Minimum Information in In Vivo Research.

Data quality, reproducibility and reliability are a matter of concern in many scientific fields including biomedical research. Robust, reproducible data and scientific rigour form the foundation on which future studies are built and determine the pace of knowledge gain and the time needed to develop new and innovative drugs that provide benefit to patients. Critical to the attainment of this is the precise and transparent reporting of data. In the current chapter, we will describe literature highlighting factors that constitute the minimum information that is needed to be included in the reporting of in vivo research. The main part of the chapter will focus on the minimum information that is essential for reporting in a scientific publication. In addition, we will present a table distinguishing information necessary to be recorded in a laboratory notebook or another form of internal protocols versus information that should be reported in a paper. We will use examples from the behavioural literature, in vivo studies where the use of anaesthetics and analgesics are used and finally ex vivo studies including histological evaluations and biochemical assays.

In Vivo Protein Complementation Demonstrates Presynaptic α-Synuclein Oligomerization and Age-Dependent Accumulation of 8-16-mer Oligomer Species.

Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson's disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide in vivo evidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated α-syn oligomers in vivo.

Effect of a change in housing conditions on body weight, behavior and brain neurotransmitters in male C57BL/6J mice.

The development of modern housing regimes such as individually ventilated cage (IVC) systems has become very popular and attractive in order to reduce spreading of pathogenic organisms and to lower the risk to develop a laboratory animal allergy for staff members. Additionally, optimal housing of laboratory animals contributes to improve animal health status and ensures high and comparable experimental and animal welfare standards. However, it has not been clearly elucidated whether 1) a change to IVC systems have an impact on various physiological phenotypic parameters of mice when compared to conventional, standard cages and 2) if this is further affected by changing from social to single housing. Therefore, we investigated the influence of a change in housing conditions (standard cages with social housing changed to standard or IVC cages combined with social or single housing) on body weight, behavior and a neurochemical fingerprint of male C57BL/6J mice. Body weight progression was significantly reduced when changing mice to single or social IVC cages as well as in single standard cages when compared to social standard housing. Automated motor activity measurement in the open field showed that mice maintained in social husbandry with standard cages displayed the lowest exploratory behavior but the highest activity difference upon amphetamine treatment. Elevated plus maze test revealed that a change to IVC single and social housing as well as single standard housing produced anxiety-related behavior when compared to maintenance in social standard housing. Additionally, postmortem neurochemical analysis of the striatum using high-performance liquid chromatography coupled to electrochemical detection showed significant differences in striatal dopamine and serotonin turnover levels. In summary, our data indicate a crucial influence of a change in housing conditions on several mouse phenotype parameters. We propose that the maintenance of well-defined housing conditions is mandatory to ensure reproducible and comparable results and contributes to the application of the 3R refinement principle in animal studies by contributing to welfare and hygienical standards.

A novel liquid chromatography/tandem mass spectrometry method for the quantification of glycine as biomarker in brain microdialysis and cerebrospinal fluid samples within 5min.

Glycine is an important amino acid neurotransmitter in the central nervous system (CNS) and a useful biomarker to indicate biological activity of drugs such as glycine reuptake inhibitors (GRI) in the brain. Here, we report how a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the fast and reliable analysis of glycine in brain microdialysates and cerebrospinal fluid (CSF) samples has been established. Additionally, we compare this method with the conventional approach of high performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). The present LC-MS/MS method did not require any derivatisation step. Fifteen microliters of sample were injected for analysis. Glycine was detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation (ESI) mode. The total running time was 5min. The limit of quantitation (LOQ) was determined as 100nM, while linearity was given in the range from 100nM to 100µM. In order to demonstrate the feasibility of the LC-MS/MS method, we measured glycine levels in striatal in vivo microdialysates and CSF of rats after administration of the commercially available glycine transporter 1 (GlyT1) inhibitor LY 2365109 (10mg/kg, p.o.). LY 2365109 produced 2-fold and 3-fold elevated glycine concentrations from 1.52µM to 3.6µM in striatal microdialysates and from 10.38µM to 36µM in CSF, respectively. In conclusion, we established a fast and reliable LC-MS/MS method, which can be used for the quantification of glycine in brain microdialysis and CSF samples in biomarker studies.

Continuous dopaminergic stimulation by pramipexole is effective to treat early morning akinesia in animal models of Parkinson's disease: A pharmacokinetic-pharmacodynamic study using in vivo microdialysis in rats.

