Valproic Acid attenuates disease symptoms and increases endogenous myelin repair by recruiting neural stem cells and oligodendrocyte progenitors in experimental autoimmune encephalomyelitis.

BACKGROUND: inefficient remyelination of demyelinated plaques in multiple sclerosis (ms) leads to secondary axon degeneration and progressive disability. therapies that potentiate remyelination would be of immense help for managing MS. OBJECTIVE: Here, we report the effects of valproic acid (VPA) on focal experimental autoimmune encephalomyelitis (fEAE). METHODS: fEAE was induced in Wistar rats by immunizing the animals with guinea pig spinal cord homogenate emulsified in complete Freund's adjuvant and with pertussis toxin (PT) injection into the spinal cord at the level of T8 vertebra on day 18 after immunization. VPA 300 mg/kg was applied for 4 days after or 8 days before PT administration. Behavioral evaluation, histological assessment and immunohistofluorescence assays were used to evaluate the outcomes. RESULTS: VPA administration had no effect on the development of symptoms, but after discontinuing VPA, animals showed faster recovery. Eight days of pretreatment with VPA accelerated the recovery phase of EAE and increased the number of remyelinated axons in the lesion area. VPA pretreatment also increased the recruitment of neural stem cells and oligodendrocyte precursors within the lesion. CONCLUSIONS: Results suggest VPA as a potential therapy for remyelinating the lesions in MS and for faster recovery from disease relapses. The effect of VPA seems to be mediated by endogenous progenitors recruitment.

The use of 3-aminophthalimide as a pro-chemiluminescent label in chemiluminescence and fluorescence-based cellular assays.

We present a labeling system for direct chemiluminescence-based cellular bioassays using the stable pro-chemiluminescent, luminol precursor, 3-aminophthalimide (API). API-coupled reporter molecules are detected chemiluminometrically after treatment with hydrazine, which converts the API label to luminol. API derivatives containing a variety of functional groups are readily synthesized, allowing for ease of coupling via the imide nitrogen to a host of reporter molecules. The fluorescent nature of APIs further allows for dual fluorescence and chemiluminescence studies. To highlight the utility of this label, we show that API-labeled insulin can be successfully utilized in cellular binding and transport assays and that an API-coupled mitochondrial probe (API-triphenylphosphonium(+)) can be used to both fluorescently and chemiluminometrically investigate mitochondrial function. We also assess the use of API as a polysaccharide and nucleic acid label, and we show that API-labeled palmitic acid undergoes cellular transport and lipid metabolism.

Nitric oxide involvement in the antidepressant-like effects of acute lithium administration in the mouse forced swimming test.

In the present study we evaluated the involvement of l-arginine/nitric oxide (NO)/cGMP pathway in the antidepressant-like effects of acute lithium administration in the mouse forced swimming test (FST). Lithium, at 30 and 100 mg/kg, significantly reduced the immobility times of mice in the FST, whereas at lower doses (0.5, 5 and 10 mg/kg) had no effect on the immobility time. The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), at 10 and 30 mg/kg, and the selective neuronal NOS inhibitor N(omega)-propyl-L-arginine (L-NPA), at 5 and 15 mg/kg, had no significant effects on the FST, whereas they significantly decreased the immobility time at 100 and 30 mg/kg, respectively. Combination of non-effective dose of lithium (10 mg/kg) with low doses of L-NAME (30 mg/kg) or L-NPA (15 mg/kg) significantly reduced the immobility times in the FST. Moreover, the guanylyl cyclase inhibitor ODQ at 50 mg/kg significantly decreased the immobility time of mice, whereas it had not significant effects on the FST at 2, 10 and 20 mg/kg. Combination of lithium (10 mg/kg) with 20 mg/kg ODQ significantly decreased the immobility times in the FST. Non-effective doses of L-arginine (750 mg/kg) or sildenafil (5 mg/kg) significantly reversed the antidepressant-like effect of 30 mg/kg lithium in the FST. Neither of the drugs had effect on the locomotor activity. These data indicate the involvement of L-arginine/NO/cGMP pathway in the antidepressant-like effect of lithium in the mouse FST and also might suggest the concurrent administration of NOS inhibitors and lithium as an appropriate strategy for treatment of depression.

Orexin A presynaptically decreases inhibitory synaptic transmission in rat locus coeruleus neurons.

Locus coeruleus nucleus (LC) is a major noradrenergic nucleus in the brain. It receives dense orexinergic projections from lateral hypothalamus. Whilst it is known that orexin A increases firing rate of LC neurons, its effect on spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs, respectively) has not been yet identified. In this research, we investigated the effect of orexin A on eIPSCs and sIPSCs in LC neurons. Whole-cell recordings revealed that orexin A suppresses eIPSCs amplitude in which this effect was blocked by an orexin type-1 receptors antagonist (SB-334867) and cannabinoid type-1 (CB1) receptors antagonist (AM251). Moreover, exposure of neurons to BAPTA (Ca2+ chelator) and U73122 (phospholipase C inhibitor) prevented orexin A-induced eIPSCs depression. On the other hand, orexin A increased pair pulse ratio and sIPSCs frequency but had no effect on sIPSCs amplitude. Our results revealed that eIPSCs suppression in the LC is mediated by CB1 receptor through a presynaptic mechanism.

In vitro and in vivo evaluation of thermosensitive chitosan hydrogel for sustained release of insulin.

