The Impact of Ghrelin on the Survival and Efficacy of Dopaminergic Fetal Grafts in the 6-OHDA-Lesioned Rat.

Ghrelin is a peptide produced in the gut with a wide range of physiological functions. Recent studies have suggested it may have potential as a neuroprotective agent in models of Parkinson's disease, reducing the impact of toxic challenges on the survival of nigral dopaminergic neurons. The presence of the ghrelin receptor (GHSR1a) on the dopaminergic neurons of the substantia nigra raises the possibility that a potential application for this property of ghrelin may be as an adjunctive neuroprotective agent to enhance and support the survival and integration of dopaminergic cells transplanted into the striatum. Thus far, inconsistent outcomes in clinical trials for fetal cell transplantation have been linked to low rates of cell survival which we hypothesize could be ameliorated by the presence of ghrelin. To explore this, we confirmed the expression of the GHSR1a and related enzymes on e14 ventral mesencephalon. To determine a functional effect, five groups of female Sprague-Dawley rats received a unilateral 6-OHDA lesion to the medial forebrain bundle and four received an intrastriatal graft of e14 ventral mesencephalic cells. Grafted rats received saline; acyl-ghrelin (10 µg/kg); acyl-ghrelin (50 µg/kg) or the ghrelin agonist JMV-2894 (160 µg/kg) i.p. for 8 weeks. An effect of ghrelin at low dose on hippocampal neurogenesis indicated blood-brain barrier penetrance and attainment of biologically relevant levels but neither acyl-ghrelin nor JMV-2894 improved graft survival or efficacy.

Increased efficacy of the 6-hydroxydopamine lesion of the median forebrain bundle in small rats, by modification of the stereotaxic coordinates.

The 6-hydroxydopamine (6-OHDA) lesion is the most widely used rat model of Parkinson's disease. A single unilateral injection of 6-OHDA into the median forebrain bundle (MFB) selectively destroys dopamine neurons in the ipsilateral substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), removing more than 95% of the dopamine innervation from target areas. The stereotaxic coordinates used to deliver 6-OHDA to the MFB have been used in our laboratory successfully for more than 25 years. However, in recent years we have observed a decline in the success rate of this lesion. Previously regular success rates of >80% of rats lesioned, have become progressively more variable, with rates as low as 20% recorded in some experiments. Having excluded variability of the neurotoxin and operator errors, we hypothesized that the change seen might be due to the use of smaller rats at the time of first surgery. An attempt to proportionally adjust the lesion coordinates base on head size did not increase lesion efficacy. However, in support of the small rat hypothesis it was observed that, using the standard coordinates, rat's heads had a "nose-up" position in the stereotaxic fame. Adjustment of the nose bar to obtain a flat head position during surgery improved lesion success, and subsequent adjustments of the lesion coordinates to account for smaller head size led to a greatly increased lesion efficacy (>90%) as assessed by amphetamine induced rotation.

Medical terminations of pregnancy: a viable source of tissue for cell replacement therapy for neurodegenerative disorders.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.

Context-driven changes in L-DOPA-induced behaviours in the 6-OHDA lesioned rat.

Both contralateral rotational behaviour and dyskinetic abnormal involuntary movements (AIMs) are induced by the administration of l-DOPA in the unilateral 6-OHDA lesioned rat model of Parkinson's disease. Since rotational responses can be conditioned to environmental cues we have investigated the extent to which drug-induced AIMS may also be conditioned by exteroceptive cues and experience. In Experiment I, 6-OHDA lesioned rats received repeated daily injections of l-DOPA either in their home cage (control) or in association with a brief (20 mins) exposure to the rotometers (paired). To assess conditioning, all animals then received two tests in the rotometer bowls. Following injection of saline the paired group both rotated more contralaterally and displayed manifest AIMs, neither of which were exhibited by the control rats. Moreover, following injection of l-DOPA, the paired group showed a trend for increased AIMs compared to controls. Two further studies provided longer exposure to the conditioning environments in counterbalanced designs. Although, using these parameters, re-exposure in the presence of saline did not induce context-dependent AIMs, a strong context-specific component of the sensitised response to l-DOPA was seen; chronic administration of drug produced a significantly stronger behavioural response in animals paired with a particular environment for drug administration than controls. This data suggests that part of the sensitisation of behavioural responding to l-DOPA administration is not solely a pharmacological phenomenon, but is also conditioned to the environmental context in which the drug is administered. This has clear implications for the clinical observation and experimental measurement of drug-induced dyskinesia in Parkinson's disease patients and animal models.

