Molecular species of prostaglandins involved in modulating luteinising hormone pulses of female rats under infectious stress conditions.

Mammalian reproductive function is controlled by the hypothalamic-pituitary-gonadal (HPG) axis, which is suppressed under infectious stress conditions. By analysing the pulsatility of luteinising hormone (LH), we have previously demonstrated that prostaglandins (PGs) in the central nervous system mediate infectious stress to suppress the activity of the HPG axis. The present study aimed to characterise the types of PGs responsible for suppression of the HPG axis. We focused on three major types of PGs: PGE2 , PGD2 and PGF2α . We used female rats overiectomised bilaterally 1 week before the experiments. Lipopolysaccharide (100 µg kg-1 ) suppressed LH pulses at the same time as enhancing the concentration of all three PGs in the cerebrospinal fluid, which was restored by indomethacin (10 mg kg-1 ). Subsequently, we observed LH pulsatility after a single injection of each PG and after co-injection of PGE2 with PGF2α into the third cerebral ventricle. A single injection of PGE2 dose-dependently induced a transient increase in mean LH concentration and LH pulse amplitude, and PGD2 significantly increased the amplitude of LH pulses, wereas PGF2α did not affect LH pulsatility. On the other hand, co-injection of PGE2 and PGF2α induced a significant suppression of both the frequency and amplitude of LH pulses. These results suggest that PGE2 and PGF2α can represent two of the mediators that suppress the HPG axis in situations of infectious stress. Moreover, the results imply that there are two contradictory effects of PGE2 on LH pulsatility: (i) enhancive when working alone and (ii) suppressive when working together with PGF2α .

Increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN) is known to play a role in the pathogenesis of neurodegenerative diseases. Recently, it has been demonstrated that patients with the homozygous mutation in the GRN gene present with neuronal ceroid lipofuscinosis, and there is growing evidence that PGRN is related to lysosomal function. In the present study, we investigated the possible role of PGRN in the lysosomes of activated microglia in the cerebral cortex after traumatic brain injury (TBI). We showed that the mouse GRN gene has two possible coordinated lysosomal expression and regulation (CLEAR) sequences that bind to transcription factor EB (TFEB), a master regulator of lysosomal genes. PGRN was colocalized with Lamp1, a lysosomal marker, and Lamp1-positive areas in GRN-deficient (KO) mice were significantly expanded compared with wild-type (WT) mice after TBI. Expression of all the lysosome-related genes examined in KO mice was significantly higher than that in WT mice. The number of activated microglia with TFEB localized to the nucleus was also significantly increased in KO as compared with WT mice. Since the TFEB translocation is regulated by the mammalian target of rapamycin complex 1 (mTORC1) activity in the lysosome, we compared ribosomal S6 kinase 1 (S6K1) phosphorylation that reflects mTORC1 activity. S6K1 phosphorylation in KO mice was significantly lower than that in WT mice. In addition, the number of nissl-positive and fluoro-jade B-positive cells around the injury was significantly decreased and increased, respectively, in KO as compared with WT mice. These results suggest that PGRN localized in the lysosome is involved in the activation of mTORC1, and its deficiency leads to increased TFEB nuclear translocation with a resultant increase in lysosomal biogenesis in activated microglia and exacerbated neuronal damage in the cerebral cortex after TBI.

Exacerbated inflammatory responses related to activated microglia after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN), a multifunctional growth factor, appears to play a role in neurodegenerative diseases accompanied by neuroinflammation. In this study, we investigated the role of PGRN in neuroinflammation, especially in the activation of microglia, by means of experimental traumatic brain injury (TBI) in the cerebral cortex of mice. The expression of GRN mRNA was increased in association with neuroinflammation after TBI. Double-immunohistochemical study showed that PGRN-immunoreactive (-IR) cells were mainly overlapped with CD68-IR cells, suggesting that the main source of PGRN was CD68-positive activated microglia. To investigate the role of PGRN in inflammatory responses related to activated microglia, we compared the immunoreactivity and expression of ionized calcium-binding adaptor molecule 1 (Iba1), CD68, and CD11b as markers for activated microglia between wild-type (WT) and GRN-deficient (KO) mice. The number of Iba1- and CD11b-IR cells and gene expression of Iba1 and CD11b were not significantly different between WT and KO mice, while the number of CD68-IR cells and CD68 expression in KO mice were significantly greater than those in WT mice. Double-immunohistochemical study showed that CD68-IR microglia were also IR for TGFß1, and TGFß1 expression and Smad3 phosphorylation in KO mice were elevated compared to WT mice. Moreover, double-immunostaining between phospho-Smad3 and glial fibrillary acidic protein suggested increased TGFß1-Smad3 signal mainly by astrocytes. The levels of protein carbonyl groups, which reflect protein oxidation, and laminin immunoreactivity, which is associated with angiogenesis, were also significantly increased in KO mice compared to WT mice. These results suggest that PGRN is produced in CD68-positive microglia and suppresses excessive inflammatory responses related to activated microglia after TBI in mice.

