Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent.

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.

Cytokines contribute to early hepatic parenchymal injury and microvascular dysfunction after bilateral hindlimb ischemia.

PURPOSE: Hepatic dysfunction may contribute to death from multiple organ dysfunction after abdominal aortic surgery. Several factors are likely responsible, and the purpose of this study was to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are involved in initiating this remote hepatic injury. METHODS: In a normotensive rat model of 4-hour bilateral hindlimb ischemia/reperfusion (I/R), we measured systemic TNF-alpha and IL-1 levels throughout the I/R period. Rats were randomly assigned to either the 3-hour control group, the 3-hour I/R group, or the I/R group with administration of a polyclonal antibody (PAb) to TNF-alpha (I/R + TNF-alpha PAb). Direct evidence of lethal hepatocyte injury through the labeling of nuclei by propidium iodide (per 10(-1)mm(3)) and altered microvascular perfusion were assessed by using intravital microscopy. RESULTS: Systemic TNF-alpha peaked at 83.97 pg/mL (P <.05, n = 5) at 30 minutes of reperfusion and returned to baseline in 60 to 90 minutes. No significant change in systemic IL-1 was detected (P <.05, n = 4). Alanine aminotransferase increased 2.5-fold in the I/R group through 3 hours of reperfusion (P <.05, n = 4), and TNF-alpha PAb did not attenuate this alanine aminotransferase increase (P <.05, n = 6). Lethal hepatocyte injury increased by 8-fold in the I/R group compared with the control group (P <.05, n = 5), whereas TNF-alpha PAb significantly reduced this injury (P <.05, n = 4). No regional differences in injury were noted within the acinus. Total perfusion within the microvascular unit did not drop; however, significant flow heterogeneity was observed. The proportion of continuously perfused sinusoids declined in the I/R group after 3 hours of reperfusion in both periportal (62.0 +/- 2.2, P <.05) and, to a lesser, although significant, degree, in the pericentral regions (73. 2 +/- 1.73, P <.05). CONCLUSION: By scavenging extracellular TNF-alpha with a PAb, we provide direct evidence that TNF-alpha contributes to, but is not solely responsible for, early remote hepatocellular injury and microvascular dysfunction. The administration of TNF-alpha PAb reduced lethal hepatocyte injury in both regions of the acinus and also improved perfusion in the periportal region (76.8 +/- 5.41, P <.05), but not in the pericentral region. This suggests that TNF-alpha released during reperfusion mediates early remote hepatocellular injury and microvascular dysfunction after a remote ischemic insult.

Microcirculatory perfusion deficits are not essential for remote parenchymal injury within the liver.

A normotensive model of hindlimb ischemia-reperfusion in Wistar rats was used to test the hypothesis that microvascular perfusion deficits contribute to the initiation of remote hepatic injury during a systemic inflammatory response. Animals were randomly assigned to one of three groups: 4 h of ischemia with 6 h of reperfusion (I/R-6; n = 4), 4 h of ischemia with 3 h of reperfusion (I/R-3; n = 5), or no ischemia (naive; n = 5). With intravital fluorescence microscopy, propidium iodide (PI; 0.05 mg/100 g body wt) was injected for the in vivo labeling of lethally injured hepatocytes (number/10(-1) mm(3)). PI-positive hepatocytes increased progressively over the 6-h period (naive 32.9 +/- 7.8 vs. I/R-3 92.8 +/- 11.5 vs. I/R-6 232 +/- 39.2), with no difference between periportal and pericentral regions of the lobule. Additionally, a significant decrease in continuously perfused sinusoids (naive 70.0 +/- 1.5 vs. I/R-3 65.0 +/- 1.0 vs. I/R-6 48.8 +/- 0.9%) was measured. Regional sinusoidal perfusion differences were only observed after 3 h of limb reperfusion. Indirect measures of hepatocellular injury using alanine transaminase levels support the progressive nature of hepatic parenchymal injury (0 h 57.8 +/- 6.5 vs. 3 h 115.3 +/- 20.7 vs. 6 h 125.6 +/- 19.5 U/l). Evidence from this study suggests that remote hepatic parenchymal injury occurs early and progresses after the induction of a systemic inflammatory response and that microvascular perfusion deficits are not essential for the initiation of such injury.

Initiation of remote hepatic injury in the rat: interactions between Kupffer cells, tumor necrosis factor-alpha, and microvascular perfusion.

