New Science Magazine Issue 1

Escrito por LDF.

new_science


A new magazine exploring the latest developments in the world of research without animals.

15 February 2008

 

 

New Science

A new magazine exploring the latest developments in the world of research without animals. Also featuring reports on the research we are funding.

 

If you are living in the UK or US and would like a free copy Esta dirección de correo electrónico está siendo protegida contra los robots de spam. Necesita tener JavaScript habilitado para poder verlo..

Microdosing: Fast forward

Introduction to the revolutionary, ultra sensitive test method, that could accelerate the development of new drugs and end the use of animals in tests…
Read more...

Mind’s Eye: The future of Neuroscience

Research we are supporting utilises cutting edge neuroimaging technology allowing static and real time brain imaging, including an LDF-sponsored Magnetic Resonance Imaging (MRI) scanner.
Read more...

MEG and auditory processing

The potential for helping people with hearing impairments and language disorders was studied using MEG to look at auditory processing. Animal model auditory research has historically focused on single-unit responses to stimuli, not the interaction of neurones. As a result of the lack of clarity which is produced by invasive measurements it will now be a challenge to understand how the non-invasive measures relate to them.
Read more...

A tissue construct model for investigating the therapeutic effects of ultrasound on cartilage

Clinical trials show that intensity pulsed ultrasound accelerates the healing of bone fractures by 30-40%. A new Lord Dowding Fund grant to researchers at the Eastman Dental Institute in London aims to result in a novel biological model of cartilage, free from animal-derived products, which can provide high quality tissue regeneration data for the establishment of clinical trial protocols. This could prevent animal suffering and benefit over 2million people in the UK alone.
Read more...

New approaches to neuro-toxicity testing

Two research projects by teams led by Dr Michael Coleman at Aston University with LDF support, have studied new approaches to testing for human neuro toxins.
Read more...

Extending the lifespan of computer–based alternatives

A new project is making our computer-assisted science teaching programmes globally accessible through the Internet, enabling multi-language versions to be created more easily than was previously possible. It will give life science and pharmacology teachers editorial control over the content of the programmes.
Read more...

A dynamic multi-cell type culture system as a model for Multiple Organ Dysfunction Syndrome (MODS)

A frequent consequence of sepsis is a syndrome known as MODS – Multiple Organ Dysfunction Syndrome – which results in the impermeable epithelial layers around the organs in the body (kidney, lungs, liver and intestines) becoming permeable, or “leaky”, causing organs to fail. A Lord Dowding Fund project aims to a cellular replacement for animal tests currently used.
Read more...



Microdosing fast forward

15 February 2008

1

From discovery of a molecule to regulatory approval, the development of a drug takes 10-12 years and now costs around $1.2billion (around £600 million).

New drugs must be evaluated in terms of how they are absorbed, distributed, metabolised and excreted (ADME) by the human body before they can advance towards the market1. Before this point extensive toxicology trials, typically using animals, are carried out.

The fact that as many as 40% of drugs fail to pass phase 1 human trials means that these preclinical studies are failing in predictability[1].

Although industry and regulators are inherently resistant to changes in the established system, the reality is that animal testing is a poor predictor of human reactions and has been estimated to have less than 60% predictive accuracy[2].

Furthermore this approach is not necessary where more relevant tests such as microdosing have the potential to replace animal use in determining suitable pharmacokinetic profiles of compounds[1].

EUMAPP stands for the European Union Microdose AMS Partnership Programme which brings together ten organisations from five countries in recognising the value of microdosing in drug development. The 30-month programme, lead by the strategic consultancy Xceleron and funded by the EU to a value of over €3million, aims to demonstrate microdosing as reliable and accurate, and in conjunction with in silico models aims to predict the human metabolism of drugs using data derived from microdosing[3].

The steering committee of EUMAPP comprises an authorised representative from each participating organisation[4].

EUMAPP will also look to verify human microdosing as a drug development approach, convincing the pharmaceutical industry of its merits and adding another seven drugs to the portfolio of compounds tested by Xceleron. The project is evidence that the value of microdosing has been recognised, and gives Europe a chance to lead the field[3].

A ‘microdose’ is defined as less than one hundredth of the proposed pharmacological dose but never exceeding 100µg[5]. Drug levels from microdosing can be measured in any biological sample such as plasma or urine to determine ADME and pharmacokinetic characteristics of a drug6.
This analysis is carried out using an Accelerator Mass Spectrometer (AMS). This is the most sensitive analytical tool available and is used to study the samples from humans, allowing early metabolism data to be obtained before going into human phase 1 trials[7].

