The magazine of the Institut national de la santé et de la recherche médicale (National Institute for Heath Care and Medical Research)
When
health comes out
into the light
N°26 l July - August 2015
©©inserm/Depardieu Michel
Light has been an integral element in biology research for
decades now but the recent, dramatic development of new
fluorescent and light-activated proteins has revolutionized its
exploitation in both fundamental and translational research.
New companies and semi-public organizations are
transforming the R&D landscape: some of these are
generating transgenic animals that express photo-sensitive
proteins; others are developing and exploiting the latest developments in
optics to map organs and tumors or watch how tens of thousands of cells
work together.
Many of these organizations propose making data obtained with their “megasystems” available in open source form. Thus, although future discoveries
based on the use of light will continue to follow classic pathways of scientific
research and industrial exploitation, at the same time the sheer volumes
of data that light-based technologies can generate are going to necessitate
reconsideration of how we conduct research.
It is no longer a futuristic idea to contemplate how to reorganize the input
of scientists when their discoveries depend on the analysis of “optical” data
generated in “high-tech” structures located on the other side of the world.
Serge Charpak
Director Inserm Unit 1128 – Université Paris-Descartes
Laboratory of Neurophysiology & Modern Microscopy
N° 26
JULY - AUGUST 2015
Free subscription, write to:
science-et-sante @ inserm.fr
Publications Director
Yves Lévy
Content Director
Arnaud Benedetti
Editor-in-Chief
Yann Cornillier
Editorial secretaries
Coralie Baud, Maryse Cournut,
Marie-Charlotte Ferran
Headlines Julie Coquart
Editorial Assistant
Coralie Baud
Collaborators on this issue
Alice Bomboy, Damien
Coulomb, Françoise Dupuy
Maury, Alexandra Foissac, Tina
Gereral, Walter Gillot, Caroline
Guignot, Amandine Henckel,
Charles Muller, Pascal Nguyên,
Julie Paysant, Hélène Perrin,
Simon Pierrefixe, Nicolas
Rigaud, Bruno Scala
Graphic design
Ghislaine Salmon-Legagneur
Art Direction
Primo&Primo
Iconography
Cécile Depot
Project Consultant
Françoise Harrois-Monin
Cover credits
Illustration:
Sébastien Thibault
Printed by
Aubin Imprimeur
N° ISSN : 2119-9051
Legal submission: July 2015
grand angle• General medicine • Entrepreneurship • Opinions • Strategies • Notepad
• leader • discoveries • Scientists • outlook on the world • Clinically yours ➜
4●
● N° 26 ● JULY - AUGUST 2015
grand angle
➜
When health
comes out into
the light
© Pasieka/SPL/PHANIE
It is summer, the part of the year when the sun shines longest
on our country. And it is the moment chosen to join UNESCO in
celebrating 2015 as the “International Year of Light”. The
Organization is supporting many initiatives to shine a light on
related applications and technologies. In our field of health,
we can shed light on the most innovative research projects
being undertaken to exploit this physical phenomenon’s unique
properties. How does science observe the infinitely small? How do living
organisms organize their most complicated tissues? How do the genes of an alga
or a bacterium help it draw attention to itself or make it invisible? How can light
be used to manage cancer? Can it stimulate the brain? In the following three
sections—Observe, Treat, Repair—find out about some of the most interesting
current research related to light ...
Compiled by Pascal Nguyên
Laser eye surgery
JULY - AUGUST 2015 ● N° 26 ●
●
5
grand angle
➜
Over the centuries, we have always tried to
tame light in the name of our health and wellbeing. While we already have many ingenious
applications, other innovations are on the way.
2
015 has been declared “International Year of
Light” by the United Nations Educational, Scientific and Cultural Organization (UNESCO). The
Organization’s Web page states that this is “a global
initiative designed to highlight the key role light and
optical technologies play in our daily lives and their
importance for our future and for the sustainable
development of the society we live in.” While light
may be important in daily life, it is vital to life period.
Firstly that emitted by the Sun which, some three or
four billion years ago, created conditions propitious to
the development of life on Earth. And now drives the
growth of plant life through photosynthesis which is
at the bottom of most of the food chains that we figure
in at higher levels. A life form that soon sought to tame
light and, over the ages, found ever-more innovative
ways of exploiting it. Today, light offers a splendid
spectrum of different applications. As highlighted
wo-photon
LTfluorescence
microscopy
Detects fluorescent
signals stimulated by
the absorption of two
photons; used to follow
in vivo changes in labeled
tissue.
LLupus vulgaris
A form of tuberculosis,
usually involving the face
©©Frédérique Koulikoff/Inserm
8 fr.unesco.org
6●
● N° 26 ● JUILLET - AOÛT 2015
©©Edmonson/SPL/Phanie
Taming
light
by UNESCO, it is exploited in a huge range of fields
from energy and construction to communications and
space exploration ... as well, of course, as health.
The first way light is used in medicine is for observation. For centuries, human beings have exploited its
properties (see Box) to study the human body and understand underlying mechanisms. We developed instruments to enhance vision and later to take its place.
The first microscope was made in the XVth or XVIth
Century. This revealed bacteria and cells measuring a
few micrometers (µm). Today, with two-photon fluorescence microscopy (L), we can generate images to a
resolution of 0.5 µm—even in three dimensions and of
living cells, to shed light on physiological components
and events in vivo. But light can be used for more than
simple observation. It can also be used in treatment.
