Malignant glioma-induced neuronal cell death in an organotypic glioma invasion model. Technical note

Rapid growth and diffuse brain infiltration are hallmarks of malignant gliomas. The underlying molecular pathomechanisms of these tumors, however, remain to be determined. The authors present a novel glioma invasion model that allows researchers to monitor consecutively tumor cell proliferation and migration in an organotypic brain environment. Enhanced green fluorescent protein-labeled F98 rat glioma cells were implanted into slice cultures obtained from a rat hippocampus, and tumor growth was microscopically documented up to 20 days in vitro. Invasion along radially oriented migratory streams could be observed 5 days after implantation of rat F98, human U87MG, and mouse GL261 glioma cells, whereas human Be(2)c neuroblastoma cells and mouse HT22 hippocampal neurons failed to invade the brain parenchyma. Following implantation of F98 glioma cells into the entorhinal cortex, cell death was observed within the infiltrated brain parenchyma as well as in the neuroanatomically connected dentate gyrus. Application of the N-methyl-D-aspartate receptor antagonist MK801 to the culture medium significantly reduced neuronal degeneration in the dentate gyrus, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist GYKI 52466 inhibited peritumoral cytotoxicity. This new model allows researchers to address in a systematic manner the molecular pathways of brain invasion as well as specific tumor-host interactions such as necrosis.     

Propofol prevents delayed neuronal death following transient forebrain ischemia in gerbils

PURPOSE: This study was conducted to ascertain whether propofol may protect against delayed neuronal death in the hippocampal CA1 subfield in gerbils. METHODS: Thirty-five gerbils were randomly assigned to five groups: Group I, the control group, a sham operation treated with physiological saline solution (PSS); Group II, ischemia/reperfusion treated with PSS; Group III, ischemia/reperfusion treated with 50 mg x kg(-1) propofol; Group IV, ischemia/reperfusion treated with 100 mg x kg(-1) propofol; Group V ischemia/reperfusion treated with 150 mg x kg(-1) propofol. Transient forebrain ischemia was induced by occluding the bilateral common carotid arteries for four minutes under N2O/O2/halothane anesthesia after administration of propofol or PSS. Five days later, histopathological changes in the hippocampal CA1 subfield were examined using a light microscope and degenerative ratio of the pyramidal cells were measured according to the following formula: (number of degenerative pyramidal cell/total number of pyramidal cells per 1 mm of hippocampal CA1 subfield) x 100. RESULTS: In group II, the pyramidal cells were atrophic and pycnotic; vacuolation and structural disruption of the radial striated zone was observed. In the other four groups, these changes were not observed. The degenerative ratios of pyramidal cells were as follows; group I: 5.9 +/- 1.9%, group II: 94.6 +/- 2.5% (P < 0.01), group III: 10.7 +/- 1.7%, group IV: 9.7 +/- 1.8%, group V: 9.2 +/- 1.9%. CONCLUSION: This study suggests that propofol may prevent delayed neuronal death in the hippocampal CA1 subfield after cerebral ischemia/reperfusion in gerbils.     

Insulin-like growth factor 1 prevents neuronal cell death and paraplegia in the rabbit model of spinal cord ischemia.

OBJECTIVE: Insulin-like growth factor 1 has been shown to be cytoprotective against ischemia-reperfusion injury in various organs. However, spinal cord protection by insulin-like growth factor 1 has not been tested. We have therefore examined the effect of insulin-like growth factor 1 on neuronal cell death and motor function after spinal cord ischemia. METHODS: Japanese white rabbits were subjected to spinal cord ischemia by clamping the abdominal aorta for 15 minutes. Insulin-like growth factor 1 (0.3 mg/kg) at a dose equipotent to insulin (0.3 IU/kg) in lowering blood glucose level or the control (phosphate-buffered saline solution as a vehicle) was administered intravenously 30 minutes before the aortic clamp. RESULTS: Hind-limb motor function had recovered normally 48 hours after the operation in all the rabbits (n = 8) treated with insulin-like growth factor 1. In contrast, all the control-treated (n = 8) and all but one of the insulin-treated (n = 6) rabbits had deteriorated to paraplegia by 48 hours after the operation. Histopathologic sections in the involved spinal cord segment showed that a significantly (P <.0001) greater number of motor neuron cells were preserved in the rabbits treated with insulin-like growth factor 1 (17.9 +/- 4.8 per section) than in those treated with the control (8.0 +/- 2.1). Although insulin was equipotent to insulin-like growth factor 1 in preserving the number of motor neuron cells (18.5 +/- 2.7), the percentage of motor neuron cells positive for terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling were significantly (P <.01) smaller in the rabbits treated with insulin-like growth factor 1 (6.0 +/- 4.6) compared with those treated with the control (54.6 +/- 33.8) and insulin (26.2 +/- 11.7). Immunohistochemical studies revealed that insulin-like growth factor 1 increased expression of the antiapoptotic Bcl-xL protein and inhibited expression of the proapoptotic Bax protein in motor neuron cells 24 and 48 hours after the operation. In contrast, expression of only Bax was increased after the operation in other groups of rabbits subjected to spinal cord ischemia. CONCLUSIONS: These results suggest that insulin-like growth factor 1, but not insulin with a conventional dose, protects motor neuron cells from ischemic spinal cord injury associated with differential regulation of Bcl-xL and Bax protein.     

