBC were increased in HS + drainage group, and the level of LA (2.060±0.810) was decreased observably (P<0.05 or P<0.01), there were no significant differences in the other indices. CONCLUSIONS: The results indicate that shock mesenteric lymph drainage plays an important role in improving the metabolism of RBC in HS rats following fluid resuscitation, subsequently, may preserve the structure and function of RBC.

Changes in renal tissue proteome induced by mesenteric lymph drainage in rats after hemorrhagic shock with resuscitation.

Kidney injury commonly occurs after hemorrhagic shock. Previous studies have shown that post-hemorrhagic shock mesenteric lymph (PHSML) return negatively affects the kidneys and may induce injury. This study investigates the effect of PHSML drainage on the proteome in renal tissue. A controlled hemorrhagic shock model was established in the shock and shock+drainage groups. After 1 h of hypotension, fluid resuscitation was implemented within 30 min. Meanwhile, PHSML was drained in the shock+drainage group. After 3 h of resuscitation, renal tissue was extracted for proteome analysis using two-dimensional fluorescence difference gel electrophoresis. Differential proteins with intensities that either increased or decreased by 1.5-fold or greater were selected for trypsin digestion and analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry and tandem TOF/TOF mass spectrometry. Enzyme-linked immunosorbent assay was used to validate the identified partial proteins. Compared with the sham group, hnRNPC and Starp decreased in the shock group, whereas Hadha, Slc25a13, Atp5b, hnRNPC, Starp, Rps3, and actin were downregulated in the shock+drainage group. Meanwhile, Atp5b and actin decreased in the shock+drainage group relative to the shock group. The identified proteins can be classified into different categories, such as cell proliferation (hnRNPC, Strap, and Rps3), energy metabolism (Hadha, Atp5b, and Slc25a13), cell motility, and cytoskeleton (actin). Moreover, enzyme-linked immunosorbent assay measurement validated the changed levels of Atp5b and Actg2. Our findings provide a starting point for investigating the functions of differentially expressed proteins in acute kidney injury induced by hemorrhagic shock. These findings hold great potential for the development of therapeutic interventions.

Role of RhoA in regulating the pump function of isolated lymphatics from hemorrhagic shock rats.

The aim of this present study was to examine changes in RhoA protein levels and the role in RhoA in lymphatic contractility and reactivity after hemorrhagic shock. Levels of RhoA and phospho-RhoA in lymphatic tissue isolated from hemorrhagic shock rats were measured, and the contractility and reactivity to substance P of lymphatics isolated from control rats and rats subjected to shock 0.5 and 2 h were determined with an isolated lymphatic perfusion system at a transmural pressure of 3 cmH2O. At the same time, lymphatics isolated from rats subjected to shock 0.5 and 2 h were incubated with agonists and antagonists of RhoA/Rho kinase signaling. Contractile frequency, end-diastolic and end-systolic diameter, and passive diameter were recorded and used to calculate lymphatic tonic index, contractile amplitude, and fractional pump flow. After stimulation with a gradient of substance P, the differences between the preadministration and postadministration values of contractile frequency, contractile amplitude, tonic index, and fractional pump flow were calculated to further assess lymphatic reactivity. RhoA protein levels were significantly increased at 0.5 h after shock but decreased at 2 and 3 h after shock; p-Rho levels were initially increased after shock and subsequently decreased. The contractility and reactivity of 0.5-h-shocked lymphatics were significantly reduced by the RhoA antagonist C3 transferase and the Rho kinase antagonist Y-27632. The RhoA agonist U-46619 increased the contractility and reactivity of 2-h-shocked lymphatics, whereas Y-27632 suppressed the effect of U-46619. Okadaic acid, an inhibitor of myosin light-chain phosphatase, had no effect on the contractility of 2-h-shocked lymphatics, but improved lymphatic reactivity. These results suggest that RhoA is involved in the modulation of lymphatic pump function during hemorrhagic shock and that its effects may be mediated by Rho kinase and MLCP.

