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Diversity of Vascular Reactivity and the Treatment Response in Diabetic, Hypertensive, Hyperlipidemic, and Healthy Rats Subjected to Hemorrhagic Shock

Wu, Yue; Zhu, Yu; Chen, Xiang-Yun; Liu, Liang-Ming; Li, Tao

Author Information
doi: 10.1097/SHK.0000000000000489

Abstract

INTRODUCTION

Trauma-induced death ranks the third place among all diseases in recent years. In addition, trauma is the most common factor leading to the death of young people (1–44 years old) in modern society. There are 3,800,000 to 5,000,000 people who die from trauma each year worldwide (1). Severe hemorrhage accounts for approximately 50% early deaths induced by trauma. Hypertension, diabetes, and hyperlipidemia are the common cardiovascular diseases which seriously threaten the human health; the associated morbidity is increasing year-by-year. There are 190 million diabetic patients and 2 billion hypertensive patients globally as of 2012 (2). In China, the morbidity of diabetes is approximately 9.6%, and the morbidity of hypertension is approximately 24% among people over 15 years old (3). In addition, the number of patients with hyperlipidemia is also rapidly increasing; the morbidity in China is greater than 20%. Few studies indicated that these cardiovascular diseases can affect the outcome of trauma. Ahmad et al. reported that patients with diabetes exposed to trauma have higher hospital morbidity and mortality and longer intensive care unit stay as compared with patients without diabetes, and increased complications (4). Lustenberger analyzed 1,272 patients with traumatic brain injury, and showed that traumatic brain injury in conjunction with diabetes has approximately a 1.5-fold increase in mortality as compared with patients without diabetes (5). However, the pathophysiological features of these patients subjected to traumatic shock and the treatment response to antishock agents are not known. It is important to elucidate this issue for the diagnosis and treatment of these diseases after traumatic shock.

Vascular hyporeactivity plays an important role in the occurrence and outcome of traumatic hemorrhagic shock, which is the important factor to induce the tissue hypoperfusion and organ function damage including mitochondrial function. Our previous studies demonstrated that improving vascular reactivity with arginine vasopressin (AVP) and phorbol-12 myristate-13-acetate (PMA) was beneficial to hemorrhagic shock, which could stabilize the hemodynamics, increase the tissue perfusion, improve the mitochondrial function, and prolong the survival time (6, 7). Some studies found that vascular reactivity was significantly increased in diabetes, hypertension, and hyperlipidemia (8, 9). However, the change pattern of vascular reactivity in these patients after traumatic hemorrhagic shock, and whether improving vascular reactivity is beneficial to these patients are not clear.

To elucidate these issues, using diabetic, hypertensive, hyperlipidemic, and healthy rats, we investigated: the characteristics of the changes in vascular reactivity, hemodynamics, tissue perfusion, and mitochondrial function of liver and kidney in diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock; the treatment response of these rats to vascular reactivity improvement with AVP and PMA in these diseased rats subjected to hemorrhagic shock; and effects of different dosage of common antishock agents (norepinephrine [NE], dopamine [DA], and AVP) on hemorrhagic shock in these diseased rats and healthy rats.

MATERIALS AND METHODS

Ethical approval

The current study and protocol were approved by the Research Council and Animal Care and Use Committee of the Research Institute of Surgery, Daping Hospital, Third Military Medical University (Chongqing, China). All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. The approval number for the animal research is DHEC-2012-020.

Experimental protocol and methods

Animal management

Healthy rats

Fourteen-week-old healthy male and female Sprague–Dawley (SD) rats without any preexisting diseases were purchased from the Animal Center of Research Institute of Surgery, Third Military Medical University. All rats were housed under controlled conditions (22°C, 55%–65% humidity, and 12 h light–dark cycle), and fed a standard rat pellet diet.

Preparation of the diabetic rat model

Six-week-old SD rats were fed a diabetic diet, including 67.5% basic forage, 2.5% yolk, 20% sucrose, and 10% lard oil. After 8 weeks of diabetic diet, rats were administered streptzotocin (35 mg/kg i.v., one time) and blood glucose was measured after 1 week. If the blood glucose level was more than 14 mmol/L, the model was considered successful. The success rate of this model was approximately 80% in the current study.