Severe trauma may initiate a systemic inflammatory response, which in turn may result in remote organ injury. After limb ischemia/reperfusion (I/R), intravital fluorescence microscopy was applied to the livers of normotensive rats to investigate the initiation of remote injury to the liver. Additionally, we determined whether Kupffer cell activation and tumor necrosis factor-alpha (TNF-alpha) were involved, via perfusion deficits, in such injury. TNF-alpha, measured by immunoassay, peaked at 30 minutes of reperfusion, but returned to baseline within 60 minutes. Limb I/R resulted in significant increases to global hepatocellular injury measured by alanine transaminase (ALT) and lethal hepatocyte injury as seen with intravital fluorescence microscopy. Although the number of perfused sinusoids went unchanged, a significantly augmented perfusion heterogeneity was measured. After 1.5 hours of reperfusion, both TNF-alpha and Kupffer cells were shown to contribute to global hepatocellular injury (e.g., ALT). After 3 hours, TNF-alpha was no longer essential for this injury, suggesting that some other mechanism(s) activated Kupffer cells and initiated hepatocellular injury. Using propidium iodide and fluorescence microscopy, we found that both TNF-alpha and Kupffer cell activation were necessary to drive hepatocytes toward lethal injury. No additional benefits were observed with a combination of TNF-alpha inhibition and Kupffer cell suppression. These results not only implicate both Kupffer cells and TNF-alpha in the initiation of remote hepatic injury, but suggest that sinusoidal perfusion deficits are not essential for the initiation of such injury. Other mechanism(s) are likely involved in the pathogenesis of remote hepatic parenchymal injury.

Prostaglandin inhibition causes an increase in reactive hyperaemia after ischaemic exercise in human forearm.

The hypothesis that prostaglandins contribute to the reactive hyperaemia after 5 min of ischaemia or 5 min of ischaemic exercise was investigated in six men by inhibiting prostaglandin production with ibuprofen (1800 mg) and indomethacin (225 mg) over 24 h before testing. Blood flow was measured continuously in the baseline and after ischaemia by combined pulsed and echo Doppler as the product of velocity and cross-sectional area. After 5 min of ischaemia, there were no differences in blood flow between placebo and the two drug conditions, except at 5 and 10 s when flow with indomethacin was greater than both placebo and ibuprofen. After 5 min of ischaemic exercise, blood flow was significantly greater as a consequence of increased vascular conductance in each of ibuprofen and indomethacin than placebo from 5 until 90 s of recovery. We conclude that prostaglandin inhibition had little or no effect on reactive hyperaemia after 5 min of circulatory occlusion alone, but that blood flow after ischaemic exercise was elevated due to increased vascular conductance when prostaglandin synthesis was inhibited.

Effects of acetylcholine and nitric oxide on forearm blood flow at rest and after a single muscle contraction.

We tested the hypothesis that ACh or nitric oxide (NO) might be involved in the vasodilation that accompanies a single contraction of the forearm. Eight adults (3 women and 5 men) completed single 1-s-duration contractions of the forearm to raise and lower a weight equivalent to approximately 20% maximal voluntary contraction through a distance of 5 cm. In a second protocol, each subject had a cuff, placed completely about the forearm, inflated to 120 mmHg for a 1-s period, then released as a simulation of the mechanical effect of muscle contraction. Three conditions were studied, always in this order: 1) control, with intra-arterial infusion of saline; 2) after muscarinic blockade with atropine; and 3) after NO synthase inhibition with NG-monomethyl-L-arginine (L-NMMA) plus atropine. Forearm blood flow (FBF), measured by combined pulsed and echo Doppler ultrasound, was reduced at rest with L-NMMA-atropine compared with the other two conditions. After the single contraction, there were no effects of atropine, but L-NMMA reduced the peak FBF and the total postcontraction hyperemia. After the single cuff inflation, atropine had no effects, whereas L-NMMA caused changes similar to those seen after contraction, reducing the peak FBF and the total hyperemia. The observation that L-NMMA reduced FBF in response to both cuff inflation and a brief contraction indicates that NO from the vascular endothelium might modulate the basal level of vascular tone and the mechanical component of the hyperemia with exercise. It is unlikely that ACh and NO from the endothelium are involved in the dilator response to a single muscle contraction.