In 1958, Richard John Cremer documented the effect
of light on neonatal jaundice. Light therapy is also
grand angle
➜
Session of
light therapy
used to manage seasonal
affective disorder. This
involves exposing the patient to light that mimics
sunshine although the
light used is not natural
sunlight. Neonatal jaundice is treated with lamps
that emit blue light. In
1903, Niels Ryberg Finsen was awarded the
Nobel Prize for his contribution to the treatment
of patients with lupus
vulgaris (L ) with an intense beam of light. This
opened a new avenue of medical research, i.e. phototherapy and treatment with simple, intense monochromatic laser light. E.g. in dermatology, UVA or
UVB phototherapy is used to treat the lesions of
psoriasis and eczema, and lasers are used to remove
tattoos. Lasers are also used in ophthalmology to treat
myopia and cataract as well as in urology to remove
calculi. Today, light comes into many sophisticated
applications and various areas of state-of-the-art research. An example: two-photon microscopy is vital
in genetic engineering strategies in which fluorescent
genes are introduced into target cells so that they can
be observed in vivo. Genetic engineering is also on
the menu when it comes to partially restoring sight.
This is the field of optogenetics which, more generally,
involves making cells photosensitive so that certain
functions—not necessarily visual—can be turned on
To each its own wavelength
Light is both an electromagnetic wave and a beam of massless
particles called photons. Unobstructed, it travels in a straight line
at a speed of 300,000 kilometers a second (in a vacuum). Light that
is visible to the human eye—the visible spectrum—is composed of
a set of monochromatic waves from violet to red, taking in all the
hues of blue, green, yellow and orange. White light is the resultant
of combination of all these waves. Each wave is physically defined
by its frequency and speed of propagation in a given medium; the
product of these two gives its wavelength. The wavelengths in the
visible spectrum go from 380 nanometers (violet) to 780 nm (red).
Shorter than 380 nm (down to 10 nm) is the ultraviolet (UV) range.
Above 780 nm (up to 1 mm) is the infrared (IR) range. Wavelength
is related to the amount of energy transported. The shorter the
wavelength, the more energy carried by the wave—and the greater
the effect on biological molecules. It is by modulating these
wavelengths that it has been possible to design certain tools and
applications, e.g. lasers are instruments that amplify and direct
light of a specific wavelength that can be used to kill cells with
perfect precision or cut tissue. Imaging techniques are based on
the property of light to reflect off certain surfaces or get offset as it
passes through media with different refractive indices. Two-photon
microscopy exploits the fluorescent properties of molecules when
stimulated by a beam of photons. Light’s properties are therefore
manifold—and its applications are as diverse.
or off. Alternatively, if malignant cells can be made
photosensitive, they can be destroyed. This is called
photodynamic therapy, a branch of phototherapy,
which should expand thanks to the invention of a new
type of lighting tissue. And within a few years, laser
bioprinting may make it possible to repair bone tissue
faster. Little by little, scientists have identified new
pathways in which light is important and affects our
physiological functions, above and beyond the simply
visual. It is these six health-related fields that we have
decided to highlight in this volume—applications of
light that are among the most innovative.
8 www.light2015.org
JUILLET - AOÛT 2015 ● N° 26 ●
●
7
grand angle
➜
OBSERVE
Fluorescent labeling
Seeing living components
in color
The introduction of one or
more exogenous genes
into a living organism
in order to study their
function or create new
variants (GMOs).
☛☛Jean Livet: Inserm Unit 968/CNRS –
Université Pierre-et-Marie-Curie
☛☛Alain Chédotal: Inserm Unit 968/CNRS –
Université Pierre-et-Marie-Curie
☛☛Marc Bajénoff: Inserm Unit 1104/CNRS –
Aix-Marseille Université
T
o study physiological mechanisms, scientists have
relied on microscopy coupled with staining techniques to highlight cells and molecules in the tissue
being examined. A trick that is essential for investigating the nervous system in which the overlapping projections from neurons (axons) massively complicate the
image. “One the first techniques used was developed by
Camillo Golgi at the end of the XIXth Century and named
after him, Golgi staining” points out Jean Livet *, Director of the Neural Network Development Group “Injecting
at the Vision Institute in fluorescent dyes
Paris. This monochromatic is still an option„
staining technique involves
treating post-mortem nervous tissue with silver nitrate and potassium bichromate prior to examination in
a light microscope. This method allowed Spanish histologist Santiago Ramon y Cajal to formulate the neuron
doctrine which defines the neuron as the fundamental
structural and functional unit of the nervous system.
A discovery that brought Golgi and Ramon y Cajal the
Nobel Prize in Medicine in 1906. “Later, fluorescent
labels were used to reveal cell anatomy and changes in
intracellular calcium concentration—and they still are.
Since the 1980’s, transgenic technology (L) has exploited
chromogenic enzymes which generate color. And then, in
the mid-1990’s, GFP was developed”, explains Jean Livet.
When illuminated with blue light, the
jellyfish Aequorea victoria fluoresces.
8●
● N° 26 ● JUILLET - AOÛT 2015
This three-letter acronym is synonymous with a revolution in the study of biology. They signify Green
Fluorescent Protein. Discovered by Japanese biologist
©©Lanting/SPL/Phanie
LTransgenesis
©©Katie Matho, LOB, Ecole Polytechnique
To see cells or in vivo events better,
they can be made to emit their own light.
This is the principle underlying fluorescent labeling methods.
They can then be “tickled”
with photons to make them light up.
