Optimization of hydrolysis conditions of amino acid analysis for UHPLC-UV antigens content determination: Bexsero vaccine a case study.

In the present study the compositional analysis of the amino acids released by the acidic hydrolysis of the vaccine antigens was approached as an alternative to the dye-binding methods, for improvement of the quality control. In particular, the Analytical Quality by Design principles were undertaken in optimizing the hydrolysis conditions of the antigens to be applied prior to the quantitation by UHPLC-UV. Bexsero was used as a case study; it is a recombinant meningococcal B vaccine and one of its critical quality attributes is the content of the three core protein antigens, namely Neisseria Heparin Binding Antigen, factor H binding protein and Neisseria adhesin A, in the final formulation. Conventionally, the proteins quantitation is carried out by dye-binding assays. Analytical Target Profile was defined as the accurate determination of amounts of the Bexsero antigens. The Critical Method Parameters were chosen by means of the cause-effect matrix. A Face Centered Design was used to select the experiments to investigate the process and finally a Method Operable Design Region with a risk of failure of 5% was defined. The selected working point for routine use was: hydrolysis time, 17 hrs; temperature, 112 °C; 6 M HCl volume, 300 µl; antioxidant 90% phenol volume, 5 µl.

The efficacy of reboxetine in preventing and reverting a condition of escape deficit in rats.

BACKGROUND: Different stress-induced experimental models of depression are currently used to study the efficacy and mechanism of action of classical or potential antidepressant compounds. We studied the effect of single and repeated administrations of reboxetine, an antidepressant that selectively inhibits noradrenaline reuptake, in the prevention and reversal of stress-induced escape deficit. Moreover, we examined the effect of chronic reboxetine on the stress-induced decrease of dopamine output in the nucleus accumbens shell (NAcS). METHODS: Rats received a single or 21-day reboxetine administration before acute unavoidable stress exposure; 24 hours later their escape response was examined. Rats were exposed to repeated unavoidable stress for 21 days, with or without reboxetine treatment, and then were tested for escape; 2 days later they were implanted with microdialysis probes in the NAcS. RESULTS: A single reboxetine administration showed a protective activity on stress-induced escape deficit development that significantly increased after 21 days of treatment. This effect was antagonized by propranolol, a selective beta-adrenergic antagonist. In rats exposed to chronic stress, a 21-day reboxetine treatment reinstated the avoidance response and NAcS dopamine output to control values. CONCLUSIONS: In these stress-induced models, long-term reboxetine administration showed a protective activity similar to that of classical antidepressants.

Effects of long-term acetyl-L-carnitine administration in rats: I. increased dopamine output in mesocorticolimbic areas and protection toward acute stress exposure.

Acetyl-L-carnitine (ALCAR) is the acetyl ester of carnitine that has been reported to be beneficial in depressive disorders and Alzheimer's disease. A 7-day administration of ALCAR in rats increased dopamine and serotonin output in the nucleus accumbens shell and it prevented the development of escape deficit produced by acute exposure to unavoidable stress. No tolerance developed to this protective effect, which appeared to be mediated by (1) the activation of 5-HT(1A) receptors, as it was antagonized by the administration of WAY100635 30 min before stress exposure; and (2) a process of neuronal plasticity dependent on NMDA receptor activity, as subcutaneous dizocilpine infusion during ALCAR treatment prevented the development of the protective effect on stress. Chronic stress exposure maintains an escape deficit condition that is reverted by a long-term treatment with antidepressants, but the same condition was not modified by long-term ALCAR administration. Thus, ALCAR cannot be defined as an antidepressant.

Effects of long-term acetyl-L-carnitine administration in rats--II: Protection against the disrupting effect of stress on the acquisition of appetitive behavior.

Long-term acetyl-L-carnitine (ALCAR) administration prevents the development of escape deficit produced by acute exposure to unavoidable stress. However, it does not revert the escape deficit sustained by chronic stress exposure. Rats exposed to chronic stress show a low dopamine (DA) output in the nucleus accumbens shell (NAcS) and do not acquire an appetitive behavior sustained by the earning of vanilla sugar (VS) made contingent on the choice of one of the two divergent arms of a Y-maze (VS-sustained appetitive behavior, VAB), while control rats consistently do. The present study shows that ALCAR treatment in rats exposed to a 7-day stress protocol prevented a decrease in DA output in the NAcS and medial prefrontal cortex (mPFC) of rats, and that it strengthened the DA response to VS consummation in the same two areas. Moreover, rats treated with long-term ALCAR or exposed to chronic stress while treated with ALCAR acquired VAB as efficiently as control rats. Moreover, VAB acquisition in stressed rats treated with ALCAR coincided with the reversal of the deficits in escape and in dopaminergic transmission in the NAcS. Thus, repeated ALCAR treatment preserved the DA response to VS in chronically stressed rats and this effect appeared to be predictive of the rat's competence to acquire VAB.

