Progression of subcortical atrophy in mild Parkinson's disease and its impact on cognition.

BACKGROUND AND PURPOSE: Mild cognitive impairment (MCI) is associated with pronounced grey matter atrophy in various brain regions. However, the association between atrophy patterns and progression from no cognitive impairment (NCI) to Parkinson's disease (PD)-MCI is not clearly known. We investigated the pattern and progression of atrophy in subcortical structures and its impact on cognition in patients with mild PD. METHODS: Sixty-five patients with mild PD with baseline and longitudinal clinical and neuropsychological assessments, and structural magnetic resonance imaging scans were studied. Movement Disorder Society Task Force criteria were used to classify patients with PD into PD-NCI (n = 54) and PD-MCI (n = 11). Based on progression over time, those who remained without cognitive impairment were classified as PD-stable (n = 42) and those who converted to MCI over 18 months were classified as PD-converters (n = 12). FreeSurfer was used to measure cortical thickness and subcortical volumes at baseline and follow-up. RESULTS: Parkinson's disease-MCI showed baseline thalamus atrophy and progressive atrophy in the thalamus, caudate, presubiculum, cornu ammonis 1 and 2-3, and significant memory and executive dysfunction compared with PD-NCI. PD-converters had greater accumbens atrophy at baseline and progressive atrophy in the thalamus, caudate and accumbens with dysfunctions in memory and executive domains. CONCLUSIONS: Progression of cognitive impairment in non-demented PD is associated with a specific pattern of subcortical atrophy. Findings from this study will allow future studies to investigate in the role of subcortical structures as a biomarker for PD dementia.

Egg production, faecal pH and microbial population, small intestine morphology, and plasma and yolk cholesterol in laying hens given liquid metabolites produced by Lactobacillus plantarum strains.

1. Various dosages of metabolite combinations of the Lactobacillus plantarum RI11, RG14 and RG11 strains (COM456) were used to study the egg production, faecal microflora population, faecal pH, small intestine morphology, and plasma and egg yolk cholesterol in laying hens. 2. A total of 500 Lohmann Brown hens were raised from 19 weeks to 31 weeks of age. The birds were randomly divided into 5 groups and fed on various treatment diets: (i) basal diet without supplementation of metabolites (control); (ii) basal diet supplemented with 0·3% COM456 metabolites; (iii) basal diet supplemented with 0·6% COM456 metabolites; (iv) basal diet supplemented with 0·9% COM456 metabolites; and (v) basal diet supplemented with 1·2% COM456 metabolites. 3. The inclusion of 0·6% liquid metabolite combinations, produced from three L. plantarum strains, demonstrated the best effect in improving the hens' egg production, faecal lactic acid bacteria population, and small intestine villus height, and reducing faecal pH and Enterobacteriaceae population, and plasma and yolk cholesterol concentrations. 4. The metabolites from locally isolated L. plantarum are a possible alternative feed additive in poultry production.

Effects of liquid metabolite combinations produced by Lactobacillus plantarum on growth performance, faeces characteristics, intestinal morphology and diarrhoea incidence in postweaning piglets.

A study was carried out to investigate the effects of feeding liquid metabolite combinations produced by Lactobacillus plantarum strains on growth performance, diarrhoea incidence, faecal pH, microfloral counts, short-chain fatty acids (SCFA) and intestinal villus height and crypt depth of postweaning piglets. A total of 120 piglets (26 days old) were randomly assigned evenly into five treatment groups treated with same basal diet: (1) -ve control (free antibiotic); (2) + ve control (0.03% of chlortetracycline); (3) Com 1 (0.3% metabolite of TL1, RG11 and RI11 strains); (4) Com 2 (0.3% metabolite of TL1, RG14 and RS5 strains); (5) Com 3 (0.3% metabolite of RG11, RG14 and RI11 strains). After 5 weeks, the average daily feed intake was not significantly different (P > 0.05) among the treatments and feed conversion ratio was the highest (P < 0.05) in the -ve control group. In addition, diarrhoea incidence was reduced when piglets were fed with metabolite combinations. Faecal lactic acid bacteria (LAB) counts were significantly higher (P < 0.05) in metabolite treatment groups than in the groups without metabolites. However, the treatment of Com 2 metabolite resulted lower (P < 0.05) faecal pH and Enterobacteriaceae (ENT) than the -ve control group. In contrast, total faecal SCFA of Com 2 were significantly higher (P < 0.05) than the -ve control group. The villus height of duodenum was higher (P < 0.05) in the + ve control and Com 2 groups as compared to -ve control group. The results obtained in this study showed that feeding metabolite combinations could improve growth performance, and increase the population of gut LAB and faecal SCFA of postweaning piglets.

