Roles Of Cyclooxygenase-2 And Cyclooxygenase-2 Inhibition NS398 On Neonatal Rats With Hypoxic-Ischemic Brain Damage

Objective: Hypoxic-ischemic brain damage (HIBD) in the human perinatal period often leads to the death of neonates or long-term neurobehavioral dysfunction. But the mechanism of HIBD has not yet been recognized and there are not efficacious means of prevention and treatment. Some researchs have been found some evidences that cyclooxygenase 2 (COX-2) may participate in Hypoxic-ischemic (HI) brain injury, but has not yet been recognized how its phase and expression played.Cyclooxygenase (COX), a rate-limiting enzyme in the synthesis of prostanoids, named prostaglandin synthase (PGS) too, has two types: COX-1 and COX-2, COX-1 reside in most of tissues, is not effected by inductor and steroid hormone; COX-2 is known to be expressed and up-regulated by inductor and steroid hormone, and with few expressed in most of normal tissues. COX-2 only has 61% autoploidy with COX-1, and has a different effective structured Researchs has been confirmed that COX-2 close participate in many diseases such as adult hypertrophic arthritis, rheumarthritis and pain syndrome. Drugs of COX-2 inhibition have been used in clinic treatments. To study roles of COX-2 and highly COX-2 selection inhibition NS398 on neonatal rats after HIBD, a model of HIBD in the left-brain of 7-day-old Wistar rats was established. The research has included observed the numbers of expression cells by immunohistochemistry, measure the numbers of remain nerve cell in the CA1 areas of hippocampus, cell apoptosis was examined with TUNEL method, and study the protective effect of NS398 on neonatal rats with HIBD in different inject times and different inject dose.Methods: 7-day-old Wistar rats were randomly divided into three groups: Normal control group, HIBD treated with saline group (HIBD group) and HIBD treated with NS398 group (treat group), respectively. NS398 group include 5, 20, 40 mg/kg three subgroups injected before HI prepared, and added 2, 6 h injected after HI prepared subgroups which injected the best dose that found in three different dose subgroups before. Rats were killed at the directed time and brain tissues were prepared. Used by the way of hematoxylin-eosin(HE) staining and light-microscopy observed the histological structure and counts cells in the CA1 areas of hippocampus of the ipsilateral hemisphere. Ischemic cerebral hemisphere areas were sampled for analysis 2, 6, 24, 72 h, and 7 d after the onset of HI, after detected COX-2 immunohistochemical cells. Cell apoptosis was examined with TUNEL method.Results: 1. (L:R)area means ratio of left-to-right hemispheric cross-sectional area. Mean (L:R)area values were calculated. Comparison of mean (R:L)area values between the normal control and HIBD group illustrated the HIBD group sustained substantial loss of tussue(p<0.01). NS398 treatment resulted in a significant reduction in tissue loss (p<0.01).2. COX-2 immunostaining is detectable on nerve cells and glial cells. Glial cells are readily identified, concentrated in the cortex and hippocampus. Positive cells gathered nears small vessels is very significantly. Compared with 5, 20, 40 mg/kg NS398 three subgroups injected before HI prepared, 20 mg/kg subgroup has barely different with 40 mg/kg subgroup (p>0.05) , significantly lower than 5 mg/kg subgroup (p<0.05) . 2 h and 6 h 20mg/kg injected after HI prepared subgroups have no significant difference of each other(p>0.05) , and have significant of difference with 20 mg/kg injected before HI prepared subgroups at 24, 72 h and 7d (p<0.05) .3. The HIBD group displayed a pronounced reduction in CA1 neurons number comparied with normal control group (p<0.01). The treat group also displayed a significant CA1 neurons number reduction, but the reduction of the existed of CA1 neurons number was significantly less than that of the HIBD group (p<0.01).4. The percentages of TUNEL-positive cells in HIBD group were higher than that of normal control group (p<0.01). The percentage of TUNEL-positive cell in treat group was significantly decreased when compared with HIBD group (p<0.01). The number of TUNEL-positive cells in 6 h 20 mg/kg injected after HI prepared subgroup > 2 h 20 mg/kg injected after HI prepared subgroup > 5 mg/kg injected before HI prepared subgroup > 20 and 40 mg/kg injected before HI prepared subgroups (p<0.05). 20 and 40 mg/kg injected before HI prepared subgroups have significance of difference each other (p<0.05) .Conclusions: 1. We induced HIBD in neonatal 7-day-old Wistar rats that consisted of unilateral carotid ligation followed by exposure to a hypoxic environment (8% O2) for 2.0 hours. Histopathologic examination of the ligated side of brains showed atrophy, regional infarction of cortex and hippocampus, and some neurons and glial cells showed signs ofnecrosis or apoptosis. These all confirmed that the establishment of neonatal rats model of HIBD was successful.2. HIBD rats have pronounced upregulation of COX-2 expression positive cells after HI prepared at 2 h, pecked between 6-24 h significantly, but declined at 72 h, keeped on to 7 d. Positive glial cells gathered nears small vessels is very significantly. It can be means COX-2 has a close-up association with vascular inflammatory factors and vascular endothelial cells, and joint action each other probablly.3. NS398 treatment can reduce neurons damage after HI treatment, which can valid to decrease the area of atrophy after HI, the lost number of CA1 neurons and TUNEL-positive cells. That means NS398 has the effective capability to treat with the brain damage in the growth period, and also explained that COX-2 close to participate HI damage.4. Compared with 5,20,40 mg/kg NS398 three subgroups injected before HI prepared, 20 mg/kg is a most rational choice in three doses.5. We can conclude that therapeutic effect of NS398 inject before HI is more than inject when HI has happened. The effect of NS398 inject after HI 2 h is more than inject after HI 6 h, means the time of therapy more close to HI is more better.6. We have confirmed that COX-2 plays a significant role in the pathological process of HIBD. Highly selection inhibition of COX-2 can inhibit COX-2 effect and effectually to treat with the brain damage when HI happened. We conclusion that drugs of the highly selection inhibition of COX-2 would be an available choice to heal clinical hypoxic-ischemic encephalopathy (HIE) patients in the future.