Cellular Cytosolic Energy Replenishment Increases Vascularized Composite Tissue Tolerance to Extended Cold Ischemia Time.

BACKGROUND: Vascularized composite allotransplantation (VCA) is a restorative surgical procedure to treat whole or partially disfiguring craniofacial or limb injuries. The routine clinical use of this VCA surgery is limited using compromised allografts from deceased donors and by the failure of the current hypothermic preservation protocols to extend the allograft's cold ischemia time beyond 4 h. We hypothesized that the active replenishment of the cellular cytosolic adenosine-5`-triphosphate (ATP) stores by means of energy delivery vehicles (ATPv) encapsulating high-energy ATP is a better strategy to improve allograft's tolerance to extended cold ischemia times. MATERIALS AND METHODS: We utilized established rat model of isolated bilateral in-situ non-cycled perfusions of both hind limbs. Ipsilateral and contralateral limbs in the anesthetized animal were randomized for simultaneous perfusions with either the University of Wisconsin (UW) solution, with/without O2 supplementation (control), or with the UW solution supplemented with the ATPv, with/without O2 supplementation (experimental). Following perfusion, the hind limbs were surgically removed and stored at 4°C for 12, 16, or 24 hours as extended cold ischemia times. At the end of each respective storage time, samples of skin, and soleus, extensor digitalis longus, and tibialis anterior muscles were recovered for assessment using tissue histology and tissue lysate studies. RESULTS: Control muscle sections showed remarkable microvascular and muscle damage associated with loss of myocyte transverse striation and marked decrease in myocyte nucleus density. A total of 1,496 nuclei were counted in 179 sections of UW-perfused control muscles in contrast to 1,783 counted in 130 sections of paired experimental muscles perfused with the ATPv-enhanced perfusate. This yielded 8 and 13 nuclei/field for the control and experimental muscles, respectively (P < .004). Oxygenation of the perfusion solutions before use did not improve the nucleus density of either the control or experimental muscles (n = 7 animals, P > .05). Total protein isolated from the muscle lysates was similar in magnitude regardless of muscle type, perfusion protocol, or duration of cold ischemia time. Prolonged static cold preservation of the hind limbs completely degraded the composite tissue's Ribonucleic acid (RNA). This supplementary result confirms the notion that that reverse transcription-Polymerase Chain Reaction, enzyme-linked immunosorbent assay, or the respiratory complex II enzyme activity techniques should not be used as indices of graft quality after prolonged static cold storage. CONCLUSIONS: In conclusion, this study demonstrates that active cellular cytosolic ATP replenishment increases hind limb composite tissue tolerance to extended cold ischemia times. Quality indicators and clinically relevant biomarkers that define composite tissue viability and function during static cold storage are warranted.

Propranolol attenuates cognitive, learning, and memory deficits in a murine model of traumatic brain injury.

BACKGROUND: ß-blockers have been shown to improve survival after traumatic brain injury (TBI); however, the impact of continuous dosage of ß-blockers on cognitive function has not been elucidated. We hypothesized that a daily dose of propranolol can improve memory, learning, and cognitive function following TBI. STUDY DESIGN: Twenty male C57BL mice were subjected to a cortical-controlled moderate TBI. Two hours after TBI, animals were randomly allocated to either the ß-blocker group (n = 10) or the placebo group (n = 10). Mice in the ß-blocker group received intraperitoneal 4 mg/kg propranolol every 24 hours for 7 days while the placebo group received 4 mg/kg normal saline. Baseline novel object recognition and classic maze tests were done prior to TBI and then daily from Day 1 through 7 after TBI. Animals were sacrificed on Day 7. Serum biomarkers were measured using ELISA and brain sections were analyzed using western blot and hematoxylin and eosin staining. RESULTS: Both the ß-blocker and placebo groups had lower recognition index scores compared with the baseline following TBI. ß-blocker mice had significantly higher novel object recognition scores compared with placebo mice 2 days after TBI. The ß-blocker group required less time to complete the maze-test compared to placebo group after Day 4. There was no difference regarding the serum levels of IL-1ß, IL-6, and TNF-α. The ß-blocker group had lower levels of UCHL-1 and higher levels of Hsp-70 in brain lysate. Hematoxylin and eosin staining revealed that more neurons in the hippocampal-CA1 area underwent apoptosis in the placebo group compared with the ß-blocker group. CONCLUSION: Postinjury propranolol administration results in improved memory, learning and cognitive functions in a murine model of moderate TBI. Propranolol increases the expression of antiapoptotic protein (Hsp-70) and decreases cell death in the hippocampal-CA1 area compared with the placebo.