Analgesia accompanying food consumption requires ingestion of hedonic foods.

Animals eat rather than react to moderate pain. Here, we examined the behavioral, hedonic, and neural requirements for ingestion analgesia in ad libitum fed rats. Noxious heat-evoked withdrawals were similarly suppressed during self-initiated chocolate eating and ingestion of intraorally infused water, sucrose, or saccharin, demonstrating that ingestion analgesia does not require feeding motivation, self-initiated food procurement, sucrose, or calories. Rather, food hedonics is important because neither salt ingestion nor quinine rejection elicited analgesia. During quinine-induced nausea and lipopolysaccharide (LPS)-induced illness, conditions when chocolate eating was presumably less pleasurable, analgesia accompanying chocolate consumption was attenuated, yet analgesia during water ingestion was preserved in LPS-injected rats who showed enhanced palatability for water within this context. The dependence of ingestion analgesia on the positive hedonics of an ingestate was confirmed in rats with a conditioned taste aversion to sucrose: after paired exposure to sucrose and LPS, rats no longer showed analgesia during sucrose ingestion but continued to show analgesia during chocolate consumption. Eating pauses tended to occur less often and for shorter durations in the presence of ingestion analgesia than in its absence. Therefore, we propose that ingestion analgesia functions to defend eating from ending. Muscimol inactivation of the medullary raphe magnus blocked the analgesia normally observed during water ingestion, showing the involvement of brainstem endogenous pain inhibitory mechanisms in ingestion analgesia. Brainstem-mediated defense of the consumption of palatable foods may explain, at least in part, why overeating tasty foods is so irresistible even in the face of opposing cognitive and motivational forces.

Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus).

AIMS: To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac-D1ae) and/or D4 (Lac-D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). METHODS AND RESULTS: The polymerase chain reaction (PCR)-amplified 250- and 750-bp sequences coding for domains D1 and D4 of aerolysin were individually cloned into pNZ8048 and electrotransformed into L. lactis. The recombinant vaccine candidates were then either orally fed or injected intraperitoneally into tilapia. The development of antibodies in sampled fish compared to control groups implied that the recombinant epitopes expressed in L. lactis were able to elicit an immunogenic response in tilapia. Interestingly, the lower doses of both Lac-D1ae and Lac-D4ae gave higher antibody levels over the study period. Fish immunized with Lac-D1ae and Lac-D4ae together showed the highest level of protection, and the mortality was reduced significantly compared to control strains in both modes of vaccination. CONCLUSIONS: The recombinant L. lactis strain expressing D1 and D4 produced aerolysin-specific serum IgM in tilapia. Both D1 and D4 promoted 55-82% relative per cent survival (RPS) against Aeromonas infection through intraperitoneal injection, whereas the RPS following oral feeding of the vaccine was 70-100%. SIGNIFICANCE AND IMPACT OF THE STUDY: The D1 and D4 regions of the aerolysin protein have been successfully identified as immunogenic regions that can elicit antibody production in tilapia and protect against challenge with Aer. hydrophila. A promising oral vaccine using L. lactis harbouring the D1 and D4 regions has been developed to control Aer. hydrophila.

Effects of feeding metabolite combinations produced by Lactobacillus plantarum on growth performance, faecal microbial population, small intestine villus height and faecal volatile fatty acids in broilers.

