In-vivo analysis of Protec™ and ß-glucan supplementation on innate immune performance and intestinal health of rainbow trout.

Functional diets are often given to fish during key stages to improve health through the interaction of the feed components with the host intestine. The additional factors added in these diets are known to modulate the immune response and as such may also offer protection against pathogenic challenges. The present study was undertaken to evaluate whether ß-glucan supplementation for 6 weeks can alter the magnitude of immune response to immunological challenges and subsequently offer an improved innate immune response to bacterial challenge in rainbow trout. Two experimental diets were used to study these effects: a basic commercial diet supplemented with ß-glucan and a commercially available functional diet (Protec™) that has ß-glucan as a functional component in addition to other components were compared to a basic commercial control diet. No significant differences were observed in biometric data. Histological analysis revealed a significantly greater number of goblet cells in the fish fed Protec™ and ß-glucan diets compared to those fed a control diet. Cell marker gene expression of distal intestine leucocytes indicated higher expression of T- and B-cells marker genes to both the ß-glucan containing diets in comparison to control. The Protec™ diet demonstrated modulation of innate immune markers after 6 weeks of feeding with key antimicrobial genes (SAA, HAMP, IL-1ß and TNFα) showing significant increases compared to the other diets. After stimulation with both PAMPs and an immune challenge with A. salmonicida fish fed the ß-glucan diet and the Protec™ exhibited modulation of the innate immune response. An immune challenge with A. salmonicida was carried out to identify if dietary composition led to differences in the innate immune response of rainbow trout. Modulation of the magnitude of response in some immune genes (SAA, IL-1ß and HAMP) was observed in both the distal intestine and head kidney in the Protec™ and ß-glucan fed fish compared to those fed the control diet.

Immune responses to prebiotics in farmed salmonid fish: How transcriptomic approaches help interpret responses.

Within aquaculture, prebiotics are composed of complex carbohydrate molecules that cannot be digested by the fish directly but are metabolised by the microbial communities within the host gut, with the desire that "healthy" bacterial species are promoted with subsequently improved performance of the fish, there are likely some direct responses of intestinal cells to these dietary components. The sources and processing of prebiotics, which fall under the overarching theme of "functional feeds" are highly varied between species and types of prebiotics administered. How these feeds exert their effect, and the host responses are hard to determine, but new technologies and the development of high-throughput technologies (omics) are enabling the mechanisms and methods of action to be further understood. The recent advances in the availability of 'omics' technologies with the transition from single gene assays to microarray and RNA-seq in fish health have enabled novel functional ingredients to be analysed. This review will focus on recent studies on targeted gene expression and 'omics' technologies to characterize immune responses. Comparisons between the immunomodulatory effect of different prebiotics have been made and specific examples of how transcriptomics techniques have been used to identify immune responses to prebiotics are given.

Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus.

The growth performance, immunological status, intestinal morphology and microbiology of tilapia, Oreochromis niloticus, were investigated after dietary administration of the commercial probiotic AquaStar(®) Growout. Tilapia (29.02 ± 0.33 g) were split into five treatments; control (CON), 1.5 g kg(-1) probiotic (PRO-1.5), 3 g kg(-1) probiotic (PRO-3), pulsed probiotic feeding (PRO-PULSE) or an initial probiotic feed followed by control feeding (PRO-INI). After six weeks of experimental feeding, fish fed PRO-3 displayed significantly higher final weight, weight gain and SGR compared to the CON or PRO-INI treatments. Supplementation of the probiotic at this dose induced an up-regulation of intestinal caspase-3, PCNA and HSP70 mRNA levels compared to the CON fed fish. Immuno-modulatory pathways were also affected; significantly higher expression of TLR2, pro-inflammatory genes TNFα and IL-1ß, and anti-inflammatory genes IL-10 and TGFß suggest that the probiotic may potentiate a higher state of mucosal tolerance and immuno-readiness. Histological appraisal revealed significantly higher numbers of intraepithelial leucocytes in the intestine of PRO-3 fed fish compared with treatments CON, PRO-PULSE and PRO-INI but not PRO-1.5. Additionally, fish receiving PRO-3 had a significantly higher abundance of goblet cells in their mid-intestine when compared with fish from all other treatments. Together, these data suggest that continuous provision of AquaStar(®) Growout at 3 g kg(-1) can improve tilapia growth and elevate the intestinal immunological status of the host.

