Plasma resuscitation improves and restores intestinal microcirculatory physiology following haemorrhagic shock.

BACKGROUND AND OBJECTIVES: Intestinal ischaemia-reperfusion injury following resuscitated haemorrhagic shock (HS) leads to endothelial and microcirculatory dysfunction and intestinal barrier breakdown. Although vascular smooth muscle machinery remains intact, microvascular vasoconstriction occurs secondary to endothelial cell dysfunction, resulting in further ischaemia and organ injury. Resuscitation with fresh frozen plasma (FFP) improves blood flow, stabilizes the endothelial glycocalyx and alleviates organ injury. We postulate these improvements correlate with decreased tissue CO2 concentrations, improved microvascular oxygenation and attenuation of intestinal microvascular endothelial dysfunction. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomly assigned to groups (n = 8/group): (1) sham, (2) HS (40% mean arterial blood pressure [MAP], 60 min) + crystalloid resuscitation (CR) (shed blood saline) and (3) HS + FFP (shed blood + FFP). MAP, heart rate (HR), ileal perfusion, pO2 and pCO2 were measured at intervals until 4 h post-resuscitation (post-RES). At 4 h post-RES, the ileum was rinsed in situ with Krebs solution. Topical acetylcholine and then nitroprusside were applied for 10 min each. Serum was obtained, and after euthanasia, tissues were harvested and snap-frozen in liquid N2 and stored at -80°C. RESULTS: FFP resuscitation resulted in sustained ileal perfusion as well as rapid sustained return to baseline microvascular pO2 and pCO2 values when compared to CR (p < 0.05). Endothelial function was preserved relative to sham in the FFP group but not in the CR group (p < 0.05). CONCLUSION: FFP-based resuscitation improves intestinal perfusion immediately following resuscitation, which correlates with improved tissue oxygenation and decreased tissue CO2 levels. CR resulted in significant damage to endothelial vasodilation response to acetylcholine, while FFP preserved this function.

Direct peritoneal resuscitation reduces inflammation in the kidney after acute brain death.

Brain death is associated with significant inflammation within the kidneys, which may contribute to reduced graft survival. Direct peritoneal resuscitation (DPR) has been shown to reduce systemic inflammation after brain death. To determine its effects, brain dead rats were resuscitated with normal saline (targeted intravenous fluid) to maintain a mean arterial pressure of 80 mmHg; DPR animals also received 30 cc of intraperitoneal peritoneal dialysis solution. Rats were euthanized at 0, 2, 4, and 6 h after brain death. Pro-inflammatory cytokines were measured using ELISA. Levels of IL-1ß, TNF-α, and IL-6 in the kidney were significantly increased as early as 2 h after brain death and significantly decreased with DPR. Levels of leukocyte adhesion molecules ICAM and VCAM increased after brain death and were decreased with DPR (ICAM 2.33 ± 0.14 vs. 0.42 ± 0.04, P = 0.002; VCAM 82.6 ± 5.8 vs. 37.3 ± 1.9, P = 0.002 at 4 h) as were E-selectin and P-selectin (E-selectin 25,605 vs. 16,144, P = 0.005; P-selectin 82.5 ± 3.3 vs. 71.0 ± 2.3, P = 0.009 at 4 h). Use of DPR reduces inflammation and adhesion molecule expression in the kidneys, and is associated with reduced macrophages and neutrophils on immunohistochemistry. Using DPR in brain dead donors has the potential to reduce the immunologic activity of transplanted kidneys and could improve graft survival.

Damage-associated molecular patterns in resuscitated hemorrhagic shock are mitigated by peritoneal fluid administration.

Conventional resuscitation (CR) of hemorrhagic shock (HS), a significant cause of trauma mortality, is intravenous blood and fluids. CR restores central hemodynamics, but vital organ flow can drop, causing hypoperfusion, hypoxia, damage-associated molecular patterns (DAMPs), and remote organ dysfunction (i.e., lung). CR plus direct peritoneal resuscitation (DPR) prevents intestinal and hepatic hypoperfusion. We hypothesized that DPR prevents lung injury in HS/CR by altering DAMPs. Anesthetized male Sprague-Dawley rats were randomized to groups ( n = 8/group) in one of two sets: 1) sham (no HS, CR, or DPR), 2) HS/CR (HS = 40% mean arterial pressure (MAP) for 60 min, CR = shed blood + 2 volumes normal saline), or 3) HS/CR + DPR. The first set underwent whole lung blood flow by colorimetric microspheres. The second set underwent tissue collection for Luminex, ELISAs, and histopathology. Lipopolysaccharide (LPS) and DAMPs were measured in serum and/or lung, including cytokines, hyaluronic acid (HA), high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 protein (MYD88), and TIR-domain-containing adapter-inducing interferon-ß (TRIF). Statistics were by ANOVA and Tukey-Kramer test with a priori P < 0.05. HS/CR increased serum LPS, HA, HMGB1, and some cytokines [interleukin (IL)-1α, IL-1ß, IL-6, and interferon-γ]. Lung TLR4 and MYD88 were increased but not TRIF compared with Shams. HS/CR + DPR decreased LPS, HA, cytokines, HMGB1, TLR4, and MYD88 levels but did not alter TRIF compared with HS/CR. The data suggest that gut-derived DAMPs can be modulated by adjunctive DPR to prevent activation of lung TLR-4-mediated processes. Also, DPR improved lung blood flow and reduced lung tissue injury. Adjunctive DPR in HS/CR potentially improves morbidity and mortality by downregulating the systemic DAMP response.