The acute inflammatory response after trauma is heightened by frailty: A prospective evaluation of inflammatory and endocrine system alterations in frailty.

BACKGROUND: Frailty is a geriatric syndrome characterized by decreased physiological reserves, increased inflammation, and decreased anabolic-endocrine response. The biomarkers associated with frailty are poorly understood in trauma. The aim of this study was to analyze the association between frailty and immune: IL-1ß, IL-6, IL-2Rα, tumor necrosis factor (TNF)-α, and endocrine biomarkers: insulin-like growth factor-1 and growth hormone in trauma patients. METHODS: We conducted a 1-year (2017-2018) prospective analysis of geriatric (≥65 years) trauma patients admitted to our Level I trauma center. Frailty was measured using the trauma-specific frailty index (TSFI) and blood samples were collected within 24 hours of admission. Patients were stratified into two groups: frail (TSFI > 0.25) and nonfrail (TSFI ≤ 0.25). We then measured the levels of immune and endocrine biomarkers by a colorimetric output that was read by a spectrophotometer (Quantikine ELISA). The outcome measures were the levels of the immune and endocrine markers in the two groups. Multivariable linear regression was performed. RESULTS: A total of 100 geriatric trauma patients were consented and enrolled. The mean age was 77.1 ± 9.8 years and 34% were female. Thirty-nine (39%) patients were frail. Frail patients were more likely to present after falls (p = 0.01). There was no difference in age (p = 0.78), sex (p = 0.77), systolic blood pressure (p = 0.16), and heart rate (p = 0.24) between the two groups. Frail patients had higher levels of TNF-α (p = 0.01), IL-1ß (p = 0.01), and IL-6(p = 0.01) but lower levels of growth hormone (p = 0.03) and insulin-like growth factor-1 (p < 0.04) compared with nonfrail patients. There was no difference in the level of IL-2Rα (p = 0.25). On regression analysis, frailty was positively correlated with the levels of proinflammatory biomarkers, that is, TNF- α, IL-1 ß, and IL-6 and negatively correlated with endocrine biomarkers. CONCLUSION: This study supports the association between frailty and immune and endocrine markers. Frailty acts synergistically with trauma in increasing the acute inflammatory response. Moreover, frail patients have lower levels of anabolic hormones. Understanding the inflammatory and endocrine response in frail trauma patients may result in better therapeutic strategies.

Glycine improves peritoneal vasoreactivity to dialysis solutions in the elderly.