Preparation of the hyperlipidemic rat model

Six-week-old SD rats were fed with a hyperlipidemic diet, including 77.6% basic forage, 10% yolk, 10% lard oil, 0.2% halocholic acid, and 0.2% propylsulfur-pyrimidine. After 8 weeks of hyperlipidemic diet, the blood lipid levels were measured. If the total cholesterol was more than 6.2 mmol/L, triglycerides were more than 6 mmol/L, and low-density lipoprotein was more than 6 mmol/L, the model was considered successful. The success rate of this model was more than 95% in the current study.

Preparation of hypertensive rats

Fourteen-week-old spontaneously hypertensive (SHR/NcrlVr) rats were purchased from Beijing WeiTongLiHua Company (Beijing, China); the mean arterial pressure (MAP) of these rats was between 140 mm Hg and 170 mm Hg.

Pathophysiological characteristics of diabetic, hypertensive, and hyperlipidemic rats after traumatic hemorrhagic shock

Two traumatic hemorrhagic shock models were adopted for this part of experiment: one was fixed pressure model, the MAP was maintained at 40 mm Hg for 2 h; another was 40% fixed blood volume hemorrhage. On the day of experiment, hypertensive, hyperlipidemic, diabetic, and healthy rats were anesthetized with sodium pentobarbital (30–50 mg/kg). The right femoral artery and vein were catheterized with polyethylene catheters for monitoring the MAP and drug administration, respectively. The left ventricle was catheterized via the right carotid artery for hemodynamics measurement. Following a 10-min equilibration, blood was withdrawn from the femoral artery catheter until the MAP decreased to 40 mm Hg and was maintained at this level for 2 h, or 40% hemorrhage within 40 min (the total estimated blood volume in healthy rat is 70 ml/kg body weight, the diseased rats were referred to this parameter). The vascular reactivities of thoracic artery (TA), superior mesenteric artery (SMA), and left renal artery (LRA), the hemodynamics including MAP, left ventricular systolic pressure (LVSP), the tissue blood flow of liver and kidney and their mitochondrial function were observed at baseline and the end of shock or hemorrhage (n = 8/group). The survival time of rats was observed with separate experiment (n = 16/group). The detailed methods for the measurement of these parameters are described below.

Effects of AVP and PMA on diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock

The fixed pressure shock model (the MAP was maintained at 40 mm Hg for 2 h) was used in following experiment. At the end of shock, rats were infused AVP (0.4 U/kg) or PMA (1 μg/kg) with two volumes of shed blood of lactated Ringer‘s solution (LR). The average infusion rate of fluid was 20 mL/h. The shed blood was not re-infused. The vascular reactivity, hemodynamics, and the blood flow of liver and kidney and their mitochondrial function were observed at the end of infusion or at 2 h after infusion (n = 8/group). The survival time was also observed with separate experiment (n = 16/group).

Effects of common antishock agents in different dosages on diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock

In this aspect of the study, we observed the effects of common antishock agents at different dosages on MAP, tissue blood flow (n = 8/group), animal survival (separate experiment, n = 16/group) in diabetic, hypertensive, hyperlipidemic, and healthy rats after hemorrhagic shock (MAP maintained at 40 mm Hg for 2 h) to compare their appropriate effective dosage. The antishock agents and the dosages we used in the current study were AVP (0.04 U/kg, 0.1 U/kg, 0.4 U/kg, 1 U/kg, and 4 U/kg); NE (5 μg/kg/min, 10 μg/kg/min, 15 μg/kg/min, 30 μg/kg/min, and 50 μg/kg/min); and DA (1 μg/kg/min, 3 μg/kg/min, 5 μg/kg/min, 10 μg/kg/min, and 15 μg/kg/min).