Valproic acid preserves motoneurons following contusion in organotypic spinal cord slice culture

Objective: Spinal cord injury (SCI) is a devastating condition causing neuronal loss. A key challenge in treatment of SCI is how to retain neurons after injury. Valproic acid (VPA) is a drug recently has been appreciated for its neuroprotective and neurotrophic properties in various SCI models. In this study the role of VPA was assessed in organotypic spinal cord slice culture following the contusion.

Design: The lumbar enlargement of adult rat was cut transversely and slices were cultured. Seven days after culturing, injury was induced by dropping a 0.5 gram weight from 3?cm height on the slice surface. One hour after injury, the VPA was administered at 1, 5 and 10?µM concentrations. Afterward, at day 1 and 3 post injury (DPI: 1 and 3) propidium iodide (PI) and immunohistochemistry staining were performed to evaluate the cell death, NeuN and β-Tubulin expression, respectively.

Results: The PI staining of slices at DPI: 1 and 3 following treatment with VPA revealed significant decreases in the cell death in all three concentrations comparing to the non-treated group. Also immunostaining showed VPA only at 5?µM concentration considerably rescued ventral horn’ MNs from death and protected the neuronal integrity.

Conclusion: The results of this study indicate applying VPA one hour after injury can prevent the death of a majority of cells, importantly MNs and preserve the neuronal integrity. Since the first 24 hours after SCI is a critical period for employing any treatment, VPA can be considered as an option for further evaluation.     

Effects of neuroprotective cocktails on hippocampal neuron death in an in vitro model of cerebral ischemia

Cocktails of neuroprotectants acting at different parts of the ischemic injury cascade may have advantages over single agents. This study investigated, singly and in combination, the neuroprotective efficacy of an energy substrate (3.5 mM fructose 1,6-bisphosphate, FBP), an antagonist of NMDA receptors (1 and 10 microM MK-801), a free-radical scavenger (100 microM ascorbate), an adenosine A1 receptor agonist (10 microM 2-chloroadenosine), and an inhibitor of neurotransmission (2% isoflurane). These agents were evaluated for their ability to prevent loss and morphologic damage of CA1 neurons in rat hippocampal slices when these agents were administered during 30 minutes in vitro ischemia (combined oxygen/glucose deprivation at 37 degrees C) followed by 5 hours of recovery. Ten microM MK-801, alone or in combination with the other compounds, prevented loss of CA1 neurons and preserved their histologic appearance. Isoflurane, which prevents glutamate receptor-dependent cell death in this model, was also protective. Protection against neuron loss was also found when a subtherapeutic concentration of MK-801 (1 microM) was combined with 2-chloroadenosine (which indirectly causes NMDA receptor suppression), but not FBP or ascorbate. The authors conclude that in this model, the strategy of antagonizing NMDA receptors appears more protective than fructose-1,6-bisphosphate, 2-chloroadenosine or ascorbate.     

