Abstract
Serrano GE, Lelutiu N, Rojas A, Cochi S, Shaw R, Makinson CD, Wang D, Fitzgerald GA, Dingledine R. J Neurosci 2011;31(42):14850–14860.
Cyclooxygenase-2 (COX-2), a source of inflammatory mediators and a multifunctional neuronal modulator, is rapidly induced in select populations of cortical neurons after status epilepticus. The consequences of rapid activity-triggered induction of COX-2 in neurons have been the subject of much study and speculation. To address this issue directly, we created a mouse in which COX-2 is conditionally ablated in selected forebrain neurons. Results following pilocarpine-induced status epilepticus indicate that neuronal COX-2 promotes early neuroprotection and then delayed neurode-generation of CA1 pyramidal neurons, promotes neurodegeneration of nearby somatostatin interneurons in the CA1 stratum oriens and dentate hilus (which themselves do not express COX-2), intensifies a broad inflammatory reaction involving numerous cytokines and other inflammatory mediators in the hippocampus, and is essential for development of a leaky blood-brain barrier after seizures. These findings point to a profound role of seizure-induced neuronal COX-2 expression in neuropathologies that accompany epileptogenesis.
Jiang J, Ganesh T, Du Y, Quan Y, Serrano G, Qui M, Speigel I, Rojas A, Lelutiu N, Dingledine R. [Published ahead of print on February 12 2012, Proc Natl Acad Sci USA.]
With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2–mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE2) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with KB 2–20 nM in Schild regression analysis and 268- to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.
Commentary
It is well established that seizures, particularly status epilepticus (SE), induce upregulation of cyclooxygenase-2 (COX-2) in both neurons and non-neuronal cells (1, 2). COX-2 plays a role in seizure-induced leukocyte infiltration, astrogliosis, microglial activation, and breakdown of the blood-brain barrier (BBB); thus, COX-2 is clearly an important component of brain inflammation. COX-2 is also thought to play an important role in seizure-induced neurodegeneration; in fact, COX-2 inhibitors generally reduce neuronal death and provide neuroprotection. Although COX-2 is thought to be expressed primarily in non-neuronal cells, it is also expressed in neurons; furthermore, seizures strongly enhance neuronal COX-2 (nCOX-2) expression. An important question addressed by the present papers is the role of nCOX-2 in brain inflammatory mechanisms, which was studied in a mouse model in which COX-2 was conditionally ablated in specific forebrain neurons. One important finding is that neuron-specific conditional COX-2 knock-out mice (nCOX-2 cKO) showed less neuronal death than wild-type controls, which suggests that nCOX-2 contributes to seizure-induced neurodegeneration. A less expected and maybe more important outcome was prevention of BBB opening. This latter result provides the beginning of a molecular understanding of how neurons control the endothelial cells and astrocytes that form the BBB.
One hypothesis investigated in these studies is that nCOX-2 has a biphasic effect on seizure-induced neuronal death. In an initial set of studies, the properties and consequences of pilocarpine-induced repetitive seizures (i.e., SE) were unaltered in the nCOX-2 cKO mice, which allowed investigation of the effects of SE on subsequent neuronal death. The data, which focused upon hippocampal CA1 pyramidal cells as the principal neurons, suggest that COX-2 mediates an early form of neuroprotection, which is then followed after a delay by neurodegeneration. This interpretation is based primarily on neuronal counts (e.g., FluroJade) at 1 day versus 4 days after SE. As the authors point out, the initial neuroprotective effect was not statistically significant and was only a trend, and there was a more robust neurodegenerative effect at the 4-day time point. Although these data suggest a neuroprotective effect, the authors make cautionary statements that deserve attention, and they emphasize the difficulty in quantitatively assessing the effects of SE on neuronal death, given the substantial variability from animal to animal. The possibility that COX-2 and other molecules in inflammatory pathways have biphasic effects on neuronal survival/death deserves additional investigation; validation of this result would add additional complexity to this already complicated pathway.
