Normalization of brain death-induced injury to rat renal allografts by recombinant soluble P-selectin glycoprotein ligand

Donor brain death has been considered a significant risk factor for both early and late organ allograft dysfunction. This central injury not only evokes an upsurge of catecholamines with resultant peripheral tissue vasoconstriction and ischemia but also promotes release of hormones and inflammatory mediators that may also affect the organs directly. One of the resultant influences of these events is the rapid upregulation of the acute-phase adhesion molecules, the selectins. These initiate leukocyte adhesion to vascular endothelium and trigger subsequent cellular and molecular changes in the compromised tissues. An established F344 --> LEW rat model of chronic rejection was used to examine (1) whether the initial inflammatory events that develop within kidney allografts from brain-dead donors could be normalized using a recombinant soluble form of P-selectin glycoprotein ligand and (2) whether amelioration of these early changes would alter the inexorable progression of chronic allograft rejection. Untreated living donor controls experienced unrelenting chronic rejection over time. This complex process was accelerated in brain-dead donor kidneys. Treatment with P-selectin glycoprotein ligand prevented the early inflammatory changes in the transplanted organs and their subsequent (200 d) functional and morphologic manifestations, particularly when the soluble ligand was administered both to the donor before organ removal and to the recipient after engraftment. This strategy of using a naturally occurring selectin ligand to prevent donor-associated chronic graft dysfunction may be of special clinical interest in cadaver donor transplantation.     

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin

BACKGROUND: Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. METHODS: Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. RESULTS: Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. CONCLUSIONS: Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.     

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.     

Reduction of severe ischemia/reperfusion injury in rat kidney grafts by a soluble P-selectin glycoprotein ligand

BACKGROUND: Inflammatory leukocyte-endothelium interactions, mediated by selectins, contribute to renal ischemia/reperfusion (I/R) injury. We examined the influence of the soluble P-selectin glycoprotein ligand 1 (sPSGL) on early I/R-induced changes in a rat kidney transplantation model with long cold ischemia. METHODS: After 24 hr of cold storage, syngeneic kidneys were grafted into bilaterally nephrectomized rats. Before transplantation, recipients received either 1 mg/kg of sPSGL or vehicle (n=8 per group). Six hours after reperfusion, grafts were removed for light microscopy and immunohistochemistry. Capillary blood flow was measured under a fluorescence microscope by using the concentric-circles method. RESULTS: A greater proportion, 74.7+/-7.2% (sPSGL) vs. 28+/-7.4% (controls), of all dye-labeled outer medullary capillaries appeared in the 12-microm radius (P<0.01), indicating dense blood flow, whereas 7.6+/-2.9% vs. 43.3+/-9.7%, respectively, appeared in the 60-microm radius (P<0.05), indicating rarefied blood flow. In the sPSGL-treated group, the extent of severe tubular damage within the inner stripe of the outer medulla was lower compared with controls (37.5+/-8.3% vs. 78.4+/-3.5%, P<0.01). Outer medullary heat shock protein 72 expression was 14.5+/-1.6% in the sPSGL-treated group compared with 9.6+/-1.4% in controls (P<0.05). The number of infiltrating polymorphonuclear leukocytes was similar in both groups. Treatment with sPSGL had no influence on the serum creatinine level. CONCLUSIONS: Our data suggest that impairment of outer medullary blood flow is crucial in I/R injury of kidney grafts with prolonged cold storage. Reduction of capillary blood flow perturbations by sPSGL protects tubular cells from severe structural damage. Blocking early selectin-mediated leukocyte adhesion may have therapeutic implications in improving the prognosis of renal transplants with severe I/R injury.     

