apyrase and Hyperoxia

Critically ill patients are routinely exposed to high concentrations of supplemental oxygen for prolonged periods of time, which can be life-saving in the short term, but such exposure also causes severe lung injury and increases mortality. To address this therapeutic dilemma, we studied the mechanisms of the tissue-damaging effects of oxygen in mice. We show that pulmonary invariant natural killer T (iNKT) cells are unexpectedly crucial in the development of acute oxygen-induced lung injury. iNKT cells express high concentrations of the ectonucleotidase CD39, which regulates their state of activation. Both iNKT cell-deficient (Jα18(-/-)) and CD39-null mice tolerate hyperoxia, compared with wild-type control mice that exhibit severe lung injury. An adoptive transfer of wild-type iNKT cells into Jα18(-/-) mice results in hyperoxic lung injury, whereas the transfer of CD39-null iNKT cells does not. Pulmonary iNKT cell activation and proliferation are modulated by ATP-dependent purinergic signaling responses. Hyperoxic lung injury can be induced by selective P2X7-receptor blockade in CD39-null mice. Our data indicate that iNKT cells are involved in the pathogenesis of hyperoxic lung injury, and that tissue protection can be mediated through ATP-induced P2X7 receptor signaling, resulting in iNKT cell death. In conclusion, our data suggest that iNKT cells and purinergic signaling should be evaluated as potential novel therapeutic targets to prevent hyperoxic lung injury.

Topics: Acute Lung Injury; Adoptive Transfer; Animals; Antigens, CD; Apoptosis; Apyrase; Cell Proliferation; Cells, Cultured; Cytokines; Hyperoxia; Lung; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Neutrophil Infiltration; Neutrophils

To determine the spatial pattern and temporal evolution of the change in retinal partial oxygen pressure (DeltaPO(2)) associated with a murine oxygen-induced retinopathy (OIR) model of retinal neovascularization (NV).. On P7, newborn C57BL/6 mice were exposed to 75% oxygen until postnatal day (P)12, followed by recovery in room air until P17 or P34. Control mice remained in room air until P17 or P34. At P17 and P34, functional magnetic resonance imaging (MRI) and a carbogen inhalation challenge was used to measure retinal DeltaPO(2). Retinal avascularity, distance from the optic nerve head to the vascular edge in the peripheral retina, and NV incidence and severity were measured in retinas stained with adenosine diphosphatase (ADPase).. In P17 and P34 controls and in P34 OIR animals, retinas were fully vascularized without evidence of NV. In P17 OIR mice, there was a large central retinal capillary-free zone (22% +/- 3% of the entire retinal area, mean +/- SD) and 4 clockhours (range 1-7) of retinal NV at the border of the peripheral vascular and central acapillary retina in 100% (36/36) of the mice. In P17 OIR mice, retinal DeltaPO(2) over the vascularized far peripheral retina was not significantly (P > 0.05) different from the P17 control but was supernormal (P < 0.05) over the central capillary-free retina. However, no differences (P > 0.05) in retinal DeltaPO(2) were found between the P34 control and OIR groups.. A reversible supernormal DeltaPO(2) was found only over the central acapillary retina during the appearance of retinal NV in a mouse OIR model. The present data show the applicability of carbogen-challenge functional MRI to the study of retinal DeltaPO(2) in vivo in eyes that are too small for the use of existing techniques.

Topics: Animals; Animals, Newborn; Apyrase; Disease Models, Animal; Female; Hyperoxia; Magnetic Resonance Imaging; Mice; Mice, Inbred BALB C; Oxygen; Partial Pressure; Retina; Retinal Neovascularization; Retinal Vessels

Vascular endothelial cell growth factor (VEGF) has been implicated in vascular development and in proliferative retinopathies. The goal of this study was to examine the immunohistochemical localization and relative levels of VEGF receptor-2 (KDR) in canine retina during postnatal vasculogenesis and during angiogenesis in oxygen-induced retinopathy (OIR) and to investigate the effects of neutralizing KDR on these processes.. Eyes from normal dogs ranging from 1 to 22 days of age and age-matched oxygen-treated animals were snap frozen for immunohistochemical analysis with antibodies against human KDR. To examine the effects of blocking KDR, 6-day-old air-reared control and oxygen-treated animals were surgically implanted with slow release polymer pellets containing control IgG or anti-KDR. Material eluted from pellets was assessed using a binding assay (measures binding to soluble KDR) to determine the kinetics of anti-KDR release and endothelial cell proliferation to measure bioactivity. Animals were killed at 22 days of age and tissues examined with adenosine diphosphatase (ADPase) histochemical staining of blood vessels.. KDR immunoreactivity was only weakly associated with developing retinal vessels and was not observed in angioblasts throughout normal postnatal development. Immunoreactivity was very strong in reforming retinal vessels and intravitreal neovascularization in oxygen-treated animals. Anti-KDR had no effect on vessel morphology or growth in air-reared control animals. In oxygen-treated animals, anti-KDR significantly inhibited revascularization of the retina (P = 0.005) and formation of intravitreal neovascularization compared with control IgG pellet eyes (P < 0.04).. KDR/Flk-1 was only weakly associated with normal developing primary retinal vessels but was strongly expressed by proliferating endothelial cells in reforming retinal vessels and intravitreal neovascularization after hyperoxic insult. Anti-KDR antibody delivered by slow-release pellets had no effect on normal vasculogenesis, but it inhibited the formation of intravitreal neovascularization and retinal vessel development in OIR. The study suggests that blocking KDR may be beneficial for treating pathologic angiogenesis in adult tissue.