Background: Peritoneal dialysis solution (PDS) dilates peritoneal microvessels predominantly by the activation of the endothelial nitric oxide (NO) pathway. We made an incidental observation of decreased PDS-induced, NO-dependent peritoneal microvascular vasoreactivity in elderly rats naïve to PDS exposure. We hypothesized that this subordinate NO-mediated peritoneal microvascular vasoreactivity is caused by increased oxidative stress in the aged endothelium, which compromises NO bioavailability in the elderly, and that peritoneal microvascular vasoreactivity can be improved by the supplementation of antioxidant glycine to PDS. Methods: We studied PDS-mediated vasoreactivity of four intestinal visceral arterioles of different orders by in vivo intravital microscopy in weaned, adult, and elderly rats to (i) confirm subordinate vasoreactivity to PDS in elderly rats; (ii) restore vasoreactivity by glycine supplementation; and (iii) establish age as an independent risk factor for endothelial cell dysfunction. Results: In a crossover series, peritoneal microvascular vasoreactivity to PDS exposure was remarkably decreased in elderly rats. This subordinate vasoreactivity was completely restored by the supplementation of glycine to PDS. In a separate series, we assessed in situ endothelial cell function in weaned and adult rats using the cumulative acetylcholine concentration-response curves. Unlike the adults, the weaned rats demonstrated remarkable sensitivity and reactivity to cumulative acetylcholine concentrations, suggesting the dependency of endothelial cell function on age. Conclusion: Aging is an independent risk factor for peritoneal microvascular endothelial cell dysfunction. Endothelial function in the elderly can be recovered by reinforcing the bioavailability of endothelial-derived NO through glycine. Dietary glycine supplementation is a potential therapeutic strategy to decrease the burden of oxidative stress on the aged endothelium.

3-Factor Versus 4-Factor PCC in Coagulopathy of Trauma: Four is Better Than Three.

INTRODUCTION: Coagulopathy of trauma (COT) is common and highly lethal. Prothrombin complex concentrate (PCC) has been advocated for correction of COT. However, the difference in efficacy between three-factor PCC (3-PCC) versus four-factor PCC (4-PCC) remains unclear. The aim of our study was to compare efficacy of 3-PCC versus 4-PCC in COT. METHODS: Five-year (2013-2017) review of coagulopathic trauma patients at our Level-I trauma center who received 3- or 4-PCC. Patients were divided into two groups (4-PCC and 3-PCC) and matched in 1:1 ratio using propensity-score-matching for demographics, injury parameters, admission vitals, and hematological parameters. Primary outcomes were time to correction of international normalized ratio (INR), blood products transfusion, thromboembolic complications, and mortality. Secondary outcomes were hospital-length of stay (LOS), intensive care unit (ICU)-LOS, cost of therapy, and total hospital cost. RESULTS: Six hundred fifty-seven patients met inclusion criteria of whom 250 patients (4-PCC:125; 3-PCC:125) were matched. The mean age was 50 ±â€Š19.4 y, 64% were male, and median-injury severity score was 24[15-33]. 4-PCC was associated with accelerated correction of INR (365 vs. 428 min, P = 0.01), decrease in red blood cells (7 units vs. 10 units, P = 0.04) and FFP (6 units vs. 8 units, P = 0.03) transfused. There was no difference in platelet transfusion, thromboembolic complications, mortality, hospital, and ICU-LOS. 4-PCC was associated with higher cost of PCC therapy, and lower cost of transfusion. There was no difference regarding the total hospital cost between the two groups. CONCLUSION: Compared with 3-factor PCC, the use of 4-factor PCC is associated with a rapid reversal of INR and reduction in transfusion requirement without increasing the overall hospital cost or the risk of thromboembolic events. 4-PCC may be preferred as an adjunct for the resuscitation of coagulopathic trauma patients.

Outcomes After Massive Transfusion in Trauma Patients: Variability Among Trauma Centers.

