Exogenous melatonin's effect on salivary cortisol and amylase: A randomized controlled trial.

This study aimed to examine the effect of acute exogenous melatonin administration on salivary cortisol and alpha-amylase (sCort and sAA) as representatives of the HPA axis and the sympathetic nervous system, respectively. A single-dose prolonged-release melatonin (2 mg) or a placebo tablet was given to healthy volunteers (n = 64) at 20:00 h in a crossover design. The saliva was collected at six time points (20:00, 21:00, awakening, 30 min after awakening, 10:00, and 12:00 h) and was measured for sCort, sAA, and salivary melatonin (sMT) levels. Pulse rates and sleep parameters were also collected. Melatonin was effective in improving sleep onset latency by 7:04 min (p = .037) and increasing total sleep time by 24 min (p = .006). Participants with poor baseline sleep quality responded more strongly to melatonin than participants with normal baseline sleep quality as they reported more satisfaction in having adequate sleep (p = .017). Melatonin administration resulted in higher sCort levels at awakening time point (p = .023) and a tendency of lower sAA levels but these were not significant. Melatonin ingestion at 20:00 h resulted in a marked increase in sMT levels at 21:00 h and remained higher than baseline up to at least 10:00 h (p < .001). Melatonin increases sCort levels at certain time point with a tendency to lower sAA levels. These opposing effects of melatonin suggested a complex interplay between melatonin and these biomarkers. Also, the results confirmed the positive acute effect of a single-dose melatonin on sleep quality.

The Use of Coffee Aroma for Stress Reduction in Postgraduate Dental Students.

OBJECTIVES: This study aimed to investigate the potential reduction of academic stress related to a graded oral presentation in postgraduate dental students using coffee aromatherapy. METHODS: Healthy postgraduate dental students in a seminar class were divided into coffee (n = 32) and control (n = 26) groups. There were 3 modes of aroma distribution: personal distribution with a coffee pad attached to a lanyard, a lanyard plus a personal fan for ventilation of the aroma, and the typical method of the diffuser to spread the aroma in the ambient air. Stress markers comprised levels of salivary alpha-amylase (sAA), cortisol (sCort), and chromogranin A (sCgA). Pulse rates were also measured. RESULTS: Levels of sAA increased 176.62% ± 30.26% between pre- and postpresentation in the control group. Inhaling coffee aroma during the presentation period significantly ameliorated sAA increase at 81.02% ± 14.90% (P = .015). sCort levels tended to decrease in the coffee group, but not significantly. Surprisingly, sCgA levels increased more in the coffee group. Also, pulse rates decreased in the coffee group (-2.07 ± 2.81 bpm) and increased in the control group (6.90 ± 3.22 bpm; P = .035). Subgroup analysis did not reveal differences in salivary markers amongst the 3 aroma distribution modes. CONCLUSIONS: Coffee aroma could have an anxiolytic effect on postgraduate dental students, as evidenced by changes in sAA levels and pulse rates. Personal aroma distribution was also a useful and effective mode of aromatherapy.

A pure compound from Curcuma comosa Roxb. protects neurons against hydrogen peroxide-induced neurotoxicity via the activation of Nrf-2.

Oxidative stress plays a pivotal role in several human diseases including Parkinson's disease (PD). Curcuma comosa, a member of Zingiberaceae, is widely known in Thailand as an alternative medicinal herb for uterine inflammation and estrogenic properties. In this study (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol or compound 092 (C-092, or ASPP 092), a pure compound isolated from ethanol extract of C. comosa, was evaluated for neuroprotective effect on hydrogen peroxide-induced toxicity in SH-SY5Y cells. C-092 demonstrated a radical scavenging effect with comparable efficacy to ascorbic acid and exhibited a neuroprotective effect via suppression of apoptotic cell death as evidenced by a reduction in phospho-p53 and cleaved caspase-3 expression. C-092 causes induction of Nrf-2, which is a transcription factor responsible for the expression of a range of antioxidant genes. Moreover, the reduction in catalase activity caused by hydrogen peroxide was also alleviated by C-092 treatment. These results suggested the therapeutic potential of this compound for neurodegenerative diseases caused by oxidative stress.

