152 Original article
Chemical sympathectomy attenuates inflammation, glycocalyx shedding and coagulation disorders in rats with acute traumatic coagulopathy Lin XuM, Wen-Kui YuM, Zhi-Liang Lin, Shan-Jun Tan, Xiao-Wu Bai, Kai Ding and Ning Li Acute traumatic coagulopathy (ATC) may trigger sympathoadrenal activation associated with endothelial damage and coagulation disturbances. Overexcitation of sympathetic nerve in this state would disrupt sympathetic– vagal balance, leading to autonomic nervous system dysfunction. The aim of this study was to evaluate the autonomic function in ATC and its influence on inflammation, endothelial and coagulation activation. Male Sprague–Dawley rats were randomly assigned to sham, ATC control (ATCC) and ATC with sympathectomy by 6-hydroxydopamine (ATCS) group. Sham animals underwent the same procedure without trauma and bleeding. Following trauma and hemorrhage, rats underwent heart rate variability (HRV) test, which predicts autonomic dysfunction through the analysis of variation in individual R-R intervals. Then, rats were euthanized at baseline, and at 0, 1 and 2 h after shock and blood gas, conventional coagulation test and markers of inflammation, coagulation, fibrinolysis, endothelial damage and catecholamine were measured. HRV showed an attenuation of total power and high frequency, along with a rise of low frequency and low frequency : high frequency ratio in the ATC rats, which both were reversed by sympathectomy in the ATCS group. Additionally, sympathetic denervation significantly suppressed the increase of proinflammatory
Introduction As important contributors to preventable deaths [1], severe injury and concomitant uncontrolled bleeding often lead to acute traumatic coagulopathy (ATC), which has been defined recently as endogenous coagulation disorders prior to fluid resuscitation [2] and could be observed in the early stage ( 0.05, data not shown). The serial changes of MAP, HR, base excess and fibrinogen are shown in Fig. 1. No significant differences in basal HR, base excess and fibrinogen were found among three groups, but basal MAPs were lower in sympathectomized rats in comparison with control and sham rats (P < 0.01, Fig. 1a). Following the end of shock, MAP in the ATCC and ATCS group both increased at 1 h and then fell at 2 h. Additionally, MAP in the ATCC group was significantly higher than in the ATCS group at 1 and 2 h (P < 0.05). There was an evident fall in base excess and fibrinogen in the ATCC and ATCS groups after shock (all P < 0.01, Fig. 1c and d). No significant differences were found in HR, base excess or fibrinogen between the ATCC and ATCS groups (P > 0.05).
Fig. 1
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Serial changes in (a) mean arterial pressure (MAP), (b) heart rate (HR), (c) base excess and (d) fibrinogen (Fib) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; ##P < 0.01 Sham vs. ATCS.
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Sympathectomy attenuates acute coagulopathy Xu et al. 155
Autonomic function before and after trauma and shock
The frequency-domain parameters of HRV are shown in Fig. 2. Basal values of HRV in three groups were similar (P > 0.05). Total power and high frequency both showed significant decline after shock in the ATCC and ATCS groups compared with the sham group (all P < 0.01), but this fall was effectively attenuated in sympathectomized rats and the values of total power (P < 0.05) and high frequency (P < 0.01) were higher in the ATCS group than the ATCC group (Fig. 2a and b). Low frequency power was significantly increased from control group values in the ATCC group (P < 0.01) and ATCS groups (P < 0.05) after shock (Fig. 2c). Moreover, a comparison between the ATCC and ATCS groups revealed that sympathectomy significantly inhibited the augmentation of low frequency in the ATCS group (P < 0.01). Low frequency : high frequency ratio, as an index of the sympathetic–vagal balance, was significantly increased in the ATCC and ATCS groups (all P < 0.01, Fig. 2d). However, this increase was evidently reduced by sympathectomy in the ATCS group when compared with ATCC group (P < 0.01, Fig. 2d). Plasma tumor necrosis factor-a and interleukin-6 levels
Compared with the sham group, trauma and shock induced a significant increase in serum TNF-a in the
ATCC and ATCS groups, which reached a peak at 1 h and decreased thereafter (P < 0.01, Fig. 3a). Additionally, the serum interleukin-6 level was significantly increased in the ATCC and ATCS groups compared with those in the sham animals after shock (P < 0.01, Fig. 3b). Chemical sympathectomy significantly decreased the induction of serum TNF-a level at 0, 1 and 2 h after shock (P < 0.01, P < 0.05, P < 0.05, respectively, Fig. 3a). Nevertheless, the serum interleukin-6 showed no significant difference between the ATCC and ATCS groups (P > 0.05, Fig. 3b). Conventional coagulation test and coagulation, anticoagulation and platelet activation parameters
Conventional coagulation tests including PT and APTT are shown in Fig. 4. Basal values of PT and APTT were similar in three groups. The ATCC and ATCS groups had significantly higher PT levels (all P < 0.01) and APTT levels (all P < 0.05) after shock, compared with the sham group. No significant differences were found in PT and APTT between the ATCC and ATCS groups (all P > 0.05). To evaluate the effects of sympathectomy on coagulation, anticoagulant and platelet activation in ATC,
Fig. 2
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Serial changes in parameters of heart rate variability including (a) total power, (b) high frequency, (c) low frequency and (d) low frequency : high frequency ratio in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
156 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 2
Fig. 3
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Serial changes in serum levels of (a) tumor necrosis factor-a (TNF-a) and (b) interlukine-6 in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; ##P < 0.01, Sham vs. ATCS.
