Introduction
Previously, we have shown that tolerance to ischemia is induced by applying hypertonic NaCl to the cerebral cortex 1 . Application of hypertonic NaCl causes a small lesion without evoking cortical spreading depression (CSD). However, little is known about the molecular mechanisms by which a cortical lesion induces tolerance in the absence of CSD. Lesions to the central nervous system are known to trigger an inflammatory response, including expression of TNF, which itself has been shown to induce tolerance to ischemia. Inflammation is normally controlled by counter-regulatory pathways, which include the induction of feedback inhibitors of proinflammatory cytokine-signaling. The objective of the present study was to determine whether application of hypertonic NaCl upregulates the expression of mRNAs encoding TNF and its feedback inhibitors, tristetraproline (TTP), suppressors of cytokine signaling 3 (SOCS3), IL-1 receptor-associated kinase-M (IRAK-M), and toll-interacting protein (TOLLIP).
Methods
A small cortical lesion was made in the left frontal cortex in anesthetized Sprague-Dawley rats by applying 5 M NaCl to the intact dura for 2 hr. After recovery for 0, 2, 4, and 24 hr (n=4 animals per time point), the animals were sacrificed, and the brains were removed for sampling. The brain was sectioned in the coronal plane 5, 10 and 15 mm behind the frontal pole. Tissue samples from the frontal cortex of both hemispheres were extracted for total RNA, and the extracts were analyzed on northern blots for mRNAs encoding TNF, TTP, SOCS3, IRAK-M and TOLLIP.
Results
TNF and SOCS3 mRNAs were significantly elevated 2, 4, and 24 hr following NaCl application in the frontal cortex of the ipsilateral hemisphere, relative to the contralateral hemisphere. TTP mRNA was significantly elevated in the ipsilateral hemisphere at all times tested. There were no significant differences in IRAK-M and TOLLIP mRNAs between the hemispheres at any time point.
Conclusions
These results demonstrate that a small cortical lesion increases the levels of mRNAs encoding the proinflammatory cytokine, TNF, and its feedback inhibitors, TTP and SOCS3. TTP is a proline-rich protein that binds to the AU-rich element present in the 3′-untranslated region of mRNAs encoding proinflammatory cytokines, including TNF 2 . SOCS3 is one member of a family of proteins that suppresses signal transduction of cytokines, thereby inhibiting inflamation 3 . Cerebral ischemia evokes a progressive inflammatory response that is believed to exacerbate ischemic damage 4 . Thus, the present results suggest that the expression of feedback inhibitors of cytokine-signaling may contribute to the tolerance to ischemia induced by a small cortical lesion.