Nathalie Percie
email: npercied@sgul.ac.uk

Du Sert

J A Rudd

C C Apfel

P L R Andrews

N Percie Du Sert

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT 3 receptor antagonists

Keywords: Cisplatin, emesis, ferret, meta-analysis, systematic review, ondansetron, 5-HT 3 receptor antagonist, granisetron, tropisetron 5-HT 3 : 5-hydroxytryptamine 3, NK 1 : neurokinin 1, i.v.: intravenous, i.p.: intraperitoneal, p.o.: per os, oral, s.c.: sub-cutaneous, R+V: retches and vomits, SD: standard deviation, CI: confidence interval

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT receptor antagonists

INTRODUCTION

It is generally accepted that nausea and vomiting (emesis) are components of a protective mechanism by which the human body defends itself against ingested toxins. However, the emetic reflex can be triggered inappropriately and nausea and vomiting are also relatively common side effects of drugs in current use (e.g. morphine, anti-cancer chemotherapy) as well as dose-limiting toxicities which may limit the development of novel chemical entities intended for the treatment of a range of diseases (e.g. phosphodiesterase-IV inhibitors for the treatment of asthma [START_REF] Spina | The potential of PDE4 inhibitors in respiratory disease[END_REF]).

The multi-system nature of the emetic reflex coordinated in the brainstem and the behavioural and sensory expression of nausea have meant that, to date, preclinical studies of the mechanisms involved and identification of novel anti-emetic agents have involved studies in whole animals (conscious, anaesthetised or decerebrate) [START_REF] Holmes | Opportunities for the replacement of animals in the study of nausea and vomiting[END_REF]. Nausea and vomiting are particularly associated with the treatment of cancer by cytotoxic drugs (e.g. cisplatin), symptoms which patients find particularly distressing and impact upon compliance with treatment. In the absence of anti-emetic prophylaxis, cisplatin induces nausea and vomiting in virtually all patients [START_REF] Roila | Prevention of chemotherapy-and radiotherapy-induced emesis: results of the 2004 Perugia International Antiemetic Consensus Conference[END_REF]; the emetic response lasts up to 5 days on each cycle and is characterised by an intense acute phase lasting ~24 hours and a less intense but more protracted delayed phase peaking during the period 48-72 hours following the administration of cisplatin [START_REF] Kris | Incidence, course, and severity of delayed nausea and vomiting following the administration of high-dose cisplatin[END_REF]. In the early 1980's the ferret (Mustela putorius furo L.) was reported to develop an acute emetic response to high dose cisplatin (8-10 mg.kg -1 ) and was proposed as an alternative model to the dog, cat and monkey (commonly used at the time) to study cytotoxic drug-induced emesis and identify potential anti-emetic agents [START_REF] Florczyk | Cisplatin-induced emesis in the Ferret: a new animal model[END_REF].

Subsequently the acute cisplatin model was modified and the dose of cisplatin lowered to 5 mg.kg -1 to investigate delayed emesis [START_REF] Rudd | Profiles of emetic action of cisplatin in the ferret: a potential model of acute and delayed emesis[END_REF]. The ferret model of cisplatin-induced emesis was rapidly adopted for the investigation of new anti-emetic agents and was pivotal in establishing the anti-emetic efficacy of 5-hydroxytryptamine 3 (5-HT 3 ) [START_REF] Miner | Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism[END_REF] and tachykinin NK 1 receptor antagonists [START_REF] Watson | The anti-emetic effects of CP-99,994 in the ferret and the dog: role of the NK1 receptor[END_REF], which are both currently in widespread use for the treatment of chemotherapy-induced nausea and vomiting [START_REF] Roila | Prevention of chemotherapy-and radiotherapy-induced emesis: results of the 2004 Perugia International Antiemetic Consensus Conference[END_REF].

The use and benefit of animal models in research is regularly questioned and anecdotal evidence or unsupported claims, as opposed to quantitative support, are too often used as justifications [START_REF] Matthews | Medical progress depends on animal models -doesn't it?[END_REF][START_REF] Pound | Where is the evidence that animal research benefits humans?[END_REF]. There has recently been a growing interest in systematic reviews and meta-analyses to assess the validity of animal models (i.e.

how preclinical research has informed clinical research) and their utility in drug discovery (i.e. evaluate data and inform the decision to carry out a clinical trial). The Nuffield Council for Bioethics [START_REF]The ethics of research involving animals[END_REF] recommends that such reviews are undertaken to "evaluate more fully the predictability and transferability of animal model". Such analyses also have implications for the application of the principles of the 3Rs (Replacement, Refinement, Reduction) to animal experimentation [START_REF] Holmes | Opportunities for the replacement of animals in the study of nausea and vomiting[END_REF][START_REF] Kilkenny | Systematic reviews of animal research[END_REF] and should inform preclinical guidelines produced by regulators [e.g . 37]. Recently, systematic reviews and meta-analysis of animal models of stroke have been carried out. A retrospective study concluded that even though individual studies had reported beneficial effects of the calcium channel blocker nimodipine, overall the preclinical data available were not conclusive [START_REF] Horn | Nimodipine in animal model experiments of focal cerebral ischemia: a systematic review[END_REF], which is consistent with the fact that this type of drug was without effect in humans [START_REF] Horn | Calcium antagonists for ischemic stroke: a systematic review[END_REF] and highlights the necessity of quantifying animal data adequately before starting clinical trials. Later studies assessed the preclinical evidence of the effect of potential treatments in experimental stroke and characterised their neuroprotective properties in order to identify research priorities [START_REF] Macleod | Systematic review and meta-analysis of the efficacy of melatonin in experimental stroke[END_REF][START_REF] Macleod | Systematic review and metaanalysis of the efficacy of FK506 in experimental stroke[END_REF][START_REF] Macleod | Pooling of animal experimental data reveals influence of study design and publication bias[END_REF].

The cisplatin-induced emesis ferret models provide a unique opportunity to assess the value of systematic reviews in specific areas, because the wealth of data available in this relatively circumscribed area allows assessment of two characteristics of a model: the response to cisplatin itself, and the anti-emetic potential of agents that are currently used in humans. The aim of this systematic review is two-fold: firstly, this study intends to provide an objective measure of the characteristics of cisplatin-induced emesis in the ferret, in terms of the latency, magnitude (number of retches and vomits) and profile of the emetic response.

Secondly, the effect of 5-HT 3 receptor antagonists in the ferret model will be quantified; the present study will assess the efficacy of ondansetron against the acute phase of emesis; additionally, we will compare the overall effect of 5-HT 3 receptors antagonists against the acute and delayed phases of emesis. This paper is the first systematic review and meta-analysis covering a model of emesis and anti-emetics. It provides evidence, which supports the predictability of the model and identifies new features of the model not apparent from individual studies.

Additionally it shows the limitations of the model and identifies opportunities for enhanced animal welfare according to the principles of the "3Rs" formulated by Russell and Burch over 50 years ago [START_REF] Russell | The Principles of Humane Experimental Technique[END_REF].

METHODS

Search strategy

Studies were identified from Pubmed (1974 to March 2007) and Embase (1980 to March 2007) using the combination of words: CISPLATIN and FERRET; hand searching of abstracts of scientific meetings and personal files. All references of newly identified publications were also screened until no further eligible references were found. Language was not restricted. Values for data expressed graphically were either requested from authors or measured from the graphs. Corresponding authors were also contacted to obtain data that was not reported clearly enough in their publications.

Inclusion criteria:

 Report of cisplatin-induced emesis in the ferret  Emetic response documented, and quantified by at least one of the following: latency to onset of emesis (retching or vomiting), number of animals developing emesis, number of retches (R), vomits (V), retches and vomits (R+V) defined according to our definition and reported as mean only or mean ± SEM or SD, and number of ferrets per group.

Exclusion criteria:

 Number of animals not stated  Emetic response investigated under anaesthesia  Emetic response not reported as the number of animals developing emesis, or mean latency, or mean number of retches and vomits compatible with the standard definition of emesis.

Emesis was defined as retching (i.e. rhythmic abdominal contractions against a closed glottis) and vomiting (i.e. rhythmic abdominal contractions associated with the oral expulsion of solid or liquid materials from the gastrointestinal tract) [START_REF] Borison | Physiology and pharmacology of vomiting[END_REF][START_REF] Costall | Emesis induced by cisplatin in the ferret as a model for the detection of anti-emetic drugs[END_REF][START_REF] Mccarthy | Respiratory mechanics of vomiting in decerebrate cats[END_REF].

Reports stating this definition in their methods section were included in this study; if the definition was absent or unclear, evaluation of the results reported and their inclusion in this quantitative review were left to the judgement of the investigator and discussed with co-workers; reports were included:

-If the same team had published other reports clearly stating this definition or a member of the team-most senior or corresponding author-was contacted to confirm the definition used to characterise emesis.

-If the report referred to publications clearly stating this definition.

-If the definition stated allowed the identification of the number of retches and/or vomits according to our definition.

The latency (time to onset of emesis) was a potential confounding factor as many publications reported the latency as a mean for all the animals in the groups, including those free of emesis, in which case the latency was taken as the total duration of the observation time. All latencies were recalculated as the mean latency to the 1 st retch or vomit in animals that developed emesis only. The latency was either measured as the time to the first retch, the time to the first vomit or the time to the first emetic episode. All the latencies reported were included combined together, as it was considered that only a minimal period of time separates the first retch from the first vomit [START_REF] Tsuchiya | Anti-emetic activity of the novel nonpeptide tachykinin NK1 receptor antagonist ezlopitant (CJ-11,974) against acute and delayed cisplatin-induced emesis in the ferret[END_REF], a more rigid approach including only the studies with either one or the other measurement and excluding those reporting the latency to the first emesis or emetic episode would have induced a greater error.