Recombinant P selectin glycoprotein ligand ameliorates chronic ischemia and reperfusion injury after rat intestinal transplantation

Previous studies have demonstrated that the blockade of P selectin through the administration of recombinant P Selectin Glycoprotein Ligand (rPSGL-1Ig) in a model of rat intestinal ischemia and reperfusion injury (IRI) afforded short-term improvement in outcome. The aim of this study was to investigate the long-term consequences of such therapy. IRI was induced in a model of isogeneic intestinal transplantation (ITx) using Lewis rats wherein intestines were procured, cold preserved in lactated Ringer solution for 6 h, and transplanted orthotopically. rPSGL-1Ig (0.4 mg/kg/dose) or saline was administered intravascularly into the intestine prior to storage and into the recipient prior to reperfusion. Separate survival and analysis groups were undertaken. For analysis, animals were sacrificed at day 3, 7, 30, and 100 after ITx. Transplanted ileal tissue was procured and stored for analysis that included histopathology, quantitative cytokine rtPCR, and hemoxygenase-1 (HO-1) and anti-apoptotic (Bcl-2, Bcl-xl) protein expression. Statistical comparison was performed using a Student’s t test. Survival at 100 days was markedly (P < 0.05) improved in the rPSGL-1Ig treated group (90%) versus saline-treated control group (40%). Histopathology was markedly improved at 100 days. At all time points, rtPCR demonstrated significantly (P < 0.05) lower mRNA production in treated animals of the cytokines: TNF-α, IL-6, TGF-β, and β-FGF. Western blot analysis demonstrated increased production of the protective proteins HO-1, Bcl-2, and Bcl-xl with rPSGL-1Ig treatment. Early blockade of P selectin at the time of ITx-associated IRI using rPSGL-1Ig led to significant improvement in survival, reductions in tissue injury on histopathology, reduction in mRNA production for chronic inflammatory cytokines, and increased protection of cellular protective proteins. This data indicate that the control of the early events associated with IRI leads to improvement in long-term isograft inflammatory parameters in a model of rat ITx.     

Cerebral water and electrolytes in experimental ischaemia following omental transposition to the brain

Summary Occlusion of the middle cerebral artery induces a local decrease in percentage of tissue dry weight in rabbit brain, associated with flux of sodium and potassium in reciprocal directions. Cortical swelling occurs also in remote non-ischaemic areas. Previous transposition of the omentum majus to the brain minimizes the onset of oedema consequent on occlusion of a major cerebral artery. Increasing experimental evidence points to the role of omental transposition in providing an effective source of collateral circulation, thus strongly affecting the threshold for infarction.     

P-selectin糖蛋白配体-1的基因克隆及其Ig融合蛋白的表达和纯化

目的:克隆P-selectin糖蛋白配体-1的基因,表达和纯化其Ig融合蛋白。方法:RT-PCR法克隆出P-选择素糖蛋白配体-1(CD162)cDNA,构建真核表达载体,DEAD-Dextran法转染COS7细胞,上清经纯化后,分别使用WesternBlot和流式细胞仪鉴定CD162-hIgFc融合蛋白的性质和活性。结果:RT-PCR克隆出801bp的CD-162cDNA片段,插入载体构建出pIg-CD162,将其转染COS7细胞后获得CD162-hIgFc融合蛋白,分子量为74KD。WesternBlot证实其为CD162-hIgFc融合蛋白,流式细胞仪证明获得的CD162-hIgFc融合蛋白能够识别血小板表面的CD62P。结论:本研究成功制备了CD162-hIgFc融合蛋白,为进一步探讨P选择素与其配体CD162之间的作用打下坚实的基础。     

Ultrastructural changes in human skeletal muscles following tendon and nerve injuries. I. Ultrastructural changes following tendon injuries]

During reconstructive procedures performed 4-16 weeks after the tendon lesion the specimens obtained from the injured muscle have been examined by the authors. It was found that after the tendon injury inactivity atrophy develops and a condition of equilibrium could be observed at this time. The most important changes in the fine structure were seen in the contractile elements: these were atrophied, homogenized, fragmentated and ragged independently from the functional unities. The number of the mitochondria was considerably decreased, the sarcoplasmic reticulum was increased, and the difference between the originally red and white muscular fibres was indistinct. The glycogen content of the musculature was decreased, or it disappeared completely. No pathologic changes have been observed in the sarcolemma, the cell nuclei and the motor nerve end-organs.     

