Effect Of Shock Mesenteric Lymph On Vascular Reactivity In Rats

During the episodes of severe shock, vascular hyporeactivity is one ofthe main reasons of refractory hypotension and organs hypoperfusion,which is believed as a vital contributor to multiple organ dysfunctionsyndrome (MODS) and ensuing multiple organ failure (MOF). Lymphcirculation, as an important component of the circulatory system and withits role in the pathogenesis of severe shock has been paid much attention toby domestic and foreign scholars. It has been documented that mesentericlymph duct ligation in rats subjected to severe hemorrhagic shock, to blockmesenteric lymph return, can reduce the production of inflammatoryfactors in organs, ameliorate the damage of multiple organ which is induceby free radicals, decline the expression of nitric oxide and attenuate theneutrophil sequestration in tissues. Simultaneously, drainage of intestinallymph from shocked animals can reduce histological damage and improveenergy metabolism of vital organs. These results suggest that intestinallymph has great significance in the pathogenesis of shock and subsequentmultiple organ injury. However, it remains to be elucidated whether thereturn of shock lymph involves the pathogenesis of vascular hyporeactivityand whether the vascular dysfunction relates to the changes of mesentericlymph properties. The elucidation for these questions may provide a novelinsight into the therapeutic strategy for shock related hypotension.Therefore, this paper, by duplicating the model of hemorrhagic shock(HS) in rats, observed the effect of intestinal lymphatic gut ligation orintestinal lymph drainage in HS rats on vascular reactivity and calciumsensitivity. Meanwhile, the effect of drainaged shock lymph on thereactivity to norepinephrine (norepinephrine, NE) of isolated vascular ringsin normal rats was observed. To investigate the role of shock intestinal lymph in the pathogenesis ofvascular hyporeactivity, seventy-two male Wistar rats were randomlydivided into Sham group (surgical procedure only), Shock (40 mmHg for180 minutes) group, Shock+Ligation group (hemorrhagic shock +mesenteric lymph ligation), Shock+Drainage group (hemorrhagic shock +mesenteric lymph drainage), all nequal 18. The changes of mean arterypressure (MAP) after administration of norepinephrine (NE, 3μg?kg-1) atdifferent time points were recorded. After maintaining the hypotension(40mmHg) for three hours (or corresponding time in Sham group rats), thesuperior mesenteric artery (SMA) vascular ring was prepared for assayingthe vascular reactivity and calcium sensitivity by determining thecontraction induced by NE and [Ca2+] in vitro. The effect of the angiotensinⅡ(AngⅡ) and insulin (Ins) on vascular reactivity and calcium sensitivitywas also measured in vitro. The results showed the△MAP which reflectedthe pressor effect of NE was no significant difference between Shock groupand Sham group before blood loss. The pressor response to NE wassignificantly enhanced at immediate hemorrhage and 0.5 h after the onsetof hemorrhagic hypotension. Thereafter, the pressor response to NEcontinuously declined throughout the hypotensive period. The mesentericlymphatic duct ligation and mesenteric lymph drainage was associated witha significant increase in the response of MAP at 0, 0.5, 1.0 h and asignificant decline at 2.5 h and 3.0 h compared with sham group. Comparedto shock group, the lymphatic duct ligation and mesenteric lymph drainageenhanced the response of MAP to NE at diverse time points afterhemorrhage of 0.5 h. After 3.0 h of shock or corresponding times, theresponse of SMA rings to NE and calcium sensitivity much moresignificantly decreased in Shock, Shock + ligation and Shock + drainagegroups than Sham group. However, the response of SMA rings to NE andcalcium sensitivity was higher in Shock + ligation and Shock + drainagegroups than Shock group. Incubation with AngⅡor Ins, the response of SMA rings to NE and calcium sensitivity in Shock, Shock + ligation andShock + drainage groups were lower than those in Sham group, andShock+ligation and Shock+ drainage groups were higher compared withShock group.Fifty-four male Wistar rats were randomly divided into four groups:sham (n equal 6), shock (n equal 6), shock+ ligation (n equal 21) and shock+ drainage group (n equal 21). After maintaining hypotension for 3 h, sixSMA vascular rings were prepared from sham-operated rats and ratssubjected to hemorrhagic shock respectively. Fourty-two SMA vascularrings were prepared from rats of shock + ligation and shock + drainagegroups respectively. Six rings from each group were applied to determinecalcium sensitivity. The remaining 36 rings from rats of shock + ligationand shock + drainage groups were treated by Rho kinase regulators (AngⅡand fasudil), protein kinase C (PKC) regulators [phorbol 12-myristate13-acetate (PMA) and staurosporine] and protein kinase G (PKG)regulators [8-Bromo-cGMP (8Br-cGMP) and KT5823] for 10 minrespectively with each group six cases. The influence of Rho kinaseregulators, PKC regulators and PKG regulators to calcium sensitivity ofSMA vascular rings were investigated in rats of shock + ligation and shock+ drainage groups, respectively. The results of ring response when ratswere subjected to shock + ligation