Adipose-derived stromal cells accelerate wound healing in an organotypic raft culture model

Adipose tissue is a known reservoir of multipotent mesenchymal stem cells, which can be manipulated in culture to produce cells with different phenotypes. The goal of this study was to determine whether the addition of these multipotential cells to organotypic, human skin equivalent cultures would accelerate wound healing after laser injury. For our initial studies, we were able to obtain 3-dimensional raft cultures from adult skin explanted directly onto the dermal equivalent containing human fibroblasts with or without adipose-derived stromal cells (ADSCs). Two days after laser injury, the raft cultures of skin explants that contained ADSCs had a completely healed multilayered epidermis, whereas the control raft culture without the adipose-derived cells still had areas of injury. With this encouraging outcome, these experiments were then repeated in a raft culture system initiated from dissociated primary adult human keratinocytes on the humanized dermal equivalent. Again, the cultures containing ADSCs healed faster than the control cultures. In conclusion, these data provide support to our hypothesis that ADSCs are an excellent and readily available source of factors necessary for accelerated wound healing and tissue regeneration.     

Isoflurane-induced neuronal degeneration: an evaluation in organotypic hippocampal slice cultures

Prolonged exposure of postnatal day (PND) 7 rat pups to anesthetics, which act via N-methyl-D-aspartate antagonism and/or gamma-amino butyric acid enhancement, causes neurodegeneration and persistent behavioral deficits. We studied these findings in vitro and determined whether the age of rat pups used for study or duration of anesthetic exposure modulates resultant neurodegeneration. Organotypic hippocampal slices (OHSs) were prepared from rat pups on PNDs 4, 7, and 14 and cultured 7 or 14 days in vitro. The slices were exposed to 1.5% isoflurane or fresh gas for durations of 1, 3, or 5 h. Hippocampal CA1, CA3, and dentate gyrus neuronal survival was assessed 3 days later. Neuronal cell death was greatest in OHSs prepared from PND 7 rat pups (P < 0.001) and was most evident after 5 h exposure to isoflurane (P < 0.001). By eliminating variables such as hemodynamics, nutrition, oxygenation, and carbon dioxide elimination, this in vitro investigation supports both an age- and duration-dependent relationship between 1.5% isoflurane exposure and perinatal neuronal death.     

Diabetes activates cell death pathway after transient focal cerebral ischemia

It is well known that diabetes aggravates brain damage in experimental and clinical stroke subjects. Diabetes accelerates maturation of neuronal damage, increases infarct volume, and induces postischemic seizures. The mechanism by which diabetes increases ischemic brain damage is still elusive. Our previous experiments indicate that mitochondria dysfunction may play a role in neuronal death. The objective of this study is to determine whether streptozotocin-induced diabetes activates cell death pathway after a brief period of focal cerebral ischemia. Both diabetic and nondiabetic rats were subjected to 30 min of transient middle cerebral artery occlusion, followed by 0, 0.5, 3, and 6 h of reperfusion. We first determined the pathological outcomes after 7 days of recovery by histopathology, and then detected key components of programmed cell death pathway using immunocytochemistry coupled with confocal laser-scanning microscopy and Western blot analysis. The results show that the cytosolic cytochrome c increased mildly after reperfusion in nondiabetic samples. This increase was markedly enhanced in diabetic rats in both ischemic focus and penumbra. Subsequently, caspase-3 was activated and poly-ADP ribose polymerase (PARP) was cleaved. Our results suggest that activation of apoptotic cell death pathway may play a pivotal role in exaggerating brain damage in diabetic subjects.     

Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia

BACKGROUND: General anesthetics reduce neuron loss following focal cerebral ischemia in rodents. The relative efficacy of this action among different anesthetics clinically used for neuroprotection is uncertain. In addition, it remains unclear how anesthetics compare to neuroprotection afforded by mild hypothermia. This study was performed to evaluate the comparative effects of isoflurane, sodium pentothal, and mild hypothermia in a hippocampal slice model of cerebral ischemia and to determine if the mechanism of neuroprotection of isoflurane involves inhibition of glutamate excitotoxicity. METHODS: Survival and morphology of CA1, CA3, and dentate gyrus neurons in rat hippocampal slices were examined after 10 or 20 min of combined oxygen-glucose deprivation (in vitro ischemia) followed by a 5-h recovery period. RESULTS: 10 or 20 min in vitro ischemia at 37 degrees C killed 35-40% of neurons in CA1 (P < 0.001), 6% in CA3 (not significant) and 18% in dentate (P < 0.05). Isoflurane (0.7 and 2.0%, approximately 0.45 and 1.5 minimum alveolar concentration), pentothal (50 microm, approximately 1 minimum alveolar concentration equivalent) and mild hypothermia (34 degrees C) all reduced CA1 cell loss and morphologic damage to similar degrees in 10- and 20-min periods of ischemia (P < 0.001). The noncompetitive N-methyl-D-aspartate antagonist MK-801 prevented cell damage, showing that N-methyl-D-aspartate receptor activation is an important mechanism of injury in this model. Glutamate (1 mm) produced cell loss similar to in vitro ischemia. Isoflurane (2%) prevented cell damage from glutamate exposure. CONCLUSIONS: In hippocampal slices, neuron death from simulated ischemia was predominately due to activation of glutamate receptors. Isoflurane, sodium pentothal, an N-methyl-D-aspartate receptor antagonist, and mild hypothermia prevented cell death to similar degrees. For isoflurane, the mechanism appears to involve attenuation of glutamate excitotoxicity.     

