Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity. IMPORTANCE: Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.

Single-genome analysis reveals heterogeneous association of the Herpes Simplex Virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. In contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. This was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity.

Complete Spontaneous Bone Regeneration following Surgical Enucleation of a Mandibular Cemento-Ossifying Fibroma.

Background: Cemento-ossifying fibroma (COF) is a type of benign fibro-osseous tumor that mainly occurs in the maxillofacial region. Bone reconstruction after the surgery is often performed with bone transplantation. However, the present case report describes the accurate diagnosis and successful surgical resection of a COF with periosteum preservation, after which the defect was completely and spontaneously filled with the newly formed bone through a natural process. Case Presentation. A 32-year-old Iranian female patient presented with a history of gradual development of painful swelling, spontaneous pain, and lower lip and chin hypoesthesia in the lower third of the left side of her face. The dome-shaped swelling was tender. The patient was suffering from renal infection and urethral prolapse and was taking folic acid. She also mentioned a positive family history of similar swellings in her mother and uncle. Intraoral examination indicated a lesion in buccal and lingual vestibules extending from the first premolar to the third molar teeth. It had a firm consistency, and the covering mucosa was normal in terms of color and texture. The aspiration test was negative. The lesion had caused severe mobility of the second premolar and first and second molar teeth. Panoramic radiography revealed an extensive well-defined unilocular radiolucency. Significant knife-edge resorption of the first and second molar roots at the involved site and thinning of the alveolar crest and inferior border of the mandible were also clear. Cone-beam computed tomography showed severe expansion in the buccal and moderate expansion in the lingual aspect, causing thinning of both the buccal and lingual cortical plates. Histopathological analysis revealed neoplastic tissue mixed with fibrous connective tissue and several round and oval-shaped calcification foci. Immunohistochemical analysis confirmed the final diagnosis (COF) with the presence of SMA-8. The lesion was removed by enucleation and curettage, while the periosteum was carefully preserved. Fixation with screw and plate was also performed. Conclusions: Correct diagnosis of COF and precise implementation of the periosteal osteogenesis technique, in this case, resulted in entirely and spontaneously bone regeneration, which was a rare and favorable outcome with minimum cost and complications for the patient.

Manufacturing and evaluation of multi-channel cylinder applicator with 3D printing technology.

PURPOSE: This study was designed to assess dosimetric characteristics of 3D-printed personalized multi-channel cylinder applicator (MCCA). MATERIAL AND METHODS: UnionTech RS Pro 600 (UnionTech, Inc., Shanghai, China) 3D printer was used for manufacturing MCCA. The geometry of MCCA was designed with Fusion 360 v.2.0.5827 (Autodesk, Inc.) software. The designed file was exported to Meshmixer v.3.5 (Autodesk, Inc.) to create three-dimensional model in stereolithography (STL) file format, which is the common file format for inputting data to 3D printers. We used high-temp resin, FLHTAM02 model (Formlabs Inc., MA, USA), as material in 3D printing process. This resin model has good resistance to high temperature and compatibility with various solvents. We created a simple cubic shape phantom for dosimetric evaluation of the applicator with Gafchromic EBT3 films. Also, Monte Carlo method was applied to simulate MCCA in the same configuration as in experimental test. RESULTS: The mean ± standard deviation (SD) difference between measured and calculated doses in treatment planning system (TPS) for all control points was 0.0860 ±0.0393 Gy, corresponding to 4.01 ±1.21%. The mean ±SD difference between doses calculated by Monte Carlo simulation and TPS for all control points was 0.0996 ±0.0471 Gy, corresponding to 4.58 ±1.05%. The mean ±SD of dose difference between film measurement and Monte Carlo simulation for all control points was 0.0136 ±0.0200 Gy, corresponding to 0.60 ±0.69%. P-value for dose difference between film measurement and TPS, Monte Carlo and TPS, and film measurement and Monte Carlo were 0.7, 0.66, and 0.95, respectively. CONCLUSIONS: Dosimetric results and mechanical accuracy of MCCA show that high-temp resin with SLA 3D printing technique can be used for producing patient-specific MCCA in brachytherapy.

Protective effect of apigenin on ethylene glycol-induced urolithiasis via attenuating oxidative stress and inflammatory parameters in adult male Wistar rats.