肝移植术后移植肝组织P-选择素、ICAM-1基因mRNA表达的实验研究

目的：研究移植肝组织P-选择素、ICAM-1基因mRNA表达变化以及供肝交感神经、枯否细胞对mRNA表达的影响。方法：使用氯化钆抑制供肝枯否细胞、六甲胺阻断供肝交感神经，观察肝移植术后4，8，16，24h移植肝P-选择素、ICAM-1 mRNA表达的变化。结果：肝移植术后肝组织P-选择素、ICAM-1基因mRNA表达增高，阻断供肝交感神经和抑制供肝枯否细胞，可下调P-选择素、ICAM-1的mRNA表达。结论：阻断供肝交感神经和抑制供肝枯否细胞能明显下调肝组织P-选择素、ICAM-1基因表达，减轻移植肝的缺血再灌注损伤。     

Winner of the ESVS Prize 1988. Effects of prostaglandin E1 (PGE1) on experimental renal ischaemia

A persisting incidence of acute renal failure has been observed after operative treatment of thoracoabdominal aortic aneurysm, ruptured abdominal aortic aneurysm and renal artery occlusive disease in patients with preoperative impairment of renal function. Because preservation of kidney function can play an important role in the outcome of these patients, the effects of prostaglandin E1 (PGE1) to prevent ischaemic renal failure were studied in an experimental model. Twenty dogs were exposed to 3 h warm ischaemia by clamping of the supra- and infrarenal aorta and both renal arteries. In 10 dogs PGE1 was given intravenously (100 ng/kg/min) for 15 min before clamping. Ten dogs treated with normal saline served as controls. Glomerular filtration rate, renal plasma flow, plasma creatinine, blood urea nitrogen, urine volume, free water clearance and renovascular resistance were calculated before and after renal ischaemia for both groups. The dogs were followed-up for 2 weeks and radionuclide studies with Tc-99m-MAG3, I-131-OIH and In-113m-DTPA were performed on the third postoperative day to calculate global and split renal clearance, tracer extraction fraction and mean transport time. After renal ischemia 9 dogs of the control group and 3 dogs of the PGE1-group developed acute renal failure (P less than 0.05 due to Fisher's exact text). PGE1 infusion significantly attenuated the postischaemic fall in glomerular filtration rate and renal concentrating ability as well as the postischaemic increase of plasma creatinine and blood urea nitrogen induced by 3 h warm renal ischaemia (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia activates neutrophils but inhibits their local and remote diapedesis.

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 /- 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemc levels of 74 +/- 14 fmDCF/cell (p less than 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10(-7) M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber were 1636 +/- 258 PMN/mm3 after three hours (n = 8) compared to 63 +/- 18 PMN/mm3 induced by sham plasma (n = 4, p less than 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 +/- 2 X 10(4) PMN/mm3 compared to 5 +/- 1 X 10(4) PMN/mm3 with sham plasma (n = 4, p less than 0.05). Diapedesis was completely prevented (0-3 PMN/mm3, p less than 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p less than 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.     

Adenosine triphosphate regeneration and function in the rat kidney following warm ischaemia.

Tissue levels of adonosine triphosphate (ATP) have been measured in rat kidneys following periods of warm ischaemia: (1) immediately after the ischaemic period and (2) after the kidney had been reperfused with blood for 10 min. ATP levels at the end of the period of ischaemia are similar for ischaemic periods of 10 to 60 min and give no indication of the kidneys subsequent functional ability. The amount of ATP regenerated in 10 min of reperfusion correlates both with the duration of the period of ischaemia and with the subsequent functional ability of the kidney.     

