Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects.

In a previous clinical study, a probiotic formulation (PF) consisting of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (PF) decreased stress-induced gastrointestinal discomfort. Emerging evidence of a role for gut microbiota on central nervous system functions therefore suggests that oral intake of probiotics may have beneficial consequences on mood and psychological distress. The aim of the present study was to investigate the anxiolytic-like activity of PF in rats, and its possible effects on anxiety, depression, stress and coping strategies in healthy human volunteers. In the preclinical study, rats were daily administered PF for 2 weeks and subsequently tested in the conditioned defensive burying test, a screening model for anti-anxiety agents. In the clinical trial, volunteers participated in a double-blind, placebo-controlled, randomised parallel group study with PF administered for 30 d and assessed with the Hopkins Symptom Checklist (HSCL-90), the Hospital Anxiety and Depression Scale (HADS), the Perceived Stress Scale, the Coping Checklist (CCL) and 24 h urinary free cortisol (UFC). Daily subchronic administration of PF significantly reduced anxiety-like behaviour in rats (P < 0·05) and alleviated psychological distress in volunteers, as measured particularly by the HSCL-90 scale (global severity index, P < 0·05; somatisation, P < 0·05; depression, P < 0·05; and anger-hostility, P < 0·05), the HADS (HADS global score, P < 0·05; and HADS-anxiety, P < 0·06), and by the CCL (problem solving, P < 0·05) and the UFC level (P < 0·05). L. helveticus R0052 and B. longum R0175 taken in combination display anxiolytic-like activity in rats and beneficial psychological effects in healthy human volunteers.

Effect of an oral supplementation with a proprietary melon juice concentrate (Extramel) on stress and fatigue in healthy people: a pilot, double-blind, placebo-controlled clinical trial.

BACKGROUND: Recent studies have demonstrated a correlation between perceived stress and oxidative stress. As SOD is the main enzyme of the enzymatic antioxidant defence system of the body, we evaluated the effect of an oral daily intake of a proprietary melon juice concentrate rich in SOD (EXTRAMEL) on the signs and symptoms of stress and fatigue in healthy volunteers. METHODS: This randomized, double blind, placebo controlled clinical study was conducted with seventy healthy volunteers aged between 30 and 55 years, who feel daily stress and fatigue. They took the dietary supplement based on the melon juice concentrate (10 mg Extramel corresponding to 140 IU SOD per capsule) or a placebo one time daily during 4 weeks. Stress and fatigue were measured using four observational psychometric scales: FARD, PSS-14, SF-12 and Epworth scale. The study was conducted by Isoclin, a clinical research organization, located in Poitiers, France. RESULTS: No adverse effect was noted. The supplementation with the proprietary melon juice concentrate bringing 140 IU SOD/day significantly improved signs and symptoms of stress and fatigue linked to performance, physical (pain, sleep troubles), cognitive (concentration, weariness, sleep troubles) or behavioural (attitude, irritability, difficulty of contact) compared to the placebo. In the same way, quality of life and perceived stress were significantly improved with SOD supplementation. CONCLUSION: This pilot study showed that an oral supplementation with a proprietary melon juice concentrate rich in SOD may have a positive effect on several signs and symptoms of perceived stress and fatigue.

Effects of lifelong intervention with an oligofructose-enriched inulin in rats on general health and lifespan.

Ageing is associated with changes in physiology and morphology; nutritional strategies to decrease morbidity and to prolong life are of high interest. The aim of the study was to investigate the effects of lifelong supplementation with an oligofructose-enriched inulin on morphological and biological markers and lifespan in male and female rats. Male and female rats, age 3 months, were randomised into two groups to receive either a diet with 10 % of an oligofructose-enriched inulin (Synergy 1) or a standard diet (control) for 27 months. The rats were weighed every 2 weeks and their food intake was evaluated on four successive days every 4-6 weeks. Samples were taken at 12, 18 and 24 months of age. During the whole intervention period, male rats receiving Synergy 1 (SYN1-M) displayed lower body weight, cholesterol and plasma triacylglycerolaemia compared with the controls (Cont-M). The survival rate at 24 months of age of SYN1-M rats was 35.3 % greater than that of Cont-M rats. In female rats, the Synergy 1 supplementation (SYN1-F) group also reduced body weight, cholesterol and triacylglycerolaemia levels, but results were less consistent over the experiment. The survival rate at 24 months of age in SYN1-F rats was 33.3 % greater compared with that of the control (Cont-F) group. To conclude, lifelong intervention with Synergy 1 improved biological markers during ageing and survival rate (lifespan) of rats.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice.

Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for alpha-tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain alpha-tocopherol in both homozygous (-30.1%, P<0.0002) and heterozygous (-18.0%, P<0.05) PLTP knocked-out mice. Alpha-tocopherol depletion in PLTP-deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus-maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP-deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

Ethological comparison of the effects of a bovine alpha s1-casein tryptic hydrolysate and diazepam on the behaviour of rats in two models of anxiety.

A bovine alpha s1-casein tryptic hydrolysate was previously demonstrated to display an anxiolytic-like activity in the conditioned defensive burying and in the elevated plus-maze models when i.p. injected. The present study assessed the anxiolytic-like effects of this tryptic hydrolysate after an oral administration in rats faced to the same behavioural situations using diazepam as a reference. In a first experiment, the behavioural effects of the hydrolysate in the conditioned defensive burying test were investigated at doses ranging 5-50 mg/kg. The results showed that the minimal dose required to elicit an anxiolytic-like activity is 15 mg/kg. In a second experiment, the alpha s1-casein tryptic hydrolysate (15 mg/kg, p.o.) was demonstrated to display an anxiolytic-like activity similar to diazepam (3 mg/kg, p.o.) in the conditioned defensive burying test and the elevated plus-maze. However, the ethological analysis of behaviour indicated that this hydrolysate has a different activity compared to diazepam. While diazepam induced a disinhibition state in rats, possibly related to the risk-taking behaviour observed after a benzodiazepine ingestion in humans, the tryptic hydrolysate did not display such a side effect. These results suggest that the mechanism of action of the bovine alpha s1-casein tryptic hydrolysate may differ from that of diazepam.

The delivery rate of dietary carbohydrates affects cognitive performance in both rats and humans.

RATIONALE: Glucose is the main metabolic fuel of the brain. The rate of glucose delivery from food to the bloodstream depends on the nature of carbohydrates in the diet, which can be summarized as the glycaemic index (GI). OBJECTIVES: To assess the benefit of a low versus high GI breakfast on cognitive performances within the following 4 h. METHODS: The influence of the GI of the breakfast on verbal memory of young adults was measured throughout the morning in parallel to the assessment of blood glucose levels. The learning abilities of rats performing an operant-conditioning test 3 h after a breakfast-like meal of various GI was also examined. RESULTS: A low GI rather than high GI diet improved memory in humans, especially in the late morning (150 and 210 min after breakfast). Similarly, rats displayed better learning performance 180 min after they were fed with a low rather than high GI diet. CONCLUSION: Although performances appeared to be only remotely related to blood glucose, our data provide evidence that a low GI breakfast allows better cognitive performances later in the morning.

Anxiolytic-like effects and safety profile of a tryptic hydrolysate from bovine alpha s1-casein in rats.

The anxiolytic activity and adverse benzodiazepine-like effects of a bovine alpha s1-casein tryptic hydrolysate (CH) were evaluated. The effects of CH orally administered at doses of 5 and 15 mg/kg were compared with those of diazepam (DZ) at 3 mg/kg in the conditioned defensive burying test. Rats treated either with CH at 15 mg/kg or with DZ showed a decrease in anxiety. A drug-related difference was observed in terms of duration, as the anxiolytic-like action of CH was maintained after 7 days with twice-daily administration, whereas that of DZ was not. CH and DZ were then evaluated for their potential effects on memory in a passive avoidance paradigm. CH-treated rats had significantly longer latencies before entering the dark compartment where they were previously delivered a shock, indicating better retention relative to DZ-treated rats. In the final test, CH and DZ were evaluated for place preference, an index of the possible addictive potential of these substances. DZ-treated rats spent more time in the compartment associated with drug exposure than control rats. This effect was not found in CH-treated rats. Thus, CH did not display side effects associated with DZ, despite its affinity for gamma-aminobutyric acid(A) (GABA(A)) receptors. Specific linking of CH on GABA(A) receptor function involved in anxiolysis, but not on that implied in memory-impairing effects, may be hypothesized to explain its specific activity. This profile might render it advantageous for nutritional purposes.