we assessed the serum levels of TAT, aPC, sTM and sPselectin (Fig. 5). These markers showed no evident difference among three groups at baseline (P > 0.05). There was no significant change in plasma TAT level during the experimental period in the sham group, indicating that the anesthesia and catheterization procedure did not induce a significant hemostatic alteration. Plasma TAT in the ATCC and ATCS groups significantly increased and reached the peak at 1 h after shock and decreased thereafter (P < 0.05, Fig. 5a). Sympathetic denervation did not influence serum level of TAT in the ATCS group compared with the ATCC group (P > 0.05). Serum levels of aPC, sTM and sP-selectin were significantly higher in the ATCC and ATCS groups compared with those in the sham group after shock (all P < 0.01, Fig. 5b, c and d). In the ATCS group, the increase of serum sTM was significantly suppressed compared with the ATCC group (P < 0.01, Fig. 5c). There were no significant differences in aPC or sP-selectin between the ATCC and ATCS groups (P > 0.05, Fig. 5b, 5d).
Fibrinolytic parameters
Markers of fibrinolysis are shown in Fig. 6. Tissue injury and hemorrhagic shock induced a rapid activation of the fibrinolytic system as reflected by an early and sustained increase in the levels of D-dimer, tPA and PAP in the ATCC and ATCS groups compared with those in the sham group (all P < 0.01). Compared with the ATCC group, chronic sympathectomy attenuated the increase in the serum tPA and PAP in the ATCS group (all P < 0.01, Fig. 6b and d). No differences in D-dimer and PAI-1 were found between the ATCC and ATCS groups (P > 0.05, Fig. 6a and c). Sympathoadrenal activation and glycocalyx shedding parameters
Serum levels of catecholamine are shown in Fig. 7. These markers were significantly higher in the ATCC and sham groups compared with those in the ATCS group through the study (P < 0.01, Fig. 7a, b). However, the ATCC and ATCS groups showed a sustained increasing tendency in the catecholamine levels after shock. Serum syndecan-1
Fig. 4
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Serial changes in (a) prothrombin time (PT) and (b) activated partial thromboplastin time (APTT) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. &P < 0.05, ATCC vs. Sham; && P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
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Sympathectomy attenuates acute coagulopathy Xu et al. 157
Fig. 5
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Serial changes in plasma levels of (a) thrombin–antithrombin complex (TAT), (b) activated protein C (aPC), (c) soluble thrombomodulin (sTM) and (d) soluble P-selectin in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &P < 0.05, ATCC vs. Sham; &&P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ## P < 0.01, Sham vs. ATCS.
in the ATCC and ATCS groups exhibited an evident increase compared with that in the sham group (all P < 0.01, Fig. 7c). Furthermore, compared with the ATCC group, chronic sympathectomy produced a significant decrease in serum syndecan-1 in the ATCS group (P < 0.01).
markers including tPA and PAP persistently increased following trauma and shock and were significantly inhibited in sympathectomized rats, reflecting the antifibrinolysis effect of chemical sympathectomy. It is also noteworthy that sympathetic denervation did not alter the values of PT, APTT and the plasma level of activated protein C.