AMS is able to directly count individual atoms and is so sensitive it has the ability to detect a liquid compound even after one litre of it has been diluted in the world’s oceans[1].

By conducting Human Phase 0 microdosing, drug candidates can be efficiently tested, directly in the relevant species, using trace doses to obtain early pharmacokinetic information.
This ultra sensitive analytical-technique allows greater predictability than animal studies and reduces the pre-clinical testing time from 18 months to 6 months. It also permits enhanced drug candidate selection and results in safer clinical trials[3].

The drug candidates selected for testing in the EUMAPP project were agreed upon because they were representative examples of drugs that exhibit properties in humans that are difficult to predict in animal or in vitro models. They were also chosen as drugs with properties that might be difficult to predict at a therapeutic dose from microdose data[8].

The preclinical animal tests which the pharmaceutical industry carries out are long, costly and unreliable. The reduced time that microdosing and AMS take to assess drugs in development will mean a reduction in the cost of producing drugs. This in turn could mean that more candidate drugs can be tested before clinical trials and potentially more drugs making it onto the market, leading to improved human health.

(1) Rowland, M (2006) Microdosing and the 3Rs, NC3Rs #5
(2) Ward, K. W and Smith, B. R (2004) A comprehensive quantitative and qualitative evalustion of extrapolation of intravenous pharmacokinetic parameters from rat, dog and monkey to humans. I. clearance, Drug metabolism and disposition, 32 (6): 603-611
(3) http://www.xceleron.com/metadot/index.pl?id=2572&isa=DBRow&op=show
(4) http://www.eumapp.com/index.php?content=committees
(5) http://www.emea.europa.eu/pdfs/human/swp/259902en.pdf
(6) http://www.xceleron.com/metadot/index.pl?iid=2494
(7) http://www.xceleron.com/metadot/index.pl?id=2502&isa=Category&op=show
(8) Lappin, G (2007) Translational medicine microdosing workshop: A General Introduction to Microdosing. http://www.xceleron.com/metadot/index.pl?id=2710&isa=Category&op=show

 

The Mind’s Eye: The present and future of Neuroscience

15 February 2008

1

Neuroimaging is contributing to the detailed mapping of the human brain, providing unprecedented understanding of functioning and development of mental ill health and neurodegenerative diseases1. The neuroscience facility at Aston University utilises cutting edge technology allowing static and real time brain imaging, including an LDF-sponsored Magnetic Resonance Imaging (MRI) scanner.

Aston was the UK’s first location to combine the emerging scanning technologies – with scientists in Japan and Germany taking the same bold step. Several years on, and the Aston Neuroimaging Research Group has gained an international reputation and as more and more research data emerges from other facilities using these techniques, these approaches are already surpassing their promise. Behold the future of neuroscience......

Aston actively seeks to provide more appropriate modes of research in the study of human perception and cognition than animal models. The study of neuroscience is important in both characterisation of neurodevelopmental disorders and is improving differential diagnostic and treatment strategies.
By studying single cell physiology and their networks, researchers at Aston University seek to determine principles of functional behaviour with non-invasive human studies. Examining the traits and attributes of neurodevelopmental disorders, such as Parkinson’s and Alzheimer’s, enables individual diagnostic and treatment strategies to be tailored to patients. Such studies offer increased clarity without the imprecision and misleading results caused by species differences during animal experiments. It is also possible to monitor the progression of neuro-degenerative disease in patients by carrying out brain imaging at different points of the illness, something which is not possible in a dissected animal brain.

There are some animal experimenters now advocating MRI to study primate brains, which illustrates the resistance in some quarters to actually shift away from the animal research habit – even when a technique for human study is available.

The Neuroimaging Research Group have gained an international reputation in developing novel approaches to modelling and analysing the biological processing of neuronal communication in the brain. These are being applied to a wide range of human studies, with the aim of improving diagnosis and aiding surgical intervention and are validated during surgery by evaluation of cortical localisation.

Aston University has secured a five year research fellowship from the Research Council UK (RCUK) allowing them to explore alternatives to pharmaceutical and physiological brain research in animals. As a result of this funding the Neuroimaging Research Group at Aston is in a unique position to make important progressions in the field of advanced research solutions.

The Lord Dowding Fund is sponsoring the annual running costs of the fMRI facility at Aston Life Sciences Academy, for the five years up to 2009. We have also supported a range of individual neuroscience research projects at Aston and elsewhere.