AIMS: Apigenin (4',5,7-trihydroxyflavone) is one of the subclasses of flavonoids and has various pharmacological effects. The present work was carried out to study the effect of apigenin on ethylene glycol-induced kidney damage in male Wistar rats. MAIN METHODS: We evaluated the effects of apigenin orally administrated in normal and urolithiatic rats. Animals were assigned to nine groups in random: normal control; apigenin alone (0.005, 0.01, and 0.02 g/kg bw); urolithiatic control (0.75% ethylene glycol and 1.0% ammonium chloride in drinking water); apigenin (0.005, 0.01, and 0.02 g/kg bw) plus ethylene glycol and ammonium chloride; and cystone (0.75 g/kg bw) plus ethylene glycol and ammonium chloride. At the end of 28th day of treatment, animals were sacrificed for biochemical and histopathological assays. KEY FINDINGS: Our results indicated that the apigenin treatment decreased the formation of urinary stones in urolithiatic rats. Also, apigenin reduced the generation of malondialdehyde and enhanced antioxidant enzymes activities in the kidney homogenate of rats. It also caused a significant decrease in the calcium oxalate crystals numbers in urinary sample of rats with ethylene glycol-induced hyperoxaluria. These findings were supported by histopathological examinations. SIGNIFICANCE: Based on the results obtained, apigenin attenuate ethylene glycol-related kidney damage in male Wistar rats. Although the underlying mechanism of apigenin effect has not been determined, reduction of urinary levels of stone-producing constituents, antioxidant activities, and inhibition of TGF-ß signaling may be involved.

Chlorogenic acid induces 4T1 breast cancer tumor's apoptosis via p53, Bax, Bcl-2, and caspase-3 signaling pathways in BALB/c mice.

Despite all the new treatments, metastatic breast cancer (BC) causes many deaths. Chlorogenic acid (CGA) is a polyphenol compound with various pharmacological traits, such as anticancer properties. Targeting apoptotic death pathways has been propounded as the most effective therapeutic method in various cancers. In the current study, apoptotic agents such as p53, Bax, Bcl-2, and caspase-3 have been investigated. The experimental groups included saline, BC, CGA, protective (PR), and treatment (TM) groups. First, 4T1 mouse BC was established and then the effects of treatment with CGA were investigated through measurement of tumor weight and volume, metastatic nodules, liver biochemical tests, hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, and real-time reverse transcription-polymerase chain reaction (RT-PCR) in experimental groups. The findings showed that CGA reduced tumor weight and volume in the PR group (P < .05) and in the TM group (P < .001). Surprisingly, it eliminated the tumors in the TM group. Metastatic nodules in the PR and TM groups were significantly reduced as compared with the BC group (P < .001). The evaluation by H&E staining showed cell apoptosis in both the PR and TM groups. The results of real-time RT-PCR showed that CGA therapy increased the expression ratio of Bax/Bcl-2 (P < .001 and P < .05, respectively) and the expression of p53 (P < .001 and P < .05, respectively) and caspase-3 genes (P < .01) in the PR and TM groups. The IHC data regarding the Bax/Bcl-2 ratio confirmed the other results (P < .001). The findings demonstrate that CGA plays a significant role in the induction of apoptosis and the treatment of 4T1 BC tumors in BALB/c mice.

The effect of tapered master gutta-percha cone on apical seal of straight and curved root canals prepared with nickel-titanium rotary files.

BACKGROUND: Gutta-percha has been the predominant root canal filling material which is developed with different taper. Canal obturation fixed with nickel-titanium (NiTi) instruments and tapered gutta-percha master cone and lateral condensation is advantageous because it is clinically effectual and appears to result in a radiographically acceptable outcome. The aim of this in vitro study was to determine the effect of tapered master gutta-percha cone on apical seal of straight and curved root canals using NiTi rotary files. MATERIALS AND METHODS: In this in vitro study total of 130 mandibular molars were selected and divided into six experimental groups (n = 20) based on the degree of root canal curvatures (0°-20°and 20°-40°) and the taper of master cones (0.02, 0.04, and 0.06). The roots were immersed in the bacterial leakage model and monitored daily for a period of 70 days. Data were analyzed using Kaplan-Meier approach, log-rank test, and Chi-square tests. P < 0.05 was considered statistically significant. RESULTS: The microleakage in the 0°-20° canal curvature using 0.02- and 0.04-tapered master cones was similar and considerably <0.06-tapered master cone (P < 0.05). However, the microleakage in the 20°-40° canal curvature using 0.02- and 0.04-tapered master cones was more than 0°-20° and for 0.06-tapered master cone was <0°-20°, but there was no statistical difference between the use of 0.02-, 0.04-, and 0.06-tapered master cones (P > 0.05). CONCLUSION: The lateral condensation filling technique using 0.02- and 0.04-tapered master cones is more effective in minimizing microbial leakage in straight canals than 0.06-tapered master cone.

