Role Of Mitochondrial Dysfunction Inducing The Hypo-reactivity Of Lymphatics Obtained From Shocked Rats

In the process of severe shock induced by ischemic hypoxia, major inflammation, acidosis and so on, the decreased microvascular vessels reactivity to vasoconstrictor and vasodilator, such as norepinephrine(NE)and substance P(SP), leads to the loss of circulating blood volume,intractable hypotension and organs hypoperfusion, which is considered, by now, as one of the main reasons of multiple organ injury, and even the death in these patients.. The context of shock impedes the treatment and prognosis of the sufferings. The lymph system is an important part of circulation. The dysfunction of lymph system involves in the progress of irreversible shock conditions. It has been reported that the rhythmic systolic and diastolic motion of lymphatics is the major propellent power of lymph circulation. Previous studies have shown that lymphatic phasic motion and the reactivity of lymphatics to vasoactivators(such as NE and SP) was elevated in early stage and reduced in late stage during hemorrhagic shock episode. These results indicated that the dysfunction of lymphatic contractility and reactivity underlies the decrease of lymph pump function. The existing research suggested that vascular hyporeactivity associates with mitochondrial structural damage inducing arteriolar smooth muscle cells(ASMCs) dysfunction, including energy metabolic disorder, KATP channel opening and hyperpolarization. Our previous studies indicated that KATP is involved in the process of NO exacerbating pump dysfunction of lymphatics in hemorrhagic shock conditions. Therefore, whether lymphatic pump dysfunction presented in hemorrhagic shock conditions is associated with mitochondrial damage remains to be further investigation.Therefore, to reveal the role of mitochondrial dysfunction playing in lymphatic hyporeactivity of shocked rats, the present study, taking a mitochondrial protective agent cyclosporine A(CsA) as an intervention factor, is to examine whether CsA influences the ultrastructure and energy metabolism of mitochondria in lymphatic vessel smooth muscle cells in hemorrhagic shock rats. Furthermore, the effect of Cs A on the contractility and reactivity to SP of lymphatics obtained form hemorrhagic shock rats is determined. The present study may provide an experimental base for modulation of lymphatic pump function.In first set of experiment, twenty-four male rats were randomly divided into the sham(the rats were performed same protocol as other groups, but no blood was withdrawn), sham+CsA(sham protocol plus CsA 6mg·kg-1), shock(blood was withdrawn from the right femoral artery to a mean arterial pressure of 40 mmHg and was maintained at this level for 3h) and shock+CsA(blood was withdrawn from the right femoral artery to a mean arterial pressure of 40 mmHg and was maintained at this level for 3h, Cs A was administrated at 1h after the hypotension) groups.The thoracic ducts were harvested at 3h of hypotension in shock group and corresponding time point in other groups and fixed in glutaraldehyde solution for the determination of mitochondrial ultrastructure using transmission electron microscopy. Results indicated that the mitochondria of lymphatic smooth muscle cells(LSMCs) in shock group featured edema, clear electron-lucent area part of which is vacuole-shape, and the formation of cystic-like substances; those in shock+CsA group appeared part of edema, main integrity structure and clear homogeneous matrix.These results suggested that CsA can attenuate structural damage of mitochondria of LSMCs in rats subjected to hemorrhagic shock.In the next set of experiment, thirty-six rats were grouped by above mentioned method. The thoracic ducts were prepared for the determination of mitochondrial energy metabolism(concentrations of ATP, ADP andAMP, and then calculation of the total amount of adenosine(TAN) and the energy charge(EC) using HPLC method. The results showed that concentrations of ATP, ADP, AMP and TAN in the shock group were significantly decreased compared with those of sham group, but the EC significantly increased. Concentrations of ATP, ADP, AMP and TAN in the shock+CsA group significantly increased compared with those of shock group, EC presented a downwards trend when compared with that of shock group. These results suggested that CsA can blunt the mitochondrial metabolic dysfunction of shocked lymphatic system.In the last set of experiment, twenty-four rats were grouped and prepared using above mentioned method. Lymphatic rings were prepared from thoracic ducts and mounted to the bath chamber of a pressure myograph system at a transmural pressure of 3 cmH2 O. Once spontaneous phasic contractions were observed, the vessel was allowed to equilibrate at3 cmH2 O for another 30 min. After the equilibration period, the contraction frequency(CF), end systolic diameter(ESD), end diastolic diameter(EDD) and passive diameters(PD) were measured, from which the tonic index(TI), contraction amplitude(CA) and fractional pump flow(FPF) were calculated for the assessment of lymphatic contractility and the effects of CsA for shocked lymphatic contractility. In addition, SP of gradient concentrations(1×10-8, 3×10-8, 1×10-7 and 3×10-7) were added into the bath chamber and incubated for 3 min, in sequence. The maximum difference value of △CF, △TI, △CA and △FPF between pre- and post- administration of SP was used to assess lymphatic reactivity.Results demonstrated that the indexes of CF, FPF, and TI of isolated shock lymphatics were significantly decreased compared with those of sham group. CsA significantly increased the indexes of CF, FPF, and TI of isolated shock lymphatics. Moreover, the reactivity of lymphatics to SP of shock lymphatics was significantly decreased compared with that of sham lymphatic at multiple concentrations. CsA significantly increased thevalue of △CF, △TI and △FPF of isolated shock lymphatics at multiple concentrations. These findings suggested that CsA can enhance the contractility and reactivity of isolated shock lymphatics.In summary, in the progress of hemorrhagic shock, the LSMCs appeared structural damage and energy metabolic dysfunction in mitochondria, by which the contractility and reactivity of isolated shock lymphatics decreased. Mitochondria protectant CsA can attenuate the damage of mitochondria and energy metabolic dysfunction, increase the lymphatic contractility and reactivity. These results suggested that mitochondria dysfunction plays a certain role in lymphatic pump dysfunction following hemorrhagic shock. Targeting mitochondria by which shock lymphatic function can be modulated may represent a novel approach for prevention and therapy of lymphatic hyporeactivity in severe shock conditions.