Hypertonic saline

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Hi All,

I know some of you requested more info on hypertonic saline uses, and here's some research in trauma which you may find interesting.

HSPTX Protects Against Hemorrhagic Shock Resuscitation-Induced Tissue Injury: An Attractive Alternative to Ringer’s Lactate

[Original Articles]

Coimbra, Raul MD, PhD, FACS; Porcides, Rafael MD; Loomis, William BS; Melbostad, Heidi BS; Lall, Rohan MD; Deree, Jessica MD; Wolf, Paul MD; Hoyt, David B. MD, FACS

From the Department of Surgery, Division of Trauma and Surgical Critical Care, University of California San Diego School of Medicine, San Diego, California.

Submitted for publication September 28, 2005.

Accepted for publication November 11, 2005.

Presented at the Annual Meeting of the American Association for the Surgery of Trauma, September 22–24, 2005. Atlanta, Georgia.

Address for Reprints: Raul Coimbra, MD, PhD, FACS, Division of Trauma and Surgical Critical Care, University of California San Diego, 200 W Arbor Drive, #8896, San Diego, CA, 92103-8896; email: rcoimbra@ucsd.edu.

]

Abstract

Background: Conventional fluid resuscitation with Ringer’s lactated (RL) activates neutrophils and causes end-organ damage. We have previously shown that HSPTX, a combination of small volume hypertonic saline (HS) and pentoxifylline (PTX), a phosphodiesterase-inhibitor, downregulates in vitro neutrophil activation and proinflammatory mediator synthesis. Herein, we hypothesized that HSPTX decreases end-organ injury when compared with RL in an animal model of hemorrhagic shock.

Methods: Sprague-Dawley rats were bled to a mean arterial pressure of 35 mm Hg for 1 hour. Animals were divided into 3 groups: sham (no shock, no resuscitation, n = 7), RL (32 mL/kg, n = 7), and HSPTX (7.5% NaCl 4 mL/kg + PTX 25 mg/kg; n = 7). Shed blood was infused after fluid resuscitation. Blood pressure was monitored until the end of resuscitation. Animals were sacrificed at 24 hour after resuscitation. Bronchoalveolar lavage fluid (BALF) was obtained for white cell count (total and differential) and TNF-[alpha] and IL-1[beta] levels were measured by ELISA. Lung and intestinal injury at 24 hour were evaluated by histopathology. Organ damage was graded by a pathologist and a score was created (0 = no injury; 3 = severe). Lung neutrophil infiltration was evaluated by MPO immune staining.

Results: There were no differences in mean arterial pressure between groups. At 24 hours, BALF leukocyte count was decreased by 30% in HSPTX animals (p < 0.01). TNF-[alpha] and IL-1[beta] levels were markedly decreased in HSPTX-resuscitated animals compared with their RL counterparts (p < 0.01). HSPTX-resuscitated animals (lung injury score = 1.0 ± 0.4) had markedly decreased acute lung injury compared with RL-treated animals (2.5 ± 0.3) (p < 0.01). RL resuscitation led to a two-fold increase in lung neutrophil infiltration whereas in HSPTX-treated animals, the number of MPO + cells was similar to sham animals (p < 0.001). Intestinal injury was markedly attenuated by HSPTX (1.1 ± 0.3) compared with RL animals (2.6 ± 0.4) (p < 0.001).

Conclusions: HSPTX, a small volume resuscitation strategy with marked immunomodulatory potential led to a marked decrease in end-organ damage. HSPTX is an attractive alternative to RL in hemorrhagic shock resuscitation.

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Hemorrhagic shock is the most important cause of early death after major trauma. The relationship between hemorrhagic shock, duration of ischemia and reperfusion, and the development of sepsis and MODS has been well studied. Splanchnic ischemia leads to translocation of bacteria or bacterial products from the intestinal lumen to the central circulation through the portal venous system and the mesenteric lymphatic circulation. Activation of inflammatory cell populations in target organs then takes place, which, if uncontrolled, causes tissue damage.1–4

Prompt fluid resuscitation shortening the duration of shock should, theoretically, decrease the inflammatory response after hemorrhage and trauma. However, there is strong evidence suggesting that the current clinical resuscitation regimen with Ringer’s lactate (RL) potentiates neutrophil activation and is associated with increased organ injury, particularly in the lung.5–8 Alternative resuscitation regimens have been studied recently. Our laboratory has investigated the anti-inflammatory and immune effects of small volume hypertonic saline resuscitation (HS) and pentoxifylline (PTX) following hemorrhagic shock. These two strategies were aimed to modulate shock-induced inflammation and end organ injury.9–14

The ideal fluid resuscitation regimen would be administered to the patient immediately after the injury or during the initial phase of care in the hospital, would attenuate the inflammatory response decreasing organ injury secondary to uncontrolled inflammation, and ultimately, would improve survival.