Measurement methods

Hemodynamics, blood flow of liver and kidney

The MAP and LVSP were determined with a polygraph physiologic recorder (SP844, Power Laboratory; AD Instruments, Castle Hill, Australia) via a pressure transducer as described. The liver and kidney (left) blood flow were measured by a Laser Doppler Blood Flowmeter (Periflux System 5000; Primed, Stockholm, Sweden) as previously described (10).

Vascular reactivity

After in vivo measurement in each experiment, TA, SMA, and LRA were obtained to make a 2 mm to 3 mm long artery ring. The responses of these artery rings to a series of concentrations of NE (1 × 10–10–1 × 10−4 mol/L) were measured using a Power Lab System via a force transducer (AD Instruments), as previously described (11). The concentration–response curve and the maximal contractile tension (Emax) to NE were used to reflect the vascular reactivity.

Mitochondrial function of liver and kidney

After measurement of in vivo parameters in each experiment, samples of liver or kidney tissues (5 g) were obtained and used to extract mitochondria. To assure a mitochondrial rich fraction, sample management must be under ice-cold condition and go through the strict centrifuge procedures according to previous report (12). Briefly, liver or kidney sample was put into 20 mL ice-cold isolation buffer (0.25 M sucrose, 0.1 mmol/L Na2EDTA, 0.01 M Tris, pH 7.6). They were cut into small pieces and washed three times to remove blood. Tissues with isolation buffer were homogenized and then centrifuged at 1,600 × g for 12 min at 4°C. The supernatant was further centrifuged twice at 25,000 × g for 15 min at 4°C. The pellet was collected and resuspended in 1 mL isolation buffer. The concentration of protein was measured using the Lowry method (13). Subsequently, the mitochondrial function was measured. Briefly, 1.4 mL of measurement buffer warmed to 30°C was added to the reaction chamber and equilibrated. The mitochondrial mixture was then put into the reaction chamber and equilibrated, and then the mitochondrial metabolic substrates sodium malate, sodium glutamate, and adenosine diphosphate (400 nmol/L) were added in succession. The rate of oxygen consumption was determined by a mitochondrial function analyzer (MT 200; Strathkelvin, United Kingdom). Mitochondrial function was represented by the respiration rate (RCR) (consumed oxygen rate with and without adenosine diphosphate (10).

Statistical analyses

Data (blood loss, MAP, LVSP, vascular reactivity, tissue blood flow, and RCR) were presented as the mean ± SD of n observations. The statistical differences of the data among groups in the current study were analyzed by one factor (different diseases) or two factors (diseases and treatments): ANOVA analyses, followed by the post hoc Tukey test (SPSS ver15.0; SPSS Incorporated, Chicago, IL) for multiple comparisons between two groups. Before ANOVA analyses, all data received Kolmogorov–Smirnov normality test and Bartlett sphericity test; results showed all data from different groups of rats satisfied the normality and hemogeneity-of-variance. The statistical analyses were corrected for multiple comparisons. The time and prevalence of survival were analyzed by median and interquartile ranges, Kaplan–Meier survival analyses, and the log-rank test. The difference at P < 0.05 was considered significant; the difference at P < 0.01 was considered very significant.

RESULTS

Pathophysiological characteristics of diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock

Total blood loss

In general, MAP maintained at 40 mm Hg for 2 h hemorrhagic shock induced a greater blood loss in diabetic (8.9 mL/rat), hypertensive (10.3 mL/rat), and hyperlipidemic (10.3 mL/rat) rats than in healthy rats (7.1 mL/rat). The blood loss rates were 69.7%, 55.8%, 52.6%, and 43.9% accounting for the total estimated blood volume in hypertensive, diabetic, hyperlipidemic, and healthy rats, respectively (Table 1).

Table 1
Table 1:
Changes of blood loss, MAP, and LVSP (mm Hg) in diabetic, hypertensive, and hyperlipidemic rats after 40 mm Hg for 2 h of hemorrhagic shock

Hemodynamics

MAP at 40 mm Hg for 2 h hemorrhagic shock induced a more remarkable decrease in LVSP in diabetic, hypertensive, and hyperlipidemic rats than in healthy rats. The decrease rates of LVSP in diabetic, hypertensive, and hyperlipidemic rats were 60.7%, 66.3%, and 59.3%, respectively; they were significantly higher than the decrease rate in healthy rats, 51.9% (P < 0.05) (Table 1).