Alterations in platelet-derived growth factor expression in the pathophysiology of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) involves impaired ileal blood flow due to alterations in vascular tone control and intestinal angiogenesis. Platelet-derived growth factor (PDGF) is a mediator of normal angiogenesis in intestinal epithelium. We hypothesized that gene dysregulation during experimental NEC results in altered PDGF expression. METHODS: Sprague-Dawley rats were randomized to groups by litter. Controls were delivered vaginally and dam-fed. NEC groups were delivered prematurely by cesarean section and subjected to an established NEC protocol. Ileum was obtained at 0, 12, 24, 48, 72, and 96 h of life from all animals (N = 108 animals). Western blot analysis was carried out for every time point, and samples were evaluated by immunohistochemistry. Antibodies against PDGF-A, PDGF-B, and their receptors, PDGFR-α and PDGFR-ß, were used. Statistical analysis was performed using two-way analysis of variance with a priori P < 0.05. RESULTS: Ileal PDGF-A concentration was higher in controls versus NEC from 24-96 h of life. Its receptor, PDGFR-α, was low in concentration in both groups at all time points. PDGF-B concentration was increased in controls at 24 and 72 h of life but decreased at the 48-h mark. Its receptor, PDGFR-ß, was also low in both groups at 12 and 24 h but increased in controls at 48 and 72 h. CONCLUSIONS: These data support our hypothesis that PDGF and PDGF receptor expression are altered in experimental NEC. Dysregulation of PDGF during intestinal maturation could contribute to the development of NEC. Further investigation into this pathway could yield new therapeutic targets for this devastating disease.

Intraperitoneal 1.5% Delflex improves intestinal blood flow in necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) alters intestinal microvascular control mechanisms causing significant vasoconstriction. Our prior work with intraperitoneal 2.5% dextrose solution demonstrated increased intestinal perfusion in experimentally induced NEC. In the current study, we examine whether a buffered solution with lower glucose and osmolar loads similarly increases intestinal blood flow. We hypothesized that buffered 1.5% dextrose solution would increase ileal blood flow compared with baseline in NEC. METHODS: We randomly assigned pregnant Sprague-Dawley rats to control (n = 103) or NEC (n = 123) groups, by litter. We induced NEC by previously published methods. Control pups were vaginally delivered and dam-fed. We used laser Doppler flowmetry to evaluate perfusion in the terminal ileum at 12, 24, 48, 72, or 96 h after delivery at baseline and after application of topical 1.5% dextrose solution. We evaluated differences between groups and time points by analysis of variance and Tukey post hoc test. RESULTS: Baseline blood flow in the terminal ileum increased with gestational age in both groups (P < 0.05). Control groups had significantly greater baseline blood flow than NEC groups (P < 0.05), and topical application of buffered 1.5% dextrose solution increased blood flow compared with baseline in both groups at all time points (P < 0.05). CONCLUSIONS: Topical 1.5% dextrose solution significantly enhanced blood flow in the terminal ileum to the same degree as 2.5% dextrose solution. Thus, the use of buffered 1.5% dextrose solution might be more beneficial in treating clinical NEC, because it places a lower glucose and osmotic load on NEC-injured intestine.

Preservation of hepatic blood flow by direct peritoneal resuscitation improves survival and prevents hepatic inflammation following hemorrhagic shock.