Progranulin enhances neural progenitor cell proliferation through glycogen synthase kinase 3ß phosphorylation.

Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3ß), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3ß-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3ß phosphorylation.

Possible involvement of microglia containing cyclooxygenase-1 in the accumulation of gonadotrophin-releasing hormone in the preoptic area in female rats.

Prostaglandins (PGs), especially PGE(2), are involved in the hypothalamic control of gonadotrophin-releasing hormone (GnRH) release, acting at least in part on the terminal of GnRH axons in the median eminence. The present study aimed: (i) to clarify the role of PG(s) in regulating GnRH cell function at the level of the perikarya in the preoptic area; (ii) to determine the cyclooxygenase (COX) isozyme responsible for producing PG(s) that regulates GnRH perikarya; and (iii) to identify cell types that contain the responsible COX isozyme in female rats. A surge of luteinising hormone (LH) secretion was induced by oestrogen and progesterone in ovariectomised rats. Treatment of the rat before the LH surge with indomethacin, a nonselective COX inhibitor, or NS-398, a selective COX-2 inhibitor, did not interfere with the surge. However, treatment with indomethacin or flurbiprofen, a selective COX-1 inhibitor, significantly reduced the number of GnRH-immunoreactive cells in the preoptic area at the time of peak LH secretion during the surge. NS-398 did not affect the GnRH immunoreactivity. Double-labelled immunofluorescent histochemistry revealed COX-1 immunoreactivity in the vicinity of, but not within, GnRH containing neurones in the preoptic area. COX-2 immunoreactivity was not found in the same area. The COX-1 immunoreactivity was almost entirely localised in microglia in the preoptic area, but not in neurones or astrocytes. These results suggest that microglia in the preoptic area containing COX-1 are responsible for producing PG(s), which, in turn, facilitates the accumulation of GnRH during the gonadotrophin surge in female rats.

Local injection of methotrexate dissolved in saline versus methotrexate suspensions for the conservative treatment of ectopic pregnancy.

To compare the local injection of methotrexate (MTX) dissolved in saline and MTX suspensions for the laparoscopic treatment of ectopic pregnancy in terms of success rate and postoperative tubal patency. A total of 26 patients with unruptured ectopic pregnancies were selected from among 60 women with ectopic pregnancies admitted to the Nagasaki University clinic. Of these patients, 12 were treated with MTX dissolved in saline solution (solution group) and 14 with MTX suspensions consisting of lipiodol (LPD) with phosphatidylcholine (PC) added as a dispersing stabilizer (suspension group). Except for one case treated under transvaginal guidance, all the patients were treated by laparoscopy. Persistent ectopic pregnancy was recognized in seven cases (58%) in the solution group but in only two cases (14%) in the suspension group. Moreover, rupture occurred in two cases in the solution group but in no case in the suspension group. A patent treated tube was found in seven of 10 cases in the saline group and in 10 of 12 cases in the suspension group. During the follow-up period of 6-31 months, five women in the saline group and three women in the suspension group had an intrauterine pregnancy. In this study, the local injection of MTX is considered to be a reasonable method for the treatment of unruptured ectopic pregnancy, and the MTX suspension seems to be more effective and useful than MTX solution.

A new approach to methotrexate and lipiodol suspensions for ectopic pregnancy: preliminary in vitro and animal experiments.

OBJECTIVES: To measure and evaluate the in vitro release of MTX from suspensions of methotrexate (MTX) and lipiodol (LPD), and to investigate the MTX concentration and fertility rate using an animal model. METHODS: This study was divided into three components: a) An in vitro Release Study, b) Animal Study No. 1 (concentration of tissue and plasma levels), and c) Animal Study No. 2 (fertility rate after MTX suspension). RESULT: The releasing rate in vitro was more rapid with MTX-S (MTX dissolved in saline) than with either MTX-LPD (MTX dissolved in lipiodol) or MTX-LPD-PC (MTX suspended in lipiodol and phosphatidylcholin, i.e., MTX suspension). The tissue concentration tended to be higher with an MTX suspension than with MTX-S. There was no significant difference in the fertility rate or the nidation index among the 3 groups (Groups 1, 2, and 3). CONCLUSION: The injection of an MTX suspension is useful for increasing the tissue concentration and maintaining the long-term effectiveness of MTX, and this technique might offer a new approach in the treatment of ectopic pregnancy (EP) or in second-line therapy for persistent EP.