WANG, Na; GUO, Qu-lian; YE, Zhi; XIA, Ping-ping; WANG, E; YUAN, Ya-jing

Stroke is a leading cause of death and disability around the world. Data published over the past decade have suggested a greater role for inflammation in the pathophysiology of ischemic stroke. Ischemia induces inflammatory cell recruitment and migration and upregulates inflammatory mediators in the brain for hours to days after the onset of injury. Due to the concerted action of phospholipases, huge amounts of free arachidonic acids are released from membrane phospholipids after an ischemic event. The arachidonic acid metabolic pathway, via cyclooxygenases (COX), is actively involved in neuroinflammation following the occlusion of cerebral blood vessels, and is a key component of the initiation and propagation of the inflammatory process after ischemia.^1,2 It has been known for decades that COX inhibitors decrease ischemic brain injury.³ Recently, it has been reported that COX-2 selective inhibitors prevent post-ischemic prostaglandin accumulation and ischemic neuronal damage, suggesting that the beneficial effects observed with non-selective COX inhibitors are probably associated with COX-2 rather than to COX-1 inhibition.^4,5 Parecoxib (parecoxib sodium), the first COX-2 specific inhibitor administered as an intravenous or intramuscular injection in clinic, has been shown to be neuroprotective when administered intraperitoneally in spontaneously hypertensive rats subjected to transient middle cerebral artery occlusion.6 In this study, we investigated the neuroprotective effects of intravenous parecoxib in a rat model of permanent middle cerebral artery occlusion (pMCAO).

METHODS

Subjects

Ninety male Sprague-Dawley rats weighing 300–350 g, were provided by the Center of Experimental Animals, Central South University. Principles of laboratory animal care and all procedures were conducted according to the Guide for the Care and Use of Laboratory Animals and the Committee of Experimental Animals of Xiangya Hospital. The animals were housed in a temperaturecontrolled room with normal 12–12 hours light-dark cycle. Food and water were freely available in the cage. All investigators directly involved in the study were blinded to the experimental design.

The animals were randomly divided into three groups: the sham group, ischemia/reperfusion (I/R) group and the parecoxib group. The animals in the parecoxib group received 4 mg/kg parecoxib (Pfizer, USA) intravenously 15 minutes before ischemia via the vena dorsalis penis and again at 12 hours after ischemia. The animals in the sham and I/R groups were treated with normal saline in a similar fashion. All rats were allowed to survive for 24 or 72 hours (n=6 for each group at each time point).

I/R surgery

The surgical procedures were performed under aseptic conditions. Animals were anesthetized with chloral hydrate (350 mg/kg, I.P.). Under deep anesthesia, the two common carotid arteries were identified and isolated through a ventral midline cervical incision. The body temperature was kept at 37°C using a thermal blanket. Focal cerebral ischemia was induced by pMCAO, as described previously.⁷ After opening a bone-window at the base of the cranium, the left MCA was permanently occluded by bipolar electrical coagulation at the lateral edge of the olfactory tract. Subsequently, both common carotid arteries were occluded with miniature clips for 60 minutes. The sham group had exposure of the left distal middle cerebral artery and the common carotid arteries, but vessels were not occluded.

Neurological deficit scores (NDSs)

Neurological evaluation was performed before ischemia and at 24 and 72 hours after reperfusion and scored on a 7-point scale.⁸ Grade 6: normal extension of both forelimbs towards the floor when lifted; Grade 5: consistent flexion of the forelimb contralateral to the injured hemisphere; Grade 4: dysfunctional rats with consistently reduced resistance to lateral push towards the paretic side; Grade 3: circling towards the paretic side if pulled and lifted by the tail; Grade 2: circling towards the paretic side if pulled by the tail; Grade 1: circling spontaneously towards the paretic side; Grade 0: no spontaneous motion.

Cerebral infarct volume

The volume of the infarct was analyzed using 2, 3, 5-triphenyltetrazolium chloride (TTC), a histological assay for determining dehydrogenase activity. Eighteen rats were sacrificed at 72 hours after reperfusion. Their brains were sliced into 2 mm-thick sections and stained with 2% TTC (Sigma, USA) at 37°C for 30 minutes. Then sections were fixed in paraformaldehyde overnight and photographed.⁹ The infarct volume was measured in each slice and summed by Photoshop CS (Adobe, USA). In order to minimize artifacts produced by post-ischemic edema in the infarcted area and correct for the individual difference in brain volumes, the percentage of infarct volume in the total brain volume was calculated.

Hematoxylin and eosin (H&E) staining and Nissl staining

At 24 and 72 hours after reperfusion, thirty-six rats were anesthetized and perfused trans-cardially with normal saline, followed by 4% paraformaldehyde in phosphate buffer saline. The brains were harvested, routinely processed, and embedded in paraffin. Coronal sections were cut for H&E staining and Nissl staining in order to determine morphological changes under light microscopy.¹⁰

Western blotting

Thirty-six rats were sacrificed at 24 and 72 hours after reperfusion. The brains were carefully removed, placed in chilled saline, dissected into the penumbra and then snap-frozen in liquid nitrogen. For sample preparation, the tissue was homogenized in buffer with a protease inhibitor (Sigma). The samples were separated by electrophoresis on 12% SDS-polyacrylamide gels (Bio-Rad, USA) and then transferred to nitrocellulose membranes (Pierce, USA). The immunoblots were incubated with anti-HMGB1 (Abcam, USA) or anti-TNF-α (Abcam). Then the blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (SantaCruz, USA). Protein signals were visualized with an enhanced chemiluminescence system (Pierce).¹¹

Statistical analysis

NDSs were presented as median and quartile range, other data were presented as mean ± standard deviation (SD). All data were analyzed by SPSS18.0 (SPSS Inc., USA) using Kruskal-Wallis H test and analysis of variance (ANOVA), respectively. P <0.05 was considered statistically significant.

RESULTS

NDSs and cerebral infarct volume

There were no significant differences in NDSs between groups before ischemia. Rats in the sham group had no neurological deficit nor infarction. Cerebral infarct volume was larger (P <0.05) and NDSs were lower (P <0.05) in the I/R group than in the sham group. The animals in the parecoxib group had smaller cerebral infarcts (P <0.05) and higher NDSs (P <0.05) than those in the I/R group (Table and Figure 1).

Table

Figure 1.

H&E staining and Nissl staining

In the sham group, neither neuronal damage nor 2006 Chin Med J 2011;124(13):2004–2008 inflammatory cell infiltration were observed. Neurons were eumorphism with normal cellular architecture. The nuclei were in the cell center and clearly stained. In the I/R group, the brain tissue was characterized by a majority of degenerated and necrotic cells, which contained pyknotic nuclei, cavitation with neuronal loss and they were disordered. In the parecoxib group, the number of normal neurons was markedly increased compared to the I/R group, and the extent of damage was significantly diminished as well (Figure 2A).

Figure 2.

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).

Western blotting

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).

Figure 3.

DISCUSSION

In the current study, we examined the neuroprotective effects of intravenous parecoxib in a rat model of pMCAO. The rats treated with intravenous parecoxib had a better neurological outcome and smaller cerebral infarct volume. The post-ischemic HMGB1 and TNF-α expression in the penumbra were consistently and significantly lower in the parecoxib-treated than in the saline-treated animals.

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.

In summary, intravenous administration of parecoxib, a selective COX-2 inhibitor, improved neurological outcome and reduced infarct size in a rat model of pMCAO. The neuroprotective effects of parecoxib may be associated with the attenuation of inflammatory reaction and the inhibition of inflammatory mediators.

REFERENCES