1. Four combinations of metabolites produced from strains of Lactobacillus plantarum were used to study the performance of broiler chickens. 2. A total of 432 male Ross broilers were raised from one-day-old to 42 d of age in deep litter pens (12 birds/pen). These birds were divided into 6 groups and fed on different diets: (i) standard maize-soybean-based diet (negative control); (ii) standard maize-soybean-based diet + Neomycin and Oxytetracycline (positive control); (iii) standard maize-soybean-based diet + 0.3% metabolite combination of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains (com3456); (iv) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RI11 and RG11 (Com246); (v) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RG14 and RG11 (Com256) and (vi) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RS5, RG14 and RG11 (Com2356). 3. Higher final body weight, weight gain, average daily gain and lower feed conversion ratio were found in all 4 treated groups. 4. The addition of a metabolite combination supplementation also increased faecal lactic acid bacteria population, small intestine villus height and faecal volatile fatty acids and faecal Enterobacteriaceae population.

Influence of postbiotic RG14 and inulin combination on cecal microbiota, organic acid concentration, and cytokine expression in broiler chickens.

This study examined the effects of different combinations of inulin and postbiotics RG14 on growth performance, cecal microbiota, volatile fatty acids (VFA), and ileal cytokine expression in broiler chickens. Two-hundred-and sixteen, one-day-old chicks were allocated into 6 treatment groups, namely, a basal diet (negative control, NC), basal diet + neomycin and oxytetracycline (positive control, PC), T1 = basal diet + 0.15% postbiotic RG14 + 1.0% inulin, T2 = basal diet + 0.3% postbiotic RG14 + 1.0% inulin, T3 = basal diet + 0.45% postbiotic RG14 + 1.0% inulin, and T4 = basal diet + 0.6% postbiotic RG14 + 1.0% inulin, and fed for 6 weeks. The results showed that birds fed T1 and T3 diets had higher (P < 0.05) final body weight and total weight gain than NC and PC birds. A lower (P < 0.05) feed conversion ratio was observed in birds fed T1 and T2 compared with those fed the NC diet. Birds fed PC, T1, T2, and T3 had higher (P < 0.05) cecum total bacteria and Bifidobacteria compared to the NC birds. Diet had no effect on cecum Lactobacilli, Enterococcus and Salmonella. The NC birds had higher (P < 0.05) Enterobacteria and E. coli than other treatments. Concentration of acetic acid was higher in birds fed PC, T1, and T4 compared to the NC birds. However, the concentration of butyric acid, propionic acid, and total VFA did not differ (P > 0.05) among diets. The NC birds had higher (P < 0.05) expression of interferon (IFN) and Lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) gene compared with those fed other diets. The mRNA expression of interluken-6 (IL-6) was up-regulated in birds fed T3 and T4 compared to the NC birds. However, the expression of interluken-8 (IL-8) gene was not influenced by diet. Postbiotic and inulin combinations are potential replacements for antibiotic growth promoters in the poultry industry.

Discharge of raphe magnus ON and OFF cells is predictive of the motor facilitation evoked by repeated laser stimulation.

Medullary raphe magnus (RM) on and off cells are thought to modulate spinal nociception by gating withdrawals evoked by noxious stimulation. To test whether withdrawal initiation is the target of RM modulation, we examined the relationship between on and off cell discharge and motor withdrawal evoked by noxious laser heat in halothane-anesthetized rats. The cellular responses of both cell types began during the 50 msec after onset of the tail flick, peaked within 200 msec, and outlasted the duration of the motor reaction. Thus, it is unlikely that the target of on and off cell modulation is withdrawal initiation; instead, on and off cells may modulate reactions to repeated noxious stimulation. We therefore tested whether laser heat-evoked changes in RM cell discharge were predictive of the modulatory effects of one noxious stimulus on the reaction to a subsequent noxious stimulus. Two pulses of laser heat were presented at interpulse intervals of 0.8, 2.0, or 10.0 sec. The motor withdrawal evoked by the second pulse was significantly enhanced relative to that evoked by the first pulse. The observed motor enhancement depended on supraspinal input because it was not present in spinalized rats. Comparison of the relative changes in motor and cellular activity preceding double laser heat stimulation revealed parallel changes between motor facilitation, decreases in off cell discharge, and increases in on cell discharge. This finding suggests a preparatory role for RM on and off cells in enhancing reactions to a noxious stimulus that closely follows another noxious stimulus.

Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis.

Epithiospecifier protein (ESP), a ferrous ion dependent protein, has a potential role in regulating the release of elemental sulphur, nitriles, isothiocyanates and cyanoepithioalkanes from glucosinolates. Two classes of ESP polypeptides were purified with molecular masses of 39 and 35 kDa, and we show that the previously reported instability was conditionally dependent. The 39 kDa polypeptide was made up of two distinct isozymes (5.00, 5.14) whilst several were present for the 35 kDa form of ESP (5.40-5.66). An anti-ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and strongly with a polypeptide corresponding to the 35 kDa ESP form in Crambe abyssinica, but did not detect any ESP in Sinapis alba or Raphanus sativus. A cytochrome P-450 mediated iron dependent epoxidation type mechanism is suggested for ESP.

Hypoalgesia elicited by a conditioned stimulus is blocked by a mu, but not a delta or a kappa, opioid antagonist injected into the rostral ventromedial medulla.

The present study investigated the role of micro, delta, and kappa receptors within the RVM in mediating expression of conditional hypoalgesia (CHA). Five groups of rats with RVM cannulae were given daily sessions of paired or unpaired presentations of an auditory CS (white noise) and foot shock across three consecutive days. On the test day, rats in the Paired condition were injected with the micro antagonist CTAP, the delta antagonist naltrindole, the kappa antagonist nor-BNI, or saline. Rats in the Unpaired condition were injected with saline. TFLs were measured before and after injections, as well as during and after presentations of the CS. The results showed that none of the drugs affected baseline TFLs. During CS presentation, rats in the Paired condition injected with saline showed longer TFLs than those in the Unpaired condition given saline, confirming the presence of CHA. Expression of this response was blocked by CTAP, but was unaffected by naltrindole or nor-BNI. These results suggest that mu, but not delta or kappa, opioid receptors in the RVM mediate expression of CHA.

Brainstem modulation of pain during sleep and waking.

Moderately painful stimuli applied during sleep evoke motor and neural responses indicative of arousal, but seldom cause awakening. Different reactions occur in response to acute pain stimulation across behavioral states; pain reactions are modulated by the activity of serotonergic and non-serotonergic cells in the raphe magnus (RM). Serotonergic RM cells have state-dependent discharge and may inhibit simple motor withdrawal responses during waking. ON and OFF cells are non-serotonergic RM neurons thought to facilitate and inhibit pain, respectively. These cells display reciprocal spontaneous discharge patterns across the sleep-wake cycle, with ON cells most active during waking and OFF cells most active during sleep. We propose that they also play an important role in modulating the alertness evoked by any brief external stimulus, either noxious or innocuous. ON cells may facilitate alertness during waking and OFF cells suppress arousals during sleep. In the presence of chronic pain, both ON and OFF cell discharge appear to increase. The increase in ON cell discharge may contribute to enhancing pain sensitivity and alertness. Future research is needed to understand why sleep is so adversely affected in chronic pain patients, whereas sleep is minimally disrupted, even by acutely painful stimuli, in humans and animals without chronic pain.

Expression of antinociception in response to a signal for shock is blocked after selective downregulation of mu-opioid receptors in the rostral ventromedial medulla.

Prior work has shown that release of endogenous ligands for mu-opioid receptors in the rostral ventromedial medulla (RVM) is critical for the modulation of spinal nociceptive reflexes observed during stress. In the present study, we used antisense oligodeoxynucleotides (AS ODN) to suppress synthesis of mu-opioid receptors in the RVM prior to activating descending antinociceptive systems with a signal for foot shock. Five groups of rats with RVM cannulae were trained with paired or unpaired exposures to white noise (WN) and foot shock. Over several days, they received RVM infusions of an AS ODN probe targeting exon 1 of the cloned MOR-1 receptor, an inactive missense (MS) ODN with the same base composition in which the sequence for four bases was changed, an AS ODN probe targeting exon 4, or saline. Tail-flick latencies (TFLs) were measured before, during, and after presentation of the auditory signal for shock. Rats given paired training and saline injections displayed longer TFLs than saline control rats given unpaired exposures to WN and shock, confirming the ability of the conditional stimuli (CS) to elicit antinociception. Expression of this conditional hypoalgesia (CHA) was attenuated by pretreatment with the AS ODN probe targeting exon 1, but was unaffected by pretreatment with AS ODN probe targeting exon 4 or MS ODN sequence for exon 1. However, pretreatment with the AS ODN probe targeting exon 1 did not affect expression of conditional freezing to other shock-associated cues. Testing of the same animals several days after the ODN injections showed that the attenuating effect on expression of CHA were reversible. These results support the idea that mu-opioid receptors in the RVM are critically involved in mediating expression of hypoalgesia following stress. They also provide further evidence for dissociation in the mechanisms mediating expression of aversive conditional responses.