Mesenteric lymph return is an important contributor to vascular hyporeactivity and calcium desensitization after hemorrhagic shock.

Vascular hyporeactivity is an important factor in irreversible shock, whereas calcium desensitization is one of the mechanisms of vascular hyporeactivity, and the intestinal lymphatic pathway plays an important role in multiple organ injury after severe hemorrhagic shock (HS). In this study, our aims were to determine the effects of mesenteric lymph on vascular reactivity during HS and the mechanisms involved. First, the in vivo pressor response was observed by intravenous injection of norepinephrine (3 µg/kg) at different time points after HS. We found that mesenteric lymph duct ligation (MLDL) and mesenteric lymph drainage (MLD) enhanced the pressor response at multiple time points after shock. Next, vascular reactivity and calcium sensitivity in superior mesenteric artery (SMA) vascular rings were examined using an isolated organ perfusion system. Vascular reactivity and calcium sensitivity were higher for SMA rings from rats that had undergone HS plus MLDL or MLD that those from rats that had undergone only HS. The effects of MLDL and MLD on vascular reactivity and calcium sensitivity were significantly increased following incubation with the calcium sensitizer angiotensin II and were reduced after incubation with the calcium sensitivity inhibitor insulin. When SMA rings from normal rats were incubated with mesenteric lymph from rats subjected to HS, lymph obtained 0 to 0.5 h after shock enhanced vascular reactivity and calcium sensitivity, whereas lymph obtained 1 to 3 h after shock blunted these effects. We finally examined vascular reactivity and calcium sensitivity in HS rats subjected to MLD at 0 to 3 h or 1 to 3 h after shock. We found that contractile activity of SMAs in response to norepinephrine or Ca was higher in HS rats subjected to MLD at 1 to 3 h after shock compared with rats subjected to MLD at 0 to 3 h after shock. These results indicate that mesenteric lymph return plays an important role in biphasic changes in vascular reactivity during HS. Even more importantly, mesenteric lymph 1 h after shock was an important contributor to vascular hyporeactivity, and its mechanism of action was related to calcium desensitization. Targeting lymph may therefore have therapeutic potential in the treatment of severe shock-induced hypotension.

[Reactivity to substance P of isolated lymphatics in hemorrhagic shock rat].

OBJECTIVE: To observe the change of lymphatic reactivity to substance P (SP) during the process of hemorrhagic shock (HS) with a technique of lymphatic perfusion in vitro in this study. METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and HS group (the rats in this group were further divided into five subgroups: shock 0 h, 0.5 h, 1 h, 2 h and 3 h groups after duplicating the HS model with method of bloodletting to mean arterial blood pressure was 40 mmHg through the femoral venous). Thoracic ducts were separated from HS rats at the corresponding time points in each group. A segment of thoracic duct was pressed and perfused in vitro at transmural pressure of 3 cm H2O, and then stimulated with gradient SP respectively. The end systolic diameter, end diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured, while the contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated, and the different values between pre- and post- administration of SP of CF, CA, TI and FPF were calculated and expressed as Delta CF, Delta TI, Delta CA and Delta FPF to further assess the reactivity of lymphatics. RESULTS: After SP incubation, the Delta CF, Delta TI, Delta CA and Delta FPF of 0 h- and 0.5 h shocked lymphatics were significantly increased when compared with that of control group on one or several concentrations. The Delta CF (at 3 x 10(-7) mol/L of SP) and Delta TI (1 x 10(-7) mol/L) of 2 h- shocked lymphatics and the Delta CF (1 x 10(-7) mol/L, 3 x 10(-7) mol/L), Delta TI (1 x 10(-7) mol/L) and Delta CA (1 x 10(-7) mol/L) of 3 h- shocked lymphatics were all significantly reduced when compared with control group. CONCLUSION: The reactivity of lymphatics to SP presented a biphasic change during the process of HS: increase in early phase and decline in later stage.