Chlorogenic acid inhibits growth of 4T1 breast cancer cells through involvement in Bax/Bcl2 pathway.

OBJECTIVE(S): Chlorogenic acid is an herbal compound with various effects such as antiviral, antioxidant, and anticancer effect with low toxicity, which inhibits cell proliferation. Clinical studies had shown that chlorogenic acid has a positive effect on the different types of cancers treatment. Hence, this study evaluates chlorogenic acid effects on 4T1 breast cancer cells. MATERIALS AND METHODS: In this study, cell proliferation was measured using an 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) on 4T1 cells. Afterwards, other assays like P53, Caspase-3 proteins expression and Annexin V/PI were detected by flow cytometry. Also; Bax and Bcl-2 were carried out by immunocytochemistry. RESULTS: 200 µM of chlorogenic acid concentration showed the highest level of cytotoxicity toward 4T1 cells. Percentage of cell viability data were significant in 100 µM (P < 0.05) and 150, 200 µM (P < 0.001) doses. The evaluation using Annexin V/PI showed cell apoptosis in 100 µM (P < 0.05), 150 µM (P < 0.01), and for 200 µM (P < 0.05 and P < 0.01). Immunocytochemistry results showed the upregulation of Bax and also the downregulation of Bcl-2 in 4T1 cells treated with chlorogenic acid (P < 0.001). The expression level of P53 and caspase-3 increased during treatment with chlorogenic acid in the 4T1 cells (P < 0.001). CONCLUSION: Our findings demonstrated that chlorogenic acid plays a notable role on apoptosis inducing in the 4T1 cells through regulation of apoptotic proteins.

Silver nanoparticles (Ag-NPs) in the central amygdala protect the rat conditioned by morphine from withdrawal attack due to naloxone via high-level nitric oxide.

Repeated injection of morphine during conditioned place preference (CPP) leads to spatial craving due to high-level nitric oxide (NO) in the central nucleus of amygdala (CeA). Silver nanoparticles (Ag-NPs) can produce oxygen-free radicals that lead to NO formation. We aimed to show the Ag-NPs protective effect on naloxone (NLX)-induced morphine withdrawal in the conditioned rats. Wistar rats (300-350 g) were implanted with cannulae in the CeA. After recovery, they were randomly divided into experimental and saline groups. CPP was conducted by three-phase unbiased program. Morphine (0.5-7.5 mg/kg) was injected subcutaneously (s.c.) once/per day during the conditioning phase. Naloxone (NLX) (0.05-0.4 µg/rat) was given, intra-CeA, 10 min before the CPP test. Ag-NPs (0.0001-0.01 µg/rat) were administered alone or prior to the NLX effective dose (0.4 µg/rat), intra-CeA. Conditioning score and withdrawal signs (wet dog shaking and scratching) were obtained and compared with saline group data. All rats' brains were collected in formalin 10% and after 48-72 h stained with NADPH-diaphorase, the NO marker. All data were analyzed by one-way or two-way ANOVA. Morphine (2.5-7.5 mg/kg, s.c.) induced a significant CPP vs. saline (1 mL/kg, s.c.). The single Ag-NPs had no significant effect, whereas the NLX caused meaningful WDS and scratching. However, the NLX pre-treatment in combination with Ag-NPs eliminated these signs. Furthermore, the NO level increased in the CeA. The Ag-NPs may protect the morphine-conditioned rats against the NLX-induced withdrawal symptoms due to high-level NO in the CeA.

Progression-Mediated Changes in Mitochondrial Morphology Promotes Adaptation to Hypoxic Peritoneal Conditions in Serous Ovarian Cancer.