Variations in illumination, closed wall transparency and/or extramaze space influence both baseline anxiety and response to diazepam in the rat elevated plus-maze.

Numerous methodological-related variables have been demonstrated to influence the baseline anxiety level of rodents exposed to the elevated plus-maze (EPM), raising questions about the sensitivity of this test for the detection of the effects of anxiolytic drugs. Thus, the present study was designed (1) to assess the combined effects of illumination (40-lx red or white light), closed wall type (walls made of translucent or opaque material) and extramaze space size (small or spacious experimental room) on rat behaviour, and (2) to investigate the effects of such parameters on the relevance of the maze for detecting the effects of diazepam orally administrated at the anxiolytic dose of 3 mg/kg. Results indicate that illumination and closed wall type are two main independent parameters that are able to modify the open arm avoidance. Moreover, the closed wall type interacts with the extramaze space size since the reduction of the open arm exploration induced by opaque closed walls is two-fold stronger in the spacious experimental room than in the small one. Finally, the diazepam anxiolytic activity is significantly detected in our laboratory in specific EPM conditions (maze with opaque walls, use of a red light, maze located in a spacious experimental room). In conclusion, the present study demonstrates that an inappropriate baseline anxiety level due to the methodological use of the EPM can dramatically reduce the sensitivity of the maze for the detection of benzodiazepine-related compounds. This study also provides new insights into the perception of the EPM open space in rats.

Neurobehavioral maturation of offspring from epileptic dams: study in the rat lithium-pilocarpine model.

In the present study, we explored the consequences of epilepsy on the neurobehavioral development of the offspring in a rat model of spontaneous epilepsy, the lithium-pilocarpine model of temporal lobe epilepsy not dependent on genetic factors and in animals not receiving any antiepileptic treatment. Status epilepticus was induced by lithium-pilocarpine in female rats. After the occurrence of spontaneous seizures the rats were mated and the neurobehavioral development of the offspring was explored. Rat pups were cross-fostered early after birth. We hence obtained pups born from or raised by epileptic or non-epileptic dams. On the dams, we performed a follow-up of maternal care during pregnancy. On the pups, we performed a follow-up of classical parameters of development such as body weight and eyelid opening, and subjected the pups to various tests representative of neurobehavioral maturation extending from postnatal day 4 (PD4) to PD30 (righting reflex, suspension time, negative geotaxis, open field, locomotor coordination and eight arm maze). Altogether our data show that rat pups born from or raised by epileptic dams develop as well as control pups raised by control dams. Intriguingly, pups born from lithium-pilocarpine exposed dams and raised by control mothers tend to have better scores than the two other groups in all tests. This indicates that the exposure to seizures during pregnancy is not harmful for the development of the fetus.

A 5-month period of epilepsy impairs spatial memory, decreases anxiety, but spares object recognition in the lithium-pilocarpine model in adult rats.

PURPOSE: In temporal lobe epilepsy (TLE), interictal behavioral disorders affect patients' quality of life. Therefore we studied long-term behavioral impairments in the lithium-pilocarpine (li-pilo) model of TLE. METHODS: Eleven li-pilo adult rats exhibiting spontaneous recurrent seizures (SRSs) during 5 months were compared with 11 li-saline rats. Spatial working memory was tested in a radial arm maze (RAM), anxiety in an elevated plus-maze (EPM), and nonspatial working memory in an object-recognition paradigm. Neuronal loss was assessed on thionine brain sections after behavioral testing. RESULTS: In the RAM, the time to complete each session and the number of errors per session decreased over a 5-day period in li-saline rats but remained constant and significantly higher in li-pilo rats. In the EPM, the number of entries in and time spent on open arms were significantly higher in li-pilo than li-saline rats. In the object-recognition task, the two groups exhibited a comparable novelty preference for the new object. Neuronal loss reached 47-90% in hilus, CA1, amygdala, and piriform and entorhinal cortex. CONCLUSIONS: In li-pilo rats having experienced SRS for 5 months, performance in the object-recognition task is spared, which suggests that object discrimination remains relatively intact despite extensive damage. Neuronal loss in regions mediating memory and anxiety, such as hippocampus, entorhinal cortex, and amygdala, may relate to impaired spatial orientation and decreased anxiety.