Data extraction and analysis

Meta-analysis: the ferret model of cisplatin-induced emesis

The number of retches (R), vomits (V), retches + vomits (R+V) and/or latency data from control groups (i.e. animals that received no other drug than cisplatin or cisplatin and an inactive-i.e. non-emetogenic-vehicle) were extracted as mean, standard deviations (SD) and number of animals per group. Weighted mean and weighted mean of the SD were calculated and a one-way ANOVA was carried out to compare the onset of emesis following different doses of cisplatin. Unless stated, all results are reported as mean ± SD. In order to identify variables modulating the emetic response, subgroup analyses were carried out according to criteria such as the vehicle used, duration of the observation period, the mode of administration of cisplatin (i.v. or i.p.), the use of anaesthesia and the recovery time prior to the emetic challenge, the strain (fitch or albino), sex and origin of the ferrets. This analysis was only carried out on the most common doses of cisplatin used to induce acute (10 mg.kg -1 ) and acute and delayed emesis in the ferret (5 mg.kg -1 ); the two doses were treated separately. Weighted means and the weighted mean of the SDs were calculated and profiles of emesis were constructed with Graphpad Prism® version 5.0, Graphpad Software Inc., San Diego, USA. Differences were assessed by a oneway analysis of variance (ANOVA) or independent sample t-tests as appropriate.

Descriptive statistics and comparisons were carried out using SPSS ® 14.00, SPSS Inc., Chicago, USA and CLINSTAT (M. Bland). Differences were considered statistically significant when p<0.05.

Meta-analysis: the effect of anti-emetics

For the meta-analysis of the effects of anti-emetics, comparisons were only included if the effect of a prophylactic anti-emetic treatment was reported, the 3 outcomes measured were: the number of R+V, proportion of animals experiencing emesis and latency to the onset of emesis. To calculate the effect size and its 95% confidence interval for the continuous outcomes (i.e. R+V and latency), the mean outcome for the treatment group, and the SDs in treatment and control groups were expressed as a proportion of the outcome in the control group [START_REF] Macleod | Pooling of animal experimental data reveals influence of study design and publication bias[END_REF]. Actual data were used for dichotomous outcomes (i.e. the number of animals with emesis). When a control group was used to assess more than one treatment group, the number of animals in the control group was divided by the number of treatment groups and if needed, adjusted to the next integer. This methodology is consistent with what has been done in another meta-analysis of animal data in a model of stroke [START_REF] Macleod | Pooling of animal experimental data reveals influence of study design and publication bias[END_REF]. The effect of ondansetron was examined for each of the 3 outcomes; subgroup analyses were carried out depending on the dose of cisplatin and duration of the observation period, dose, timing and mode of administration of ondansetron, mode of administration of cisplatin, origin of the ferrets, and quality score of the study. Additional analyses examined the effects of 5-HT 3 receptor antagonists on the latency to the onset of cisplatin (10 mg.kg -1 )-induced emesis and on the acute and delayed R+V induced by 5 mg.kg -1 cisplatin (criteria for variation: individual compound). In the later analysis, comparisons on a given day were only included if the anti-emetic treatment started before or at the start of the 24h period and was continued throughout the day.

Methodological quality of individual studies was assessed according to criteria chosen to evaluate the reliability of the data extracted. These criteria were: no duplicate publication identified-confirmed or suspected-, retch and vomit clearly defined or definition confirmed by authors, latency to the 1 st retch or vomit given, SEM/SD given for the mean latency, number of retches and vomits or R+V given, SEM/SD given for the mean R+V, number of ferrets completely protected given (1 point per criterion fulfilled), origin, sex, strain and body weight of the ferrets given (1/2 point per criterion fulfilled). Each study was given a quality score out of a possible total of 9 points. The DerSimonian and Laird method was used to combine dichotomous (risk difference [RD]) and continuous data (weighted mean difference

[WMD]). The random-effects model was chosen over the fixed effect assumption because it incorporates inter-study differences into the analysis of the overall treatment efficacy [START_REF] Dersimonian | Random-effects model for meta-analysis of clinical trials: an update[END_REF]. The data were analyzed with Review Manager (RevMan. Version 5.0 for Macintosh. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2008). All effect estimates are reported as mean and 95% confidence intervals. Z-tests were used to assess the overall effect of treatments and Chi-squared (χ 2 ) tests were used to assess the heterogeneity along with I 2 , which describes the percentage of the variability in effect estimates that is due to heterogeneity rather than chance (a value greater than 50% may be considered substantial heterogeneity). The differences between the treatment effects of subcategories of a particular outcome were assessed by a Z-test [START_REF] Altman | Practical Statistics for Medical Research[END_REF][START_REF] Matthews | Interaction 3: How to examine heterogeneity[END_REF], potential publication biases were assessed by Funnel plots [START_REF]Open Learning Material[END_REF].

Anti-emetic drugs examined

Ondansetron: 9-methyl-3-[(2-methylimidazol-1-yl)methyl]-2,3-dihydro-1H-carbazol-4- one. Granisetron: 1-methyl-N-(9-methyl-9-azabicyclo[3.3.1]nonan-7-yl)indazole-3- carboxamide. Indisetron: N-(3,9-dimethyl-3,9-diazabicyclo[3.3.1]nonan-7-yl)-1H- indazole-3-carboxamide. Zacopride: 4-amino-N-(1-azabicyclo[2.2.2]octan-8-yl)-5- chloro-2-methoxybenzamide. FK1052: (7S)-10-methyl-7-[(5-methyl-1H-imidazol-4- yl)methyl]-8,9-dihydro-7H-pyrido[1,2-a]indol-6-one hydrochloride. Dolasetron: (3R)- 10-oxo-8-azatricyclo[5.3.1.0 3,8 ]undec-5-yl 1H-indole-3-carboxylate. L-683,877: 2 1 -(1- Methyl-lH-indol-3-yl)-)spiro(1-azabicyclo[2.2.2]octane-3,5 1 (4 1 H)-oxazole. Ramosetron: (1-methylindol-3-yl)-[(5R)-4,5,6,7-tetrahydro-3H-benzimidazol-5- yl]methanone. Azasetron: N-(1-azabicyclo[2.2.2]octan-8-yl)-6-chloro-4-methyl-3-oxo- 1,4-benzoxazine-8-carboxamide. Renzapride: 4-amino-N-[(5S,6S)-1- azabicyclo[3.3.1]nonan-6-yl]-5-chloro-2-methoxybenzamide. Bemesetron: [(1S,5R)-8- methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate.

RESULTS

Publications

As of March 2007, 182 publications were retrieved, 115 publications describing cisplatin-induced emesis in the ferret were identified, 32 publications were excluded and 83 publication contained usable data (Figure 1, Tables 1 and2). A further 15 publications were excluded on the grounds that data was already reported elsewhere. The remaining 68 publications were either fully or partly included as some papers presented original data and duplicate data together, in this case only the original data was extracted and the duplicate data was ignored.

Out of the 68 publications from which at least one outcome was extracted, 44, 10 and 9 publications reported the effect of at least one 5-HT 3 , NK 1 receptor antagonist and glucocorticoid, respectively. In terms of outcome, 63 studies reported the latency, which was either measured as the latency to the first retch (13% of the publications reporting the latency), the latency to the first vomit (16%) or the latency to the first emetic episode (75%). Fifty-one studies reported the number of retches and vomits (R+V) in a given observation time, 37 and 36 studies respectively reported the number of retches and vomits separately. The number of animals with emesis during the duration of the observation time was reported in 45 publications.

Meta-analysis: the ferret model of cisplatin induced emesis

Dose of cisplatin

The latency to the onset of emesis was dose-dependant; the time of onset was significantly delayed following a dose of cisplatin of 5 mg.kg -1 , compared with higher doses (6-20 mg.kg -1 , one-way ANOVA followed by Bonferroni post-tests, see Figure 2). Additionally differences between the doses of 6 to 20 mg.kg -1 were detected (p<0.001, one way ANOVA) and the latency shortened when the dose increased.

The 10 mg.kg -1 cisplatin model

The duration of the observation period varied from 2 to 40h (at which time the animals were killed). The most common observation period was 4h, during which 107 ± 48 R+V (n=199, 22 studies) were observed. In studies using 2h, 3h, 6h, 24h and 40h observation periods, 79 ± 35 R+V (n=40, 6 studies), 101 ± 48 R+V (n=29, 3 studies), 170 ± 56 R+V (n=73, 8 studies), 204 ± 52 R+V (n=16, 3 studies) and 188 R+V (n=4, 1 study) were observed, respectively. The number of R+V significantly increased as the observation period increased up to 24h (one-way ANOVA, p<0.001). The intensity of emesis reached a maximum after 2h and decreased to a very low level (less that 5 R+V per hour) from 3h onward post cisplatin (Figure 3A).

The latency to the onset of emesis was shorter when cisplatin was administered intravenously (1.16 ± 0.35 h, n=277) compared to intraperitoneally (1.51 ± 0.29 h, n=134, p<0.001, independent sample t-test). However, the route of administration was directly related to the use of anaesthesia, and all the animals injected with cisplatin intravenously (i.v.) had prior anaesthesia during which an i.v. line was implanted or were anaesthetised at the time of cisplatin injection, and prior anaesthesia was not reported in any of the animals that received an intraperitoneal (i.p.) injection of cisplatin. The recovery period between anaesthesia and the administration of cisplatin did not make a difference as ferrets that received cisplatin under anaesthesia developed emesis with a latency of 1.21 ± 0.32 h (n=93), and those allowed up to 3 and more than 3 days recovery developed emesis with a latency of 1.11 ± 0.31 h (n=51) and 1.13 ± 0.46 h (n=89), respectively (one-way ANOVA, p=0.21). The use of an injectable anaesthetic (sodium pentobarbital 30-35 mg.kg -1 i.p.) further shortened the latency (0.95 ± 0.14 h, n=46) compared to volatile anaesthetics (1.21 ± 0.39 h, n=176, p<0.001, independent sample t-test).

