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.

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate-limiting enzyme in the ectonucleotidase pathway is ecto-5'-nucleotidase (e-5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e-5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e-5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double-immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e-5NT-positive astrocytes, accounting for up-regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e-5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.

The weak regenerative capacity of self-repair after injury to the adult brain is caused by the formation of glial scar due to reactive astrogliosis. In the present study the beginning of reactive astrogliosis in the adult, as shown immunocytochemically by upregulation of glial fibrillary acidic protein (GFAP) and vimentin, was seen two days after the left sensorimotor cortex lesion, being maximal during the first two weeks and declining by 30 days after the lesion. This was accompanied by intensive glial scarring. Conversely, after the neonatal lesion a lack of gliotic scar was seen until 30 days postsurgery, although the pattern of GFAP and vimentin expression during recovery period was the same. The aim of the study was to define an appropriate therapeutic intervention that could modulate astrocyte proliferation and diminish glial scar formation after adult brain lesion. For this purpose the effects of an antiproliferative agent, the purine nucleoside analogue ribavirin was examined. It was shown that daily injection of ribavirin for 5 and 10 days considerably decreased the number of reactive astrocytes, while slight GFAP labeling was restricted to the lesion site. Obtained results show that ribavirin treatment downregulates the process of reactive astrogliosis after adult brain injury, and thus may be a useful approach for improving neurological recovery from brain damage.

No abstract

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering multiple cellular responses to injury. Here, we evaluated changes in extracellular ATP, ADP and AMP hydrolysis at different times (0-24 hours) after unilateral cortical stab injury (CSI) in adult rats. Results demonstrated that 24 hours following CSI, ATP and ADP hydrolyzing activities were not significantly altered in injured cortex. Based on calculated V (ATP)/V (ADP) ratio it was concluded that ATP/ADP hydrolysis was primarily catalyzed by NTPDase1 enzyme form. In contrast, AMP hydrolysis, catalyzed by 5'-nucleotidase, was significantly reduced at least 4 hours following CSI. Kinetic analysis and Lineweaver-Burk transformation of the enzyme velocities obtained over the range of AMP concentrations (0.05-1.50 mM) revealed that inhibition of 5'-nucleotidase activity after CSI was of the uncompetitive type. Taken together our data suggest that injured tissue has reduced potential for extracellular metabolism of adenine nucleotides in early stages after CSI.