Somatostatin-expressing GABAergic interneurons are known to be highly sensitive to repetitive seizures, and their loss is a common feature of epileptic tissue in both animal models and resected samples from humans with intractable temporal lobe epilepsy. The nCOX-2 cKO mice showed preservation of somatostatin-expressing interneurons after pilocarpine-induced SE, unlike the wild-type mice. Therefore, although COX-2 did not appear to be expressed in GABAergic interneurons, nCOX-2 surprisingly appeared to be involved in their degeneration. This suggests that COX-2 expression in principal cells, such as CA1 pyramidal cells and dentate granule cells, somehow plays a role in the seizure-induced degeneration of GABAergic interneurons. This observation leads to a new level of complexity in the inflammatory response to repetitive seizures, because these interneurons are one of the few cell types that apparently do not express COX-2. The mechanism by which nCOX-2 contributes to—or even mediates—sensitivity to repetitive seizures in these interneurons also deserves more study.
An important product of COX-2 is prostaglandin E2. This prostaglandin activates four different receptors, EP1 through EP4. Other evidence has already suggested that activation of EP2 is neuroprotective (3). In pilocarpine-treated rats, in-traventricular injection of butaprost, a selective EP2 agonist, immediately after termination of SE reduced the number of FluoroJade-stained neurons by 50%. These data support the hypothesis that EP2 receptors are important in nCOX-2–mediated neurodegeneration. Recent data show that delayed administration of an EP2 antagonist, timed to match the peak of COX-2 induction (approximately 5 hours after the seizures), is strongly neuroprotective when examined 4 days after SE. Thus, EP2 receptor modulators, similar to other anti-inflammatory compounds (4), may be efficacious neuroprotective drugs.
The field of brain inflammation continues to show increasing levels of complexity, but how this will be used to suppress epileptogenesis after a brain injury or treat pharmacoresistant epilepsy remains unclear. When considering global inhibition of COX-2 by systemic administration of inhibitors, data from several laboratories point to a role in seizure-induced neurodegeneration. The degree to which neuronal death leads to inflammation, or inflammation contributes to neuronal death, or both needs additional clarification, particularly in regard to the mechanisms of epileptogenesis. For example, an in vitro model of epileptogensis that is based on the damage inherent in preparation of organotypic slices has little or no inflammatory response but consistently develops robust spontaneous recurrent seizure-like events in a time-dependent manner (5). Although injury-induced epileptogenesis can occur in vitro with little or no inflammatory response, inflammation may still contribute to epileptogenesis in the intact animal or human. One could speculate that the severity and type of inflammation (i.e., which cytokines are released, by which cells, and when) may influence the amount of neuronal death, which then secondarily affects epileptogenesis and/or its rate of progression.
What is needed is the determination of whether experimental manipulation of inflammatory mechanisms after a brain injury (e.g., severe SE) affects the time-dependent development of spontaneous recurrent seizures in an animal model of acquired epilepsy (i.e., disease modification). If inflammatory mechanisms are important in the generation of the spontaneous recurrent seizures that define epilepsy, experimental manipulation of these mechanisms after a brain insult ought to alter the frequency and severity of the spontaneous seizures. In fact, if disruption of the BBB and associated inflammatory mechanisms are important in epilepsy, a direct investigation of the role of anti-inflammatory agents in the development of chronic epilepsy and the maintenance of spontaneous epileptic seizures in patients with acquired epilepsy ought to be no more difficult (and ought to be associated with no more risk) than a standard clinical trial of an antiepileptic drug. One difficulty is that inflammation is multidimensional, with beneficial as well as detrimental consequences. An analogy with infection might be appropriate: The inflammatory process that causes chemotaxis of immune cells to the site of infection is initially beneficial in scavenging the invading bacteria, but prolonged inflammation with consequent tissue swelling can cause structural changes that impair normal function.