Vascular reactivity

Without hemorrhage shock insult, hypertensive, diabetic, and hyperlipidemic rats had higher vascular reactivity than in healthy rats, while following hemorrhagic shock, the diseased rats had greater decrease rate than in healthy rats. For example, the decrease rates of vascular reactivity in TA in hypertensive, diabetic, hyperlipidemic, and healthy rats were 53.1%, 57.2%, 42.1%, and 31.2%, respectively, after hemorrhagic shock (Fig. 1).

Fig. 1
Fig. 1:
The contractile responses of the SMA, TA, and LRA from hypertensive, diabetic, and hyperlipidemic rats before and after hemorrhagic shock.Data are mean ± standard deviation (n = 8/group). Differences between experimental groups were analyzed using two-factor ANOVA. A, B, C: The contractile response of thoracic aorta (TA) to norepinephrine (NE); D, E, F: the contractile response of superior mesenteric artery (SMA) to NE; G, H, I: the contractile response of left renal artery (LRA) to NE. DM, diabetes; HyL, hyperlipidemia; HyP, hypertension. ** P < 0.01 versus healthy rats before shock; ## P < 0.01 versus before shock in healthy, diabetic, hypertensive, or hyperlipidemic rats; @ P < 0.05, @@ P < 0.01 versus healthy rats after shock.

Blood flow of liver and kidney and their mitochondrial function

Hemorrhagic shock (40 mm Hg for 2 h) induced a significant decrease in blood flow and the mitochondrial function in liver and kidney in hypertensive, diabetic, hyperlipidemic, and healthy rats. Diseased rats had a more significant decrease than in healthy rats when subjected to hemorrhagic shock. For example, the decrease rates in tissue blood flow and mitochondrial function (represented by mitochondrial respiratory control rate) of liver and kidney in healthy rats were approximately 30%, whereas the decrease rates of these parameters in these diseased rats were all more than 50% (Fig. 2, A–D).

Fig. 2
Fig. 2:
The changes of blood flow, mitochondrial function in the liver and kidneys in hypertensive, diabetic, and hyperlipidemic rats before and after hemorrhagic shock and survival.Data are mean ± standard deviation. Differences between experimental groups were analyzed using one-factor or two-factor ANOVA. A, B: Blood flow of the liver and kidneys (n = 8/group); C, D: mitochondrial function of the liver and kidneys (n = 8/group); E, F: the changes of survival time, survival rate in healthy and diseased rats following shock (n = 16/group). DM, diabetes; HyL, hyperlipidemia; HyP, hypertension; RCR, respiratory control rate. ** P < 0.01 versus healthy rats; ## P < 0.01 versus before shock in healthy, diabetic, hypertensive, or hyperlipidemic rats; @ P < 0.05, @@ P < 0.01 versus healthy rats after shock.

Survival time and survival rate

After hemorrhagic shock, the natural death time in hypertensive, diabetic, and hyperlipidemic rats was less than in healthy rats. In 40 mm Hg 2-h hemorrhagic shock model, the natural death time (survival time) in diabetic, hypertensive, and hyperlipidemic rats was 2.65 h, 3.75 h, and 5.45 h, respectively, which were significantly shorter than in healthy rats (8.95 h). There was no significant difference in natural death time among hypertensive, diabetic, and hyperlipidemic rats (Fig. 2, E and F).