Vision research

A series of projects is providing a bridge between microscopic descriptions of cell functioning and macroscopic, behavioural observations, by relating behavioural performance to neural functioning in the cortex. Researchers achieved this by using tests involving visual stimuli and the resulting mental state and behaviour. In order to achieve high temporal and high spatial resolution for cortical processes, MEG and fMRI are used in combination. Modelling of cellular level networks can then be designed using the observation of this dynamic activity.

This research has provided the opportunity to compare measures of visual brain activity in humans to published data collected in invasive primate procedures. The recorded gamma activity can be reconstructed from MEG using sophisticated processing techniques which show the stimulus related dynamics over time in a human subject.

It has been claimed that certain detailed readings cannot be obtained from non-invasive human study, but can be obtained for example using micro-electrodes in the heads of primates. This human research has shown that comparable readings can be obtained from the human studies. The human-based data is immeasurably superior to the monkey data because it is from the correct species.

Investigating neuronal networks in visual information processing tasks: Concordance between fMRI and MEG scanning

In order to study the neuronal networks involved in sustained attention and vigilance, participants carrying out the Rapid Visual Information Processing (RVIP) task were measured with MEG and fMRI. A previous study using fMRI found two networks, one involved in sustained attention and one corresponding to working memory. Results of this study, in agreement with previous work, found various areas of the brain significantly active during the RVIP task. The images resulting from the two neuroimaging techniques were found to be concordant.

Auditory, Speech and Language

Speech and language research looks at functions measurable only in the human brain. Research at Aston has ranged from simple tone to complex higher level speech experiments involving semantic processing. Low level auditory processing projects seek to make a correlation between Magneto-encephalogram (MEG) data, which shows measures of the magnetic field in the brain, and the functions of the neuronal network.

One project has been investigating the neurophysiology of speech perception. Sinewave speech is a form of artificially degraded speech which is used as a tool for measuring speech perception. Once familiarised with the phonetic content the participant can recognise it as speech. Participants listening to sinewave speech were measured using MEG to explore the characteristics of the neurophysiological networks involved in the perception of phonetic information in auditory stimuli. Adding to knowledge from functional Magnetic Resonance Imaging (fMRI) research, it is possible to begin to understand the biological underpinnings of speech perception – a uniquely human process.

The brain and pharmaceuticals

We are also gaining a remarkable insight into the impacts of drugs on the human brain.

Psychotropic agents are drugs which affect mental activity, perception or behaviour. In a study of cognition and neuropharmacokinetics, the effects of these drugs on attention and working memory are being measured at Aston. These can be monitored for changes extensively using tasks which measure rapid visual information and offers much for the future study of nutrition and drug development and testing.

Another project has investigated brain changes as a result of drug uptake. Using a combination of MEG and structural MRI, neuronal changes were studied in relation to administration of a low dose of the tranquilliser diazepam with a focus on finding which loci of the brain are affected in all participants. Researchers also monitored neuronal changes in response to diazepam over the whole cortex. The researchers now have an MEG drug profile of diazepam which can be compared to other studies in order to discuss and hopefully develop an entirely new approach to pharmacological imaging. The specifics of diazepam induced changes in therapy were considered as well as the potential application of this method in drug development, neuronal network investigation and microdosing.

Pain processing

One of the earliest LDF projects utilising neuro-imaging, was an examination of the areas of the brain connected with pain in the gut. The result of the collaboration between Hope Hospital, Salford and Aston University was a new brain imaging technique to non-invasively record nerve cell activity in the human brain – known as Synthetic Aperture Magnetometry (SAM). This important field of research remains high on the agenda at Aston.
Working with gastro intestinal (GI) physiologists, researchers have characterised activation in the cortex of the brain, associated with abdominal pain such as in the gut. This information can provide a valuable model for the evaluation of pain incorporating autonomic nervous system measures. These can then be used to further explore measures of pain sensitisation and regulation.

Around a tenth of the UK population suffer Functional Gastrointestinal Disorder (FGD) the commonest symptom of which is GI pain. However differences between members of this group and a lack of clinical investigation mean that the origin of the pain often goes undiscovered. It has been suggested that these pains are a result of either an actual sensitivity which exists due to previous injury or inflammation in the gut, or that there is abnormal processing of abdominal sensation in the brain as a result of psychological abnormalities. Researchers plan to test, using models of oesophageal hypersensitivity, for the individual differences between patients with unexplained GI pain in whole human studies. MEG will be used to measure cortical activity in order to distinguish activity evoked by an actual stimulus from outside the body, from activity which is induced in anticipation of a stimulus.

