Maier, Marcus‡; Ströbele, Hubert*; Voges, Jaqueline*; Bauer, Clemens†; Marzi, Ingo‡
Hemorrhagic shock induces ischemia/reperfusion injury through activation of inflammatory mediators and increased leukocyte adhesion. Hypoxia leads to anaerobic glycolysis and depletion of cellular ATP (1). Increased hypoxanthine concentrations and restored oxygen delivery during resuscitation induces the production of toxic superoxide radicals and chemotactic peptides in polymorphonuclear granulocytes (2). Leukocytes and endothelial cells are further activated by these mediators to express adhesion molecules. L-selectins on leukocytes and E-selectins on endothelial cells are activated by histamine, tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), or lipopolysaccharide (LPS) to form a reversible binding of leukocytes to the endothelium. This process correlates to the intravital microscopic observation of slow rolling (3). Irreversible adhesion and migration of leukocytes into the interstitium involves integrins CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150/95) on leukocytes and integrins intercellular adhesion molecule (ICAM)-1 and ICAM-2 on endothelial cells (4,5).
Overwhelming activation of the immune system is known to lead to systemic inflammatory response syndrome (SIRS), which may involve temporary organ dysfunction or even organ failure (6). The liver plays an extraordinary role in this response. Intestinal ischemia, with disruption of the mucosal barrier, can lead to high systemic levels of endotoxins (7). As a filtering organ, the liver is largely responsible for the production of high levels of TNF-α by activated liver macrophages (8). Whereas low TNF levels might have a protective effect, sustained endotoxinemia induces inadequately enhanced cytokine production, resulting in remote organ damage (e.g., in the lungs, kidneys; 9).
Earlier studies using intravital microscopy have demonstrated microcirculatory disturbances after hemorrhagic shock, with reduced blood flow and increased leukocyte-endothelial interaction. Reduction of microcirculatory damage has been previously discussed for a variety of anti-inflammatory mediators including an IL-1 receptor antagonist, pentoxifylline and superoxide dismutase (10–12).
Among the large number of cytokines participating in the complex pathophysiology of shock-induced inflammation, TNF-α plays a central role by initiating the production of further cytokines and the expression of adhesion molecules on endothelial cells (9,13). In experimental settings, TNF-α is able to produce most of the clinically observed symptoms that typically follow hemorrhagic shock. Monoclonal antibodies against TNF-α, as well as against recombinant, soluble TNF receptors and physiologically produced TNF receptors, are supposed to antagonize the effects of TNF-α (14). Beneficial effects on the liver microcirculation and survival after hemorrhagic shock have been reported with monoclonal TNF antibody (15,16).
The purpose of this study was to investigate the effects of a novel, recombinant TNF-α receptor blocker (TNF-BP) on the liver microcirculation and on leukocyte-endothelial adhesion after hemorrhagic shock. To this end, the effects of TNF-BP on the hepatic microcirculation were examined using intravital microscopy at 5 and 48 h after hemorrhagic shock.
MATERIALS AND METHODS
Experiments were performed with the approval of the local ethics committee. Sprague-Dawley rats (HAN, Hannover, Germany) weighing 200–240g were randomly assigned to one of six groups (n = 6 each). Throughout the whole experiment, the animals were anesthetized with an initial i.p. injection of 50 mg/kg pentobarbital. After 48 h, three groups received a second application of pentobarbital.
Recombinant TNF-BP was purified, sequenced and cloned at Amgen (Boulder, CO). A dimeric form was engineered by attaching a bifunctional 20,000-Da polyethylene glycol (PEG) reagent to a cysteine that was substituted at residue 105. The resulting PEG-linked dimeric form, TNF-BP, is a potent inhibitor of TNF (17).
Preparation for hemorrhagic shock
The tail vein was cannulated for blood and fluid application. A polyethylene catheter (0.8 mm in diameter, Braun, Melsungen, Germany) was inserted into the left femoral artery for measurement of mean arterial blood pressure and heart rate (Hellige SMK 154-9, Freiburg, Germany). The animal was placed on a warming pad to maintain a constant temperature of 37°C. Blood samples of 150 μL were taken from the femoral artery for analysis (hematocrit, WBC, platelets, acid-base state) before shock induction, at the beginning of reperfusion and before each intravital microscopy at 5 and 48 h after shock.