Inhibition of LPS-Induced Microglial Activation by the Ethyl Acetate Extract of Pueraria mirifica.

Microglial activation has been found to play a crucial role in various neurological disorders. Proinflammatory substances overproduced by activated microglia, such as cytokines, chemokines, reactive oxygen species, and nitric oxide (NO), can result in neuroinflammation that further exacerbates the course of the diseases. This study aimed to explore the anti-inflammatory effect of the ethyl acetate extract of Pueraria mirifica on microglial activation. Lipopolysaccharide (LPS)-induced inflammation was used as a model to investigate the effects of P. mirifica on HAPI (highly aggressive proliferating immortalized), a rat microglial cell line. Administration of ethyl acetate extract from the tuberous roots of P. mirifica to HAPI cells dose-dependently reduced NO production and iNOS expression induced by LPS. Attenuation of IRF-1 (interferon regulatory factor-1) induction, one of the transcription factors governing iNOS expression, suggested that the inhibitory effect on NO production by the plant extract was at least partially mediated through this transcription factor. In addition, LPS-stimulated mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) was also suppressed with P. mirifica extract pretreatment. This study indicates that the ethyl acetate extract of P. mirifica could potentially serve as an anti-inflammatory mediator and may be useful in relieving the severity of neurological diseases where microglia play a role.

Baseline states of mind differentially affected diurnal salivary stress biomarkers: A preliminary study.

The study aimed to determine how different baseline states of mind in each day (relaxed, ordinary, or stressful) affected the diurnal patterns of three commonly investigated salivary stress biomarkers: cortisol (sCort), alpha-amylase (sAA), and chromogranin A (sCgA). A total of 32 healthy volunteers collected saliva on three different mood days at six time points each day (awakening, 30 min after, 10:00, 12:00, 16:00, and 19:00 h). Pulse rates and subjective feeling of stress using a visual analog scale (VAS) were also recorded. The levels of sCort and sAA were highest on a stressful day at certain time points. The levels of sAA were lowest on a relaxing day in the afternoon. Surprisingly, sCgA levels showed an opposite pattern with the highest level seen on a relaxing day. Of note was that the majority of the participants chose a day during a meditation retreat as their relaxing day and participants practicing mindfulness manifested lower levels of sCort (p = 0.003) and sAA (p = 0.043) at 19:00 h compared with those choosing a general leisure day as their relaxing day. Different states of mind were associated with different courses of salivary stress biomarkers. sCort and sAA are the most reliable markers showing the expected trend with higher levels on a stressful day and lower levels on a relaxing day. While the current result cast doubt on the use of sCgA as a stress marker since it was the only marker that showed the opposite trend compared with those of the other two markers as well as pulse rates and VAS. Furthermore, this is the first study to demonstrate that mindfulness practice might have different effects on these biomarkers from just a general relaxed state of mind.

Comparison of salivary cortisol, amylase, and chromogranin A diurnal profiles in healthy volunteers.

OBJECTIVE: This study aimed to compare the diurnal patterns of three salivary biomarkers (cortisol, amylase, and chromogranin A), as well as the factors affecting their levels. DESIGN: A total of 110 participants with mean age of 26.93 ± 6.05 years old took part in the study. The saliva was collected at awakening, 30 min after awakening, 10:00, 12:00, 16:00, and 19:00 h. Cortisol, amylase, chromogranin A, and total protein levels were determined. RESULTS: The diurnal patterns of three biomarkers were different with cortisol showing the least variance and chromogranin A showing the highest variance among individuals. Participants with lower BMI exhibited higher cortisol levels (p = 0.044). Age oppositely affected amylase and chromogranin A, as being older was associated with higher amylase (p = 0.029) and lower chromogranin A levels (p < 0.01) even though both markers represent the sympathetic activity. Male participants also showed lower chromogranin A levels than females (p = 0.045). Total protein concentration affected chromogranin A levels only around awakening period but not at other time points suggesting that protein adjustment may not be necessary if the experiment is performed during the day. The afternoon was the period where all three biomarkers showed rather stable levels. CONCLUSIONS: These results illustrate the nature of cortisol, amylase, and chromogranin A patterns throughout the day in a normal physiological state and help in choosing the right condition to perform the experiment with these biomarkers.