Several similarities comparing the effects of HS and PTX on the inflammatory response have been observed. The effects of HS in the macro and microcirculation associated with the anti-inflammatory effects of both HS and PTX makes their combination an attractive strategy for hemorrhagic shock resuscitation.15,16

We recently proposed that a combination of HS and PTX (HSPTX) would down-regulate neutrophil activation and inflammatory mediator synthesis ex vivo, possibly by acting at different steps of the signaling pathways, ultimately leading to an enhanced effect with both drugs. We have previously shown that HSPTX attenuates neutrophil activation by decreasing oxidative burst and adhesion molecule _expression as well as proinflammatory mediator production.17

Therefore, we hypothesized that HSPTX, as opposed to RL, would attenuate end-organ injury without compromising hemodynamics. In the present study we investigated the effects of HSPTX as a novel hemorrhagic shock resuscitation strategy on lung and gut injury and on restoration of blood pressure.

MATERIALS AND METHODS

The experiments were approved by the University of California, San Diego Animal Subjects Committee and in accordance with guidelines established by the National Institutes of Health.

Experimental Model

Male Sprague-Dawley rats (300–400 g) were purchased from Harlan Sprague-Dawley (San Diego, CA). A 12 hour light and dark cycle was used and food and water were provided ad libitum. Animals were anesthetized with ketamine and xylazine by intraperitoneal injection. A right inguinal incision was performed and the femoral vessels (artery and vein) were cannulated with a polyethylene catheter (PE50). The venous catheter was used for injection of resuscitation fluids and the arterial catheter was used for monitoring mean arterial pressure (MAP) and for blood withdrawal. Blood was withdrawn over a period of 10 minutes until a MAP of 35 mm Hg was reached. This level of hypotension was then maintained for 1 hour by withdrawing or re infusing blood as necessary to maintain the MAP at 35 ± 5 mm Hg.

At the end of the shock period animals were divided into three groups according to the treatment received as follows: sham (cannulation only; no shock, no resuscitation, n = 5); RL (32 mL/kg, n = 7); and HSPTX (7.5% NaCl 4 mL/kg + 25 mg/Kg PTX, n = 7). Shed blood was reinfused after RL and HSPTX resuscitation. The body temperature of the animals was maintained constant at 37°C throughout the experiment.

PTX was purchased from Sigma (St. Louis, Mo). The volume of RL was chosen to give the animals the same sodium load received by animals in the HSPTX group. The dose of PTX was chosen based on multiple studies from our laboratory demonstrating its safety and lack of hypotension.13,14,18 At the end of volume resuscitation and after the final MAP measurement, catheters were removed, the incision was closed, and the animals were returned to their cages.

Twenty-four hours after shock and resuscitation the animals were killed. Blood, organs (lungs and intestines), and bronchoalveolar lavage fluid (BALF) were collected as described below.

White Cell Count in the Bronchoalveolar Lavage Fluid

Bronchoalveolar lavage fluid was obtained at 24 hour after resuscitation. Briefly, the animals were anesthetized and exsanguinated by cardiac puncture. Immediately after sacrifice, the trachea was cannulated and the lungs were washed with 10 mL of sterile normal saline. The lavage fluid was collected and centrifuged at 250 × g for 10 minutes. The supernatant was collected and stored at -70°C for further measurements of proinflammatory cytokines and protein content. The resultant cell pellet was resuspended in saline and total cell counts were obtained using a hemocytometer. Differential cell counts were obtained after counting 200 cells on a cytospin prepared slide stained with Giemsa.

BALF Proinflammatory Cytokine Levels

Tumor necrosis factor alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) were measured in the BALF 24 hours after shock and resuscitation using a commercially available sandwich enzyme immunoassay technique (ELISA kit, R&D Systems, Minneapolis, Minn). Results were expressed as pg/mL.

Histopathological Examination

Lungs and a segment of distal ileum were collected 24 hours after shock and resuscitation. Rat lung tissue, stored in 10% PBS buffered formalin, was embedded with paraffin using an automated processing unit (Autotechnicon). There were 5 µm sections cut from paraffin blocks and transferred onto glass slides. Lung histologic specimens stained with hematoxylin and eosin (Richard Allen Scientific, Kalamazoo, Mich).

A pathologist blinded to the groups subjected the lungs to histopathological examination, and a score was calculated. A scale from 0 to 3 was graded (0 = normal, 3 = most severe). Separate ratings were estimated for pulmonary edema, intra-alveolar hemorrhage, congestion, neutrophil, and mononuclear cell infiltration.