Role of prostaglandins in delayed cerebral ischemia after subarachnoid hemorrhage.

Prostaglandin E2, thromboxane B2, and 6-oxo-prostaglandin F1 alpha were assayed in blood and cerebrospinal fluid samples from patients after subarachnoid hemorrhage (SAH) and from a control population. The levels found in samples obtained from patients after SAH were compared with those found in controls and were also correlated with a number of clinical and radiological variables, many of which are either significantly associated with or represent evidence of cerebral ischemia. The levels of prostaglandin E2, thromboxane B2, and 6-oxo-prostaglandin F1 alpha in blood samples from patients after SAH and from controls were below the level of sensitivity of the assays. Levels of prostaglandin E2, thromboxane B2, and 6-oxo-prostaglandin F1 alpha in cerebrospinal fluid from patients after SAH were significantly elevated when compared with those found in control samples. There was no significant correlation, however, between the level of each prostaglandin measured and the following variables: clinical grade on admission as assessed by the Glasgow Coma Score and the World Federation of Neurological Surgeons grading system; the amount of subarachnoid blood seen on computed tomographic scan; the occurrence of ischemic deterioration; the occurrence of low density change on computed tomographic scan; the presence of vasospasm on angiography; clinical outcome as assessed by the Glasgow Coma Score 3 months after the ictus; and the incidence of ischemia as a cause of death or disability as assessed 3 months after the ictus. A primary role for prostaglandins in the etiology of delayed cerebral ischemia after SAH is not therefore confirmed.     

Isoflurane provides long-term protection against focal cerebral ischemia in the rat

BACKGROUND: Long-term neuroprotection by isoflurane has been questioned. The authors examined factors in experimental models potentially critical to definition of enduring isoflurane neuroprotection. METHODS: Rats were prepared for temporary middle cerebral artery occlusion (MCAO). Pericranial normothermia was maintained. Neurologic deficits (range, 0-48; 0=no deficit) and cerebral infarct volumes were measured. In experiment 1, rats underwent 50 or 80 min MCAO while awake or anesthetized with 1.8% isoflurane. Blood pressure was controlled with phenylephrine. Outcome was evaluated 2 weeks later. In experiment 2, rats underwent 50 min MCAO while awake or anesthetized with isoflurane, with outcome evaluated 8 weeks later. In experiment 3, rats underwent 50 min MCAO while awake or anesthetized with isoflurane and 2 weeks recovery. Effects of phenylephrine and the mitochondrial adenosine triphosphate-sensitive K channel antagonist 5-hydroxydecanoate were studied. In experiment 4, isoflurane-anesthetized rats underwent 50 min MCAO with permanent or temporary common carotid artery occlusion, with outcome evaluated 2 weeks later. RESULTS: In experiment 1, isoflurane reduced neurologic deficit (median+/-interquartile range; awake vs. isoflurane: 11+/-12 vs. 8+/-6 for 80 min and 13+/-4 vs. 3+/-9 for 50 min; P=0.0006) and infarct size (160+/-97 vs. 84+/-62 mm for 80 min and 169+/-78 vs. 68+/-61 mm for 50 min; P<0.0001). In experiment 2, isoflurane protection persisted at 8 weeks after ischemia. In experiment 3, there was no effect of phenylephrine or 5-hydroxydecanoate. In experiment 4, permanent common carotid ligation increased infarct size threefold versus temporary occlusion. CONCLUSIONS: Isoflurane repeatedly improved long-term neurologic and histologic outcome from focal ischemia independent of ischemia duration, perfusion pressure, or pretreatment with 5-hydroxydecanoate.     

