Involvement of NOS/NO in the development of chronic dental inflammatory pain in rats.

Nitric oxide (NO) is believed to be an important messenger molecule in nociceptive transmission. To assess the possible roles of NO in trigeminal sensory system, we examined the distribution and density of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), and immunohistochemical staining for c-Fos, a neuronal activity marker, in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) following pulp exposure (PX) injured rats. The neurons innervating injured tooth in TG were labeled by the retrograde transport of fluoro-gold (FG). Teeth were processed for H&E staining. We found that NADPH-d activity increased significantly in the TG and Vc following PX pretreatment (7-28 days, especially in 21-28 days). Such changes were closely corresponding to the pattern of c-Fos detected by immunocytochemistry. The results demonstrate that PX-induced chronic pulpal inflammation results in significant alterations in the TG cells and in the Vc, and such changes may underlie the observed NADPH-d activity. It suggests that NOS/NO may play an active role in both peripheral and central processing of nociceptive information following chronic tooth inflammation.

Expression and colocalization of NADPH-diaphorase and heme oxygenase-2 in trigeminal ganglion and mesencephalic trigeminal nucleus of the rat.

Carbon monoxide (CO) and nitric oxide (NO) are two endogenously produced gases that can function as second messenger molecules in the nervous system. The enzyme systems responsible for CO and NO biosynthesis are heme oxygenase (HO) and nitric oxide synthase (NOS), respectively. The present study was undertaken to examine the distribution of HO-2 and NOS of the trigeminal primary afferent neurons of the rat, located in the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN), using histochemistry and immunohistochemistry. NADPH-d staining was found in most neurons in TG. The intensely NADPH-d-stained neurons were small- or medium-sized, while the large-sized neurons were less intensely stained. Immunocytochemistry for HO-2 revealed that almost all neurons in TG expressed HO-2, but they did not appear cell size-specific pattern. NADPH-d and HO-2 positive neurons appeared the same pattern, which was NADPH-d activity and HO-2 expression progressively declined from the caudal to rostral part of the MTN. A double staining revealed that the colocalization of NADPH-d/HO-2 neurons was 97.3% in TG and 97.6% in MTN. The remarkable parallels between NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and mediate the orofacial nociception and sensory feedback of the masticatory reflex arc together.

Differential effects of melatonin on hippocampal neurodegeneration in different aged accelerated senescence prone mouse-8.

OBJECTIVES: A purpose of this study is to compare the differential effects of melatonin on hippocampal neurodegeneration in accelerated senescence prone mouse-8 (SAMP8) which is initiated treatment at different age. METHODS: The 4-months old SAMP8 mice were injected subcutaneously with melatonin (1 mg/kg/day) for 4 months. Similar treatments were performed in the 7-months old mice. When the animals were complete 11-months old, a series of tests were performed. Y maze test and Eight-arm radial maze task were used to assess cognitive performance. Hippocampal pyramidal cells were estimated by Nissl's staining. By using Gomori's methenamine silver methods, the methenamine silver staining granules (MSSG) were observed in area CA1 of hippocampus. A computer-assisted morphometric study was carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria. The volume density (Vv), surface density (Sv), numerical density (Nv) and mean volume (V) of the mitochondria were calculated. RESULTS: Melatonin treatment obviously reduced the deposition of MSSG and elevated hippocampal pyramidal cell number while improving the learning and memory deficits of SAMP8. The mice initiated treatment from 4-months old exhibited a greater response to melatonin supplementation than 7-months old mice. It also decreased mean volume (V) and significantly elevated the Sv and Nv of the mitochondria in hippocampal CA1 region. However, 7-months old mice showed little effects on it. CONCLUSIONS: Our results indicate that the protective effects of melatonin on hippocampal neurodegeneration of SAMP8 are age dependent.

Effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-San on rat pineal melatonin synthesis.

OBJECTIVES: The purpose of this study is to investigate if the aqueous extract of the Chinese medicine Danggui-Shaoyao-San (DSS) can increase the plasma level of melatonin and enhance the function of the pineal gland of naturally aged rats. METHODS: The rats were treated with DSS at doses of 3ml or same volume of distilled water by oral administration at 11 p.m. for three weeks. The plasma level of melatonin were measured by radioimmunoassay. The function of pineal gland were measured through three parameters: pineal beta adrenergic receptor binding investigated by [3H]DHA binding; pineal expression of NAT mRNA detected by real-time RT-PCR; phosphorylation of CREB (P-CREB) and total level of CREB (T-CREB) measured by western blot analysis. RESULTS: DSS significantly increased melatonin level at night after oral administration for 3 weeks. By measurement of pineal [3H]DHA binding, it was found DSS improved the beta-adrenergic receptors binding in pineals. The stimulatory effect of DSS on the expression of NAT mRNA in the old rat pineal gland has been demonstrated in this study. Western blot analysis showed that DSS significantly increased phosphorylation of CREB. CONCLUSIONS: Our results indicate that a downstream pathway for DSS induction of melatonin synthesis in the rat pineal gland acts via cyclic AMP-dependent cascade and transcription mechanism.