Ileum was fixed in 10% PBS buffered formalin and embedded with paraffin using an automated processing unit. There were 7 µm sections cut from paraffin blocks, transferred onto glass slides and stained with hematoxylin-eosin (Richard Allen Scientific). A pathologist blinded to the groups subjected the lungs to histopathological examination, and a score was calculated. A modification of the scale proposed by Cuzzocrea et al. from 0 to 3 was used as follows: 0 = normal, no damage; 1 = mild; focal epithelial edema and necrosis; 2 = moderate; diffuse swelling or necrosis of the villi; 3 = severe; diffuse necrosis of the villi with evidence of neutrophil infiltration in the submucosa and/or hemorrhage.19

Lung MPO Immunohistochemistry

PMN accumulation in the lung was assessed by staining tissue sections for myeloperoxidase (MPO) and counting positively stained cells. After deparaffinization, lung slides were incubated in Target Retrieval Solution (DAKO, Carpinteria, Calif) for 20 minutes at 95°C and cooled at room temperature. On every slide, each section was encircled with a PAP Pen (Sigma, St. Louis, Mo). All subsequent steps were conducted at room temperature in a humid chamber. Endogenous peroxide activity was quenched with 1.5% H2O2 for 5 minutes.

Tissue sections were blocked for 20 minutes (1.5% goat serum in PBS) and incubated for 2 hours with rabbit polyclonal MPO antibody (Lab Vision Corporation, Fremont, Calif) diluted 1:100 in blocking solution. Sections were washed with PBS and incubated with a biotinylated rabbit secondary antibody diluted 1:400 for 30 minutes. Specific labeling was detected with an Elite ABC peroxidase kit and DAB substrate (Vector Laboratories, Burlingame, Calif). To ensure specific staining of each primary antibody, a negative control was run in parallel with each set of experiments. A RL section was incubated with PBS, instead of the primary antibody, to evaluate for the possible nonspecific staining associated with each antibody. There was no staining evident in these samples (data not shown). MPO positive control slides, obtained from Lab Vision Corporation, were utilized to determine the efficacy of our laboratory’s staining protocol.

Statistical Analysis

Data are presented as Mean ± SEM. One-way ANOVA was used to test for significant differences between experimental groups. Differences were considered statistically significant at p values of < 0.05.

RESULTS

MAP and Shed Blood Volume

Initial MAP was higher than 100 mm Hg in all groups. There were no differences regarding the MAP between groups before or during shock. Fluid resuscitation restored blood pressure in RL- and HSPTX-treated animals to levels comparable and no different from sham animals (p > 0.20; Fig. 1).

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[Email Jumpstart To Image] Fig. 1. MAP measurements. MAP was measured continuously throughout the experiment. Data shown preshock MAP levels (baseline), measurements after initial blood drawing when the target blood pressure was achieved (T15 minutes), MAP measurements during shock (T30 minutes), and MAP measurements after fluid resuscitation (Resus). No differences were observed in MAP after fluid resuscitation (Resus) comparing HSPTX and RL.

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No differences were observed in total shed blood volume comparing RL (11.9 ± 0.7 mL) to their HSPTX (11.4 ± 0.5) counterparts.

White Cell Count in the Bronchoalveolar Lavage Fluid

Total leukocyte count in the BAL 24 hour after treatment was significantly elevated in RL treated animals (5.8 × 106 ± 0.3) compared with HSPTX (3.6 × 106 ± 0.7; p = 0.014) and sham animals (2.4 × 106 ± 0.4; p < 0.0001). No differences were observed comparing HSPTX to sham animals (Fig. 2). In addition, the higher total leukocyte count observed in the RL group was correlated with an increased number of neutrophils (15%), as compared with HSPTX (8%) or sham animals (5%) (p < 0.05).

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[Email Jumpstart To Image] Fig. 2. Total White Cell Count in BALF. Bronchoalveolar lavage fluid was collected at 24 hour after resuscitation. Total and differential cell counts were obtained. HSPTX treatment markedly decreased the number of white cells in the BALF compared with RL treated animals. HSPTX animals had cells counts similar to sham animals. *, p < 0.0001).

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BALF Proinflammatory Cytokine Levels

A marked increase in TNF-[alpha] levels was observed in the BALF after shock and resuscitation, regardless of the resuscitation fluid used. However, HSPTX-treated animals had TNF-[alpha] levels significantly lower than their RL counterparts (p = 0.003) (Fig. 3).

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[Email Jumpstart To Image] Fig. 3. TNF-[alpha] Levels in BALF. TNF-[alpha] levels were measured in BALF at 24 hour after resuscitation by ELISA as described in Methods. TNF-[alpha] levels were significantly decreased in HSPTX compared with RL-treated animals. *, p = 0.003 RL versus HSPTX; $, p < 0.0001 RL versus sham; # p < 0.01 HSPTX versus sham.