Intraperitoneal cisplatin induced a more severe emetic response and 156 ± 41 R+V (n=19) and 172 (n=65, SD not available) were quantified over 4h and 6h, respectively compared to 102 ± 52 R+V (n=180, p<0.001, independent sample t-test) and 152 (n=8, SD not available) following i.v. cisplatin injection (with prior anaesthesia). No differences were observed over an observation period of 2h (i.p.: 72 ± 41, n=22; i.v.: 88 ± 32, n=18, p=0.194, independent samples t-test). There was not enough data available on the emetic response quantified for 3, 24 and 40h to investigate the influence of the mode of administration of cisplatin and the use of anaesthesia.

In animals that did not receive any vehicle, the latency was 1.39 ± 0.20 h (n=18) and ferrets had 92 ± 15 R+V (n=5). Different vehicles such as saline/distilled water (1.30 ± 0.38 h, n=296; 97 ± 31 R+V, n=125), propylene glycol (1.23 ± 0.06 h, n=4), PEG 300 (1.79 ± 0.18 h, n=5), Tween 80 (1.22 ± 0.19 h, n=8) and methyl cellulose (1.13 ± 0.25 h, n=5) impacted on the latency (p<0.0001, one-way ANOVA) but no significant differences were found compared to the group that received no vehicle (p>0.05, Bonferroni post-tests). Glucose (1.33 ± 0.24 h, n=7; 148 ± 100 R+V, n=7) was also found to increase the number of R+V (p<0.05, one-way ANOVA and Bonferroni posttests).

No differences were detected in latency or number of R+V over 4h in groups of male ferrets only compared to groups of males and females (p>0.05, independent sample t-tests). The latency was significantly reduced in albino ferrets (1.07 ± 0.14h, n=29) compared to fitch ferrets (1.34 ± 0.20h, n=70) and mixed groups of albino and fitch ferrets (1.25 ± 0.36h, n=108, p<0.05, one-way ANOVA and Bonferroni post-tests) but not enough data were available to compare the number of R+V.

The latency to the onset of emesis was significantly longer in ferrets bred in New Zealand (1.73 ± 0.37h, n=11) compared to animals bred in the UK (1.25 ± 0.44h, n=170) and in the USA (1.31 ± 0.19h, n=176, p<0.05, one-way ANOVA and Bonferroni post-tests). No differences were detected in the number of R+V over 4h (p>0.05) but the number of R+V over 2h was reduced in New Zealand ferrets (40 ± 41 R+V, n=6) compared to U.K. animals (88 ± 32 R+V, n=18, p=0.007, independent sample t-tests). As all the ferrets originating from New Zealand were challenged with an intraperitoneal dose of cisplatin, a sub-analysis was carried out only in animals administered cisplatin i.p. and the delay in latency was still significant in ferrets bred New Zealand compared to animals bred in the U.K. (1.43 ± 0.46 h, n=29, p<0.05) but not to animals bred in the U.S.A (1.51 ± 0.21 h, n=84, p>0.05, one-way ANOVA and Bonferroni post-tests).

The 5 mg.kg -1 cisplatin model (acute and delayed emesis model)

Fourteen studies investigated cisplatin-induced acute and delayed emesis in the ferret; cisplatin was administered i.p. in all studies and no study reported prior use of anaesthesia. A biphasic profile of emesis was observed; the acute phase started 10.51 ± 0.58 h (n=156) after cisplatin administration, peaked after 12h and a nadir was reached after 24h (Figure 3B). The delayed phase was more intense than the acute phase and reached a peak 48h post cisplatin before gradually decreasing in intensity during the next 24h, until 72h post cisplatin, at which time a small amount of emesis still persisted. 105 ± 83 R+V (n=215) and 340 ± 171 (n=153) were observed during the acute and delayed phase, respectively (161 ± 98 R+V during day 2 and 179 ± 94 R+V during day 3, n=130). Overall, 448 ± 231 R+V (n=153) were observed during the entire 72h period.

In animals that did not receive any vehicle, the latency was 11.76 ± 9.86 h (n=98) and animals that received i.p. injections of vehicles such as saline, distilled water and 10% NaHCO 3 had a latency of 5.52 ± 3.32 h (n=12), 8.74 ± 8.61 h (n=41) and 5.71 ± 5.66 h (n=5), respectively. Overall the injection of a vehicle had an impact on the latency (p<0.0001, one-way ANOVA) but no specific differences were detected compared to the group that did not receive any vehicle (p>0.05, Bonferroni posttests). None of the vehicles had a significant impact on the number of R+V during the acute or the delayed phase (p>0.05, one-way ANOVA). Neither the strain nor the sex of the ferrets had an impact on the latency or the number of R+V (p>0.05, one-way ANOVA).

Meta-analysis: the effect of anti-emetics

Effect of ondansetron on the acute phase of emesis

Outcome: number of retches and vomits (R+V) Data for the number of R+V was extracted from 11 full papers, 23 comparisons involving 107 ferrets assessed the efficacy of ondansetron vs. control during the acute phase of emesis induced by 5 and 10 mg.kg -1 cisplatin. As shown in Figure 4 the overall effect of ondansetron was a reduction of the R+V by about 70% (-0.69, -0.82 to -0.55) and this was highly significant (Z=10.05, p<0.0001). A small statistical heterogeneity was detected (χ 2 =37.15, df=23, p=0.03; I 2 =38.1%).

Five variants of the acute model of cisplatin-induced emesis were combined in this meta-analysis: emesis induced by 5 mg.kg -1 cisplatin and R+V quantified for 24h and emesis induced by 10 mg.kg -1 and R+V quantified for 24h, 6h, 4h and 2h.

Ondansetron significantly reduced the R+V in all variants of the model but one; as shown Table 3, the reduction of R+V did not reach statistical significance when emesis was induced by 10 mg.kg -1 cisplatin and quantified for 24h. There was a trend for the effect of ondansetron to increase with shorter observation times (41, 68, 70 and 93% reduction for 24, 6, 4 and 2h observation periods, respectively) but this did not reach statistical significance. The R+V reduction was dose-dependent, doses of 1-10 mg.kg -1 afforded a more effective protection than lower doses of 0.1-0.5 mg.kg -1 (40% and 83%, respectively, Z=2.52, p=0.010). The regimen of ondansetron administration did not change the outcome; the effect of ondansetron was similar with i.v., i.p., and s.c. injections (Z tests, p>0.05) and ferrets treated 30 min prior to cisplatin administration received the same degree of protection as ferrets treated at the time of cisplatin injection (Z=0.04, p=0.965, see Table 3). With longer observation periods, ondansetron three times daily was as effective as twice-daily injections (Z=0.05, p=0.959). Ondansetron had the same efficacy on the R+V induced by an i.p. or an i.v. dose of cisplatin (Z=1.07, p=0.287) and the origin of the animals or the quality score of the studies (see Table 1) did not influence the outcome (Z tests, p>0.05).

Outcome: number of animals with emesis

Data on the number of animals with emesis was extracted from 14 publications; 28 comparisons involving 256 ferrets were identified. The global estimate of the effect of ondansetron was -0.33 (-0.48 to 0.17) indicating that following the administration of cisplatin 5 or 10 mg.kg -1 , ondansetron abolished the emetic response during the observation time in one third of the ferrets (see Figure 5). Overall, this effect was significant (Z=4.09, p<0.001) but a substantial statistical heterogeneity (χ 2 =115.13, df=27, p<0.001, I 2 =76.5%) was detected between comparisons. Subgroup analyses revealed differences in estimates of efficacy between variants of the model. Whereas ondansetron had a significant effect against emesis induced by 10 mg.kg -1 cisplatin and quantified over short observation times (2-4h) it did not show any significant effect against 10 mg.kg -1 cisplatin-induced emesis quantified for 6 and 24h and 5 mg.kg -1 cisplatin-induced emesis quantified for 24h (see Table 4). Efficacy increased as the dose increased, and only doses of ondansetron higher than 0.1 mg.kg -1 had a significant effect but there was significant heterogeneity within those two subgroups (see Table 4). No statistical differences were detected between i.p., i.v. and oral (p.o.) administration of ondansetron (Z tests, P>0.05) but 2 and I 2 tests revealed a high degree of heterogeneity in all 3 groups. Administered s.c., ondansetron was ineffective but this could potentially be misleading as only 2 comparisons were included in that group and both reported the number of animals completely protected for 24h in which setting none of the animals were completely protected (see Table 4 and Figure 5). The regimen used did not influence significantly the number of animals completely protected by ondansetron. There were no differences between the subgroup that received ondansetron at the time of cisplatin injection and the subgroups that received it 30 min and 1h prior to cisplatin (Z-tests, p>0.05) but once again, these results should be taken with caution as the heterogeneity within each subgroup was high. With longer observation times (24h), ondansetron was equally ineffective given 2 or 3 times a day. Ondansetron appeared slightly more effective when cisplatin was injected i.v. compared to i.p. but this did not reach statistical significance (Z=1.757, p=0.079) and could be biased by the fact that in all the comparisons where animals were observed for 24h, cisplatin was injected i.p. Furthermore, heterogeneity was, once again, highly significant in both groups (see Table 4). The origin of the animals and the quality score of the studies did not influence the effect of ondansetron (Z tests, p>0.05), however heterogeneity was high in all subgroups (see Table 4).