Neurodevelopment and clinical research

The Aston University Neuroimaging Research Centre considers two aspects of translational research; to apply basic research to address clinical questions and to seek alternative approaches to animal experimentation. Studying neurodevelopment through linking brain changes to behaviour allows the improvement of diagnosis and treatment in disorders unique to humans.
Studying cortical networks which underlie languages and assessing language functions in both the left and right hemispheres would prove valuable in pre-surgical evaluation. Current invasive techniques cause temporary paralysis and speech arrest which can be distressing and complications do occur in a small percentage of patients. The need for individual evaluation of patients is evident as large differences were detected between adult participants in a word generation task when analysed with SAM. This method allows easy estimates of laterality in brain hemispheres by simple visual inspection of SAM analysis without having to rely on a calculated index. This is valuable to safeguard the language areas during brain surgery.
1.Human brain: The next frontier: http://humanitieslab.stanford.edu/2/372 (accessed 9/1/08)

 

MEG and auditory processing

15 February 2008

The potential for helping people with hearing impairments and language disorders was studied using MEG to look at auditory processing. Animal model auditory research has historically focused on single-unit responses to stimuli, not the interaction of neurones.

As a result of the lack of clarity which is produced by invasive measurements it will now be a challenge to understand how the non-invasive measures relate to them.

Researchers studied how neurones encode types of sounds, which make up speech, such as amplitude (AM) and frequency modulations (FM) by examining the long-term temporal dynamics of auditory responses.

Testing the null hypothesis that AM and FM are encoded by the same neural mechanism in the auditory cortex, psychometric functions were measured for detection of AM and FM for each participant using a two-alternative forced-choice task. Analysis using a spatial filtering technique, Synthetic Aperture Magnetometry (SAM), revealed that peak neuronal activations in response to AM and FM stimuli exist in the same region of the auditory cortex. The magnitude of responses was found to be unstable over the duration of the stimuli, which was reflected in the patterns of the phases and power of the evoked response.

This research puts MEG researchers a step closer to being able to describe their data in ways that reflect the temporal characteristics of responses. This research also uncovers the physiological role that induced oscillatory activity of neurones play in auditory perception. This large scale physiological measurement provides information which has not been generated by single unit studies in animals. Overall, this study provides the opportunity to begin understanding the auditory physiology in atypical developmental disorders eg developmental dyslexia, the information for which could simply not be provided by animal studies.

 

A tissue construct model for investigating the therapeutic effects of ultrasound on cartilage

15 February 2008

1

Clinical trials show that intensity pulsed ultrasound accelerates the healing of bone fractures by 30-40%.

The American Food and Drug Administration have approved the use of an ultrasonic osteogenic healing device, to augment normal fracture healing and treat bone non-union by daily application of a twenty minute localised ultrasound treatment.

Unfortunately, without a recognised human-based in vitro test model, human clinical trials are delayed whilst animal tests are conducted – and inevitably, these prove to be contradictory.

A new Lord Dowding Fund grant to researchers at the Eastman Dental Institute in London aims to result in a novel biological model of cartilage, free from animal-derived products, which can provide high quality tissue regeneration data for the establishment of clinical trial protocols.

Importantly, this model will be able to assess therapeutic response within a human-derived biological system, thereby avoiding species differences.
This could prevent animal suffering and benefit over 2,000,000 people in the UK alone.

Background

Cartilage degeneration and damage results in over two million annual visits to the local doctor in the UK, and is the leading cause of joint replacement surgery.

The expense and donor tissue limitations of surgical treatment mean that a non-invasive, ultrasound-based technique that enhances cartilage self-regeneration could be hugely important.

An increasing body of literature suggests that, like bone cells, chondrocytes, (the cells that make up cartilage), respond beneficially to ultrasound. This results in up-regulation of key extracellular matrix-associated proteins and genes in vitro. Thus, ultrasound treatment has been suggested as a potential clinical tool for the repair of damaged cartilage tissue.

Unfortunately, studies to date have revealed variations in ultrasonic effects on chondrocyte proliferation, depending on the studies and cell species.
The establishment of an experimental method of chondrogenic cells within an animal derivative-free matrix, will create a biological model from which histological, cell proliferation and other data can be gathered without animal dissection.

The model will also provide a high degree of control over ultrasound dose delivery. This avoids some of the problems with previous animal studies, such as variation in tissue thickness, as well as potential for poor dose delivery from misalignment of ultrasound.

Additionally, the use of human-derived biological specimens, rather than animal tissues, avoids justifiable concern regarding the extrapolation of animal findings to human subjects.