The changes of hemodynamics and natural death time in diabetic, hypertensive, and hyperlipidemic rats subjected to 40% hemorrhage

As the same trend to fixed pressure hemorrhagic shock, 40% hemorrhage induced a more severe decrease in MAP and LVSP in diabetic, hypertensive, and hyperlipidemia rats than in healthy rats. Although the absolute values of MAP and LVSP in hypertensive rats were higher than in healthy rats, the decrease rates were greater in these diseased rats than in healthy rats (see Table, Supplemental Digital Content 1, at http://links.lww.com/SHK/A350). The natural death time in hypertensive, diabetic, and hyperlipidemic rats after 40% hemorrhage was also less than in healthy rats. The natural death time (survival time) in diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock was 4.5 h, 5.2 h, and 7.4 h, respectively, which were significantly shorter than in healthy rats (12.2 h) (see Figure, Supplemental Digital Content 2, at http://links.lww.com/SHK/A351). Because the change trend of vascular reactivity and tissue blood flow in healthy rats and diabetic, hypertensive, and hyperlipidemic rats after 40% hemorrhage were the same to fixed pressure hemorrhagic shock, so their data were not shown here.

Effects of AVP and PMA on diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock

Vascular reactivity

AVP (0.4 U/kg) or PMA (1 μg/kg) treatment significantly improved the contractile responses of TA, SMA, and LRA to NE both in diseased rats (diabetes, hypertension, and hyperlipidemia) and healthy rats after hemorrhagic shock. The improving effect in healthy rats was better than in diseased rats. The beneficial effect of AVP was better than PMA (Fig. 3).

Fig. 3
Fig. 3:
Effects of AVP and PMA on vascular reactivity in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock.Data are mean ± standard deviation (n = 8/group). Differences between experimental groups were analyzed using one-factor ANOVA. A, B, C: Maximum vascular reactivity of thoracic artery (TA), superior mesenteric artery (SMA), and left renal artery (LRA) to norepinephrine (NE) after 2 h of administration; D, E, F: increase rate of TA, SMA, and LRA contraction as compared with shock. AVP, arginine vasopressin; DM, diabetes; HyL, hyperlipidemia; HyP, hypertension; PMA, phorbol-12-myristate-13-acetate. * P < 0.05, ** P < 0.01 versus LR group with same disease status; # P < 0.05 versus healthy rats with same treatment.

Hemodynamics

As the same vascular reactivity, AVP (0.4 U/kg) or PMA (1 μg/kg) treatment significantly improved the MAP and LVSP both in diseased (diabetes, hypertension, and hyperlipidemia) and healthy rats after hemorrhagic shock. AVP and PMA improving the hemodynamic parameters in healthy rats were better than in diabetic, hypertensive, and hyperlipidemic rats. The effect of AVP was better than PMA (Table 2).

Table 2
Table 2:
Effects of PMA and AVP infusion on MAP and LVSP in diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock

Blood flow and mitochondrial function of liver and kidney

Similar to the changes of vascular reactivity and hemodynamics, AVP and PMA treatment significantly improved the tissue blood flow and mitochondrial function of the liver and kidneys in diseased rats (diabetes, hypertension, and hyperlipidemia) and healthy rats after hemorrhagic shock. The improving effects of AVP and PMA on the two parameters in healthy rats were better than in diseased rats (Fig. 4).

Fig. 4
Fig. 4:
Effects of AVP and PMA on blood flow and mitochondrial function of the liver and kidneys in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock.Data are mean ± standard deviation (n = 8/group). Differences between experimental groups were analyzed using one-factor ANOVA. A, B: Blood flow of the liver and kidneys after 2 h of administration; C, D: respiratory control rate of mitochondria of the liver and kidneys after 2 h of administration (n = 8/group). AVP, arginine vasopressin; DM, diabetes; LR, lactated Ringer‘s solution; HyL, hyperlipidemia; HyP, hypertension; PMA, phorbol-12-myristate-13-acetate. # P < 0.05, ## P < 0.01 versus LR group in the same disease rats.

Survival

As compared with LR, AVP and PMA treatment significantly improved the animal survival, and prolonged the survival time of diseased (diabetes, hypertension, and hyperlipidemia) and healthy rats subjected to hemorrhagic shock. The beneficial effects of AVP and PMA treatment in healthy rats were superior to in the diseased rats; AVP had a better effect than PMA on animal survival (Fig. 5).