BACKGROUND: Exsanguinating trauma patients often require massive blood transfusion (defined as transfusion of 10 or more pRBC units within first 24 h). The aim of our study is to assess the outcomes of trauma patients receiving massive transfusion at different levels of trauma centers. METHODS: Two-y (2013-2014) retrospective analysis of the American College of Surgeons Trauma Quality Improvement Program. We included all adult trauma patients who received massive transfusion (MT) of blood. Outcome measures were mortality, hospital length of stay, intensive care unit-free and ventilator-free days, blood products received, and complications. RESULTS: We analyzed a total of 416,957 patients, of which 2776 met the inclusion criteria and included in the study. Mean age was 40.6 ± 20 y, 78.3% were males and 33.1% of the injuries were penetrating. Median injury severity score [IQR] was 29 [18-40], median [IQR] Glasgow Coma Scale 10[4-15]. Mean packed red blood cells transfusion in the first 24 h was 20 ± 13 units and mean plasma transfusion was 13 ± 11 units. Overall in-hospital mortality was 43.5%. Receiving MT in level I trauma center was independently associated with lower rates of mortality (odds ratio [OR]: 0.75 [0.46-0.96], P < 0.001). Higher injury severity score (OR: 1.020 [1.010-1.030], P < 0.001) and increased units of packed red blood cells transfused (OR: 1.067 [1.041-1.093], P < 0.001) were independently associated with increased mortality. However, there was no association between teaching status, age, gender, emergency department vitals, and units of plasma transfused. CONCLUSIONS: Hemorrhage continues to remain one of the most common cause of death after trauma. Almost half of the patients who received massive transfusion died. Patients who receive massive blood transfusion in a level I trauma centers have improved survival compared with level II trauma centers.

Direct Oral Anticoagulants vs Low-Molecular-Weight Heparin for Thromboprophylaxis in Nonoperative Pelvic Fractures.

BACKGROUND: Patients with pelvic fractures are prone to venous thromboembolic (VTE) complications. Recent literature shows superiority of direct oral anticoagulants (DOACs) over low-molecular-weight heparin (LMWH) for thromboprophylaxis in patients undergoing orthopaedic operations. The aim of our study was to compare in-hospital outcomes for DOACs vs LMWH in patients with nonoperative pelvic fractures. STUDY DESIGN: We performed a 2-year (2015 to 2016) analysis of the American College of Surgeons-Trauma Quality Improvement Program (ACS-TQIP) database. We included all adult patients with isolated blunt pelvic fractures who were managed nonoperatively and received thromboprophylaxis with either LMWH or DOACs (Factor-Xa inhibitor or direct thrombin inhibitor). Patients were divided into 2 groups based on receipt of DOACs vs LMWH and were propensity-score-matched in a 1:2 ratio to control for possible confounding factors. Primary outcomes were deep venous thrombosis (DVT) and/or pulmonary embolism (PE). Secondary outcomes were pRBC transfusions, intervention for hemorrhage control, and in-hospital mortality after initiation of thromboprophylaxis. RESULTS: We identified 20,692 patients with pelvic fractures. There were 7,312 patients with isolated pelvic fractures included, 852 of whom were matched (DOACs: 284; LMWH: 568). Mean age was 43.2 ± 15 years, median Injury Severity Score was 14 (range 10 to 18). Matched groups were similar in demographics, vital signs, injury parameters, and timing of initiation of thromboprophylaxis. Overall, 5.2% of patients had DVT, 1.4% PE, and 1.3% died. Patients who received DOACs were less likely to develop DVT (1.8% vs 6.9%, p < 0.01) compared with LMWH. There was no difference in PE (p = 0.85) or in-hospital mortality (p = 0.79) between the 2 groups. Similarly, there was no difference in post-prophylaxis blood transfusion, and post-prophylaxis intervention for hemorrhage control. CONCLUSIONS: In patients with nonoperative pelvic fractures, DOACs were associated with a reduced rate of DVT vs LMWH without increasing the risk of bleeding complications. No association was found between the type of thromboprophylactic agent and rates of PE or in-hospital mortality.

The burden of excess length of stay in trauma patients.

BACKGROUND: Disposition of trauma patients frequently results in excessive hospital-stay. The aim of this study was to assess the risk of developing complications due to excessive stay in the hospital. METHODS: Over a period of 4 years (2012-2015) we analyzed all trauma patients with hospital length-of-stay (h-LOS) >30 days. Outcome measures were complications after termination of medical care. RESULTS: 416 patients were identified having h-LOS>30 days of which 61.0% (254) had an excess hospital stay and were included. The most common causes of excess hospital stay were placement in SNiF followed by placement in Inpatient-Rehabilitation. Excessive hospital-stay was independently associated with the development of complications (p = 0.004). Each excess day in the hospital after completion of medical care was associated with 5% higher odds of complications (OR [95%CI]: 1.05[1.02-1.09]) independent of presenting condition of the patient. CONCLUSION: Each extra day spent in the hospital after completion of medical care was associated with higher odds of developing complications.