Conventional resuscitation (CR) from hemorrhagic shock (HS) results in gut and liver hypoperfusion, organ and cellular edema, and vital organ injury. Adjunct direct peritoneal resuscitation (DPR) with dialysate prevents gut vasoconstriction, hypoperfusion, and injury. We hypothesized that DPR might also improve hepatocellular edema, inflammation, and injury. Anesthetized male SD rats were assigned to groups (n = 8/group): 1) sham (no HS); 2) HS (40% MAP/60 min) + intravenous fluid conventional resuscitation [CR; shed blood + 2 vol saline (SAL)/30 min]; 3) HS+CR+DPR (30 ml ip 2.5% glucose dialysate); or 4) HS+CR+SAL (30 ml ip saline). Histopathology showed lung and liver injury in HS+CR and HS+CR+SAL up to 24-h postresuscitation (post-RES) that was not in shams and which was prevented by adjunct DPR. Wet-to-dry weight ratios in HS+CR revealed organ edema formation that was prevented by adjunct DPR. HS+CR and HS+CR+SAL had 34% mortality by 24-h post-RES, which was absent with DPR (0%). Liver IFN-γ and IL-6 levels were elevated in CR compared with DPR or shams. TNF-α mRNA was upregulated in CR/sham and DPR/sham. IL-17 was downregulated in DPR/sham. CXCL10 mRNA was upregulated in CR/sham but downregulated in DPR/sham. Despite restored central hemodynamic performance after CR of HS, liver blood flow was compromised up to 24 h post-RES, and the addition of DPR restores and maintains liver perfusion at 24-h post-RES. DPR prevented liver injury, histological damage, and edema formation compared with CR alone. DPR provided a mitigating anti-inflammatory dampening of the systemic inflammatory response. In all, these effects likely account for improved survivorship in the DPR-treated group.

Effect of Plasma Resuscitation with Adjunctive Peritoneal Resuscitation on Hepatic Blood Flow and End-Organ Damage after Hemorrhagic Shock.

BACKGROUND: Intestinal injury from resuscitated hemorrhagic shock (HS) disrupts intestinal microvascular flow and causes enterocyte apoptosis, intestinal barrier breakdown, and injury to multiple organs. Fresh frozen plasma (FFP) resuscitation or directed peritoneal (DPR) resuscitation protect endothelial glycocalyx, improve intestinal blood flow, and alleviate intestinal injury. We postulated that FFP plus DPR might improve effective hepatic blood flow (EHBF) and prevent associated organ injury (liver, heart). STUDY DESIGN: Anesthetized Sprague-Dawley rats underwent HS (40% mean arterial pressure, 60 minutes) and were randomly assigned to groups (n = 8 per group): Sham; crystalloid resuscitation (CR; shed blood + 2 volumes CR); DPR (intraperitoneal 2.5% peritoneal dialysis fluid); FFP (shed blood + 1 vol IV FFP); FFP + DPR. EHBF was measured at postresuscitation timepoints. Organ injury was evaluated by serum ELISA (fatty acid-binding protein [FABP]-1 [liver], FABP-3 [heart], Troponin-I [heart], and Troponin-C [heart]) and hematoxylin and eosin. Differences were evaluated by 1-way ANOVA and 2-way repeated-measures ANOVA. RESULTS: CR resuscitation alone did not sustain EHBF. FFP resuscitation restored EHBF after resuscitation (2 hours, 3 hours, and 4 hours). DPR resuscitation restored EHBF throughout the postresuscitation period but failed to restore serum FABP-1 VS other groups. Combination FFP + DPR rapidly and sustainably restored EHBF and decreased organ injury. CR and DPR alone had elevated organ injury (FABP-1 [hepatocyte], FABP-3 [cardiac], and Troponin-I/C), whereas FFP or FFP + DPR demonstrated reduced injury at 4 hours after resuscitation. CONCLUSION: HS decreased EHBF, hepatocyte injury, and cardiac injury as evidenced by serology. FFP resuscitation improved EHBF and decreased organ damage. Although DPR resuscitation resulted in sustained EHBF, this alone failed to decrease hepatocyte or cardiac injury. Combination therapy with DPR and FFP may be a novel method to improve intestinal and hepatic blood flow and decrease organ injury after HS/resuscitation.

Glucose-induced intestinal vasodilation via adenosine A1 receptors requires nitric oxide but not K(+)(ATP) channels.

BACKGROUND: Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that adenosine triphosphate (ATP) utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K(+)(ATP)) to dilate intestinal microvessels. METHODS: Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles, proximal (p3A), and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K(+)(ATP) channel inhibitor (glibenclamide). RESULTS: As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K(+)(ATP) channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. CONCLUSION: These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K(+)(ATP) channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure.

Plasma resuscitation with adjunctive peritoneal resuscitation reduces ischemia-induced intestinal barrier breakdown following hemorrhagic shock.