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.

Frequency-selective electrokinetic enrichment of biomolecules in physiological media based on electrical double-layer polarization.

Proteomic biomarkers of interest to the early diagnosis of diseases and infections are present at trace levels versus interfering species. Hence, their selective enrichment is needed within bio-assays for speeding binding kinetics with receptors and for reducing signal interferences. While DC fields can separate biomolecules based on their electrokinetic mobilities, they are unable to selectively enrich biomarkers versus interfering species, which may possess like-charges. We present the utilization of AC electrokinetics to enable frequency-selective enrichment of nanocolloidal biomolecules, based on the characteristic time constant for polarization of their electrical double-layer, since surface conduction in their ion cloud depends on colloidal size, shape and surface charge. In this manner, using DC-offset AC fields, differences in frequency dispersion for negative dielectrophoresis are balanced against electrophoresis in a nanoslit channel to enable the selective enrichment of prostate specific antigen (PSA) versus anti-mouse immunoglobulin antibodies that cause signal interferences to immunoassays. Through coupling enrichment to capture by receptors on graphene-modified surfaces, we demonstrate the elimination of false positives caused by anti-mouse immunoglobulin antibodies to the PSA immunoassay.

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis.

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5-15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery.

Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels.

The neuroprotective potential of sinapic acid in the 6-hydroxydopamine-induced hemi-parkinsonian rat.

Parkinson's disease (PD) is a neurodegenerative movement disorder due to selective loss of dopaminergic neurons of mesencephalic substantia nigra pars compacta (SNC) with debilitating motor symptoms. Current treatments for PD afford symptomatic relief with no prevention of disease progression. Due to the antioxidant and neuroprotective potential of sinapic acid, this study was conducted to evaluate whether this agent could be of benefit in an experimental model of early PD in rat. Unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats were pretreated p.o. with sinapic acid at doses of 10 or 20 mg/kg. One week after surgery, apomorphine caused significant contralateral rotations, a significant reduction in the number of Nissl-stained and tyrosine hydroxylase (TH)-positive neurons and a significant increase of iron reactivity on the left side of SNC. Meanwhile, malondialdehyde (MDA) and nitrite levels in midbrain homogenate significantly increased and activity of superoxide dismutase (SOD) significantly reduced in the 6-OHDA-lesioned group. In addition, sinapic acid at a dose of 20 mg/kg significantly improved turning behavior, prevented loss of SNC dopaminergic neurons, lowered iron reactivity, and attenuated level of MDA and nitrite. These results indicate the neuroprotective potential of sinapic acid against 6-OHDA neurotoxicity that is partially due to the attenuation of oxidative stress and possibly lowering nigral iron level.

Comparison of outcome of femoral shaft fracture fixation with intramedullary nail in elderly patient and patients younger than 60 years old.

BACKGROUND: Although intramedullary nailing (IMN) is an established and accepted operative treatment for femoral shaft fracture in patients younger than 60, there is a lack of data on the results of this treatment on those over 60. The purpose of this study was to determine if the outcome of IMN for femoral shaft fracture in elderly patients is also acceptable. Particular challenges in this group of patients included osteopenia and other associated multiple medical problems frequently observed. METHODS: The outcome of 84 patients who had IMN for femoral shaft fracture was reviewed and the results were compared between two groups of patients (younger than 60 and over 60 year old patients). Complications and mortality was analyzed for each group, and then compared between the two groups by testing the null hypothesis that the outcome of treatment in the two groups are similar (P>0.05). RESULTS: The mean duration of follow up was 57.3 months (range: 10-94 months). Incidence of malunion, nonunion, infection, DVT, and dependence on walker/crutch in the groups were similar and differences were not significant (P>0.05). However, incidence of mortality (P<0.05), knee pain, loss of motion, and dependence on cane were significantly higher in elderly patients (P<0.05). CONCLUSIONS: There is no significant difference between the outcomes of femoral shaft fracture treatment with IMN fixation in younger patients when compared with elderly patients. However, elderly patients with IMN have more symptoms when compared with younger patients.

Aromatase inhibition exacerbates pain and reactive gliosis in the dorsal horn of the spinal cord of female rats caused by spinothalamic tract injury.