Fig. 5
Fig. 5:
Effects of AVP and PMA on animal survival in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock.Survival time and survival rate were analyzed by median and interquartile range and Kaplan–Meier survival analysis and log-rank test (n = 16/group). A, B, C: Survival rate after LR infusion, PMA, and AVP administration; D: survival time. AVP, arginine vasopressin; DM, diabetes; LR, lactated Ringer‘s soluiton; HyL, hyperlipidemia; HyP, hypertension; PMA, phorbol-12-myristate-13-acetate. * P < 0.05, ** P < 0.01 versus healthy rats with same treatment. # P < 0.05, ## P < 0.01 versus LR group in the same disease rats.

Effects of different dosage of common antishock agents (NE, DA, AVP) on diabetic, hypertensive, and hyperlipidemic rats after hemorrhagic shock

MAP

AVP, NE, and DA treatment significantly increased the MAP in healthy rats; there was no significant difference among different doses of AVP, NE, and DA. Although in diabetic, hypertensive, and hyperlipidemic rats only lower doses of AVP (0.01 U/kg, 0.1 U/kg, and 0.4 U/kg), NE (5–15 μg/kg/min), and DA (1–5 μg/kg) increased the MAP, higher doses of AVP, NE, and DA did not further increase but decreased the MAP (Table 3).

Table 3
Table 3:
The changes of MAP after different concentration of vasoactivator (mm Hg)

Survival

As compared with LR control group, AVP significantly increased the survival rate and survival time both in healthy rats and diseased rats. In healthy rats, five doses of AVP (0.04–4 U/kg) all increased the animal survival of shock rats. Although in diabetic, hypertensive, and hyperlipidemic rats only two lower doses of AVP (0.01 U/kg and 0.1 U/kg) increased the animal survival, more than 0.4 U/kg of AVP decreased the animal survival. The trend of NE and DA on animal survival in healthy and diseased rats was similar to AVP, whereas AVP had a better effect on animal survival than NE and DA, NE had the worst effect among AVP, NE, and DA (Fig. 6, A–C).

Fig. 6
Fig. 6:
Effects of different dosages of AVP, NE, and DA on animal survival and blood flow in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock.Survival time was analyzed by median and interquartile range and Kaplan–Meier survival analysis and log-rank test (n = 16/group). Blood flow was expressed by mean ± standard deviation (n = 8/group). A, B, C: Effects of arginine vasopressin (AVP), norepinephrine (NE), and dopamine (DA) on survival time; D, E, F: effects of AVP, NE, and DA on blood flow of liver; G, H, I: effects of AVP, NE, and DA on blood flow of kidney. DM, diabetes; HyL, hyperlipidemia; HyP, hypertension. * P < 0.05, ** P < 0.01 versus healthy group.

Blood flow of liver and kidney

AVP, NE, and DA treatment significantly increased the blood flow in healthy rats, whereas there was no significant difference among different doses of AVP, NE, and DA. Although in diabetic, hypertensive, and hyperlipidemic rats only two lower doses of AVP (0.01 U/kg and 0.1 U/kg), NE (5–10 μg/kg/min), and DA (1–3 μg/kg) increased the blood flow of liver and kidney, higher doses of AVP, NE, and DA did not further increase but decreased the blood flow of liver and kidney. Effects of AVP on blood flow were superior to NE and DA both in healthy and diseased rats (Fig. 6, D–I).

DISCUSSION

Trauma-induced death ranks the third place among all diseases in recent years. And in modern society, metabolic cardiovascular diseases, such as hypertension, diabetes, and hyperlipidemia, are more and more. Few studies, however, focused on the diagnosis and treatment for these diseases when suffering from severe trauma and hemorrhagic shock. The current study primarily investigated the pathophysiological features and treatment responses using hypertensive, diabetic, and hyperlipidemic rats subjected to hemorrhagic shock. The results showed that two hemorrhagic shock models (40% fixed hemorrhage or 40 mm Hg for 2 h fixed pressure hemorrhagic shock) all induced a more severe damage on vascular reactivity, hemodynamics, tissue perfusion, and mitochondrial function of vital organs, and led to a more rapid death in hypertensive, diabetic, and hyperlipidemic rats than in health rats. Improving vascular reactivity with AVP and PMA prolonged the survival time both in diseased rats and healthy rats. AVP and PMA had better effect in healthy rats than in diseased rats. Similarly, common antishock agents, NE, DA, and AVP, had better effect on hemorrhagic shock in healthy rats than in diseased rats. AVP, DA, and NE had wider effective dosages in healthy rats than in diseased rats following hemorrhagic shock.

