SHOCK, Vol. 42, No. 5, pp. 464Y471, 2014
PROTECTION AGAINST INTESTINAL INJURY FROM HEMORRHAGIC SHOCK BY DIRECT PERITONEAL RESUSCITATION WITH PYRUVATE IN RATS Jing-Jing Zhang, Zong-Ze Zhang, Jian-Juan Ke, Xiang-Hu He, Jia Zhan, Dong-Ling Chen, Yi-Peng Wang, and Yan-Lin Wang Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China Received 9 May 2014; first review completed 27 May 2014; accepted in final form 25 Jun 2014 ABSTRACT—Objective: We explored the effects of direct peritoneal resuscitation with pyruvate-peritoneal dialysis solution (PDS) following intravenous resuscitation (VR) on intestinal ischemia-reperfusion injury in rats with hemorrhagic shock (HS). Methods: Fifty rats were randomly assigned equally to five groups. In group sham, a surgical operation was performed on rats without shock or resuscitation. In group VR, rats were subjected only to VR. In groups NS, LA, and PY, direct peritoneal resuscitation was performed with normal saline (NS), lactate-based PDS (Lac-PDS), and pyruvate-based PDS (Pyr-PDS), respectively, after VR. Mean arterial pressure was monitored in the right common carotid artery. Two hours after resuscitation, the lactate level in arterial blood and the wet weight/dry weight ratio of the intestine were determined. The intestinal mucosal damage index was estimated, and ultrastructural changes in the intestinal mucosa were observed. Malondialdehyde, myeloperoxidase, nitric oxide, and tumor necrosis factor ! levels were also measured. Results: Two hours after HS and resuscitation, the increase in arterial blood lactate and intestinal wet weight/dry weight ratio declined significantly in rats from Groups LA and PY compared with groups VR and NS, whereas group PY was more advantageous in the changes of these parameters. The intestinal mucosal damage index and ultrastructural changes were also improved in groups LA and PY when compared with groups VR and NS. Protection was more apparent with Pyr-PDS than Lac-PDS. Hemorrhagic shock resulted in a significant increase in malondialdehyde levels and myeloperoxidase activity and was accompanied by overexpression of tumor necrosis factor ! and a reduction in nitric oxide levels. These changes were significantly attenuated by Lac-PDS and Pyr-PDS at 2 h after resuscitation, and Pyr-PDS showed more effective protection for the intestine than Lac-PDS. Conclusions: Direct peritoneal resuscitation with Lac-PDS and Pyr-PDS after VR alleviated intestinal injury from HS in rats, and Pyr-PDS was superior to Lac-PDS in its protective effect. Mechanisms of action might include the elimination of free oxygen radicals, reduction of neutrophil infiltration, inhibition of the inflammatory response, and regulation of intestinal mucosal blood flow and barrier function. KEYWORDS—Peritoneal dialysis solution, lactate, intestinal mucosa, ischemia-reperfusion injury, oxygen free radicals, inflammatory mediators ABBREVIATIONS—DPRVdirect peritoneal resuscitation; VRVintravenous resuscitation; PDSVperitoneal dialysis solution; HSVhemorrhagic shock; MAPVmean arterial pressure; MDAVmalondialdehyde; MPOVmyeloperoxidase; NOVnitric oxide; TNF-!Vtumor necrosis factor !; IMDIVintestinal mucosal damage index
pyruvate protects splanchnic organ function from HS in rats (6), and pyruvate prolongs the survival time of animals experiencing HS and resuscitation (7). Pyruvate Ringer_s solution effectively treats hypoxic lactic acidosis and significantly increases the survival rate of rats with lethal HS (8). Conventional resuscitation has the potential to improve conventional indicators such as blood pressure, heart rate, central blood volume, and urine volume. However, the stomach, intestine, liver, and kidney experience continuous vasoconstriction and hypoperfusion. As a result, patients often die of systemic inflammation and multiple organ dysfunction syndrome (MODS) after severe HS and conventional resuscitation. As a new therapy of HS, intraperitoneal resuscitation or direct peritoneal resuscitation (DPR) can improve blood circulation of organs such as the intestine. Furthermore, this treatment can avoid MODS and increase the survival rate (9Y12) The intestine plays a key role in the pathophysiologic changes that follow severe HS and intestinal ischemiareperfusion injury (IRI) caused by HS, and conventional resuscitation plays a critical role in the development of systemic inflammation and MODS (1, 13). In this study, we evaluated the effects of DPR with pyruvate-based peritoneal dialysis
Hemorrhagic shock (HS) is a major cause of death caused by trauma. Hemorrhagic shock causes a series of pathophysiologic changes, including microcirculation disturbance, a local reduction in blood perfusion, a lack of oxygen supply in tissues, and metabolic disorder. Recovery of blood perfusion and the maintenance of oxygen supply for tissues are the major goals of resuscitation after HS. Therefore, effective hemorrhage control and optimal resuscitation are the primary objectives for the treatment of severely injured patients (1). However, resuscitation, if not performed properly, can exacerbate cellular injury caused by HS. The type of fluid used for resuscitation plays an important role in the pattern of injury (2). Pyruvic acid is an important substrate of the tricarboxylic acid cycle, and pyruvate is an effective antioxidant and free radical scavenger (3Y5). Recent research has demonstrated that
Address reprint requests to Yan-Lin Wang, MD, PhD, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, No. 169 Donghu Rd, Wuhan 430071, Hubei, China. E-mail: [email protected]. This research supported by the Fundamental Research Funds for the Central Universities (no. 2012103030042). DOI: 10.1097/SHK.0000000000000230 Copyright Ó 2014 by the Shock Society
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SHOCK NOVEMBER 2014 solution (Pyr-PDS) following intravenous resuscitation (VR) on intestinal IRI from HS and resuscitation in rats compared with DPR with lactate-based PDS (Lac-PDS), normal saline (NS), and VR alone. We evaluated major parameters and explored the possible mechanism of DPR with Pyr-PDS. MATERIALS AND METHODS Materials Sodium pyruvate and sodium lactate were purchased from Sigma (St Louis, Mo). Lactate, malondialdehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO) detection kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Streptavidin/peroxidase (SP-9003) kits were purchased from Zymed Laboratories (San Diego, Calif).
Preparation of solution In this study, we used 2.5% Glu-Pyr-PDS (Pyr-PDS; 396 mOsm/L, pH 5.2), which contained 40 mmol/L of pyruvate, 132 mmol/L of Na+, 1.75 mmol/L of Ca2+, 0.25 mmol/L of Mg2+, 96 mmol/L of Clj, and 2.5 g/dL of glucose, and 2.5% Glu-Lac-PDS (Lac-PDS; 396 mOsm/L, pH 5.2), which contained 40 mmol/L of lactate, 132 mmol/L of Na+, 1.75 mmol/L of Ca2+, 0.25 mmol/L of Mg2+, 96 mmol/L of Clj, and 2.5 g/dL of glucose. Both DPR solutions were prepared fresh in the laboratory. The pH was adjusted to 5.2 with HCl or NaOH. The DPR solutions were kept in the refrigerator at 4-C and warmed up to room temperature before use. The Pyr-PDS stability was verified by determining pyruvate concentrations with the high-performance liquid chromatography analysis (6). The Lac-PDS prepared herein was the same as the standard PDS available commercially.
Animals Male Sprague-Dawley rats (200Y250 g) were purchased from The Center for Animal Experiment of Wuhan University (Wuhan, China). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Experimental procedures were approved by the Animal Experiment Committee of Wuhan University (China). All the animals were acclimatized to 12-h light-dark cycles for 1 week with free access to food and water. Rats were fasted overnight and allowed water until 4 h before experimentation.