Discussion
ANS consists of the sympathetic and vagus nerve, which typically have opposing effects to each other [24]. Severe injury and hemorrhage are reportedly associated with autonomic dysfunction [18,25], which has been defined as a risk factor for poorer prognosis and death [26]. However, among early counter-regulatory stress responses to severe injury, the sympathetic nerve system plays the central role [15]. According to our observations, sympathetic tone significantly increased in the early posttraumatic period and led to sympathetic–vagal imbalance, reflected by increased low frequency and low frequency : high frequency ratio, respectively [25]. However, chemical sympathectomy markedly diminished the augmentation of low frequency and the low frequency : high frequency ratio, suggesting the key role of sympathetic tone in the neural responses in ATC. Furthermore, our results showed that autonomic function
In this in-vivo rat model of ATC, we observed hypocoagulability (prolongation of PT and APTT) and hyperfibrinolysis (examined by tPA and PAP), demonstrating that ATC was induced by the combination of trauma and hemorrhage. The analysis of HRV parameters revealed the autonomic dysfunction in ATC, which involves the concurrent sympathetic overexcitation and vagal suppression. In addition, sympathectomy effectively counteracted these alterations and attenuated autonomic neuropathy. The production of proinflammatory cytokines (TNF-a) was also significantly inhibited by sympathectomy. Sympathetic denervation significantly suppressed serum levels of endothelial activation and injury markers including sTM and syndecan-1, suggesting that sympathetic nervous system plays a key role in endothelial damage. Furthermore, fibrinolysis
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158 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 2
Fig. 6
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Serial changes in serum levels of (a) D-dimer, (b) tissue type plasminogen activator (tPA), (c) plasminogen activator inhibitor 1 (PAI-1) and (d) plasmin–antiplasmin complex (PAP) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. group; ##P < 0.01, Sham vs. ATCS.
alteration in ATC involves parasympathetic suppression as well, evidenced by the significant decline of high frequency after trauma and shock. This is similar to previous reports showing that vagal activation could be inhibited by sympathetic overexcitation [27]. And this result is reinforced by a report of Norris et al. [26] who found marked decrease in high frequency power in the trauma patients with higher risk of death. In short, these results suggested that sympathetic activation and parasympathetic inhibition synergistically, rather than independently, together lead to autonomic neuropathy in ATC. Following trauma and hemorrhage, serum TNF-a level increased to the peak at 1 h and decreased thereafter, whereas serum interleukin-6 level persistently increased. This result is consistent with the findings from earlier studies suggesting that sympathetic activation evidently mediates acute inflammatory response following trauma [24]. Furthermore, the enhanced expression of TNF-a was significantly reduced by sympathectomy, indicating the anti-inflammatory effect of sympathectomy. However, the parasympathetic system could inhibit proinflammatory cytokines and exert physiological anti-inflammatory effect through a specific cholinergic anti-inflammatory pathway
[28]. Thus, ANS may play an important role in regulating inflammation through the release of different neurotransmitters from both sympathetic and vagus nerve [16]. Meanwhile, the influence of the immune system on ANS was indicated by several studies [16], which reported that TNF-a as a proinflammatory cytokine could potently induce autonomic dysfunction by acting on vago-vagal reflex circuits in the brainstem [29]. We therefore speculated that enhanced inflammatory response facilitates the effect of the sympathetic nerve in mediating autonomic deficits. Hyperfibrinolysis, as an important part of ATC, has been reported to exist in more than half of trauma patients and is associated with poor outcome and high mortality [30]. We observed an apparent increase in the levels of D-dimer, tPA and PAP and an evident fall in fibrinogen in the ATC rats, reflecting the rapid activation of the fibrinolytic system induced by tissue injury and hypoperfusion. Moreover, our results showed that sympathetic denervation markedly attenuated the increase in the serum tPA and PAP in the ATC rats following trauma and shock, suggesting the antifibrinolysis effect of sympathectomy. On the one hand, besides injury and shock, hyperfibrinolysis is also attributed to massive release of
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Sympathectomy attenuates acute coagulopathy Xu et al. 159
Fig. 7
only adrenal gland but also the sympathetic axons in small vessel [32].