Topics: Animals; Animals, Newborn; Antibodies, Blocking; Apyrase; Disease Models, Animal; Dogs; Endothelium, Vascular; Humans; Hyperoxia; Immunoenzyme Techniques; Immunoglobulin G; Infant, Newborn; Neovascularization, Physiologic; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity

Hypercarbia has been suggested as a risk factor for retinopathy of prematurity (ROP). We investigated the effect of hypercarbia on the retinal vasculature of the neonatal rat to determine whether hypercarbia alone could induce preretinal neovascularization analogous to ROP.. In a preliminary blood gas study, 8-11-day-old rats were exposed to specific levels of inspired O2 and either 0.2% CO2 or 10% CO2. Arterial blood gases were obtained, and a level of inspired O2 was determined that, when combined with inspired 10% CO2, would produce a PaO2 equivalent to room air (pure hypercarbia). In the formal retinopathy study, 300 newborn rats raised in 12 expanded litters (n = 25 each) were exposed for 7 days to either room air, 10% CO2 in 21% O2 and nitrogen (high-inspired CO2 group) or 10% CO2 in 12.5% O2 and nitrogen (pure-hypercarbia group). Each type of exposure was followed by recovery in room air for 5 days. Animals were sacrificed on day 13 and retinae were analyzed, using fluorescein perfusion and ADPase staining techniques.. Neovascularization occurred in 19% of rats in the high-inspired CO2 group, and 14% of rats in the pure-hypercarbia group, compared to 0% of rats exposed to room air alone (p = 0.001, Chi square).. In the neonatal rat model, exposure to hypercarbia alone, followed by room air recovery, results in preretinal neovascularization similar to that seen in oxygen-induced retinopathy. Our results support the suggestion that hypercarbia may be a risk factor for retinopathy of prematurity.

Topics: Animals; Animals, Newborn; Apyrase; Blood Gas Analysis; Carbon Dioxide; Humans; Hypercapnia; Hyperoxia; Infant, Newborn; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Risk Factors

In previous studies the morphologic features of the acute vaso-obliterative and vasoproliferative stages of oxygen-induced retinopathy (OIR) were quantified and described in the dog model of retinopathy of prematurity (ROP). In the present study the sequelae of these events were examined using fluorescein angiography and histologic, enzyme, and immunohistochemical techniques.. Thirty newborn animals were exposed to 95% to 100% oxygen for 4 days and returned to room air until they were 22 to 45 days of age. Before death some animals were anesthetized, and fluorescein angiography was performed. Retina and vitreous from some animals were processed for adenosine diphosphatase (ADPase) flat-embedding. In other cases, eyes were prepared for full-thickness eyewall sectioning or frozen for histochemical analysis.. Fluorescein angiography, funduscopic examination, and ADPase preparations showed dilated and tortuous retinal vessels, pigmentary changes, incomplete vascularization of peripheral retina, vitreous hemorrhage, and persistence of massive intravitreal neovascularization. Full-thickness eyewall sections showed tractional retinal folds, tented intravitreal vascularized membranes, and vitreous synchysis. Immunohistochemical analysis showed inner retinal astrogliosis. Enzyme histochemistry showed high alpha glycerophosphate dehydrogenase activity in poorly differentiated neovascular formations and low activity in formations with mature pericytes and endothelial cells.. End-stage OIR in the neonatal dog shares many features with the chronic human disease. These results provide additional support for the use of this model in experimental studies of ROP.

Topics: Animals; Animals, Newborn; Apyrase; Astrocytes; Disease Models, Animal; Dogs; Fluorescein Angiography; Fundus Oculi; Glial Fibrillary Acidic Protein; Glycerolphosphate Dehydrogenase; Humans; Hyperoxia; Immunoenzyme Techniques; Infant, Newborn; Oxygen; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Vitreous Hemorrhage; von Willebrand Factor

To quantify the acute constrictive response of developing retinal blood vessels to hyperoxia and to examine the vaso-obliterative phase of sustained oxygen breathing in the neonatal dog model of retinopathy of prematurity.. Seven littermates were used to examine the acute constrictive response of the developing retinal vessels to hyperoxia (30 minutes to 96 hours of 100% oxygen). ADPase retinal flatmounts were prepared, and morphometric measurements were made using computer-assisted analysis. Vaso-obliteration also was examined in three animals killed after prolonged exposure to hyperoxia (4 days of 100% oxygen) and in three room air controls using ADPase flat-embedded retinas and cross-sections. Choroids were processed for alkaline phosphatase flat-embedding.. After 1 hour of oxygen breathing, all vascular components showed a reduction in diameter: Arteries were reduced 27%, veins 18.3%, and capillaries 27.7%. Capillary constriction peaked by 24 hours (69.4% reduction), whereas arteries and veins continued to close. Although capillary diameters did not decrease significantly after 24 hours, the number of capillaries, as determined by percent vascular area calculations, continued to decrease in all areas through the additional 3 days of oxygen breathing. In contrast, after 4 days of hyperoxia the choriocapillaris lumenal diameters and percent vascular area did not vary significantly from controls. Analysis of sections taken through various retinal regions of these animals revealed significant decreases (40%) in the volume of the extracellular spaces available for blood vessel formation. Hyperoxia also reduced in a 55.6% decrease in the total number of cells (endothelial cells, ablumenal cells, perivascular cells) within the inner retina; however, there was no significant difference in ganglion cell counts in the two groups.. This study demonstrates that the pattern and severity of the reaction of developing retinal vessels to hyperoxia in the newborn dog is similar to that described for the kitten and the premature human. This response is unlike that exhibited by the newborn rat or mouse.

Topics: Animals; Apyrase; Cell Count; Choroid; Disease Models, Animal; Dogs; Humans; Hyperoxia; Infant, Newborn; Mice; Oxygen; Rats; Retinal Artery Occlusion; Retinal Vein Occlusion; Retinal Vessels; Retinopathy of Prematurity