Modulation of the intestinal microbiota and morphology of tilapia, Oreochromis niloticus, following the application of a multi-species probiotic.

The intestinal microbiota and morphology of tilapia (Oreochromis niloticus) were investigated after the application of a multi-species probiotic containing Lactobacillus reuteri, Bacillus subtilis, Enterococcus faecium and Pediococcus acidilactici (AquaStar(®) Growout). Tilapia (55.03 ± 0.44 g) were fed either a control diet or a probiotic diet (control diet supplemented with AquaStar(®) Growout at 5 g kg(-1)). After four and eight weeks, culture-dependent analysis showed higher levels of lactic acid bacteria (LAB), enterococci and Bacillus spp. in the mucosa and digesta of fish fed AquaStar(®) Growout. At week four, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed a higher similarity within the probiotic fed replicates than replicates of the control group; after eight weeks, the compositional dissimilarity of the microbiome profiles between the groups was greater than the dissimilarities within each group (P < 0.05). High-throughput sequencing revealed that the probiotic treatment significantly reduced the number of operational taxonomic units and species richness in the digesta. Significantly higher proportions of reads belonging to Proteobacteria and Cyanobacteria were detected in the control group whereas the probiotic-fed fish displayed a significantly higher abundance of reads assigned to the Firmicutes (which accounted for >99 % of reads). Bacillus, Cetobacterium and Mycobacterium were the dominant genera in the digesta of control fish whereas Bacillus, Enterococcus and Pediococcus were the largest constituents in probiotic-fed fish. The addition of AquaStar(®) Growout to tilapia diets led to increased populations of intraepithelial leucocytes, a higher absorptive surface area index and higher microvilli density in the intestine. These data suggest that AquaStar(®) Growout can modulate both the intestinal microbiota and morphology of tilapia.

Neural mechanisms underlying peak-dose dyskinesia induced by levodopa and apomorphine are distinct: evidence from the effects of the alpha(2) adrenoceptor antagonist idazoxan.

Dyskinesia, secondary to dopamine replacement therapy, is the major complication of currently available therapies for Parkinson's disease. Alpha(2) adrenoceptor antagonists, such as idazoxan, can significantly reduce levodopa-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned, nonhuman primate model of Parkinson's disease and in human. This action of adrenoceptor antagonists may involve blockade of the actions of noradrenaline synthesised from levodopa. We hypothesise that, because dopamine receptor agonists, such as apomorphine, cannot be metabolised to produce noradrenaline, activation of adrenoceptors may not be involved in dyskinesia produced by such agents. If this were the case, idazoxan would not be expected to reduce apomorphine-induced dyskinesia. MPTP-lesioned marmosets with stable dyskinesia induced by prolonged levodopa therapy were given an acute challenge with apomorphine (0.3 mg/kg subcutaneously) or levodopa (8.0 mg/kg orally), these doses produced equivalent peak-dose dyskinesia. Idazoxan (2.5 mg/kg p.o.), or vehicle, was then administered with either apomorphine or levodopa. Idazoxan abolished levodopa-induced dyskinesia but did not affect apomorphine-induced dyskinesia (P < 0.05 and P > 0.05, respectively, Wilcoxon matched pairs test). Idazoxan also extended the anti-parkinsonian actions of levodopa but did not affect those of apomorphine. The pharmacological characteristics of the neural mechanisms underlying levodopa-induced dyskinesia and apomorphine-induced dyskinesia in parkinsonism thus appear to be distinct, at least with respect to the involvement of alpha(2) adrenoceptors. Specifically, levodopa, but not apomorphine-induced dyskinesia, involves activation of adrenoceptors. This finding may have major implications for understanding dyskinesia and should be borne in mind when designing clinical studies in which levodopa or dopamine receptor agonist challenges are employed to assess potential anti-dyskinetic properties of drugs.

Antiparkinsonian actions of ifenprodil in the MPTP-lesioned marmoset model of Parkinson's disease.

