Under normal conditions and because it cannot store oxygen, the brain cannot withstand being deprived of oxygen for more than a few minutes without risking serious consequences. After an accident (cranial trauma or stroke), emergency teams therefore try to restore cerebral oxygenation as quickly as possible. The faster and more precisely physicians work, the greater the chances of recovery. A multi-disciplinary team at the Grenoble Institute of Neuroscience (GIN, Inserm/ Grenoble-Alps University/Grenoble teaching hospital) comprising physicists, biologists and physicians (neurologists and anaesthetists) has developed a new method for measuring cerebral oxygenation using MRI. Besides being non-invasive, this technique identifies the least oxygenated areas of the brain with precision. Ultimately, it could be used to guide therapeutic interventions and make them more precise, less risky and more effective.
These results are published in the Journal of Cerebral Blood Flow and Metabolism.

Although the brain accounts for just 2% of bodyweight, it uses 20% of the total oxygen consumed by the body. It is unable to create ‘reserves’ and the slightest shortage of oxygen can result in serious consequences (e.g. loss of language functions and motor skills) and even death if not restored very quickly. Ideally in a trauma situation, it should be possible to immediately identify the areas of the brain most affected by a lack of oxygen and where possible avoid physically operating on already weakened bodies.

After an accident, it is vital to monitor oxygenation of the brain. Nowadays, standardized international methods, notably the installation of a probe in the brain, allow brain oxygenation to be estimated locally. This operation entails a neurosurgical procedure and only allows a highly localized measurement of cerebral oxygenation. The only means of mapping the brain’s oxygen levels is by a radioactive measurement of oxygen using functional imaging (a technique developed in the 80s). However, this method also has its limits as it is expensive and requires PET (positron emission tomography) equipment which is scarce in France. Finally, while the brain’s total oxygen consumption remains constant, it varies between the various regions of the brain.

The new MRI method developed by Inserm researchers measures brain tissue oxygen saturation or the quantity of oxygen present in the tissue microvascularization (expressed as StO2).

Thanks to this technique, researchers have observed a major reduction in cerebral oxygenation in an animal model ischemic stroke and, to a lesser extent, following cranial trauma. By mapping cerebral oxygenation, it has been possible to identify regional heterogeneity of StO2 in strokes, cranial trauma and a model brain tumour.

This innovative approach offers a number of benefits including the possibility of immediate use with humans. Unlike positron emission imaging (or PET imaging), which is the gold standard technique, no radioactive tracer needs to be injected when performing an MRI examination. StO2 maps with enhanced spatial resolution can also be produced by MRI. Moreover, MRI examinations are less expensive than PET examinations and MRI imaging devices are more widespread in France than PET imaging devices.

The results of this preclinical trial match those achieved with the two gold standard methods (measurement of blood gas levels and histological mapping of oxygen deprived areas) for different oxygenation levels.

From a clinical perspective, several applications may be foreseen, which are illustrated in this project by 3 situations:

• In patients suffering from cranial trauma, this map could help identify the least oxygenated areas of the brain. Follow-up and therapeutic strategy could therefore be adapted to optimal effect.
• In stroke patients, this technique should enable more effective identification of the area of ischemic brain tissue which is likely to recover after treatment.
• Finally, certain malignant brain tumours contain poorly oxygenated areas which are known to be resistant to treatments such as chemotherapy and radiotherapy. It is strongly suspected that these poorly oxygenated areas contain tumour stem cells which cause tumour recurrence. By mapping them more effectively, it should be possible to adapt and customize treatments.

Although the reliability of this system has now been established, it remains to be seen to what extent it is capable of guiding the work of neurologists and anaesthetists. “In several years’ time, it is possible that a precise map of brain oxygenation will enable us to supply drugs to the right place or more effectively configure surgical operations to reduce intracranial hypertension” explains Emmanuel Barbier, Inserm research director.

This work was carried out using the IRMaGe platform, which is a platform of the ‘France Life Imaging’ national life imaging infrastructure. It was funded notably by the Agence Nationale pour la Recherche [French National Research Agency] (IMOXY project).