Central pain syndrome is characterized by severe and excruciating pain resulting from a lesion in the central nervous system. Previous studies have shown that estradiol decreases pain and that inhibitors of the enzyme aromatase, which synthesizes estradiol from aromatizable androgens, increases pain sensitivity. In this study we have assessed whether aromatase expression in the dorsal horns of the spinal cord is altered in a rat model of central pain syndrome, induced by the unilateral electrolytic lesion of the spinothalamic tract. Protein and mRNA levels of aromatase, as well as the protein and mRNA levels of estrogen receptors α and ß, were increased in the dorsal horn of female rats after spinothalamic tract injury, suggesting that the injury increased estradiol synthesis and signaling in the dorsal horn. To determine whether the increased aromatase expression in this pain model may participate in the control of pain, mechanical allodynia thresholds were determined in both hind paws after the intrathecal administration of letrozole, an aromatase inhibitor. Aromatase inhibition enhanced mechanical allodynia in both hind paws. Because estradiol is known to regulate gliosis we assessed whether the spinothalamic tract injury and aromatase inhibition regulated gliosis in the dorsal horn. The proportion of microglia with a reactive phenotype and the number of glial fibrillary acidic protein-immunoreactive astrocytes were increased by the injury in the dorsal horn. Aromatase inhibition enhanced the effect of the injury on gliosis. Furthermore, a significant a positive correlation of mechanical allodynia and gliosis in the dorsal horn was detected. These findings suggest that aromatase is up-regulated in the dorsal horn in a model of central pain syndrome and that aromatase activity in the spinal cord reduces mechanical allodynia by controlling reactive gliosis in the dorsal horn.

N-Methyl-D-aspartate receptors in the ventral tegmental area are involved in retrieval of inhibitory avoidance memory by nicotine.

The interaction of opiate, cholinergic, glutamatergic and (possibly) dopaminergic inputs in the ventral tegmental area (VTA) influencing a learned behavior is certainly a topic of great interest. In the present study, the effect of intra-VTA administration of N-methyl-d-aspartate (NMDA) receptor agents on nicotine's effect in morphine state-dependent learning was investigated. An inhibitory avoidance (IA) task was used for memory assessment in male Wistar rats. Subcutaneous (s.c.) administration of morphine (5 and 7.5mg/kg) immediately after training decreased IA response on the test day, which was reinstated by pre-test administration of the same doses of the opioid; this is known as state-dependency. Moreover, pre-test administration of nicotine (0.2, 0.4 and 0.6 mg/kg, s.c.) also reversed the decrease in IA response because of post-training morphine (5mg/kg). Here, we also show that when infused into the VTA before testing, NMDA (0.01 and 0.1 microg/rat) reverse the post-training morphine effect on memory. In addition, the sub-effective doses of NMDA (0.0001 and 0.001 microg/rat) in combination with a low dose of nicotine (0.1mg/kg) which had no effects by themselves, synergistically improved retrieval of IA memory on the test day. In contrast, pre-test administration of a competitive NMDA receptor antagonist D-AP5 (0.5, 1 and 2 microg/rat) which had no effect alone prevented the nicotine reversal of morphine effect on memory. Our data indicate that NMDA receptors in the VTA are involved in the reversing effect of nicotine on morphine induced state-dependency.

Nicotine improves morphine-induced impairment of memory: possible involvement of N-methyl-D-aspartate receptors in the nucleus accumbens.

The possible involvement of N-methyl-D-aspartate (NMDA) receptors in the nucleus accumbens (NAc) in nicotine's effect on impairment of memory by morphine was investigated. A passive avoidance task was used for memory assessment in male Wistar rats. Subcutaneous (s.c.) administration of morphine (5 and 10 mg/kg) after training impaired memory performance in the animals when tested 24 h later. Pretest administration of the same doses of morphine reversed impairment of memory because of post-training administration of the opioid. Moreover, administration of nicotine (0.2 and 0.4 mg/kg, s.c.) before the test prevented impairment of memory by morphine (5 mg/kg) given after training. Impairment of memory performance in the animals because of post-training administration of morphine (5 mg/kg) was also prevented by pretest administration of a noncompetitive NMDA receptor antagonist, MK-801 (0.75 and 1 microg/rat). Interestingly, an ineffective dose of MK-801 (0.5 microg/rat) in combination with low doses (0.075 and 0.1 mg/kg) of nicotine, which had no effects alone, synergistically improved memory performance impaired by morphine given after training. On the other hand, pretest administration of NMDA (0.1 and 0.5 microg/rat), which had no effect alone, in combination with an effective dose (0.4 mg/kg, s.c.) of nicotine prevented the improving effect of nicotine on memory impaired by pretreatment morphine. The results suggest a possible role for NMDA receptors of the NAc in the improving effect of nicotine on the morphine-induced amnesia.

