apyrase and Brain-Injuries

Microglial cells invade the brain as amoeboid precursors and acquire a highly ramified morphology in the postnatal brain. Microglia express all essential purinergic elements such as receptors, nucleoside transporters and ecto-enzymes, including CD39 (NTPDase1) and CD73 (5'-nucleotidase), which sequentially degrade extracellular ATP to adenosine. Here, we show that constitutive deletion of CD39 and CD73 or both caused an inhibition of the microglia ramified phenotype in the brain with a reduction in the length of processes, branching frequency and number of intersections with Sholl spheres. In vitro, unlike wild-type microglia, cd39-/- and cd73-/- microglial cells were less complex and did not respond to ATP with the transformation into a more ramified phenotype. In acute brain slices, wild-type microglia retracted approximately 50% of their processes within 15 min after slicing of the brain, and this phenomenon was augmented in cd39-/- mice; moreover, the elongation of microglial processes towards the source of ATP or towards a laser lesion was observed only in wild-type but not in cd39-/- microglia. An elevation of extracellular adenosine 1) by the inhibition of adenosine transport with dipyridamole, 2) by application of exogenous adenosine or 3) by degradation of endogenous ATP/ADP with apyrase enhanced spontaneous and ATP-induced ramification of cd39-/- microglia in acute brain slices and facilitated the transformation of cd39-/- and cd73-/- microglia into a ramified process-bearing phenotype in vitro. These data indicate that under normal physiological conditions, CD39 and CD73 nucleotidases together with equilibrative nucleoside transporter 1 (ENT1) control the fate of extracellular adenosine and thereby the ramification of microglial processes.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Animals; Animals, Newborn; Antigens, CD; Apyrase; Brain; Brain Injuries; Cell Count; Cells, Cultured; Chemotaxis; Dipyridamole; Disease Models, Animal; Equilibrative Nucleoside Transporter 1; Metabolic Networks and Pathways; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microglia; Phenotype; Receptors, Purinergic P2Y12

Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while 2-arachidonoylglycerol (2-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain.

Topics: Adenosine Triphosphate; Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents; Apyrase; Arachidonic Acids; Astrocytes; Brain; Brain Injuries; Connexin 43; Disease Models, Animal; Endocannabinoids; Ethanolamines; Flufenamic Acid; Glycerides; Lasers; Mice; Mice, Knockout; Mice, Transgenic; Microglia; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

Traumatic CNS injury activates and mobilizes resident parenchymal microglia (MG), which rapidly accumulate near injured neurons where they transform into phagocytes. The mechanisms underlying this rapid 'homing' in situ are unknown. Using time-lapse confocal imaging in acutely excised neonatal hippocampal slices, we show that rapid accumulation of MG near somata of injured pyramidal neurons in the stratum pyramidale (SP) results from directed migration from tissue regions immediately adjacent to (<200 microm from) the SP. Time-lapse sequences also reveal a 'spreading activation wave' wherein MG situated progressively farther from the SP begin to migrate later and exhibit less directional migration toward the SP. Because purines have been implicated in MG activation and chemotaxis, we tested whether ATP/ADP released from injured pyramidal neurons might account for these patterns of MG behavior. Indeed, application of apyrase, which degrades extracellular ATP/ADP, inhibits MG motility and homing to injured neurons in the SP. Moreover, bath application of exogenous ATP/ADP disrupts MG homing by inducing directional migration toward the slice exterior and away from injured neurons. These results indicate that extracellular ATP/ADP is both necessary and sufficient to induce directional migration and rapid homing of neonatal MG to injured neurons in situ. Rapid, ATP/ADP-dependent MG homing may promote clearance of dead and dying cells and help limit secondary damage during the critical first few hours after neuronal injury.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Animals, Newborn; Apyrase; Brain Injuries; Cell Communication; Cell Count; Cell Movement; Hippocampus; Mice; Mice, Inbred C57BL; Microglia; Microscopy, Confocal; Organ Culture Techniques; Phagocytosis; Pyramidal Cells; Rats; Rats, Sprague-Dawley

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5'-nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5'-nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5'-nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5'-nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5'-nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up-regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5'-nucleotidase were predominantly astrocytes.

Topics: 5'-Nucleotidase; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Astrocytes; Brain; Brain Injuries; Male; Microglia; Rats; Up-Regulation

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

Topics: Adenosine Triphosphate; Animals; Apyrase; Astrocytes; Brain; Brain Injuries; Cell Communication; Chemotaxis; Connexins; Gliosis; Green Fluorescent Proteins; Mice; Mice, Transgenic; Microglia; Phagocytosis; Purinergic P2 Receptor Antagonists; Reaction Time; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Signal Transduction

We evaluated the effects of two consecutive concussive injuries on brain energy metabolism and N-acetylaspartate (NAA) to investigate how the temporal interval between traumatic events influences overall injury severity.. Rats were injured to induce diffuse traumatic brain injury (TBI) (mild, 450 g/1 m; severe, 450 g/2 m). In two groups, two mild TBIs were delivered in 3- or 5-day intervals. Three additional animal groups were used: single mild TBI, single severe TBI, and sham. All animals were killed 48 hours postinjury. Adenosine 5'-triphosphate (ATP), adenosine diphosphate, and NAA concentrations were analyzed with high-performance liquid chromatography on deproteinized whole brain extracts.. In control animals, the NAA concentration was 9.17 +/- 0.38 micromol/g wet weight, the ATP concentration was 2.25 +/- 0.21 micromol/g wet weight, and the ATP-to-adenosine diphosphate ratio was 9.38 +/- 1.23. These concentrations decreased to 6.68 +/- 1.12 micromol/g wet weight, 1.68 +/- 0.24 micromol/g wet weight, and 6.10 +/- 1.21 micromol/g wet weight, respectively, in rats that received two mild TBIs at a 5-day interval (P < 0.01; not different from results in rats with single mild TBI). When a second TBI was delivered after 3 days, the NAA concentration was 3.86 +/- 0.53 micromol/g wet weight, the ATP concentration was 1.11 +/- 0.18 micromol/g wet weight, and the ATP-to-adenosine diphosphate ratio was 2.64 +/- 0.43 (P < 0.001 versus both controls and 3-day interval; not different from rats receiving a single severe TBI).. The biochemical modification severity in double TBI is dependent on the interval between traumatic events, which demonstrates the metabolic state of the vulnerable brain after mild TBI. These data support the hypothesis of the application of proton magnetic resonance spectroscopy to measure NAA as a possible tool to monitor the full recovery of brain metabolic functions in the clinical setting, particularly in sports medicine.

Topics: Adenosine Triphosphate; Animals; Apyrase; Aspartic Acid; Blood Gas Analysis; Brain; Brain Injuries; Chromatography, High Pressure Liquid; Disease Models, Animal; Energy Metabolism; Male; Rats; Rats, Wistar; Time Factors