Short-acting dopamine (DA) agonists are usually administered several times a day resulting in fluctuating plasma and brain levels. DA agonists providing continuous dopaminergic stimulation may achieve higher therapeutic benefit for example by alleviating nocturnal disturbances as well as early morning akinesia. In the present study continuous release (CR) of pramipexole (PPX) was maintained by subcutaneous implantation of Alzet minipumps, whereas subcutaneous PPX injections were used to mimic PPX immediate release (IR) in male Wistar rats. In the catalepsy bar test, PPX-CR (1 mg/kg/day) reversed the haloperidol-induced motor impairment in the morning and over the whole observation period of 12h. In contrast, PPX-IR (tid 1 mg/kg, pre-treatment the day before) was not effective in the morning but catalepsy was reduced for 6 h after PPX-IR (1 mg/kg) injection. In the reserpine model, early morning akinesia indicated by the first motor activity measurement in the morning was significantly reversed by PPX-CR (2 mg/kg/day). Again, PPX-IR (tid 0.3 mg/kg, pre-treatment the day before) was not able to antagonise early morning akinesia. These results are in agreement with in vivo microdialysis measurements showing a continuous decrease of extracellular DA levels and a continuous PPX exposure in the PPX-CR (1 mg/kg/day) group. In contrast, PPX-IR (0.3 mg/kg) produced a transient decrease of extracellular DA levels over 6 h and showed maximum PPX levels 2 h after dosing which decreased over the following 6-8 h. The present study demonstrates that PPX-CR may offer a higher therapeutic benefit than PPX-IR on early morning akinesia and confirms earlier reports that PPX-IR reverses motor impairment for several hours.

The alpha(2) adrenoceptor antagonist idazoxan alleviates L-DOPA-induced dyskinesia by reduction of striatal dopamine levels: an in vivo microdialysis study in 6-hydroxydopamine-lesioned rats.

L-DOPA-induced dyskinesia is characterised by debilitating involuntary movement, which limits quality of life in patients suffering from Parkinson's disease. Here, we investigate effects of the a2 adrenoceptor antagonist idazoxan on L-DOPA-induced dyskinesia as well as on alterations of extracellular L-DOPA and dopamine (DA) levels in the striatum in dyskinetic rats. Male Wistar rats were unilaterally lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA/benserazide to induce stable dyskinetic movements.Administration of idazoxan [(9 mg/kg, intraperitoneal (i.p.)]significantly alleviated L-DOPA-induced dyskinesia, whereas idazoxan (3 mg/kg, i.p.) did not affect dyskinetic behaviour.Bilateral in vivo microdialysis revealed that idazoxan 9 mg/kg reduces extracellular peak L-DOPA levels in the lesioned and intact striatum as well as DA levels in the lesioned striatum. In parallel, the exposure to idazoxan in the striatum was monitored.Furthermore, no idazoxan and L-DOPA drug-drug interaction was found in plasma, brain tissue and CSF. In conclusion, the decrease of L-DOPA-derived extracellular DA levels in the lesioned striatum significantly contributes to the anti-dyskinetic effect of idazoxan.

Rapid analysis of GABA and glutamate in microdialysis samples using high performance liquid chromatography and tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been established for the rapid and reliable determination of gamma-aminobutyric acid (GABA) and glutamate in brain microdialysates. The microdialysis samples were analysed using a HILIC (hydrophilic interaction liquid chromatography) column, which is able to retain the polar amino acid neurotransmitters. The mobile phase consisted of a binary gradient elution profile comprising 0.1% formic acid in water and acetonitrile. GABA, glutamate as well as the respective internal standards [D(6)]-GABA and [D(5)]-glutamate were detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation mode within a running time of 3 min. The linearity ranged from 1 nM to 10 microM for GABA and 10 nM to 10 microM for glutamate. The limit of quantitation was found to be 1 nM for GABA and 10nM for glutamate (injection volume 10 microl). The present LC-MS/MS method was compared to the classical method for analysis of GABA and glutamate using high performance liquid chromatography (HPLC) and fluorescence detection (FD). Eventually, the feasibility of the LC-MS/MS method was demonstrated using in vivo microdialysis in rats by monitoring changes of the extracellular concentrations of GABA and glutamate in the globus pallidus following stimulation with potassium.