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Time (h) Serial changes in serum levels of (a) epinephrine, (b) norepinephrine and (c) syndecan-1 in the Sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &P < 0.05, ATCC vs. Sham; &&P < 0.01, ATCC vs. Sham; # P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
tPA derived from endothelial cells, which is directly damaged by circulating catecholamine [9]. On the other hand, several studies demonstrated that sympathetic axons in resistance vessel walls could store neuron-generated tPA and release it into the plasma resulting in fibrinolysis activation [12,31]. Taking together, we considered that the antifibrinolysis effect of sympathectomy involves the reduction of catecholamine derived from not
Recent studies have found that glycocalyx exerts antiadhesive, anticoagulant and endothelial protective effects under normal condition and its degradation is associated with inflammation and hyperfibrinolysis in trauma [10]. Our study showed that serum levels of sTM and syndecan-1 in the ATC rats exhibited an evident and sustained increase after trauma and shock, showing the endothelial damage and glycocalyx degradation in ATC [9]. But levels of these two markers were significantly suppressed by sympathectomy, confirming the endothelial protective effect of sympathetic denervation. According to our results, sympathetic hyperactivity and subsequent ANS dysfunction are closely related to endothelial injury and glycocalyx degradation. In addition, a recent study concerning the interaction between inflammation and glycocalyx showed that TNF-a is closely related to evident degradation of the glycocalyx and augmented vascular leak [33]. Hence, sympathectomy may clearly preserve the endothelial glycocalyx partly through the reduction of plasma level of TNF-a. Following trauma and hemorrhage, plasma TAT increased and reached the peak at 1 h after shock and decreased thereafter, indicating initial coagulation activation and subsequent hypocoagulability [3], whereas serum aPC continuously raised after shock, demonstrating the role of protein C activation in the pathogenesis of ATC [2]. Sympathectomy, however, did not influence serum level of TAT and aPC. Additionally, PT and APTT in rats with ATC remained unaltered after chemical sympathectomy. These results suggested that the activation of protein C pathway is mainly induced by hypoperfusion [20] rather than the sympathetic overexcitation, suggesting the primary pathogenesis of ATC deserves further study. As severe hypoperfusion remains the central driver in pathogenesis of ATC [2], reversal of tissue hypoperfusion and other hemostatic resuscitation measures are equally important in the treatment. In addition, several limitations exist in our study. We did not set an additional group of sham rats received sympathectomy because animal studies have demonstrated that 6-OHDA would not alter the basal autonomic function of rats [19]. To avoid the influence of hemodilution, rats in our study did not receive any resuscitation, leading to a short observation period. As various studies have shown that ATC occurs in the early stage following trauma (
Chemical sympathectomy attenuates inflammation, glycocalyx shedding and coagulation disorders in rats with acute traumatic coagulopathy Lin XuM, Wen-Kui YuM, Zhi-Liang Lin, Shan-Jun Tan, Xiao-Wu Bai, Kai Ding and Ning Li Acute traumatic coagulopathy (ATC) may trigger sympathoadrenal activation associated with endothelial damage and coagulation disturbances. Overexcitation of sympathetic nerve in this state would disrupt sympathetic– vagal balance, leading to autonomic nervous system dysfunction. The aim of this study was to evaluate the autonomic function in ATC and its influence on inflammation, endothelial and coagulation activation. Male Sprague–Dawley rats were randomly assigned to sham, ATC control (ATCC) and ATC with sympathectomy by 6-hydroxydopamine (ATCS) group. Sham animals underwent the same procedure without trauma and bleeding. Following trauma and hemorrhage, rats underwent heart rate variability (HRV) test, which predicts autonomic dysfunction through the analysis of variation in individual R-R intervals. Then, rats were euthanized at baseline, and at 0, 1 and 2 h after shock and blood gas, conventional coagulation test and markers of inflammation, coagulation, fibrinolysis, endothelial damage and catecholamine were measured. HRV showed an attenuation of total power and high frequency, along with a rise of low frequency and low frequency : high frequency ratio in the ATC rats, which both were reversed by sympathectomy in the ATCS group. Additionally, sympathetic denervation significantly suppressed the increase of proinflammatory
Introduction As important contributors to preventable deaths [1], severe injury and concomitant uncontrolled bleeding often lead to acute traumatic coagulopathy (ATC), which has been defined recently as endogenous coagulation disorders prior to fluid resuscitation [2] and could be observed in the early stage ( 0.05, data not shown). The serial changes of MAP, HR, base excess and fibrinogen are shown in Fig. 1. No significant differences in basal HR, base excess and fibrinogen were found among three groups, but basal MAPs were lower in sympathectomized rats in comparison with control and sham rats (P < 0.01, Fig. 1a). Following the end of shock, MAP in the ATCC and ATCS group both increased at 1 h and then fell at 2 h. Additionally, MAP in the ATCC group was significantly higher than in the ATCS group at 1 and 2 h (P < 0.05). There was an evident fall in base excess and fibrinogen in the ATCC and ATCS groups after shock (all P < 0.01, Fig. 1c and d). No significant differences were found in HR, base excess or fibrinogen between the ATCC and ATCS groups (P > 0.05).
