cAMP/PKA Agonist Restores the Fasting-Induced Down-Regulation of nNOS Expression in the Paraventricular Nucleus

Gene expression of neuronal nitric oxide synthase (nNOS) changes in the hypothalamic paraventricular nucleus (PVN) depending on feeding conditions, which is decreased during food deprivation and restored by refeeding, and phosphorylated cAMP response element binding protein (pCREB) was suggested to play a role in its regulation. This study was conducted to examine if the fasting-induced down-regulation of the PVN-nNOS expression is restored by activation of cAMP-dependent protein kinase A (cAMP/PKA) pathway. Freely moving rats received intracerebroventricular (icv) injection of cAMP/PKA activator Sp-cAMP (40 nmol) or vehicle (sterilized saline) following 48 h of food deprivation. One hour after drug injections, rats were transcardially perfused with 4% paraformaldehyde, and the PVN tissues were processed for nNOS or pCREB immunohistochemistry. Sp-cAMP significantly increased not only nNOS but also pCREB immunoreactivities in the PVN of food deprived rats. Fasting-induced down-regulation of the PVN-nNOS was restored by 1 h after the icv Sp-cAMP. Results suggest that cAMP/PKA pathway may mediate the regulation of the PVN-nNOS expression depending on different feeding conditions.

Effects of oropharyngeal taste stimuli in the restoration of the fasting-induced activation of the HPA axis in rats

INTRODUCTION: This study examined the regulatory mechanism underlying the meal-induced changes in the hypothalamic-pituitary-adrenal gland (HPA) axis activity. MATERIALS AND METHODS: Male Sprague-Dawley rats (250-300 g) were hired for two different experiments as follows; 1) rats received either 8% sucrose or 0.2% saccharin ad libitum after 48 h of food deprivation with the gastric fistula closed (real feeding) or opened (sham feeding). 2). rats received 5 ml of intra-oral infusion with 0.2% saccharin or distilled water after 48 h of food deprivation. One hour after food access, all rats were sacrificed by a transcardiac perfusion with 4% paraformaldehyde. The brains were processed for c-Fos immunohistochemistry and the cardiac blood was collected for the plasma corticosterone assay. RESULTS: Real feedings with sucrose or saccharin and sham feeding saccharin but not sucrose, following food deprivation decreased the plasma corticosterone level. c-Fos expression in the nucleus tractus of solitarius (NTS) of the fasted rats was increased by the consumption of sucrose but not saccharin, regardless of the feeding method. On the other hand, the consumption of sucrose or saccharin with real feeding but not the sham, induced c-Fos expression in the paraventricular nucleus (PVN) of the fasted rats. The intra-oral infusion with saccharin or water decreased the plasma corticosterone level of the fasted rats. Intra-oral water infusion increased c-Fos expression in both the PVN and NTS, but saccharin only in the NTS in the fasted rats. CONCLUSION: Neither restoration of the fasting-induced elevation of plasma corticosterone nor the activation of neurons in the PVN and NTS after refeeding requires the palatability of food or the post-ingestive satiety and caloric load. In addition, neuronal activation in the hypothalamic PVN may not be an implication in the restoration of the fasting-induced elevation of the plasma corticosterone by oropharyngeal stimuli of palatable food.

5-hydroxy-L-tryptophan Suppressed Food Intake in Rats Despite an Increase in the Arcuate NPY Expression

This study was conducted to define the underlying mechanism of hypophagia induced by increased central serotonergic action. Rats received 3 daily injections of 5-hydroxy-L-tryptophan (5-HTP), a serotonin precursor, at a dose of 100 mg/kg/10 ml saline at 1 h before lights off. A significant suppression in food intake was observed shortly after the 5-HTP injection and persisted during 3 daily 5-HTP injections. Neuropeptide Y (NPY) expression in the arcuate nucleus increased after 3 days of 5-HTP treatment, as high as in the pair-fed group. Immunoreactivity of phosphorylated extracellular signal-regulated protein kinase (pERK1/2) in the hypothalamic paraventricular nucleus (PVN) was increased markedly by 3 days of 5-HTP treatment, but not by 3 days of pair-fed. mRNA expression levels of serotonin reuptake transporter (5-HTT) was increased in the dorsal raphe nucleus of the 5-HTP treated rats, but not in the pair-fed group. Results suggest that increased pERK1/2 in the PVN of 5-HTP injected rats may be a part of serotonergic anorectic signaling, perhaps blunting the orectic action of NPY; i.e., 5-HTP injected rats showed hypophagia despite of increased NPY expression in the arcuate nucleus.