In recent years, 3D printing has developed in the area of health. Custom medical devices and prostheses were the first applications for this new technology. In 2011, for example, the first prosthesis (a titanium jaw) made with the help of 3D printing was implanted. Two years later, a 3D printed cranium (the largest ever placed) was implanted into a 22-year-old woman in the Netherlands.

In addition to these medical devices made of inert materials, researchers took on a challenge of an entirely different nature : bioprinting involves printing living cellular materials!

In contrast to traditional 3D printing, bioprinting involves consideration of a 4th dimension: the time dimension, in which the printed cells assemble themselves, migrate and differentiate autonomously to form functional tissues.

Bioprinting 3D © Inserm/ Guillemot, Fabien – Alphanov / Lescieux, Ludovic

Laser bioprinting in Bordeaux, an innovative approach

One of the goals of the laboratory is to develop laser and microfabrication technologies with the aim of printing tissues in vitro and in vivo. The researchers in the laboratory were pioneers in Europe, developing laser-assisted bioprinting from 2005. This Inserm/University of Bordeaux joint research unit is one of a very few worldwide to use this process. The objective of Fabien Guillemot’s team is therefore not only to position cells in 3D, but to define and model the self-assembly dynamic of the printed cells.

What are the results using the laser approach?

In the laboratory, bioprinting employs the principles of 3D printing to assemble the components of biological tissues (such as the cells and the extracellular matrix) layer by layer in digitally designed predefined patterns.

Laser-assisted bioprinting enables the manufacture of complex tissues with the help of printing using bioinks with cell concentrations close to physiological conditions, with a high degree of resolution (micron scale, pL volume) and a high operating speed (.> 10,000 droplets per second).

“In the area of bioprinting, laser technology offers the highest resolution,” explains Fabien Guillemot, Inserm Research Fellow.

Since 2005, the research team has succeeded in printing different structures and cell types with multiple layers of keratinocytes (cells of the superficial layer of the skin and appendages—nails and body and head hair) and collagen.

Impression de peau réalisée en juin 2014
© Ludovic Lescieux Alphanov / Fabien Guillemot Inserm.

At the moment, researchers are working on printing corneal and skin tissues in order to meet the needs of regenerative medicine, pharmacology, cosmetics, etc.

At the same time, the research team also conducts in vivo experiments in mice. In 2010 it succeeded in printing mesenchymal stem cells in the bone of live mice. The next step will involve testing computer-aided surgery which would allow in vivo printing of tissues directly where required.

What is the outlook for tomorrow?

The challenge of bioprinting remains the production of functional tissues with the aim of creating:

• Here and now….Predictive models that reproduce the physiology of healthy human tissues or diseased tissues, enabling the more predictive testing of drugs, components and candidate drugs. These physiological models will be used in the pharmaceutical field. (For cosmetic applications, the overall market for alternative methods has been estimated at €1 billion in 2015 (Source: Transparency Market Research), with an annual growth of 13.1%.)
  
  

• In the next 3-5 years…Individualised tissues, made using patient cells, that allow in vitro selection of treatment based on these tissues, and development of personalised treatment solutions. Fabien Guillemot’s team hopes to include bioprinting in the developments of the new Cancer Plan concerning individualised medicine.
  
  

• In the next 7-10 years…Implantable tissues for regenerative medicine. The development and manufacture of biological tissues represent major socioeconomic challenges. The market for tissue engineering was valued at $15 billion in 2014, and should double by 2018 (source: MedMarket Diligence, LLC.). Moreover, because of the increase in life expectancy, and in the incidence of major diseases such as cancer and diabetes, the number of people waiting for an organ transplant is constantly increasing (51,000 people in Europe in 2013).
  
  

Despite advances in research, it is not presently possible to print functioning organs. 

“Once researchers are able to create functional tissues, they will then be able to modify these tissues to improve them. Ethical debate will be needed to determine the extent to which tissue modification is possible, and for what purposes,” emphasises the researcher.