INTRODUCTION: Hemorrhagic shock (HS) and resuscitation (RES) cause ischemia-induced intestinal permeability due to intestinal barrier breakdown, damage to the endothelium, and tight junction (TJ) complex disruption between enterocytes. The effect of hemostatic RES with blood products on this phenomenon is unknown. Previously, we showed that fresh frozen plasma (FFP) RES, with or without directed peritoneal resuscitation (DPR) improved blood flow and alleviated organ injury and enterocyte damage following HS/RES. We hypothesized that FFP might decrease TJ injury and attenuate ischemia-induced intestinal permeability following HS/RES. METHODS: Sprague-Dawley rats were randomly assigned to groups (n = 8): sham; crystalloid resuscitation (CR) (HS of 40% mean arterial pressure for 60 minutes) and CR (shed blood plus two volumes of CR); CR and DPR (intraperitoneal 2.5% peritoneal dialysis fluid); FFP (shed blood plus one volume of FFP); and FFP and DPR (intraperitoneal dialysis fluid plus two volumes of FFP). Fluorescein isothiocyanate-dextran (molecular weight, 4 kDa; FD4) was instilled into the gastrointestinal tract before hemorrhage; FD4 was measured by UV spectrometry at various time points. Plasma syndecan-1 and ileum tissue TJ proteins were measured using enzyme-linked immunosorbent assay. Immunofluorescence was used to visualize claudin-4 concentrations at 4 hours following HS/RES. RESULTS: Following HS, FFP attenuated FD4 leak across the intestine at all time points compared with CR and DPR alone. This response was significantly improved with the adjunctive DPR at 3 and 4 hours post-RES (p < 0.05). Resuscitation with FFP-DPR increased intestinal tissue concentrations of TJ proteins and decreased plasma syndecan-1. Immunofluorescence demonstrated decreased mobilization of claudin-4 in both FFP and FFP-DPR groups. CONCLUSION: Fresh frozen plasma-based RES improves intestinal TJ and endothelial integrity. The addition of DPR can further stabilize TJs and attenuate intestinal permeability. Combination therapy with DPR and FFP to mitigate intestinal barrier breakdown following shock could be a novel method of reducing ischemia-induced intestinal permeability and systemic inflammation after trauma. LEVEL OF EVIDENCE: Prognostic/Epidemiologic, Level III.

Direct Peritoneal Resuscitation Reduces Lung Injury and Caspase 8 Activity in Brain Death.

Background: Acute brain death (ABD) is associated with inflammation and lung injury. Direct peritoneal resuscitation (DPR) improves blood flow to the vital organs after ABD. DPR reduces lung injury, but the mechanism for this is unknown. Methods: Male Sprague-Dawley rats were randomized to five groups (n = 8/group): (1) Sham (no ABD); (2) Targeted intravenous fluid (TIVF) (ABD plus enough IVF to maintain a MAP of 80 mmHg) at 2 hours post-resuscitation (RES); (3) ABD + TIVF + DPR (TIVF and 30 cc intraperitoneal 2.5% Delflex) at 2 hours post-RES; (4) ABD + TIVF at 4 hours post-RES; and (5) ABD + TIVF + DPR at 4 hours post-RES. Messenger RNA (mRNA) levels were measured using Qiagen qRT PCR. Protein levels were assessed using quantitative ELISAs and the Luminex MagPix system. Results: Use of DPR caused 5.8-fold downregulation of mRNA expression for TNF-α and 2.7-fold decrease for the TNF receptor compared to TIVF alone. Caspase 8 mRNA was also downregulated. Protein levels for TNF-α, TNF receptor, caspase 8, NFκB, and NFκB inhibitor kinase, which promotes dissociation of NFκB inhibitor, were reduced by DPR. Cell death markers M30 and M65 were also decreased with DPR. Conclusions: Use of DPR caused changes in the expression of multiple mRNAs and proteins in the caspase 8 apoptotic pathway. These data represent a mechanism through which DPR exerts its beneficial effects within the lung tissue.

Plasma resuscitation with adjunctive peritoneal resuscitation reduces ischemic intestinal injury following hemorrhagic shock.

INTRODUCTION: Impaired intestinal microvascular perfusion following resuscitated hemorrhagic shock (HS) leads to ischemia-reperfusion injury, microvascular dysfunction, and intestinal epithelial injury, which contribute to the development of multiple organ dysfunction syndrome in some trauma patients. Restoration of central hemodynamics with traditional methods alone often fails to fully restore microvascular perfusion and does not protect against ischemia-reperfusion injury. We hypothesized that resuscitation (RES) with fresh frozen plasma (FFP) alone or combined with direct peritoneal resuscitation (DPR) with 2.5% Delflex solution might improve blood flow and decrease intestinal injury compared with conventional RES or RES with DPR alone. METHODS: Sprague-Dawley rats underwent HS (40% mean arterial pressure) for 60 minutes and were randomly assigned to a RES group (n = 8): sham, HS-crystalloid resuscitation (CR) (shed blood + two volumes CR), HS-CR-DPR (intraperitoneal 2.5% peritoneal dialysis fluid), HS-FFP (shed blood + two volumes FFP), and HS-DPR-FFP (intraperitoneal dialysis fluid + two volumes FFP). Laser Doppler flowmeter evaluation of the ileum, serum samples for fatty acid binding protein enzyme-linked immunosorbent assay, and hematoxylin and eosin (H&E) staining were used to assess intestinal injury and blood flow. p Values of <0.05 were considered significant. RESULTS: Following HS, the addition of DPR to either RES modality improved intestinal blood flow. Four hours after resuscitated HS, FABP-2 (intestinal) and FABP-6 (ileal) were elevated in the CR group but reduced in the FFP and DPR groups. The H&E staining demonstrated disrupted intestinal villi in the FFP and CR groups, most significantly in the CR group. Combination therapy with FFP and DPR demonstrated negligible cellular injury in H&E graded samples and a significant reduction in fatty acid binding protein levels. CONCLUSION: Hemorrhagic shock leads to ischemic-reperfusion injury of the intestine, and both FFP and DPR alone attenuated intestinal damage; combination FFP-DPR therapy alleviated most signs of organ injury. Resuscitation with FFP-DPR to restore intestinal blood flow following shock could be an essential method of reducing morbidity and mortality after trauma.