Injectable In situ gel-forming chitosan/ß-glycerol phosphate (CS/ß-Gp) solution can be introduced into the body in a minimally invasive manner prior to solidifying within the target tissue. This hydrogel is a good candidate for achieving a prolonged drug delivery system for insulin considering its high molecular weight. In addition to the physicochemical characterization of this hydrogel, in vitro and in vivo applications were studied as a sustained insulin delivery system. In the in vitro release studies, 19-63% of total insulin was released from the CS/ß-Gp hydrogel within 150 h at different ß-Gp and insulin concentrations. The best formulation was selected for in vivo experimentation to control the plasma glucose of diabetic mice models. The hypoglycemic effect of this formulation following subcutaneous injection in diabetic mice lasted 5 d, significantly longer than that of free insulin solution which lasted several hours.

Assessment of 6-sulfatoxymelatonin rhythms and melatonin response to light in disease states: lessons from cirrhosis.

Circadian rhythmicity and non-visual sensitivity to light can be assessed, in healthy subjects, by measuring the rhythm of the urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) and by determining the response of plasma melatonin to nocturnal retinal light exposure, respectively. However, the validity of these techniques has not been assessed in disease states in which disruption of the circadian rhythm is known or suspected to occur. Thus, the aims of this study were as follows: (i) to assess the reliability of circadian aMT6s profile estimates derived from 36 h versus 56 h urine collections and (ii) to test different models for calculating melatonin suppression in response to light in healthy volunteers and patients with cirrhosis. Twenty patients with biopsy-proven cirrhosis and 10 matched healthy volunteers undertook: (i) separate 36 - and 56-h urine collections, under controlled conditions, for cosinor analysis of the urinary aMT6s profile; (ii) a melatonin suppression test, comprising of a baseline night, during which subjects were woken and asked to sit in front of a switched off light sphere, and an experimental night, identically executed, except that the light sphere was switched on and the subjects were exposed to white light (4.1 × 10(14) photons/cm(2)/s) for 30 min. Alternative approaches to the calculation of melatonin suppression were taken, with/without inclusion of the baseline night. Eighteen patients and eight healthy volunteers had matched analysable 36 - and 56-h urinary samples. Cosinor analysis showed a significant fit in 88% of the remaining 56 h collections, and 48% of the remaining 36-h collections. Thus, eight patients and five healthy volunteers had matched analysable samples for cosinor analysis. In the healthy volunteers, aMT6s profile indices obtained using the 36 - and the 56-h collections did not differ significantly. In contrast, considerably more variability was observed in patients [i.e. the difference in the aMT6s peak time was 0.5 ± 1.7 h (limits of agreement: -3.9; +2.9 h)]. No difficulties were encountered in obtaining suppression estimates by use of the experimental night only. In contrast, suppression estimates obtained by use of both nights were considered inaccurate in one (11%) healthy volunteer and in 5 (28%) patients, primarily because: (i) melatonin concentrations at the beginning of light administration were significantly different on baseline and experimental night; (ii) the rise in melatonin was inconsistent on baseline night; and (iii) the shape of the rising phase of melatonin was different on baseline and experimental night. In conclusion, shorter urine collections lead to a higher number of profiles with no significant cosinor fit, and differences in cosinor indices obtained from the 36 - and 56-h collections were considerable, especially in patients. Thus, 56-h collections are probably advisable. Use of both baseline and experimental nights to calculate melatonin suppression often resulted in increased variation and confounding, due to point oscillations in melatonin concentration and lack of repeatability of the melatonin profiles on the two nights. Thus, use of the experimental night only is probably advisable.

Do endogenous opioids contribute to the bradycardia of rats with obstructive cholestasis?

Endogenous opioids have nitric oxide (NO)-dependent cardiovascular actions. In the light of biological evidence of accumulation of endogenous opioids in cholestasis and also existence of NO-dependent bradycardia in cholestatic subjects, this study was carried out to evaluate the role of endogenous opioids in the generation of bradycardia in a rat model of cholestasis. Male Sprague-Dawley rats were used to induce cholestasis by surgical ligation of the bile duct, with sham-operated animals serving as a control. The animals were divided into six groups which received naltrexone [20 mg/kg/day, subcutaneously (s.c.)], N(G)-L-nitro-arginine methyl ester (L-NAME, 3 mg/kg/day, s.c.), aminoguanidine (200 mg/kg/day, s.c.), L-arginine (200 mg/kg/day, s.c.), naltrexone + L-NAME (20 and 3 mg/kg/day, s.c) or saline. One week after the operation, a lead II electrocardiogram (ECG) was recorded and the spontaneously beating atria of the animals were then isolated and the chronotropic responses to epinephrine evaluated. The plasma L-nitro-tyrosine level and alanine amino transferase and alkaline phosphatase activities were also measured. The heart rate of cholestatic animals was significantly lower than that of control rats in vivo and this bradycardia was corrected with daily adminstration of naltrexone or L-NAME. The basal spontaneous beating rate of atria in cholestatic animals was not significantly different from that of sham-operated animals in vitro. Cholestasis induced a significant decrease in the chronotropic effect of epinephrine. This effect was corrected by daily injection of naltrexone or L-NAME, or concurrent administration of naltrexone + L-NAME, and was not corrected by aminoguanidine. L-arginine had an equivalent effect to L-NAME and increased the chronotropic effect of epinephrine in cholestatic rats but not in control animals. Bile duct ligation increased the plasma activity of liver enzymes as well as the level of L-nitro-tyrosine. L-arginine and naltrexone treatment significantly decreased the elevation of liver enzymes in bile duct-ligated rats. Pretreatment of cholestatic animals with naltrexone or L-NAME decreased the plasma L-nitro-tyrosine level. The results suggest that either prevention of NO overproduction or protection against liver damage is responsible for recovery of bradycardia after naltrexone administration.