nNOS Levels Research Articles

Activation of Sigma-1 Receptor Alleviates Postpartum Estrogen Withdrawal-Induced "Depression" Through Restoring Hippocampal nNOS-NO-CREB Activities in Mice.

Postpartum depression affects approximately 15% of mothers; however, its pathological mechanisms still remain unclear. Ovariectomized adult mice received the administration of estrogen (E2) and progesterone with a subsequent alone E2, termed hormone-simulated pregnancy (HSP). Affective behaviors as assessed by forced swim and tail suspension tests, hippocampal neuronal nitric oxide synthase (nNOS), nitric oxide (NO), cyclic AMP (cAMP) response element binding protein (CREB) phosphorylation (phosphor-CREB), and neurosteroidogenesis were examined before E2 withdrawal (EW; HSP mice) and on days 2-4 (early-EW mice) and days 8-10 (late-EW mice) after EW. Depressive-like behaviors were observed in early-EW mice but not in late-EW mice. Levels of nNOS, NO, and phosphor-CREB were increased in HSP mice followed by a significant decline in early-EW mice with a subsequent restoration in late-EW mice. The treatment of early-EW mice with NO donor alleviated depressive-like behaviors and decline of phosphor-CREB. The nNOS inhibitor and NO scavenger caused depressive-like behaviors and reduced phosphor-CREB in HSP mice and late-EW mice. Notably, the levels of steroidogenic enzymes StAR and P450scc were elevated in late-EW mice. The sigma-1 receptor (σ1R) agonist could alleviate depressive-like behaviors and decline of nNOS-NO-CREB in early-EW mice. The pharmacological blockade or deficiency of σ1R in late-EW mice caused depressive-like behaviors with decline of nNOS-NO-CREB. The reduction of hippocampal brain-derived neurotrophic factor (BDNF) or N-methyl-D-aspartic acid (NMDA) receptor NR2B phosphorylation in early-EW mice could recover in late-EW mice, which was sensitive to the blockade of σ1R. The NMDA receptor agonist, but not TrkB receptor activator, could correct the decline of nNOS-NO-CREB in early-EW mice. The findings indicate that the activation of σ1R can alleviate postpartum "depression" through increasing nNOS-NO-CREB activities.

Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this “toughening” effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with “toughening” and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor, Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice.

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal-lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.

Ameliorative effects of pentoxifylline on NOS induced by diabetes in rat kidney

Objectives Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide. The NO system has been implicated in the pathogenesis of DN. In this study, we aimed to evaluate the healing effect of pentoxifylline on NOS in STZ-induced diabetic rat’s kidney. Material and methods In this study, 50 Wistar albino male rats were used. The rats were divided into five groups; Group C control; Group D only diabetes; Group D + PI and D + PII diabetes + pentoxifylline; Group P only pentoxifylline. Group DPI rats received just pentoxifylline from the beginning of the experiments. However, Group DPII rats received saline in the first month and 50 mg/kg/day of pentoxifylline for the following month. At the end of two months, NOS expressions in kidney tissue were assessed using qRT-PCR and immunohistochemistry analysis. Results At the end of the experiments, desquamation of the epithelial cells of the tubules, clear glycogen-filled distal tubules and increased number of apoptotic cells were seen in Group D. Diabetic rats’ nNOS immunoreactivity had increased and eNOS and iNOS immunoreactivity had decreased; nNOS, iNOS and eNOS mRNA levels tended to decrease compared to the control group. PTX ameliorated eNOS, iNOS and nNOS protein levels and apoptotic cells, but did not affect mRNA levels. Conclusion In conclusion, PTX has a healing effect on this damage by affecting NOS expression.

Region-specific localization of NOS isoforms and NADPH-diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus).

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage-dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)-NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH-d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH-d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell-specific localization in the efferent ductules and region- and cell-specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation.

Effect of nitrergic system on colonic motility in a rat model of irritable bowel syndrome.