Constructs can be manufactured with reproducible physical, chemical and mechanical properties to match human tissue properties such as viscosity, density and elasticity. These properties, which may differ in animal tissues, fundamentally define the acoustic properties of tissue.

Project Aims

The LDF’s policy is to encourage the replacement of cell culture media originating from animals – for example foetal calf serum. This study will therefore use an animal-free alginate culture system, hitherto unused in ultrasound studies but successfully applied in other physical therapy studies.
The alginate matrices can be manufactured with specific mechanical properties and cell populations dispersed, cultured over several weeks and sectioned or digested for investigation by a variety of methods.

There are five stages to the project, encompassing different areas of expertise.
In the first six months an hMSC-alginate model of cartilage tissue is to be established. Using human patient cells, initial experiments will confirm the progress of each cell population down a chondrogenic pathway, following pelleting by centrifugation and application of specific chemical reagents. The protein and gene expression of macromolecules constituting the cartilage matrix will be determined over culture time. Gels of varying weights and volumes will be manufactured and their mechanical properties investigated against alginate content. Gel matrix concentrations will be tailored, as much as possible, to match the properties of cartilage tissue.

Simultaneously, a dosimetric evaluation of new ultrasound exposure system is planned with the construction of an ultrasound exposure device at the Open University. The ultrasound fields will be characterised with a needle hydrophone to ensure no reflections or standing waves are formed within the system. Measurements will be taken across each of the six piezoelectric ultrasound transducers and the dose distribution and mean dose will be established. Following this, hydrophone measurements will be made at the entrance, exit and at intermediate path lengths through the alginate gel to establish the mean ultrasound dose per cell within the cartilage model.
This will be followed by extensive examination of the effects of standard SAFHS treatment on developed cartilage model at the Eastman Dental Institute. The bulk of the work will examine the effects of standard SAFHS exposure parameters, which are currently used in clinical treatment of bone fracture, on the cartilage model. Twenty minute exposures will be applied, which is what occurs in the clinical protocol.

This study will represent the first SAFHS investigation of human cells within a tissue substrate.

Studies will initially address effects on cytotoxicity and cell growth, by using an established staining technique and also by measuring the incorporation of radiolabeled DNA precursors into the newly synthesised genetic material. DNA content will then be analysed to determine the proportion of cells at various stages of multiplication providing crucial data about whether, and which parameters of the ultrasound influence the cell cycle.

Thereafter the expression of key cartilage proteins and generic growth factors will be investigated for SAFHS exposure using flow cytometry. Gel sectioning and staining of notable changes over extended time periods will follow. If sufficient progress is made, the effects of ultrasound on apoptosis (programmed cell death will be examined due to its importance in regenerative processes.

Work at the Eastman will also examine the ultrasound intensity which initiates beneficial effects in cartilage. This will be performed by insertion of a thin urethane disc within the exposure assembly to provide controlled attenuation of ultrasound from the prototype exposure unit.

This work will determine the maximum attenuation (thus calculated tissue depth dependant on anatomical site) that can be applied to SAFHS therapy in order to induce a positive effect on cartilage repair. This will generate new information as to whether cartilage, located at deep sites, is a legitimate choice for treatment by ultrasound, information useful within the medical community. Keeping all human radiation exposure “As Low As Reasonably Achievable” is a key aspect of Medical Devices legislation (the ALARA principle). This may permit future therapy protocols where exposure time can be increased if the device output is lowered.

To optimise the parameters of the therapy, a new prototype unit will be supplied by Smith and Nephew by the later stages of the study. This unit will emit, provisionally, a choice of three ultrasound frequencies and a range of pulsing characteristics. Pulsing is particularly useful for cartilage tissues where blood supply is poor and so there is potential to thermally damage tissue. Studies will focus on determining if pulsing regimes can use greater beam-off time, to reduce thermal damage, without diminishing the benefits of ultrasound exposure. This will be measured by gene and cartilage matrix production.

At the Open University, the final part of the study will examine the acoustic mechanisms that may elicit such biological effects within the alginate gels. The extent of the three main acoustic mechanisms, which will induce heating, cavitation and radiation force, will each be examined by experimental techniques to assess their likelihood and impact on biological response within the ultrasound exposure system.

The magnitude and presence of these three acoustic mechanisms will be examined using previously described methods. These are infra-red thermometry for heating, spectral analysis to determine sub-harmonic emission for cavitation presence and Doppler laser or Doppler ultrasound measurement for bulk streaming effects.