Fig. 1
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Serial changes in (a) mean arterial pressure (MAP), (b) heart rate (HR), (c) base excess and (d) fibrinogen (Fib) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; ##P < 0.01 Sham vs. ATCS.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Sympathectomy attenuates acute coagulopathy Xu et al. 155
Autonomic function before and after trauma and shock
The frequency-domain parameters of HRV are shown in Fig. 2. Basal values of HRV in three groups were similar (P > 0.05). Total power and high frequency both showed significant decline after shock in the ATCC and ATCS groups compared with the sham group (all P < 0.01), but this fall was effectively attenuated in sympathectomized rats and the values of total power (P < 0.05) and high frequency (P < 0.01) were higher in the ATCS group than the ATCC group (Fig. 2a and b). Low frequency power was significantly increased from control group values in the ATCC group (P < 0.01) and ATCS groups (P < 0.05) after shock (Fig. 2c). Moreover, a comparison between the ATCC and ATCS groups revealed that sympathectomy significantly inhibited the augmentation of low frequency in the ATCS group (P < 0.01). Low frequency : high frequency ratio, as an index of the sympathetic–vagal balance, was significantly increased in the ATCC and ATCS groups (all P < 0.01, Fig. 2d). However, this increase was evidently reduced by sympathectomy in the ATCS group when compared with ATCC group (P < 0.01, Fig. 2d). Plasma tumor necrosis factor-a and interleukin-6 levels
Compared with the sham group, trauma and shock induced a significant increase in serum TNF-a in the
ATCC and ATCS groups, which reached a peak at 1 h and decreased thereafter (P < 0.01, Fig. 3a). Additionally, the serum interleukin-6 level was significantly increased in the ATCC and ATCS groups compared with those in the sham animals after shock (P < 0.01, Fig. 3b). Chemical sympathectomy significantly decreased the induction of serum TNF-a level at 0, 1 and 2 h after shock (P < 0.01, P < 0.05, P < 0.05, respectively, Fig. 3a). Nevertheless, the serum interleukin-6 showed no significant difference between the ATCC and ATCS groups (P > 0.05, Fig. 3b). Conventional coagulation test and coagulation, anticoagulation and platelet activation parameters
Conventional coagulation tests including PT and APTT are shown in Fig. 4. Basal values of PT and APTT were similar in three groups. The ATCC and ATCS groups had significantly higher PT levels (all P < 0.01) and APTT levels (all P < 0.05) after shock, compared with the sham group. No significant differences were found in PT and APTT between the ATCC and ATCS groups (all P > 0.05). To evaluate the effects of sympathectomy on coagulation, anticoagulant and platelet activation in ATC,
Fig. 2
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Total power (ms2)
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&&
2
ATCC ATCS SHAM
##
4
ATCC ATCS SHAM
&&
&&
0.6 0.4
**
**
**
##
##
##
0.2 0.0
0.0 Baseline
0
1 Time (h)
2
Baseline
0
1
2
Time (h)
Serial changes in parameters of heart rate variability including (a) total power, (b) high frequency, (c) low frequency and (d) low frequency : high frequency ratio in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
156 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 2
Fig. 3
(b)
&&
20 15
ATCC ATCS SHAM
* *
10
**
Serum TNF-α (ng/ml)
&&
25
5 ##
##
##
&&
&&
Baseline
0 1 Time (h)
2
ATCC ATCS SHAM
&&
##
0
0
Serum IL-6 (ng/ml)
&&
30
30 60 90 120 150 180
(a)
Baseline
##
0 1 Time (h)
##
2
Serial changes in serum levels of (a) tumor necrosis factor-a (TNF-a) and (b) interlukine-6 in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.05, ATCC vs. ATCS; P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. Sham; ##P < 0.01, Sham vs. ATCS.