Aim:The aim of this study is to investigate whether nitric oxide (NO)-mediated colonic motility was altered in rat irritable bowel syndrome (IBS) model, using different isoforms of NO-synthase (NOS) inhibitors.Materials and Methods:The animal model of IBS-like visceral hypersensitivity was induced by intra-colonic infusion of 0.5% acetic acid (AA) in saline once daily from postnatal days 8 to 21. Control animals received saline instead of AA. Experiments were performed at the end of 8 weeks. Distal colon tissues were resected and direct effects of different NOS inhibitors; N-omega-nitro-L-arginine methyl ester hydrochloride, (L-NAME), ARL-17477 dihydrochloride hydrate (ARL 17477), N-[3-(Aminomethyl) phenyl] methyl]-ethanimidamidedihydrochloride (1400 W), and N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO) were evaluated concentration-dependently in vitro tissue bath. Besides, morphology of both groups was assessed with hematoxylin and eosin (H and E) staining and the impact of NO antibodies was determined using the immunohistochemical method.Results:The mean pressure values of spontaneous contractions and KCL (80 mmol/L) responses of distal colonic segments were similar in normal and IBS rats. L-NAME and ARL-17477 significantly increased the mean pressure of spontaneous colonic contractions in normal rats versus own base values (P < 0.05), but this increase did not significantly different when compared to IBS rats. In H and E staining, there was no difference with regard to morphology between two groups. Neuronal NOS (nNOS) immunoreactivity was found to be significantly decreased in IBS when compared to control groups (P < 0.05).Conclusion:L-NAME and ARL-17477 mediated mean pressure values were found to be slightly decreased in IBS rats. These findings may be related to a decrease in nNOS level in IBS.

Anti-nociceptive effects of Paecilomyces hepiali via multiple pathways in mouse models.

Paecilomyces hepiali (PH), a well-known medicinal fungus, has various pharmacological efficacies. In our study, the antinociceptive effects of PH and underlying mechanisms were evaluated using various mouse models. An acetic acid-induced writhing test, hot plate test, and formalin test were employed to evaluate the antinociceptive activities of PH. The levels of neuronal nitric oxide synthase (nNOS) in the hypothalamus and monoamine neurotransmitters in the serum and hypothalamus of experimental mice were examined. Additionally, hot plate tests using mice pretreated with various antagonists were used to determine the mechanisms of PH-mediated antinociception. The PH-enhanced latency period of mice in the hot plate test was significantly blocked by pretreatment with atropine and glibenclamide. PH shortened the phase I and phase II reaction times of formalin-treated mice. Strongly reduced writhing and stretching induced by acetic acid were observed in PH-treated mice, indicating that PH mainly exerts antinociceptive activity on neurogenic pain. After thermal pain stimulation for 30 s, compared to control mice, 7-day PH-treated mice had lower nNOS and dopamine levels, and increased levels of serotonin in both the serum and hypothalamus. Collectively, our data showed that PH mediated antinociceptive activities via multiple pathways, including monoamines, nNOS/ATP-sensitive K+ channels, and M-type acetylcholine receptors.

Effects of Acupuncture at Varying Depths at the Connecting Point on the Changes of Levels of nNOS, No and Norepinephrine in Rats

Objectives : This study was intended to observe the changes in the expression of neurotransmitters, such as nNOS, NO and NE upon the needle insertion at varying depths at the connecting point. Methods : Needles were inserted into rats, on both left and right sides of the connecting point, including the LI6, SI7 and TE5 acupoints which are three yang meridians of the hand. After insertion, needles were retained for five minutes. Each acupuncture groups were treated acupuncture at each acupoint and at the depths of superficial, middle and deep layer. After the retention, blood was drawn via cardiac puncture, and tissues of each point near meridian vessels were extracted to examine the changes in the expression of nNOS, NO and NE. Results : In terms of the effect in nNO production, there was a significant increase only in the middle and deep layer at SI7 acupoint, but there was no significant change in the expression of NO. Regarding the formation of norepinephrine within tissues, the middle layer on LI6 acupoint, the middle layer and the deep layer on TE5 acupoint showed a significant increase, while production of plasma norepinephrine was significantly decreased at the middle layer and the deep layer on LI6 acupoint and the deep layer on SI7 acupoint. Conclusions : The effect of needles applied at the connecting point of three yang meridians on the activities of nNOS, and NE could be observed, and it can be induced that the effect of needle stimulation on disrupted nervous system can be examined through additional researches based on this one.

Alterations of Synaptic Proteins in the Hippocampus of Mouse Offspring Induced by Developmental Lead Exposure.