Vasoactive components of dialysis solution.

BACKGROUND: Conventional peritoneal dialysis (PD) solutions elicit vasodilation, which is implicated in the variable rate of solute transport during the dwell. The components causing such vasoactivity are still controversial. This study was conducted to define the vasoactive components of conventional and new PD solutions. METHODS: Three visceral peritoneal microvascular levels were visualized by intravital video microscopy of the terminal ileum of anesthetized rats. Anesthesia-free decerebrate conscious rats served as control. Microvascular diameter and blood flow by Doppler measurements were conducted after topical peritoneal exposure to 4 clinical PD solutions and 6 prepared solutions designed to isolate potential vasoactive components of the PD solution. RESULTS: All clinically available PD solutions produced a rapid and generalized vasodilation at all intestinal microvascular levels, regardless of the osmotic solute. The pattern and magnitude of this dilation was not affected by anesthesia but was determined by arteriolar size, the osmotic solute, and the solution's buffer anion system. The greatest dilation occurred in the small precapillary arterioles and was elicited by conventional PD solution and heat re-sterilized solution containing low glucose degradation products (GDPs). Hypertonic mannitol solutions produced a dilation that was approximately 50% less than the dilation obtained with glucose solutions with identical osmolarity and buffer. Increasing a solution's osmolarity did not produce a parallel increase in the magnitude of dilation, suggesting a nonlinear relationship between the two variables. Lactate dissolved in an isotonic solution was completely non-vasoactive unless the solution's H(+) concentration was increased. At low pH, isotonic lactate produced a rapid but transient vasodilation. This vascular reactivity was similar in magnitude and pattern to that obtained with the isotonic 7.5% icodextrin solution (Extraneal; Baxter Healthcare, Deerfield, Illinois, USA). CONCLUSIONS: (1) Hyperosmolarity is the major vasoactive component of PD solution. (2) Hyperosmolarity and active intracellular glucose uptake account together for approximately 75% of PD solution-induced dilation, whereas GDPs contribute to approximately 25%. (3) Lactate is vasoactive only at low pH (high [H(+)]). (4) The magnitude of PD solution-mediated vasodilation is partially dependent on the nature of the osmotic solute, the GDP contents, and the [H(+)], which determine the vasoactivity of the lactate-buffer anion system. Studies are required to define the molecular mechanisms of PD-induced vasodilation and to determine the vasoactive properties of these solutions after chronic infusion.

Obesity, inflammation, and the potential application of pharmaconutrition.

Obesity is an emerging problem worldwide. Hospitalized obese patients often have a worse outcome than patients of normal weight, particularly in the setting of trauma and critical care. Obesity creates a low-grade systemic inflammatory response syndrome (SIRS) that is similar (but on a much smaller scale) to gram-negative sepsis. This process involves up-regulation of systemic immunity, is characterized clinically by insulin resistance and the metabolic syndrome, and puts the patient at increased risk for organ failure, infectious morbidity, and mortality. Through lipotoxicity and cytokine dysregulation, obesity may act to prime the immune system, predisposing to an exaggerated subsequent immune response when a second clinical insult occurs (such as trauma, burns, or myocardial infarction). Specialized nutrition therapy for such patients currently consists of a hypocaloric, high-protein diet. However, this approach does not address the putative pathophysiologic mechanisms of inflammation and altered metabolism associated with obesity. A number of dietary agents such as arginine, fish oil, and carnitine may correct these problems at the molecular level. Pharmaconutrition formulas may provide exciting innovations for the nutrition therapy of the obese patient.

Chronic infusion of sterile peritoneal dialysis solution abrogates enhanced peritoneal gene expression responses to chronic peritoneal catheter presence.

