Cyclooxygenase-2 inhibition provides lasting protection against neonatal hypoxic-ischemic brain injury

Abstract

Objective: To investigate whether inhibition of cyclooxygenase-2, a critical component of the inflammatory pathway, is neuroprotective in a neonatal rat model of cerebral hypoxia-ischemia. The development of brain inflammation largely contributes to neonatal brain injury that may lead to a lifetime of neurologic deficits.

Design: Laboratory investigation.

Setting: University research laboratory.

Subjects: Postnatal day ten Sprague-Dawley rats.

Interventions: Neonatal hypoxia-ischemia was induced by ligation of the right common carotid artery followed by 2 hrs of hypoxia (8% oxygen). The pups in treatment groups were administered 10 mg/kg (low dose) or 30 mg/kg (high dose) of a known selective cyclooxygenase-2 inhibitor (NS398). Animals were euthanized at three time points: 72 hrs, 2 wks, or 6 wks. Inflammation outcomes were assessed at 72 hrs; brain damage was assessed at 2 wks and 6 wks along with other organs (heart, spleen). Detailed neurobehavioral examination was performed at 6 wks.

Measurements and main results: Pharmacologic inhibition of cyclooxygenase-2 markedly increased survivability within the first 72 hrs compared with untreated rats (100% vs. 72%). Low- and high-dose NS398 significantly attenuated the loss of brain and body weights observed after hypoxia-ischemia. Neurobehavioral outcomes were significantly improved in some parameters with low-dose treatment, whereas high-dose treatment consistently improved all neurologic deficits. Immunohistochemical results showed a marked decrease in macrophage, microglial, and neutrophil abundance in ipsilateral hemisphere of the NS398-treated group along with a reduction in interleukin-6 expression.

Conclusions: Selective cyclooxygenase-2 inhibition protected neonatal rats against death, progression of brain injury, growth retardation, and neurobehavioral deficits after a hypoxic-ischemic insult.

Figure 1

Long-term effects of NS398 on body weight. Postnatal day-10 rats were induced with a hypoxic-ischemic (HI) event: ligation of right common carotid artery and 2hrs hypoxia (8% O2) [Vehicle], then treated with 6 intraperitoneal injections (1, 6, 24, 36, 48, and 60hrs post-hypoxia) of a selective cyclooxygenase-2 (COX-2) inhibitor at either 10mg/kg [NS-10] or 30mg/kg [NS-30] dosage. Sham animals had same anesthesia and surgical procedure, except that the common carotid artery was not ligated. A, Vehicle animals had a significantly lower mean body weight at 2-, 3- and 6wks, compared to sham (*p < .05). NS-30 had long-term lasting effects as it maintained body weight compared to vehicle 6wks after HI (#p < .05). Only mean body weights of animals kept through the 6wk time-point are represented (n = 9/group). B, Differences in fur texture and appearance were detected as early as 2wks between treated (T) and untreated rats (U). C, Is a close-up picture demonstrating the somatic differences between treated and untreated rats at 2wks.

Figure 2

Dose-dependent effect of NS398. Brain and organ tissue of postnatal day-10 rats induced with a hypoxic-ischemic (HI) event [Vehicle], then treated with a selective cyclooxygenase-2 (COX-2) inhibitor at either 10mg/kg [NS-10] or 30mg/kg [NS-30] dosage were examined. Sham animals were used as control. A, NS-10 and NS-30 maintained the gross morphology of the rat brains at 2- and 6wks post-HI. Representative pictures of heart and spleen are shown of all groups. B, Right to left hemispheric (RH:LH) weight ratio is representative of brain atrophy. At 2wks post-insult, vehicle rats had a significantly reduced RH:LH ratio compared to sham (0.67 ± .05 vs. 1.00 ± .01). This was attenuated by NS-10 (0.88 ± .04) and NS-30 (0.93 ± .03). At 6wks post-insult, vehicle rats had a significantly reduced RH:LH ratio compared to sham (0.49 ± .04 vs. 1.00). This was attenuated by treatment (NS-10:0.78 ± .08; NS-30:0.87 ± .06). C, Vehicle rats had a significantly reduced heart to body weight ratio (0.0037 ± .0001 vs. 0.0041 ± .0001) and spleen to body weight ratio (0.0026 vs. 0.0030 ± .0001) as compared to sham, at 6wks post-insult. Treatment increased the heart to body weight ratio (NS-10:0.0045 ± .0001; NS-30:0.0040 ± .0001) and the spleen to body weight ratio (NS-10: 0.0029 ± .0002; NS-30: 0.0029 ± .0002) at 6wks post-insult. Data represent mean ± SEM; *p < .05 versus sham, #p < .05 versus vehicle. Numbers in bars indicate animals/group.