indicated that after being treated by AngⅡ, the calcium sensitivity was enhanced significantly when compared withrats subjected to shock and shock + ligation and declined significantlywhen compared with rats subjected to sham operation, after incubation withfasudil, the calcium sensitivity declined significantly when compared withsham operation, shock + ligation and AngⅡgroups, after application ofPMA, the calcium sensitivity was enhanced significantly when comparedwith rats subjected to shock and shock + ligation and declined significantlywhen compared with sham group, after administration of staurosporine, thecalcium sensitivity declined significantly when compared with sham operation, shock + ligation and PMA incubation, after being treated with8Br-cGMP, the calcium sensitivity declined significantly when comparedwith sham operation, shock and shock + ligation, after incubation withKT5823, the calcium sensitivity increased obviously compared with shock,shock + ligation and 8Br-cGMP incubation and declined significantly whencompared with sham operation. The results of rings response when wererats subjected to shock + drainage indicated that after being treated by AngⅡthe calcium sensitivity was enhanced significantly when compared withrats subjected to shock and shock + drainage and declined significantlywhen compared with rats subjected to sham operation, after the incubationwith fasudil, the calcium sensitivity declined significantly when comparedwith sham operation, shock + drainage and AngⅡgroups, afterapplication of PMA, the calcium sensitivity was enhanced significantlywhen compared with rats subjected to shock and shock + drainage anddeclined significantly when compared with sham group, afteradministration of staurosporine, the calcium sensitivity declinedsignificantly when compared with sham group, shock + drainage and PMAincubation, after being treated with 8Br-cGMP, the calcium sensitivitydeclined significantly when compared with sham, shock and shock +drainage groups, after the incubation with KT5823, the calcium sensitivityincreased obviously compared with shock, shock + drainage and8Br-cGMP incubation groups and declined significantly when comparedwith shamgroup.The hemorrhagic shock was established, and the mesenteric lymphwas harvested at different time points during the process of experiments.The samples deriving from the animals before hemorrhage were served asnormal lymph. Samples collected from animals subjected to hemorrhagicshock during 0 h to 0.5 h were served as 0.5 h shock lymph, during 0.5 h to1.0 h served as 1 h shock lymph and during 1.0 h to 3.0 h served as 3.0 hshock lymph, respectively. Twenty-four male Wistar rats were anesthetized with pentobarbital sodium (50mg?kg-1) intramuscularly to prepare 48 SMAvascular rings under aseptic conditions. The response of vascular rings forNE was determined after incubating by the final concentration of 10% 0.5h shock lymph, 10% 1.0 h shock lymph, 5% 3.0 h shock lymph, 10%3.0 h shock lymph, 15% 3.0 h shock lymph and 10% normal lymph, for20 min, respectively (n equal 6 in each group). And the effect of differentincubated times on SMA vascular rings reactivity for NE was observedafter incubating by 5% 3.0 h shock lymph for 40 min and 60 min. After theSMA was treated by shock lymph of different times, the results showedthat the 0.5 h shock lymph enhanced the contractile activity and Emaxvalue of SMA rings at the concentration of 10-7, 10-5, 10-4 mol?L-1 of NEsignificantly increased when compared with the effect of normal lymph.There was no significant difference between shock lymph for 1.0 h and thenormal lymph. The contractile activity was blunted significantly by 3.0 hshock lymph at the concentration of 10-9, 10-7, 10-6 mol?L-1 of NEcompared with the effect of 0.5 h shock lymph, 1.0 h shock lymph andnormal lymph. Meanwhile, After the SMA was treated by 3.0 h shocklymph of different consistency, 15% 3.0 h shock lymph reduced thecontraction and Emax value of SMA rings significantly compared to theeffect of normal lymph, 10% 3.0 h shock lymph and 5% 3.0 h shocklymph respectively. However, no significant difference in the effect ofmesenteric lymph was observed between the normal lymph and 10% 3.0 hshock lymph group. The reactivity to NE of normal SMA rings was treatedby 3.0 h shock lymph 40 min and 60 min for was reduced than that of 20min group, and that of 60 min group was lower that of 40 min group at 10-5mol?L-1 of NE, observably.By blockading the return of shock mesenteric lymph, mesentericlymph duct ligation or mesenteric lymph drainage can enhance the vascularreactivity of rats subjected to hemorrhagic shock, whose mechanisminvolves the elevation of calcium sensitivity. And the effect of blocking mesenteric lymph return improving the calcium sensitivity is related to Rhokinase, PKC and PKG. Mesenteric lymph from rats subjected to severehemorrhagic shock for 3.0 h can blunt the vascular reactivity of normalanimals, and the shock lymph of 0.5 h can enhance the vascular reactivity.These results suggested that the effect of shock lymph was one of theimportant contributors of the vascular hyporeactivity after hemorrhagicshock. Targeting shock lymph, therefore, may find a potential therapeuticapproach in severe shock inducing vascular hypotension.