Differential responses of salivary cortisol, amylase, and chromogranin A to academic stress.

Salivary biomarkers have been widely used to help diagnose stress, anxiety, and/or depression. This study aimed to compare the responses of three commonly investigated salivary stress biomarkers that represent the hypothalamic-pituitary-adrenal activity (cortisol; sCort) and the sympathetic activity (alpha-amylase; sAA and chromogranin A; sCgA), using academic oral presentation as a model of stress. Twenty postgraduate dental students attended the seminar class as presenter and audience. The presenters' performances were evaluated by the instructors suggesting more stress than the audience. The saliva was collected two times: before attending class and after an academic presentation (for presenters) or during the class (for audience). The pulse rates (PR) were also recorded. The results showed that the levels of all three biomarkers, as well as PR, were significantly higher in the presenter group compared with the audience group; however, the changes were most prominent with sCort and sAA (99.56 ± 12.76% for sCort, 93.48 ± 41.29% for sAA, 16.86 ± 6.42% for sCgA, and 15.06 ± 3.41% for PR). When compared between pre-post presentation, the levels of sCgA were not different, while those of sCort and sAA were significantly increased. These results suggest more sensitive reactivity to academic stress of sCort and sAA compared with sCgA and that the response of sCgA did not necessarily follow sAA pattern even though both are claimed to reflect the sympathetic activity. More studies are needed to elucidate the roles of sCgA in stress.

The calming effect of roasted coffee aroma in patients undergoing dental procedures.

Coffee beverage consumption is well-known to exert various health benefits; however, the effects of coffee aroma are rarely explored. This study aimed to investigate the calming effect of inhaling coffee aroma while the patients underwent dental procedures (probing and scaling). Salivary α-amylase (sAA) and cortisol (sCort) levels were measured as proxies of sympathetic nervous system and hypothalamic-pituitary-adrenal axis responses to stress respectively. Blood pressures and pulse rates were recorded. The results showed that undergoing dental procedures could increase sAA and sCort levels of the patients inhaling sham aroma while those inhaling coffee aroma had significantly decreased sAA and sCort levels (40% and 25% differences, respectively). The pulse rates of those inhaling coffee aroma were also lower. Subjective assessment using visual analog scale was in line with objective measures as well. The preference for coffee aroma or the frequency of coffee drinking had no effect on the sAA and sCort responses. This is the first study to provide evidence on the effect of coffee aroma on sAA and sCort levels in patients undergoing dental procedures.

Sterility maintenance of reused disposable paper/plastic sterilization pouches in actual clinical practice.

Paper/plastic sterilization pouches are commonly used packaging material for steam sterilization. Reuse of these pouches is a general practice in Thailand despite a single-use recommendation. This study aimed to determine microbial contamination after reusing paper/plastic sterilization pouches in a dental clinic and storage in a closed environment for 6 months. Three hundred and twenty pouches underwent 3 times of clinical use in terms of packaging, autoclave sterilization, handling, and unpacking. A mouth mirror was packed in each pouch to be used in a clinic. After each use, a pouch would be carefully inspected for reusability and undergone packaging, sterilization, handling again. In all steps, sterilization monitoring was rigorously applied. After 3 times of use, a piece of filter paper was placed inside each pouch (instead of a mouth mirror), the pouch was autoclaved and stored in a closed environment for 6 months. Then the filter paper was retrieved for microbial cultivation. A negative control group comprised new pouches containing filter paper without storage and a positive control group comprised pouches with impaired integrity. All samples in both the reuse and the negative control groups had no microbial contamination. All samples in the positive control group showed contamination. These results suggested that reusing paper/plastic sterilization pouches could be a safe practice provided careful monitoring and inspection were employed.