This is two-photon microscopy.
grand angle
➜
Axons and neuronal synapses in
the brain stem
of a brainbow mouse
ing strategies, we managed to express random different
colors inside cells of interest,” he explains. “With a mix of
blue, green, yellow and red, we can get a series of different
colors—up to one hundred different hues.” This makes
it possible to distinguish individual cells by means of
the color of the signal they emit. Applied to neurons,
this approach can be used to draw a complete map
of their connections to one another. Recently, Alain
Chédotal * of the Vision Institute used this technique to study living oligodendrocytes, the cells that
synthesize the myelin which is an essential component
of the sheath that protects nerve fibers.
and chemist Osamu Shimomura in the early 1960’s,
it is a protein found in the jellyfish Aequorea victoria
that emits a fluorescent signal when stimulated by blue But this multicolor labeling method has yielded anlight. In the 1980’s, scientists isolated the GFP gene other application. “Brainbow makes it possible to folfrom the jellyfish so that it could be
low tissue development and turnover,
introduced it into other organisms. In “Using tissue
in other words homeostasis (L). Like,
a cell in which GFP is being synthefor example, the epithelium(L) of the
engineering strategies, gut,” specifies Jean Livet. This techsized, it emits a signal when exposed
to blue light. This labeling method we managed to express nique makes it possible to investigate
was perfected in the 1990’s, earning random different
the mechanisms underlying cell prothe 2008 Nobel Prize in Chemistry for colors inside cells of
liferation, notably how stem cells are
Osamu Shimomura who discovered interest„
regulated in a tissue, if they all behave
GFP, Martin Chalfie who succeeded
in the same way or have different fates
in inserting the corresponding gene
... They appear labeled blue, green, ...
into the nematode (roundworm) genome, and Roger colors that are passed into their progeny cells so lineages
Tsien who developed variants of GFP that emit differ- and cell differentiation can be followed.
ent colors. These variants emit cyan and yellow signals, Marc Bajénoff * of the Immunology Center in Marextending the spectrum of the green-emitting jellyfish seille is using these fluorescent techniques: “GFP has
protein. And a fluorescent protein derived from a coral allowed us to watch how lymphocytes move around in
adds red to the pallet.
the lymph node of a live mouse. In 2009, we focused on
Using these labels has made it possible to make direct architecture, the stromal cells in lymph nodes (L).” To go
observations (notably using two-photon microscopy, further, his team did experiments in a mouse that had
as detailed later in this volume) of the structure and been genetically modified to express brainbow markers
behavior of cells in living tissue, e.g. the anatomy of neu- in cells of interest at specific times. “We have been able
rons and their interconnections inside the brain, and to follow the line of descent of a sub-type of stromal cells
how they get remodeled. Neuroscientists extensively and understand how these cells do what they do”, celeused GFP for one-color imaging until a new technique brates the scientist. “It is not the same thing if 100 cells
was developed that makes it possible to express differ- divide in two as if 90 cells do nothing and just 10 generate
110 new cells. Ultimately, there are still 200 cells but the
ent-colored fluorescent proteins in the same animal.
This method called brainbow—for brain and rain- process is different.” bow—was developed in 2007 at Harvard University, by Further evidence that a colorful life is better for research
a team that included Jean Livet. “Using genetic engineer- than a monotonous world.
LHomeostasis
Physiological processes
that maintain the balance
necessary to normal
functioning.
LEpithelium
The tissue that covers
external body surfaces
(the skin) and internal
surfaces (the pleura,
peritoneum, gut wall,
etc.), constituted by tightly
juxtaposed cells devoid
of extracellular matrix,
vessels and fibers.
LLymph node
Part of the immune
system where immune
cells proliferate and
differentiate.
JUILLET - AOÛT 2015 ● N° 26 ●
●
9
grand angle
➜
OBSERVE
Two-photon
fluorescence microscopy
Two photons are better than one!
©©Patrick Delapierre/inserm
Aligning a laser
beam for multiplexed
two-photon
microscopy
☛☛Emmanuel Beaurepaire: Inserm Unit
1182/École polytechnique/CNRS –
École polytechnique
☛☛Serge Charpak: Inserm Unit 1128/CNRS
– Université Paris-Descartes
10 ●
● N° 26 ● JUILLET - AOÛT 2015
©©Barker/A.I.P/SPL/Phanie
S
ince the construction of the first light microscope
(either around 1590 by Janssen father and son in
Holland or in 1609 by Galileo) technologies for
the investigation of cells—and later physiological and
biochemical mechanisms—have been steadily evolving and feeding off one another. One of the most recent techniques: two-photon microscopy. As its name
indicates, this technique involves delivering pulses of
photons into a tissue expressing fluorescent proteins
or into which fluorescent dyes have been injected.
When a fluorescent molecule absorbs two photons at
the same time, it gets excited to a higher energy state.
To return back down to its resting state, it emits a photon of variable color—green, red, blue, ...—depending
on its fluorescence spectrum. The emitted photons
can be detected with a highly sensitive detector, e.g.
to monitor activity in a mouse brain, a gene for a flu-
Maria Goeppert
Mayer
(1906-1972)
orescent protein could be
introduced into certain
neurons. When the area
of interest is examined
in a two-photon microscope, proteins excited at
the focal point emit a fluorescent signal that can be
acquired. Such two-photon imaging of fluorescent
proteins can be used to
study cell events in vivo
such as the division of a
human malignant cell,
changes in neuronal connectivity with time, nerve
cell activity or blood flow.
“Now in widespread use”,
says Emmanuel Beaurepaire * of the Palaiseau
Laboratory of Optics &
Biological Sciences, “this
technique looks at fluorescent signals from labels
like GFP (see p. 24-25) or
from naturally fluorescent
proteins like elastin and
keratin.”