Two hemorrhagic shock models were adopted in the current study. Many factors can affect the total blood volume such as age, sex, obesity, and so on. There is no evidence to exactly show the blood volumes in normal, hyperlipidemic, hypertensive, and diabetic rats are similar. To avoid the diversity of the blood volumes among diseased rats and healthy rats bringing the bias of results, two types of hemorrhagic shock model were used in current study. The results showed that the change tendencies of each parameter in these two hemorrhagic shock models were accordant in same diseases rats, whereas the severity between healthy rats and diseased rats was different. The reason may be because metabolic-cardiovascular diseases have preexisting damage before hemorrhage, especially cardiovascular complications. These results suggest that the severities of organ damage in traumatic or hemorrhagic shock patients with basic disease are different from those without basic disease although their MAP or estimated blood loss is the same, so more attentions should be paid to the diagnosis and treatment on these patients when subjected to severe trauma and shock.

Previous study demonstrated that vascular hyporeactivity is a very important pathophysiological feature in many critical illnesses such as severe trauma, shock, and sepsis (14). Good effects have been achieved in recent years aimed at improving vascular hyporeactivity for severe trauma, shock, and sepsis (15). Our previous studies demonstrated that AVP and PMA have beneficial effects on severe trauma and shock by improving decreased vascular reactivity (7, 11). The current study showed that AVP and PMA were also beneficial to diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock via improving vascular reactivity, and via improving the tissue perfusion and hemodynamics. AVP had better effect than PMA. Among many PKC isoforms, our previous studies demonstrated that PKCα and PKCε are the main isoforms participated in the regulation of vascular reactivity after hemorrhagic shock; their agonists can improve the decreased vascular reactivity (16). Nevertheless, we found that the nonspecific agonist of PKC, phorbol ester (PMA), could better improve the vascular reactivity of shock animal and via this action improving the survival of shock animal than specific agonist of PKC did (6). So, in the current study, phorbol ester (PMA) was used to activate PKC to improve the vascular reactivity.

Our previous studies demonstrated that the vascular reactivity of blood vessel from different place or organ had some differences after hemorrhagic shock. For example, Liu et al. reported the vascular reactivity of celiac artery and left femoral artery (LFA) lost more severe and faster than SMA and LRA after hemorrhagic shock (17). Zhu et al. found that over 30 min of hemorrhagic shock could damage the vascular reactivity in many blood vessels including LFA, SMA, right renal artery (RRA), pulmonary (PA), and middle cerebral artery (MCA). LFA had the highest loss rate of vascular reactivity (64.51%), followed by the SMA (44.69%), TA (36.06%), PA (37.83%), and RRA (32.33%), MCA had the lowest loss rate (18.45%) (18). In addition, many studies found that the blood vessels were damaged in metabolic diseases such as diabetes and hyperlipidemia. Different blood vessels had different extent of damage. Research showed, for example, diabetes mainly damages renal artery and retinal artery (19). To avoid the result bias using only one blood vessel, we adopted three arteries including the TA (representing main conductive blood vessel) and SMA and LRA (representing blood vessels to organ) as the representatives in the current study. The results showed that the decreased rate of vascular reactivity in TA, SMA, and LRA in diabetic, hypertensive, and hyperlipedemic rats had a similar trend.