we assessed the serum levels of TAT, aPC, sTM and sPselectin (Fig. 5). These markers showed no evident difference among three groups at baseline (P > 0.05). There was no significant change in plasma TAT level during the experimental period in the sham group, indicating that the anesthesia and catheterization procedure did not induce a significant hemostatic alteration. Plasma TAT in the ATCC and ATCS groups significantly increased and reached the peak at 1 h after shock and decreased thereafter (P < 0.05, Fig. 5a). Sympathetic denervation did not influence serum level of TAT in the ATCS group compared with the ATCC group (P > 0.05). Serum levels of aPC, sTM and sP-selectin were significantly higher in the ATCC and ATCS groups compared with those in the sham group after shock (all P < 0.01, Fig. 5b, c and d). In the ATCS group, the increase of serum sTM was significantly suppressed compared with the ATCC group (P < 0.01, Fig. 5c). There were no significant differences in aPC or sP-selectin between the ATCC and ATCS groups (P > 0.05, Fig. 5b, 5d).
Fibrinolytic parameters
Markers of fibrinolysis are shown in Fig. 6. Tissue injury and hemorrhagic shock induced a rapid activation of the fibrinolytic system as reflected by an early and sustained increase in the levels of D-dimer, tPA and PAP in the ATCC and ATCS groups compared with those in the sham group (all P < 0.01). Compared with the ATCC group, chronic sympathectomy attenuated the increase in the serum tPA and PAP in the ATCS group (all P < 0.01, Fig. 6b and d). No differences in D-dimer and PAI-1 were found between the ATCC and ATCS groups (P > 0.05, Fig. 6a and c). Sympathoadrenal activation and glycocalyx shedding parameters
Serum levels of catecholamine are shown in Fig. 7. These markers were significantly higher in the ATCC and sham groups compared with those in the ATCS group through the study (P < 0.01, Fig. 7a, b). However, the ATCC and ATCS groups showed a sustained increasing tendency in the catecholamine levels after shock. Serum syndecan-1
Fig. 4
(a)
(b) 30
ATCC &&
25
&&
20 ##
15
Baseline
0
30
##
##
1 Time (h)
2
&
ATCS SHAM
APTT (s)
PT (s)
&&
20
#
#
0 1 Time (h)
2
#
Baseline
ATCC ATCS SHAM
&
25
15
&
Serial changes in (a) prothrombin time (PT) and (b) activated partial thromboplastin time (APTT) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. &P < 0.05, ATCC vs. Sham; && P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Sympathectomy attenuates acute coagulopathy Xu et al. 157
Fig. 5
(a)
(b) 100 ATCS SHAM
20
15 &
10
ATCC
ATCC
Plasma aPC (ng/ml)
Plasma TAT complex (ng/ml)
25
&&
80 &&
60 40 20
##
##
##
0
1
2
0 Baseline
0
1
Baseline
2
Time (h)
Time (h)
(d) 200 && &&
150
ATCS
&&
SHAM
**
**
100
ATCC
**
50 0
Baseline
##
##
0
1
##
2
Time (h)
Plasma sP-selectin (ng/ml)
(c)
Plasma sTM (ng/ml)
ATCS SHAM
&&
150 &&
120
&&
ATCC ATCS
&&
SHAM
90 60 ##
##
##
0
1
2
30 0 Baseline
Time (h)
Serial changes in plasma levels of (a) thrombin–antithrombin complex (TAT), (b) activated protein C (aPC), (c) soluble thrombomodulin (sTM) and (d) soluble P-selectin in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &P < 0.05, ATCC vs. Sham; &&P < 0.01, ATCC vs. Sham; #P < 0.05, Sham vs. ATCS; ## P < 0.01, Sham vs. ATCS.
in the ATCC and ATCS groups exhibited an evident increase compared with that in the sham group (all P < 0.01, Fig. 7c). Furthermore, compared with the ATCC group, chronic sympathectomy produced a significant decrease in serum syndecan-1 in the ATCS group (P < 0.01).
markers including tPA and PAP persistently increased following trauma and shock and were significantly inhibited in sympathectomized rats, reflecting the antifibrinolysis effect of chemical sympathectomy. It is also noteworthy that sympathetic denervation did not alter the values of PT, APTT and the plasma level of activated protein C.