By approaching this study from a biological angle and a mechanistic one too, it is hoped that this will greatly increase the multi-disciplinary interest and impact of the published work.

 

New approaches to neuro-toxicity testing

15 February 2008

1

Two research projects by teams led by Dr Michael Coleman at Aston University with LDF support, have studied new approaches to testing for human neuro toxins.

Reactions in brain cells and their use in assessments of toxicity

This project has laid the groundwork for the development of a simple system, using human astrocytic cell lines for the high throughput screening of toxins.
The aim was to develop a co-culture model which would examine the relationship between human neuronal cells and astrocytic cells because the full role of astrocytes is not reflected in current neuro-toxicity models. Astrocytes have high levels of antioxidant systems compared to other brain cells, so they are important in fighting toxins in the central nervous system. These systems act to maintain normal levels of signalling molecules, detoxify chemicals and reduce the products of toxic exposure.

Co-cultures can show how two different types of cells behave, but individual cell types can only be observed if there are markers that are individual to each cell type, so the co-culture should be evaluated in terms of each cell type and collectively. There is a paucity of astrocytic-specific markers that give insight into how astrocytes respond to toxins etc. Astrocytes respond in a physically unique way, called astrogliosis, one component of which is an increase in the cellular levels of Glial Fibrillary Acidic Protein (GFAP) and cytokines IL-1 and IL-6.

Astrocytes can synthesise a number of chemical factors, the levels of which increase after the astrocytes are activated. These include growth factors, which have been shown to stimulate the recovery and regeneration of neurones, following trauma.

The project intended to extend the knowledge of astrogliosis and it’s effect on astrocytes specific markers, so studies were designed to investigate the changes in GFAP and cytokines.

The main role of GFAP is thought to be in providing structural stability to glial cells by maintaining cytoskeleton integrity.

The use of GFAP as a marker for toxic effects in the CNS has been suggested since 1991 in vivo work. Various toxins have dose, time and region dependent effects on GFAP, so it can be used to indicate toxicity even in the absence of cytopathology. The increase in GFAP is not permanent and it was shown that astrocytes increase in volume rather than cell number.

By using human cell lines, the problem of extrapolation of the results is avoided. Prior to this study, the authors only knew of one previous study in this area using human astrocytes to monitor GFAP levels. Due to the lack of previous studies, there was no comparison of human cell lines – which can differ greatly in phenotype and genotypes & possibly in response to toxins.
The aims of the project were to select a suitable cell line that could then use toxins that have previously induced reactivity in the cells and hence an increase in GFAP. The relative cell toxicities will be determined and the sensitivities of the cells to them. The data can then be used as a reference for non-cytotoxic effects of the toxins. When a method for quantifying the GFAP is found, the effects of each toxin can be determined and related to existing assays. The effects of exposure time etc can be determined and also further investigation into the other markers of astrocytes reactivity.

Two potential cell lines were selected and it came to light that one of them had an extremely low level of GFAP. This is of interest to note, rather than implying that the particular line is not of any use as a marker of toxicity.
It was observed that GFAP was expressed at levels of toxin far lower than those which would be sufficient to cause cytotoxicity, so GFAP is an important indicator of the presence of a toxic stimuli rather than a crude indicator of serious damage. The ability to detect effects at such low concentrations, which mimic real-life, where exposure is a constant low-level and causes subtle effects on cellular function, is especially important.

With the cytokine response, one of the cell lines had a strong response to two of the chemicals at very low doses. This reaction supports the model’s further development as a human astrocytic model in it’s own right, and as part of a co-culture system. There were certain limitations, in that one chemical, which was expected, was not detected, but this re-iterates the importance of a battery of cell models overcome any lack of response in the other models in certain areas.

There are now the foundations for a simple system, using human astrocytic cell lines for the high throughput screening of toxins. Using GFAP as a marker, the astrocytes response can be distinguished from other cell types.
The introduction of neurons would create a more accurate representation of the interaction between different cell types in the central nervous system (CNS) and how each cell type responds to toxic exposure

A 3-dimensional system to develop tests of neurotoxicity using human brain cells

Neurones and glial cells are fundamental components of the brain and are susceptible to damage due to various factors. Neurones in particular, have little regenerative capacity, which can lead to permanent impairment of the CNS.

Progress in human CNS research has been slow; there is a lack of clinical knowledge of even the basic pathology of many neurodegenerative diseases.
This LDF grant researched the use of human embryonal carcinoma cell line (NT2) which can be differentiated into neurons and astrocytes upon exposure to retinoic acid.