The novel oral anticoagulants (NOACs) have worse outcomes compared with warfarin in patients with intracranial hemorrhage after TBI.

INTRODUCTION: Novel oral anticoagulant (NOAC) use is increasing in trauma patients. The reversal of these agents after hemorrhage is still evolving. The aim of our study was to evaluate outcomes after traumatic brain injury in patients on NOACs. METHODS: 3-year (2014-2016) analysis of our prospectively maintained traumatic brain injury (TBI) database. We included all TBI patients with intracranial hemorrhage (ICH) on anticoagulants. Patients were stratified into two groups, those on NOACs and on warfarin, and were matched in a 1:2 ratio using propensity score matching for demographics, injury and vital parameters, type, and size of ICH. Outcome measures were progression of ICH, mortality, skilled nursing facility (SNF) disposition, and hospital and intensive care unit (ICU) length of stay (LOS). RESULTS: We analyzed 1,459 TBI patients, of which 210 patients were matched (NAOCs, 70; warfarin, 140). Matched groups were similar in age (p = 0.21), mechanism of injury (p = 0.61), Glasgow Coma Scale (GCS) score (p = 0.54), Injury Severity Score (p = 0.62), and type and size of ICH (p = 0.09). Patients on preinjury NOACs had higher rate of progression (p = 0.03), neurosurgical intervention (p = 0.04), mortality (p = 0.04), and longer ICU LOS (p = 0.04) compared with patients on warfarin. However, there was no difference in hospital LOS (p = 0.22) and SNF disposition (p = 0.14). On sub-analysis of severe TBI patients (GCS ≤ 8), rate of progression (p = 0.59), neurosurgical intervention (p = 0.62), or mortality (p = 0.81) was similar in both groups. CONCLUSIONS: The use of NOACs generally carries a high risk of bleeding and can be detrimental in head injuries with ICH. NOAC use is associated with increased risk of progression of ICH, neurosurgical intervention, and mortality after a mild and moderate TBI. Primary care physicians and cardiologists need to reconsider the data on the need for anticoagulation and the type of agent used and weigh it against the risk of bleeding. In addition, development of reversal agents for the NOACs and implementation of a strict protocol for the reversal of these agents may lead to improved outcomes. LEVEL OF EVIDENCE: Therapeutic studies, level III.

The role of four-factor prothrombin complex concentrate in coagulopathy of trauma: A propensity matched analysis.

BACKGROUND: Coagulopathy is a common complication after severe trauma. The efficacy of 4-factor prothrombin complex concentrate (4-PCC) as an adjunct to fresh frozen plasma (FFP) in reversal of coagulopathy of trauma (COT) has not been studied. The aim of our study is to compare 4-PCC + FFP versus FFP alone for the treatment of COT. METHODS: We reviewed all trauma patients older than 18 years who received PCC + FFP or FFP alone at our Level I trauma center from 2015 to 2016. We excluded patients on preinjury oral anticoagulants. Patients were divided into two groups (4-PCC + FFP: FFP alone) and were matched in a 1:2 ratio using propensity score matching for demographics, vital and injury parameters, and initial international normalized ratio (INR). COT was defined as admission INR > 1.5. Corrected INR was defined as an INR of 1.5 or less. Outcome measures were time to correction of INR, packed red blood cells units transfused, thromboembolic complications, and mortality. RESULTS: We analyzed 516 trauma patients, of which 120 patients (4-PCC + FFP: 40, FFP: 80) were matched. Mean age was 58 ± 20 years; 60% were male, median Injury Severity Score was 29 (14-38). Mechanism of injury was blunt in 87% patients. 4-PCC + FFP was associated with an accelerated correction of INR (373 minutes vs. 955 minutes; p = 0.001), a decrease in packed red blood cells units (7 units vs. 9 units; p = 0.04), and FFP units (5 units vs. 7 units; p = 0.03) transfused compared to FFP alone. 4-PCC + FFP was associated with a lower mortality (25% vs. 33% p = 0.04) compared with FFP alone; however, there was no difference in the thromboembolic complications (2.5% vs. 1.2%, p = 0.5) between the two groups. Administration of PCC + FFP led to an earlier correction of the INR compared with FFP alone. CONCLUSION: Results of our study demonstrated that the use of 4-PCC in conjunction with FFP is associated with the rapid reversal of INR and reduction in transfusion requirements as compared with FFP alone. Four-factor PCC as a component therapy along with FFP is superior to FFP alone for the reversal of COT. LEVEL OF EVIDENCE: Therapeutic studies, level IV.