Chronic exposure to sterile peritoneal dialysis (PD) solutions is associated with microvascular and interstitial changes within the blood-peritoneal barrier (peritoneum). These changes are commonly linked to loss of peritoneal function over time, presumably because of angiogenesis-related increased vascular area. However, the effects on peritoneal microvascular function of chronic peritoneal exposure to PD solutions are unknown. The present study examined peritoneal microvascular function after chronic exposure to sterile PD solution. Six rats underwent permanent catheter insertion under anesthesia. Three rats were treated with approximately 16 mL conventional PD solution daily for 6 weeks; catheter insertion controls received 1 mL saline daily. At 6 weeks, visceral peritoneal microvascular function was assessed in vivo using intravital microscopy. Endothelial cell functions were assessed using messenger RNA (mRNA) gene microarray analysis. In both groups, significant angiogenesis was seen, predominantly in the base of the mesentery. Sensitivity and reactivity of the intestinal visceral peritoneal pre-capillary arterioles (A3 arterioles, 8 - 15 microm in diameter) were decreased in the catheter controls, but not in the chronic PD infusion rats. Chronic catheter presence increased the expression of 18 genes in the controls as compared with 12 genes in the chronic infusion rats. In both groups, expression of fibronectin, integrin-beta, integrin-alpha5, collagen type XVIII-alpha1, and matrix metalloproteinase was enhanced. Endothelial expression of proinflammatory genes (interleukin-1beta, tissue pathway inhibitor, chemokine ligand 2) was enhanced by chronic catheter insertion, but not after chronic PD fluid infusion. Increased expression of genes encoding proteins involved in inflammation and tissue remodeling results from peritoneal catheter-related endothelial cell activation. Chronic exposure of the nonuremic peritoneum to sterile PD solutions overrides the catheter-related endothelial cell proinflammatory phenotype to restore peritoneal microvascular function.

Plasma appearance rate of intraperitoneal macromolecular tracer underestimates peritoneal lymph flow.

The magnitude of peritoneal lymph flow is an issue of great controversy in peritoneal dialysis (PD) research. Because no single lymphatic duct drains the entire peritoneal cavity, peritoneal lymph flow is indirectly measured as lymphatic removal of intraperitoneal macromolecular tracer. In rats, the peritoneal clearance (K) of such a tracer is 5 times the approximately 8 microL/min determined from the tracer appearance rate in blood (Cl). The fractional contribution of tissues bordering the peritoneal cavity to the overall Cl was determined to be diaphragm, 55%; viscera, 30%; and abdominal wall, 15%. The present study determines whether direct measurement of visceral peritoneal lymph flow matches the 30% (approximately 2.5 microL/min) contribution of the visceral peritoneal lymph flow as measured indirectly by the Cl method. The mesenteric lymph duct that exclusively drains lymph from the gut, liver, and mesentery was cannulated in 15 rats, and lymph flow from the duct was collected at hourly intervals up to 6 hours under near-normal physiologic conditions and under conditions of simulated PD. Changes in mesenteric lymph flow that resulted from a challenge with 3 mL intravenous saline were captured using real-time video. We observed no significant differences between the hourly lymph volumes collected over 6 hours in naïve animals (n = 5, p > 0.05). Under conditions of simulated PD with dialysis fluid in the peritoneal cavity, the mesenteric duct lymph flow averaged 8.67 +/- 1.41 microL/min (n = 10). That flow is similar to reported data on total peritoneal Cl in rats; and 4 times the 2.5 microL/min visceral peritoneal contribution to the total peritoneal Cl. The intravenous saline challenge significantly increased mesenteric lymph duct output to 30.9 +/- 1.6 microL/min (n = 5, p < 0.01) and reduced the lymph-to-plasma concentration ratio (L/P) by 43%. The reflection coefficient for total proteins (sigma(prot)) across the intestinal capillaries as calculated from the filtration rate-dependent L/P ratio when the transcapillary fluid escape rate and the mesenteric lymph flow were both high was more than 0.87. We concluded that (A) under near-normal physiologic conditions, the mesenteric lymph duct flow is steady, but quite low; (B) under conditions of simulated PD, the mesenteric lymph duct flow increases significantly from the physiologic norm; (C) mesenteric lymph duct flow is sensitive to the peritoneal fill volume; (D) during simulated PD, the fractional visceral peritoneal lymph flow measured indirectly from plasma appearance of intraperitoneal tracer underestimates the directly measured mesenteric duct lymph flow; and (E) the increased transcapillary fluid escape rate is rapidly buffered by augmentation of mesenteric lymph duct output.

Direct Peritoneal Resuscitation Alters Leukocyte Infiltration in the Lung After Acute Brain Death.