Outcome: latency

Latency data was extracted from 10 full papers. Fifteen comparisons involving 131 ferrets assessed the effect of ondansetron on emesis induced by 10 mg.kg -1 cisplatin and 3 comparisons assessed the effect of ondansetron on the latency to the onset of emesis induced by 5 mg.kg -1 cisplatin (see Figure 6). The global estimate of the effect of ondansetron on the latency was 0.86 (0.49-1.24), which means that the latency was nearly twice as long in the groups treated with ondansetron compared to the control groups. This effect was significant (Z=4.45, p<0.001) but χ 2 test revealed a high degree of heterogeneity (χ 2 =314.35, df=14, p<0.001), which was corroborated by the I 2 (96%). Subgroup analysis revealed that ondansetron statistically delayed the latency to the onset of emesis induced by 10 mg.kg -1 cisplatin but not 5 mg.kg -1 cisplatin (Table 5 and Figure 6). The effect of ondansetron was dose-dependant and doses of 1 mg.kg -1 conferred a significantly higher protection, increasing the latency by about 200% (Z tests, p<0.05). Ondansetron was more effective when given i.p. than p.o. or i.v. (Z tests, p<0.05); however, this result needs to be taken with caution as ondansetron was injected i.p. in all the comparisons where the highest dose was given (see Figure 6). No significant differences were observed with different treatment times, there was no difference in outcome if cisplatin was injected i.p. or i.v. and the origin of the ferrets did not influence the outcome (Z tests, p>0.05). The outcome was not influenced by the quality of the study (see Table 1) as no differences were detected between studies scoring less than 5 out of 9, between 5 and 7/9 and 7.5 or higher (Z tests, p>0.05).

Funnel plots for the effect of ondansetron on the R+V and the number of animals with emesis were relatively symmetrical (see Figure 7). The funnel plot for the latency was slightly asymmetrical, which reflects the high degree of heterogeneity detected for this outcome. Overall no associations between treatment effect and sample size were detected, suggesting no evidence of publication bias.

Effect of 5-HT 3 receptor antagonists on the latency to the onset of emesis induced by 10 mg.kg -1 cisplatin.

The effects of 5-HT 3 receptor antagonists on the latency to the onset of 10 mg/kg cisplatin-induced emesis were investigated with 11 different anti-emetics (ondansetron, granisetron, tropisetron, indisetron, dolasetron, L-683,877, renzapride, zacopride, bemesetron, azasetron and ramosetron), this data was extracted from 22 studies; 76 comparisons were reported, involving 587 ferrets. However, for 14 comparisons, because only one animal developed emesis in the treatment group, the point estimate and confidence interval could not be computed and was therefore not included in the calculation of the effect estimate. Altogether, 5-HT 3 receptor antagonists increased the latency by 72% (effect estimate: 0.72, 95% CI: 0.56-0.87); this effect was highly significant (Z=9.08, p<0.0001) but a high degree of heterogeneity ( 2 =1001.57, df=61, p<0.0001, I 2 =93.9%) was detected. Dolasetron (0.02, -0.76-0.80) renzapride (1.11, -0.18-2.40), bemesetron (0.29, -0.17-120) and ramosetron (0.19, -0.02-0.39) did not delay the onset of emesis in a statistically significant manner (Z-tests, P>0.05) whereas ondansetron (0.88, 0.49-1.27), granisetron (0.67, 0.41-0.93), tropisetron (0.49, 0.17-0.81), indisetron (0.92, 0.28-1.57), L-683,877 (0.72, 0.11-1.33), zacopride (0.79, 0.37-1.20) and azasetron (0.52, 0.37-0.67) significantly increased the latency by 49-92% (Z-tests, p<0.05).

Effect of 5-HT 3 receptor antagonists on the acute and delayed phases of emesis induced by 5 mg.kg -1 cisplatin.

Data for the effects of 5-HT 3 receptor antagonists on the daily emetic response induced by 5 mg.kg -1 i.p. cisplatin were extracted from 5 studies; 8 comparisons were identified, involving 72 animals. 5-HT 3 receptor antagonists reduced the number of R+V by 68% (-0.68, -0.91 to -0.45) on day 1, 67% (-0.67, -0.86 to -0.48) on day 2 and 53% (-0.53, -0.85 to -0.38) on day 3; this was statistically significant on each of the 3 days (Z=5.74, Z= 6.81 and Z=4.44 for day 1, 2 and 3 respectively; p<0.0001, see Figure 8). There was no difference between the effect of 5-HT 3 receptor antagonists on each of the 3 days (Z tests, P>0.05); no statistical heterogeneity was detected ( 2 tests, p>0.05 and I 2 =0% on each of the 3 days). The effects of granisetron and ondansetron were significant for each of the 3 days, whereas indisetron did significantly reduce the R+V during day 1 and 3 but not day 2. No significant differences were detected between the effects of ondansetron, granisetron and indisetron for each of the 3 days (Z-tests, p>0.05).

DISCUSSION AND CONCLUSIONS

Cisplatin-induced emesis in the ferret

We found that the latency to the onset of the cisplatin-induced emesis was dosedependent, which is consistent with findings in other species such as humans [START_REF] Kris | Are more antiemetic trials with a placebo necessary? Report of patient data from randomized trials of placebo antiemetics with cisplatin[END_REF], dogs [START_REF] Bhandari | Emetic profile of cisplatin in dogs[END_REF] and pigeons [START_REF] Preziosi | The effects of 5-HT3 receptor antagonists on cisplatin-induced emesis in the pigeon[END_REF]. A step-change was observed between 5 and 6 mg.kg -1 , suggesting a difference in the mechanisms triggering emesis at low and high dose, possibly the activation of an additional mechanism (e.g. recruitment of less sensitive vagal afferent branches, area postrema). Prior anaesthesia and the route of administration of cisplatin were identified as confounding factors; the emetic response was also modulated by some of the vehicles used and factors inherent to the ferrets such as strain and origin. The difference between intravenous and intraperitoneal cisplatin may only reflect prior anaesthesia, as these two factors were dependent in the present study. Certainly, anaesthesia had an impact in its own right as differences were detected between injectable and volatile anaesthetics. The impact of the ferrets' origin could reflect a genuine difference between populations of ferrets but it might also indicate differences between laboratories rather than a difference between animals; this cannot be determined from the present study. These findings however stress the relevance of choosing appropriate controls (e.g. vehicle control, sham-operated) and homogeneous groups of animals when using the ferret cisplatin model of emesis.

The profile of emesis induced by 5 mg.kg -1 i.p. cisplatin in the ferret was clearly biphasic, which is consistent with the profile of emesis observed in the clinic [START_REF] Martin | The severity and pattern of emesis following different cytotoxic agents[END_REF] but differences were observed in the timing and magnitude of the two phases. Whereas the acute phase is more severe than the delayed phase in humans [START_REF] Kris | Incidence, course, and severity of delayed nausea and vomiting following the administration of high-dose cisplatin[END_REF][START_REF] Martin | The severity and pattern of emesis following different cytotoxic agents[END_REF], with an onset 1-6h following cisplatin infusion [START_REF] Harris | Mechanisms and Treatment of Cytotoxic-Induced nausea and Vomiting[END_REF][START_REF] Kris | Are more antiemetic trials with a placebo necessary? Report of patient data from randomized trials of placebo antiemetics with cisplatin[END_REF][START_REF] Martin | The severity and pattern of emesis following different cytotoxic agents[END_REF], the latency to the onset of the acute phase was greater in the ferret (>10h) and its relative intensity compared to the delayed phase was lower. The incidence of emesis on each day could not be investigated in the ferret as all reports stated that 100% of the animals developed an emetic response but no distinction was made between the acute and delayed phases. A few studies have however suggested that the acute phase was not observed in all animals [START_REF] Rudd | The action of the NK1 tachykinin receptor antagonist, CP 99,994, in antagonizing the acute and delayed emesis induced by cisplatin in the ferret[END_REF][START_REF] Watanabe | Long-lasting anti-emetic effect of T-2328, a novel NK(1) antagonist[END_REF], which is also consistent with our recent observations [START_REF] Du Sert | The delayed phase of cisplatin-induced emesis is mediated by the area postrema and not the abdominal visceral innervation in the ferret[END_REF] and suggests that whereas the incidence of emesis during the delayed phase is close to 100% the incidence of acute emesis at this dose is lower.

In humans treated with placebo anti-emetics, however, the incidence of emesis during the acute phase [98%, 71] is higher than during the delayed phase (44-89% [START_REF] Gandara | Delayed emesis following high-dose cisplatin: a double-blind randomised comparative trial of ondansetron (GR 38032F) versus placebo[END_REF][START_REF] Kris | Incidence, course, and severity of delayed nausea and vomiting following the administration of high-dose cisplatin[END_REF]).

Effect of 5-HT 3 receptor antagonists

The efficacy of ondansetron in the acute model of cisplatin-induced emesis was assessed measuring 3 outcomes: the number of R+V, the number of animals with emesis and the latency to the onset of emesis. Overall, all 3 outcomes permitted the detection of significant anti-emetic protection, which is consistent with findings in humans [START_REF] Cubeddu | Efficacy of ondansetron (GR 38032F) and the role of serotonin in cisplatin-induced nausea and vomiting[END_REF], but different variants of the model resulted in different levels of antiemetic protection. Whereas, all variants of the acute model reflected a similar reduction of R+V, ondansetron only delayed significantly the onset of emesis following 10 but not 5 mg.kg -1 cisplatin, and the number of animals with emesis was only reduced with observation periods no longer than 4h. In the 10 mg.kg -1 4h variant of the model, half of the animals were completely protected from emesis, which was comparable to percentage of patients free of emesis during the acute phase of high dose (>50 mg.m -2 ) cisplatin-induced emesis in human patients [START_REF] Hesketh | GR 38032F (GR-C507/75): a novel compound effective in the prevention of acute cisplatin-induced emesis[END_REF][START_REF] Jantunen | An overview of randomised studies comparing 5-HT3 receptor antagonists to conventional anti-emetics in the prophylaxis of acute chemotherapy-induced vomiting[END_REF][START_REF] Marty | Is Navoban (tropisetron) as effective as Zofran (ondansetron) in cisplatin-induced emesis? The French Navoban Study Group[END_REF][START_REF] Ruff | Ondansetron compared with granisetron in the prophylaxis of cisplatin-induced acute emesis: a multicentre double-blind, randomised, parallel-group study. The Ondansetron and Granisetron Emesis Study Group[END_REF].