Discussion
ANS consists of the sympathetic and vagus nerve, which typically have opposing effects to each other [24]. Severe injury and hemorrhage are reportedly associated with autonomic dysfunction [18,25], which has been defined as a risk factor for poorer prognosis and death [26]. However, among early counter-regulatory stress responses to severe injury, the sympathetic nerve system plays the central role [15]. According to our observations, sympathetic tone significantly increased in the early posttraumatic period and led to sympathetic–vagal imbalance, reflected by increased low frequency and low frequency : high frequency ratio, respectively [25]. However, chemical sympathectomy markedly diminished the augmentation of low frequency and the low frequency : high frequency ratio, suggesting the key role of sympathetic tone in the neural responses in ATC. Furthermore, our results showed that autonomic function
In this in-vivo rat model of ATC, we observed hypocoagulability (prolongation of PT and APTT) and hyperfibrinolysis (examined by tPA and PAP), demonstrating that ATC was induced by the combination of trauma and hemorrhage. The analysis of HRV parameters revealed the autonomic dysfunction in ATC, which involves the concurrent sympathetic overexcitation and vagal suppression. In addition, sympathectomy effectively counteracted these alterations and attenuated autonomic neuropathy. The production of proinflammatory cytokines (TNF-a) was also significantly inhibited by sympathectomy. Sympathetic denervation significantly suppressed serum levels of endothelial activation and injury markers including sTM and syndecan-1, suggesting that sympathetic nervous system plays a key role in endothelial damage. Furthermore, fibrinolysis
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
158 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 2
Fig. 6
(b) 40 ATCS
&&
SHAM
40 30 20 ##
##
##
10 0
Serum tPA (ng/ml)
50
&& &&
30
0
1
20 10
##
Baseline
2
0
##
##
1
2
Time (h)
Time (h)
ATCS
24
SHAM
20
&& &&
16
&&
## ##
##
1
2
12 Baseline
0
Time (h)
25 ATCC &&
20
&&
ATCS SHAM
15
&&
10
**
ATCC
5
**
28
Serum PAP complex (ng/ml)
(d)
(c) Serum PAI-1 (ng/ml)
SHAM
&&
0 Baseline
ATCC ATCS
##
##
**
Serum D-dimer (ug/l)
ATCC
**
&& &&
**
60
**
(a)
##
0 Baseline
0
1
2
Time (h)
Serial changes in serum levels of (a) D-dimer, (b) tissue type plasminogen activator (tPA), (c) plasminogen activator inhibitor 1 (PAI-1) and (d) plasmin–antiplasmin complex (PAP) in the sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &&P < 0.01, ATCC vs. group; ##P < 0.01, Sham vs. ATCS.
alteration in ATC involves parasympathetic suppression as well, evidenced by the significant decline of high frequency after trauma and shock. This is similar to previous reports showing that vagal activation could be inhibited by sympathetic overexcitation [27]. And this result is reinforced by a report of Norris et al. [26] who found marked decrease in high frequency power in the trauma patients with higher risk of death. In short, these results suggested that sympathetic activation and parasympathetic inhibition synergistically, rather than independently, together lead to autonomic neuropathy in ATC. Following trauma and hemorrhage, serum TNF-a level increased to the peak at 1 h and decreased thereafter, whereas serum interleukin-6 level persistently increased. This result is consistent with the findings from earlier studies suggesting that sympathetic activation evidently mediates acute inflammatory response following trauma [24]. Furthermore, the enhanced expression of TNF-a was significantly reduced by sympathectomy, indicating the anti-inflammatory effect of sympathectomy. However, the parasympathetic system could inhibit proinflammatory cytokines and exert physiological anti-inflammatory effect through a specific cholinergic anti-inflammatory pathway
[28]. Thus, ANS may play an important role in regulating inflammation through the release of different neurotransmitters from both sympathetic and vagus nerve [16]. Meanwhile, the influence of the immune system on ANS was indicated by several studies [16], which reported that TNF-a as a proinflammatory cytokine could potently induce autonomic dysfunction by acting on vago-vagal reflex circuits in the brainstem [29]. We therefore speculated that enhanced inflammatory response facilitates the effect of the sympathetic nerve in mediating autonomic deficits. Hyperfibrinolysis, as an important part of ATC, has been reported to exist in more than half of trauma patients and is associated with poor outcome and high mortality [30]. We observed an apparent increase in the levels of D-dimer, tPA and PAP and an evident fall in fibrinogen in the ATC rats, reflecting the rapid activation of the fibrinolytic system induced by tissue injury and hypoperfusion. Moreover, our results showed that sympathetic denervation markedly attenuated the increase in the serum tPA and PAP in the ATC rats following trauma and shock, suggesting the antifibrinolysis effect of sympathectomy. On the one hand, besides injury and shock, hyperfibrinolysis is also attributed to massive release of
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Sympathectomy attenuates acute coagulopathy Xu et al. 159
Fig. 7
only adrenal gland but also the sympathetic axons in small vessel [32].
