Changjong Moon

Cyclophosphamide impairs hippocampus-dependent learning and memory in adult mice: Possible involvement of hippocampal neurogenesis in chemotherapy-induced memory deficits

Cyclophosphamide (CYP) is an anti-neoplastic agent as well as an immunosuppressive agent. In order to elucidate the alteration in adult hippocampal function following acute CYP treatment, hippocampus-related behavioral dysfunction and changes in adult hippocampal neurogenesis in CYP-treated (intraperitoneally, 40 mg/kg) mice (8-10-week-old ICR) were analyzed using hippocampus-dependent learning and memory tasks (passive avoidance and object recognition memory test) and immunohistochemical markers of neurogenesis (Ki-67 and doublecortin (DCX)). Compared to the vehicle-treated controls, mice trained at 12h after CYP injection showed significant memory deficits in passive avoidance and the object recognition memory test. The number of Ki-67- and DCX-positive cells began to decrease significantly at 12h post-injection, reaching the lowest level at 24h after CYP injection; however, this reverted gradually to the vehicle-treated control level between 2 and 10 days. We suggest that the administration of a chemotherapeutic agent in adult mice interrupts hippocampal functions, including learning and memory, possibly through the suppression of hippocampal neurogenesis.

The involvement of Nrf2 in the protective effects of diallyl disulfide on carbon tetrachloride-induced hepatic oxidative damage and inflammatory response in rats

This study investigated the potential effect of diallyl disulfide (DADS) against carbon tetrachloride (CCl4)-induced oxidative hepatic damage and inflammatory response in rat liver. DADS at doses of 50 and 100 mg/kg/day was administered orally once daily for 5 days, prior to CCl4 administration. Pretreatment with DADS attenuated CCl4-induced elevated serum transaminase activities and histopathological alterations in liver. It prevented the hepatocellular apoptotic changes with induction of Bcl-2-associated X (Bax), cytochrome c, and caspase-3 caused by CCl4. An increase in the nuclear translocation of nuclear factor-kappaB (NF-κB) and phosphorylation of I kappaB alpha (IκBα) was observed in the livers of CCl4-treated rats that coincided with induction of inflammatory mediators or cytokines. In contrast, DADS inhibited NF-κB translocation and IκBα phosphorylation, and that subsequently decreased inflammatory mediators. Furthermore, DADS prevented CCl4-induced depletion of cytosolic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2, which, in turn, up-regulated phase II/antioxidant enzyme activities. Taken together, these results demonstrate that DADS increases the expression of phase II/antioxidant enzymes and simultaneously decreases the expression of inflammatory mediators in CCl4-induced liver injury. These findings indicate that DADS induces antioxidant defense mechanism by activating Nrf2 pathway and reduces inflammatory response by inhibiting NF-κB activation.

Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was analyzed. Western blot analysis showed that three isotypes of caveolins including caveolin-1, -2 and -3 increased significantly in the spinal cords of rats during the early stage of EAE, as compared with the levels in control animals (p<0.05); the elevated level of each caveolin persisted during the peak and recovery stage of EAE. Immunohistochemistry demonstrated that caveolin-1 and -2 were expressed constitutively in the vascular endothelial cells and ependymal cells of the normal rat spinal cord, whereas caveolin-3 was almost exclusively localized in astrocytes. In EAE lesions, the immunoreactivity of caveolin-1 was increased in the ependymal cells, some astrocytes, and some inflammatory cells of the spinal cord, while that of caveolin-2 showed an intense immunoreactivity. Caveolin-3 was expressed constitutively in some astrocytes, but not in endothelial cells; its immunoreactivity was increased in reactive astrocytes in EAE lesions. The results of the Western blot analysis largely confirmed the observations obtained with immunohistochemistry. Taking all the findings into consideration, we postulate that the expression levels of each caveolin begin to increase when EAE is initiated, possibly contributing to the modulation of signal transduction pathways in the affected cells.

Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Arginase-1, a marker for M2 phenotype alternatively activated macrophages, inhibits inflammation and is associated with phagocytosis of cell debris and apoptotic cells. We analyzed the expression of arginase-1, a competitive enzyme of inducible nitric oxide synthase (iNOS), in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that both arginase-1 and iNOS significantly increased in the spinal cords of rats at the peak stage of EAE compared with the expression level in control animals (p<0.05) and declined thereafter. Immunofluorescent staining demonstrated that increased expression of arginase-1 in EAE spinal cords was confirmed in macrophages as well as in some neurons and astrocytes that were constitutively positive for arginase-1 in normal spinal cords. A semiquantitative analysis by immunofluorescence showed that in EAE lesions, an increased level of arginase-1 immunoreactivity was matched with ED1-positive macrophages, which were also positive for activin A, a marker for the M2 phenotype. Taking all of these findings into consideration, we postulate that the increased level of arginase-1, which is partly from M2 macrophages, contributes to the modulation of neuroinflammation in EAE lesions, possibly through the reduction of nitric oxide in the lesion via competition with iNOS for the use of L-arginine.