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Unlike TNF-[alpha], BALF IL-1[beta] levels were not upregulated by hemorrhagic shock per se. A marked increase in IL-1[beta] levels were observed in RL-treated animals compared with HSPTX and sham groups (p = 0.021) (Fig. 4).

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[Email Jumpstart To Image] Fig. 4. IL-1[beta] Levels in BALF. IL-1[beta] levels were measured in BALF at 24 hour after resuscitation by ELISA as described in Methods. IL-1[beta] levels were significantly decreased in HSPTX compared with RL-treated animals. *, p = 0.021 RL versus HSPTX; $, p < 0.01 RL versus sham.

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Lung Injury Score

At 24 hour, RL-treated animals demonstrated significantly more severe lung injury when compared with sham and HSPTX-treated animals (p = 0.01). The lung injury score was 1.0 ± 0.4 in the HSPTX group and 2.5 ± 0.3 in the RL group. Lung specimens from RL-treated animals displayed significant histologic changes including cellular inflammatory infiltrate, alveolar-capillary membrane thickening, hyaline membrane formation, hemorrhage, and edema. In contrast, the histologic appearance of lung specimens from HSPTX-treated animals was similar to sham animals (Fig. 5 A–C).

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[Email Jumpstart To Image] Fig. 5. Lung injury. Lungs were harvested at 24 hour after shock and resuscitation and evaluated by a pathologist unaware of the groups the animals belonged to (P.W.). Panel A shows the normal appearance of lung specimens from sham animals. Panel B shows the appearance of lungs from RL-treated animals. Note the marked inflammatory infiltrate, alveolar-capillary membrane thickening, hyaline membrane formation, and some areas of hemorrhage and alveolar edema. Panel C shows the histologic appearance of lungs form HSPTX-treated animals. Note the similarity compared with sham animals.

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Lung Myeloperoxidase Immune Staining

PMN infiltration into the lung tissue, a marker of tissue-injury, was evaluated by MPO immune staining. RL treatment led to a two-fold increase in the number of positive MPO stained cells when compared with sham and HSPTX groups (p < 0.001) (Fig. 6). The number of positive MPO stained cells in HSPTX-treated animals was similar to those seen in sham animals.

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[Email Jumpstart To Image] Fig. 6. Lung myeloperoxidase immune staining. MPO immune staining was used as a marker of neutrophil infiltration into the lung parenchyma. RL-treated animals had a two-fold increase in the number of MPO+ cells (neutrophils) in the lung parenchyma compared with HSPTX and sham animals. *, p < 0.001 RL versus HSPTX and RL versus sham).

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Gut Injury Score

At 24 hour, the histopathological appearance of the distal ileum of HSPTX-treated animals was similar to sham controls, indicating the protective effects of HSPTX on the development of intestinal injury after shock and resuscitation. The degree of intestinal injury was markedly increased in RL-treated animals (2.6 ± 0.4) when compared with sham and HSPTX-treated animals (1.1 ± 0.3; p < 0.001) (Fig. 7 A–C).

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[Email Jumpstart To Image] Fig. 7. Gut injury. Samples of distal ileum were harvested at 24 hour after shock and resuscitation and evaluated by a pathologist unaware of the groups the animals belonged to (P.W.). Panel A represents the normal appearance of gut specimens from sham animals. Panel B shows the gut appearance in RL-treated animals. Note the striking epithelial necrosis, inflammatory infiltrate, and destruction of the vili. Panel C shows the histologic appearance of intestines form HSPTX-treated animals. Note the similarity compared with sham animals.

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DISCUSSION

Hemorrhagic shock is the most frequent cause of death after trauma. Early deaths are generally because of uncontrollable bleeding. However, those initially presenting in shock that survive the initial insult will go on to develop systemic inflammation and organ dysfunction at a later time.

Conventional fluid resuscitation with RL has been shown to be deleterious because of the induction of inflammation and organ injury.5–8 Therefore, several laboratories have focused on investigating alternatives to RL resuscitation. However, the debate of which fluid resuscitation is ideal is far from being resolved.

We have investigated the immunomodulatory effects of HS and phosphodiesterase inhibition with PTX resuscitation for more than one decade. We have demonstrated that both strategies are effective in downregulating hemorrhagic shock-induced inflammation and end-organ injury.9–14,20,21 The mechanisms of action of both strategies have also been investigated in our laboratory and others. Collectively, it seems that PTX has a more potent downregulatory effect than HS on TNF-[alpha] synthesis and similar effect on neutrophil activation.15,16,20–26 However, HS seem to intercept intracellular signals at steps upstream from protein kinase C (PKC) whereas PTX seems to act at steps downstream from PKC. In addition, there is evidence that PTX decreases neutrophil MAPK p38 and ERK 1/2 activation, while HS affects mainly MAPK 38.27–31 NF-kB nuclear translocation and subsequent proinflammatory gene transcription is clearly downregulated by PTX. HS decreases proinflammatory cytokine synthesis in some cell populations, such as alveolar macrophages, likely by decreasing NF-kB DNA binding, but its effects on peripheral blood mononuclear cells seems to be less pronounced that that of PTX.30–38

Because uncontrolled activation and extravasation of neutrophils may be harmful to the host, modification of neutrophil responses driven by proinflammatory cytokines is an attractive pharmacological target.