Hemorrhagic shock and resuscitation protocols Rats were anesthetized with pentobarbital sodium (45 mg/kg, i.p.). Hemorrhagic shock and resuscitation in rats were induced as previously described with slight modifications (14). Rats were allowed to breathe spontaneously. PE-50 catheters were placed in the right carotid artery and connected to a pressure transducer for continuous arterial blood pressure monitoring with a Surgivet Advisor Vital Signs (Smiths Medical, Norwell, Mass). Catheters were also placed in the left femoral artery and connected to a syringe for blood withdrawal to induce shock and in the right femoral vein for heparinization (heparin sodium, 300 IU/kg) and infusion of fluid. Surgery was performed with aseptic technique. All catheters were filled with NS containing heparin (100 IU/mL). A heat lamp was used to maintain normal body temperature. The rats were allowed to stabilize for 15 to 30 min after surgery. Hemorrhagic shock was induced by withdrawing blood within 10 min using the syringe connected to the left femoral artery. Mean arterial pressure (MAP) was maintained at 35 T 5 mmHg for 60 min by further withdrawal or reinfusion of blood as required before fluid resuscitation. All animals were randomly divided into five groups (n = 10 each). In group sham, animals underwent surgical preparation, but not HS or resuscitation. At the end of the HS period in group VR, NS equal to twice the volume of the withdrawn blood was reinjected into the rats followed by return of the withdrawn blood. The withdrawn blood and resuscitation fluid were given over the course of 30 min. Three groups were provided with DPR. At the end of the HS period in groups NS, LA (Lac-PDS), and PY (Pyr-PDS), animals received VR. Simultaneous with VR, the DPR groups were intraperitoneally infused with 20 mL of NS, Lac-PDS, or Pyr-PDS, with a microinfusion pump over the course of 30 min. Mean arterial pressure was monitored and recorded continuously from the beginning of the exsanguination until 2 h after fluid resuscitation terminated.
Detection of arterial blood lactate levels and the wet weight/dry weight ratio of the intestine Two hours after resuscitation, blood samples were drawn from the left femoral artery. The lactate level in arterial blood was detected to evaluate oxygen deficit in the blood using a lactate detection kit (Jiancheng Bioengineering Institute), according to the manufacturer_s instructions. After drawing blood samples, the rats were killed humanely by exsanguination. Tissue samples from
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the small intestine were removed 10 cm from the ileocecal valve and rinsed with ice-cold NS. Ten centimeters of the intestine was frozen immediately at j70-C for future detections. Five centimeters of the intestine was fixed with 4% paraformaldehyde, and 0.5 cm was fixed with 2.5% glutaraldehyde for further studies. Another 10 cm of the intestine was harvested and weighed immediately after death to determine the wet weight. The intestinal samples were then placed into an electronic oven at 80-C for 3 days to dry the tissues for the determination of the dry weight. The intestinal WW/DW was calculated as the wet weightYdry weight ratio and measured to evaluate the tissue edema level.
Determination of the intestinal mucosal damage index using light microscopy Intestinal tissues fixed in 4% paraformaldehyde were cut into paraffin sections. Hematoxylin-eosin stain was applied to the intestinal paraffin sections to observe morphological changes in the small intestine mucosa using a light microscope. The intestinal mucosal damage index (IMDI) was evaluated using Chiu and colleagues_ method (15) by three pathologists blinded to the treatment groups. The following standards for evaluation were used: 0 points were assigned to intestinal mucosal villi with normal tissue structure; 1 point was assigned for widening of the subepithelial space in the top of intestinal villi; 2 points were assigned for a further widening of the subepithelial space in the top of intestinal villi, raising of the villi apex and separation from the lamina propria; 3 points were assigned for serious abscissions of the epithelium beside the intestinal villi; 4 points were assigned when abscission of the epithelium was complete and there was only lamina propria left; 5 points were assigned when the lamina propria of the intestinal mucosa disintegrated and bleeding and ulceration appeared.
Evaluation of ultrastructural changes of intestinal mucosa using transmission electron microscopy Intestinal tissue fixed in 2.5% glutaraldehyde was rinsed three times with 0.1 M phosphate-buffered saline, postfixed with osmic acid, rinsed three more times with 0.1 M phosphate-buffered saline, and dehydrated twice with graded ethanol for 15 min each. Then the intestinal tissue was embedded in epoxy resin as follows: the sample was dehydrated twice for 15 min with pure acetone, soaked in EPON812:acetone (1:1) for 30 min, soaked in pure embedding solution for 1 h, and solidified with pure embedding solution at 37-C for 24 h and then at 60-C for 48 h. After this treatment, the intestinal tissues were sliced with an ultramicrotome (LKB-V; BROMMA Co, Stockholm, Sweden) and stained with uranium and plumbum (uranyl acetate and lead citrate). Finally, ultrastructural changes in the intestinal mucosa were observed using transmission electron microscopy (TEM) (H-600 TEM; Hitachi Ltd, Tokyo, Japan).
Detection of intestinal MDA, NO, and MPO levels Intestine samples stored in an ultralow temperature freezer were taken out and homogenized at 4-C with a tissue homogenizer. The MDA, NO, and MPO content were determined to evaluate the extent of mucosal injury in the intestine using the relevant detection kit (Jiancheng Bioengineering Institute) according to the manufacturer_s instructions. Malondialdehyde levels were measured using the thiobarbituric acid reaction method, NO levels were measured with the nitrate reductase method, and MPO activity was detected using colorimetry.
Detection of the intestinal tumor necrosis factor ! expression level Intestinal tissue fixed in 4% paraformaldehyde was cut into paraffin sections. To evaluate the inflammatory response, the expression level of tumor necrosis factor ! (TNF-!) was determined using immunohistochemistry with a streptavidin/peroxidase (SP-9003) kit (Zymed Laboratories, San Diego, Calif). Photographs of sections were analyzed with Image Pro Plus 6.0 software. The average optical density was measured to reflect the TNF-! expression level.
Statistical analysis All continuous variables were expressed as the mean T SD. The statistical significance of differences between groups was analyzed using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons using commercial statistical software (SPSS statistics 17.0; SPSS Inc, Chicago, Ill). P G 0.05 was considered to be statistically significant.
RESULTS Mean arterial blood pressure
Compared with group sham, MAP promptly declined to 35 T 5 mmHg when rats from the other four groups experienced HS and then rose gradually following fluid resuscitation.
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Intestinal mucosal damage index
FIG. 1. The effect of DPR on MAP after HS and resuscitation in rats (n = 10). aP G 0.05 versus group sham; bP G 0.05 versus group VR; cP G 0.05 versus group NS; dP G 0.05 versus group LA.
At 60 min after HS (H60), there was no significant difference in MAP in the groups that experienced shock and resuscitation (P 9 0.05). However, at 5, 30, 60, 90, and 120 min after fluid resuscitation (R5, R30, R60, R90 and R120, respectively), the MAP in groups LA and PY was higher than that in groups VR and NS (P G 0.05), and group PY had a higher MAP than did group LA (P G 0.05). Changes in MAP are shown in Figure 1. Arterial-blood lactate level
To evaluate oxygen deficit in the blood circulation, the arterial-blood lactate level was determined (Table 1). At 2 h after resuscitation (R120), the lactate level in the arterial blood of rats from the four shock and resuscitation groups was higher than that for group sham (P G 0.05), which certified that rats from the four groups indeed suffered from HS. Compared with groups VR and NS, the arterial blood lactate level of rats was significantly lower in groups LA and PY (P G 0.05). Group PY had a lower arterial blood lactate level than did group LA (P G 0.05). WW/DW of the intestine
The WW/DW of the intestine, which reflects the water content of intestinal tissue, is presented in Table 1. At R120, the intestinal WW/DW was higher in the four groups that underwent HS and fluid resuscitation than in group sham (P G 0.05). Compared with groups VR and NS, WW/DW was significantly lower in groups LA and PY (P G 0.05), and group PY had a lower WW/DW than did group LA (P G 0.05).