The idea of two-photon
excitation was first proposed in 1931 by the German-American physicist
Maria Goeppert-Mayer
who won the Nobel Prize
in Physics in 1963. Her
work predicted that, in
theory, a single molecule
could absorb two photons at the same time.
But it was not until 1990
that a report from scientists at Cornell University in the United States
was published in Science
grand angle
➜
©©LOB Polytechnique-CNRS-Inserm / Institut de la Vision
Brain tissue in a
brainbow mouse
(multicolor two-photon
microscopy)
on a concrete application of the theoretical concept
to microscopy. “Implementation of this technique required the development of femtosecond laser pulsing”
explains Serge Charpak *, Director of the Laboratory of Neurophysiology & Modern Microscopy in
Paris. This means devices that emit very short, intense
pulses of laser light, thereby increasing the likelihood
that two photons might be absorbed at the same time
by a fluorescent molecule without delivering too much
energy overall. The photons in these pulses are in the
infrared (IR) range of 690-1,300 nm, corresponding
to half of the energy of those in a continuous beam of
ultraviolet (UV) or visible light.
“The advantage of this type of microscopy lies in the IR
pulses which, by virtue of their longer wavelength, penetrate more deeply into tissue,” explains Serge Charpak,
“of the order of a millimeter compared with hundreds
of micrometers for UV or visible light.” But that is not
all. The advantage of using two low-energy photons
to excite a molecule instead of one more powerful
one is that only molecules located at the focal point
get excited and emit light. Others, struck by a single
IR photon remain in a resting state. In consequence,
images are sharper because they are not parasitized
by “noise” due to the excitation of distant molecules
as is the case with continuous UV or visible light. “As
few molecules outside of the focal area are excited, there
is less light-induced damage or destruction of the biological structures being examined”, adds the scientist.
With its capacity for deep penetration into tissue
(0.5-1 mm) without interfering with cells that
are not being examined
coupled with the high
resolution of the images
(of the order of one micrometer), two-photon
fluorescence microscopy
is the ideal way of studying tissue structure and
cell functioning in vivo.
By scanning the surface
and deep inside, scientists obtain three-dimensional images or optical
slices on organisms that
are entirely alive. “This
creates three-dimensional
images of big volumes of
intact tissue”, confirms
Jean Livet, “which is not
possible with conventional
microscopy on sectioned tissue.”
And neuroscientists have rushed in to make the most
of the advantages of this non-invasive technique. In
2011, Serge Charpak and his colleagues measured
blood flow and oxygen tension in capillary vessels in
the rodent olfactory bulb(L). They demonstrated that
the concentration of oxygen fluctuates in the course of
sensory activation and, more recently, that the passage
of every red cell through a capillary in the brain is
accompanied by a transient rise in oxygen tension*.
But to put a brake on, two-photon microscopy cannot
be used to study brain activity in humans—for both
technical and ethical reasons. “This technology applies
above all to fundamental and applied research in animal
disease models,” emphasizes Serge Charpak.
However, two-photon microscopy has not finished
developing. We are looking at how to combine it with
other techniques like second harmonic generation microscopy to provide complementary information, or
light sheet fluorescence microscopy to speed up image
acquisition. “Excitation in light sheet fluorescence microscopy yields images about 100 times faster [Editor’s
Note: currently one image takes about a second and a
3D image takes a minute] or over a broader field, with
still less interference,” explains Emmanuel Beaurepaire.
He also talked about miniaturizing microscopes so
that they can be used to explore internal tissues like
an endoscope. In conclusion, photons are not ready
to retire from light microscopy.
LOlfactory bulb
The part of the brain that
first processes olfactory
information coming from
neurons in the epithelium
of the nasal cavities.
W. Denk et al. Science, April 1990;
248: 73-6
J. Lecoq et al. Nature Medicine, June
2011 ; 17(7): 893-8
D. A. Dombeck et al. Nature Neuroscience,
October 2010; 13: 1433-40
JUILLET - AOÛT 2015 ● N° 26 ●
●
11
grand angle
➜
TREAT
Linking light with genetics—a feat called optogenetics, a
new scientific discipline that is opening huge possibilities,
especially in neurodegenerative disease. Another
accomplishment now within reach thanks to photodynamic
therapy—fighting cancer with light.
Optogenetics
A technique to switch neurons
on and off
©©Hanen Khabou
of degenerative disease of the retina, light is directly
delivered to neural tissue via the retina. But in the experiments on neurons in laboratory rodents, relatively
non-invasive devices based on optic fibers were used
to target specific deep tissues.
É. Burguière et al. Science, 7 June 2013 ;
340 (6137) : 1243-6
H. Ye et al. Science, 24 June 2011 ;
332 (6037) : 1565-8
M. Choi et al. Nature Photonics,
20 October 2013 ; 7 : 987-94
K. Deisseroth et al. Science,
17 April 2009 ; 324 (5925) : 354-9
Z. H. Pan et al. Neuron, 6 April 2006 ;
50 (1) : 23-33
☛☛Deniz Dalkara: Inserm Unit 968/CNRS –
Université Pierre-et-Marie-Curie
☛☛Éric Burguière: Inserm UMRS 1127/CNRS
– Université Pierre-et-Marie-Curie
12 ●
S
ince the early 2000’s, optogenetics* involves inserting a gene into a neuron to make it sensitive
to light. Why? To stimulate or inhibit specific
functions when the cells are illuminated. “To insert the
gene, we use adeno-associated virus (AAV),” explains
Deniz Dalkara * of the Vision Institute. “These
vectors readily cross plasma membranes making the
method relatively non-invasive and, most importantly,
applicable in vivo.” This non-pathogenic virus transports the gene right inside the cell without eliciting
a significant immune response. Depending on the
cell-type in question, some function may be stimulated or inhibited by exposure to light. In the case
● N° 26 ● JUILLET - AOÛT 2015
In the Institute in which Deniz Dalkara works, optogenetics is seen as a promising way of restoring sight
in people with degenerative disease of the retina, the
most common of which is retinitis pigmentosa (L).