Based on our previous experience evaluating the anti-inflammatory effects of HS and PTX in vitro and in vivo, we proposed that a combination of both (HSPTX) would further down-regulate neutrophil activation and inflammatory mediator synthesis, possibly by acting at distinct steps of the signaling pathways, ultimately leading to an enhanced effect of both drugs. We have recently demonstrated that HSPTX attenuates neutrophil oxidative burst, downregulates neutrophil CD11b _expression, and decreases TNF-[alpha] production. These effects were more pronounced than those observed with HS alone.17

Although PTX and HS attenuate the inflammatory response, PTX is not a volume expander itself. Because of the beneficial effects seen with the use of HS and PTX on inflammatory events and attracted by the possibility of obtaining additional anti-inflammatory effects with a low-volume hypertonic resuscitation strategy, we decided to investigate whether a combination of both would provide a salutary effect on animals undergoing hemorrhagic shock.

Intravascular volume expansion was similar when comparing RL to HSPTX-treated animals because all animals had restoration of blood pressure to levels comparable to sham controls and to prehemorrhage levels. HS has proven to be safe and effective in restoring MAP and microcirculatory blood flow (reviewed in 16). Similarly, we have shown, at least in animal models that PTX is safe, improves cardiac performance, and contrary to old concepts, does not cause hypotension.18 Therefore, it is expected that HSPTX is as safe and effective in achieving restoration of hemodynamic parameters as each of the treatments alone.

We observed a marked attenuation of lung and gut injury in animals resuscitated with HSPTX compared with conventional RL treatment, despite both groups having achieved hemodynamic stability. The appearance of the lung tissue in HSPTX-treated animals was very similar to that seen after HS and PTX treatment and reported by us in previous publications.12–14 To confirm the critical role played by neutrophils in the development of acute lung injury, the cellularity of the BALF as well as lung MPO staining were analyzed. RL-treated animals had significantly more white blood cells and a higher percentage of neutrophils in the BALF when compared with their HSPTX counterparts.

In addition, a significantly higher number of positive MPO stained cells were seen in the lungs of RL-treated animals. Neutrophil infiltration into the lung is a determinant factor for the development of lung injury after hemorrhagic shock.39 Neutrophil migration and sequestration into the tissue involves a complex process of cell rolling and adherence to the endothelium regulated by the _expression of adhesion molecules, and transendothelial migration through a chemo attractant gradient.40,41

Using an intravital microscopy approach, we have previously demonstrated that both HS and PTX decrease neutrophil adhesion to the endothelium following hemorrhagic shock.14 Rizoli et al. showed that animals treated with HS had less lung injury and less neutrophil infiltration in the BALF when compared with animals receiving RL after hemorrhagic shock and intratracheal endotoxin injection. They have also demonstrated that HS diminishes adhesion molecule _expression such as L-selectin, CD 11b and ICAM-I.25 Angle at al., from our laboratory, showed that HS reduces neutrophil margination by suppressing neutrophil L-selectin _expression.21

Shock leads to the release of proinflammatory mediators from multiple cell populations. TNF-[alpha] is released very early after shock. It is considered a proximal mediator of the inflammatory cascade, and is responsible for the regulation and synthesis of several cytokines and chemokines such as IL-1, IL-6, and IL-8.42,43 The rapid release of TNF-[alpha] plays a central role in the _expression of adhesion molecules in neutrophils (CD11b/CD18) and endothelial cells (ICAM-1).44 The up-regulation of proinflammatory cytokine synthesis alters the interactions between leukocytes and endothelial cells, resulting in the sequestration and migration of neutrophils to various tissues.

Herein, we reported increased levels of TNF-[alpha] and IL-1-[beta] in BALF of RL-resuscitated animals compared with HSPTX-treated animals, which had cytokine levels similar to sham controls. Because TNF-dependent adhesion molecule _expression is key to neutrophil-endothelial cell interactions before diapedesis occurs, direct or indirect (through downregulation of TNF-[alpha]) inhibition of the _expression of such molecules may constitute one of the mechanisms by which HSPTX resuscitation attenuated lung injury after hemorrhagic shock resuscitation in the present study. In a previous study, we reported that hemorrhagic shock resuscitation with either HS or PTX decreased ICAM-1 _expression in lung vessels. Furthermore, increased PMN adherence to the endothelium in RL-treated animals was observed, paralleling the peak of ICAM-1 _expression in the lung.14