A significant breakthrough could revolutionize surgical practice and regenerative medicine. A team led by Ludwik Leibler from the Laboratoire Matière Molle et Chimie (CNRS/ESPCI Paris Tech) and Didier Letourneur from the Laboratoire Recherche Vasculaire Translationnelle (INSERM/Universités Paris Diderot and Paris 13), has just demonstrated that the principle of adhesion by aqueous solutions of nanoparticles can be used in vivo to repair soft-tissue organs and tissues. This easy-to-use gluing method has been tested on rats. When applied to skin, it closes deep wounds in a few seconds and provides a esthetic, high quality healing. It has also been shown to successfully repair organs that are difficult to suture, such as the liver. Finally, this solution has made it possible to attach a medical device to a beating heart, demonstrating the method’s potential for delivering drugs and strengthening tissues. This work has just been published on the website of the journal Angewandte Chemie.

In an issue of Nature published in December last year, a team led by Ludwik Leibler 1 presented a novel concept for gluing gels and biological tissues using nanoparticles 2. The principle is simple: nanoparticles contained in a solution spread out on surfaces to be glued bind to the gel’s (or tissue’s) molecular network. This phenomenon is called adsorption. At the same time the gel (or tissue) binds the particles together. Accordingly, myriad connections form between the two surfaces. This adhesion process, which involves no chemical reaction, only takes a few seconds. In their latest, newly published study, the researchers used experiments performed on rats to show that this method, applied in vivo , has the potential to revolutionize clinical practice.

In a first experiment, the researchers compared two methods for skin closure in a deep wound: traditional sutures, and the application of the aqueous nanoparticle solution with a brush. The latter is easy to use and closes skin rapidly until it heals completely, without inflammation or necrosis. The resulting scar is almost invisible.

Phase 1 Skin injury
Phase 2 Application of the solution
Phase 3 Using pressure to hold the edges together
Phase 4 Skin closure

Illustration of the first experiment conducted by the resear chers on rats: a deep wound is repaired by applying the aqueous nanoparticle solution. The wound closes in thirty seconds.
© “Matière Molle et Chimie” Laboratory (CNRS/ESPCI Paris Tech)

In a second experiment, still on rats, the researchers applied this solution to soft-tissue organs such as the liver, lungs or spleen that are difficult to suture because they tear when the needle passes through them. At present, no glue is sufficiently strong as well as harmless for the organism. Confronted with a deep gash in the liver with severe bleeding, the researchers closed the wound by spreading the aqueous nanoparticle solution and pressing the two edges of the wound toget her. The bleeding stopped. To repair a sectioned liver lobe, the researchers also used nanoparticles: they glued a film coated with nanoparticles onto the wound, and stopped the bleeding. In both situations, organ function was unaffected and the animals survived.

“Gluing a film to stop leakage” is only one example of the possibilities opened up by adhesion brought by nanoparticles. In an entirely different field, the researchers have succeeded in using anoparticles to attach a biodegradable membrane used for cardiac cell therapy, and to achieve this despite the substantial mechanical constraints due to its beating. They thus showed that it would be possible to attach various medical devices to organs and tissues for therapeutic, repair or mechanical strengthening purposes.

This adhesion method is exceptional because of its potential spectrum of clinical applications. It is simple, easy to use and the nanoparticles employed (silica, iron oxides) can be metabolized by the organism. It can easily be integrated into ongoing research on healing and tissue regeneration and contribute to the development of regenerative medicine.

Researchers at IGS, the genomic and structural information laboratory (CNRS/Aix-Marseille University), working in association with the large-scale biology laboratory (CEA/Inserm/Grenoble Alpes University) have just discovered two giant viruses which, in terms of number of genes, are comparable to certain eukaryotes, microorganisms with nucleated cells. The two viruses – called “Pandoravirus” to reflect their amphora shape and mysterious genetic content – are unlike any virus discovered before. This research appeared on the front page of Science on July 19, 2013.