Overall the acute phase of emesis induced by 5 mg.kg -1 cisplatin represented poorly the clinical situation and the 4h-10 mg.kg -1 model was more predictive of cisplatininduced emesis in humans.

Overall, 5-HT 3 receptor antagonists reduced the emetic response to the same extent during the acute and delayed phases, which contrasts with findings reported in the majority of clinical studies, describing limited or non significant effect of 5-HT 3 receptor antagonists during the delayed phase [START_REF] Hesketh | Management of Nausea and Vomiting in Cancer and Cancer Treatment[END_REF][START_REF] Kubota | Effectiveness of anti-emetics for the prophylaxis of cisplatin-induced delayed emesis: a systematic review[END_REF]. This discrepancy may be explained by a difference in outcome usually measured in humans (daily incidence, percentage of patients developing emesis) and ferrets (severity, number of R+V).

Even though daily incidence and severity (measured by visual analogue scale) of the delayed phase emesis appear to be positively correlated in the absence of antiemetic therapy [START_REF] Kris | Incidence, course, and severity of delayed nausea and vomiting following the administration of high-dose cisplatin[END_REF], they can be uncoupled following anti-emetic treatment and the number of emetic episodes may be reduced while the incidence remains unchanged [START_REF] Navari | Oral ondansetron for the control of cisplatin-induced delayed emesis: a large, multicenter, double-blind, randomized comparative trial of ondansetron versus placebo[END_REF]. Alternatively, it is conceivable that the delayed phase of emesis in the ferret is more sensitive to 5-HT 3 receptor antagonists than it is in humans. The latter would be consistent with a longer acute phase in the ferret [see 110 for details], implying that the mechanism regulating the acute phase (i.e. 5-HT-mediated activation of the abdominal vagal afferents) remains activated longer. The ferret model thus correctly identified the anti-emetic potential of 5-HT 3 receptor antagonists against both the acute and delayed phases of cisplatin-induced emesis but the magnitude of the antiemetic effect during the delayed phase appears greater in the ferret than it is in humans.

Methodology

In the present study, the methodology used in the meta-analysis of human clinical trials was modified and adapted to animal research, and several concessions had to be made. First of all, the criteria used to select studies for inclusion into the systematic review and meta-analysis, and assess quality, did not include randomization and blinding. Whereas these two parameters are considered essential for human clinical trials and it has been suggested that their absence favours positive findings in animal research [START_REF] Bebarta | Emergency medicine animal research: does use of randomization and blinding affect the results?[END_REF], emesis is an objective measurement, which is not investigator-dependent and we have no reason to believe that the inclusion of such studies in our analysis biased our findings. Additionally, randomization and blinding in the identified studies were too rarely reported to be used as inclusion criteria. The exclusion criteria were chosen to ensure the collection of reliable, clearly defined data. The majority of excluded studies (see Table 2) were removed because emesis was not quantified as latency, retches, vomits and/or incidence. We chose not to include outcomes such as "emetic episodes" or "bouts of emesis" in the present analysis because of the disparity of definitions and possible interpretations. This may however restrict the conclusions of our study.

Secondly, whereas clinical trials usually reports tens or hundreds of patients in each study arm, we found that in the cisplatin ferret model, typically 4 to 8 animals were allocated in each treatment group. This may limit the relevance of such an analysis, designed to compare much bigger samples. Additionally, because a majority of studies compared one control group to several treatment groups-typically different doses of a compound-in order to maintain the data from different doses as distinct comparisons, the number of animals in the control group was divided by the number of treatment groups it was compared to. The limitation of such an approach is that in the effect estimate calculation, the weight of such comparisons is reduced, which benefits comparisons extracted from studies that only compared one control group to one treatment group and is not justified by the quality of the studies.

Conclusions

We demonstrated the potential of a meta-analysis to address the 3R's (Replacement, Refinement and Reduction), developed by Russel and Burch as criteria for a humane use of animals in research [START_REF] Russell | The Principles of Humane Experimental Technique[END_REF]. By maximising the utilisation of animal data, thus extracting novel scientific information without increasing the number of animals, such analysis addresses Reduction, as this reduces the future use of animals. Additionally, the effects of ondansetron on the 3 outcomes highlighted a logical Refinement of the model by reducing the observation period. The cisplatin 10 mg/kg-4h variant of the model stands out as the most appropriate to study the acute phase of cisplatin-induced emesis, whereas 5 mg/kg-24 to 72h remains the model of choice to study cisplatin-induced delayed emesis.

The present study is a proof of concept. In an attempt to focus the scope of the analysis, it was limited to an animal model with one emetic: cisplatin, one species: the ferret and one class of anti-emetic drugs: 5-HT 3 receptor antagonists; the drugs investigated are already successfully used in humans against chemotherapy-induced emesis. Globally, the effects in the ferret were consistent with clinical findings, which was expected as they were originally developed in the ferret model; this demonstrates that a meta-analysis is an appropriate method to identify the antiemetic potential of a drug and confirms that the ferret model of cisplatin-induced emesis is truly predictive and relevant to humans. This method can now be applied to investigate the effects of NK 1 receptor antagonists, which were also developed using the ferret and which have recently been introduced into the clinic. However, recent studies suggest that there may be a discrepancy between the "broad spectrum" antiemetic efficacy in the ferret and the efficacy in the clinic [START_REF] Andrews | The role of tachykinins and the tachykinin NK1 receptor in nausea and emesis[END_REF][START_REF] Dando | Aprepitant: a review of its use in the prevention of chemotherapy-induced nausea and vomiting[END_REF]. This method can also be used in other "model" species (e.g. dog) to reassess "older" compounds such as dopamine receptor antagonists or opioids, whose effects on cisplatin-induced emesis are less well characterized. It can also be directly applied to other emetic models (different emetogen and/or different species) and adapted to assess the relevance of models arguably predictive of nausea and emesis such as pica in the rat and conditioned taste aversion [START_REF] Andrews | Signals for nausea and emesis: Implications for models of upper gastrointestinal diseases[END_REF][START_REF] Liu | Ghrelin alleviates cancer chemotherapy-associated dyspepsia in rodents[END_REF]. Reason for exclusion is indicated as: duplicate: data published elsewhere; group number: number of animals in the group missing; emesis:

emesis not quantified as number of animals free of emesis, number of retches, number of vomits or latency, or emesis not defined; anaesthesia: emetic response investigated in anaesthetized animals.

Figure 1 .

 1 Figure 1. Flow chart of identified studies. Reproduced and adapted from the

Figure 2 .

 2 Figure 2. Latency to the onset of emesis (first retch, vomit or vomiting episode)

Figure 3 .

 3 Figure 3. Profile of emesis induced by 10 mg.kg -1 (A) and 5 mg.kg -1 (B) cisplatin in the ferret. A: data plotted as mean vomits ± SD per 30 min periods, collected from

Figure 4 .Figure 5 .

 45 Figure 4. Efficacy of ondansetron number of retches + vomits during the acute phase of emesis induced by cisplatin 5 or 10 mg.kg -1 . Point estimates and 95%

Figure 6 :

 6 Figure 6: Efficacy of ondansetron on the latency to emesis induced by cisplatin

Figure 7 :

 7 Figure 7: Funnel plots for the effect of ondansetron on the number of retches + vomits (A), number of animals with emesis (B) and latency (C). For each

Figure 8 :

 8 Figure 8: Efficacy of 5-HT 3 receptor antagonists on the daily number of retches + vomits (R+V) induced by 5 mg.kg -1 i.p. cisplatin during the acute (day 1) and

Table 1 .

 1 List of 68 included publications.

	Study	Quality score	Cisplatin dose	Obs. period	Study	Quality score	Cisplatin dose	Obs. period

Table 2 .

