Role Of Nitric Oxide On Isolated Lymphatic Contractile Activity In Hemorrhagic Shock Rats And Its Mechanism

During the process of hemorrhagic shock (HS), refractory hypotension and multiple organs injury induced by circulatory failure is the main reason of death of critical suffering. The lymph circulation, as an important component of circulatory system, plays an important role in the regulation of body homeostasis. It has been documented that, as well as blood circulation disturbance, lymph microcirculation dysfunction characterized by reduced lymphatic contractile activity also presented in the process of HS, moreover, further exacerbated the progress of irreversible shock. From this perspective, the function of lymph circulation is strongly associated with the development of severe shock. As we have already learned, the spontaneous contractions of lymphatic vessels are the major propelent power of lymph circulation. So, the role of reduced lymphatic contractile activity playing in the pathogenesis of shock remains a concern and its mechanisms remains to be further addressed.The bioactive molecule nitric oxide (NO) plays an important role in the development of shock and involves in the pathological process of vascular hypo-reactivity and organ damage. It has been confirmed that periodical changes of NO were responsible for the physiologic accommodation of lymphatic contraction, relaxation and tension. Therefore, we wonder whether NO involves in the regulation of reduced contractile activity of lymphatic after shock. Our previous research indicated contractile response of lymphatic to vasoconstrictors reduced, so called"lymphatic hypo-reactivity". So, whether NO is also related to the lymphatic hypo-reactivity is still a problem to be solved. In order to research the contractile activity of lymphatic during the process of shock effectively, a platform of technique for shock lymphatic investigation in vitro is established through a pressure myograph and used to observe the changes of contractile activity and response to substance P, one of the modulators of lymphatic contractile activity, of lymphatic obtained from rats at different time points of HS. Meanwhile, the effects of tool agents of NO/NOS on contractile activity and response to substance P (SP) of lymphatic were observed in an attempt to provide evidence for the regulation of lymphatic contractile activity during the process of shock.In the first set of experiments, forty-eight male Wistar rats were randomized control (only anesthetization and laparotomy) and shock group (the mean arterial pressure was maintained at 40 mmHg by withdrawing or reinfusing shed blood as needed; rats in this group were further divided into shock 0h, shock 0.5h, shock 1h, shock 2h and shock 3h subgroups with each 8 cases). The thoracic ducts were isolated after a 30min equilibration period following laparotomy in control group and at different time points after HS. The thoracic duct was dissected free, transferred to the chamber of pressure myograph, cannulated and tied on to two glass pipettes, in physiological saline buffer (37°C). The buffer is gassed with 95% O2 -5% CO2 Once spontaneous phasic contractions were observed, the lymphatic vessel was allowed to equilibrate at 3 cmH₂O for 30 min. After the equilibration period, the contraction frequency (CF), end systolic diameter (ESD), end diastolic diameter (EDD) and passive diameters (PD) at different transmural pressure (1 cmH₂O, 3 cmH₂O, 5 cmH₂O, 7 cmH₂O and 9 cmH₂O) were measured, from which the tonic index (TI), contraction amplitude (CA) and fractional pump flow (FPF) were calculated by the following formulas: TI=(PD-EDD)/PD×100%; CA= (EDD-ESD)/PD×100%; FPF=(EDD²-ESD²)/EDD²Ã—CF. The results suggested that the values of CF, TI and FPF of 0h- and 0.5h- shocked lymphatic were significantly increased at multiple transmural pressures when compared with that of sham-operated control. With the development of shock, the values of CF, TI and FPF from 2h- and 3h- shocked lymphatic were significantly decreased when compared with that of sham-operated control. No significant difference was found in CA of