Continuous intrathecal glyceryl trinitrate prevents delayed cerebral vasospasm in the single-SAH rabbit model in vivo

Background  

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) is a major cause of high morbidity and mortality. The reduced availability of nitric oxide (NO) in blood and cerebrospinal fluid (CSF) is well established as a key mechanism of vasospasm. Systemic administration of glyceryl trinitrate (GTN), an NO donor also known as nitroglycerin, has failed to be established in clinical settings to prevent vasospasm because of its adverse effects, particularly hypotension. The purpose of this study was to analyze the effect of intrathecally administered GTN on vasospasm after experimental SAH in the rabbit basilar artery.     

Cimetidine prevents suppression of delayed hypersensitivity in an animal model of haemorrhagic shock

BACKGROUND: Cimetidine reverses immunosuppression following trauma, however, its effect on pure haemorrhagic shock is unknown. METHODS: Mice sensitized by injection of sheep red blood cells (SRBCs), were subjected to cardiac puncture and randomized to a control group-A (n=11) and three shock groups (35% of blood volume extracted): group-S had no treatment (n=16), group-CP received cimetidine 50mg/kg intraperitoneally (n=16), group-CW received oral cimetidine (200mg/kg per day, n=16). After 5 days, animals were challenged by injection of SRBCs into the foot-pad of the right hind paw (same volume of saline was injected into left paw). Foot-pad thickness ratios (FPTRs) were determined at 16 and 40 h, and inflammatory response was assessed histologically. RESULTS: At 16 h, FPTRs were greater in group-CW than group-S (P=0.01). There were no differences at 40 h. More animals in groups-CP and -CW had grade 3/4 inflammation, whilst group-S had the least inflammatory response (NS). CONCLUSIONS: Cimetidine prevents suppression of delayed hypersensitivity in this model.     

The rat lung organotypic culture: an in vitro model for studying surfactant metabolism abnormalities

The amount of surfactant present in the intra-alveolar space is mainly regulated by the synthesis and recycling of surfactant by type II pneumocytes. Biochemical analyses have shown that the surfactant level is frequently diminished and that protein-rich exudate can further interfere with surfactant function in the lungs of adult respiratory distress syndrome (ARDS) patients. The microenvironmental changes that occur in the alveoli of burned patients, who are prone to developing ARDS, are unclear. Therefore, using an in vitro rat lung organotypic culture, we showed that the sera of rats with a 3-day old, third-degree thermal injury (25-30% total body surface area) inhibited surfactant synthesis in organotypically cultured rat lung cells. Surfactant precursor, 3H-choline, incorporation into the surfactant was 58% of control. Using liposomes made of dipalmitoyl phosphatidylcholine and phosphatidylglycerol (8:1, v/v) or surfactant we showed that surfactant endocytosis by purified type II alveolar cells is an active, temperature-dependent process, and correlates with the quantity of surfactant present in the milieu. We also found that plasma protein-rich fluid interfered with surfactant endocytosis by the purified type II pneumocytes. These two processes of inhibition of surfactant synthesis and its reutilization by these cells may contribute to the pathogenesis of ARDS.     

Isoflurane produces delayed preconditioning against myocardial ischemia and reperfusion injury: role of cyclooxygenase-2

