To our knowledge, this is the first systematic study performed to characterize the expression and cellular localization of COX and PGES isozymes in the hemorrhagic brain of mice. Using immunofluorescence staining, we observed constitutive expression of COX-1, mPGES-2, and cPGES in neurons; COX-1 was also constitutively expressed in microglia. In contrast, COX-2 and mPGES-1 immunoreactivity, which was minimal in the normal brain, underwent distinct time-dependent changes in neurons and astrocytes of the perihematomal region during the first 3 days post-ICH. Our data support the premise that the COX/PGES signaling pathway contributes to ICH pathology.
COX-1 is constitutively expressed in most tissues . We demonstrated for the first time that COX-1 is constitutively expressed in neurons and microglia in the hemorrhagic brain. Although involvement of COX-1 in ICH pathology has not been studied, the evidence that COX-1 is produced in microglia of the perihematomal region implies a toxic role of COX-1 in the pathophysiology of the disease. This hypothesis is based on the fact that activated microglia/macrophages are the major sources of proinflammatory mediators [1, 3] and that inhibition of microglial activation before or early after ICH decreases neuronal death and improves neurologic function [21, 25]. We therefore propose that microglial COX-1 might immediately initiate synthesis of prostaglandins in response to microglial activation and could be considered one of the major players in mediating neuroinflammation after ICH.
In the normal brain, COX-2 is constitutively expressed in neurons of the cortex, hippocampus, and striatum . COX-2 is mainly induced in response to inflammatory stimuli; deletion of the COX-2 gene or selective COX-2 inhibition reduces infarction volume and neuronal death after cerebral ischemia [26–28]. To our knowledge, only two studies have investigated post-ICH COX-2 expression, and the results were conflicting [18, 19]. Zhao et al.  demonstrated that COX-2 mRNA and protein were increased within 3 h after ICH and that COX-2 immunoreactivity was increased in blood vessels and neurons in the perihematomal region at 4 h; the increase in immunoreactivity was transient, followed by a significant down-regulation at days 1 and 3. In the present study, we found that the immunoreactivity of COX-2 in astrocytes increased gradually in the perihematomal region from 5 h to 3 days, whereas neuronal COX-2 increased only transiently at 5 h after ICH. Most of our results are consistent with the findings by Gong et al. , except that we found COX-2 to be increasingly induced in astrocytes from 1 to 3 days, whereas Gong et al. reported that COX-2 was induced in endothelial cells, perivascular cells, and infiltrating leukocytes at 1 day after blood infusion. The reason for the discrepancy between the previous studies and our own is not clear, but it may be a result of differences in ICH models and species used and the size of the intrastriatal hematoma formed. In the two previous studies, investigators modeled ICH by injecting rats intrastriatally with differing amounts of autologous blood. In contrast, we used a collagenase-induced ICH model in mice that may cause gradual hematoma growth over the first few hours with subsequent inflammation. Our data suggest that astrocytic COX-2 in concert with microglial COX-1 might contribute to collagenase-induced post-hemorrhagic neuroinflammation. More studies are warranted to understand whether the functions of neuronal and astrocytic COX-2 differ after ICH.
Induction of mPGES-1 expression has been observed in various conditions, such as inflammation, fever, pain, tissue repair, and cancer, in which COX-2-derived PGE2 plays a critical role . In the ischemic brain, mPGES-1 and COX-2 are both induced in neurons, microglia, and endothelial cells in the ipsilateral cerebral cortex and striatum . It has been confirmed that mPGES-1 and COX-2 are co-localized and co-induced in the infarct region of the cortex, and it has been suggested that they act together to exacerbate stroke injury . At 1 day post-ICH in our model, mPGES-1 expression in the ipsilateral cortex was elevated primarily in neurons whereas in the perihematomal region, it was elevated in astrocytes. Astrocytic expression continued to increase for at least 3 days. However, we observed no apparent changes in the expression of neuronal mPGES-2 or cPGES. The different cellular expression profiles for mPGES-1 between cortex and striatum suggests that mPGES-1 may be involved in different signaling pathways within the cortical neurons and striatal astrocytes after ICH. In line with the results from a lipopolysaccharide-induced inflammation model , we found that the induction of COX-2 protein expression was more rapid than that of mPGES-1 in the hemorrhagic brain. These results suggest that the sequential up-regulation and co-induction of COX-2 and mPGES-1 in astrocytes in the perihematomal region might contribute to inflammation-mediated secondary brain injury after ICH, possibly through excessive PGE2 production.
In conclusion, our data provide novel evidence that COX-1, mPGES-2, and cPGES are constitutively expressed in the hemorrhagic brain; COX-2 is induced early in neurons and later in astrocytes. Although neuronal COX-2 is induced earlier than astrocytic COX-2, the latter is induced in parallel with astrocytic mPGES-1. Together, our data suggest that microglial COX-1, neuronal COX-2, astrocytic COX-2, and astrocytic mPGES-1 may work sequentially to affect ICH outcomes. Based on our previous observations that neuroinflammation affects the normal function of the entire brain in patients with lethal ICH , these findings have implications for efforts to develop anti-inflammatory strategies that target the COX/PGES pathway to reduce ICH-induced secondary brain damage. Indeed, a recent study showed that inhibition of COX-2 attenuated inflammation, neuronal death, and gliosis and promoted long-term recovery in motor function and myelination in rabbit pups with intraventricular hemorrhage .