Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation.

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1ß (IL-1ß), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1ß synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.

Elevated beta1,4-galactosyltransferase-I induced by the intraspinal injection of lipopolysaccharide.

Beta1,4-galactosyltransferase-I (beta1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that beta1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of beta1,4-GalT-I deficient mice were impaired. In this study, we investigate the expression of beta1,4-GalT-I in lipopolysaccharide (LPS)-induced neuroinflammatory processes. The results of this study demonstrated that beta1,4-GalT-I was strongly induced by intraspinal administration of LPS. More than 90% galactose-containing glycans and beta1,4-GalT-I were expressed in immune cells. The ELISA assay shows focal injection LPS also induces TNF-alpha alteration. Double staining indicated beta1,4-GalT-I overlapped with TNF-alpha. Moreover, RT-PCR for beta1,4-GalT-I mRNA showed that beta1,4-GalT-I mRNA in microglia in vitro was affected in a dose- and time dependent manner in response to LPS or TNF-alpha stimulation. All these results indicated that the increase of beta1,4-GalT-I might attribute to the effect of TNF-alpha excreting during inflammation. E-selectin, which ligand was modified by beta1,4-GalT-I, was correlated with galactose-containing glycans following injecting LPS into spinal cord. We therefore suggest that beta1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site.

Persistent accumulation of cyclooxygenase-1 (COX-1) expressing microglia/macrophages and upregulation by endothelium following spinal cord injury.

Acute inflammation following spinal cord injury results in secondary injury and pathological reorganisation of the central nervous system (CNS) architecture. Cyclooxygenases (Prostaglandin Endoperoxide H Synthases, PGH) are key enzymes in the conversion of arachidonic acid into prostanoids which mediate immunomodulation, mitogenesis, apoptosis, blood flow, secondary injury (lipid peroxygenation) and inflammation. Here, we report cyclooxygenase-1 (COX-1) expression following spinal cord injury. In control spinal cords, COX-1 expression was localized by immunohistochemistry to ependymal cells, some neurons, inclusive dorsal and ventral root ganglion cells, few endothelial cells but rarely to brain microglia/macrophages. In injured spinal cords, COX-1(+) microglia/macrophages accumulated highly significantly (P<0.0001) at peri-lesional areas and in the developing necrotic core early after injury. Here numbers of COX-1(+) cells remained persistently elevated up to 4 weeks following injury. Further, COX-1(+) cells were located in perivascular Virchow-Robin spaces, between spared axons and in areas of Wallerian degeneration. Double labeling experiments confirmed co-expression of COX-1 by ED-1(+) and OX-42(+) microglia/macrophages. Transiently after infarction most COX-1(+) microglia/macrophages coexpress the activation antigen OX-6 (MHC class II). However, the prolonged accumulation of COX-1(+) microglia/macrophages at the lesion site enduring the acute post injury inflammatory response points to a role of COX-1 in tissue remodeling or secondary injury. We have identified and localized persistent accumulation of COX-1 expressing cells which might be a potential pharmacological target following spinal cord injury. Therefore, we suggest that approaches based on: (i) short-term; and (ii) selective COX-2 blocking alone might not be a sufficient tool to suppress the local synthesis of prostanoids.

Expression and action of cyclic GMP-dependent protein kinase Ialpha in inflammatory hyperalgesia in rat spinal cord.

Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an important role in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.