Continuous remote ischemic conditioning attenuates cognitive and motor deficits from moderate traumatic brain injury.

BACKGROUND: While studies show that single-dose remote ischemic conditioning (RIC) improves outcomes, the effect of continuous (daily) RIC is unknown. Thus, we aimed to investigate the role of continuous RIC on cognitive and motor function following traumatic brain injury (TBI). METHODS: We subjected 24 male C57BL mice to a cortical-controlled TBI. Two hours after TBI, the animals were randomly allocated to the RIC group (n = 12) or the sham group (n = 12). Remote ischemic conditioning was induced by noninvasive external compression of the hind limb using an occlusive band (six 4-minute cycles/24 hours) for six consecutive days. Before TBI, a baseline rotarod test and novel object recognition were performed. Post-TBI rotarod and novel object recognition tests were performed on Days 1 to 5, 7, 14, and 21. After the animals were sacrificed on Day 21, brain sections were analyzed using hematoxylin and eosin and glial fibrillary acidic protein staining to evaluate the hippocampal CA1 area for neuronal injury. RESULTS: Both the RIC and sham groups had lower latency to fall compared with the baseline post-TBI. The RIC animals had a higher latency to fall compared with the sham animals at all time points, statistically significant after Day 3, until Day 21 post-TBI. Both the RIC and sham groups had lower recognition index compared with the baseline post-TBI. The RIC animals had a significantly higher recognition index than the sham animals after Day 1, until Day 21 post-TBI. Hematoxylin and eosin and glial fibrillary acidic protein staining of the brain samples of the sham group revealed that more neurons in the hippocampal CA1 area appeared shrunken with eosinophilic cytoplasm and pyknotic nuclei compared with the brain samples of the RIC group. CONCLUSION: Postinjury continuous RIC resulted in improved cognitive functions and motor coordination in a mouse model of moderate TBI. Further studies are required to determine optimum dosage and frequency of this novel therapy to maximize its beneficial effects following TBI.

Perioperative glycemic control and postoperative complications in patients undergoing emergency general surgery: What is the role of Plasma Hemoglobin A1c?