Intestinal mucosal damage index, which characterizes morphological changes (Fig. 2) in the intestinal mucosa, is presented in Table 1. At R120, the IMDI was significantly higher in the four groups subjected to HS and fluid resuscitation than that in group sham (P G 0.05). Compared with groups VR and NS, the IMDI was significantly lower in groups LA and PY (P G 0.05), and group PY had a significantly lower IMDI than did group LA (P G 0.05). Morphological changes in the intestinal mucosa were identified using a light microscope. In rats from group sham, the structure of the intestinal mucosal epithelium was integrated, and there were very few lymphocytes and no neutrophil infiltration. In groups VR and NS, the subepithelial space at the top of intestinal villus clearly widened, and there was serious abscission of the epithelium next to parts of the intestinal villus, erosion caused by cell necrosis and cell loss, edema of the lamina propria, and infiltration of a large number of neutrophils and lymphocytes. In groups LA and PY, pathological injury was alleviated. The subepithelial space at the top of the intestinal villus widened slightly with a slight edema of the lamina propria. Injury in group PY was alleviated further with infiltration of few neutrophils and lymphocytes. Ultrastructural changes in intestinal mucosa
Ultrastructural changes in the intestinal mucosa observed with TEM are shown in Figure 3. In rats from group sham, epithelial cells of the intestinal mucosa had abundant organelles, and all cellular structure was normal. In groups VR and NS, we observed swollen mitochondria with breaks in or disappearance of cristae, dilation of the endoplasmic reticulum, sparse irregular microvilli, and compaction of nuclear chromatin gathered in the cell border in intestinal mucosal epithelial cells. In the intestinal mucosal epithelial cells of rats from group LA, the mitochondria and endoplasmic reticulum were slightly swollen, and microvilli were slightly irregular. In the epithelial cells from the intestinal mucosa of rats in group PY, the mitochondria and endoplasmic reticulum were nearly normal, and all cellular structures were intact with regular microvilli. Intestinal tissue parameters
Intestinal MDA levels, which are accumulated as a product of lipid peroxidation, are presented in Table 1. At R120, intestinal MDA levels were elevated in the four HS and fluid
TABLE 1. The effect of DPR on arterial blood lactate level, intestinal WW/DW, MDA, NO, and the IMDI at 2 h after resuscitation in rats (n = 10) Group
Lactate, mmol/L
WW/DW
IMDI
MDA, 2mol/g
NO, 2mol/g
Sham
1.7 (0.5)
4.01 (0.24)
0.3 (0.5)
1.16 (0.13)
0.341 (0.035)
VR
8.0 (0.9)*
5.25 (0.37)*
3.4 (0.5)*
3.03 (0.24)*
0.192 (0.025)*
NS
7.5 (0.7)*
5.12 (0.34)*
3.1 (0.7)*
2.87 (0.26)*
0.212 (0.023)*
LA
4.9 (0.9)*†‡
4.68(0.27)*†‡
2.0 (0.9)*†‡
2.25 (0.17)*†‡
0.279 (0.026)*†‡
PY
2.5 (0.8)*†‡§
4.31 (0.23)*†‡§
1.1 (0.6)*†‡§
1.55 (0.14)*†‡§
0.311 (0.031)*†‡§
Values are presented as the mean T SD of 10 animals. *P G 0.05 versus group sham. † P G 0.05 versus group VR. ‡ P G 0.05 versus group NS. § P G 0.05 versus group LA.
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FIG. 2. The effect of DPR on morphological changes in the intestinal mucosa of rats (n = 10). Photomicrographs of hematoxylin-eosinYstained sections of the small intestine in rats 2 h after resuscitation (R120) (original magnification, 200 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY.
resuscitation groups relative to group sham (P G 0.05). Intestinal MDA levels in groups LA and PY were significantly lower than in groups VR and NS (P G 0.05), and group PY had lower levels of MDA than group LA (P G 0.05). Intestinal NO levels, which reflect the barrier function of the intestinal mucosa, are presented in Table 1. At R120, NO levels in the intestine were lower in the four HS and fluid resuscitation groups (P G 0.05) than those in group sham. In groups LA and PY, intestinal NO levels were significantly higher relative to groups VR and NS (P G 0.05), and group PY had significantly higher NO levels than did group LA (P G 0.05). Intestinal MPO activity, which reflects intestinal inflammatory injury, is presented in Figure 4. At R120, intestinal MPO activity was higher in the four HS and fluid resuscitation groups (P G 0.05) than that in group sham. In groups LA and PY, intestinal MPO activity was significantly lower than that in groups VR and NS (P G 0.05), and group PY had a lower MPO activity when compared with group LA (P G 0.05). Intestinal TNF-! levels
Immunohistochemical staining of TNF-! in the intestinal mucosa at 2 h after resuscitation is presented in Figure 5. As an inflammatory mediator, the expression of intestinal TNF-!
reflects the inflammatory response level (Fig. 6). At R120, the level of TNF-! in the intestine was significantly higher in the four HS and fluid resuscitation groups than that in group sham (P G 0.05). In groups LA and PY, the intestinal TNF-! level was significantly lower than that in groups VR and NS (P G 0.05), and group PY had a significantly lower TNF-! level relative to group LA (P G 0.05). DISCUSSION In surgery, intestinal IRI is a common form of organ damage that occurs during resuscitation after HS, and it is also the pathophysiological basis for numerous other diseases. As an organ with active metabolism, the intestine has a substantial immunological function and is the largest store of bacteria in the body. Research has indicated that MODS begins in the intestine and proceeds as follows: first, there are metabolic disorders of oxygen and energy, leukocyte adhesion, and endothelial cell injury, and injury caused by oxygen radicals and then intracellular calcium overload occur. When IRI occurs in vivo, the intestine becomes a source of early-stage inflammatory cytokines, and this organ may play a significant role in the development of systemic inflammatory response syndrome (SIRS), MODS, and even death (13).
FIG. 3. The effect of DPR on ultrastructural changes in the intestinal mucosa in rats (n = 10). Transmission electron microscopy photomicrographs of uranyl acetate and lead citrateYstained sections of the small intestine in rats at 2 h after resuscitation (R120) (original magnification, 10,000 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY. In B and C, the arrows highlighted the swollen mitochondria with breaks in or disappearance of cristae, dilation of the endoplasmic reticulum, sparse irregular microvilli, compaction of nuclear chromatin gathered in the cell border, and damaged cellular tight junctions in intestinal mucosal epithelial cells. In Figure D and E, the arrows highlighted the slightly swollen mitochondria and endoplasmic reticulum.
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FIG. 4. Effects of DPR on MPO activity of intestine 2 h after resuscitation in rats (n = 10). a P G 0.05 versus group sham; b P G 0.05 versus group VR, c P G 0.05 versus group NS; d P G 0.05 versus group LA.
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The superiority of adjunct DPR with Pyr-PDS in rats with HS
FIG. 6. The effects of DPR on the TNF-! expression level of the intestine at 2 h after resuscitation in rats (n = 10). Photographs of the sections were analyzed with Image Pro Plus 6.0 software. The TNF-! expression level was indicated by the average optical density. aP G 0.05 versus group sham; bP G 0.05 versus group VR; cP G 0.05 versus group NS; dP G 0.05 versus group LA.
Mean arterial pressure shows blood circulation and reflects the condition of HS and resuscitation in the body. Our results indicated that DPR following VR restored blood pressure and improved blood circulation. Also, Pyr-PDS improved blood circulation and stabilized blood pressure. Previous research supports the view that pyruvate-enriched solutions stabilize hemodynamics, suppress myocardial nitrosative stress, minimize systemic inflammation, and suppress the apoptotic cascade after HS more effectively than do lactate Ringer_s solution (16). Direct peritoneal resuscitation also improves intestinal blood flow(17). Arterial blood lactate reflects anaerobic metabolism. As one of the parameters indicating hypoperfusion in the body, an increase in arterial blood lactate indicates tissue
hypoxia, and the lactate level is a predictor of morbidity and mortality (18). Moreover, it is one of the parameters used to evaluate the effect of resuscitation. In this study, arterial blood lactate levels in group VR indicated serious hypoxia. However, DPR with Lac-PDS and Pyr-PDS following VR ameliorated hypoperfusion and hypoxia in the body. These results demonstrated that Pyr-PDS promoted recovery from tissue hypoxia more effectively than Lac-PDS. As a novel PDS, Pyr-PDS would be more effective than conventional methods in correction of metabolic acidosis. Because of its various cytoprotective effects, Pyr-PDS not only corrects acidosis, but also protects against the underlying dysfunction of vital organs (19). Evidence also shows that Pyr-PDS is superior to Lac-PDS in biocompatibility
FIG. 5. Immunohistochemical staining of TNF-! in the intestinal mucosa 2 h after resuscitation in rats (n = 10) (original magnification, 400 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY. The arrows highlighted the expression of intestinal TNF-!.