lymphatic among sham-operated control and different time points of shock process. These results indicated that lymphatic contractile activity possesses a biphasic change during the development of HS. The contractile activity of lymphatic vessels increased in early phase of hemorrhagic shock and declined in later stage. The lymphatic contractile activity can't restore to the baseline levels after working at an excessive transmural pressure. Moreover, we found, the transmural pressure of 3 cmH₂O is the best working condition of thoracic duct, which lay the foundation of methodology for further experiments.In the second set of experiments, thirty-six male Wistar rats were randomly assigned to one of six groups with each six cases: sham control, shock 0h, shock 0.5h, shock 1h, shock 2h and shock 3h groups. According to the methods described earlier, the thoracic duct preparation was isolated at the corresponding time points from rats in each group. A segment of thoracic duct was pressured and equilibrated at transmural pressure of 3 cmH₂O, and then stimulated with gradient SP (10^-8 mol·L^-1, 3×10^-8 mol·L^-1, 10^-7 mol·L^-1, 3×10^-7 mol·L^-1). To observe the moduation of SP on shock lymphatic contractile activity, the ESD, EDD, CF and PD of isolated lymphatic vessels were measured, from which the CA, TI and FPF were further calculated. The results indicated that the CF, TI and FPF of lymphatic vessels were significantly elevated by SP and these effects escalated with the concentration of administration. Started from the concentration of 3×10^-8 mol·L^-1, the CF, TI and FPF of 2h- and 3h-shocked lymphatic vessels elevated by SP exceeded the value of baseline levels before the experiment. At the same concentration of SP, the CA of lymphatic showed no significant difference in each group, but with the increase of SP concentration, the lymphatic CA had a downward trend in each group. These data suggested that, as well as increasing the pump activity of lymphatic in physiological condition, SP can increase the pump function of lymphatic in shock with different seriousness. The results showed that as well as increasing the pump activity of lymphatic in physiological condition, SP can increase the pump function of lymphatic in shock with different seriousness. Meanwhile, these data demonstrated that SP can be used as a lymphatic modulator to study the lymphatic reactivity in the next experiments, which also provides an experimental basis to research the reactivity of shocked lymphatic vessels.To further assess the reactivity of lymphatic, the different values between pre- and post- administration of SP of CF, CA, TI and FPF were calculated and expressed asâ–³CF,â–³TI,â–³CA andâ–³FPF. The results showed that, after SP incubation, theâ–³CF,â–³TI,â–³CA andâ–³FPF of 0h- and 0.5h shocked lymphatic were significantly increased when compared with that of control group on one or several concentrations. Theâ–³CF (at 3×10^-7 mol·L^-1 of SP) andâ–³TI (1×10^-7 mol·L^-1) of 2h- shocked lymphatic and theâ–³CF (1×10^-7mol·L^-1, 3×10^-7mol·L^-1),â–³TI (1×10^-7mol·L^-1) andâ–³CA (1×10^-7mol·L^-1) of 3h- shocked lymphatic were all significantly lowered when compared with control group. The results suggested that the response of lymphatic to SP presented a biphasic change during the process of HS: increase in early phase (shock 0.5h) and decline in later stage (shock 2h). The data suggested that the change of lymphatic reactivity was an important mechanism of the changed contractile activity of shocked lymphatic and the hypo-reactivity of lymphatic following severe shock was one of pivotal mechanisms of reduced lymphatic contractile activity.To investigate the effects of NO on lymphatic contractility and reactivity in HS rats, male Wistar rats were randomized to sham and HS groups (after HS established, rats were further divided into shock 0h, shock 0.5h, shock 1h, shock 2h and shock 3h subgroups). The thoracic ducts isolated from sham-operated and shocked rats were used to detect the protein expression of iNOS. The result showed that the expression of iNOS protein were decreased gradually with the development of