Based on the pathophysiological features of traumatic hemorrhagic shock, the main measures by which to resuscitate traumatic hemorrhagic shock include fluid resuscitation and hemodynamic support. AVP, NE, and DA are the common antishock agents in clinic. Our current study found that although AVP, NE, and DA were effective both in healthy rats and in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock, their effective dosage range were different. AVP, NE, and DA had a wider effective dosage range in healthy rats than in diabetic, hypertensive, and hyperlipidemic rats. The results showed that in diabetic, hypertensive, and hyperlipidemic rats, only lower doses of AVP, NE, and DA increased the MAP; the blood flow of liver and kidney and the animal survival; higher doses of AVP, NE, and DA did not further increase the beneficial effects, while decreased the beneficial effects. There were other similar reports; for instance, Abe et al. found that high-dose intravenous administration of DA led to further hemodynamic deterioration in patients with tabotsubo cardiomyopathy (20). Our previous study found that increasing NE dose in septic shock patients did not further increase the MAP, whereas MAP was decreased from 75 mm Hg to 60 mm Hg (21). The mechanisms responsible for high doses of vasoactives inducing MAP and other hemodynamic parameters decrease may be related to following reasons: high dose of vasoactives can speed up the heart rate and increase the oxygen consumption, damaging the cardiac function; long-time stimulation of high dose of vasoactives can result in the adrenergic receptor desensitization. Sandrini et al. reported that the amount of α- and β-adrenergic receptors in heart and α2-adrenergic receptors in spleen were significantly reduced for high concentration of catecholamine stimulation after hemorrhagic shock (22). Maisel et al. demonstrated that intraperitoneal injection of epinephrine (0.25 mg/kg) resulted in a 75% total α1-receptor and 50% β-adrenergic receptor density loss in guinea pig hearts (23).

Dopamine and NE are commonly recommended as the first-line antishock agents in clinic (24). Dopamine is considered beneficial in the treatment of hemorrhagic shock with LR solution or hydroxyethyl starch (25). There are some controversies about the effect of NE in the treatment of hemorrhagic shock; the focus of the concerns is the strong vasoconstriction effect of NE (26). The current study showed that NE, DA, and AVP all had some beneficial effects in diabetic, hypertensive, and hyperlipidemic rats subjected to hemorrhagic shock. Although among the three antishock agents, AVP had the best effect, DA was the next. Other studies support the notion that AVP can increase tissue perfusion and oxygen delivery. For example, Meybohm et al. reported that during hemodynamics decompensation, AVP infusion significantly increased the cerebral perfusion pressure and cerebral venous partial pressure of oxygen as compared with NE after 10 min of therapy (27). Taken together, AVP may be recommended as the first-line antishock agent at the early stage for some chronic cardiovascular diseases when subjected to hemorrhagic shock.

There are many diabetic models including spontaneous, chemical- or operation-inducing diabetic model. Hyperlipidemia combined with streptozotocin-induced diabetic rat model is a common diabetic model. Before streptozotocin administration, rats received 8 weeks of hyperlipidemia diet. Streptozotocin is a DNA alkylation agent which can target to damage langerhans’ β cell and duplicate type-2 diabetic model. As compared with other chemical agent such as alloxan, streptozotocin has more specificity to langerhans’ β cell, less toxicity, and higher successful rate. This model is similar to diabetic patients in clinical (28–32). So hyperlipidemia combined with streptozotocin diabetic model was used in current study.

Some limitations need further investigation. First, this study was limited to small animals (rats), whether these findings can be extrapolated to large animals and humans need further investigation. Second, this study only explored the diversity of treatment response to AVP, NE, and DA, what are the responses to other measures such as fluid resuscitation and other antishock agents need further investigation. Third, vascular reactivity was determined with in vitro technique; in vivo measurement should be paid attention to.

CONCLUSIONS

A same severity of hemorrhagic shock induces a more severe damage on cardiovascular function in metabolic disease rats than in healthy rats, and this change results in a not good treatment effect in these diseased rats to vascular reactivity improving measures and common antishock agents. Because of these pathophysiological features and treatment responses, more attention should be paid to the early diagnosis and application of antishock agents for such patients with hemorrhagic shock.

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Keywords:

Diabetes; hemorrhage shock; hyperlipidemia; hypertension; vascular reactivity

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