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SHOCK NOVEMBER 2014 (20). The ratio of wet weight to dry weight reflects the tissue water content and the degree of tissue and cellular edema. This study showed that DPR with Lac-PDS or Pyr-PDS following VR reduced intestinal edema, and Pyr-PDS had an advantage in recovery from tissue edema relative to Lac-PDS. Intestinal mucosal damage index, which we evaluated with light microscopy, and ultrastructural changes in the intestinal mucosa, which we observed using TEM, both showed that DPR following VR alleviated pathological injury in the intestinal mucosa, and the protection from intestinal mucosal injury promoted by Pyr-PDS was superior to Lac-PDS. Potential mechanisms of DPR with Pyr-PDS
Previous research supports the view that sodium pyruvate has a protective effect for IRI after HS that may be related to the scavenging of oxygen free radicals and a reduction in the neutrophil and anti-inflammatory response (21). Pyruvate is an energy substrate that has both inotropic and antioxidant properties (7). As is the case for the production of oxygen free radicals and lipid peroxidation, an increase in MDA levels indicates that oxygen free radicals attack the cytomembrane and lead to lipid peroxidation damage. Myeloperoxidase is a specific marker enzyme for neutrophils, and an increase in MPO activity indicates the infiltration of neutrophils that cause lipid peroxidation damage. Meanwhile, MPO catalyzes an excess of oxidant production that causes oxidative damage of tissues. Our results from this study demonstrated that intestinal injury in rats was related to MDA and MPO, which indicated that the protective effects of Pyr-PDS on intestinal IRI were related to a reduction in oxygen free radical levels and the inhibition of neutrophil infiltration. As the end-product of glycolysis and the starting substrate for the tricarboxylic acid cycle, pyruvate plays a key role in intermediary metabolism (3). Reactive oxygen species have been implicated in the pathogenesis of structural and functional alterations to tissues that are associated with a variety of pathological processes, including sepsis and septic shock, thermal injury, doxorubicininduced cardiomyopathy, HS, and mesenteric IRI. Pyruvate (CH3COCOOj) is also a potent and effective reactive oxygen species scavenger (4, 5). Meanwhile, pyruvate can cause systemic alkalinization. When lactate is replaced by pyruvate, acute toxic injuries caused by DPR can be avoided (22). Pyruvatebased PDS has general protective effects for glycometabolism, excellent systemic alkalinization, and potent antioxidant capacity. It has a unique advantage for the development of fluid therapy and is applicable to the prevention and treatment of refractory HS. Tumor necrosis factor ! is an early and central inflammatory mediator and an initial factor for the induction of SIRS and MODS (23). The intestine is one of the major organs that release TNF-!. Many studies have demonstrated that TNF-! and IL-6 play important roles in the occurrence and development of sepsis and MODS. Neutrophils function in the immune defense in HS. However, an excessive stress response would occur if there were a large number of neutrophils to be activated, and activated neutrophils play an important role in SIRS and MODS, because they induce production of intestinal inflammatory mediators, including TNF-! (24). Tumor necrosis
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factor ! and MPO levels indicate the release of intestinal inflammatory mediators and the degree of the inflammatory response. In this study, our results indicated that the protective effects of Pyr-PDS on intestinal IRI were related to a reduction in inflammatory mediators and an alleviation of the inflammatory response. Studies have shown that DPR not only can significantly increase IL-10 level and decrease TNF-! and IL-6 levels, but also can inhibit the inflammatory response in the liver and other internal organs (12). Pyruvate pretreatment of the rat small intestine also inhibited IRI and neutrophil infiltration (25). This shows that DPR with Pyr-PDS alleviates the systemic inflammatory response and improves systemic immune status. All of these effects improve patient prognosis. It has been suggested that the balance between locally produced NO and endothelin 1 plays an important role in the maintenance of intestinal mucosal microcirculation (26). As a significant factor in the regulation of intestinal mucosal blood flow and barrier function, NO has positive effects on intestinal IRI. Recent evidence has demonstrated that the preservation of the intestinal barrier can attenuate intestinal IRI (27). This study demonstrated that microcirculation disturbances after intestinal IRI were related to reduction in NO. Evidence also suggests that an endothelin-receptor antagonist can relieve continuous intestinal vasoconstriction and hypoperfusion after conventional intravenous recovery, and DPR can also promote continuous intestinal vasodilation and hypertransfusion (28). Direct peritoneal resuscitation increases the perfusion of internal organs by a related mechanism based on the vascular endothelium, and in this way, PyrPDS is more effective than Lac-PDS. These observations may be related to the release of NO and activation of the adenosine A1 receptor subtype. Previous studies have shown that PyrPDS significantly improves NO generation under both physiological pH and acidic pH and also under high glucose conditions (29). Pyruvate infusion clearly protects the small intestine against IRI. This protection primarily results from local effects on the small intestine (30). Conventional VR from HS results in hypoperfusion of the gut and liver, edema of organs and cells, and injury to vital organs. Adjunct DPR with dialysate prevents gut vasoconstriction, hypoperfusion, and injury (31); shortens the interval to definitive fascial closure; and reduces intraabdominal complications (32). For these purposes, Pyr-PDS is superior to Lac-PDS. Pyruvate-based PDS provides antiinflammatory dampening of the systemic inflammatory response and improves the survival rate. Pyruvate-based PDS slightly inhibits peritoneal macrophages and maintains the integrity of peritoneal mesothelial cells. There are changes in peritoneal solute transport resulting from pyruvate treatment, accompanied by a reduction in both peritoneal membrane angiogenesis and fibrosis, indicating novel mechanisms that could potentially reduce glucose-driven alterations to the peritoneal membrane in vivo (33). In addition, pyruvate enables the cells to remain viable during prolonged hypoxia. Clinical significance
We demonstrated in this study that blood circulation improved, hypoperfusion and hypoxia were ameliorated, and IRI
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of the intestine was alleviated by DPR with Pyr-PDS following VR from lethal HS in rats. Although the blood base excess and survival rates that are associated with the therapeutic effect of this novel resuscitation were not observed in this study, these parameters were shown to be significantly improved with adjunct DPR from severe shock (34). Recently, DPR with PyrPDS and hypertonic pyruvate have been shown to be an efficient therapy in resuscitation from HS in rats, and this treatment significantly improved organ function and the blood flow in various organ surfaces, including the liver, kidney, and intestinal mucosa (6). All of the present results demonstrated that DPR with Pyr-PDS combined with VR may be superior to NS or lactate-containing fluids or only VR in clinical resuscitation from severe HS. Further investigation is warranted. Limitations
In this study, animals were heparinized before HS and resuscitation for anticoagulation. Various studies have indicated that heparin can significantly improve hemodynamics and microcirculation during mesenteric reperfusion after hemorrhage (35), diminish clinical risks of gastrointestinal complications, and thus influence clinical outcomes. Therefore, this is a limitation for this study. Because heparinization was necessary in the experimentation, it may interfere with the effects of DPR and pyruvate on intestinal injury from HS in rats. Perhaps this problem can be solved in future studies by using lowmolecular-weight heparin or adjusting the heparin dose. The period after HS and resuscitation was chosen to be 2 h (9Y11, 14) for a preliminary study about DPR with pyruvate on animals. To obtain more potent evidences of Pyr-PDS_s effects, further research is needed, and the postresuscitation period can be prolonged in the future. Because it is likely that the data would have been more differentiated if they were normalized to milligrams of protein of intestine, the magnitude of changes in the inflammatory markers could be another possible limitation. CONCLUSIONS This study demonstrates that DPR with Pyr-PDS following VR alleviates intestinal IRI significantly after HS and resuscitation in rats and has a protective effect on HS rats. We provide evidence that mechanisms by which Pyr-PDS attenuates intestinal IRI may include the elimination of oxygen free radicals, reduction of neutrophil infiltration, inhibition of the inflammatory response, and regulation of intestinal mucosal blood flow and barrier function. Our results indicate that Pyr-PDS has significant advantages in the treatment of HS resuscitation and thus requires further investigation in the future. ACKNOWLEDGMENT The authors thank Fang-Qiang Zhou, MD, from Fresenius Dialysis Centers at Chicago for his assistance with pharmacology in this research.
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Flaherty DC, Hoxha B, Sun J, Gurji H, Simecka JW, Mallet RT, OlivenciaYurvati AH: Pyruvate-fortified fluid resuscitation improves hemodynamic stability while suppressing systemic inflammation and myocardial oxidative stress after hemorrhagic shock. Mil Med 175(3):166Y172, 2010. 18. Maki AC, Matheson PJ, Shepherd JA, Garrison RN, Downard CD: Intestinal microcirculatory flow alterations in necrotizing enterocolitis are improved by direct peritoneal resuscitation. Am Surg 78(7):803Y807, 2012. 19. Peerless J: Fluid management of the trauma patient. Curr Opin Anesthesiol 14(2):221Y225, 2001. 20. Zhou FQ: Pyruvate in the correction of intracellular acidosis: a metabolic basis as a novel superior buffer. Am J Nephrol 25(1):55Y63, 2005. 21. Zhou FQ: Advantages of pyruvate over lactate in peritoneal dialysis solutions. Acta Pharmacol Sin 22(5):385Y392, 2001. 22. 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SHOCK NOVEMBER 2014 30. Wu YT, Wu ZL, Jiang XF, Li S, Zhou FQ: Pyruvate improves neutrophilic nitric oxide generation in peritoneal dialysis solutions. Artif Organs 29(12): 976Y980, 2005. 31. Petrat F, Ro¨nn T, de Groot H: Protection by pyruvate infusion in a rat model of severe intestinal ischemia-reperfusion injury. J Surg Res 167(2):e93Ye101, 2011. 32. Hurt RT, Matheson PJ, Smith JW, Zakaria el R, Shaheen SP, McClain CJ, Garrison RN: Preservation of hepatic blood flow by direct peritoneal resuscitation improves survival and prevents hepatic inflammation following hemorrhagic shock. Am J Physiol Gastrointest Liver Physiol 303(10):G1144YG1152, 2012.