With the discovery of Mimivirus ten years ago and, more recently, Megavirus chilensis[1], researchers thought they had reached the farthest corners of the viral world in terms of size and genetic complexity. With a diameter in the region of a micrometer and a genome incorporating more than 1,100 genes, these giant viruses, which infect amoebas of the Acanthamoeba genus, had already largely encroached on areas previously thought to be the exclusive domain of bacteria. For the sake of comparison, common viruses such as the influenza or AIDS viruses only contain around ten genes each.

In the article published in Science, the researchers announced they had discovered two new giant viruses:
Pandoravirus salinus, on the coast of Chile;
Pandoravirus dulcis, in a freshwater pond in Melbourne, Australia.

Detailed analysis has shown that these first two Pandoraviruses have virtually nothing in common with previously characterized giant viruses. What’s more, only a very small percentage (6%) of proteins encoded by Pandoravirus salinus are similar to those already identified in other viruses or cellular organisms. With a genome of this size, Pandoravirus salinus has just demonstrated that viruses can be more complex than some eukaryotic cells[2]. Another unusual feature of Pandoraviruses is that they have no gene allowing them to build a protein like the capsid protein, which is the basic building block of traditional viruses.

Despite all these novel properties, Pandoraviruses display the essential characteristics of other viruses in that they contain no ribosome, produce no energy and do not divide.

This groundbreaking research included an analysis of the Pandoravirus salinus proteome, which proved that the proteins making it up are consistent with those predicted by the virus’ genome sequence. Pandoraviruses thus use the universal genetic code shared by all living organisms on the planet.

This shows just how much more there is to learn regarding microscopic biodiversity as soon as new environments are considered. The simultaneous discovery of two specimens of this new virus family in sediments located 15,000 km apart indicates that Pandoraviruses, which were completely unknown until now, are very likely not rare. It definitively bridges the gap between viruses and cells — a gap that was proclaimed as dogma at the very outset of modern virology back in the 1950s. It also suggests that cell life could have emerged with a far greater variety of pre-cellular forms than those conventionally considered, as the new giant virus has almost no equivalent among the three recognized domains of cellular life, namely eukaryota (or eukaryotes), eubacteria, and archaea.

Notes
[1] Arslan D, Legendre M, Seltzer V, Abergel C, Claverie JM (2011) “Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae.” PNAS. 108:17486-91
[2] Parasitic microsporidia of the Encephalitozoon genus in particular.

Although excessive quantities of cholesterol in the body are known to have adverse effects on health, researchers might polish up its reputation via one of its derivatives. The research team from Inserm and the CNRS led by Marc Poirot and Sandrine Silvente-Poirot at the “Centre de recherche en cancérologie de Toulouse” (Inserm / CNRS / Université Toulouse III – Paul Sabatier), has not only discovered a new molecule derived from cholesterol (known as dendrogenine A), but has also provided proof in mice that this molecule has anti-cancer properties.

This work has been published in the journal Nature Communications.

Cholesterol is involved in different chronic pathologies, such as cardiovascular diseases and cancer. Current knowledge led us to suppose that cholesterol had a negative effect on cancers, mainly for two reasons. Firstly, cholesterol is a precursor of androgens and estrogens, both of which are involved in the development of cancers known as “hormone-dependent” cancers. Secondly, the synthesis process of cholesterol (that contains over 20 different stages) activates tumour-related genes.

Blocking the upstream biosynthesis pathway using inhibitors such as statins should have provided protection against cancer or some measure of anti-cancer efficiency, but this was not confirmed by clinical studies involving very large patient cohort studies; This suggests that its metabolism is much more complicated[1].

So the team of researchers led by Marc Poirot and Sandrine Silvente-Poirot concentrated particularly on the metabolism of cholesterol. Using screening and chemical synthesis techniques, the researchers were able to show that the product of the chemical reaction between a cholesterol derivative and a histamine generates a new class of sterols known as Dendrogenine A (DDA). This molecule, obtained in laboratory conditions, shows remarkable properties for inducing differentiation and death of cancer cells.