Dopamine-replacement strategies form the basis of most symptomatic treatments for Parkinson's disease. However, since long-term dopamine-replacement therapies are characterized by many side effects, most notably dyskinesia, the concept of a nondopaminergic therapy for Parkinson's disease has attracted great interest. To date, it has proved difficult to devise a nondopaminergic therapy with efficacy comparable to that of dopamine replacement. In animal models of Parkinson's disease, loss of striatal dopamine leads to enhanced excitation of striatal NR2B-containing NMDA receptors. This is responsible, in part at least, for generating parkinsonian symptoms. Here we demonstrate that, in the MPTP-lesioned marmoset, monotherapy with the NR2B-selective NMDA receptor antagonist, ifenprodil, administered de novo, has antiparkinsonian effects equivalent to those of l-DOPA (administered as its methyl ester form). In MPTP-lesioned marmosets, median mobility scores, following vehicle-treatment were 12.5/h (range 6-21), compared to 61/h (range 26-121) in normal, non-MPTP-lesioned animals. Following ifenprodil (10 mg/kg) treatment in MPTP-lesioned marmosets, the median mobility score was 66/h (range 34-93), and following l-DOPA (10 mg/kg i.p.) treatment 89/h (range 82-92). The data support the proposal that NR2B-selective NMDA receptor antagonists have potential as a nondopaminergic monotherapy for the treatment of parkinsonian symptoms when given de novo.

The alpha2-adrenergic receptor antagonist idazoxan reduces dyskinesia and enhances anti-parkinsonian actions of L-dopa in the MPTP-lesioned primate model of Parkinson's disease.

Dopamine replacement therapy in patients with Parkinson's disease is plagued by the emergence of abnormal involuntary movements known as L-dopa-induced dyskinesias. It has been demonstrated that yohimbine can reduce L-dopa-induced dyskinesia in the MPTP-lesioned primate model of Parkinson's disease. Yohimbine is, among other things, an alpha-adrenergic receptor antagonist. In this study, we demonstrate that the selective and potent alpha2-adrenergic receptor antagonist idazoxan reduces L-dopa-induced dyskinesia in the MPTP-lesioned marmoset model of Parkinson's disease. The alpha2-adrenergic receptor antagonists rauwolscine and yohimbine also reduce L-dopa-induced dyskinesia. Furthermore, we demonstrate that coadministration of idazoxan with L-dopa can provide an anti-parkinsonian action more than twice the length of that seen with L-dopa alone. However, idazoxan as a monotherapy displayed no anti-parkinsonian actions. We propose that idazoxan in combination with L-dopa may provide a novel approach to the treatment of Parkinson's disease that will not only reduce the dyskinetic side effects, but extend the anti-parkinsonian actions of L-dopa. Idazoxan, as an adjunct to dopamine replacement, may prove useful in the treatment of parkinsonian patients at all stages of disease progression.

Subthalamic nucleotomy alleviates parkinsonism in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed primate.

Research into the neural mechanisms underlying the symptoms of parkinsonism utilizing the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed primate model have shown that the subthalamic nucleus (STN) occupies a central role. As a logical development of this theory, we have studied the effects of thermocoagulative lesions of the STN in the primate model. Such lesions can cause remarkable symptom reversal in the experimental primate model.

Lesion of the subthalamic nucleus for the alleviation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in the primate.

Central to the neural mechanism of MPTP-induced parkinsonism is overactivity of the subthalamic nucleus. Lesion of the subthalamic nucleus has therefore been performed in two primates with this condition and found to produce a remarkable amelioration of the disorder.

Induction of chorea and dystonia in parkinsonian primates.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in primates induced a parkinsonian syndrome that could be reversed by levodopa treatment. Animals quickly developed an apparent restlessness ("akathisia") of the lower limbs after as little as five doses. After 4-10 weeks of regular levodopa therapy, animals developed "peak dose" choreiform movements in the lower limbs that spread, with time, to involve the upper limbs and orofacial musculature. With further treatment (5-21 months), animals developed "peak dose" dystonia that variably involved the limbs and orofacial musculature. These conditions represent novel models of levodopa-induced chorea and dystonia in humans. They depend on the same underlying neuropathology and treatment regimen as their human counterparts. It is to be anticipated that these models of dyskinesia will be useful in determining the mechanisms underlying chorea and dystonia in humans and are ideally suited for experimental evaluation of new treatment strategies.