Effects of ultra-low doses of nicotine on the expression of morphine-induced conditioned place preference in mice.

In the present study, the effects of acute administration of nicotine, as well as nicotinic and muscarinic acetylcholine receptor antagonists, on the expression of morphine-induced conditioned place preference, have been investigated in male Swiss-Webster mice. Animals received different doses of morphine 5 days after surgical cannulation in the lateral ventricle. Subcutaneous injections of morphine (2-5 mg/kg) in mouse produced place preference in a dose-dependent manner. Furthermore, both intraperitoneal (0.0006-0.1 mg/kg) and intracerebroventricular (0.007-25 ng) nicotine administration significantly reduced the expression of morphine-induced place preference, in a dose-dependent manner. Nicotine, however, was effective over narrow ultra-low dose ranges (0.0012, 0.0025, 0.005 and 0.01 mg/kg; intraperitoneal) and (0.03, 0.1, 0.3 and 0.6 ng/mouse; intracerebroventricular). In addition, locomotor activity was reduced when higher doses of nicotine [both intraperitoneal (0.02, 0.03 and 0.1 mg/kg) and intracerebroventricular (10 and 24 ng/mouse)] were used. Nicotine alone, however, did not cause motivational effects. Intracerebroventricular injection of hexamethonium (0.03, 0.1 and 0.3 mug/mouse; 10 min before nicotine) diminished the effects of nicotine on morphine-induced conditioned place preference. This effect could neither be obtained by intraperitoneal administration of hexamethonium (1, 5 and 10 mg/kg; 30 min before nicotine), nor be reproduced after either intracerebroventricular or intraperitoneal injection of atropine (a muscarinic receptor antagonist). The antagonists, themselves, did not show any motivational effects when used alone and were unable to affect the expression of morphine-induced conditioned place preference. It appears that ultra-low doses of nicotine can reduce the expression of morphine-induced place preference, and that central nicotinic acetylcholine receptors play a role in this regard.

Dorsal hippocampal muscarinic and nicotinic receptors are involved in mediating morphine reward.

In the present study, the effects of bilateral injections of cholinergic agents into the hippocampal CA1 region on morphine-induced conditioned place preference (CPP) were investigated in male Wistar rats. Subcutaneous (s.c.) administration of different doses of morphine sulphate (0.5-6 mg/kg) produced a dose-dependent CPP. Using a 3-day schedule of conditioning, it was found that intra-CA1 administration of the anticholinesterase, physostigmine (2, 4 and 8 microg/rat) significantly potentiated the morphine (0.5 mg/kg)-induced CPP. Moreover, intra-CA1 administration of the muscarinic receptor antagonist, atropine (1, 4 and 7 microg/rat) inhibited the morphine (6 mg/kg)-induced CPP dose-dependently. On the other hand, atropine (7 microg/rat, intra-CA1) reversed the physostigmine-induced potentiation of the morphine response. Furthermore, intra-CA1 administration of nicotine (0.5, 0.75 and 1 microg/rat) with an ineffective dose of morphine (0.5 mg/kg) elicited a significant CPP. Bilateral injections of different doses of the nicotinic receptor antagonist, mecamylamine (2, 4 and 8 microg/rat) into the CA1 regions significantly inhibited the morphine (6 mg/kg)-induced CPP. Moreover mecamylamine (8 microg/rat, intra-CA1) decreased the effect of nicotine-induced potentiation of the morphine response. Intra-CA1 injections of physostigmine, atropine, nicotine or mecamylamine alone did not induce a significant place preference or place aversion. It may be concluded that the muscarinic and nicotinic receptors of the hippocampal CA1 regions play an important role in morphine reward.