Prediction of the delayed complications of intestinal and mesenteric injuries following experimental blunt abdominal trauma

Injuries to the intestine and mesentery are often found in patients undergoing laparotomy for blunt abdominal trauma. Although treatment of perforations is relatively straightforward, the same is not true for contusions. Few guidelines exist at present to aid the surgeon in deciding which injuries require resection in order to avoid the complications of delayed perforation and late stricture formation. The natural history of these non-perforating intestinal and mesenteric injuries has been examined in an experimental model to identify possible criteria on which future management can be based. In the immediate postinjury period peristalsis and local mesenteric pulsation were absent in the majority of injuries which went on to full recovery and these observations are thus of little predictive value in predicting outcome. The initial size of contusion (length of contusion along longitudinal axis of bowel) relative to bowel wall circumference (BWC) was related to complications as follows: contusion less than BWC (n = 47)--one complication; contusion greater than BWC (n = 8)--three complications (P = 0.02). Similarly, six mesenteric injuries which produced an initial ischaemia (assessed by fluorescein) less than twice the BWC did not result in any complications, compared with four complications which occurred in ten cases when the initial ischaemia was greater than twice the BWC. These results go some way towards providing a better understanding of these injuries and in turn may help the emergency surgeon in deciding which injuries require resection.     

Histology and histomorphometry of bone regeneration after experimental injuries.

Reparative callus formation upon tubular bone was studied after surgical injuries of different degrees. Thirty-seven young rats were divided into three groups. In the periosteum group the bone was scraped, in the fissure group we made a slit reaching the medulla, and in the defect group a standard defect was sawn. Rats were killed at 6, 12 and 18 days. The formed callus was studied histologically and histomorphometrically. The results suggest than even the primary osteochondrogenic callus formation is dependent on the mode of injury. At six days the three groups differed both qualitatively and quantitatively in regard to callus formation. At 12 and 18 days the total area of woven bone was proportional to the degree of trauma according to the linear regression line Y = 0.883 x X + 0.226 (r = 0.876, P less than 0.001). In the fissure and defect groups reparative bone filled the hole proportionally to the same extent. Periosteal and endosteal woven bone formed round the bone. Medullary bone formation was limited mainly to the area immediately adjacent to the trauma, the result being a cylinder-shaped callus. Cartilage formation was most abundant at six days and was related to the amount of woven bone in the external callus (r = 0.854, P less than 0.001) and to the trauma area (r = 0.707, P less than 0.05).     

Alpha-1-acid glycoprotein and its potential impact on local anesthetic dose in neonates

Background

Alpha-1-acid glycoprotein is an acute-phase protein with a high affinity for amide local anesthetics. Compared to adults, neonates have lower concentrations of this glycoprotein in plasma, and are therefore at higher risk of developing local anesthetic toxicity. Alpha-1-acid glycoprotein concentrations rise in adults after surgery as a response to stress as well as in inflammatory conditions. Previous studies have shown that concentrations of alpha-1-acid-glycoprotein in neonates vary postpartum, influenced by gestational age and mode of delivery.

Aim

This study aims to determine the concentrations of alpha-1-acid glycoprotein pre- and postoperatively in neonates undergoing major surgery. This information is important for determining safe and effective dosage of local anesthetic in this vulnerable group of patients.

Methods

In this prospective observational study, 25 neonates (median 3 days of age) undergoing major surgery were included. Blood sampling was performed preoperatively and at four occasions postoperatively. Alpha-1-acid-glycoprotein plasma concentrations were analyzed using an immunoturbidimetric assay. Mann–Whitney U test, Kruskal–Wallis and Spearman ranking correlation test were used for the statistical analysis.

Results

Higher plasma concentrations of alpha-1-acid-glycoprotein were found 48 h postoperatively compared to preoperatively [median (inter-quartile range) 0.815 g L⁻¹ (0.663–0.983 g L⁻¹) vs. 0.300 g L⁻¹ (0.205–0.480 g L⁻¹ p < 0.001)], respectively. It was not possible to detect any influence of sex, postnatal age, gestational age, or delivery mode on alpha-1-acid-glycoprotein concentrations in our data.

Conclusions

Alpha-1-acid-glycoprotein concentrations increase in neonates as a response to surgery regardless of gestational age, sex, or mode of delivery.