shock. Another twenty-four male Wistar rats were randomly divided into 0.5h- and 2h- shocked group (n=12) and used to prepare isolated thoracic duct. The isolated thoracic duct was pressured and equilibrated at transmural pressure of 3 cmH₂O. Once spontaneous phasic contractions were observed, the 0.5h- shocked lymphatic vessels were incubated for 5 min with NO donor L-Arg (1×10^-3 mol·L^-1, as shock 0.5h+L-Arg group) and L-Arg+soluble guanylate cyclase inhibitor ODQ (1×10-5 mol·L^-1, as shock 0.5h+L-Arg+ODQ group), respectively. The 2h- shocked lymphatic vessels were incubated with 5 min by NOS inhibitor L-NAME (1×10^-8mol·L^-1, as shock 2h+L-NAME group), L-NAME+phosphodiesterase inhibitor aminophylline (AP, 1×10^-5 mol·L^-1, as shock 2h+L-NAME+AP group), respectively. After the equilibration period, the ESD, EDD, CF and PD were measured, from which the CA, TI and FPF were calculated. The indicators of contractile activity of sham, 0.5h- and 2h- shocked untreated lymphatic vessels served as control to assess the effects of NO on lymphatic contraction. The results suggested that L-Arg reduced the CF, TI and FPF of 0.5h- shocked lymphatic to the control levels. ODQ suppressed the effect of L-Arg and significantly increased the CF, TI and FPF, and, however, the CF and FPF remains lower than that of 0.5h- shocked lymphatic. L-NAME elevated the CF, TI and FPF of 2h- shocked lymphatic to the control levels. AP suppressed the effect of L-NAME and declined the elevated CF, TI and FPF of lymphatic by L-NAME. Moreover, the elevated CF and TI by L-NAME were lowered by AP than that of 2h- shocked lymphatic. These tool agents have no significant influence on the CA of lymphatic. These data suggested that NO plays a major regulating role in the biphasic change of lymphatic contraction after HS, its mechanism may involve in the modulation to cGMP levels.After the baseline values of 0.5h- and 2h- shocked and tool agents treated lymphatic registered, the response of lymphatic to SP (1×10-8 mol·L^-1, 3×10^-8 mol·L^-1, 1×10^-7 mol·L^-1, 3×10^-7 mol·L^-1) was measured after a 10 min of incubated period by tool agents. The indexes of CF, EDD, ESD and PD in the first 3 min after administration of SP were recorded, from which the TI, CA and FPF were calculated. Taken the values ofâ–³CF,â–³TI,â–³CA andâ–³FPF after administration of SP as control, the influence of NO on reactivity of lymphatic was assessed during the process of HS. The results suggested that L-Arg reduced theâ–³CF,â–³TI andâ–³FPF of 0.5h- shocked lymphatic at various concentration conditions of SP. This effect of L-Arg was suppressed by ODQ obviously. At certain concentration conditions of SP, theâ–³CF,â–³TI andâ–³FPF increased strikingly compared with that of shock 0.5h + L-Arg group, and theâ–³CF andâ–³FPF increased remarkably compared with that of control group. L-NAME elevated theâ–³CF,â–³TI andâ–³FPF of 2h- shocked lymphatic at various concentration conditions of SP and the manifestation of lymphatic vessels exceeded the values of control levels. In the experiment of 2h- shocked lymphatic treated by the combination of L-NAME and AP, the effect of L-NAME was supressed significantly, which manifested by the decreasedâ–³CF,â–³TI andâ–³FPF when compared to the values of shock 2h+L-NAME group at 1×10^-8 mol·L^-1 and 3×10-8 mol·L^-1 of SP. These data indicated that NO involves in the biphasic modulation of shocked lymphatic and its effect might be achieved by cGMP.In summary, the contractile activity and reactivity of lymphatic both presents a biphasic change in the process of HS, which is manifested by an increase at early shock and decrease at late shock. The change of lymphatic reactivity is the main mechanism underling the damage of contractile activity during the shock conditions. NO involves in the biphasic modulation of shocked lymphatic and its effect might be achieved by cGMP. Targeting NO, the contractile activity of shocked lymphatic can be modulated, which provides a novel insight to advance our understanding of the contractile mechanisms of lymphatic and may eventually lead to therapeutic strategies for treating lymphatic dysfunction.