In these diseases, photosensitive cells like cones (L)
and rods (L) gradually lose their responsiveness to
light, sometimes leading to complete blindness: they
are referred to as sleeping cells. The idea is to insert
a gene from the bacterium Natronomonas pharaonis
that codes for halorhodopsin, a photosensitive transmembrane protein, into
“sleeping” cone cells to
Mouse photoreceptor
restore their responcells expressing
siveness to light. When
halorhodopsin (showing
exposed to light from
in green).
outside, these cells send
the electrical signals required for vision, thereby compensating for the impairment. An optogenetic strategy that has already
yielded results: “In 2006, a group at Wayne State University in Detroit isolated the gene that codes for channelrhodopsin-2, a photosensitive protein, from green
alga Chlamydomonas reinhardtii and inserted it into
the retinas of blind mice and managed to restore some of
their sight,” recounts the scientist. “After treatment, the
animals were able to distinguish between light and dark.
In 2010, Swiss researchers from the Friedrich Miescher
Institute in Basel working together with scientists from
the Vision Institute observed promising results after
inserting the Natronomonas pharaonis gene in mice
grand angle
©©inserm/delapierre patrick
➜
with retinitis pigmentosa. The animals recovered so- togenetics, we can envisage thousands of photoreceptor
phisticated visual functions like the capacity to detect cells with at least 100 times better definition.” movement and contrast, and were able to move around
according to the light. This approach is now being in- And partial or complete sight restoration is not the
vestigated in macaques whose endogenous responses to only possibility offered by this new approach. The
light have been simply blocked (rather than their having ability to activate and inhibit specific physiological
been rendered blind).” functions by light, i.e. at a distance, appeals to scienAccording to Deniz Dalkara, early results on the ef- tists in diverse fields, including Éric Burguière *, a
ficacy and safety of this treatment modality are very neuroscientists from the Brain & Spinal Cord Institute
promising. Although these results are ready for pub- in Paris, who works on obsessive compulsive disorlication, she nevertheless adds a proviso: “The bac- der (OCD) (L). In 2013, he reported the results of
terial gene acts as a short-cut to the light. It responds experiments on mice showing compulsive behavior
to it through the expression of a single protein. In con- in the form of excessive grooming. Using an optotrast, as a result of more complicated
genetic strategy based the channelevolution, our bodies respond in a far
rhodopsin-2 gene from green alga
more complex way.” In human beings, “Good outcomes in
C. reinhardtii, his group managed to
sight is the result of the expression of mice with retinitis
mitigate their compulsive behavior by
many different proteins. In a pathway pigmentosa„
light stimulation via a device containreferred to as the phototransduction
ing optic fibers implanted into the
cascade which is composed of a series
heads of the mice. This confirmed
of biochemical reactions that convert a light signal into their hypothesis that compulsive behavior patterns
a set of nervous impulses. “Optogenetics is just a crutch like those seen in OCD are due to impaired inhibitory
for the time being,” she adds. “It just restores colorless mechanisms. In practice, although optogenetics may
vision. Moreover, it requires intense illumination and lead to novel treatment modalities, it is above all else
the image’s definition is poor. In the next few years, we a powerful aid to understanding fundamental physihope to be able to help blind people see something but ological mechanisms.
we will not be able to restore their sight straight away.” Deniz Dalkara nevertheless promises that this tech- This technique combining genetics and optics is also
nology will be improved. Although other strategies are being used to identify which cells are involved in disin development, including approaches based on stem ease. In 2009, scientists from Stanford University did
cells and electrical implants, it seems that optogenet- interesting work on a murine model of Parkinson’s
ics could see success in the field. “It is more difficult disease. Using the genes for halorhodopsin and later
to generate stem photoreceptor cells than to make cells channelrhodopsin, they identified novel areas that
photoreceptive by means of optogenetics. As for retinal might respond to deep brain stimulation. Blindness,
implants which are already commercially available [Ed- OCD, Parkinson’s disease, ... All targets for optogenetic
itor’s Note: Argus II from the Second Sight company], approaches. But it is not only the neuroscientists who
they carry 60 electrodes which generate the same num- are excited. In 2011, a group at the École Polytechnique
ber of pixels so resolution is poor,” he notes. “With op- in Palaiseau used this strategy to induce insulin secretion in diabetic mice with flashing light. Other
scientists have applied optogenetics to cardiology.
In optogenetics, light can be directed through
In 2010, a group from the
optic fibers.
University of California
controlled the activity of
the heart (tachycardia,
bradycardia and cardiac
arrest) in Zebrafish with
different types of light
emission by implanting
the genes that code for
channelrhodopsin and
halorhodopsin. Above
and beyond the scientific and ethical problems
posed by modification
of the genome, there
remains the problem of
how to deliver the light.
“For delivery, we cur- 
etinitis
LRpigmentosa
Degeneration of the retina
that can cause blindness.
LCones
Photoreceptor cells in
the retina responsible
for color perception and
visual acuity in the middle
of the visual field.