(a)
&
&&
&&
ATCC ATCS SHAM
25 ##
#
##
##
20
**
**
1 Time (h)
2
** **
Epinephrine (ng/ml)
30
15
Baseline
0
(b) &&
&&
ATCC
&&
ATCS SHAM
15 10
5
##
#
##
**
##
**
** **
Norepinephrine (ng/ml)
20
0
Baseline
0
1
2
Time (h) (c) &&
Syndecan-1 (ng/ml)
200
&& &&
150
**
**
ATCC ATCS SHAM
** 100 ##
50
##
##
0
Baseline
0
1
2
Time (h) Serial changes in serum levels of (a) epinephrine, (b) norepinephrine and (c) syndecan-1 in the Sham group, acute traumatic coagulopathy (ATC) control (ATCC) group (ATCC rats), and ATC sympathectomized group (ATCS rats) are shown. Values represent means standard error mean. n ¼ 5 at every time point in the Sham group and n ¼ 7–10 at every time point in the ATCC and ATCS groups. P < 0.01, ATCC vs. ATCS; &P < 0.05, ATCC vs. Sham; &&P < 0.01, ATCC vs. Sham; # P < 0.05, Sham vs. ATCS; ##P < 0.01, Sham vs. ATCS.
tPA derived from endothelial cells, which is directly damaged by circulating catecholamine [9]. On the other hand, several studies demonstrated that sympathetic axons in resistance vessel walls could store neuron-generated tPA and release it into the plasma resulting in fibrinolysis activation [12,31]. Taking together, we considered that the antifibrinolysis effect of sympathectomy involves the reduction of catecholamine derived from not
Recent studies have found that glycocalyx exerts antiadhesive, anticoagulant and endothelial protective effects under normal condition and its degradation is associated with inflammation and hyperfibrinolysis in trauma [10]. Our study showed that serum levels of sTM and syndecan-1 in the ATC rats exhibited an evident and sustained increase after trauma and shock, showing the endothelial damage and glycocalyx degradation in ATC [9]. But levels of these two markers were significantly suppressed by sympathectomy, confirming the endothelial protective effect of sympathetic denervation. According to our results, sympathetic hyperactivity and subsequent ANS dysfunction are closely related to endothelial injury and glycocalyx degradation. In addition, a recent study concerning the interaction between inflammation and glycocalyx showed that TNF-a is closely related to evident degradation of the glycocalyx and augmented vascular leak [33]. Hence, sympathectomy may clearly preserve the endothelial glycocalyx partly through the reduction of plasma level of TNF-a. Following trauma and hemorrhage, plasma TAT increased and reached the peak at 1 h after shock and decreased thereafter, indicating initial coagulation activation and subsequent hypocoagulability [3], whereas serum aPC continuously raised after shock, demonstrating the role of protein C activation in the pathogenesis of ATC [2]. Sympathectomy, however, did not influence serum level of TAT and aPC. Additionally, PT and APTT in rats with ATC remained unaltered after chemical sympathectomy. These results suggested that the activation of protein C pathway is mainly induced by hypoperfusion [20] rather than the sympathetic overexcitation, suggesting the primary pathogenesis of ATC deserves further study. As severe hypoperfusion remains the central driver in pathogenesis of ATC [2], reversal of tissue hypoperfusion and other hemostatic resuscitation measures are equally important in the treatment. In addition, several limitations exist in our study. We did not set an additional group of sham rats received sympathectomy because animal studies have demonstrated that 6-OHDA would not alter the basal autonomic function of rats [19]. To avoid the influence of hemodilution, rats in our study did not receive any resuscitation, leading to a short observation period. As various studies have shown that ATC occurs in the early stage following trauma (