Effect of the NMDA antagonist MK-801 on MPTP-induced parkinsonism in the monkey.

Current evidence suggests that the motor symptoms of parkinsonism are due to abnormal overactivity of the medial segment of the globus pallidus, brought about by overactivity of the subthalamic nucleus, from which it receives an excitatory amino acid-mediated projection. The possibility exits, therefore, that excitatory amino acid antagonists might have an anti-parkinson effect by normalising medial pallidal activity. The NMDA antagonist MK-801 was administered i.m. to a single cynomolgus monkey with parkinsonism induced by the neurotoxin MPTP. In fact, MK-801 exacerbated the symptoms of parkinsonism. When administered after a therapeutic dose of L-DOPA it antagonised the anti-parkinson action of L-DOPA. The results suggest that any potential anti-parkinson action of excitatory amino acid antagonists will depend upon an action at non-NMDA sites. The administration of the selective neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce a primate model of Parkinson's disease is well-documented (Burns, Markey, Phillips & Chiuch, 1984; Crossman, 1987; Langston, Forno, Rebert & Irwin, 1984). Intravenous injection of MPTP, titrated judiciously over a period of several weeks, can produce a stable manifestation of the motor disability seen in the idiopathic disease of man, with a remarkable correspondence of both symptoms and pathology. Additionally, primates rendered parkinsonian by MPTP respond well to L-DOPA treatment. As in human Parkinson's disease, long-term L-DOPA therapy of MPTP-induced parkinsonism tends to be complicated by the emergence of choreiform movements and dystonic postures (Boyce, Clarke, Luquin, Peggs, Robertson, Mitchell, Sambrook & Crossman, 1989; Clarke, Sambrook, Mitchell & Crossman, 1987).(ABSTRACT TRUNCATED AT 250 WORDS)

Neural mechanisms underlying parkinsonian symptoms based upon regional uptake of 2-deoxyglucose in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The 2-deoxyglucose metabolic mapping technique has been used to investigate the neural mechanisms which underlie the symptoms of Parkinsonism in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine primate model of Parkinson's disease. In six cynomolgus monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was either (a) administered intravenously to induce generalized Parkinsonism, or (b) infused into one carotid artery to induce unilateral Parkinsonism. Post-mortem examination revealed profound cell loss from the substantia nigra, pars compacta either bilaterally or unilaterally in the two groups, respectively. In addition, there was pathological involvement of the ventral tegmental area and locus coeruleus in animals receiving intravenous 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. 2-Deoxyglucose autoradiography revealed widespread changes in 2-deoxyglucose uptake in the brains of parkinsonian animals when compared to controls. Most of these changes were in basal ganglia and related structures and were qualitatively similar in the two groups of experimental animals. Prominent increases in 2-deoxyglucose uptake were observed in the lateral segment of the globus pallidus (24-27%), the ventral anterior and ventral lateral nuclei of the thalamus (14-22%) and the nucleus tegmenti pedunculopontinus of the caudal midbrain (17-69%). A profound decrease (17-26%) in 2-deoxyglucose uptake was observed in the subthalamic nucleus. We propose these data to indicate that in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism there is the following pattern of abnormal neuronal activity in basal ganglia circuitry: (i) increased activity in the projection from the putamen to the lateral segment of the globus pallidus; (ii) decreased activity in the projection from the putamen to the medial segment of the globus pallidus; (iii) decreased activity in the projection from the lateral segment of the globus pallidus to the subthalamic nucleus; (iv) increased activity in the projection from the subthalamic nucleus to the globus pallidus; and (v) increased activity in neurons of the medial segment of the globus pallidus projecting to the ventral anterior/ventral lateral thalamus and the pedunculopontine nucleus. These results are compared to the 2-deoxyglucose uptake findings in previous studies from this laboratory in hemiballism and hemichorea in the monkey. The central importance of the subthalamic nucleus in all three conditions is proposed, and supportive evidence for the excitatory nature of subthalamic efferent fibres is adduced.