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33. Smith JW, Garrison RN, Matheson PJ, Franklin GA, Harbrecht BG, Richardson JD: Direct peritoneal resuscitation accelerates primary abdominal wall closure after damage control surgery. J Am Coll Surg 210(5):658Y664, 2010. 34. van Westrhenen R, Zweers MM, Kunne C, de Waart DR, van der Wal AC, Krediet RT: A pyruvate-buffered dialysis fluid induces less peritoneal angiogenesis and fibrosis than a conventional solution. Perit Dial Int 28(5): 487Y496, 2008. 35. Luo X, Jian D, Lv Z: The beneficial effect of direct peritoneal resuscitation on septic shock in rats. J Biomed Biotechnol 2011:743763, 2011.
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PROTECTION AGAINST INTESTINAL INJURY FROM HEMORRHAGIC SHOCK BY DIRECT PERITONEAL RESUSCITATION WITH PYRUVATE IN RATS Jing-Jing Zhang, Zong-Ze Zhang, Jian-Juan Ke, Xiang-Hu He, Jia Zhan, Dong-Ling Chen, Yi-Peng Wang, and Yan-Lin Wang Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China Received 9 May 2014; first review completed 27 May 2014; accepted in final form 25 Jun 2014 ABSTRACT—Objective: We explored the effects of direct peritoneal resuscitation with pyruvate-peritoneal dialysis solution (PDS) following intravenous resuscitation (VR) on intestinal ischemia-reperfusion injury in rats with hemorrhagic shock (HS). Methods: Fifty rats were randomly assigned equally to five groups. In group sham, a surgical operation was performed on rats without shock or resuscitation. In group VR, rats were subjected only to VR. In groups NS, LA, and PY, direct peritoneal resuscitation was performed with normal saline (NS), lactate-based PDS (Lac-PDS), and pyruvate-based PDS (Pyr-PDS), respectively, after VR. Mean arterial pressure was monitored in the right common carotid artery. Two hours after resuscitation, the lactate level in arterial blood and the wet weight/dry weight ratio of the intestine were determined. The intestinal mucosal damage index was estimated, and ultrastructural changes in the intestinal mucosa were observed. Malondialdehyde, myeloperoxidase, nitric oxide, and tumor necrosis factor ! levels were also measured. Results: Two hours after HS and resuscitation, the increase in arterial blood lactate and intestinal wet weight/dry weight ratio declined significantly in rats from Groups LA and PY compared with groups VR and NS, whereas group PY was more advantageous in the changes of these parameters. The intestinal mucosal damage index and ultrastructural changes were also improved in groups LA and PY when compared with groups VR and NS. Protection was more apparent with Pyr-PDS than Lac-PDS. Hemorrhagic shock resulted in a significant increase in malondialdehyde levels and myeloperoxidase activity and was accompanied by overexpression of tumor necrosis factor ! and a reduction in nitric oxide levels. These changes were significantly attenuated by Lac-PDS and Pyr-PDS at 2 h after resuscitation, and Pyr-PDS showed more effective protection for the intestine than Lac-PDS. Conclusions: Direct peritoneal resuscitation with Lac-PDS and Pyr-PDS after VR alleviated intestinal injury from HS in rats, and Pyr-PDS was superior to Lac-PDS in its protective effect. Mechanisms of action might include the elimination of free oxygen radicals, reduction of neutrophil infiltration, inhibition of the inflammatory response, and regulation of intestinal mucosal blood flow and barrier function. KEYWORDS—Peritoneal dialysis solution, lactate, intestinal mucosa, ischemia-reperfusion injury, oxygen free radicals, inflammatory mediators ABBREVIATIONS—DPRVdirect peritoneal resuscitation; VRVintravenous resuscitation; PDSVperitoneal dialysis solution; HSVhemorrhagic shock; MAPVmean arterial pressure; MDAVmalondialdehyde; MPOVmyeloperoxidase; NOVnitric oxide; TNF-!Vtumor necrosis factor !; IMDIVintestinal mucosal damage index
pyruvate protects splanchnic organ function from HS in rats (6), and pyruvate prolongs the survival time of animals experiencing HS and resuscitation (7). Pyruvate Ringer_s solution effectively treats hypoxic lactic acidosis and significantly increases the survival rate of rats with lethal HS (8). Conventional resuscitation has the potential to improve conventional indicators such as blood pressure, heart rate, central blood volume, and urine volume. However, the stomach, intestine, liver, and kidney experience continuous vasoconstriction and hypoperfusion. As a result, patients often die of systemic inflammation and multiple organ dysfunction syndrome (MODS) after severe HS and conventional resuscitation. As a new therapy of HS, intraperitoneal resuscitation or direct peritoneal resuscitation (DPR) can improve blood circulation of organs such as the intestine. Furthermore, this treatment can avoid MODS and increase the survival rate (9Y12) The intestine plays a key role in the pathophysiologic changes that follow severe HS and intestinal ischemiareperfusion injury (IRI) caused by HS, and conventional resuscitation plays a critical role in the development of systemic inflammation and MODS (1, 13). In this study, we evaluated the effects of DPR with pyruvate-based peritoneal dialysis
Hemorrhagic shock (HS) is a major cause of death caused by trauma. Hemorrhagic shock causes a series of pathophysiologic changes, including microcirculation disturbance, a local reduction in blood perfusion, a lack of oxygen supply in tissues, and metabolic disorder. Recovery of blood perfusion and the maintenance of oxygen supply for tissues are the major goals of resuscitation after HS. Therefore, effective hemorrhage control and optimal resuscitation are the primary objectives for the treatment of severely injured patients (1). However, resuscitation, if not performed properly, can exacerbate cellular injury caused by HS. The type of fluid used for resuscitation plays an important role in the pattern of injury (2). Pyruvic acid is an important substrate of the tricarboxylic acid cycle, and pyruvate is an effective antioxidant and free radical scavenger (3Y5). Recent research has demonstrated that
Address reprint requests to Yan-Lin Wang, MD, PhD, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, No. 169 Donghu Rd, Wuhan 430071, Hubei, China. E-mail: [email protected]. This research supported by the Fundamental Research Funds for the Central Universities (no. 2012103030042). DOI: 10.1097/SHK.0000000000000230 Copyright Ó 2014 by the Shock Society
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SHOCK NOVEMBER 2014 solution (Pyr-PDS) following intravenous resuscitation (VR) on intestinal IRI from HS and resuscitation in rats compared with DPR with lactate-based PDS (Lac-PDS), normal saline (NS), and VR alone. We evaluated major parameters and explored the possible mechanism of DPR with Pyr-PDS. MATERIALS AND METHODS Materials Sodium pyruvate and sodium lactate were purchased from Sigma (St Louis, Mo). Lactate, malondialdehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO) detection kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Streptavidin/peroxidase (SP-9003) kits were purchased from Zymed Laboratories (San Diego, Calif).
Preparation of solution In this study, we used 2.5% Glu-Pyr-PDS (Pyr-PDS; 396 mOsm/L, pH 5.2), which contained 40 mmol/L of pyruvate, 132 mmol/L of Na+, 1.75 mmol/L of Ca2+, 0.25 mmol/L of Mg2+, 96 mmol/L of Clj, and 2.5 g/dL of glucose, and 2.5% Glu-Lac-PDS (Lac-PDS; 396 mOsm/L, pH 5.2), which contained 40 mmol/L of lactate, 132 mmol/L of Na+, 1.75 mmol/L of Ca2+, 0.25 mmol/L of Mg2+, 96 mmol/L of Clj, and 2.5 g/dL of glucose. Both DPR solutions were prepared fresh in the laboratory. The pH was adjusted to 5.2 with HCl or NaOH. The DPR solutions were kept in the refrigerator at 4-C and warmed up to room temperature before use. The Pyr-PDS stability was verified by determining pyruvate concentrations with the high-performance liquid chromatography analysis (6). The Lac-PDS prepared herein was the same as the standard PDS available commercially.