 2 List of 47 excluded publications.

	Study	Reason for exclusion	Study	Reason for exclusion	Study	Reason for exclusion
	Blum et al., 1992 [13]	emesis	Gooch et al. 1998 [48]	anaesthesia	Rudd et al., 2001 [116]	group number
	Bountra et al., 1993 [15]	emesis	Haga et al., 2000 [51]	emesis	Rudd et al., 2006b [123]	duplicate
	Chevalier et al., 1998 [16]	emesis	Hale et al., 1998 [53]	duplicate	Rupniak et al., 1997 [125]	emesis
	Clark et al., 1993 [17]	duplicate	Hale et al., 2000 [52]	duplicate	Shiroshita et al., 1992 [133]	emesis
	Costall et al., 1987a [18]	duplicate	Hargreaves et al., 1994 [54]	duplicate	Stables et al., 1987 [136]	duplicate
	Eglen et al., 1993 [31]	emesis	Hollingworth et al., 2006 [60] group number	Tattersall et al., 1990 [138]	duplicate
	Eglen et al., 1994 [29]	emesis	Ito et al., 1990 [64]	emesis	Tattersall et al., 1996 [141]	group number
	Eglen et al., 1995 [30]	emesis	Kim et al., 2005 [69]	emesis	Twissell et al., 1993 [145]	duplicate
	Endo et al., 1990a [32]	duplicate	Lasheras et al., 1996 [74]	emesis	Van Sickle et al., 2003 [146]	emesis
	Endo et al., 1992 [34]	group number	Matsui et al., 1992 [86]	emesis	Yoshida et al., 1991 [154]	emesis
	Endo et al., 1995 [35]	emesis	Minami et al., 1991 [92]	duplicate	Yoshida et al., 1992b [152]	emesis
	Fitzpatrick et al., 1990 [39]	emesis	Miner et al. 1986b [95]	duplicate	Yoshikawa et al., 1996 [155]	emesis
	Florczyk, et al., 1981 [41]	duplicate	Monkovic et al., 1988 [98]	emesis	Yoshikawa et al., 2001a [156]	emesis
	Gardner et al. 1994 [45]	emesis	Ohta et al., 1996 [102]	emesis	Youssefyeh et al., 1992a [159]	duplicate
	Gardner et al. 1995 [46]	emesis	Price et al., 1990 [107]	emesis	Youssefyeh et al., 1992b [158]	duplicate
	Gardner et al. 1996 [44]	emesis	Rudd et al., 1998a [121]	emesis

cisplatin administration), the animal origin (country animals were bred) and the quality score assigned to the study where comparisons were extracted.

ACKNOWLEDGEMENTS

The authors would like to thank Carol Kilkenny (NC3Rs/MRC), Fiona Reid (SGUL) and Toby Lasserson (SGUL) and for statistical advice, and Dr John Watson (Pfizer, USA), Dr Masahiko Hirafuji (University of Hokkaido, Japan) and Dr Hisashi Yamakuni (Astellas Pharma Inc., Japan), who provided original data. NPdS was supported by a PhD studentship from Merck Research Laboratories.

Table 3. Sensitivity analyses of the effect of ondansetron on the number of retches + vomits (R+V) induced by cisplatin (5 or 10 mg.kg -1 ). The effect estimate was computed as the weighted mean difference (WMD) and expressed as the proportion of retches and vomits in the control group. An effect estimate of -1 indicates that emesis was abolished in the treatment group, 0 indicates that the treatment had no effect on the R+V response and an effect estimate >0

indicates that the treatment increased the number of R+V. The variables examined were the variant of the cisplatin model (5 or 10 mg.kg-1 cisplatin and the duration of the observation period), the mode of administration of cisplatin, the dose of ondansetron, the regimen of ondansetron administration (mode of delivery and timing relative to cisplatin administration), the animal origin (country animals were bred) and the quality score assigned to the study where comparisons were extracted. The effect estimate was computed as the risk difference (RD) and represents the proportion of animals with emesis during the duration of the observation period. An effect estimate of 0 indicates that the treatment had no effect on the number of animals with emesis, -1 indicates maximal effect. The variables examined were the variant of the cisplatin model (5 or 10 mg.kg -1 cisplatin and the duration of the observation period), the mode of administration of cisplatin, the dose of ondansetron, the regimen of ondansetron administration (mode of delivery and timing relative to Table 5. Sensitivity analyses of the effect of ondansetron on the latency to the onset of emesis induced by cisplatin (5 or 10 mg.kg -1 ). The effect estimate was computed as the weighted mean difference (WMD) and represents the impact of the treatment on the latency expressed as a proportion of the latency in the control group. An effect estimate <0 indicates that the latency was shorter in the control group than in the treatment group, 0 indicates that the treatment had no effect on the latency and an effect estimate of 1 indicates that the treatment increased the latency by 100%. The variables examined were the dose and mode of administration of cisplatin, the dose of ondansetron, the regimen of ondansetron administration (mode of delivery and timing relative to cisplatin administration), the animal origin (country animals were bred) and the quality score assigned to the study where comparisons were extracted.


B Gamper

Z Uddin

M Jahangir

O Allard

H Knöckel

E Tiemann

L Windholz
email: windholz@tugraz.at

Investigation of the hyperfine structure of Pr I and Pr II lines based on highly resolved Fourier transform spectra

We report the recording of new highly resolved Fourier transform spectra of the neutral praseodymium atom. With the help of the new spectra we found about 9000 new lines, from which -in the region 3260 to 9880 Å -1194 could be classified as transitions between energy levels of the Pr atom and 19 as transitions of the Pr ion. 23 new atomic energy levels of odd parity and one of even parity were discovered during this first examination. The spectra might be very helpful for further laser spectroscopic investigations of the hyperfine structure of Pr I and Pr II transitions.

Introduction

Praseodymium is a metal and belongs to the group of lanthanides. The 141 Pr atom has a non zero nuclear spin I = 5/2 due to the odd number of protons (Z=59). Like other lanthanides also Pr has a very rich and complex optical spectrum; it has a few ten thousands lines from which a big part is not classified. The praseodymium nucleus is almost spherically symmetric, thus it has a very small electric quadrupole moment and the hyperfine constants B are very small. The first studies of the hyperfine (hf) structure of spectral lines of Pr were done by King in 1928 [1]. The next extensive continuation of this work was done by Ginibre in 1988; she presented many new energy levels [2], [3] and [4] and listed approximately 17000 spectral lines. Nevertheless, her list is far from being complete. The Poznań group around Dembczyński performed theoretical interpretation of the hyperfine structure of Pr and could solve some of the configuration mixing problems [5]. Furmann, also part of the group in Poznań, investigated the hf structure of Pr via the method of laser-induced fluorescence (LIF) spectroscopy since 2005 [6] and [7] and he could enlarge the level list of Pr I and Pr II. Studies of the hf structure of neutral praseodymium have been performed by the group of Guthöhrlein in Hamburg [START_REF] Guthöhrlein | Universität der Bundeswehr[END_REF] and by our group since 2004 [START_REF] Uddin | [END_REF][START_REF] Gamper | [END_REF]. Even though the enormous work of Ginibre was based on an evaluation of Fourier transform (FT) spectra, the primary spectral data are not available to us. Nevertheless, the line list of Ginibre, containing approximately 17000 lines, was the basis of all laser spectroscopic work until 2007. In these laser spectroscopic experiments, lines which are given by Ginibre but could not be classified by her were excited by laser light, setting the laser wavelength close to the listed wavelength of the center of gravity (cg), and searching for LIF signals.

However, if someone wants to perform LIF spectroscopy more efficiently, a precise setting of the excitation laser frequency is desired. Thus, we decided to take new FT spectra, having a high resolution in order to resolve at least the widely splitted hf patterns. The goal of our investigation is to classify as many lines as possible and to find up to now unknown energy levels in order to increase the knowledge of the stationary states of the electron shells as a basis for a better theoretical description of these states.

Experimental details

FT spectroscopy has a lot of advantages over other spectroscopic techniques: one can record many thousands lines by one scan under Doppler-limited conditions. With a FT spectrometer it is possible to measure very weak signals and to cover a large wave number range per scan. It has high resolution, large dynamic range of the intensity and a relatively low scan time.

The FT spectra were recorded at the Institute of Quantum Optics at the Leibniz University in Hannover with a continuously scanning high resolution FT-IR spectrometer (IFS 120 HR, Bruker Corp.). An example of a sequence of hf structure patterns recorded is shown in figure 1. As a light source we used a home made hollow cathode lamp which was the same as we use in Graz for performing laser-induced fluorescence spectroscopy. In the hollow cathode lamp (detailed scheme of the apparatus see [START_REF] Jaritz | [END_REF]) a cloud of free praseodymium atoms is produced by cathode sputtering. The copper cathode is lined with praseodymium and has a typical inner diameter of 3 mm and a length of 19 mm. The anodes on both sides of the cathode consist of aluminum and are spaced with a ceramic insulator. The discharge was operated with a current of about 65 mA (dc) using argon as carrier gas at a pressure of about 0.6 mbar. The lamp was cooled by liquid nitrogen to reduce the Doppler width for better hf resolution. The advantage of a hollow cathode lamp is that it produces praseodymium atoms not only in the ground state but also in higher excited states. The emission spectrum of the Pr-Ar plasma under the applied conditions contains mainly Pr I lines, but also Pr II and Ar I and Ar II lines.

The light emitted by the discharge was collected and focused with a set of lenses on the entrance aperture of the FT spectrometer in order to get a Fourier transformed emission spectrum over the very large region from 2380 to 12500 Å. As beam splitters we employed the commercial ones provided by the manufacturer: for the near infrared region between 12500 Å and 10000 Å CaF 2 coated with silicon, for the visible range from 10000 Å to 4000 Å quartz with dielectric coating and for the UV quartz with Al coating. The resolution of the spectra is given by the convolution of the instrumental profile and the width of the spectral lines. The entrance aperture was varied from 0.5 to 1.3 mm diameter. To optimize the signal-to-noise ratio (SNR) the spectra were recorded in parts limiting the recorded ranges by optical dielectric low pass and high pass filters. For the near infrared range a silicon photodiode was used as a detector, while for the visible and UV ranges we employed a photomultiplier. Multiple scans were averaged at a typical resolution of 0.03 cm -1 for an improved SNR.

The original data files of the FT spectrometer contained intensity versus wave number. The specification of the instrument is given to be better than 5.10 -8 relative and 5.10 -7 absolute. This is 0.025 cm -1 at 50000 cm -1 or 0.001 Å at 2000 Å; 0.0125 cm -1 at 25000 cm -1 or 0.002 Å at 4000 Å; 0.005 cm -1 at 10000 cm -1 or 0.005 Å at 10000 Å. Spectra from different regions from IR to UV were combined to form one spectrum. Their correct overlapping was checked by comparing the hf structures of the recorded spectral lines. Then the spectrum was converted to intensity versus air wavelength using the dispersion formula given by Peck and Reader [12]. This spectrum can be handled in graphic form by the existing classification program [13].

