In the sham group, neurons in the cortex showed no morphological changes. In the I/R group, most neurons showed loss of Nissl's bodies, chromosome condensation, nuclear pyknosis, or lack of cellular structure. Compared to the I/R group, the parecoxib treatment reduced the degeneration of neurons and significantly increased the number of intact neurons (Figure 2B).
High morbility group box 1 protein (HMGB1) and TNF-α levels were not significantly different in the sham group over the time course. In the I/R group, the HMGB1 protein level gradually decreased (P <0.05), but the TNF-α level increased at 24 hours (P <0.05) and was still high at 72 hours after reperfusion, compared with the sham group. Parecoxib decreased theHMGB1 level (P <0.05) and TNF-α level (P <0.05) at 24 and 72 hours after reperfusion, compared with the I/R group (Figure 3).
The importance of the inflammatory response in the pathophysiology of ischemic stroke is well recognized. Ischemia induces inflammatory cell recruitment and migration (neutrophils followed later by monocytes) and upregulates inflammatory mediators (cytokines, chemokines, and adhesion molecules) in the brain for hours to days after the onset of ischemia.12 In this early phase, the endothelium promotes inflammation and recruits circulating leukocytes through the upregulation of adhesion molecules. These recruited leukocytes then release metalloproteinases, which participate in the breakdown of the neurovascular matrix with consequent blood-brain barrier disruption, edema, and/or hemorrhage.
HMGB1 is a nonhistone DNA-binding protein, which participates in nucleosome formation and regulation of gene transcription, having both nuclear and extracellular functions.13 HMGB1 has recently been characterized as a key cytokine, which may contribute to the delayed death of brain cells in the ischemic peri-infarct region. HMGB1 receptors such as RAGE, TLR2, and TLR4 are expressed in neurons, glia, and endothelial cells. RAGE appears to be upregulated in the cortical peri-infarct region. Recombinant HMGB1 or HMGB1 released by injured neurons in culture induces proinflammatory cytokine expression in neurons, astrocytes, and endothelial cells. Extracellular HMGB1 binds to its receptors, and may function as a proinflammatory cytokine and activate microglia and other inflammation-related cells, thus stimulating the release of other cytokines and aggravating brain injury.11 HMGB1 proinflammatory signaling might begin early, possibly in the first hour after focal ischemia, but may continue for hours thereafter.14 Kim et al15 found that HMGB1 expression in the postischemic brain was regulated differentially in the ischemic hemisphere. HMGB1 gradually decreased in the penumbras, whereas it notably decreased immediately after MCAO and then slowly but significantly increased in the infarction cores. Within the ischemic core, HMGB1 appears to be released from the nucleus and cytoplasm into the extracellular space, whereas peri-infarct regions appear to maintain translocation of HMGB1 from the nucleus into the cytoplasm.16 Our results are consistent with the findings of Kim et al15 and indicate that attenuation of HMGB1 expression may be a potential mechanism of parecoxib's neuroprotection in cerebral ischemia.
TNF-α is one of the major causes of neuronal damage in ischemia. It can activate polymorphic neutrophils, induce the release of cytokines, and increase leukocyteendothelial cell adhesion thus aggravating inflammatory responses. TNF-α can also damage the microvascular endothelium, disrupt the brain blood barrier, and accelerate the infiltration of leucocytes to infarct lesion and the formation of cerebral edema. The level of TNF-α in human brain increased after cerebral infarction and appears sequentially in the infarct core and peri-infarct areas before expression in tissue within the unaffected hemisphere.17 In animal models of cerebral ischemia, TNF-α expression increased after ischemic injury.18,19 TNF-α, which binds to receptor, such as TNF-R1 or TNF-R2, is a pleiotropic cytokine suspected to enhance or deter cellular survival through activation of receptor-mediated signal transduction.20,21 TNF-α expressed by angiogenic blood vessels after pMCAO is likely to have a detrimental effects on the survival of neuroblasts in the peri-infarct region, possibly through activation of TNF-R1 signaling. In our study, intravenous parecoxib administration reduced ischemic injury and attenuated TNF-α expression. However, Kelsen et al6 found that TNF-α mRNA levels were unaffected by parecoxib treatment. This is due to the differences in the cerebral ischemia models, tissue sampling, and/or experimental technique for detecting TNF-α expression.
The neuroprotective effects of COX-2 inhibitors have been demonstrated in several cerebral ischemia models, even when the COX-2 inhibitor is administered in a delayed fashion. Furthermore, the neuroprotection of the COX-2 inhibitors is long-lasting. However, clinical trials evaluating efficacy of COX-2 inhibitors in patients with stroke have become more difficult because of recent concerns about the increased cardiovascular risk after chronic treatment with this class of pharmacological agents. A recent clinical study indicates that COX-2 selective inhibitors may differ in their potential to cause ischemic cerebrovascular events. An increased risk of ischemic stroke may be influenced by additional pharmacological properties of individual COX-2 inhibitors. Odds ratios of ischemic stroke appeared to increase with higher daily dose and longer duration of rofecoxib and etoricoxib.22 It is noteworthy to mention that the increased toxicity of COX-2 inhibitors is observed after prolonged administration in patients at risk to develop cardiovascular events.23 In the present study, we used a lower dose of parecoxib for a short period of time. It may have little effect on platelet function and may not increase the incidence of cerebrovascular events, while lessening initial ischemic brain damage.
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