Animals Male Sprague-Dawley rats (200Y250 g) were purchased from The Center for Animal Experiment of Wuhan University (Wuhan, China). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Experimental procedures were approved by the Animal Experiment Committee of Wuhan University (China). All the animals were acclimatized to 12-h light-dark cycles for 1 week with free access to food and water. Rats were fasted overnight and allowed water until 4 h before experimentation.
Hemorrhagic shock and resuscitation protocols Rats were anesthetized with pentobarbital sodium (45 mg/kg, i.p.). Hemorrhagic shock and resuscitation in rats were induced as previously described with slight modifications (14). Rats were allowed to breathe spontaneously. PE-50 catheters were placed in the right carotid artery and connected to a pressure transducer for continuous arterial blood pressure monitoring with a Surgivet Advisor Vital Signs (Smiths Medical, Norwell, Mass). Catheters were also placed in the left femoral artery and connected to a syringe for blood withdrawal to induce shock and in the right femoral vein for heparinization (heparin sodium, 300 IU/kg) and infusion of fluid. Surgery was performed with aseptic technique. All catheters were filled with NS containing heparin (100 IU/mL). A heat lamp was used to maintain normal body temperature. The rats were allowed to stabilize for 15 to 30 min after surgery. Hemorrhagic shock was induced by withdrawing blood within 10 min using the syringe connected to the left femoral artery. Mean arterial pressure (MAP) was maintained at 35 T 5 mmHg for 60 min by further withdrawal or reinfusion of blood as required before fluid resuscitation. All animals were randomly divided into five groups (n = 10 each). In group sham, animals underwent surgical preparation, but not HS or resuscitation. At the end of the HS period in group VR, NS equal to twice the volume of the withdrawn blood was reinjected into the rats followed by return of the withdrawn blood. The withdrawn blood and resuscitation fluid were given over the course of 30 min. Three groups were provided with DPR. At the end of the HS period in groups NS, LA (Lac-PDS), and PY (Pyr-PDS), animals received VR. Simultaneous with VR, the DPR groups were intraperitoneally infused with 20 mL of NS, Lac-PDS, or Pyr-PDS, with a microinfusion pump over the course of 30 min. Mean arterial pressure was monitored and recorded continuously from the beginning of the exsanguination until 2 h after fluid resuscitation terminated.
Detection of arterial blood lactate levels and the wet weight/dry weight ratio of the intestine Two hours after resuscitation, blood samples were drawn from the left femoral artery. The lactate level in arterial blood was detected to evaluate oxygen deficit in the blood using a lactate detection kit (Jiancheng Bioengineering Institute), according to the manufacturer_s instructions. After drawing blood samples, the rats were killed humanely by exsanguination. Tissue samples from
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the small intestine were removed 10 cm from the ileocecal valve and rinsed with ice-cold NS. Ten centimeters of the intestine was frozen immediately at j70-C for future detections. Five centimeters of the intestine was fixed with 4% paraformaldehyde, and 0.5 cm was fixed with 2.5% glutaraldehyde for further studies. Another 10 cm of the intestine was harvested and weighed immediately after death to determine the wet weight. The intestinal samples were then placed into an electronic oven at 80-C for 3 days to dry the tissues for the determination of the dry weight. The intestinal WW/DW was calculated as the wet weightYdry weight ratio and measured to evaluate the tissue edema level.
Determination of the intestinal mucosal damage index using light microscopy Intestinal tissues fixed in 4% paraformaldehyde were cut into paraffin sections. Hematoxylin-eosin stain was applied to the intestinal paraffin sections to observe morphological changes in the small intestine mucosa using a light microscope. The intestinal mucosal damage index (IMDI) was evaluated using Chiu and colleagues_ method (15) by three pathologists blinded to the treatment groups. The following standards for evaluation were used: 0 points were assigned to intestinal mucosal villi with normal tissue structure; 1 point was assigned for widening of the subepithelial space in the top of intestinal villi; 2 points were assigned for a further widening of the subepithelial space in the top of intestinal villi, raising of the villi apex and separation from the lamina propria; 3 points were assigned for serious abscissions of the epithelium beside the intestinal villi; 4 points were assigned when abscission of the epithelium was complete and there was only lamina propria left; 5 points were assigned when the lamina propria of the intestinal mucosa disintegrated and bleeding and ulceration appeared.
Evaluation of ultrastructural changes of intestinal mucosa using transmission electron microscopy Intestinal tissue fixed in 2.5% glutaraldehyde was rinsed three times with 0.1 M phosphate-buffered saline, postfixed with osmic acid, rinsed three more times with 0.1 M phosphate-buffered saline, and dehydrated twice with graded ethanol for 15 min each. Then the intestinal tissue was embedded in epoxy resin as follows: the sample was dehydrated twice for 15 min with pure acetone, soaked in EPON812:acetone (1:1) for 30 min, soaked in pure embedding solution for 1 h, and solidified with pure embedding solution at 37-C for 24 h and then at 60-C for 48 h. After this treatment, the intestinal tissues were sliced with an ultramicrotome (LKB-V; BROMMA Co, Stockholm, Sweden) and stained with uranium and plumbum (uranyl acetate and lead citrate). Finally, ultrastructural changes in the intestinal mucosa were observed using transmission electron microscopy (TEM) (H-600 TEM; Hitachi Ltd, Tokyo, Japan).
Detection of intestinal MDA, NO, and MPO levels Intestine samples stored in an ultralow temperature freezer were taken out and homogenized at 4-C with a tissue homogenizer. The MDA, NO, and MPO content were determined to evaluate the extent of mucosal injury in the intestine using the relevant detection kit (Jiancheng Bioengineering Institute) according to the manufacturer_s instructions. Malondialdehyde levels were measured using the thiobarbituric acid reaction method, NO levels were measured with the nitrate reductase method, and MPO activity was detected using colorimetry.
Detection of the intestinal tumor necrosis factor ! expression level Intestinal tissue fixed in 4% paraformaldehyde was cut into paraffin sections. To evaluate the inflammatory response, the expression level of tumor necrosis factor ! (TNF-!) was determined using immunohistochemistry with a streptavidin/peroxidase (SP-9003) kit (Zymed Laboratories, San Diego, Calif). Photographs of sections were analyzed with Image Pro Plus 6.0 software. The average optical density was measured to reflect the TNF-! expression level.
Statistical analysis All continuous variables were expressed as the mean T SD. The statistical significance of differences between groups was analyzed using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons using commercial statistical software (SPSS statistics 17.0; SPSS Inc, Chicago, Ill). P G 0.05 was considered to be statistically significant.
RESULTS Mean arterial blood pressure
Compared with group sham, MAP promptly declined to 35 T 5 mmHg when rats from the other four groups experienced HS and then rose gradually following fluid resuscitation.
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Intestinal mucosal damage index
FIG. 1. The effect of DPR on MAP after HS and resuscitation in rats (n = 10). aP G 0.05 versus group sham; bP G 0.05 versus group VR; cP G 0.05 versus group NS; dP G 0.05 versus group LA.
At 60 min after HS (H60), there was no significant difference in MAP in the groups that experienced shock and resuscitation (P 9 0.05). However, at 5, 30, 60, 90, and 120 min after fluid resuscitation (R5, R30, R60, R90 and R120, respectively), the MAP in groups LA and PY was higher than that in groups VR and NS (P G 0.05), and group PY had a higher MAP than did group LA (P G 0.05). Changes in MAP are shown in Figure 1. Arterial-blood lactate level
To evaluate oxygen deficit in the blood circulation, the arterial-blood lactate level was determined (Table 1). At 2 h after resuscitation (R120), the lactate level in the arterial blood of rats from the four shock and resuscitation groups was higher than that for group sham (P G 0.05), which certified that rats from the four groups indeed suffered from HS. Compared with groups VR and NS, the arterial blood lactate level of rats was significantly lower in groups LA and PY (P G 0.05). Group PY had a lower arterial blood lactate level than did group LA (P G 0.05). WW/DW of the intestine
The WW/DW of the intestine, which reflects the water content of intestinal tissue, is presented in Table 1. At R120, the intestinal WW/DW was higher in the four groups that underwent HS and fluid resuscitation than in group sham (P G 0.05). Compared with groups VR and NS, WW/DW was significantly lower in groups LA and PY (P G 0.05), and group PY had a lower WW/DW than did group LA (P G 0.05).