Effects of a tryptic hydrolysate from bovine milk alphaS1-casein on hemodynamic responses in healthy human volunteers facing successive mental and physical stress situations.

BACKGROUND: Preclinical results in rats have demonstrated anxiolytic-like effects of a tryptic bovine alphaS1-casein hydrolysate. AIM OF THE STUDY: We investigated the putative effects of this tryptic hydrolysate on systolic (SBP), diastolic (DBP) blood pressures, heart rate (HR) values and plasma cortisol concentrations (CC) in human healthy volunteers facing successive stress situations. METHODS: The subjects were (double blind) randomly allocated to ingest three times, 12 hours apart, two capsules containing either 200 mg of alphaS1-casein hydrolysate (TS) or bovine skimmed milk powder as a placebo (CS). On the morning of the test day, a first blood sample for baseline measurement of CC was taken before the subjects were submitted to the Stroop test (ST) and, after a 30-min rest, to a Cold Pressor test (CPT). SBP, DBP, and HR were continuously recorded for 5 min before the ST and during each stress situation. A second blood sample was taken 15 min after the end of the CPT condition. RESULTS: ST and ST + CPT combined test situations increased SBP, DBP and HR. The significant "Treatment x SBP" and "Treatment x DBP" interactions indicated the lower percentage changes in SBP and DBP of the TS. In addition, the results showed a significant decrease of the CC in the TS but not in the CS throughout the ST + CPT combined stress tests. HR remained stable in TS between the initial rest period and the CPT unlike what happened in CS. CONCLUSION: On the basis of blood pressure and cortisol changes, these results suggest an antistress profile of this alphaS1-casein hydrolysate in human subjects.

Behavioural and cognitive effects of oligofructose-enriched inulin in rats.

The behavioural and cognitive effects of oligofructose-enriched inulin at the doses of 5 and 10 % in the diet, orally ingested daily during 2 weeks, were investigated using a functional observational battery (FOB) and the light extinction test in male Wistar rats. Control rats received a standard diet and were tested in the same test situations. The behavioural effects were assessed 2 d before and 14 d after the beginning of the treatment period and the cognitive effects were investigated after the administration period by lever-pressing activity and learning discrimination using the light extinction test paradigm. In general, the study demonstrated that oligofructose-enriched inulin at 5 % in the diet, and particularly at 10 % in the diet, caused relaxing-like effects, stimulated and increased the general activity and interest of the rats to the test environment. In addition, both doses of oligofructose-enriched inulin showed significant effects on learning discrimination in male rats, in comparison with the control diet. These results suggest that oligofructose-enriched inulin, particularly at the dose of 10 %, improves cognitive performances in the light extinction test and the well-being of male rats using the FOB.

Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity.

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4-1 microM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100-200 mcicrog/kg doses of sialorphin required the activation of mu- and delta-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

Histopathological alterations and functional brain deficits after transient hypoxia in the newborn rat pup: a long term follow-up.

To assess temporal brain deficits consecutive to severe birth hypoxia, newborn rats were exposed for 20 min to 100% N2. This treatment induced a long-term growth retardation and a delayed, but only transient, neuronal loss (approximately 25%) in the CA1 hippocampus and parietal cortex, starting from 3 days and peaking at 6 days post-hypoxia. The expression profiles of various apoptosis-regulating proteins (including Bcl-2, Bax, p53 and caspase-3) were well correlated to the alterations of nuclear morphology depicted by 4,6-diamidino-2-phenylindole (DAPI). Whereas they confirmed a gradual histological recovery, specific DNA fragmentation patterns suggested that birth hypoxia may transiently reactivate the developmental programme of neuronal elimination. Although they successfully achieved various behavioral tests such as the righting reflex, negative geotaxis, locomotor coordination, and the eight-arm maze tasks, both developing and adult hypoxic rats were repeatedly slower than controls, suggesting that birth hypoxia is associated to moderate but persistent impairments of functional capacities.