Studies evaluating the effects of PTX on adhesion molecule _expression have reported decreased P-selectin _expression after hemorrhagic shock, decreased proinflammatory cytokine-induced ICAM-1 _expression in human pulmonary epithelial cells, and polymorphonuclear leukocytes.45–47 HS resuscitation has also been shown to decrease leukocyte adhesion to the endothelium after hemorrhage. Pascual et al. showed that HS resuscitation after hemorrhagic shock decreases PMN rolling and adherence to the endothelium and reduces vascular leakage compared with RL-treated animals.48 In a two-hit model of hemorrhagic shock and infection, HS resuscitation attenuated PMN lung sequestration and transmigration by decreasing leukocyte-endothelium interactions.49

RL resuscitation has been shown to activate neutrophils and its use in hemorrhagic shock is associated with increased organ injury, particularly in the lungs.5–7 Recent studies have shown that RL adequately improves hemodynamic parameters after hemorrhagic shock, but it may cause detrimental effects on the immune response by altering leukocyte function. These effects are most likely related to the composition of the racemic solution, which includes D- and L-lactate.8 Therefore, RL may up-regulates lung ICAM-1 _expression after hemorrhagic shock and HSPTX may reduce it, possibly to the same extent that eliminating D-lactate from the racemic RL solution minimizes some of the “proinflammatory” properties of RL. Alternatively, other events such as ischemia and reperfusion, may be more important than the resuscitation solution in the initiating the activation of the inflammatory cascade and up-regulating adhesion molecules. To that effect, several lines of evidence have documented that the production of reactive oxygen species (ROS) after ischemia and reperfusion primes macrophages, resulting in increased NF-kB activation, proinflammatory mediator production, augmented neutrophil recruitment, and organ injury.36,50,51

The gut has been implicated in the pathogenesis of MODS and as a major source of ROS and proinflammatory mediators which reach the central circulation mainly through the lymphatic system and portal vein.52,53 Evidence suggests that proinflammatory mediators originating in the gut after shock and resuscitation will prime lung alveolar macrophages to produce additional proinflammatory mediators such as TNF-[alpha] and IL-1. These, in turn, will amplify the inflammatory response by stimulating the release of chemoattractant factors by alveolar macrophages and airway epithelial cells.54,55 HS and PTX resuscitation have been shown to improve splanchnic perfusion and to decrease ischemia/reperfusion-induced intestinal damage.38,56–58

Therefore, in the present study, we evaluated the degree of gut injury after shock and resuscitation. HSPTX completely abrogated the mucosal destruction, necrosis, and cellular infiltration seen in RL-resuscitated animals. The hemodynamic as well as anti-inflammatory effects of HSPTX may have contributed to the maintenance of normal intestinal architecture and preservation of the mucosal barrier.

In conclusion, we have shown that compared with RL, HSPTX is superior as a fluid resuscitation strategy in hemorrhagic shock. HSPTX decreased proinflammatory mediator production in the lung and attenuated lung and gut injury.

Despite new findings and corroboration of the results of our preliminary in vitro studies, further work evaluating the cellular and molecular mechanisms by which HSPTX acts is necessary to characterize its protective effects as a resuscitation strategy.

The attractiveness of this novel resuscitation strategy is based on two aspects: its anti-inflammatory and possible immunomodulatory properties and its hypertonic small-volume resuscitation characteristic. Modulating the inflammatory response that follows major hemorrhage with novel resuscitation regimens may prove beneficial in clinical practice.

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DISCUSSION

Dr. David G. Burris (Bethesda, Maryland): Ah, the most elusive of prizes, the elixir of life bubbling forth from the ever-youthful fountain, the sweet ambrosia that quaffed and the magic draft will impart extended life to under-perfused, nearly dead cells, and while restoring life, ameliorate the immunologic backlash to perfusion from a near-death experience and all offered in the holy grail demitasse of a small volume resuscitation that will prevent rebleeding and restore life quickly in keeping with our evermore rapid existence before the next commercial break. The talented group from San Diego has, in a series of studies, offered us excellent results from two such solutions in the past: hypertonic saline rapidly restores perfusion and by temporarily tanning white cells prevents their non-laudable actions during reperfusion, pentoxifylline, a phosphodiasterase-inhibitor, moderates inflammatory response in other ways.

If each is good, how could the combination not be better? One fluid to find them; one fluid to rule them all, and in one vial to bind them. We saw beautiful, youthful looking pictures of tissue restored with HSPTX. While we were not suspicious, experience teaches us to fear unintended or unmeasured consequences, to seek a closeted painting of Dorian Grey where the hidden consequences of ages are displayed, so I must ask a few questions.