Intestinal mucosal damage index, which characterizes morphological changes (Fig. 2) in the intestinal mucosa, is presented in Table 1. At R120, the IMDI was significantly higher in the four groups subjected to HS and fluid resuscitation than that in group sham (P G 0.05). Compared with groups VR and NS, the IMDI was significantly lower in groups LA and PY (P G 0.05), and group PY had a significantly lower IMDI than did group LA (P G 0.05). Morphological changes in the intestinal mucosa were identified using a light microscope. In rats from group sham, the structure of the intestinal mucosal epithelium was integrated, and there were very few lymphocytes and no neutrophil infiltration. In groups VR and NS, the subepithelial space at the top of intestinal villus clearly widened, and there was serious abscission of the epithelium next to parts of the intestinal villus, erosion caused by cell necrosis and cell loss, edema of the lamina propria, and infiltration of a large number of neutrophils and lymphocytes. In groups LA and PY, pathological injury was alleviated. The subepithelial space at the top of the intestinal villus widened slightly with a slight edema of the lamina propria. Injury in group PY was alleviated further with infiltration of few neutrophils and lymphocytes. Ultrastructural changes in intestinal mucosa
Ultrastructural changes in the intestinal mucosa observed with TEM are shown in Figure 3. In rats from group sham, epithelial cells of the intestinal mucosa had abundant organelles, and all cellular structure was normal. In groups VR and NS, we observed swollen mitochondria with breaks in or disappearance of cristae, dilation of the endoplasmic reticulum, sparse irregular microvilli, and compaction of nuclear chromatin gathered in the cell border in intestinal mucosal epithelial cells. In the intestinal mucosal epithelial cells of rats from group LA, the mitochondria and endoplasmic reticulum were slightly swollen, and microvilli were slightly irregular. In the epithelial cells from the intestinal mucosa of rats in group PY, the mitochondria and endoplasmic reticulum were nearly normal, and all cellular structures were intact with regular microvilli. Intestinal tissue parameters
Intestinal MDA levels, which are accumulated as a product of lipid peroxidation, are presented in Table 1. At R120, intestinal MDA levels were elevated in the four HS and fluid
TABLE 1. The effect of DPR on arterial blood lactate level, intestinal WW/DW, MDA, NO, and the IMDI at 2 h after resuscitation in rats (n = 10) Group
Lactate, mmol/L
WW/DW
IMDI
MDA, 2mol/g
NO, 2mol/g
Sham
1.7 (0.5)
4.01 (0.24)
0.3 (0.5)
1.16 (0.13)
0.341 (0.035)
VR
8.0 (0.9)*
5.25 (0.37)*
3.4 (0.5)*
3.03 (0.24)*
0.192 (0.025)*
NS
7.5 (0.7)*
5.12 (0.34)*
3.1 (0.7)*
2.87 (0.26)*
0.212 (0.023)*
LA
4.9 (0.9)*†‡
4.68(0.27)*†‡
2.0 (0.9)*†‡
2.25 (0.17)*†‡
0.279 (0.026)*†‡
PY
2.5 (0.8)*†‡§
4.31 (0.23)*†‡§
1.1 (0.6)*†‡§
1.55 (0.14)*†‡§
0.311 (0.031)*†‡§
Values are presented as the mean T SD of 10 animals. *P G 0.05 versus group sham. † P G 0.05 versus group VR. ‡ P G 0.05 versus group NS. § P G 0.05 versus group LA.
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FIG. 2. The effect of DPR on morphological changes in the intestinal mucosa of rats (n = 10). Photomicrographs of hematoxylin-eosinYstained sections of the small intestine in rats 2 h after resuscitation (R120) (original magnification, 200 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY.
resuscitation groups relative to group sham (P G 0.05). Intestinal MDA levels in groups LA and PY were significantly lower than in groups VR and NS (P G 0.05), and group PY had lower levels of MDA than group LA (P G 0.05). Intestinal NO levels, which reflect the barrier function of the intestinal mucosa, are presented in Table 1. At R120, NO levels in the intestine were lower in the four HS and fluid resuscitation groups (P G 0.05) than those in group sham. In groups LA and PY, intestinal NO levels were significantly higher relative to groups VR and NS (P G 0.05), and group PY had significantly higher NO levels than did group LA (P G 0.05). Intestinal MPO activity, which reflects intestinal inflammatory injury, is presented in Figure 4. At R120, intestinal MPO activity was higher in the four HS and fluid resuscitation groups (P G 0.05) than that in group sham. In groups LA and PY, intestinal MPO activity was significantly lower than that in groups VR and NS (P G 0.05), and group PY had a lower MPO activity when compared with group LA (P G 0.05). Intestinal TNF-! levels
Immunohistochemical staining of TNF-! in the intestinal mucosa at 2 h after resuscitation is presented in Figure 5. As an inflammatory mediator, the expression of intestinal TNF-!
reflects the inflammatory response level (Fig. 6). At R120, the level of TNF-! in the intestine was significantly higher in the four HS and fluid resuscitation groups than that in group sham (P G 0.05). In groups LA and PY, the intestinal TNF-! level was significantly lower than that in groups VR and NS (P G 0.05), and group PY had a significantly lower TNF-! level relative to group LA (P G 0.05). DISCUSSION In surgery, intestinal IRI is a common form of organ damage that occurs during resuscitation after HS, and it is also the pathophysiological basis for numerous other diseases. As an organ with active metabolism, the intestine has a substantial immunological function and is the largest store of bacteria in the body. Research has indicated that MODS begins in the intestine and proceeds as follows: first, there are metabolic disorders of oxygen and energy, leukocyte adhesion, and endothelial cell injury, and injury caused by oxygen radicals and then intracellular calcium overload occur. When IRI occurs in vivo, the intestine becomes a source of early-stage inflammatory cytokines, and this organ may play a significant role in the development of systemic inflammatory response syndrome (SIRS), MODS, and even death (13).
FIG. 3. The effect of DPR on ultrastructural changes in the intestinal mucosa in rats (n = 10). Transmission electron microscopy photomicrographs of uranyl acetate and lead citrateYstained sections of the small intestine in rats at 2 h after resuscitation (R120) (original magnification, 10,000 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY. In B and C, the arrows highlighted the swollen mitochondria with breaks in or disappearance of cristae, dilation of the endoplasmic reticulum, sparse irregular microvilli, compaction of nuclear chromatin gathered in the cell border, and damaged cellular tight junctions in intestinal mucosal epithelial cells. In Figure D and E, the arrows highlighted the slightly swollen mitochondria and endoplasmic reticulum.
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FIG. 4. Effects of DPR on MPO activity of intestine 2 h after resuscitation in rats (n = 10). a P G 0.05 versus group sham; b P G 0.05 versus group VR, c P G 0.05 versus group NS; d P G 0.05 versus group LA.
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The superiority of adjunct DPR with Pyr-PDS in rats with HS
FIG. 6. The effects of DPR on the TNF-! expression level of the intestine at 2 h after resuscitation in rats (n = 10). Photographs of the sections were analyzed with Image Pro Plus 6.0 software. The TNF-! expression level was indicated by the average optical density. aP G 0.05 versus group sham; bP G 0.05 versus group VR; cP G 0.05 versus group NS; dP G 0.05 versus group LA.
Mean arterial pressure shows blood circulation and reflects the condition of HS and resuscitation in the body. Our results indicated that DPR following VR restored blood pressure and improved blood circulation. Also, Pyr-PDS improved blood circulation and stabilized blood pressure. Previous research supports the view that pyruvate-enriched solutions stabilize hemodynamics, suppress myocardial nitrosative stress, minimize systemic inflammation, and suppress the apoptotic cascade after HS more effectively than do lactate Ringer_s solution (16). Direct peritoneal resuscitation also improves intestinal blood flow(17). Arterial blood lactate reflects anaerobic metabolism. As one of the parameters indicating hypoperfusion in the body, an increase in arterial blood lactate indicates tissue
hypoxia, and the lactate level is a predictor of morbidity and mortality (18). Moreover, it is one of the parameters used to evaluate the effect of resuscitation. In this study, arterial blood lactate levels in group VR indicated serious hypoxia. However, DPR with Lac-PDS and Pyr-PDS following VR ameliorated hypoperfusion and hypoxia in the body. These results demonstrated that Pyr-PDS promoted recovery from tissue hypoxia more effectively than Lac-PDS. As a novel PDS, Pyr-PDS would be more effective than conventional methods in correction of metabolic acidosis. Because of its various cytoprotective effects, Pyr-PDS not only corrects acidosis, but also protects against the underlying dysfunction of vital organs (19). Evidence also shows that Pyr-PDS is superior to Lac-PDS in biocompatibility
FIG. 5. Immunohistochemical staining of TNF-! in the intestinal mucosa 2 h after resuscitation in rats (n = 10) (original magnification, 400 for the five panels). A, Group sham. B, Group VR. C, Group NS. D, Group LA. E, Group PY. The arrows highlighted the expression of intestinal TNF-!.