Effects of hypophysectomy and adrenalectomy on naloxone-induced analgesia.

Experiment 1 demonstrated that pairings of the opiate antagonist, naloxone, with a heated floor came to induce analgesia, as indexed by the latencies with which rats licked their paws. This analgesia appears to be neurally mediated because it is unaffected by either hypophysectomy (experiment 2) or adrenalectomy (experiment 3). However, there was evidence for a pituitary involvement, as its removal potentiated the analgesic effect accruing from naloxone-stressor pairings.

Activation of kappa opioid receptors in the rostral ventromedial medulla blocks stress-induced antinociception.

Prior work has shown that kappa opioids may attenuate the effects of analgesic mu receptor agonists in some neural circuits related to pain modulation. This study examined whether hypoalgesia following exposure to a signal for shock is attenuated by infusions of the kappa agonist U69593 into the rostral ventromedial medulla (RVM). Rats were trained with paired or unpaired presentations of white noise and foot shock. On test days, tail flick latencies were measured before, during, and after exposure to the auditory conditioned stimulus (CS). One of three doses of U69593 (0.0445, 0.178 and 1.00 microg) or an equivalent volume of saline was injected into the RVM. Rats that had received noise-shock pairings displayed conditional hypoalgesia (CHA) compared to those given unpaired presentations. Expression of CHA was completely blocked by the highest dose of U69593 (1.00 microg) injected 20 min before testing, indicating an antagonistic effect of U69593 on expression of CHA. These findings are discussed in terms of the evidence for antagonism of morphine- and DAMGO-induced hypoalgesia by kappa agonists.

Glucose-mediated alteration of cellular function in human periodontal ligament cells.

Because diabetic patients are easily led to manifest severe periodontitis, we wanted to determine whether various glucose levels interfere with normal cellular function. Human periodontal ligament (PDL) cells were cultured in glucose-free medium, or in medium containing either 1100 mg/L of glucose (normal-glucose medium) or 4500 mg/L of glucose (high-glucose medium). Cells cultured in glucose-free medium changed their morphology from spindle-shaped to round, and incorporated trypan blue in a time-dependent manner. The incorporation rate was much faster in cells with shorter cell cycles than in those with longer cycles, suggesting the involvement of cell-cycle progression in cell death. However, fragmented DNA, which suggests apoptotic cell death, was not observed in these cells. We reasoned that initial cell rounding and detachment from the culture plate might be due to the conformational changes in cell-surface receptors to fibronectin, a major extracellular matrix for fibroblasts. Western blot analysis revealed that cells cultured in glucose-free medium lost their fibronectin receptor in a time-dependent manner. In addition, fibronectin receptor expression was much higher in cells cultured in high-glucose medium than in cells cultured in normal-glucose medium. Furthermore, the over-expression of the fibronectin receptor resulted in a suppressed chemotactic response of these cells to platelet-derived growth factor. On the basis of these data, it was hypothesized that a high glucose level induced over-expression of these receptors. This might be the mechanism by which a high glucose level compromises wound healing in diabetic patients and, at least in part, might be the reason diabetic patients are subject to severe periodontal destruction.

Acquisition and expression of conditioned hypoalgesia in morphine-naive and morphine-tolerant rats.