Hemorrhagic shock was induced by blood withdrawal and a mean arterial blood pressure of 40 mmHg was maintained for 90 min. Resuscitation was started with intravenous application of 4 mg/kg TNF-BP in 0.5 mL of Lactated Ringers (LR) solution or vehicle followed by 60% of the shed blood and a standardized volume of LR. In the first hour of reperfusion, LR was administered as twice the volume of shed blood; in the second and third h, LR was administered each hour at a volume equal to that of the shed blood. Thereafter, LR was infused at a rate of 10 mL/kg/h. Sham operated animals underwent the same treatment, except blood withdrawal for shock induction, and received a continuous infusion of LR at a rate of 10mL/kg/h.
Intravital Fluorescence Microscopy of the liver
Intravital microscopy of the liver microcirculation was performed at 5 h and at 48 h after induction of reperfusion. At the end of the reperfusion period, the abdomen of the rat was opened by a midline laparotomy. For epi-il luminescence intravital microscopy (Nikon-MM-11, 100 W Hg lamp, 10× ocular, zoom objective 0.7-2.25, 546nm filter, 10x/0.4 water immersion objective, magnification 330×), the upper left liver lobe was exteriorized onto a specially prepared Plexiglas stage. Lactated Ringers solution at 37°C was then continuously poured onto the liver surface to prevent desiccation. After fractionated injection of acridine orange (0.1 mL of 1 mg/kg BW; Sigma Chemical Co., St. Louis, MO) to dye the leukocytes, five liver lobules and five central veins were recorded for 30 s on a S-VHS video system according to the technique previously described by Marzi et al. (2). Evaluation was performed with a computer-assisted imaging system (Lobulus, medvis, Saarlouis, Germany).
The following parameters were determined:
1. Leukocyte flow resulting from leukocyte velocity and sinusoidal diameter.
2. The number of temporary adherent leukocytes in liver sinusoids and central veins (0.2–20 s).
3. The mean adhesion time of the temporary leukocytes in liver sinusoids and central veins.
4. The number of permanent adherent leukocytes in liver sinusoids and central veins (>20 s).
Two animals from each group were taken for immunohistochemical evaluation for ICAM-1-expression. For this investigation, liver tissue was covered with Tissue Tec frozen to −70°C. The tissue was prepared with acetone and 0.1 M Tris-HCl Buffer (pH 8.2) and covered with purified mouse anti-rat CD54 monoclonal antibody (Pharmingen, Hamburg, Germany). After 30 min, the secondary antibody DAKO-EnVision-detection system (DAKO Diagnostik GmbH, Hamburg, Germany) was added. The tissue was stained with Fast-Red-solution (Fast Red TR, Sigma Chemical Co., Germany) and Haemalaun solution.
The results are given as mean ± SEM. Statistical evaluation was performed using analysis of variance and post-hoc t test with Bonferroni correction. The level of significance of the data was determined at P < 0.05.
Macrohemodynamic and laboratory findings after hemorrhagic shock
At the end of the reperfusion period and before microscopy at 5 and 48 h after induction of hemorrhagic shock, mean arterial blood pressure was comparable between the placebo and TNF-BP-treated groups. Forty-eight hours after shock, at the beginning of intravital microscopy, heart rate was significantly higher in the placebo group (442 ± 13/min; 435 ± 15/min; 427 ± 15/min) than in TNF-BP subjects (391 ± 117/min; 381 ± 13/min; 376 ± 15/min) and the sham group (361 ± 11/min; 354 ± 13/min; 342 ± 11/min) as shown in Fig. 1. Furthermore, 48 h after shock, a significantly improved base deficit was found in the TNF-BP group (−1.8 ± 0.9 mmol/L) compared with the placebo group (−4.8 ± 2.3 mmol/L). Five hours after shock, the base deficit did not differ between the two groups. No significant differences were found at 5 or at 48 h after hemorrhagic shock concerning hematocrit and white blood cell count (Table 1).