Lead exposure can cause cognitive dysfunction in children, thus it still raises important public health concerns in China and other countries. However, the underlying molecular mechanisms are still not well defined. In this study, we aimed to elucidate the mechanisms underlying lead neurotoxicity by focusing on alterations of synaptic proteins in the mouse hippocampus at the early life. Mother mice and their offspring were exposed to 0, 0.5, 1.0, and 2.0g/L lead via drinking water from the first day of gestation until postnatal day (PND) 40. Synaptic ultrastructure and expressions of postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS) and synaptophysin (SYP) at both protein and gene levels in the hippocampus were analyzed. The results revealed that developmental lead exposure caused a diminished postsynaptic density in the hippocampus. Moreover, the protein levels of PSD-95, nNOS, and SYP decreased significantly due to developmental lead exposure. On the other hand, the messenger RNA (mRNA) levels of PSD-95 and SYP decreased significantly in PND 40 mice exposed to lead. Collectively, developmental lead exposure might result in decreased protein and gene expressions of both presynaptic and postsynaptic proteins. Our findings raised a possibility that alterations of synaptic proteins in the hippocampus induced by lead exposure at the early life might serve an important role for the subsequent intellectual impairments, e.g., deficits in spatial learning and memory ability at later ages shown in our recently published paper.

Nitric oxide synthase in diabetic rat testicular tissue and the effects of pentoxifylline therapy

Diabetes is known to be associated with erectile dysfunction, retrograde ejaculation, level of testicular hormone, and a decrease in semen quality, respectively. In this project, we aimed to investigate at the molecular level, the effects of NOS on testes pathology in diabetes and examine the effects of pentoxifylline on healing. A total of 50 Wistar albino male rats were divided into five groups: Group I control; Group II only diabetes; Group III and IV diabetes + pentoxifylline; Group V only pentoxifylline. Group III rats received 50 mg/kg/day pentoxifylline during two months. In comparison, Group IV rats received saline in the first month followed by 50 mg/kg/day of pentoxifylline for the following month. NOS expression in testicular tissue was assessed using qRT-PCR, western blot, and immunohistochemistry. The mean seminiferous tubule diameter, Johnsen's testicular biopsy score, and serum testosterone levels decreased compared to controls. In contrast, the number of apoptotic cells, the levels of nNOS, iNOS and eNOS mRNA, and protein increased when compared to the control. Upon pentoxifylline therapy NOS decreased suggesting that it contributes to this damage and treatment with pentoxifylline may be effective in reversing this damage.

Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats.

A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR.

Nitric oxide production increases during Toxoplasma gondii encephalitis in mice

Toxoplasma gondii is an intracellular parasite with the potential of causing severe encephalitis among immunocompromised human and animals. The aim of this experimental study was to investigate the immunomodulatory and immunopathological role of nitric oxide (NO) in central nervous systems and to identify any correlation between toxoplasmosis neuropathology and investigate the consequences of the cellular responses protect against T. gondii. Mice were infected with ME49 strain T. gondii and levels of endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS, iNOS), glial fibrillary acidic protein (GFAP) and neurofilament (NF) were examined in brain tissues by immunohistochemistry, during the development and establishment of a chronic infection at 10 30 and 60 days post infection. Results of the study revealed that the levels of eNOS (p < 0.05), nNOS (p < 0.05), iNOS (p < 0.005), GFAP (p < 0.005) and NF (p < 0.005) were remarkably higher in T. gondii-infected mice than in uninfected control. The most prominent finding from our study was 10 and 30 days after inoculation data indicating that increased levels of NO not only a potential neuroprotective role for immunoregulatory and immunopathological but also might be a molecular trigger of bradyzoite development. Furthermore, this findings were shown that high expressed NO origin was not only inducible nitric oxide synthase but also endothelial and neuronal. We demonstrated that activation of astrocytes and microglia/macrophages is a significant event in toxoplasma encephalitis (TE). The results also clearly indicated that increased levels of NO might contribute to neuropathology related with TE. Furthermore, expression of NF might gives an idea of the progress and critical for diagnostic significance of this disease.

Protective effect of telmisartan on rats with renal failure and its mechanism.