Understanding graft-induced dyskinesia.

The transplantation of dopaminergic cells for the treatment of symptoms of Parkinson’s disease has several hurdles to overcome before it can be considered a successful therapeutic approach. One issue is the development of abnormal involuntary movements in the absence of L-3,4-dihydroxyphenylalanine following the transplantation of fetal ventral mesencephalon identified in three different clinical trials. Hypotheses as to the cause of these movements include: the composition of the graft, size of the graft, L-3,4-dihydroxyphenylalanine exposure and L-3,4-dihydroxyphenylalanine-induced dyskinesia prior to transplantation and inflammatory responses in and around the graft. We evaluate the clinical evidence supporting these hypotheses and the preclinical models upon which experiments are being based to resolve them.

Priming for L-DOPA-induced abnormal involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats.

In some patients, graft-induced dyskinesia develops following intrastriatal transplantation of embryonic neural tissue for the treatment of Parkinson's disease. The mechanisms underlying these involuntary movements need to be clarified before this approach to clinical cell therapy can be developed further. We previously found that rats with 6-OHDA lesions, primed with L-DOPA treatment and that have subsequently undergone intrastriatal graft surgery exhibit involuntary movements when subjected to amphetamine. This model of amphetamine-induced AIMs reflects a pattern of post-graft behaviours that in the absence of robust spontaneous GID in the rat is the closest approximation that we currently have available. We now show that they are associated with the chronic administration of L-DOPA prior to the transplantation surgery. We also demonstrate that neither changes in c-fos nor FosB/DeltaFosB expression in the lateral striatum are associated with the expression of these behaviours. Taken together, these data reveal that the severity of abnormal movements elicited by amphetamine in grafted animals may relate to previous L-DOPA exposure and dyskinesia development, but they develop through mechanisms that are independent of FosB/DeltaFosB upregulation.

Neuroinflammation in the generation of post-transplantation dyskinesia in Parkinson's disease.

The observation that neural grafts can induce dyskinesias has severely hindered the development of a transplantation therapy for Parkinson's disease (PD). We addressed the hypothesis that inflammatory responses within and around an intrastriatal graft containing dopamine neurons can trigger dyskinetic behaviors. We subjected rats to unilateral nigrostriatal lesions with 6-hydroxydopamine (6-OHDA) and treated them with L-DOPA for 21 days in order to induce abnormal involuntary movements (AIMs). Subsequently, we grafted the rats with allogeneic embryonic ventral mesencephalic tissue in the dopamine-denervated striatum. In agreement with earlier studies, the grafted rats developed dyskinesia-like AIMs in response to amphetamine. We then used two experimental approaches to induce an inflammatory response and examined if the amphetamine-induced AIMs worsened or if spontaneous AIMs developed. In one experiment, we challenged the neural graft hosts immunologically with an orthotopic skin allograft of the same genetic origin as the intracerebral neural allograft. In another experiment, we infused the pro-inflammatory cytokine interleukin 2 (IL-2) adjacent to the intrastriatal grafts using osmotic minipumps. The skin allograft induced rapid rejection of the mesencephalic allografts, leading to disappearance of the amphetamine-induced AIMs. Contrary to our hypothesis, the rejection process itself did not elicit AIMs. Likewise, the IL-2 infusion did not induce spontaneous AIMs, nor did it alter L-DOPA-induced AIMs. The IL-2 infusions did, however, elicit the predicted marked striatal inflammation, as evidenced by the presence of activated microglia and IL2Ralpha-positive cells. These results indicate that an inflammatory response in and around grafted dopaminergic neurons is not sufficient to evoke dyskinetic behaviors in experimental models of PD.

Striatal output markers do not alter in response to circling behaviour in 6-OHDA lesioned rats produced by acute or chronic administration of the monoamine uptake inhibitor BTS 74 398.