Sinusoidal perfusion and leukocyte adhesion after hemorrhagic shock
Compared with the sham group, leukocyte flow velocity and sinusoidal width were significantly reduced 48 h after hemorrhagic shock. Sinusoidal width and leukocyte flow were also reduced after 5 h (P < 0.05). However, post-shock treatment with TNF-BP could not significantly change leukocyte flow velocity or sinusoidal width compared with the placebo (Table 2).
Leukocyte adhesion was measured in liver sinusoids and central veins. Treatment with TNF-BP significantly reduced temporary leukocyte adhesion (0.2–20 s) in liver sinusoids, but not in central veins compared with the placebo (Fig. 2). Forty-eight hours after hemorrhagic shock, temporary leukocyte adhesion in both liver sinusoids and central veins was only slightly attenuated.
Five hours after shock, mean adhesion time of leukocytes to the endothelium was significantly shorter in central veins of the TNF-BP group (1.65 ± 0.17 s) compared with the placebo group (2.26 ± 0.11 s;Fig. 3). In contrast, at 48 h, mean adhesion time between TNF-BP and placebo groups did not differ. Mean adhesion time in liver sinusoids was similar in TNF-BP and placebo groups at both 5 and 48 h after hemorrhagic shock.
The number of permanent adherent leukocytes did not differ between placebo and TNF-BP after 5 h of reperfusion. At 48 h, however, permanent leukocyte adhesion in the central hepatic vein was significantly reduced in the TNF-BP group (19 ± 1%) compared with the placebo group (28.3 ± 2.7%;Fig. 4).
A substantial increase in ICAM-1 expression was observed at 5 and at 48 h after shock and resuscitation. Treatment with TNF-BP (Fig. 5A) led to a substantial reduction after 48 h in liver sinusoids and central veins compared with the placebo group (Fig. 5B).
Intravital microscopy is a well-established tool for assessing the hepatic microcirculation (18,19). It has been shown that hemorrhagic shock leads to microcirculatory disturbances with reduced sinusoidal blood flow and increased leukocyte-endothelial adhesion. Various mediators, such as platelet-activating factor antagonist (PAF antagonist), interleukin-1-receptor antagonist (IL-1ra), monoclonal TNF-antibody, and so on have revealed beneficial anti-inflammatory effects (10,15,20). In this study we examined the influence of a recombinant TNF binding protein, applied at the time of resuscitation, on the liver microcirculation at 5 and at 48 h after hemorrhagic shock. The time intervals chosen are relevant because clinical complications, such as SIRS and multiple organ failure, can develop within days after shock. In addition, initiating treatment is clinically only possible at the time of resuscitation. Systemic circulatory measurements (e.g., mean arterial blood pressure) showed comparable results in the TNF-BP and placebo groups. However, 48 h after shock, heart rates in the placebo group were significantly higher whereas systemic blood pressure was lower in comparison with the TNF-BP group. This could be the result of the typical hyperdynamic state that is often observed during SIRS, leading to multiple organ failure, which was partly attenuated in the TNF-BP-treated group (21).
Determination of white blood cell concentration and blood gas analysis revealed comparable results in both the placebo and TNF-BP groups. At 48 h after shock, the base deficit was significantly reduced in the TNF-BP group compared with the placebo group. This indicates that TNF-BP partly prevents metabolic acidosis after hemorrhagic shock, as previously shown with a monoclonal TNF antibody (15).
After hemorrhagic shock, sinusoidal width was reduced in treated animals compared with sham controls due to cell swelling and vasoactive factors, such as endothelins, thromboxanes, oxygen radicals, prostacyclins, and PAF (22,23). In this study, no significant difference of sinusoidal width was observed in the TNF-BP group compared with the placebo group. Furthermore, recombinant TNF-BP did not notably influence microvascular blood flow after hemorrhagic shock in the rat. A similar observation was made during previous experimentation using TNF-α monoclonal antibody (15). Microvascular blood flow is determined in part by sinusoidal diameter. One of the reasons why TNF-BP failed to improve the microvascular blood flow may be because it only exerts its influence on the vascular endothelium indirectly, via endothelins and nitric oxide.