LRods
Photoreceptor cells in
the retina responsible
for night vision (low light)
and the detection of
movement.
bsessive
LOcompulsive
disorder
An anxiety disorder
characterized by certain
recurrent thoughts
(sometimes phobic)
JUILLET - AOÛT 2015 ● N° 26 ●
●
13
grand angle
➜
LHydrogel
A matrix made of a fluid
component and a solid
component (notably
used to make soft
contact lenses).
 rently use optic fibers,” specifies Éric Burguière. “Although
they are thin, the devices still necessitate an invasive procedure in
a living organism.” But scientists
are mobilizing on this too. A team
at the Korea
A d v a n c e d “Fiber optic
Institute of
Science and remains an
Technology in invasive
Daejeon has device„
developed an
implant composed of a hydrogel (L) which
can conduct light inside a mouse’s
body. All these experiments are
far from leading to treatments
for human beings but, after just
ten years, this new discipline is
already shining light in a number
of medical fields.
Light delivery
to neurons
in the brain via an implanted
bundle of optic fibers.
©©Inbal Goshen and Karl Deisseroth
TREAT
Photodynamic therapy
The destructive power
of light
I
☛☛Serge Mordon: Inserm Unit 1189/CHRU
de Lille/CNRS – Université de Lille 2
14 ●
magine a good fairy hovering over a patient with
cancer with light streaming out of the end of her
magic wand making all the cancer cells disappear!
The only fictional bits in this story are the fairy and the
magic. Because the rest is possible for doctors with the
technology. It is indeed possible to treat some forms
of cancer with light, including skin cancer. Photodynamic therapy (PDT) involves applying a topical
photosensitizing agent, 5- aminolevulinic acid and
methyl-aminolevulinate, that is absorbed over a longer
time frame by malignant cells than healthy cells. After
a certain period when most of the healthy cells have
cleared the product, the area to be treated is exposed
to light and the cancer cells die. Any photosensitizing agent only responds to a certain wavelength. “It’s
a chemical reaction between the product and oxygen,
● N° 26 ● JUILLET - AOÛT 2015
induced by light,” explains Serge Mordon *, Director of the Onco-THAI (Image-assisted laser therapy
in oncology) Unit, one of the world leaders in PDT.
“Unlike chemotherapy which indiscriminately affects
both diseased and healthy tissue, PDT is targeted,” he
emphasizes. “In some cases, it can be used instead of or
as well as surgery, before or after the operation.” But what types of cancer can be treated in this way?
First example: actinic keratitis, pre-malignant lesions
on the scalp due to prolonged exposure to ultraviolet
light. “Many lesions can be surgically removed but some
are so small that they cannot be seen by eye,” points out
Serge Mordon. “PDT treats the whole skull, missing
nothing.” There is therefore no need for surgery and
the risk of recurrence is substantially lower because
of the thoroughness of the treatment. Other forms of
grand angle
©©Inserm Unit 1189 ONCO-THAI
skin cancer like carcinoma (L) can also be treated.
The principle is the same: the photosensitizing agent
is applied to the patch of skin to be treated which is
then illuminated with light of the right wavelength.
Through the work of Serge Mordon, PDT could eventually be used on other forms of cancer, including of
the prostate, brain and cervix or the peritoneal and
pleural cavities (e.g. asbestosis). With a significant
contribution from the National School of Arts & the
Textile Industry in Lille, he has developed a major
innovation in the form of a fabric containing optic
fibers coupled with a laser, to deliver uniform light of
a precise wavelength over
a large area. Because, as
“Photosensitizing
we know, photosensitizagents respond to
ing agents only respond
light of a specific
to light of a specific wavewavelength and a
length and given intengiven intensity„
sity. “If the signal is too
weak, there is no reaction.
If it is too strong, the product breaks down too quickly.
In both cases, the result is inefficacy,” affirms the scientist. The malleable fabric fits snugly over the surface
to be treated to deliver a perfectly uniform stimulus.
Since September 2014, Onco-THAI has been testing a
prototype cap made of this fabric to treat patients with
©©Inserm Unit 1189 ONCO-THAI
➜
actinic keratitis in the Flexitheralight Project. Results
will be published soon. Serge Mordon is co-ordinating another project, Phos-Istos, which is developing
a mobile PDT machine so that
patients with actinic keratitis
can be treated at home. Which
would save the four hours of
hospital time required today.
And this fabric that can now
be produced on an industrial
scale could also be used to
treat neonatal jaundice (L ).
This does not require any photosensitizing agent but simple
exposure to blue light to break
down the bilirubin that causes
the yellowness. The fabric can
be connected to any kind of
light source and its flexibility
makes it possible to produce a
sheet or even comfortable pajamas. A possibility not available
today—and one that combines
French expertise in textile technology and medicine.
Treating neonatal
jaundice with
phototherapy
delivered via a
fabric made of
fiber optic
LCarcinoma
Cancer derived from
epithelial cells like those
of the skin or mucous
membranes
LNeonatal
jaundice
Jaundice characterized by
yellow skin and mucous
membranes as a result
of the breakdown of
hemoglobin in
the blood.
A patient with
actinic keratitis
being treated with
the Flexitheralight
device.
JUILLET - AOÛT 2015 ● N° 26 ●
●
15
grand angle
➜
Repair
Light can stimulate the brain and treat certain forms of
depression. How does it work? Through a light-sensitive
photopigment called melanopsin found in the retina of the
eye. And as well as for cutting and cauterizing, laser light
can be used to generate living tissue in a process called
bioprinting.
