POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH IMPAIRS SPLENIC DENDRITIC CELL FUNCTION IN MICE.

ABSTRACT: Dendritic cell (DC)-mediated immune dysfunction is involved in the process of severe hemorrhagic shock that leads to sepsis. Although post-hemorrhagic shock mesenteric lymph (PHSML) induces immune organs injuries and apoptosis, whether PHSML exerts adverse effects on splenic DCs remains unknown. In this study, we established a hemorrhagic shock model (40 ± 2 mm Hg for 60 min) followed by fluid resuscitation with the shed blood and equal Ringer's solution and drained the PHSML after resuscitation. At 3 h after resuscitation, we harvested the splenic tissue to isolate DCs using anti-CD11c immunomagnetic beads and then detected the necrotic and apoptotic rates in splenocytes and splenic DCs. We also detected the levels of TNF-α, IL-10, and IL-12 in the culture supernatants and surface marker expressions of MHC-II, CD80, and CD86 of splenic DCs following LPS stimulation for 24 h. Second, we purified the DCs from splenocytes of normal mice to investigate the effects of PHSML treatment on cytokine production and surface marker expression following LPS stimulation. The results showed that PHSML drainage attenuated LPS-induced cell death of splenocytes and DCs. Meanwhile, PHSML drainage enhanced the DC percentage in splenocytes and increased the TNF-α and IL-12 production by DCs and the expressions of CD80, CD86, and MHCII of DCs treated by LPS. Furthermore, PHSML treatment reduced the productions of TNF-α, IL-10, and IL-12 and the expressions of CD80 and CD86 in normal DCs after treatment with LPS. In summary, the current investigation demonstrated that PHSML inhibited the cytokine production and surface marker expressions of DCs stimulated by LPS, suggesting that PHSML plays an important role in hemorrhagic shock-induced immunosuppression through the impairment of DC function and maturation.

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.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro.

BACKGROUND: Immune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. METHODS: The normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. RESULTS: Hemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. CONCLUSIONS: The results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

[Role of mesenteric lymph drainage in the balance of ACE/ACE2 in murine myocardium following hemorrhagic shock].

OBJECTIVE: To observe the change of angiotensin converting enzyme (ACE) and ACE2 in the murine myocardium followed hemorrhagic shock and the role of post-hemorrhagic shock mesenteric lymph (PHSML) drainage. METHODS: Twenty-four male mice were ran-domly divided into control, sham, shock, and shock + drainage groups. A hemorrhagic shock model was established and then fluid resuscita-tion was performed to the mice in the shock and shock + drainage groups, and the PHMSL was drained in the shock + drainage group after fluid resuscitation. After 6 h of resuscitation in the shock and shock + drainage groups or corresponding time in the sham group, or after anesthesia in the control group, the myocardial tissues were harvested for the determination of the mRNA expressions of ACE, ACE2, angiotensin Ⅱ (Ang Ⅱ) type 1 receptor (AT1R), and Mas related G protein coupled receptor (Mas1R) using the method of qRT-PCR, and the levels of Ang Ⅱ and Ang (1-7) using the method of ELISA. RESULTS: In the myocardial tissue of shock group, the ACE and AT1R mRNA expressions and Ang Ⅱ level were significantly increased than those of the control and sham groups, the ACE2 and Mas1R mRNA expressions were significantly de-creased than that of the control and sham groups, the Ang (1-7) level was decreased compared with the control group, the ratios of ACE/ACE2, Ang Ⅱ/Ang (1-7), and AT1R/Mas1R in the shock group were significantly increased than the control and sham groups. Meanwhile, PHSML drainage obviously suppressed the effects of hemorrhagic shock on these indices. CONCLUSIONS: Hemorrhagic shock up-regulated the ACE-Ang Ⅱ-AT1R axis, down-regulated the ACE2-Ang (1-7)-Mas1R axis, and induced the unbalance of ACE and ACE2 in myocardial tis-sue. PHSML drainage decreased the ACE-Ang Ⅱ-AT1R axis and increased the ACE2-Ang (1-7)-Mas1R axis, resulted in the balance of ACE and ACE2.

Reduction of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation.

PURPOSE: To evaluate the changes of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation. METHODS: Six rats in the shock group suffered hypotension for 90 min by hemorrhage, and resuscitation with shed blood and equal ringer's solution. Then, the contractility of lymphatics, obtained from thoracic ducts in rats of the shock and sham groups, were evaluated with an isolated lymphatic perfusion system using the indices of contractile frequency (CF), tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF). The lymphatic reactivity to substance P (SP) was evaluated with the different volume of CF, CA, TI and FPF between pre- and post-treatment of SP at different concentrations. RESULTS: The CF, FPF, and TI of lymphatics obtained from the shocked rats were significantly decreased than that of the sham group. After SP stimulation, the ∆CF (1 × 10(-8), 3 × 10(-8), 1 × 10(-7), 3 × 10(-7) mol/L), ∆FPF (1 × 10(-8), 3 × 10(-8), 1 × 10(-7) mol/L), and ∆TI (1 × 10(-8) mol/L) of lymphatics in the shock group were also obviously lower compared with the sham group. In addition, there were no statistical differences in CA and ∆CA between two groups. CONCLUSION: Lymphatic contractility and reactivity to substance P appears reduction following hemorrhagic shock with resuscitation.

Reduction of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation

PURPOSE: To evaluate the changes of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation. METHODS: Six rats in the shock group suffered hypotension for 90 min by hemorrhage, and resuscitation with shed blood and equal ringer's solution. Then, the contractility of lymphatics, obtained from thoracic ducts in rats of the shock and sham groups, were evaluated with an isolated lymphatic perfusion system using the indices of contractile frequency (CF), tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF). The lymphatic reactivity to substance P (SP) was evaluated with the different volume of CF, CA, TI and FPF between pre- and post-treatment of SP at different concentrations. RESULTS: The CF, FPF, and TI of lymphatics obtained from the shocked rats were significantly decreased than that of the sham group. After SP stimulation, the ∆CF (1×10-8, 3×10-8, 1×10-7, 3×10-7 mol/L), ∆FPF (1×10-8, 3×10-8, 1×10-7 mol/L), and ∆TI (1×10-8 mol/L) of lymphatics in the shock group were also obviously lower compared with the sham group. In addition, there were no statistical differences in CA and ∆CA between two groups. CONCLUSION: Lymphatic contractility and reactivity to substance P appears reduction following hemorrhagic shock with resuscitation. .