Leukocyte-endothelial adhesion is seen as one important factor that may account for a reduction in microvascular blood flow (1,11). Thrombosis of the small vessels in the gut or reduced post-hepatic blood flow after shock could also be responsible mechanisms.
Leukocyte-endothelial adhesion is a known part of the inflammatory response and is one important factor that can initiate tissue injury and organ dysfunction after hemorrhagic shock. Moreover, migration of neutrophils to the tissue is a major factor contributing to irreversible organ failure (24). Monoclonal antibodies to adhesion receptors have shown protective effects during this inflammatory response (25). In earlier studies, two different types of adhesion were observed in liver sinusoids and in central veins (3,24,25). Evaluation of intravital microscopy showed either short-term adhesion of leukocytes, sticking to the endothelium for 0.2–20 s, or long-term adhesion of leukocytes, sticking >20 s. Short-term adhesion can be observed even under physiologic conditions and is the first step in the cascade of leukocyte-endothelial interactions. Long-term adhesion, a second step, is necessary for firm leukocyte adhesion and migration to the interstitium (3,5). This sequential adhesion process is analogous to the adhesion receptor types described in earlier studies. Expression of LECAM-1 and sialyl-LewisX (L-selectin) on leukocytes, as well as ELAM-1 (E-selectin), GMP-140 (P-selectin), and hyaluronic acid (CD44) on endothelial cells, is upregulated by oxygen radicals, PAF, histamine, and thrombins, starting just minutes after shock is induced lasting for several h afterwards (3,4,24–26). These previous observations are consistent with the results of intravital microscopy seen in this study: Temporary adhesion was significantly reduced 5 h after shock in the sinusoids but not in central veins, whereas permanent adhesion was significantly reduced 48 h after shock in central veins but not in the liver sinusoids. IL-1 and TNF-α induce shedding of these adhesion receptors and at the same time support upregulation of integrins like ICAM-1 (CD54) on endothelium and CD11a, CD11b, CD11c/CD18 on leukocytes (9,24,25). The significant reduction in leukocyte-endothelial adhesion in different sublobular regions 5 and 48 h after shock might be caused by a time-dependent shift of TNF receptors in the liver. The significant attenuation in leukocyte adhesion in TNF-BP treated animals 48 h after shock parallels the tremendous reduction in ICAM expression observed in liver sinusoids and central veins. It has been shown that leukocyte adhesion is attenuated most effectively if treatment is started early. TNF-α inhibition before shock induction and reperfusion reduces permanent adherent leukocytes significantly, whereas starting treatment 15 min after shock is less effective (19). Because it is widely accepted that TNF-alpha is crucial for the activation of the inflammatory cytokine cascade, neutrophil recruitment and upregulation of adhesion molecule expression, this study examined in particular the effect of TNF-BP, administered after shock, on lasting alterations of the liver microcirculation over a 2-day period. Significant reduction of permanent adherent leukocytes 48 h after hemorrhagic shock in the TNF-BP group emphasizes a lasting benefit of anti-inflammatory treatment when it is given at the beginning of the resuscitation period. This is underlined in the study by Bahrami et al. that showed protection from organ injury and increased survival after post-shock treatment with TNF antibody (16). It seems remarkable that delaying the start of treatment until the beginning of resuscitation still reduces the inflammatory response, indicating a long-lasting effect of TNF-BP.
There is evidence that the concentration of TNF-α that is endogenously produced is important in the maintenance of physiological homeostasis (27). Low concentrations of TNF-α have an obvious protective effect against endotoxinemia. TNF-BP seems to form a complex with TNF-α, after subsequent slow release of TNF-α after shock. However, because of the complexity of the cytokine cascade involved, the function of TNF-α is still not precisely defined. The results of this study indicate an anti-inflammatory effect of TNF-BP that can be observed up to 48 h after hemorrhagic shock. However, because of the redundancy of multiple cytokine actions, it is unlikely that monotherapy with TNF-BP could altogether reverse the manifestations of an inflammatory response.
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