Melanopsin
Another perception
of light
LfMRI
Medical imaging
technique that yields a
two- or three-dimensional
image used to study how
the brain works.
☛Howard
☛
Cooper : Inserm Unit 846/Inra –
Université Claude-Bernard Lyon 1
Seasonal affective
disorder can be treated
by light therapy.
S. Laxhmi Chellappa et al. PNAS, 22 April
2014; 111 (16): 6087-91
16 ●
● N° 26 ● JUILLET - AOÛT 2015
to which they had been exposed previously. When
they were first exposed to orange light (589 nm), they
performed better under the test light than after exposure to blue light (461 nm). Evidence that “colder”
light enhances cognitive function on the one hand
and on the other, that this improvement is associated
©©Frank MULLER/HH-REA
A
good tickle with light and we’re off? This is essentially the conclusion from experiments carried
out last year in the Chronobiology Department
of the Inserm Stem Cells & Brain Institute at Bron near
Lyon, working in collaboration with the Cyclotron
Research Center at Liège University. The researchers managed to show
that exposure to light
can enhance cognitive
functions like memory,
speech, concentration
and reasoning. The subjects were first exposed
to light of various colors and wavelengths for
an hour and were then
asked to perform a series
of auditory tests under
green test illumination
(515 nm). Cerebral activity was monitored
by functional magnetic
re s on anc e i m ag i ng
(fMRI) ( L ) . Results
were better when the tests
were performed under a
“colder” light than that
grand angle
➜
with “photic memory”. And this is what most
interests these scientists. Photic memory (a
delayed light effect) manifests when preliminary exposure to light affects responses to
subsequent exposure. “Melanopsin underlies
this mechanism,” reveals Howard Cooper *,
Director of the Chronobiology Department
who co-authored this publication.
Melanopsin is a photopigment discovered
fifteen years ago by Ignacio Provencio, a researcher at Virginia University. “It is not found
in the photoreceptor cells of the eye’s retina—the
rods and cones—but in ganglion cells, neurons
that are responsible for receiving visual information
from these photoreceptor cells and relaying it to the
brain, mainly via the optic nerve,” stipulated Howard
Cooper. Until the
discovery of this
“Some types of light
pigment in 1998,
have a beneficial effect
scientists believed
that light percepon cognitive function„
tion depended
exclusively on rod
and cone cells, the only photoreceptive cells known
in vertebrates.
“However, light-dependent physiological phenomena
were clearly in operation in blind mice and some blind
human beings,” he remembers. Subsequent experiments showed that this photopigment supported a
non-visual, light-dependent pathway that affected various phenomena like circadian rhythm (L). Melanopsin regulates the cyclical secretion of melatonin (the
sleep hormone) and cortisone (which is important in
carbohydrate metabolism).
“How light affects the brain is still poorly understood,”
states Howard Cooper in mitigation, “but melanopsin
seems to be important. As we showed, some types of light
can enhance cognitive function.” The scientists are now
investigating how to capitalize on this phenomenon.
One aim is to optimize lighting in offices to enhance
employee efficiency. “We know that melanopsin is more
sensitive to blue light,” explains the chronobiologist.
“In our experiments, we vary the light throughout the
day to adapt to the physiology of the subjects, namely
bluer light in the early morning for stimulation, blue
light all day to sustain concentration, and then warmer
light towards the end of the day to prepare for sleep.” In
everyday life, you see the effects: watching television
or looking at the screen of a computer, tablet or smart
phone—all of which tend towards the blue end of the
spectrum—tends to make falling asleep more diffi-
©©Inserm/ Howard Cooper
A cross-section through the retina
of a mouse showing cone cells in the
outer layer (green) and a ganglion cell
expressing melanopsin in the innermost
layer (red).
cult. However, “although different types of light have
been observed to have disparate effects, the underlying
mechanisms have not been elucidated,” says Howard
Cooper. “And research in this field is complicated in
the real world because so many complex parameters
need to be taken into account.” In consequence, such
projects tend to take a long time to set up and are
difficult to fund.
But enhanced productivity may not be the only benefit. “Melanopsin may also be important in the effects
of light on the onset and treatment of seasonal affective disorder,” adds Howard Cooper. This condition
comes on when the days shorten, affecting some 3%
of the population, according to the scientist. It is often
treated with light therapy in which the patient is exposed to artificial white light at an intensity of about
20,000 lux (L) for half-an-hour to two hours a day
(depending on the intensity). An attenuated form of
the problem commonly referred to as “Winter Blues”
may affect 30% of people according to Howard Cooper. While just one sunny day may be enough to chase
this problem away. “Light can act very fast,” he points
out. “A day skiing on a beautiful winter’s day can have
immediate beneficial effects.” To investigate how light modulates different physiological functions, Howard Cooper is launching into
a long-term project. In partnership with the Salk Institute for Biological Studies in San Diego, California
and the Institute for Primate Research in Nairobi,
Kenya, he plans to study the effects of varying light
over the course of a day on gene expression in primates. The scientist is enthusiastic: “Instead of studying a single brain structure, we are going to look at the
impact of light on all the body’s brain structures and
organs.” Knowing which physiological processes are
stimulated by light and which are inhibited, and how
these processes are coordinated over the day could
open up new avenues of research on melanopsin.
ircadian
LCrhythm
A biological cycle lasting
24 hours like the sleepwake cycle
LLux
The measurement unit
for illumination. Outside
in the daytime, we may
perceive between 500 and
100,000 lux, depending
on whether the sky is
covered or there is bright
sunshine.