NT2 cells can be grown either in mono-layers or as cell aggregates. To differentiate in mono-layers takes 6 weeks, the aggregates take 24-28 days. Cell differentiation is a complex process, mediated by the exchange of ions and molecules through GAP junctions. GAP junctions allow electrical and metabolic communication. The major protein in GAP junctions is connexion, the different types of which are specific to different types of cells. To ensure that there was a mix of neurons and astrocytes in the cell aggregates, a number of connexins were looked at. This ensured different cell types were present and cell-cell communication could take place. The team have also been investigating how Gap junctions develop over time in the aggregates.
As well as looking at the connexins in the cells, the team will investigate changes within the cell.

The results so far show that the connexins being studied are present at all stages of differentiation, so astrocytes and neurons are present throughout the differentiation process. The lack of a specific marker has also highlighted that as soon as the retinoic acid is added the cells undergo differentiation and thus lose the pluripotency of the undifferen-tiated NT2 cells.

In the future the project will study the effect of toxins on the cell aggregates. They will also study any differences in the cells when they are differentiated in free-fall in the bio-reactor. The cells will then be exposed to a number of toxins.

 

Extending the lifespan of computer–based alternatives

15 February 2008

1

Over twenty years ago, LDF began financing the development of computer programmes to replace the use of animals in higher education practicals.
The physiology and pharmacology programmes developed then, and subsequently, by Professor David Dewhurst and his team have had an enormous impact, saving millions of animals’ lives, and a fresh approach to research and learning has been engendered.

However, technology does not stand still. Having started out in an era when computers were almost an exotic novelty, we have seen almost constant development, rewriting and evolution of these resources to keep up with modern technology. Two years ago we embarked on one of our most ambitious projects yet, ReCAL with a grant to extend the lifespan of our now ageing computer-assisted learning (CAL) resources. This funding has now been extended up to the end of 2009 with a further commitment.
The aim is to make our computer-assisted science teaching programmes globally accessible through the Internet, to enable multi-language versions to be created more easily than was previously possible. It will give life science and pharmacology teachers editorial control over the content of the programmes.

The basis of ReCAL is to break down existing programmes into their learning components, make these available as discrete objects and provide online tools to enable teachers to reassemble them and tailor them to their own courses or different curriculums.

It is increasingly accepted that computer-based alternatives, many of which were developed over 10 years ago, can provide viable and cost effective alternatives to animal models and meet the learning objectives of undergraduate pharmacology and physiology classes. However, technological advances over that period have rendered many of these almost unusable. Regular rewrites is expensive and resource intensive so the ReCAL approach has been to develop a creative solution to this problem by separating the content of the programs from the delivery mechanism which we believe will greatly extend their life-span. The methodology we have developed also has the added bonus of making the resources easier to modify by teachers with little technical knowledge so that they can be adapted to different teachers’ needs and meet broader global communities.

The project has worked with a number of existing, proven computer-based alternatives. Each has been disaggregated into its component learning objects (text, images, animations, questions, data traces) and many have been rejuvenated where this has been necessary. These building blocks are then stored, with associated metadata (a set of basic terms which describe the data and enable search and browse functions), in an online repository and made available to teachers along with easy-to-use authoring tools with which they can create their own learning resources.

To date seventeen alternative computer programmes have been subjected to the ReCAL process. The metadata includes the data on the file type, location, associated key words, intellectual property rights, dimensions and size. This metadata is in line with various common specifications making it compatible with initiatives which organise data, such as the Dublin Core Metadata Initiative.

The web-based repository allows authorised individuals to search for, view, download, edit and upload learning objects.

Each programme generates around 100 learning objects and there are now more than 2000 catalogued into the online repository. New learning objects can be added and we have already demonstrated the ease with which different language versions may be created. Should the delivery mechanism become obsolete, the content can simply be linked to the next generation technology without the need for extensive rewrites.

New learning objects such as Chinese or Spanish versions of learning objects have already been created and this expansion of the repository is central to long term sustainability and building learning communities.
The project has made significant progress in the development of effective authoring and aggregation tools. The Labyrinth authoring tool has gone beyond the original scope of the ReCAL project to develop templates in Adobe Flash. It provides authors with the flexibility to express a high level of creativity and originality in their work.

Early testing has been carried out on the creation of new learning objects, for example translation of text into different languages. Professor Dewhurst has, for example, established contacts in Eastern Europe interested in the translation of textual learning objects into different Baltic languages.
Dissemination of the programmes has included presentations at the 15th World Congress of Pharmacology in China, the Annual Conference of the Indian Pharmacological Society and the 6th World Congress on Alternatives and Animal use in Life Sciences, in Japan.