Effects of Saccharin Intake on Hippocampal and Cortical Plasticity in Juvenile and Adolescent Rats

The sensory system is developed and optimized by experiences given in the early phase of life in association with other regions of the nervous system. To date, many studies have revealed that deprivation of specific sensory experiences can modify the structure and function of the central nervous system; however, the effects of sensory overload remains unclear. Here we studied the effect of overloading the taste sense in the early period of life on the synaptic plasticity of rat hippocampus and somatosensory cortex. We prepared male and female Sprague Dawley rats with ad libitum access to a 0.1% saccharin solution for 2 hrs per day for three weeks after weaning on postnatal day 22. Saccharin consumption was slightly increased in males compared with females; however, saccharin intake did not affect chow intake or weight gain either in male or in female rats. We examined the effect of saccharin-intake on long term potentiation (LTP) formation in hippocampal Schaffer collateral pathway and somatosensory cortex layer IV - II/III pathways in the 6-week old saccharin-fed rats. There was no significant difference in LTP formation in the hippocampus between the control group and saccharin-treated group in both male and female rats. Also in the somatosensory cortex, we did not see a significant difference in LTP among the groups. Therefore, we conclude that saccharin-intake during 3~6 weeks may not affect the development of physiological function of the cortical and hippocampal synapses in rats.

Sinus floor grafting using calcium phosphate nano-crystal coated xenogenic bone and autologous bone

or=2mm and < or=6mm. Lateral window approach was used, with grafting using Bio-ceraTM only(n=1) or mixed with autogenous bone from ramus and/or maxillary tuberosity(n=13). After 6 months of healing, implant sites were created with 3mm diameter trephine and biopsies taken for histomorphometric analysis. The parameters assessed were area fraction of new bone, graft material and connective tissue. Immediate and 6 months after grafting surgery, and 6 months after implantation, computed tomography (CT) was taken and the sinus graft was evaluated morphometric analysis. After implant installation at the grafted area, the clinical outcome was checked.RESULTS:Histomorphometry was done in ten patients. Bio-ceraTM particles were surrounded by newly formed bone. The graft particles and newly formed bone were surrounded by connective tissue including small capillaries in some fields. Imaging processing revealed 24.86+/-7.59% of new bone, 38.20+/-13.19% connective tissue, and 36.92+/-14.51% of remaining Bio-ceraTM particles. All grafted sites received an implant, and in all cases sufficient bone height was achieved to install implants. The increase in ridge height was about 15.9+/-1.8mm immediately after operation (from 13mm to 19mm). After 6 months operation, ridge height was reduced about 11.5+/-13.5%. After implant installation, average marginal bone loss after 6 months was 0.3+/-0.15mm.CONCLUSION: Bio-ceraTM showed new bone formation similar with Bio-Oss(R) histomorphometrically and appeared to be an effective bone substitute in maxillary sinus augmentation procedure with the residual bone height from 2 to 6mm.

Experience of Neonatal Maternal Separation May Lead to a Long-term Modulation in the Neuronal Activity of Nucleus Accumbens in the Offspring

Dysfunction of the nucleus accumbens (NAcb) is implicated in the development of anhedonia, a core symptom of major depressive disorder. In order to define the neural basis of depression-like behaviors induced by experience of neonatal maternal separation (MS), both basal and stress-induced neuronal activations in the NAcb of adolescent rats with MS experience were examined parallel with palatable food intake. Rat pups were separated from dam daily for 180 min during the first two weeks of age (MS), and non-handled control (NH) pups were left undisturbed. After weaning on postnatal day (PND) 22, a half of NH or MS pups were subjected to 1 h of restraint stress every even day during PND 28~40 (NH/R or MS/R), and then had free choices of chow and chocolate cookie for 1 h immediately after returned to home cage. The rest half of NH and MS pups (NH/C or MS/C) received free choices of chow and cookie in the same time schedule with stress group, just omitting restraint stress. Cookie intake was significantly decreased in MS/C, whereas c-Fos expression in the NAcb and plasma corticosterone increased, compared to NH/C. Restraint stress suppressed cookie intake and increased the NAcb c-Fos expression in NH/R, but not in MS/R. The plasma corticosterone of NH/R, but not of MS/R, increased following repeated restraint stress. These results suggest that the increased neuronal activation in the NAcb of MS/C may be implicated in the development of anhedonia by MS experience, perhaps, in relation with a blunted responsivity of the hypothalamic-pituitary- adrenal axis to stress.

Construction of hNGF-betarecombinant adenovirus and screening of its expression after transfection into various cell lines

Sciatic nerve regeneration using calcium phosphate coated conduit and brain-derived neurotrophic factor gene-transfected schwann cell in rat

PURPOSE OF STUDY: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. MATERIALS AND METHODS: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with beta-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x10(6)) or BDNF-Ad infected Schwann cells(1x10(6)) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. RESULTS: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54+/-4.0*10(6) and 9.66+/-9.6*10(6). 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 microgram/microliter of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 microgram/microliter of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. CONCLUSION: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.