BACKGROUND: Plasma hemoglobin A1c (HbA1c) reflects quality of glucose control in diabetic patients. Literature reports that patients undergoing surgery with an elevated HbA1c level are associated with increased postoperative morbidity and mortality. The aim of our study was to evaluate the impact of HbA1c level on outcomes after emergency general surgery (EGS). METHODS: We performed a 3-year analysis of our prospectively maintained EGS database. Patients who had HbA1c levels measured within 3 months before surgery were included. Patients were divided into two groups (HbA1c < 6 and HbA1c ≥ 6). Our primary outcome measures included in-hospital complications (major and minor complications), hospital and intensive care unit length of stay, and mortality. Secondary outcomes measures were 30-day complications, readmissions, and mortality. Multivariate and linear regressions were performed. RESULTS: Of the 402 study patients, mean age was 61 ± 12 years, 53% were females, and 63.8% were diabetics. Overall, 49% had an HbA1c ≥ 6%; the mortality rate was 6%. Those with hypertension, history of coronary artery disease, and body mass index of 30 kg/m or greater were more likely to have HbA1c of 6.0% or greater. 7.9% patients experienced major complications. Patients with HbA1c of 6% or greater had a higher complication rate (36% vs 11%, p < 0.001) than those with HbA1c less than 6%. However there was no difference in mortality between two groups (p = 0.09). After controlling for confounders, HbA1c ≥ 6.0% (odds ratio [OR], 2.9; p < 0.01) and a postoperative random blood sugar (RBS) of 200 mg/dL or greater (OR, 2.3; p < 0.01) were independent predictors of major complications. Patients with both HbA1c of 6.0% or greater and postoperative RBS of 200 or greater had higher odds (OR, 4.2; p < 0.01) of developing major complication. After adjusting for confounders, a higher HbA1c was independently correlated with a higher postoperative RBS (b = 0.494, [19.7-28.4], p = 0.02), but there was no correlation with the preoperative RBS. CONCLUSION: Patients with HbA1c of 6.0% or greater and a postoperative RBS of 200 mg/dL or greater have a four times higher risk of developing major complications after EGS. A preoperative HbA1c can stratify patients prone to develop postoperative hyperglycemia, regardless of their preoperative RBS. LEVEL OF EVIDENCE: Prognostic, level III.

Hyperosmolality-mediated peritoneal microvascular vasodilation is linked to aquaporin function.

Glucose-based peritoneal dialysis (PD) solutions dilate the parietal and visceral peritoneal microvasculature by endothelium-dependent mechanisms that primarily involve hyperosmolality. This PD-mediated dilation occurs by active intracellular glucose uptake and adenosine Al receptor activation, and by hyperosmolality-stimulated glibenclamide-sensitive potassium channels. Both pathways invoke NO as a second messenger for vasodilation. We hypothesized that during crystalloid-induced osmosis, the osmotic water flux through the transendothelial water-exclusive aquaporin 1 (AQP1) channels is the primary mechanism whereby the endothelium is being stimulated to instigate hyperosmolality-driven vasodilation. Four microvascular levels (diameters in the range 6 - 100 microm) were visualized by intravital videomicroscopy of the terminal ileum in anesthetized rats. Microvascular diameters and flow were measured after topical exposure to a 5% hypertonic mannitol or 2.5% glucose-based PD solution, at baseline and after brief tissue pre-treatment (with 0.1% glutaraldehyde for 10 seconds) or after combined tissue pre-treatment and pharmacologic blockade of AQP1 with HgCl2 (100 micromol/L). Vascular endothelial integrity was verified by the response to acetylcholine (10(-4) mol/L) and sodium nitroprusside (10(-4) mol/L). The hyperosmolar solutions both caused rapid and sustained vasodilation at all microvascular levels, which was not altered by tissue pre-treatment. Inhibition of AQP1 completely abolished the mannitol-induced vasodilation and markedly attenuated the PD fluid-mediated vasodilation. Neither glutaraldehyde pre-treatment nor HgCl2 affected tissue integrity or endothelial cell function. We conclude that the peritoneal microvascular vasodilation caused by hyperosmolar PD fluid is instigated by the osmotic water flux through AQP1. Clinical PD solutions have components other than hyperosmolality that can induce endothelium-dependent peritoneal microvascular vasodilation independent of the AQP1-mediated osmosis.

Molecular mechanisms of peritoneal dialysis-induced microvascular vasodilation.