To study the protective effect of telmisartan on rats with renal failure and its mechanism. 60 Wistar rats were chosen as study objective, and were divided into 4 groups randomly: 15 in group A (sham operation group), 15 in group B (model group), 15 in group C (telmisartan group) and 15 in group D (telmisartan+GW9962 group). The difference of survival rate, blood-urine biochemical indexes, renal pathological change, and the expression level of PPARγ and nNOS were compared. After 12 weeks, the survival rate of group A was 93.33% (14/15), that of group B was 46.67% (7/15), that of group C was 86.67% (13/15), that of group D was 60.00% (9/15), and the difference among 4 groups had statistical significance (P<0.05). After 1 week, the difference of Scr, that of BUN and that of 24h protein urine among 4 groups was not statistical significant (P>0.05); after 3 weeks, 6 weeks and 12 weeks, these difference was statistical significant (P<0.05). The difference of blood-urine biochemical indexes, that of renal pathological change, and that of the expression level of PPARγ and nNOS was statistical significant (P<0.05). Telmisartan has protective effect on renal failure caused by 5/6 nephrectomy, which might be relative to the expression level of PPARγ and nNOS.

Loss of nNOS Sarcolemmal Localization and Activity During Hindlimb Unloading is Mitigated by Losartan Administration

Spaceflight and immobilization lead to weakening and atrophy of antigravity skeletal muscles. Recently we observed that angiotensin II type 1 receptor (AT1R) blockade partially prevented the decrease in muscle fiber cross sectional area (CSA) and the type I to II fiber type shift that occurs in solei during 7 days of hindlimb unloading. Recent studies reveal that translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the sarcoplasm is a causal event to atrophy during disuse. PURPOSE: Determine whether the AT1R blocker Losartan prevents the translocation of nNOS from the sarcolemma to the sarcoplasm in soleus muscle fibers during 7 days of hindlimb unloading. METHODS: Rats were divided into 4 groups: ambulatory control (CON), ambulatory + Losartan (40 mg/kg/day) (CON-L), 7 days hindlimb unloaded (HU), and HU + Losartan (HU-L). Solei were collected and analyzed. Localization of nNOS was assessed through immunohistochemistry. We measured protein content and localization via subcellular fractionation and immunoblotting. nNOS activity was observed through NADPH-diaphorase histochemistry. RESULTS: Hindlimb unloading decreased sarcolemmal-localized nNOS compared to CON muscles, an effect ameliorated by Losartan. Subcellular fractionization and immunoblotting revealed significantly elevated levels of sarcoplasmic nNOS in the HU group compared to CON (p < 0.05). Sarcoplasmic nNOS content in HU-L was not significantly different from CON. nNOS activity in the CON solei displayed activity primarily near the sarcolemma, while sarcoplasmic nNOS activity and lack of sarcolemmal activity was detected in HU muscle fibers. Losartan administration during hindlimb unloading partially preserved nNOS activity at the sarcolemma. CON-L findings were no different than CON findings in any of the experiments. CONCLUSIONS: AT1R blockade with Losartan during 7 days of hindlimb unloading aided in the retention of sarcolemmal nNOS, which partially maintained sarcolemmal nNOS activity, thus, limiting the reduction in fiber CSA and the fiber type shift that occurs during disuse-induced atrophy. Supported by NASA (NNX13AE45G), Predoctoral Space Physiology Grant from ACSM / NASA, and the Sydney and J.L. Huffines Institute.

Nitric oxide signaling is recruited as a compensatory mechanism for sustaining synaptic plasticity in Alzheimer's disease mice.

Synaptic plasticity deficits are increasingly recognized as causing the memory impairments which define Alzheimer's disease (AD). In AD mouse models, evidence of abnormal synaptic function is present before the onset of cognitive deficits, and presents as increased synaptic depression revealed only when synaptic homeostasis is challenged, such as with suppression of ryanodine receptor (RyR)-evoked calcium signaling. Otherwise, at early disease stages, the synaptic physiology phenotype appears normal. This suggests compensatory mechanisms are recruited to maintain a functionally normal net output of the hippocampal circuit. A candidate calcium-regulated synaptic modulator is nitric oxide (NO), which acts presynaptically to boost vesicle release and glutamatergic transmission. Here we tested whether there is a feedforward cycle between the increased RyR calcium release seen in presymptomatic AD mice and aberrant NO signaling which augments synaptic plasticity. Using a combination of electrophysiological approaches, two-photon calcium imaging, and protein biochemistry in hippocampal tissue from presymptomatic 3xTg-AD and NonTg mice, we show that blocking NO synthesis results in markedly augmented synaptic depression mediated through presynaptic mechanisms in 3xTg-AD mice. Additionally, blocking NO reduces the augmented synaptically evoked dendritic calcium release mediated by enhanced RyR calcium release. This is accompanied by increased nNOS levels in the AD mice and is reversed upon normalization of RyR-evoked calcium release with chronic dantrolene treatment. Thus, recruitment of NO is serving a compensatory role to boost synaptic transmission and plasticity during early AD stages. However, NO's dual role in neuroprotection and neurodegeneration may convert to maladaptive functions as the disease progresses.