Factors affecting salivary α-amylase levels measured with a handheld biosensor and a standard laboratory assay.

OBJECTIVES: A handheld biosensor for measuring salivary α-amylase (sAA) was developed for convenient on-site measurement. Previous studies reported some discrepancies in sAA levels measured with a biosensor and a standard assay. This study aimed to compare sAA levels measured with three different methods and the factors affecting its levels. METHODS: Thirty-eight participants collected saliva two times for three measurements. First, the collector strip was placed under the tongue for 2 minutes, then the strip was used to measure sAA level on-site immediately (intraoral biosensor; method 1). Then, a participant pooled the saliva for 4 minutes and collected the saliva into the tube which was aliquoted to measure in a laboratory with a handheld biosensor (extraoral biosensor; method 2) and with a standard enzyme kinetic assay (EKA; method 3). Additional experiments were carried out to compare the levels of sAA measured with differences in pooling time and positioning of the collector strip. RESULTS: A high correlation of sAA levels between an extraoral and an EKA measurement (r = 0.989) was observed, while sAA levels measured with an intraoral method showed a significant but weaker correlation with either an EKA (r = 0.475) or an extraoral method (r = 0.436). Saliva pooling time and positioning of the collector strip significantly affected sAA levels. CONCLUSIONS: A handheld biosensor is valid to measure sAA levels extraorally. For an intraoral measurement, pooling time and positioning of the collector strip need to be taken into account.

Effects of resterilization and storage time on sterility of paper/plastic pouches.

OBJECTIVES: Paper/plastic pouches are widely used packaging materials for autoclaving medical and dental equipment. Although these pouches are recommended for single use, they are generally reused in Thailand. This study aimed to determine the ability of paper/plastic pouches to maintain sterility after multiple sterilization processes and stored in a closed environment for up to 6 months. MATERIALS AND METHODS: A total of 6720 paper/plastic pouches were divided into four experimental groups: new pouches, 1 time, 3 times, and 5 times resterilized pouches. A piece of filter paper was placed inside each pouch, and the pouch was sealed, sterilized, and stored for up to 6 months. At the end of each storage period, the pouch was opened, and the filter paper was transferred to culture broth for microbial cultivation to determine sterility. Negative and positive controls were also used to validate the procedures. RESULTS: All filter papers in the experimental groups, as well as the negative control group, remained sterile for up to 6 months of storage in a closed environment. On the contrary, all filter papers in the positive control group showed microbial contamination. CONCLUSIONS: In a closed storage condition, the paper/plastic pouches that passed multiple sterilization processes (up to 5 times resterilization) still maintained good barrier efficacy and remained sterile for up to 6 months.

Concurrent suppression of NF-κB, p38 MAPK and reactive oxygen species formation underlies the effect of a novel compound isolated from Curcuma comosa Roxb. in LPS-activated microglia.

OBJECTIVE: We investigated the molecular mechanisms underlying the effect of (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol, also known as compound 092, isolated from Curcuma comosa Roxb on the production of pro-inflammatory mediators and oxidative stress in lipopolysaccharide (LPS)-activated highly aggressive proliferating immortalized (HAPI) microglial cell lines. METHOD: Nitric oxide (NO) production was determined using the Griess reaction, and reverse transcription polymerase chain reaction was used to measure the expression of inducible nitric oxide synthase (iNOS) mRNA. Western blotting was used to determine the levels of pro-inflammatory mediators and their related upstream proteins. KEY FINDING: Compound 092 suppressed NO production and iNOS expression in LPS-stimulated HAPI cells. These effects originated from the ability of compound 092 to attenuate the activation of nuclear factor (NF)-κB as determined by the reduction in p-NF-κB and p-IκB kinase (IKK) protein levels. Compound 092 also significantly lowered LPS-activated intracellular reactive oxygen species production and p38 mitogen-activated protein kinase (MAPK) activation. CONCLUSION: Compound 092 suppresses microglial activation through attenuation of p38 MAPK and NF-κB activation. Compound 092 thus holds the potential to treat neurodegenerative disorders associated with neuroinflammation and oxidative stress.