BACKGROUND: Whether volatile anesthetics produce a second window of preconditioning is unclear. The authors tested the hypothesis that isoflurane causes delayed preconditioning against infarction and, further, that cyclooxygenase (COX)-2 mediates this beneficial effect. METHODS: Rabbits (n = 43) were randomly assigned to receive 0.9% intravenous saline, the selective COX-2 inhibitor celecoxib (3 mg/kg intraperitoneal) five times over 2 days before coronary artery occlusion and reperfusion, or isoflurane (1.0 minimum alveolar concentration) 24 h before acute experimentation in the absence or presence of celecoxib pretreatment. Two additional groups of rabbits received a single dose of celecoxib either 30 min before or 21.5 h after administration of isoflurane. Rabbits were then instrumented for measurement of hemodynamics and underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Myocardial infarct size was measured using triphenyltetrazolium staining. Western immunoblotting to examine COX-1 and COX-2 protein expression was performed in rabbit hearts that had or had not been exposed to isoflurane. RESULTS: Isoflurane significantly (P < 0.05) reduced infarct size (22 +/- 3% of the left ventricular area at risk) as compared with control (39 +/- 2%). Celecoxib alone had no effect on infarct size (36 +/- 4%) but abolished isoflurane-induced cardioprotection (36 +/- 4%). A single dose of celecoxib administered 2.5 h before coronary occlusion and reperfusion also abolished the delayed protective effects of isoflurane (36 +/- 4%), but celecoxib given 30 min before exposure to isoflurane had no effect (22 +/- 4%). Isoflurane did not alter COX-1 and COX-2 protein expression. CONCLUSIONS: The results indicate that the volatile anesthetic isoflurane produces a second window of preconditioning against myocardial ischemia and reperfusion injury. Furthermore, COX-2 is an important mediator of isoflurane-induced delayed preconditioning.     

Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia

BACKGROUND: Several investigations have shown that volatile anesthetics can reduce ischemic cerebral injury. In these studies, however, neurologic injury was evaluated only after a short recovery period. Recent data suggest that injury caused by ischemia is a dynamic process characterized by continual neuronal loss for a prolonged period. Whether isoflurane-mediated neuroprotection is sustained after a longer recovery period is not known. The current study was conducted to compare the effect of isoflurane on brain injury after short (2-day) and long (14-day) recovery periods in rats subjected to focal ischemia. METHODS: Fasted Wistar-Kyoto rats were anesthetized with isoflurane and randomly allocated to an awake (n = 36) or an isoflurane (n = 34) group. Animals in both groups were subjected to focal ischemia by filament occlusion of the middle cerebral artery. Pericranial temperature was servocontrolled at 37 degrees C throughout the experiment. In the awake group, isoflurane was discontinued and the animals were allowed to awaken. In the isoflurane group, isoflurane anesthesia was maintained at 1.5 times the minimum alveolar concentration. After 70 min of focal ischemia, the filament was removed. Animals were killed 2 days (awake, n = 18; isoflurane, n = 17) and 14 days (awake, n = 18; isoflurane, n = 17) after ischemia. The volumes of cerebral infarction and selective neuronal necrosis in the animals were determined by image analysis of hematoxylin and eosin-stained coronal brain sections. RESULTS: Cortical and subcortical volumes of infarction were significantly less in the isoflurane 2-day group (26 +/- 23 mm3 and 17 +/- 6 mm3, respectively) than in the awake 2-day group (58 +/- 35 mm3, P < 0. 01; and 28 +/- 12 mm3, P < 0.01, respectively). By contrast, cortical and subcortical volumes of infarction in the awake (41 +/- 31 mm3 and 28 +/- 16 mm3, respectively) and isoflurane (41 +/- 35 mm3 and 19 +/- 8 mm3, respectively) 14-day groups were not different (cortex, P = 0.99; subcortex, P = 0.08). The volume of cortical tissue in which selective neuronal necrosis was observed, however, was significantly less in the isoflurane 14-day group (5 +/- 4 mm3) than in the awake 14-day group (17 +/- 9 mm3, P < 0.01). The total number of necrotic neurons in the region of selective neuronal necrosis was significantly smaller in the isoflurane 14-day group than in the awake 14-day group (P < 0.01). CONCLUSION: Compared with the awake state, isoflurane reduced the extent of infarction assessed 2 days after focal ischemia in rats. At 14 days, however, only selective neuronal necrosis, but not infarction, was reduced by isoflurane. These results suggest that isoflurane delays but does not prevent cerebral infarction caused by focal ischemia. Isoflurane may attenuate the delayed development of selective neuronal necrosis in periinfarct areas in this animal model.     

Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement‐driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement‐blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43‐48 hours prior to implantation into ABO‐compatible, nonsensitized, MHC‐mismatched recipients. Animals were divided into 3 donor‐treatment groups: G1 ‐ vehicle, G2 ‐ rhC1INH+heparin, and G3 ‐ heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b‐9, and reduced urinary neutrophil gelatinase–associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor‐management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD‐induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.