At the moment Flash versions of the new programs are available on CD-ROM which contains:

  • the revitalised learning objects and learning design from one of the computer based learning programmes in physiology and pharmacology
  • the extensible markup language code which explains each programmes learning design
  • an Adobe Flash player which enables the original computer programme to be delivered on a number of different computer platforms such as stand-alone computers and via institutional intranet.

Future developments will provide teachers with access to the online authoring program, Labyrinth which allows editing of the XML code and re-aggregation of the learning objects. Different language versions will also be available (Mandarin Chinese, Spanish and one other European language).

The option to gain access to online versions is also part of the longer term plan and good progress has already been made in this respect.
In summary ReCAL has provided creative solutions to extending the lifespan of existing proven computer-based alternatives in the face of rapidly developing new technologies and methods to enable teachers to edit the programs to meet local needs. The future of science learning without animal suffering is is looking bright.

 

A dynamic multi-cell type culture system as a model for Multiple Organ Dysfunction Syndrome (MODS)

15 February 2008

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Sepsis is a life threatening illness, usually caused by the body’s response to infection. The infection can be caused by various factors, including burns, bacteria etc. A frequent consequence of sepsis is a syndrome known as MODS – Multiple Organ Dysfunction Syndrome. MODS results in the impermeable epithelial layers around the organs in the body (kidney, lungs, liver and intestines) becoming permeable, or “leaky”. This causes the organs to fail.

The aim of this project was to bypass the unethical and unreliable use of animals to model this organ breakdown and to look for a cellular replacement. The current cell culture studies are very simplified versions of what happens in the body, which cannot mimic the conditions of diseased human tissue.
This study therefore represented a development of the existing protocols
The aim was to develop a cell culture system whereby different types of human cell could be cultured together, in a dynamic fluid environment to better model the human tissues affected by MODS. Better understanding of the molecular mechanisms which cause the change in permeability in epithelial membranes during MODS will help identify new therapeutic strategies or prophylactics to combat this condition.

For practical reasons some researchers employ mono-layers of a single cell type, but these do not accurately reflect the complex in vivo environs. To use the cells in dynamic systems, they must be cultured under static conditions then transferred to chambers where they are measured under flow. The change in culture conditions is expected to cause artefactural changes in cell behaviour. Other systems allow more than one cell type to be cultured, separated by a membrane, but they are static, so a new system allowing continuous flow based cell culture & co-culture of separated mono-layers of different cell types was the aim.

The group developed an endothelial-epithelial bi-layer that mimics the blood brain barrier (BBB).

This was done using human brain microvascular endothelial cells (specialised endothelial cells) and human epithelial meningeal cells (derived from brain tumours). This model was then exposed to Neisseria meningitides, an organism that can cause septic shock and MODS.

For our projects, the LDF encourages the use of alternatives to FCS (foetal calf serum), also known as FBS (foetal bovine serum). Thus, the first six months of this project were spent adapting the cell lines to grow in serum free (SF) media – this was successful and it was confirmed by monitoring IL-8 (a protein produced when infection is present) that infection had occurred, and the cells were responding characteristically. In addition to this mono-layers and bi-layers were cultured under static conditions, challenged with bacteria and examined using various modes of microscopy. The results showed that, under SF conditions, bi-layers could be successfully cultured. It was the first time that these two cell types had been cultured together in a bi-layer and neither cell line was previously cultured in SF media

Due to problems with the cell system for holding the samples, it was deemed that it was too early to introduce a flowing media to the culture system, so characterising the model under static conditions was made the priority.
The second half of this project entailed testing the bi-layer to ensure it had the same integrity as an in vivo BBB – due to tight junctions, which as their name suggests, stop substances crossing the BBB.

This was done by testing the electric resistance of the layers. The profiles of various chemicals associated with infection were also monitored to ensure that the bi-layer behaved in the same way as a live human BBB.

The results for a chemical called TNF (tumour necrosis factor) differed between the monolayer and the bi-layer.

This project had two important achievements:

  • The first, a very important milestone, was that it was the first time that these cell types had been cultured together in a bi-layer. Prior to this group’s work, neither cell line was previously cultured in serum free media. The ability of a group to grow cells in serum free media means that there is less reliance on FCS.
  • The results for the tumour necrosis factor differed between the mono- and bi- layers. This was an important finding as it shows that cells behave differently when they are in different layers.