The interface of depression and obesity.

Depression and obesity are both highly prevalent and are leading public health problems. These foregoing disorders independently have great impact on morbidity and mortality affecting patients' health and well-being as well as on the socioeconomic aspect of functional impairment and healthcare expenditure. Results from epidemiological studies, clinical trials and recent meta-analyses support the association between mood disorders and obesity as both frequently co-occur in all races of populations examined. It is now well-established through longitudinal studies that obesity is a risk factor for mood disorders and vice versa. In the current review, we aim to address the evidence regarding 4 questions: (1) does obesity moderate response to antidepressants among patients with depressive disorders?, (2) does the presence of depressive disorders moderate the progression or outcome of obesity?, (3) does treatment of obesity moderate outcomes among patients with depressive disorders?, and (4) does treatment of depressive disorders moderate outcomes of obesity? In order to improve the interpretability of the results we confined the evaluations to studies where patients met the criteria for depressive disorders or obesity (i.e. BMI>30). Extant evidence supports the association between obesity and adverse health outcomes among individuals with depressive disorders. In addition, the treatment of one condition (i.e. obesity or depressive disorders) appears to improve the course of the other condition. It might be beneficial to check for the other condition in patients presenting with one condition and treatment should be administered to treat both conditions.

Protective Effects of a Diarylheptanoid from Curcuma comosa Against Hydrogen Peroxide-Induced Astroglial Cell Death.

Oxidative stress is one of the major mechanisms causing neuronal and astroglial cell death in various neurological disorders such as Alzheimer's disease, Parkinson's disease, and brain ischemia. Two diarylheptanoids, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (ASPP 049) and (3S)-7-(3,4-dihydroxyphenyl)-1-phenyl-(1E)-1-hepten-3-ol (ASPP 092), isolated from Curcuma comosa were investigated for cytoprotective effects on C6 astroglial cells using hydrogen peroxide (H2O2) exposure as a model of oxidative stress. ASPP 092 demonstrated free radical scavenging activity comparable to that of vitamin C, while ASPP 049 showed no antioxidant activity. Treatment with H2O2 at 400 µM for 12 h caused 79 % C6 astroglial cell death which was significantly reduced to 37 % by pretreatment with ASPP 092 (5 µM). In addition, ASPP 092 attenuated the increase in reactive oxygen species production and the decrease in total glutathione level induced by H2O2. The mechanism of ASPP 092 protection against H2O2-induced apoptotic signaling appeared to involve prevention of increase in the level of phosphorylated p53 and the Bax/Bcl-2 ratio as well as cleaved caspase-3. These findings provide new evidence that the diarylheptanoid ASPP 092 from C. comosa possesses antiapoptotic properties and could be further developed as a potential treatment for oxidative stress-related neuronal diseases.

Autophagy inhibition by caffeine increases toxicity of methamphetamine in SH-SY5Y neuroblastoma cell line.

Methamphetamine (METH) is a highly addictive CNS stimulant that its long-term use is associated with the loss of neurons in substantia nigra and development of Parkinson's disease later in life. Common form of METH is Ya-Ba tablet, in which, large portion of caffeine is added to the mass to enhance the stimulatory effect. Previous study demonstrated that caffeine potentiates the toxic effect of METH in association with the production of reactive oxygen species and the induction of apoptosis. Since METH causes induction of autophagy, the question was raised whether this pathway participates in the potentiating effect of caffeine on METH neurotoxicity. We used SH-SY5Y, a neuroblastoma cell line, as an in vitro model to study the effect of METH and caffeine. Co-treatment of non-toxic concentrations of METH, at 0.5 mM, and caffeine, at 1 mM, caused reduction of the cell viability. Reduction of the cell viability was associated with attenuation of autophagy, demonstrated by reduction of LC3-II levels and the number of autophagosome puncta, together with increase of caspase-3 activation. Similar effect was produced by treatment with autophagy inhibitors, 3-MA and wortmanin. Our results suggested that caffeine potentiates METH toxicity through inhibition of autophagy and that autophagy serves as a protective mechanism. In conclusion, we proposed the augmented hazard associated with caffeine and METH combination in Ya-Ba abusers.