Results and discussion

The amount of spectral lines in Pr is enormous. An example of the FT spectrum in the visible region is given in Figure 1, which shows only a very narrow part of 2.5 Å of the whole FT spectrum. In this region there are noticeable at least 12 spectral lines with partly resolved hf structure, which are marked by brackets. Meaning of the designations: a: line already classified in ref. [4]; b: mentioned in ref. [4], but not classified; c: investigated by laser excitation and classified in ref. [START_REF] Uddin | [END_REF]; d: new lines, discovered with help of this spectrum. Around 5675.75 Å a typical blend situation can be found: Two of the lines appear to be overlapping. Sometimes even more than two lines with practically the same cg wavelength can be found. This is due to the very big number of electronic energy levels of the Pr atom: Transition frequencies between certain different pairs of levels are nearly or exactly degenerate. For such blend situations the assignment of the correct level pair (this assignment we call "classification") is not possible using only the wave number difference of the levels in comparison with the cg wave number from the FT hf pattern. In addition, the shape of the hf pattern has to be considered. For a huge number of lines the classification is possible since the hf pattern predicted from already known hf constants of the involved levels coincides with the hf pattern observed in the FT spectrum. Of course, this procedure provides that the hf constants of the combining levels are already known from earlier measurements.

In other cases hf constants can be determined using the FT pattern, if the transition is known but the hf constants not. The correctness of the hf constants can be proven by comparing the hf patterns of other lines combining with the level under investigation in the FT spectrum. For this purpose it is necessary to extend the FT spectrum over a range as wide as possible.

If the situation in the observed spectrum is very complex, so that the observed spectral structures can not be clearly distinguished, laser spectroscopic experiments are necessary to divide the excited levels by their characteristic fluorescence lines. In this case the FT spectrum can be used conveniently to set the desired laser excitation wavelength.

All components of the hf patterns of the spectral lines appearing in Figure 1 are also possible excitation wavelengths for laser-induced fluorescence (LIF) spectroscopy. The excitation wavelengths can be determined from FT spectra with an uncertainty of few 0.001 Å.

Originally, our database contained approximately 17000 lines of Pr I and Pr II. Up to now we could enlarge this list to about 26000 lines. Many of the 26000 lines are not yet classified, meaning that not every line listed in Ginibres tables [4] or discovered in the present FT spectrum is related to a transition between two already known energy levels. It should be emphasized that our additions to the line list of Ginibre (ref. [4]) are not done automatically by a computer program, but by comparing structures in the graphically depicted FT spectrum with predicted (and graphically represented) hf patterns belonging to transitions which fit in their wave number to the considered part of the FT spectrum. This comparison is possible since in the FT spectrum the relative intensities of the components of an hf pattern fit quite good to the theoretical intensity ratios.

In table 1 all new spectral lines of the praseodymium atom (Pr I) which clearly could be assigned to a transition between two energy levels, are given. This table, containing more than 1200 lines, is available in its full length only in electronic form. In table 2 the Pr I lines of table 1 with an SNR larger than 50, are listed again. Table 3 gives all the newly classified ionic lines (Pr II), respectively. The classification was performed using the cg wave numbers and the shape of the hf patterns, resolved well enough to identify the combining levels without any doubts.

All three tables have the same structure: Column 1 contains the cg wavelength in air in Å. Information about the SNR is given in column 2 (per definition, noise is treated to have SNR = 1. The SNR is a guide to the relative intensities of the lines, since the intensities depend strongly on the type of light source and the conditions under which a discharge is operated). If there is written "nl" in front of the SNR it means that this line is a new one, not known before these investigations. All the other lines are mentioned in the list of Ginibre [4] but they are not classified, either because the energy levels involved in the transition were found after she did her work or because the hf patterns were not sufficiently resolved in her spectra. In columns 3 and 5 the J-values of the involved levels are listed. The corresponding energies in cm -1 are given in columns 4 and 6. The energy levels are listed according to their parities, in the column 4 the even ones and in the column 6 the odd ones. If a new level is involved, it is marked with an upper index 'a'.

The cg wavelength for each line given in the tables is obtained by overlapping two graphs: the hf structure obtained from the FT spectrum and a synthetical hf pattern, calculated from the known hf constants of the combining levels. The intensities are normalized for this comparison, and the Gaussian width of the synthetical spectrum has to be chosen close to the width obtained in the FT spectrum. Of course this width is wavelength-dependent. One graph can be shifted against the other in steps of 0.001 Å. The correct position gives then the cg wavelength. Since human brain is quite sensitive to a mismatch of two graphs, it is really possible to notice a mismatch of 0.001 Å over the whole wavelength range (3260 to 9880 Å). Thus, the given wavelengths are good to 0.001 Å for a wide range of SNR. For lines with SNR < 10 the uncertainty is estimated to be 0.002 Å. The absolute wavelength accuracy could be checked in the range 5145 to 3729 Å against a list of Ar-lines used in FT spectrometry for wavelength calibration (see ref. [14]). Agreement was better than 0.001 Å. In the UV and IR region Ar-lines of the present spectrum were compared with Ar-lines of earlier used FT spectra of Ta and Ar, taken at Imperial College London and Kitt Peak National Observatory ( [15]). Again the agreement was better than 0.003 Å. Thus the absolute accuracy of the given wavelengths in our tables is better than 0.003 Å.

Pr I energy levels given in Tables 1,2 (and their hf constants, used for determination of the cg wavelengths) are taken either from the tables of Ginibre [4] or from measurements performed by the group in Hamburg [START_REF] Guthöhrlein | Universität der Bundeswehr[END_REF] (private communication). The wave numbers have been fine adapted by means of the present FT spectrum. Under assumption that the energy of the lowest even state, 4432.24 cm -1 , as given in ref. [START_REF] Zalubas | Atomic Energy Levels -The Rare Earth Elements[END_REF], is correct, we have first energy corrected high lying odd levels, combining with this lower level. Then we have used transitions from these levels to the second lower even energy state to correct its wave number. From this level we were going up to other odd levels and so on. The same procedure was done beginning with the odd ground state level.

The differences of the here given level energies fit to the wave numbers, calculated from the given wavelengths, better than 0.01 cm -1 . Energy values given in Table 3 are from Ginibre [3].

For some well resolved lines, which could not be classified as transitions between known levels, it was possible to determine the hf constants and to identify one of the involved levels by its hf constants. Then a new level could be introduced combining the wave number of the identified level and the wave number of the transition. The proof that such a level exists is done by calculating all possible decay wavelengths and looking into the corresponding regions of the FT spectrum for the predicted pattern. If observed structures fit with the expected pattern and the expected cg wavelength, the level is assumed to be really existing. This method of finding new levels is especially important for spectral regions where laser excitation is difficult, e.g. in the IR or UV.

In this way, we could find several new energy levels. The hf constants were determined by fitting the hf pattern of the line under investigation, using the program "Fitter" ( [START_REF]Program package "Fitter[END_REF]). These levels are marked in table 1 and table 2 with subscript "a". The results are shown in table 4. Column 1 of this table gives the energies in cm -1 of the newly discovered levels; the accuracy depends mainly on the SNR of the investigated hf patterns. Their J-values are listed in column 2; in the next column the hyperfine constants A (in MHz) are given. Due to the limited resolution of the FT spectrum the value of B was assumed to be zero. The 5 th column tells us which lines (wavelengths in air in Å) can be explained with the new levels. The corresponding transitions can be found in table 1.

As an example we discuss the line with the cg wavelength 9319.006 Å. The relative intensities and distribution of the components (Figure 2) suggest a transition with ∆J = -1 with almost equal values of the hyperfine constants A of both levels. The small sharp peak at the high frequency side of this hf structure pattern is generated under this hypothesis by the coincidence of the five off diagonal (∆F = 0) hyperfine components within the Doppler width. We fitted the structure assuming J = 17/2 and J = 15/2 (the prime marks the upper level of the transition), and got A = 970 MHz and A = 961 MHz. Then we looked in our level data base for a level with J and A as determined in the fitting process. We found an even level with the energy 11714.367 cm -1 , with A = 970.2(3) MHz, B = -16(4) MHz and J = 17/2. Assuming that this level is the lower level involved in this transition, we fitted the structure again with fixed hyperfine constants of the lower level and added the cg wave number of the investigated line to the wave number of the lower level. Finally we determined the new level to have 22442.185(5) cm -1 , J = 15/2, A = 959(9) MHz with odd parity. Fig. 3 shows the hf structure calculated with J, A and J', A' in comparison with the pattern in the FT spectrum. The new level explains 5 other lines in the IR region and 3 lines in the visible region (see Table 4).

We have to emphasize that such a fit is only possible if some of the small components (or a convolution of them) are clearly resolved in the FT spectra. Otherwise, the splitting of the diagonal components is more sensitive to the difference (A -A ) than to the absolute values of A, A , and one cannot identify one of the involved levels by its Avalue.

Altogether 24 new levels could be found by the analysis of FT patterns (mainly located in the IR region); each level explains other previously unclassified lines. The value of the hyperfine constant B could not be determined with significance therefore it is not taken into account in this work. B was set to zero when determining A. 2 The line was already classified in reference [4], but this classification is wrong.