The present study used a within-subject design to examine acquisition and expression of conditioned hypoalgesia in 50 male Wistar rats. Morphine-naive rats preexposed to a heat stressor with saline were hypoalgesic when subsequently tested for latencies to tail flick or paw lick. However, morphine-tolerant rats preexposed to the heat stressor with saline failed to display hypoalgesia when tested for latencies to tail flick, but showed hypoalgesia when tested for latencies to paw lick. Taken together, these findings suggest that expression of conditioned hypoalgesic responses in morphine-tolerant rats may depend on the nociceptive test used. Both morphine-naive and morphine-tolerant rats preexposed to the heat stressor with morphine failed to display hypoalgesia on either the tail-flick or the hot-plate test, demonstrating that morphine's ability to block acquisition of conditioned hypoalgesia is independent of the test used to assess nociceptive sensitivity.

Naloxone-induced hypoalgesia: effects of noradrenergic antagonists and agonist.

The present experiments confirmed that rats injected with naloxone and exposed to a heated floor acquired a hypoalgesic response, as indexed by the latencies to lick their paws. The expression of these latencies were unaffected by yohimbine, clonidine, propranolol, or by relatively moderate doses of prazosin, suggesting that the conditioned hypoalgesic response induced by pairings of naloxone and a heated floor is not mediated by the release and turnover of noradrenaline.

Naloxone-induced hypoalgesia: evidence from the formalin test.

Two experiments examined the hypoalgesic effects that accrue from pairing exposure to a heat stressor with the opioid antagonist naloxone. Experiment 1 confirmed that rats given separate exposures to a heated floor and to naloxone are hypoalgesic when then tested on that floor. Further, the results confirmed that pairing the initial exposure to the heat stressor with naloxone resulted in a more profound hypoalgesic response. Experiment 2 provided new evidence for the hypoalgesic effects of separate exposures to the heat stressor and to naloxone, and for naloxone's enhancement of acquisition of the hypoalgesia, in rats tested for responsiveness to formalin. These results, therefore, demonstrate generalisability of the hypoalgesic effects across assays for acute and chronic pain.

Acute effects of kava, alone or in combination with alcohol, on subjective measures of impairment and intoxication and on cognitive performance.

Kava (Piper methysticum) and alcohol were administered either separately or in combination to human subjects. Self-reports of their levels of impairment and intoxication were collected, and performance skills on a number of cognitive and visuomotor tests were determined, before and three times after consumption of the experimental drink. Kava alone had no effect on reported condition. In contrast, alcohol produced marked changes in each of the five subjective measures, all of which were in the direction of lowered ability. The combination of these two substances produced even larger negative changes on these measures. In the cognitive tests, kava produced a decrement in performance on Digit Symbol Coding. Alcohol produced a significant decrease in performance on a divided attention test, which was almost entirely on the peripheral, discontinuous component of the test. The combination of kava and alcohol produced an even greater decrease in performance on this test, and in the same component. The present findings suggest that kava alone has little effect on reported condition and cognitive performance, but appears to potentiate both perceived and measured impairment when combined with alcohol.

Concentration and state dependent reductions in corn oil intakes after glossopharyngeal nerve transections in rats.

Previous studies indicate a role for the glossopharyngeal nerve (GL) in the detection of dietary fats. The present experiments examined the effects of bilateral glossopharyngeal nerve transections (GLx) on the intake of low (4.8%), moderate (16%), and full-fat (100%) corn oil in non-deprived, food-deprived, and water-deprived rats. The rats had access to oils, 0.3 M sucrose, and water in a gustometer that measured number of licks and latency to the first lick during brief access trials. The behavioral measures were used as indices of the amount consumed and the motivation to ingest, respectively. After baseline intakes had stabilized, the rats received GLx or sham transections (Sham) and were then re-tested. Pre and post-surgery responses were compared to determine the impact of GLx on intake and the motivation to ingest. In non-deprived rats, GLx reduced the intake of 4.8% and 16% oils and decreased the motivation to ingest these oils. In food-deprived rats, GLx prevented increases in the ingestion of 4.8% and 16% oils and in the motivation to ingest these oils. In water-deprived rats, GLx reduced the intake of 100% oil and produced a general decrease in the motivation to consume low, moderate, and full-fat emulsions. These results indicate that GL is partially involved in corn oil intake and suggest an interactive effect of oil concentration with homeostatic state.