Differences in immunoreactivities of Ki-67 and doublecortin in the adult hippocampus in three strains of mice.

Neurogenesis in the adult hippocampus is differentially influenced by the genetic background. We examined the differences in Ki-67 (a proliferating cell marker) and doublecortin (DCX; an immature progenitor cell marker) immunolabelling in the dentate gyrus (DG) of the adult hippocampus in three strains of mice (ICR, C57BL/6, and BALB/c) to evaluate the effect of genetic background on adult hippocampal neurogenesis. All strains showed constitutive immunoreactivity of either Ki-67 or DCX in the DG of the adult hippocampus. C57BL/6 mice showed significantly higher levels of Ki-67-immunopositive cells in the subgranular zone (SGZ) of the DG (approximately 2.2-fold) compared to ICR and BALB/c mice. The greatest number of DCX-immunopositive cells was found in C57BL/6 (approximately 1.6-fold), which differed significantly from ICR and BALB/c mice. However, there was no significant difference in the number of Ki-67- and DCX-immunopositive cells between BALB/c and ICR mice. Genetic differences with respect to certain aspects of hippocampal neurogenesis in adult mice may influence hippocampal functions, including learning and memory.

Ca-Stimulated Type 8 Adenylyl Cyclase Is Required for Rapid Acquisition of Novel Spatial Information and for Working/Episodic-Like Memory

Ca-stimulated adenylyl cyclases (ACs) transduce neuronal stimulation-evoked increase in calcium to the production of cAMP, which impinges on the regulation of many aspects of neuronal function. Type 1 and type 8 AC (AC1 and AC8) are the only ACs that are directly stimulated by Ca. Although AC1 function was implicated in regulating reference spatial memory, the function of AC8 in memory formation is not known. Because of the different biochemical properties of AC1 and AC8, these two enzymes may have distinct functions. For example, AC1 activity is regulated by both Ca and G-proteins. In contrast, AC8 is a pure Ca sensor. It is neither stimulated by Gs nor inhibited by Gi. Recent studies also suggested that AC1 and AC8 were differentially concentrated at different subcellular domains, implicating that Ca-stimulated signaling might be compartmentalized. In this study, we used AC8 knock-out (KO) mice and found behavioral deficits in memory retention for temporal dissociative passive avoidance and object recognition memory. When examined by Morris water maze, AC8 KO mice showed normal reference memory. However, the acquisition of newer spatial information was defective in AC8 KO mice. Furthermore, AC8 KO mice were severely impaired in hippocampus-dependent episodic-like memory when examined by the delayed matching-to-place task. Because AC8 is preferentially localized at the presynaptic active zone, our results suggest a novel role of presynaptic cAMP signaling in memory acquisition and retention, as well as distinct mechanisms underlying reference and working/episodic-like memory.

Temporal patterns of the embryonic intermediate filaments nestin and vimentin expression in the cerebral cortex of adult rats after cryoinjury

The expression of two embryonic intermediate filaments, nestin and vimentin, in the rat brain at days 0 (control), 1, 4, 7 and 14 post-cryoinjury was studied to elucidate their roles in brain injury. Western blot analysis showed that both nestin and vimentin expressions in the ipsilateral cerebral cortex were significantly increased at 4 and 7 days post-cryoinjury, and were decreased at day 14 after cryoinjury. Immunohistochemistry showed that there were few nestin- and vimentin-positive cells in the cerebral cortex in normal controls. On days 4 and 7 post-injury, abundant glial cells in the periphery of the lesion were immunostained for nestin and/or vimentin; only vimentin was detected in the majority of inflammatory cells in the core lesion. These findings suggest that nestin and vimentin contribute to the repair of brain injury through the migration of activated cells and the formation of a glial scar.

N-methyl-D-aspartate-stimulated ERK1/2 signaling and the transcriptional up-regulation of plasticity-related genes are developmentally regulated following in vitro neuronal maturation.