The combination fluid is compared to the old nemesis RL. You compared them each to each other in vitro. Where are the groups comparing them to each other here? The effect seems laudable. Are they really additive? Can we know that the combination is better than either alone? Some groups have shown that different isomers of lactate behave differently. Was this the old standby racemic RL? How does HSPTX compare to improved, single isomer RL?

We seem to have been taught that deaths after shock and resuscitation are divided into two phases: early, from persistent under-perfusion leading to necrosis; and later, up to several days, resulting from immunology gone wild, sometimes involving a second hit and leading to death by multi-organ dysfunction. Yet, the RL group intestinal tissue looks very bad fairly early. In addition, resuscitation with hypertonic saline took one-quarter the volume of resuscitation with Ringer’s lactate, so it is presumably finished more quickly. Also, the blood pressure, when you give hypertonic saline, seems to jump up more quickly than with Ringer’s. So, due to these effects, even though there is no difference at the end resuscitation blood pressure in the groups, was there some period of time where they were not, in fact, at the same BP and not, in fact, equally resuscitated?

Do you have lactates or other markers showing they were equally well perfused at the end of resuscitation and during the entire resuscitation phase? Phrased differently, why would this not be gut necrosis from an unrecognized under-resuscitation in the RL group, rather than the immunologic consequences of the resuscitation that we would normally expect somewhat later?

Dr. Raul Coimbra (San Diego, California): Regarding your first question, if one is good, how could the combination not be better? There is some evidence showing that hypertonic saline alone and pentoxifylline alone have similar effects in terms of neutrophil activation and end-organ injury however, the mechanisms by which these two drugs work—and I like to call hypertonic saline a drug not, a solution—might be different. There are data from our laboratory showing that hypertonic saline actually leads to increased activation of MAP kinase P38; whereas, pentoxifylline down regulates P38 activation.

So, perhaps the end result is the same, but the mechanisms by which the end results are achieved are different. Your second question, how do we know that the combination is additive? While we didn’t have hypertonic saline alone and pentoxifylline alone in this study, but we have several studies from our laboratory looking at hypertonic saline and pentoxifylline side-to-side.

Most of the things that we evaluated in this study, combining the traditional resuscitation strategy with this novel strategy, have been investigated by us and by others in several other studies. Are they additive? I don’t know if they are completely additive, but I know that some of the in vitro effects that we’ve seen in hypertonic saline plus pentoxifylline are better than hypertonic saline alone.

Was this racemic lactated Ringer’s, yes. Perhaps, we should do a study comparing HSPTX to the single isomer lactated Ringer’s. Regarding whether or not a second hit is involved, actually, in the past, we looked at a two-hit model of hemorrhagic shock and sepsis 24 hours later, and we’ve shown that the resuscitation of animals with hypertonic saline protected them from a second hit that occurred 24 hours later without any further intervention.

Your last question, did we measure lactate levels or other markers? Yes, we did. Actually, in previous studies we looked at lactate, we looked at base deficit. All these groups have been resuscitated to the same extent. Now would this be necrosis of the gut? That’s hard to say, but I think that during the reperfusion phase, although it’s too early, there is some degree of necrosis in the gut. We were not able to identify necrosis at 4 hours in our pilot studies, but we were able to identify necrosis at 24 hours.

Dr. Charles E. Lucas (Detroit, Michigan): I have a couple of questions. Now, why is it that we continue to use Ringer’s lactate in our animal studies and not use a fluid, which has a sodium concentration of 140, like I think most of us do in the clinical setting? Secondly, what is the molecular weight of PTX? Thirdly, what is the rate by which it gets into the lymphatic system?

Dr. Raul Coimbra (San Diego, California): I will start with the third question. I don’t know the rate that it gets to the lymphatic system. Actually, I was talking to Dr. Hoyt before my presentation, and we were talking about cannulating the lymphatics and looking at lymph activity in this model, because that would be a nice complement to justify what we’ve seen in the lungs and in the intestines. Regarding molecular weights, it’s a very small molecule that goes inside the cells directly and functions by activating one of the intracellular signaling cascades.

Cyclic AMP is a second messenger that is produced by inhibiting phosphodiesterase. That’s the mechanism by which pentoxifylline acts, increasing intracellular cyclic AMP and activating protein kinase A. It’s a small molecule.

Now, why we continue to use lactated Ringer’s in our research instead of normal saline, that’s a great question. I think that the only reason not to use normal saline is because lactated Ringer’s is a solution we use in our clinical practice. In our experiments we corrected for the amount of sodium given to the animals by adjusting the volume of lactated Ringer’s. So, in terms of sodium load, the two groups receive the same amount of sodium.

Dr. Hiroshi Homma (Tokyo, Japan): Did you measure TNF Alpha and IL1 beta in serum? If you did not measure, why did you not measure it, because I think it’s difficult to get the same concentration using bronchial lavage.