Relationship between inflammatory reaction and ischemic injury of caudate-putamen in rats: inflammatory reaction and brain ischemia.

Inflammatory response after middle cerebral artery occlusion (MCAO) has been a focus of research recently, but the effect of inflammatory cells on ischemic neurons remains unclear. In order to study the effect of the inflammatory reaction on brain ischemic injury, we observed the morphology, number and distribution of CD3-, CD8-, ED1- and ED2-positive cells systematically in the caudate-putamen of rats in a MCAO model. The present results show that all four types of inflammatory cells first infiltrated the ischemic penumbra and then migrated into the center of the ischemic area, but the morphological changes and infiltration processes differed significantly; the infiltration of CD3- and CD8-positive cells into the ischemic area started at 3 days postischemia, and their number peaked at 1 week; however, although ED1- and ED2-positive cells were also observed at 3 days after ischemia, they reached their maximum number at 2 and 4 weeks, respectively. Moreover, ED1-and ED2-positive cells showed evident hyperplasia and hypertrophy in morphology. Our results also showed that the response of CD3-, CD8-, ED1- and ED2-positive cells in the ischemic area and the pathological changes in ischemic brain tissue could be inhibited by cyclosporine A. The results suggest that the infiltration and reaction of inflammatory cells are involved in the pathological process of ischemic brain injury.

WITHDRAWN: Transplantation of bone mesenchymal stem cells containing the nerve growth factor gene in adult rat brain with Fimbria Fornix lesion.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising source for cell-based treatment of brain injury, but the therapy of BMSCs is restricted by low cell survival. We examined whether nerve growth factor (NGF) improve BMSCs viability in the brain with Fimbria-Fornix lesion (FF). After transduction of NGF gene via recombinant retroviral vectors, the rat BMSCs were transformed into the NGF-GFP positive BMSCs, nearly 100% of cells expressed NGF. After transplanted into basal forebrain of rat with FF, the NGF-GFP positive BMSCs expressed the exogenous NGF gene in the host brain, and interesting, the survival number of BMSCs in the NGF group was significant more than that of the void plasmid group. Furthermore, the number of choline acetyltransferase (ChAT) immunoreactive neurons of NGF group was also significant higher than those of the void plasmid group (p<0.05) or the PBS group (p<0.01). Performance in the water maze test was improved in these rats in NGF group. These results indicate that NGF increased BMSCs survival in brain with FF, which results in better improvement of brain function than injected with BMSCs alone.

[Antibody production and its neutralization of Abeta42's cytoxicity in BALB/c mice induced by Abeta42 and its subunit vaccines].

AIM: To explore the production of anti-Abeta42 antibody after immunization with Abeta42 and its subunit peptide vaccines. METHODS: Seventy five male BALB/c with the age of 6 weeks were randomly divided into 5 groups, namely, control group, Abeta42 group, Abeta(36-42) group, Abeta(1-15)group and F Abeta(1-15) group. The BALB/c mice were immunized four times with PBS+MF59 adjuvant, Abeta42+MF59, Abeta(36-42)+heptalysine (MAP)+MF59, Abeta(1-15)+MAP+MF59 and Abeta(1-15)+MAP+Freud's adjuvant, respectively. The titers of specific antibodies in sera and supernatants of brain tissue homogenates from the immunized mice in every group were detected by indirect ELISA. After Abeta42, Abeta(36-42), Abeta(1-15) and F Abeta(1-15) were co-cultured together with cultured PC12 cells for 7 days, the cytotoxicity of the 3 antigen peptides to PC12 cells were determined by MTT colorimetry. In addition, after immune sera from each group were added to culture medium containing 20 mg/L Abeta42 and co-cultured with PC12 cells for 7 days, the survival rate of PC12 cells were examined by MTT assay. RESULTS: The production of anti-Abeta42 antibodies was detected in sera of each experimental group after the second time immunization, and the titer of antibody rose with the increase of immunizing times. In addition, the anti-Abeta42 antibody with low titer was also detected in supernatants of brain tissue homogenates. The Abeta42 could reduce the survival rate of PC12 cells, whereas Abeta(36-42) and Abeta(1-15) had no obvious effect on survival rate of PC12 cells. After immune sera from 4 experimental groups and Abeta42 were co-cultured with PC12 cells, their survival rate was found improved. CONCLUSION: Combination of Abeta42 and its subunit (Abeta(36-42) and Abeta(1-15)) vaccines with MF59 adjuvant can induce BALB/c mice to produce anti-Abeta42 antibody. The antibody may neutralize the cytotoxicity of Abeta42.