Conclusion

In this work, we have concentrated on investigations of the hyperfine structure of the Pr atom and ion based on new FT spectra. With the help of these FT spectra we could enlarge the wavelength list of Pr from around 17000 up to 26000 lines. Furthermore we assigned more than 1200 lines to a transition between two known energy levels. By fitting several lines in the IR region we were also able to determine 24 new atomic levels. Even though this number is small compared to the approximately 2000 known energy levels, it shows the potential of FT spectra in classifying lines and finding new energy levels. The new FT spectra of Pr I and Pr II will be very helpful for further laser induced fluorescence spectroscopy experiments because now one can determine exactly possible excitation wavelengths for an investigation of promising structures. The full list of all more than 26000 lines is available on request. . . line given in the tables of Ginibre (ref. [4]), but not classified c . . . line classified before having the FT spectrum by laser spectroscopy (ref. [START_REF] Uddin | [END_REF]) d . . . new line, discovered with help of this spectrum Around 5675.75 Å a typical blend situation can be noticed: a spectral line with a wide hf splitting (d 1 ) is overlapped by another line with a small hf splitting (b 1 ). The most intense component of the widely splitted line was noticed by Ginibre (ref. [4] and treated to be a spectral line with small hf splitting (b 2 ).

Figure 1 .

 1 Figure 1. Part of the FT spectrum in the visible region. a . . . line classified by Ginibre (ref. [4]) b .. . line given in the tables of Ginibre (ref.[4]), but not classified c . . . line classified before having the FT spectrum by laser spectroscopy (ref.[START_REF] Uddin | [END_REF]) d . . . new line, discovered with help of this spectrum Around 5675.75 Å a typical blend situation can be noticed: a spectral line with a wide hf splitting (d 1 ) is overlapped by another line with a small hf splitting (b 1 ). The most intense component of the widely splitted line was noticed by Ginibre (ref.[4] and treated to be a spectral line with small hf splitting (b 2 ).

Figure 2 . 2 AFigure 3 .

 223 Figure 2. Appearance of the line 9319.006 Å in the FT spectrum

Table 1 :

 1

		Spectral lines of Pr I which clearly could be assigned to a
		transition between two energy levels
				Even		Odd
	λ/ Å	S N R	J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1
	3264.301 nl 15	9/2	30625.599	9/2	0
	3308.390 nl 4	11/2	30217.535	9/2	0
	3363.938 nl 53	11/2	29718.524	9/2	0
	3437.597 nl 36	13/2	30458.359	11/2	1376.605
	3482.081 nl 7	11/2	30086.879	11/2	1376.605
	3500.325 nl 20	11/2	28560.604	9/2	0
	3518.513 nl 16	7/2	28412.960	9/2	0
	3551.415 nl 9	11/2	28149.750	9/2	0
	3617.638 nl 13	11/2	27634.464	9/2	0
	3650.240 nl 7	11/2	28764.269	11/2	1376.605
	3654.616 nl 44	9/2	4432.240	11/2	31787.102
	3670.398 nl 7	9/2	8029.290	11/2	35266.525
	3676.089 nl 6	11/2	27195.110	9/2	0
	3681.669 nl 26	11/2	27153.870	9/2	0
	3693.608 nl 15	11/2	27066.095	9/2	0
	3728.799 nl 5	9/2	9105.035	11/2	35915.701
	3732.473 nl 48	11/2	26784.270	9/2	0
	3734.024 nl 24	11/2	28149.750	11/2	1376.605
	3738.704 nl 7	11/2	6313.239	13/2	33052.863
	3744.240 nl 8	15/2	9745.420	17/2	36445.505
	3751.945 nl 4	9/2	4432.240	11/2	31077.525
	3764.900 nl 8	9/2	4432.240	9/2	30985.825
	3782.853 nl 8	9/2	4432.240	11/2	30859.807
	3785.058 nl 16	11/2	4866.530	13/2	31278.701
	3789.689 nl 17	7/2	6535.587	9/2	32915.484
	3789.738 nl 10	13/2	6603.606	15/2	32983.162
	3792.514 14	11/2	4866.530	13/2	31226.830
	3792.953 nl 10	11/2	26357.240	9/2	0
	3796.162 nl 8	11/2	4866.530	13/2	31201.444
	3822.768 nl 13	9/2	30583.869	11/2	4432.240
	3832.285 nl 14	11/2	4866.530	13/2	30953.221
	3836.285 nl 12	15/2	28906.235	13/2	2846.744
	3844.099 nl 11	11/2	6313.239	13/2	32319.759
	3845.830 nl 9	15/2	8363.910	17/2	34358.775
	3850.364 nl 23	9/2	5822.905	11/2	31787.102
	3895.616 nl 20	9/2	4432.240	11/2	30094.849
	3920.839 nl 5	9/2	4432.240	9/2	29929.763
	3955.220 nl 12	11/2	4866.530	13/2	30142.420
	3966.271 nl 19	9/2	25205.463	9/2	0
	nl ... new line, a previously unknown energy level

J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1 J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1 J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1

Table 2 :

 2 Spectral lines of Pr I with an intensity larger than 50 which clearly could be assigned to a transition between two energy levels nl ... new line, a previously unknown energy level

				Even		Odd
	λ/ Å	S N R	J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1
	3363.938 nl 53	11/2	29718.524	9/2	0
	4216.768 nl 53	11/2	4866.530	13/2	28574.696
	4519.626 nl 112	15/2	8363.916	15/2	30483.514
	4598.951 nl 178	11/2	24584.745	13/2	2846.744
	4692.652 55	11/2	4866.530	9/2	26170.480
	4795.241 50	7/2	30427.864	9/2	9579.832
	4802.883 55	11/2	6313.239	11/2	27128.251
	4825.813 70	9/2	4432.240	7/2	25148.347
	nl ... new line, a previously unknown energy level

a

Table 2 -

 2 ContinuedEven Odd λ/ Å S N R J -v a l u e L e v e lE n e r g y / cm -1 J-value Level Energy/cm -1 nl ... new line, a previously unknown engergy level

	5987.819 nl 94	9/2	8320.255	11/2	25016.201
	5994.418 nl 82	9/2	18054.202	11/2	1376.605
	6023.642	15/2	27265.622	15/2	10668.960
	6030.234	15/2	28342.772	17/2	11764.250
	6061.992	9/2	27145.754	7/2	10654.110
	6074.993	9/2	24706.499	9/2	8250.170
	6083.735	11/2	6714.199	13/2	23146.917
	6085.810	9/2	24677.280	9/2	8250.170
	6107.014	9/2	25205.463	11/2	8835.380
	6118.016	11/2	24590.800	9/2	8250.170
	6236.807	19/2	29309.852	17/2	13280.440
	6238.372	7/2	6535.587	9/2	22560.965 a
	6280.999	11/2	27662.982	13/2	11746.340
	6440.364	13/2	13146.599	11/2	28669.174
	6440.366	15/2	26191.740 a	15/2	10668.960
	6441.818	15/2	27265.622	13/2	11746.340
	6486.560	17/2	25059.089	15/2	9646.850
	6539/224	19/2	30098.998	19/2	14799.880
	6616.025	7/2	15110.661	9/2	0
	6617.336	15/2	24759/250	15/2	9646.850
	6725.360	5/2	6451.823	7/2	21316.810
	6749.209	15/2	8765.556	15/2	23578.018 a
	6845.305	5/2	6451.823	5/2	21056.348 a
	6931.411	11/2	6313.239	9/2	20736.338 a
	6995.508	7/2	6535.587	5/2	20826.530 a
	7083.429	13/2	9464.460	15/2	23578.018 a
	7255.175	13/2	15156.070	11/2	1376.605
	7278.726	11/2	15111.480	11/2	1376.605
	7889.311	17/2	9770.330	15/2	22442.185 a
	8395.932	11/2	8829.078	9/2	20736.338 a
	8986.243	19/2	13626.720	19/2	24751.780 a
	8993.509	9/2	19196.472	11/2	8080.440
	9144.025	5/2	9710.615	3/2	20643.800
	9293.861	5/2	9710.640	5/2	20467.450 a
	9319.006	17/2	11714.367	15/2	22442.185 a
	9583.331	11/2	11282.870	11/2	21714.802 a

Table 3 .

 3 Spectral lines of Pr II which clearly could be assigned to a transition between two energy levels

				Even		Odd
	λ/ Å	SNR J-v alue Lev elEnergy/cm -1 J-value Level Energy/cm -1
	3898.837 nl 11	7	14035.46	8	39676.86
	3956.753 nl 49	6	5854.54	6	31120.63
	3974.851 nl 9	9	13639.13	9	38790.18
	4010.602 nl 14	7	11253.46	7	36180.33
	4014.331 nl 3	6	10137.93	7	35041.68
	4022.712 nl 10	8	13021.33	8	37873.15
	4062.220 nl 6	8	12237.15	8	36847.30
	4087.209 nl 5	9	14330.52	9	38790.18
	4133.612 nl 11	6	9211.95	7	33397.06
	4176.318 nl 7	6	11283.14	5	35220.93
	4213.554 nl 8	6	9670.82	7	33397.06
	4223.466 nl 6	6	10454.26	5	34124.85
	4306.037 nl 9	5	9733.44	6	32950.10
	4382.390 nl 6	6	10137.93	6	32950.10
	4394.984 nl 6	7	10650.24	7	33397.06
	4446.986 nl 5	5	8379.46	6	30860.28
	5201.579 nl 15	4	14167.90	5	33387.64
	5219.048 38	7	25569.19	7	6413.93
	5567.521 nl 2	8	13021.33	7	30977.67

Table 4 -

 4 Continuedenergy/cm -1 J-value A 1 /MHz parity lines which can be explained ( Å)

	26191.740(10)	15/2	478(3)	e	6920.705, 6440.366, 6334.990, 5726.350
	26692.000(5)	17/2	837(5)	o	9956.092, 9216.883 2 , 8069.405 2 , 7651.767,
					7163,745
	1