These observations led the team to search for this molecule in the tissues of mammals. And they discovered that DDA is present in healthy tissue and cells in humans, whereas it is not detectable in tumour cells. In women, its level is 5 times lower in breast tumour tissue than in normal tissue.

“Our results suggest that DDA protects the cells from carcinogenic processes” points out Marc Poirot.

In order to test this hypothesis, the researchers then attempted to repair the DDA deficiency in tumours implanted in animals. And in these animals, the administration of DDA controlled the proliferation of the tumour and extended the life expectancy.

For the researchers, this is a major discovery, because it provides the proof of the existence of a new metabolic pathway in humans, at the interface between the cholesterol metabolism and the histamine metabolism, and also because, thanks to its anti-cancer properties, DDA could be used to treat different cancers.

A discovery by INSERM Unit U1105 “Groupe de Recherche sur l’Analyse Multimodale de la Fonction Cérébrale” [Research Group for Analysis of the Multimodal Cerebral Function] of the University of Picardy Jules Verne (UPJV) and INSERM Research Unit U992 “Neuroimagerie cognitive” [Cognitive neuroimagery], NeuroSpin/CEA

The immature brain of a premature infant is capable, at the age of three months pre-term, of distinguishing syllables uttered by male and female voices. These results obtained by INSERM researchers at the University of Picardy Jules Verne and the CEA’s NeuroSpin Imaging Centre, were published in the PNAS journal dated 25 February 2013. They highlight the very early sophisticated organisation of the regions of the brain involved in language-processing and social communication in humans.

At birth, infants are capable of distinguishing between similar syllables, of recognising their mother’s voice and of differentiating between several human languages. Are these abilities in the human infant due to the presence of innate mechanisms specific to the human race for processing speech or do they indicate swift learning of the characteristics of the mother’s voice during the final weeks of pregnancy?

To discover the truth, Fabrice Wallois, Director of the mixed UPJV/INSERM research unit known as the “Research Group for Analysis of the Multimodal Cerebral Function” (GRAMFC), and Ghislaine Dehaene-Lambertz, (INSERM, NeuroSpin/Atomic Energy and Alternative Energies Commission (CEA), University of Paris-Sud) in collaboration with hospital doctors at the Amiens Picardy University Hospital, tested the auditory discrimination abilities of twelve premature babies, born at 28 to 32 weeks of amenhorræa, i.e. born two to three months’ pre-term.

At this stage of development, the brain is immature since the neurones are still in the process of migrating to their final destination. Yet the first connections between the brain and the outside world are being set up, especially those that enable the fœtus to hear sounds, thus enabling it to record the first brain responses to external stimulation.

The authors of this study stimulated premature infants by using audio stimulation, exposing them to the sounds of two similar syllables (“ga” and “ba”) pronounced by either a man or a woman. They recorded the brain responses by using functional optical imaging (near infrared spectroscopy (NIS)). The researchers were thus able to show that despite the fact that their brains were still immature, the premature babies were receptive to changes in the voice (male or female) and changes in phonemes ( “ba” or “ga”) (figure 1).

Furthermore the sets or networks of neurones involved in premature babies are very similar to those described in the adult when performing the same type of task. They are asymmetrical and especially involve the frontal areas. As in adults, the right frontal area responds to newness, regardless of the change involved, whereas the left frontal area or Broca’s region, only responds to a change of phoneme.

Figure 1: Projection of brain activation in a premature baby born at 30 weeks of amenorrhæa. The change of phoneme produces an increase in brain activity in the temporal and frontal areas, especially the left. The response to a change in the voice is more limited and only involves the change of voice is more limited, and only involves the lower right frontal area.

©Wallois

These results show very early, as soon as the first cerebral connections are established (three months before term) and above all before any learning process has occurred, that the human brain is capable of processing special characteristics of human speech thanks to the sophisticated organisation of certain language areas of the brain (the right and left perisylvian regions). Since the organisation of the regions of the brain being governed by genetic expression during the development of the fœtus, the authors suggest that the emergence of language is largely influenced by genetics and thus by innate mechanisms.