Effects of the essential oil from citrus lemon in male and female rats exposed to a persistent painful stimulation.

The ability of olfaction to modulate behavior in mammalian species has repeatedly been demonstrated. Here we tested the properties of the volatile components of lemon essential oil. Male and female rats were allowed to inhale the aroma while experiencing a persistent nociceptive input (50 microl formalin, 5%); in the same animals the c-Fos immunohistochemistry was used to test the degree of neuronal activation of areas belonging to the limbic system. In formalin-treated animals, lemon essential oil decreased licking the injected paw, in both sexes; flinching and flexing were decreased in males and increased in females in the interphase (5-20 min) of the formalin test. Essential oil increased the c-Fos expression in the arcuate n. of the hypothalamus. Essential oil and formalin increased c-Fos in the paraventricular n. of the hypothalamus and in the dentate gyrus of the hippocampus. In the paraventricular n. of the thalamus formalin induced higher c-Fos than control in both sexes; when formalin treatment was carried out in presence of essential oil, c-Fos further increased in males, but remained at control levels in females. The present results clearly indicate the ability of lemon essential oil to modulate the behavioral and neuronal responses related to nociception and pain.

Sardinian alcohol-preferring and non-preferring rats show different reactivity to aversive stimuli and a similar response to a natural reward.

Sardinian alcohol-preferring (sP) and non-preferring (sNP) rats were studied to ascertain whether some behavioral and/or neurochemical traits, beyond ethanol preference, differentiated the two lines. Spontaneous reactivity of Wistar, sP and sNP rats to aversive or pleasurable stimuli was examined in an avoidance test, an elevated plus maze test, and in response to palatable food presentation. As the mesocorticolimbic dopaminergic system plays a relevant role in the response to rewarding or aversive stimuli, extraneuronal dopamine levels and cocaine-induced dopamine accumulation in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC) were studied by microdialysis in the three groups of rats. Moreover, rats were exposed to repeated unavoidable stress and their avoidance response and NAcS dopamine output were determined. Finally, the capacity of sP, sNP, and Wistar rats to learn a palatable food-sustained appetitive behavior was studied. The present study shows that, beyond ethanol preference, there are several behavioral and neurochemical distinctions between sP and sNP rats. The sP rats displayed an increased level of anxiety-like behavior and sNP rats showed a reduced avoidance of noxious stimuli, compared to Wistar rats. Moreover, in the NAcS and PFC, extraneuronal dopamine levels were higher in sP rats and lower in sNP rats compared to Wistar rats; cocaine-induced dopamine accumulation in the NAcS was higher in sP rats than in sNP and Wistar rats. However, sP and sNP rats showed a similar behavioral and neurochemical response to chronic unavoidable stress. Interestingly, they also showed similar behavioral and neurochemical responses to a natural rewarding stimulus and a similar ability to learn an appetitive behavior.

Sex differences in the citrus lemon essential oil-induced increase of hippocampal acetylcholine release in rats exposed to a persistent painful stimulation.

The microdialysis technique was used to study the ability of essential oil from citrus lemon to modulate hippocampal acetylcholine (ACh) release in male and female rats. Animals were allowed to inhale this odor while experiencing a persistent nociceptive input (50 microl formalin, 5%) or under control conditions (sham-injection). In males, exposure to the essential oil did not change the time course and magnitude of the ACh increase induced by pain. In females, the pain-induced increase of ACh was delayed and increased by exposure to lemon essential oil. The present results indicate that lemon essential oil affects the ACh release differently in male and female rats during a painful condition.

Acquisition of an appetitive behavior prevents development of stress-induced neurochemical modifications in rat nucleus accumbens.

In rats, exposure to chronic unavoidable stress produces a decrease in dopamine output in the nucleus accumbens shell that is accompanied by a decreased density of the dopamine transporter and an increased activity of the dopamine-D(1) receptor complex. These modifications have been hypothesized to be adaptive to decreased dopamine output in stressed rats. We investigated whether the learning of an appetitive behavior sustained by palatable food, which is associated with increased dopamine output in the nucleus accumbens shell as measured by microdialysis experiments, would affect the modifications induced by chronic stress exposure on dopamine transporter density and dopamine-D(1) receptor complex activity in the nucleus accumbens. Rats exposed to chronic unavoidable stress after acquisition of the appetitive behavior showed a higher dopamine extraneuronal release in the nucleus accumbens shell than that of stressed animals, and similar to that of control rats. Moreover, previous acquisition of the appetitive behavior prevented development of a stress-induced decrease in dopamine transporter density, measured by [(3)H]-WIN 35428 binding, a stress-induced increase in dopamine-D(1) receptor density, measured by binding of [(3)H]-SCH 23390, and SKF 38393-stimulated adenylyl cyclase activity in the nucleus accumbens. These results support the hypothesis that changes induced in pre- and postsynaptic dopaminergic transmission by chronic stress exposure are related to decreased dopamine output.

Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress.

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.