Protective roles of N-benzylcinnamide on cortex and hippocampus of aged rat brains.

Brain aging has been associated with oxidative stress leading to inflammation and apoptosis. The protective effects and underlying mechanisms of N-benzylcinnamide (PT-3), purified from Piper submultinerve, on brains of 90-week-old Wistar rats were investigated following daily intraperitoneal injection with 1.5 mg of PT-3/kg of body weight for 15 days. PT-3 treatment improved spatial learning and memory of aged rats and caused significant changes in brain frontal cortex, hippocampus, and temporal cortex in parameters associated with oxidative stress (decreased reactive oxygen species production and iNOS and nNOS levels), inflammation (reduced levels of IL-1β and IL-6), apoptosis (reduced levels of Bax and activated caspase-3, and elevated level of Bcl-2), and signaling pathways related to inflammation and apoptosis (decreased amounts of phospho-JNK and -p38, increased phospho-Akt level and no change in phospho-ERK1/2 content) compared to controls. PT-3 treatment also inhibited aged rat brain AChE activity. These results suggest that PT-3 with its intrinsic antioxidant and AChE inhibitory properties has therapeutic potential in ameliorating, in part, age-associated damages to the brain.

Increased expressions of ADAMTS-13, neuronal nitric oxide synthase, and neurofilament correlate with severity of neuropathology in Border disease virus-infected small ruminants.

Border Disease (BD), caused by Pestivirus from the family Flaviviridae, leads to serious reproductive losses and brain anomalies such as hydranencephaly and cerebellar hypoplasia in aborted fetuses and neonatal lambs. In this report it is aimed to investigate the expression of neuronal nitric oxide synthase (nNOS), A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13 (ADAMTS-13), and neurofilament (NF) in the brain tissue in small ruminants infected with Border Disease Virus (BDV) and to identify any correlation between hypomyelinogenesis and BD neuropathology. Results of the study revealed that the levels of ADAMTS-13 (p<0.05), nNOS (p<0.05), and NF (p<0.05) were remarkably higher in BDV-infected brain tissue than in the uninfected control. It was suggested that L-arginine-NO synthase pathway is activated after infection by BDV and that the expression of NF and nNOS is associated with the severity of BD. A few studies have focused on ADAMTS-13 expression in the central nervous system, and its function continues to remain unclear. The most prominent finding from our study was that ADAMTS-13, which contain two CUB domains, has two CUB domains and its high expression levels are probably associated with the development of the central nervous system (CNS). The results also clearly indicate that the interaction of ADAMTS-13 and NO may play an important role in the regulation and protection of the CNS microenvironment in neurodegenerative diseases. In addition, NF expression might indicate the progress of the disease. To the best of the authors’knowledge, this is the first report on ADAMTS-13 expression in the CNS of BDV-infected small ruminants.

Which Biomarker is most Effective to Determine Severity of Acute Head Trauma in the Experimental Animal Head Trauma Model?

Because of the need for effective method to determine the severity of head trauma, the importance of biomarkers is recognized recently. This study aims to analyze the values of sera levels of some biomarkers and the relation with their tissue levels in acute head injury. In this study, rats were divided into three groups (mild head trauma, severe head trauma and control group). All rats were anaesthetized. Weightdrop method was used as trauma method. Blood samples were obtained five minutes after trauma when the acute effects of trauma occurred. Then whole brains of rats were excised. Levels of biomarkers were investigated in the sera samples and homogenized brain tissues biochemically. Significant differences in the sera GFAP (p=0.015) and insulin (p=0.011) levels were observed. Very significant difference in the sera nNOS level was observed. Extremely significant difference in the tissue IL-6 (p < 0.001) level was observed between all groups. Sera nNOS and tissue IL-6 are the best biomarkers to predict trauma severity. Sera GFAP and insulin are also capable to show trauma severity in the very acute period of postinjury. Tissue levels of the biomarkers except insulin are higher than their sera levels.