This study received support from the Picard Regional Council and the ERDF (European Regional Development Fund). 

When research, medicine and high tech work together in Rennes

Imagine, in this hybrid operating theatre, surgeons, physicians and engineers surrounded by control screens, using enhanced and roboticised reality systems, enabling interventions that are even more accurate and safe. At the Signal and Image Processing Laboratory (University of Rennes 1 / Inserm), researchers, engineers and doctors at the Rennes CHU Cardio-pneumological Centre have been working together to bring the TherA-Image platform into existence.

They have designed and implemented interventional cardiology and mini-invasive surgery techniques, using image-guidance and computer-assistance through the TherA-Image platform. These procedures are designed to reduce to a minimum the time taken for the intervention and trauma from the operation. This means that the frailest patients will be able to have access to these innovative medical techniques that are designed to improve post-operative and the prognosis thanks to instruments and skills that are unique in Europe.

TherA-Image is a hybrid operating theatre, used for both patient care and de research in the field of health technology. It is a medico-technical platform located at the interface between the Rennes University Hospital, the University of Rennes 1, INSERM and the medical industry. It is here that computerised approaches are devised and deployed for planning interventions, assistance with operating procedures and assessment of these procedures.

TherA-Image possesses state-od-the-art imaging equipment (3D intra-operating observation, enhanced reality, cardiac electrophysiology), operating assistance (endovascular navigation and a catheterisation robot) as well as video broadcasting (to obtain remote expertise, provide training, etc.)

Treating cardiac insufficiency through resynchronisation therapy /©L. Després

This combination of equipment and skills in the same operating theatre in Rennes is unique in Europe. It is the result of a convergence of views that originated long ago in the LTSI multidisciplinary teams, that included doctors, researchers and engineers, and a solid partnership has been formed for the long-term, including industry leaders in their own field of interest.

©Replacing the thoracic aorta

Today, TherA-Image makes it possible to explore new approaches to cardiovascular treatment, in order to:

• Treat cardiac insufficiency, especially through the type of treatment known as cardiac resynchronisation. Patients sometimes present with defects in the synchronisation of the heart ventricles. This means that the chambers of the heart do not all contract at the same time, so the heart no longer works efficiently and the patient becomes out of breath when making the slightest effort. Thanks to the TherA-Image platform, doctors and researchers can optimise the techniques and the implantable devices for electrically stimulating the heart.

• Eliminate the sources of electrical disturbances inside the heart muscle that are the cause of cardiac dysrhythmia. Techniques and intra-corporeal navigation models have been developed for this purpose, as well as diagrams of the electrical current inside the heart muscle. The aim is to identify the sources of electrical disturbance and check that they have been eliminated after treatment, through localised heating of the tissue.

• Promote the development of less invasive surgical techniques. For example, it should become possible to reliably replace heart valves percutaneously (passing through an artery), without having to open up the chest cavity, relying on location and algorithms and guiding the surgical instruments through the body.

• Treating aneurisms (abnormal dilatation of the walls of a blood vessel) and stenoses (shrinking or narrowing of a major blood vessel). Current mini-invasive techniques are becoming increasingly complex, but thanks to TherA-Image, the surgeon can guide his/her instruments through the blood vessels using tools for planning the route (as for GPS) and effective assisted-imaging methods (enhanced reality) to reach the lesion and install a prosthesis at the site in complete safety.

Simplified dynamic 3D modelling of the Théra-Image room/©LTSI

TherA-Image is an instrument that has caused a major development in medical research and the occupational culture associated therewith, making it possible to design, deploy and assess the surgical interventions of tomorrow, to the benefit of the patient.

TherA-Image is financed through the State-Region Project Contract 2007-2013 in an amount of €5.2 million and has received support from the European Union (FEDER: €1.7 million), and the State (€2 million), the Brittany Region (€370,000), the General Council of Ille-et-Vilaine (€640,000) and from Rennes Métropole (€526,000).