The monoamine uptake inhibitor BTS 74 398 induces ipsilateral circling in 6-hydroxydopamine (6-OHDA) lesioned rats without induction of abnormal motor behaviours associated with L-dopa administration. We examined whether this was reflected in the expression of peptide mRNA in the direct and indirect striatal output pathways.6-OHDA lesioning of the nigrostriatal pathway increased striatal expression of PPE-A mRNA and decreased levels of PPT mRNA with PPE-B mRNA expression remaining unchanged. Acute L-dopa administration normalised PPE-A mRNA and elevated PPT mRNA while PPE-B mRNA expression remained unchanged. Acute administration of BTS 74 398 did not alter striatal peptide mRNA levels. Following chronic treatment with L-dopa, PPE-A mRNA expression in the lesioned striatum continued to be normalised and PPT mRNA was increased compared to the intact side. PPE-B mRNA expression was also markedly increased relative to the non-lesioned striatum. Chronic BTS 74 398 administration did not alter mRNA expression in the 6-OHDA lesioned striatum although small increases in PPT mRNA expression in the intact and sham lesioned striatum were observed. The failure of BTS 74 398 to induce changes in striatal neuropeptide mRNA correlated with its failure to induce abnormal motor behaviours or behavioural sensitisation but does not explain how it produces a reversal of motor deficits. An action in another area of the brain appears likely and may explain the subsequent failure of BTS 74 398 and related compounds to exert anti-parkinsonian actions in man.

The impact of graft size on the development of dyskinesia following intrastriatal grafting of embryonic dopamine neurons in the rat.

Intrastriatal transplants of embryonic ventral mesencephalon can cause dyskinesia in patients with Parkinson's disease (PD). We assessed the impact of transplant size on the development of graft-induced dyskinesia. Rats with unilateral 6-hydroxydopamine lesions were primed to exhibit L-DOPA-induced dyskinesia. They were then intrastriatally grafted with different quantities of embryonic ventral mesencephalic tissue to give small and large grafts. Without drug treatment, discrete dyskinetic-like movements were observed in most rats with large grafts 2-6 weeks after transplantation, but disappeared later. Amphetamine evoked severe abnormal involuntary movements (AIMs) in grafted animals, which were more striking with large grafts. The AIMs coincided with contralateral rotation, but displayed a different temporal profile and pharmacological properties. Thus, selective dopamine uptake blockade elicited rotational behavior, whereas coadministration of both dopamine and serotonin uptake blockers was required to evoke significant orolingual and limb AIMs. In conclusion, robust and reproducible AIMs were evoked in rats with large grafts by blockade of monoamine reuptake. These AIMs may provide a new tool for assessing dyskinetic effects of neural grafting.

Does contraversive circling in the 6-OHDA-lesioned rat indicate an ability to induce motor complications as well as therapeutic effects in Parkinson's disease?

Circling behaviour in unilateral 6-OHDA-lesioned rats is interpreted as being opposite in direction to the side of the brain with highest striatal dopaminergic activation. Ipsiversive rotation indicates an action on the intact striatum, while contraversive rotation demonstrates an effect on dopamine receptors in the denervated striatum and is taken as predictive of symptomatic benefit in Parkinson's disease. But does an equivalent behavioural outcome result from stimulating the intact and denervated striatum to the same degree? We report on the behavioural responses produced by administration of L-dopa and the monoamine uptake inhibitor BTS 74,398. These were given alone, or in combination, at doses equivalent to their ED(25), ED(50) and ED(75) for inducing circling in unilateral 6-OHDA-lesioned rats. L-dopa administered alone induced dose-dependent contraversive circling while BTS 74,398 produced ipsiversive rotation. However, L-dopa and BTS 74,398 in combination, produced the same contraversive circling response as when L-dopa was administered alone. Little or no ipsiversive rotation occurred, irrespective of the combination of doses employed. This surprising finding suggests that there are major differences in the outcome of stimulating the intact and denervated striatum with the latter dominating the behavioural response. Since repeated administration of L-dopa but not BTS 74,398, sensitises rotational responses and induces abnormal movements, it may be that contraversive rotation is predictive of both clinical response in PD and the ability to induce motor complications.

Repeated administration of the monoamine reuptake inhibitor BTS 74 398 induces ipsilateral circling in the 6-hydroxydopamine lesioned rat without sensitizing motor behaviours.