Abstract 15532: Altered Ubiquitination and Stability of Protein Inhibitor of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Chronic Heart Failure Rats: Role of Angiotensin II

Introduction and Hypothesis: Expression of neuronal nitric oxide synthase (nNOS) is decreased in the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF), however the underlying molecular mechanism/s remain unclear. Recently, we demonstrated, Angiotensin II (Ang II) mediated increase in PIN: protein inhibitor of nNOS (0.76±0.10 Sham vs 1.12±0.09* CHF) which is known to down-regulate nNOS through disruption of active dimers (~60% decrease in dimer/monomer ratio) in the PVN of rats with CHF. Functionally impeded monomeric enzyme is degraded by ubiquitin proteasome system. Interestingly, PIN transcript levels remain unchanged in the PVN in CHF (1.00±0.23 Sham vs. 1.1±0.28 CHF). This observation prompted us to elucidate the molecular mechanism for the accumulation of PIN post-transcriptionally in the PVN in CHF Methods and Results: We used coronary artery ligation model of CHF in rats (6-8 weeks past ligation) and neuronal NG108-15 hybrid cell line. PIN translation was inhibited using cyclohexamide (CHX) for 0-4h after 20h of pretreatment with Ang II in NG108 cells. CHX mediated decrease in PIN expression was ameliorated with Ang II (0.19±0.04 vs 0.41±0.06* 4h). Proteasome inhibitor lactacystin (LC) treatment dramatically elevates PIN level suggesting the involvement of proteasome system in PIN regulation. Immunoprecipitation with ubiquitin antibody showed decrease PIN-Ub conjugates in Ang II-treated cells (1.04 ± 0.05 LC vs. 0.62 ± 0.07* LC AngII). In vitro ubiquitination assay in cells transfected with pCMV-(HA-Ub)8 vector revealed reduction of HA-Ub-PIN conjugates after Ang II treatment (9.2 ± 2.2 LC vs. 4.5 ± 0.6* LC Ang II). Furthermore, there was decreased accumulation of PIN-Ub conjugates in the PVN of CHF rats compared to Sham as revealed by immunohistochemistry. Conclusions: Taken together, our studies revealed that PIN is targeted for rapid degradation by the ubiquitin-proteasome pathway and Ang II delays the rate of degradation resulting in accumulation of PIN. We conclude that post-translational accumulation of PIN, mediated by Ang II, leads to a decrease in the dimeric active form of nNOS as well as protein levels of nNOS, which may lead to reduced nitric oxide resulting in over-activation of sympathetic drive during CHF.

B2 receptor blockage prevents Aβ-induced cognitive impairment by neuroinflammation inhibition

Background and purposeAβ-induced neuronal toxicity and memory loss is thought to be dependent on neuroinflammation, an important event in Alzheimer's disease (AD). Previously, we demonstrated that the blockage of the kinin B2 receptor (B2R) protects against the memory deficits induced by amyloid β (Aβ) peptide in mice. In this study, we aimed to investigate the role of B2R on Aβ-induced neuroinflammation in mice and the beneficial effects of B2R blockage in synapses alterations. Experimental approachThe selective kinin B2R antagonist HOE 140 (50pmol/site) was given by intracerebroventricular (i.c.v.) route to male Swiss mice 2h prior the i.c.v. injection of Aβ1–40 (400pmol/site) peptide. Animals were sacrificed, at specific time points after Aβ1–40 injection (6h, 1 day or 8 days), and the brain was collected in order to perform immunohistochemical analysis. Different groups of animals were submitted to behavioral cognition tests on day 14 after Aβ1–40 administration. Key resultsIn this study, we report that the pre-treatment with the selective kinin B2R antagonist HOE 140 significantly inhibited Aβ-induced neuroinflammation in mice. B2R antagonism reduced microglial activation and the levels of pro-inflammatory proteins, including COX-2, iNOS and nNOS. Notably, these phenomena were accompanied by an inhibition of MAPKs (JNK and p38) and transcription factors (c-Jun and p65/NF-κB) activation. Finally, the anti-inflammatory effects of B2R antagonism provided significant protection against Aβ1–40-induced synaptic loss and cognitive impairment in mice. Conclusions and implicationsCollectively, these results suggest that B2R activation may play a critical role in Aβ-induced neuroinflammation, one of the most important contributors to AD progression, and its blockage can provide synapses protection.