Upregulation and expression patterns of the angiogenic transcription factor ets-1 in Alzheimer's disease brain.

Immunohistochemical staining has been used to determine expression patterns of the angiogenic transcription factor, Ets-1, in the brains of Alzheimer's disease (AD) individuals. Brain tissue from non-demented controls showed little expression of Ets-1 whereas in AD brain tissue, Ets-1 was ubiquitously expressed in cortex and hippocampus. Double immunostaining with von Willerbrand factor demonstrated prominent Ets-1 intravascular immunoreactivity (ir) in AD cortical microvessels. In addition, Ets-1 also exhibited extravascular expression characterized by a diffuse pattern of Ets-1 ir in AD brain. Double staining also showed Ets-1 colocalization in microvasculature with the potent angiogenic agent, vascular endothelial growth factor (VEGF). Cell-associated tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with pro-angiogenic activity, was primarily associated with diffuse extravascular Ets-1 ir. Clusters of HLA-DR positive microglia, resident immune cells of brain which release TNF-α, were also localized with diffuse Ets-1. Intravascular Ets-1 ir was maximally co-localized with soluble amyloid-ß peptide (Aß), Aß1-40, in microvasculature whereas diffuse extravascular Ets-1 ir appeared in proximity to Aß plaques in brain parenchyma. Similar overall results were obtained for patterns of Ets-1 staining in AD hippocampal tissue. This work provides novel findings on expression of the angiogenic transcription factor, Ets-1, in vascular remodeling and its association with pro-angiogenic factors, reactive microglia, and Aß deposition in AD brain.

Phytoestrogens mediated anti-inflammatory effect through suppression of IRF-1 and pSTAT1 expressions in lipopolysaccharide-activated microglia.

Microglial activation has been implicated in various neurological disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and HIV encephalopathy. Phytoestrogens have been shown to be neuroprotective in neurotoxicity models; however, their effect on microglia has not been well established. In the current study, we report that the soy phytoestrogens, genistein, daidzein, and coumestrol, decreased nitric oxide (NO) production induced by lipopolysaccharide (LPS) in the rat microglial cell line (HAPI). The levels of inducible NO synthase (iNOS) mRNA and protein expression were also reduced. Transcription factors known to govern iNOS expression including interferon regulatory factor-1 (IRF-1) and phosphorylated STAT1 were down regulated. These observations explain, at least in part, the inhibitory effect of phytoestrogens on NO production. The levels of monocyte chemoattractant protein-1 and interleukin-6 mRNA, proinflammatory chemokine and cytokine associated with various neurological disorders, were also reduced following LPS stimulation when HAPI cells were pretreated with phytoestrogens. Hence, genistein, daidzein, and coumestrol could serve as anti-inflammatory agents and may have beneficial effects in the treatment of neurodegenerative diseases.

Actions of the anti-angiogenic compound angiostatin in an animal model of Alzheimer's disease.

We have examined the anti-angiogenic compound, angiostatin as a modulator of inflammatory reactivity and vascular responses and for neuroprotection in an animal model of Alzheimer's disease (AD). Intra-hippocampal amyloidbeta (Aß1₋42) injection, relative to controls phosphate buffer saline (PBS) or reverse peptide Aß42₋1, increased gliosis in the molecular layer (ML) of rat hippocampus. Vascular remodeling was indicated from increased microvessel immunoreactivity (ir) in ML suggesting the possibility of an angiogenic response to peptide injection. Administration of Aß1₋42 also induced a loss of neurons in the granule cell region of hippocampus relative to controls. Treatment of peptide-injected rats with angiostatin was associated with a spectrum of modulatory effects including reduced microgliosis (by 34%), diminished microvessel ir (by 36%) and increased neuronal viability (by 31%) compared with peptide injection alone. Angiostatin treatment was ineffective in reducing astrogliosis induced by Aß1₋42 and applied alone the compound had no significant effect to alter gliosis, microvessel ir or neuronal viability compared with PBS control. In vitro, angiostatin significantly attenuated secretion of the pro-angiogenic agent, vascular endothelial growth factor (VEGF) in lipopolysaccharide (LPS)-stimulated THP-1 cells. Our findings provide novel evidence for a broad spectrum of angiostatin effects in an animal model of AD including actions to reduce inflammatory reactivity, stabilize vascular remodeling and confer neuroprotection. The overall effects of angiostatin are consistent with actions of the compound to inhibit microglial secretion of VEGF.