The general features of neuroplasticity are developmentally regulated. Although it has been hypothesized that the loss of plasticity in mature neurons may be due to synaptic saturation and functional reduction of N‐methyl‐D‐aspartate receptors (NMDAR), the molecular mechanisms remain largely unknown. We examined the effects of NMDAR activation and KCl‐mediated membrane depolarization on ERK1/2 signaling following in vitro maturation of cultured cortical neurons. Although NMDA stimulated a robust increase in intracellular calcium at both DIV (day in vitro) 3 and 14, the activation of ERK1/2 and cAMP responsive element‐binding protein (CREB) was impaired at DIV 14. Specifically, the phosphorylation of ERK1/2 was stimulated by both NMDA and KCl at DIV 3. However, at DIV 14, NMDA‐ but not KCl‐stimulated ERK1/2 and CREB phosphorylation was significantly diminished. Consistently, the NMDA‐induced transcription of ERK/CREB‐regulated genes Bdnf exon 4, Arc, and zif268 was significantly attenuated at DIV 14. Moreover, in comparison with 3 DIV neurons, the phosphorylated‐ERK1/2 in 14 DIV neurons displayed a tremendous increase following maturation and was more susceptible to dephosphorylation. Blocking calcium channels by nifedipine or NMDAR by APV caused a more dramatic ERK dephosphorylation in 14 DIV neurons. We further demonstrate that the loss of plasticity‐related signaling is unrelated to NMDA‐induced cell death of the 14 DIV neurons. Taken together, these results suggest that the attenuation of certain aspects of neuroplasticity following maturation may be due to the reduction of NMDAR‐mediated gene transcription and a saturation of ERK1/2 activity.

Maternal Exposure to Multi-Wall Carbon Nanotubes Does Not Induce Embryo-Fetal Developmental Toxicity in Rats

BACKGROUND Although the potential risk of carbon nanotubes (CNTs) to humans has recently increased due to expanding production and widespread use, the potential adverse effects of CNTs on embryo-fetal development have not yet been determined. METHODS This study investigated the potential effects of multi-wall CNTs (MWCNTs) on pregnant dams and embryo-fetal development in rats. MWCNTs were administered to pregnant rats by gavage at 0, 40, 200, and 1,000 mg/kg/day. All dams were subjected to Cesarean section on day 20 of gestation, and the fetuses were examined for any morphological abnormalities. RESULTS All animals survived to the end of the study. A decrease in thymus weight was observed in the high dose group in a dose-dependent manner. However, maternal body weight, food consumption, and oxidant-antioxidant balance in the liver were not affected by treatment with MWCNTs. No treatment-related differences in gestation index, fetal deaths, fetal and placental weights, or sex ratio were observed between the groups. Morphological examinations of the fetuses demonstrated no significant difference in incidences of abnormalities between the groups. CONCLUSIONS The results show that repeated oral doses of MWCNTs during pregnancy induces minimal maternal toxicity and no embryo-fetal toxicity at 1,000 mg/kg/day in rats. The no-observed-adverse-effect level of MWCNTs is considered to be 200 mg/kg/day for dams and 1,000 mg/kg/day for embryo-fetal development. In this study, the dosing formulation was not analyzed to determine the degree of reaggregation (or not), nor were blood levels of CNTs measured in the dosed animals to verify or characterize absorption.

Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions

Expression of osteopontin and CD44 in the brain was studied after cryolesioning to understand how osteopontin and its receptor, CD44, are involved in processes in the brains of rats with cryolesions. Western blot analysis showed that osteopontin increased significantly at days 4 and 7 post-injury and declined slightly thereafter in cryolesioned brains in comparison with levels in sham-operated controls. An immunohistochemical study localized osteopontin in activated microglia/macrophages in the core lesions, where the majority of macrophages proliferate. Osteopontin was also detected temporarily in some neurons and a few astrocytes in the lesion periphery on days 4 and 7 post-injury, but the immunoreactivity in macrophages, neurons, and astrocytes disappeared by day 14 post-injury. There was some CD44, a receptor for osteopontin, in the brain cells of sham-operated rats. After injury, intense CD44 immunostaining was seen in the majority of macrophages and in reactive astrocytes, but not in neurons, in the ipsilateral lesions after day 4 post-injury, and this immunoreactivity remained on day 14 post-injury. These findings suggest that activated microglia/macrophages and some neurons are major sources of osteopontin during the early stage of brain damage induced by a cryolesion and that osteopontin interacts with CD44 expressed on astrocytes and activated microglia/macrophages in the damaged cerebral cortex, possibly mediating cell migration after cryolesioning in the rat brain.