Figure 3

Long-term neurobehavioral effects of postnatal day-10 rats induced with a hypoxic-ischemic (HI) event [Vehicle] then treated with a selective cyclooxygenase-2 (COX-2) inhibitor at either 10mg/kg [NS-10] or 30mg/kg [NS-30] dosage were examined at 6wks post-insult. Sham animals were used as control. A, Rats received a raw score of 100 for immediate and correct placement; 50 for delayed and/or incomplete placement; 0 for no placement (n = 9/group). Administration of NS-30 significantly improved all assessed behavior deficits; while, NS-10 significantly improved deficits associated with the postural reflex test. Data represent *p < .05 versus sham, #p < .05 versus vehicle. B, Vehicle rats alternated significantly less between the two arms of the maze as compared to sham (18.52% ± 5.56 vs. 82.72% ± 2.69). Percent alternations significantly rose with administration of NS-10 (53.09% ± 3.09) and more so by NS-30 (81.48% ± 2.62). Data represent mean ± SEM; *p < .05 versus sham, #p < .05 versus vehicle, ^◆p < 0.05 versus NS-10. C, Vehicle rats averaged the greatest number of foot-faults (32.33 ± 3.49), while treatment significantly reduced the deficit (NS-10:12.29 ± 1.70; NS-30:10.86 ± .86). Sham animals had an average of 10.67 ± 1.05 foot-faults. Data represent mean ± SEM; *p < .05 versus sham, #p < .05 versus vehicle. Numbers in bars indicate animals/group.

Figure 4

Cyclooygenase-2 (COX-2) expression in postnatal day-10 rats induced with a hypoxic-ischemic (HI) event [Vehicle], then treated with a selective COX-2 inhibitor at 30mg/kg [NS-30] dosage were examined at 72hrs post-insult. Sham animals were used as control. A, Diaminobenzidine (DAB) stain for COX-2 in ipsilateral cerebral cortex (D-F) and CA1 region of hippocampus (G-I) qualitatively appears less in sham (A,D,G) and NS-30 (C,F,I), as compared to vehicle (B,E,H). Six non-adjacent coronal sections per brain (n = 5/group) were analyzed. B, Western blotting supported a statistically significant reduction in COX-2 expression in the ipsilateral hemisphere of NS-30 compared to vehicle (106.92 ± 10.48 vs. 185.20 ± 19.54). Sham (120.88 ± 9.30) also had significantly less COX-2 expression than the vehicle group. Data represent *p < .05 versus sham, #p < .05 versus vehicle. Numbers in bars indicate animals/group.

Figure 5

Co-localization of cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) in neuronal cells (NeuN) of postnatal day-10 rats induced with a hypoxic-ischemic (HI) event [Vehicle], then treated with a selective COX-2 inhibitor at 30mg/kg [NS-30] dosage were examined at 72hrs post-insult. Sham animals were used as control. Six non-adjacent coronal sections per brain (n = 5/group) were analyzed. Brain slice in upper right corner of figure denotes the specific cortical and hippocampal area the immunofluorescent pictures represent. A, Immunoreactivity is shown of COX-2, IL-6 and NeuN in ipsilateral CA1 region of hippocampus. Vehicle (A-H) demonstrated strong neuronal fluorescence for COX-2 and IL-6; NS-30 (I-L) demonstrated strong fluorescence for NeuN, but weak fluorescence for COX-2 and IL-6. Sham animals are shown in subsets of (A-C). B, Immunoreactivity is shown of COX-2, IL-6 and NeuN in ipsilateral cerebral cortex. Vehicle (A-H) demonstrated a strong co-localization of COX-2, IL-6, and NeuN. NS-30 (I-L) treatment reduced signals of COX-2 and IL-6. Sham animals are shown in subsets of (A-C).

Figure 6

Interleukin-6 (IL-6) protein levels and inflammatory cell infiltration in the ipsilateral brain of postnatal day-10 rats induced with a hypoxic-ischemic (HI) event [Vehicle], then treated with a selective COX-2 inhibitor at 30mg/kg [NS-30] dosage were examined at 72hrs post-insult. Sham animals were used as control. A, Analysis by ELISA technique showed significant increase of IL-6 in the ipsilateral cerebral hemisphere of vehicle rats as compared to sham (22.61pg/mg ± 4.60 vs. 4.54pg/mg ± .77). Treatment with NS-30 markedly reduced IL-6 concentration (7.42pg/mg ± 1.44). Data represent *p < .05 versus sham, #p < .05 versus vehicle. Numbers in bars indicate animals/group. B, Vehicle (A-F) pups showed marked activation of microglia (Iba1; A and D), and infiltration of macrophages (CD68; B and E) and neutrophils (MPO; C and F) in the ipsilateral cerebral cortex. NS-30 qualitatively reduced expression of all three cell markers of inflammation (G-I). Sham animals are shown in subsets of (A-C). Six non-adjacent coronal sections per brain (n = 5/group) were analyzed. Brain slice in upper left corner of Fig. 3B denotes the specific cortical area the immunofluorescent pictures represent.