Mechanisms of Mg2+ inhibition of BzATP-dependent Ca2+ responses in THP-1 monocytes.

We have recently reported effects of Mg2+ to confer neuroprotection against toxicity of purinergic stimulated microglia and THP-1 monocytes. To examine mechanisms underlying neuroprotection, we have studied Mg2+ modulation of transient changes in intracellular Ca2+ ([Ca2+]i) in THP-1 cells induced by P2X7R agonist 2',3'-[benzoyl-4-benzoyl]-ATP (BzATP). Application of BzATP caused a rapid transient increase in [Ca2+]i followed by a prolonged component. The time course of the secondary slower phase was significantly reduced with Ca2+-free extracellular solution, with treatment of THP-1 cells by the P2X7R antagonist, oxATP or with exposure of cells to the store-operated channel (SOC) inhibitor, SKF96365. These results suggest that Ca2+ influx, mediated by both the P2X7R or by SOC, contribute to the slow component of [Ca2+]i. Treatment of THP-1 cells with 10 mMMg2+ was highly effective in reducing the time course of BzATP-induced Ca2+ decay; unlike the other modulatory protocols, Mg2+ markedly inhibited the amplitudes of slow and rapid components. In addition, acute application of Mg2+ during BzATP-induced responses elicited in the presence of either oxATP or SKF96365 to block respective P2X7R and SOC contributions, rapidly attenuated [Ca2+]i to baseline levels. Priming of cells with the inflammatory stimulus LPS/IFN-γ markedly enhanced the slower, but not rapid, phase of BzATP-induced [Ca2+]i with application of 10 mMMg2+ inhibiting both components of response. A model is proposed to account for BzATP stimulation of both ionotropic P2XR and metabotropic P2YR which provides a mechanistic basis for elevated Mg2+ anti-inflammatory and neuroprotective actions in inflamed brain.

Comparison of Vascular Perturbations in an Aß-Injected Animal Model and in AD Brain.

The validity of amyloid-ß peptide (Aß(1-42)) intrahippocampal injection, as an animal model of Alzheimer's disease (AD), has previously been considered in terms of inflammatory reactivity and neuronal damage. In this work, we have extended the testing of the animal model to vasculature by comparison of selected properties of microvessels in vivo with those in human AD brain tissue. The injection of Aß(1-42), relative to control PBS (phosphate buffered saline), increased the mean number of microvessels and diminished the mean length of microvessels in the molecular layer of dentate gyrus. The animal model showed Aß(1-42), but not PBS, injection was associated with abnormalities in morphology of microvessels which were characterized as looping, fragmented, knob-like, uneven, and constricted. In particular, numbers of constricted microvessels, defined as vessels with diameters less than 3 µm, were considerably enhanced for Aß(1-42), compared to PBS, injection. In comparison, human AD brain demonstrated an elevated number of microvessels with a diminished mean length relative to nondemented (ND) brain. Additionally, microvessel perturbations in AD brain showed a similar pattern of morphological abnormalities to those observed in Aß(1-42)-injected rat hippocampus. Constricted microvessels were a prominent feature of AD brain but were rarely observed in ND tissue. These results provide the first evidence that a peptide-injection animal model exhibits a commonality in perturbations of microvessels compared with those evident in AD brain.