BACKGROUND: Brain death is associated with significant lung injury and inflammation. This has been associated with worse long-term outcomes for transplanted lungs. Direct peritoneal resuscitation (DPR) reduces systemic inflammation in brain death and improves lung procurement rate. The effect of DPR on macrophage and neutrophil infiltration in the lungs is not known. METHODS: Male Sprague-Dawley rats had a 4F Fogarty catheter inserted into the skull and the balloon inflated until brain death was achieved. Rats were resuscitated with normal saline to maintain a mean arterial pressure of 80 mmHg (targeted intravenous fluid, TIVF) and DPR animals received an intraperitoneal injection of commercial peritoneal dialysis solution. Rats were sacrificed at 0, 2, 4, and 6 h after brain death. Protein levels were assessed using quantitative ELISA. Leukocytes were quantified using flow cytometry and immunohistochemistry. RESULTS: At all time points, DPR downregulated multiple inflammatory cytokines including IFN-γ, TNF-α, IL-1α, and IL-6. Adhesion molecules ICAM, E-selectin, and P-selectin were increased above sham at 4 and 6 h after brain death and reduced with DPR, whereas VCAM was reduced at 2 and 6 h. Infiltration of macrophages and neutrophils were trended downward at 6 h with DPR, though this difference was not statistically significant. CONCLUSIONS: Animals that received TIVF alone had significant increases in inflammatory cytokines within the lung tissue, leading to adhesion molecule expression and ultimately leukocyte infiltration. Each stage of inflammation was affected by DPR. Using DPR in brain dead organ donors shows promise as a way to reduce lung injury and inflammation.

Direct peritoneal resuscitation reduces intestinal permeability after brain death.

BACKGROUND: The profound inflammatory response associated with brain death is frequently cited as the reason organs procured from brain dead donors are associated with worse graft function. The intestine releases inflammatory mediators in other types of shock, but its role is brain death has not been well-studied. Direct peritoneal resuscitation (DPR) improves visceral organ blood flow and reduces inflammation after hemorrhagic shock. We hypothesized that use of DPR would maintain intestinal integrity and reduce circulating inflammatory mediators after brain death. METHODS: Brain death was induced in male Sprague-Dawley rats by inserting a 4F Fogarty catheter into the epidural space and slowly inflating it. After herniation, rats were resuscitated with normal saline to maintain a mean arterial pressure of 80 mm Hg and killed with tissue collected immediately (time 0), or 2 hours, 4 hours, or 6 hours after brain death. Randomly selected animals received DPR via an intraperitoneal injection of 30-mL commercial peritoneal dialysis solution. RESULTS: Levels of proinflammatory cytokines, including IL-1ß and IL-6, as well as high-mobility group box 1 protein and heat shock protein 70, were all increased after brain death and decreased with DPR. Fatty acid binding protein and lipopolysaccharide, both markers of intestinal injury, were increased in the serum after brain death and decreased with DPR. Immunohistochemistry staining for zona occludin-1 showed decreased intestinal tight junction integrity after brain death, which improved with DPR. CONCLUSIONS: Intestinal permeability increases after brain death, and this contributes to the increased inflammation seen throughout the body. Using DPR prevents intestinal ischemia and helps preserve intestinal integrity. This suggests that using this novel therapy as an adjunct to the resuscitation of brain dead donors has the potential to reduce inflammation and potentially improve the quality of transplanted organs.

Cellular edema regulates tissue capillary perfusion after hemorrhage resuscitation.

BACKGROUND: Hemorrhage-induced activation of endothelial cell Na+/H+ -exchanger results in cellular swelling, which physically impedes capillary filling and compromises gut perfusion. We hypothesized that correction of the vascular volume deficit by conventional resuscitation does not improve capillary filling unless cellular swelling is prevented. Also, we hypothesized that adjunctive direct peritoneal resuscitation (DPR) with topical peritoneal dialysis solution (Delflex; Fresenius USA, Inc., Ogden, Ut) enhances capillary filling and gut perfusion by mechanisms that are independent of the Na+/H+ function. METHODS: In vivo intravital videomicroscopy and Doppler velocimeter were used by us to measure microvascular diameter and flow, capillary filling (index of functional capillary density, FCD), and endothelial cell function in the terminal ileum of anesthetized rats. Rats were bled to 50% mean arterial pressure for 60 min and resuscitated with the shed blood plus 2 volumes of saline (conventional resuscitation). Prevention of endothelial cell swelling was achieved with topical amiloride (specific Na+/H+ inhibitor) in the tissue bath before hemorrhage or simultaneously with conventional resuscitation. DPR was simulated by instillation of Delflex in the tissue bath as adjunctive to conventional resuscitation. Sham no hemorrhage group and a simulated DPR group that received topical amiloride treatment served as controls. RESULTS: Conventional resuscitation from hemorrhagic shock restored and maintained central hemodynamics but caused progressive and persistent intestinal vasoconstriction and hypoperfusion associated with low FCD and endothelial cell dysfunction. Prevention of endothelial cell swelling when combined with conventional resuscitation, preserved endothelial cell function, and restored local intestinal microvascular variables to near-prehemorrhage levels. Simulated adjunctive DPR produced rapid, sustained, and generalized vasodilation associated with restoration of endothelial cell function, and maximum recruitment of FCD independent of the Na+/H+ -exchanger function. CONCLUSIONS: Paradoxical endothelial cell swelling occurs early during hemorrhagic shock because of activation of the Na+/H+ exchanger. This cellular edema, which is not resolved by correction of the vascular volume deficit, explains the persistent postresuscitation endothelial cell dysfunction and gut hypoperfusion. Simulated adjunctive DPR in this study reversed endothelial cell swelling and enhanced gut perfusion by mechanisms that are independent of the Na+/H+ exchanger activity.