Dr. Raul Coimbra (San Diego, California): Yes, we measure in the serum. However, this measurement was done in 24 hours in the BAL, when there is maximal lung inflammation. It’s a closed compartment and whatever is produced, some of it may be metabolized locally, but most of what is produced is there so it’s easy to measure. Although, you are absolutely correct, the concentration is very, very low. If you read my slides, it’s about 150–180 picograms in the BAL, which is very low concentration compared to 1,000 or 2,000 picograms in the serum. To determine the peak of IL1 beta and TNF alpha in this model, you’ve got to measure it in the serum at the very, very early time points.

Dr. Ajai K. Malhotra (Richmond, Virginia): Since you used a model in which the bleeding was related to blood pressure, I’d like to know how much blood was actually withdrawn. Secondly, did you do anything to say that, physiologically, the shock level was equal, not just by pressure? You used quite a high volume of LR, 35 ml per kilogram. How does it relate to the amount of blood that was withdrawn?

Now you justify it by saying the sodium load was the same. But if you give massive LR to almost anybody, why not use the minimum amount necessary to resuscitate, rather than necessary equivalent sodium loads? Finally, why did you choose to return the shed blood?

Dr. Raul Coimbra (San Diego, California): Great questions. We’ve been working with this model for many, many years, and we looked into the relationship between the amount of blood that you withdraw and the amount of fluid you’ve got to give to provide the same sodium load. It’s amazing that if you decide to go for a fixed strategy, let’s say 3:1, you’re going to end up giving the same amount of lactated Ringer’s. So, to give you an example, the total shed blood in the lactated Ringer’s group was 11.9 ml and in the HSPTX group it was 11.4 ml.

If you use the 3:1 rule, you’re going to end up giving 33 mL per animal of lactated Ringer’s, and because we decided to adjust for the sodium load we ended up giving 32, which is very close to the amount based on the 3:1 rule. Therefore, based on our previous experience, we decided that would be a reasonable strategy, because either way you go, you will be giving the same amount. We didn’t use in this model any other measure of adequacy of resuscitation such as lactate or base deficit, because in our previous experiments, using the same amounts of resuscitation and using the same animal model, we had the data.

As I demonstrated in our preliminary data here all three groups, either pentoxifylline alone, hypertonic saline alone or lactated Ringer’s, had the same lactate levels and the same base deficit levels.

Hope this helps,

ACE844

[Ace844]

(Hypertonic saline resuscitation prevents hydrostatically induced intestinal edema and ileus.

Laboratory Investigations

Critical Care Medicine. 34(6):1713-1718 @ June 2006.

Radhakrishnan, Ravi S. MD; Xue, Hasen MD; Moore-Olufemi, Stacey D. MD; Weisbrodt, Norman W. PhD; Moore, Frederick A. MD; Allen, Steven J. MD; Laine, Glen A. PhD; Cox, Charles S. Jr MD)

Abstract:

Objective: We have shown that acute edema induced by mesenteric venous hypertension (MV-HTN) impairs intestinal transit and reduces the standardized engineering measures of intestinal stiffness (elastic modulus) and residual stress (opening angle). We hypothesized that hypertonic saline (7.5%) would reverse these detrimental effects of acute edema.

Design: Laboratory study.

Setting: University laboratory.

Subjects: Male Sprague Dawley rats (270-330 g).

Interventions: Rats were randomized to five groups: sham, MV-HTN alone, MV-HTN with 4 mL/kg normal saline resuscitation (equal volume), MV-HTN with 33 mL/kg normal saline resuscitation (equal salt), and MV-HTN with 4 mL/kg hypertonic saline. Intestinal edema was measured by wet to dry tissue weight ratio. A duodenal catheter was placed and, 30 mins before death, fluorescein isothiocyanate Dextran was injected. At death, dye concentrations were measured to determine intestinal transit. Segments of distal ileum were hung to a fixed point in a tissue bath and to a force displacement transducer and stretched in increments of 1 mm; we recorded the new length and the corresponding force from the force displacement transducer to determine elastic modulus. Next, two transverse cuts were made yielding a 1- to 2-mm thick ring-shaped segment of tissue which was then cut radially to open the ring. Then the opening angle was measured.

Measurements and Main Results: MV-HTN, MV-HTN with 4 mL/kg normal saline, and MV-HTN with 33 mL/kg normal saline caused a significant increase in tissue edema and a significant decrease in intestinal transit, stiffness, and residual stress compared with sham. Hypertonic saline significantly lessened the effect of edema on intestinal transit and prevented the changes in stiffness and residual stress.

Conclusions: Hypertonic saline prevented intestinal tissue edema. In addition, hypertonic saline improved intestinal transit, possibly through more efficient transmission of muscle force through stiffer intestinal tissue.