Peritoneal dialysis (PD) solutions dilate microvessels by undefined mechanisms. This vasodilation directly affects ultrafiltration and solute exchange during a PD dwell and is thought to account for the variable mass transfer area coefficient for small solutes during a glucose-based hypertonic dwell. We hypothesized that PD-mediated vasodilation occurs by endothelium-dependent mechanisms that involve endothelium energy-dependent K+ channels (K(ATP)), adenosine A1 receptor activation, and NO release. We used intravital videomicroscopy to study 3 levels of microvessels (A1 inflow arterioles about 100 microm diameter to pre-capillary A3 arterioles 10 - 15 microm diameter) in the terminal ileum of anesthetized rats under control conditions in vivo in a tissue bath. Ileum was bathed with hypertonic mannitol or 2.5% glucose-based PD solution (Delflex: Fresenius Medical Care North America, Waltham, MA, U.S.A.) with or without topical application of individual or combined specific inhibitors of the endothelium-dependent dilation pathways.: NO (L-NMMA), prostaglandin I2 (mefenamic acid), endothelium hyperpolarizing factor (glibenclamide), and adenosine A1 receptor antagonist (DPCPX). The mannitol and PD solutions induced rapid and sustained peritoneal vasodilation whose magnitude depended on microvascular level and osmotic solute. Combined inhibition of endothelium-dependent dilation pathways completely abolished the mannitol-induced hyperosmolality-mediated dilation at all microvascular levels, but selectively eliminated the PD solution-mediated A3 dilation. The K(ATP) and adenosine receptor antagonists, individually or combined, remarkably attenuated dilation in the smaller pre-capillary arterioles; NO inhibition, alone or combined with K(ATP) and adenosine receptor antagonists, eliminated the PD solution-induced dilation. The cyclooxygenase pathway is not involved in PD-induced dilation. Solutions for PD dilate the visceral peritoneal microvasculature by endothelium-dependent mechanisms, primarily the NO pathway. Adenosine receptor-activated NO release and K(ATP) channel-mediated endothelium hyperpolarization significantly contribute to vasodilation in the smaller peritoneal pre-capillary arterioles.

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.

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.

Hemorrhage-induced hepatic injury and hypoperfusion can be prevented by direct peritoneal resuscitation.

BACKGROUND: Crystalloid fluid resuscitation after hemorrhagic shock (HS) that restores/maintains central hemodynamics often culminates in multi-system organ failure and death due to persistent/progressive splanchnic hypoperfusion and end-organ damage. Adjunctive direct peritoneal resuscitation (DPR) using peritoneal dialysis solution reverses HS-induced splanchnic hypoperfusion and improves survival. We examined HS-mediated hepatic perfusion (galactose clearance), tissue injury (histopathology), and dysfunction (liver enzymes). METHODS: Anesthetized rats were randomly assigned (n = 8/group): (1) sham (no HS); (2) HS (40% mean arterial pressure for 60 min) plus conventional i.v. fluid resuscitation (CR; shed blood + 2 volumes saline); (3) HS + CR + 30 mL intraperitoneal (IP) DPR; or (4) HS + CR + 30 mL IP saline. Hemodynamics and hepatic blood flow were measured for 2 h after CR completion. In duplicate animals, liver and splanchnic tissues were harvested for histopathology (blinded, graded), hepatocellular function (liver enzymes), and tissue edema (wet-dry ratio). RESULTS: Group 2 decreased liver blood flow, caused liver injuries (focal to submassive necrosis, zones 2 and 3) and tissue edema, and elevated liver enzymes (alanine aminotransferase (ALT), 149 +/- 28 microg/mL and aspartate aminotransferase (AST), 234 +/- 24 microg/mL; p < 0.05) compared to group 1 (73 +/- 9 and 119 +/- 10 microg/mL, respectively). Minimal/no injuries were observed in group 3; enzymes were normalized (ALT 89 +/- 9 microg/mL and AST 150 +/- 17 microg/mL), and tissue edema was similar to sham. CONCLUSIONS: CR from HS restored and maintained central hemodynamics but did not restore or maintain liver perfusion and was associated with significant hepatocellular injury and dysfunction. DPR added to conventional resuscitation (blood and crystalloid) restored and maintained liver perfusion, prevented hepatocellular injury and edema, and preserved liver function.

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.

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.