BTS 74 398 (1-[1-(3,4-dichlorophenyl)cyclobutyl]-2-(3-diaminethylaminopropylthio)ethanone monocitrate) is a monoamine reuptake inhibitor that reverses motor deficits in MPTP-treated (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) common marmosets without provoking established dyskinesia. However, it is not known whether BTS 74 398 primes the basal ganglia for dyskinesia induction. In this study, the ability of BTS 74 398 to sensitize 6-hydroxydopamine (6-OHDA)-lesioned rats for the production of abnormal motor behaviours and the induction of striatal DeltaFosB were determined in comparison with l-3,4-dihydroxyphenylalanine methyl ester (L-dopa). Acute administration of BTS 74 398 induced a dose-dependent ipsilateral circling response in unilaterally 6-OHDA-lesioned rats whereas L-dopa produced dose-dependent contraversive rotation. The ipsilateral circling response to BTS 74 398 did not alter during 21 days of administration. In contrast, L-dopa treatment for 21 days caused a marked increase in rotational response. Repeated administration of both L-dopa and BTS 74 398 increased general motor activity and stereotypic behaviour. In L-dopa-treated rats, orolingual, locomotive, forelimb and axial abnormal movements developed whereas BTS 74 398 produced only locomotion with a side bias but no other abnormal movements. Sensitization of circling responses and the development of abnormal movements in 6-OHDA-lesioned rats have been associated with the potential of dopaminergic drugs to induce dyskinesia. Furthermore, striatal DeltaFosB immunoreactivity, shown to correlate with dyskinesia induction, was increased by L-dopa but was unaffected by repeated BTS 74 398 administration. The lack of such changes following repeated BTS 74 398 treatment suggests that it may be an effective antiparkinsonian therapy that is unlikely to produce involuntary movements.

Dopamine uptake inhibitor-induced rotation in 6-hydroxydopamine-lesioned rats involves both D1 and D2 receptors but is modulated through 5-hydroxytryptamine and noradrenaline receptors.

Dopamine uptake inhibitors may provide a means of sustaining endogenous and exogenous striatal dopamine levels in Parkinson's disease, but most are not selective and also inhibit the noradrenaline and 5-hydroxytryptamine (5-HT) transporters. To determine the involvement of the individual monoamine transporters in the production of motor activity, the effect of the nonselective monoamine uptake inhibitor BTS 74 398 1-([1-(3,4-dichlorophenyl)cyclobutyl]-2-(3-diaminethylaminopropylthio) ethanone monocitrate) and the selective dopamine, GBR 12909 [1-(2-(bis-(4-fluorphenyl)-methyl)ethyl)-4-(3-phenylpropyl)piperazine) dihydrochloride], noradrenaline (nisoxetine), and 5-HT (fluvoxamine) reuptake inhibitors on circling in the unilateral 6-hydroxydopamine-lesioned rat was investigated. GBR 12909 induced ipsilateral circling, but fluvoxamine and nisoxetine were without effect. However, when administered with GBR 12909, fluvoxamine enhanced rotation, whereas nisoxetine had no effect. The results suggest that 5-HT, but not noradrenaline, reuptake inhibition facilitates dopamine-mediated motor activity. To test this hypothesis, BTS 74 398 was administered in combination with selective dopamine, 5-HT, and noradrenaline receptor antagonists. Both D(1) and D(2) receptor antagonists, SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] and raclopride, inhibited BTS 74 398-induced circling. In contrast, the 5-HT(1A) 5-HT(1A/B) antagonists, WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexane-carboxamide maleate) and pindolol, and the 5-HT(2A) antagonist, ketanserin, had no effect. The nonspecific 5-HT((1/2)) antagonists, methysergide and metergoline, and the specific 5-HT(2C) antagonist, N-desmethylclozapine, enhanced BTS 74 398-induced circling, as did the alpha(2)-adrenoceptor antagonist idazoxan. Overall, the data suggest that inhibition of the 5-HT and noradrenaline transporters modulate dopamine uptake inhibitor-mediated motor activity. However, the mechanism of this interaction is complex, involving opposing effects of noradrenaline and 5-HT agonism and antagonism.

The cranial meninges: anatomic considerations.

The anatomy of the cranial dura and leptomeninges is both intricate and complex. A thorough discussion of the protective covering of the brain including the dura, arachnoid, and pia is provided on both gross and microscopic levels. An attempt to include issues of clinical relevance is made, highlighting the Virchow-Robin spaces and the optic sheath. In addition, the normal appearance of the dura and leptomeninges on MRI is presented to establish a framework for the discussion of leptomeningeal pathology.