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SHOCK NOVEMBER 2014 (20). The ratio of wet weight to dry weight reflects the tissue water content and the degree of tissue and cellular edema. This study showed that DPR with Lac-PDS or Pyr-PDS following VR reduced intestinal edema, and Pyr-PDS had an advantage in recovery from tissue edema relative to Lac-PDS. Intestinal mucosal damage index, which we evaluated with light microscopy, and ultrastructural changes in the intestinal mucosa, which we observed using TEM, both showed that DPR following VR alleviated pathological injury in the intestinal mucosa, and the protection from intestinal mucosal injury promoted by Pyr-PDS was superior to Lac-PDS. Potential mechanisms of DPR with Pyr-PDS
Previous research supports the view that sodium pyruvate has a protective effect for IRI after HS that may be related to the scavenging of oxygen free radicals and a reduction in the neutrophil and anti-inflammatory response (21). Pyruvate is an energy substrate that has both inotropic and antioxidant properties (7). As is the case for the production of oxygen free radicals and lipid peroxidation, an increase in MDA levels indicates that oxygen free radicals attack the cytomembrane and lead to lipid peroxidation damage. Myeloperoxidase is a specific marker enzyme for neutrophils, and an increase in MPO activity indicates the infiltration of neutrophils that cause lipid peroxidation damage. Meanwhile, MPO catalyzes an excess of oxidant production that causes oxidative damage of tissues. Our results from this study demonstrated that intestinal injury in rats was related to MDA and MPO, which indicated that the protective effects of Pyr-PDS on intestinal IRI were related to a reduction in oxygen free radical levels and the inhibition of neutrophil infiltration. As the end-product of glycolysis and the starting substrate for the tricarboxylic acid cycle, pyruvate plays a key role in intermediary metabolism (3). Reactive oxygen species have been implicated in the pathogenesis of structural and functional alterations to tissues that are associated with a variety of pathological processes, including sepsis and septic shock, thermal injury, doxorubicininduced cardiomyopathy, HS, and mesenteric IRI. Pyruvate (CH3COCOOj) is also a potent and effective reactive oxygen species scavenger (4, 5). Meanwhile, pyruvate can cause systemic alkalinization. When lactate is replaced by pyruvate, acute toxic injuries caused by DPR can be avoided (22). Pyruvatebased PDS has general protective effects for glycometabolism, excellent systemic alkalinization, and potent antioxidant capacity. It has a unique advantage for the development of fluid therapy and is applicable to the prevention and treatment of refractory HS. Tumor necrosis factor ! is an early and central inflammatory mediator and an initial factor for the induction of SIRS and MODS (23). The intestine is one of the major organs that release TNF-!. Many studies have demonstrated that TNF-! and IL-6 play important roles in the occurrence and development of sepsis and MODS. Neutrophils function in the immune defense in HS. However, an excessive stress response would occur if there were a large number of neutrophils to be activated, and activated neutrophils play an important role in SIRS and MODS, because they induce production of intestinal inflammatory mediators, including TNF-! (24). Tumor necrosis
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factor ! and MPO levels indicate the release of intestinal inflammatory mediators and the degree of the inflammatory response. In this study, our results indicated that the protective effects of Pyr-PDS on intestinal IRI were related to a reduction in inflammatory mediators and an alleviation of the inflammatory response. Studies have shown that DPR not only can significantly increase IL-10 level and decrease TNF-! and IL-6 levels, but also can inhibit the inflammatory response in the liver and other internal organs (12). Pyruvate pretreatment of the rat small intestine also inhibited IRI and neutrophil infiltration (25). This shows that DPR with Pyr-PDS alleviates the systemic inflammatory response and improves systemic immune status. All of these effects improve patient prognosis. It has been suggested that the balance between locally produced NO and endothelin 1 plays an important role in the maintenance of intestinal mucosal microcirculation (26). As a significant factor in the regulation of intestinal mucosal blood flow and barrier function, NO has positive effects on intestinal IRI. Recent evidence has demonstrated that the preservation of the intestinal barrier can attenuate intestinal IRI (27). This study demonstrated that microcirculation disturbances after intestinal IRI were related to reduction in NO. Evidence also suggests that an endothelin-receptor antagonist can relieve continuous intestinal vasoconstriction and hypoperfusion after conventional intravenous recovery, and DPR can also promote continuous intestinal vasodilation and hypertransfusion (28). Direct peritoneal resuscitation increases the perfusion of internal organs by a related mechanism based on the vascular endothelium, and in this way, PyrPDS is more effective than Lac-PDS. These observations may be related to the release of NO and activation of the adenosine A1 receptor subtype. Previous studies have shown that PyrPDS significantly improves NO generation under both physiological pH and acidic pH and also under high glucose conditions (29). Pyruvate infusion clearly protects the small intestine against IRI. This protection primarily results from local effects on the small intestine (30). Conventional VR from HS results in hypoperfusion of the gut and liver, edema of organs and cells, and injury to vital organs. Adjunct DPR with dialysate prevents gut vasoconstriction, hypoperfusion, and injury (31); shortens the interval to definitive fascial closure; and reduces intraabdominal complications (32). For these purposes, Pyr-PDS is superior to Lac-PDS. Pyruvate-based PDS provides antiinflammatory dampening of the systemic inflammatory response and improves the survival rate. Pyruvate-based PDS slightly inhibits peritoneal macrophages and maintains the integrity of peritoneal mesothelial cells. There are changes in peritoneal solute transport resulting from pyruvate treatment, accompanied by a reduction in both peritoneal membrane angiogenesis and fibrosis, indicating novel mechanisms that could potentially reduce glucose-driven alterations to the peritoneal membrane in vivo (33). In addition, pyruvate enables the cells to remain viable during prolonged hypoxia. Clinical significance
We demonstrated in this study that blood circulation improved, hypoperfusion and hypoxia were ameliorated, and IRI
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of the intestine was alleviated by DPR with Pyr-PDS following VR from lethal HS in rats. Although the blood base excess and survival rates that are associated with the therapeutic effect of this novel resuscitation were not observed in this study, these parameters were shown to be significantly improved with adjunct DPR from severe shock (34). Recently, DPR with PyrPDS and hypertonic pyruvate have been shown to be an efficient therapy in resuscitation from HS in rats, and this treatment significantly improved organ function and the blood flow in various organ surfaces, including the liver, kidney, and intestinal mucosa (6). All of the present results demonstrated that DPR with Pyr-PDS combined with VR may be superior to NS or lactate-containing fluids or only VR in clinical resuscitation from severe HS. Further investigation is warranted. Limitations
In this study, animals were heparinized before HS and resuscitation for anticoagulation. Various studies have indicated that heparin can significantly improve hemodynamics and microcirculation during mesenteric reperfusion after hemorrhage (35), diminish clinical risks of gastrointestinal complications, and thus influence clinical outcomes. Therefore, this is a limitation for this study. Because heparinization was necessary in the experimentation, it may interfere with the effects of DPR and pyruvate on intestinal injury from HS in rats. Perhaps this problem can be solved in future studies by using lowmolecular-weight heparin or adjusting the heparin dose. The period after HS and resuscitation was chosen to be 2 h (9Y11, 14) for a preliminary study about DPR with pyruvate on animals. To obtain more potent evidences of Pyr-PDS_s effects, further research is needed, and the postresuscitation period can be prolonged in the future. Because it is likely that the data would have been more differentiated if they were normalized to milligrams of protein of intestine, the magnitude of changes in the inflammatory markers could be another possible limitation. CONCLUSIONS This study demonstrates that DPR with Pyr-PDS following VR alleviates intestinal IRI significantly after HS and resuscitation in rats and has a protective effect on HS rats. We provide evidence that mechanisms by which Pyr-PDS attenuates intestinal IRI may include the elimination of oxygen free radicals, reduction of neutrophil infiltration, inhibition of the inflammatory response, and regulation of intestinal mucosal blood flow and barrier function. Our results indicate that Pyr-PDS has significant advantages in the treatment of HS resuscitation and thus requires further investigation in the future. ACKNOWLEDGMENT The authors thank Fang-Qiang Zhou, MD, from Fresenius Dialysis Centers at Chicago for his assistance with pharmacology in this research.
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