Randomized Controlled Trial Evaluating the Efficacy of Peritoneal Resuscitation in the Management of Trauma Patients Undergoing Damage Control Surgery.

BACKGROUND: Peritoneal resuscitation (PR) represents a unique modality of treatment for severely injured trauma patients requiring damage control surgery. These data represent the outcomes of a single institution randomized controlled trial into the efficacy of PR as a management option in these patients. STUDY DESIGN: From 2011 to 2015, one hundred and three patients were enrolled in a prospective randomized controlled trial evaluating the use of PR in the treatment of patients undergoing damage control surgery compared with conventional resuscitation (CR) alone. Patient demographics, clinical variables, and outcomes were collected. Univariate and multivariate analysis was performed with a priori significance at p ≤ 0.05. RESULTS: After initial screening, 52 patients were randomized to the PR group and 51 to the CR group. Age, sex, initial pH, and mechanism of injury were used for randomization. Method of abdominal closure was standardized across groups. Time to definitive abdominal closure was reduced in the PR group compared with the CR group (4.1 ± 2.2 days vs 5.9 ± 3.5 days; p ≤ 0.002). Volume of resuscitation and blood products transfused in the initial 24 hours was not different between the groups. Primary fascial closure rate was higher in the PR group (83% vs 66%; p ≤ 0.05). Intra-abdominal complications were lower in the PR compared with the CR group (8% vs 18%), with abscess formation rate (3% vs 14%; p < 0.05) being significant. Patients in the PR group had a lower 30-day mortality rate, despite similar Injury Severity Scores (13% vs 28%; p = 0.06). CONCLUSIONS: Peritoneal resuscitation enhances management of damage control surgery patients by reducing time to definitive abdominal closure, intra-abdominal infections, and mortality rates.

Immune-enhancing diet and cytokine expression during chronic sepsis: an immune-enhancing diet containing L-arginine, fish oil, and RNA fragments promotes intestinal cytokine expression during chronic sepsis in rats.

Chronic feeding with enteral immune-enhancing diets (IEDs) provides benefits based on composition of the diet, route of feeding, and timing of feeding in relation to timing of trauma or surgery. Our prior studies of acute feeding in naïve rats demonstrated that IED promotes blood flow and proinflammatory cytokines in the ileum. We hypothesized that chronic feeding with IED would shift gut immune status to an anti-inflammatory state during chronic sepsis, resulting in an altered state of cytokine expression in the gut. Five days prior to feeding, gauze was implanted subcutaneously in the backs of male Sprague-Dawley rats, which were fed for 3 days with either control diet (CD, Boost; Mead-Johnson, Evansville, IL) or IED (Impact; Novartis) and randomly assigned to one of four groups: saline control (NS) + control diet (CD), sepsis (EC) + CD, NS + IED, or EC + IED. EC rats were inoculated with 10(9) CFU Escherichia coli and 10(9) CFU Bacteroides fragilis in 2 ml normal saline into the back sponge while NS rats received 2 mL normal saline alone. After 3 days, animals were anesthetized and gut tissue samples were harvested and frozen at -80 degrees C. Tissue protein was extracted and ELISA was performed for interleukin (IL-1beta, IL-5, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In saline controls, IED feeding decreased IL-1beta, IL-5, IL-6, TNF-alpha, and IFN-gamma and increased IL-10 compared with CD-fed animals. In septic animals, IED feeding increased IL-5 and IL-6, while decreasing IFN-gamma and IL-10 in the distal third of the small intestine compared with CD-fed septic rats, whereas IL-1beta and TNF-alpha levels were unchanged. Chronic IED feeding produced a anti-inflammatory state via decreased IFN-gamma and increased IL-5 and IL-6, which both promote gut IgA class switching, suggesting that the gut is shifted toward humoral immunity during chronic IED feeding in septic rats.