JUILLET - AOÛT 2015 ● N° 26 ●
●
17
grand angle
➜
••
••
Repair
Bioprinting
When light creates life
“Multicomponent”
bioprinting machine
that can print five
different cell-types at
the same time.
©©Inserm Unit 1026
in the way we are used
to printing out a page
of text, this technology
nevertheless holds great
promise.
The principle is based on
applying the methods of
three-dimensional printing to build up, layer by
layer, the various constituents of biological tissues.
This makes it possible to
create extremely complex
structures using biologiedical lasers can make finer
cal inks containing high-density cells,
cuts than any scalpel or burn “Using a laser,
quickly and at high resolution. “Using
more precisely than any other the positions of cells
a laser, the positions of cells in space can
instrument. But they also have less “ag- in space
be precisely controlled,” explains Joëlle
gressive” uses. The Tissue Engineering can be precisely
Amédée. Biological components are
Laboratory in Bordeaux, called Bio- controlled„
transferred to create one layer, then
Tis, uses the most innovative aspects
another, and then yet another, until
of laser technology. “We use lasers to
the desired structure has been assemtransfer structures, both biological and non-biological bled. Another advantage of this technology: “The
elements,” says Joëlle Amédée *, Director of Bio- laser’s settings can be adjusted to ensure printing conTis. In other words, they can print out living cells, ditions that do not damage living cells.” The groups of
growth factors, extracellular matrix proteins (L) Fabien Guillemot and then Raphaël Devillard *
and other biological elements, to reconstruct whole have shown that it is possible to print human stem
tissues! This team has been working on this for some cells that still perform the same functions after the
ten years and have produced some impressive results printing process. This is a crucial point because the
on reconstructing bone tissue in mice. It is on the basis aim is to create living tissues.
of such results that Fabien Guillemot *, one of the “Laser printing is being applied to bone tissue, blood vespioneers of research in this field, founded Poietis*, an sels ...,” says Joëlle Amédée. And skin, one of the special
Inserm spin-off specializing in laser-assisted bioprint- interests of Poietis. As for BioTis, they are working on
ing. Although neither BioTis nor Poeitis are yet ready damaged bones. Although Joëlle underlines that her
to print out a piece of bone tissue or a patch of skin team is far from reconstituting complete bones, “on
M
xtracellular
LEmatrix
Complex structure
composed of various
macromolecules that fills
the space between cells,
facilitating their mutual
binding and supporting
tissue architecture
18 ●
● N° 26 ● JUILLET - AOÛT 2015
grand angle
damaged bones, we can already print cells
out in situ which, when combined with
other biological materials, are capable
of reconstructing missing bone tissue.”
This does not therefore mean printing
out a piece of bone and “sticking it on”
but rather directly implanting cellular
material on the body so that recovery is
faster and better.
At Poietis, another order of developments is under way. Their priority is
not surgical repair. Rather, the directors’ ambitions concern making tissue
for pharmacological testing. This would
allow the pharmaceutical industry to
test responses to its products, especially
their toxicity, on living tissue created ex
nihilo. A cheaper solution that would
preclude the need for tests on human
beings and animals. The cosmetic industry, also being targeted by Fabien
Guillemot, would also be happy to have
such an option, all the more since it has
lost the right to carry out its tests on animals. Finally, a third possibility being
investigated by the biotechnology company is reproducing a patient’s tissues
to predict responses to drugs, e.g. using
tumor cells to predict the likelihood of
success with a given chemotherapy protocol. A strategy which could minimize the risk of administering
useless treatment. Savings in terms of money but,
more importantly in time and danger for the patient.
“Being able to prescribe an effective drug straight away
will afford faster cure and avoid side effects due to useless treatments,” emphasizes Fabien Guillemot.
We are on the right track although the technology
is not yet mature. “The challenge right now in bioprinting, including laser bioprinting, is how to make
functional tissue by correctly assembling different cells
to create successive layers,” explains Joëlle Amédée.
Although the scientists at the Bordeaux Tissue En-
©©Inserm Unit 1026
➜
Laying down a network between
bioprinted corneal matrix cells
gineering Laboratory are managing to create superimposed layers of cells, they are always asking the
questions: “Are they in the right order?” And “How
stable are these stacks?” The Director of BioTis draws
attention to another obstacle to be overcome, namely
bulk production. How to generate significant quantities of accurately replicated living tissues is the double
challenge being taken on the scientists at BioTis and
Poeitis.
☛☛Joëlle Amédée, Fabien Guillemot,
Raphaël Devillard: Inserm Unit 1026 –
Université de Bordeaux
A Light Show
For the Year of Light, Inserm is sponsoring “Shining the Light on Health
Care” A traveling exhibition that highlights the most innovative research
projects based on the use of light in Observation, Treatment and Repair
of the human body. It can be visited at a series of Inserm events: on 25
September in Toulouse at the Cité de l’Espace; from 28 September to 18
October at the Chapelle des Pénitents Bleus at La Ciotat; and from 7-11
October in various towns, starting in Paris, Brest, Lille, Montpellier and
Strasbourg during the Fête de la Science. It will also be presented at the International Science
Fiction festival at Les Utopiales in Nantes, from 28 October to 2 November 2015.
www.inserm.fr
JUILLET - AOÛT 2015 ● N° 26 ●
●
19
INSERM-affiche-215x252-3-vecto.indd 1
11/06/15 16:09
101, rue de Tolbiac
75654 Paris Cedex 13
www.inserm.fr
free magazine
not for sale