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Taking Brain Imaging Even Deeper

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Thanks to yet another amazing advance made possible by the NIH-led supported the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative, I can now take you on a 3D fly-through of all six layers of the part of the mammalian brain that processes external signals into vision. This unprecedented view is made possible by three-photon microscopy, a low-energy imaging approach that is allowing researchers to peer deeply within the brains of living creatures without damaging or killing their brain cells.

The basic idea of multi-photon microscopy is this: for fluorescence microscopy to work, you want to deliver a specific energy level of photons (usually with a laser) to excite a fluorescent molecule, so that it will emit light at a slightly lower energy (longer wavelength) and be visualized as a burst of colored light in the microscope. That’s how fluorescence works. Green fluorescent protein (GFP) is one of many proteins that can be engineered into cells or mice to make that possible.

But for that version of the approach to work on tissue, the excited photons need to penetrate deeply, and that’s not possible for such high energy photons. So two-photon strategies were developed, where it takes the sum of the energy of two simultaneous photons to hit the target in order to activate the fluorophore.

That approach has made a big difference, but for deep tissue penetration the photons are still too high in energy. Enter the three-photon version! Now the even lower energy of the photons makes tissue more optically transparent, though for activation of the fluorescent protein, three photons have to hit it simultaneously. But that’s part of the beauty of the system—the visual “noise” also goes down.

This particular video shows what takes place in the visual cortex of mice when objects pass before their eyes. As the objects appear, specific neurons (green) are activated to process the incoming information. Nearby, and slightly obscuring the view, are the blood vessels (pink, violet) that nourish the brain. At 33 seconds into the video, you can see the neurons’ myelin sheaths (pink) branching into the white matter of the brain’s subplate, which plays a key role in organizing the visual cortex during development.

This video comes from a recent paper in Nature Communications by a team from Massachusetts Institute of Technology, Cambridge [1]. To obtain this pioneering view of the brain, Mriganka Sur, Murat Yildirim, and their colleagues built an innovative microscope that emits three low-energy photons. After carefully optimizing the system, they were able to peer more than 1,000 microns (0.05 inches) deep into the visual cortex of a live, alert mouse, far surpassing the imaging capacity of standard one-photon microscopy (100 microns) and two-photon microscopy (400-500 microns).

This improved imaging depth allowed the team to plumb all six layers of the visual cortex (two-photon microscopy tops out at about three layers), as well as to record in real time the brain’s visual processing activities. Helping the researchers to achieve this feat was the availability of a genetically engineered mouse model in which the cells of the visual cortex are color labelled to distinguish blood vessels from neurons, and to show when neurons are active.

During their in-depth imaging experiments, the MIT researchers found that each of the visual cortex’s six layers exhibited different responses to incoming visual information. One of the team’s most fascinating discoveries is that neurons residing on the subplate are actually quite active in adult animals. It had been assumed that these subplate neurons were active only during development. Their role in mature animals is now an open question for further study.

Sur often likens the work in his neuroscience lab to astronomers and their perpetual quest to see further into the cosmos—but his goal is to see ever deeper into the brain. His group, along with many other researchers supported by the BRAIN Initiative, are indeed proving themselves to be biological explorers of the first order.

Reference:

[1] Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Yildirim M, Sugihara H, So PTC, Sur M. Nat Commun. 2019 Jan 11;10(1):177.

Links:

Sur Lab (Massachusetts Institute of Technology, Cambridge)

The Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)

NIH Support: National Eye Institute; National Institute of Neurological Disorders and Stroke; National Institute of Biomedical Imaging and Bioengineering


‘Nanoantennae’ Make Infrared Vision Possible

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Nanoparticles for infrared vision
Caption: Nanoparticles (green) bind to light-sensing rod (violet) and cone (red) cells in the mouse retina. Dashed lines (white) highlight cells’ inner/outer segments.
Credit: Ma et al. Cell, 2019

Infrared vision often brings to mind night-vision goggles that allow soldiers to see in the dark, like you might have seen in the movie Zero Dark Thirty. But those bulky goggles may not be needed one day to scope out enemy territory or just the usual things that go bump in the night. In a dramatic advance that brings together material science and the mammalian vision system, researchers have just shown that specialized lab-made nanoparticles applied to the retina, the thin tissue lining the back of the eye, can extend natural vision to see in infrared light.

The researchers showed in mouse studies that their specially crafted nanoparticles bind to the retina’s light-sensing cells, where they act like “nanoantennae” for the animals to see and recognize shapes in infrared—day or night—for at least 10 weeks. Even better, the mice maintained their normal vision the whole time and showed no adverse health effects. In fact, some of the mice are still alive and well in the lab, although their ability to see in infrared may have worn off.

When light enters the eyes of mice, humans, or any mammal, light-sensing cells in the retina absorb wavelengths within the range of visible light. (That’s roughly from 400 to 700 nanometers.) While visible light includes all the colors of the rainbow, it actually accounts for only a fraction of the full electromagnetic spectrum. Left out are the longer wavelengths of infrared light. That makes infrared light invisible to the naked eye.

In the study reported in the journal Cell, an international research team including Gang Han, University of Massachusetts Medical School, Worcester, wanted to find a way for mammalian light-sensing cells to absorb and respond to the longer wavelengths of infrared [1]. It turns out Han’s team had just the thing to do it.

His NIH-funded team was already working on the nanoparticles now under study for application in a field called optogenetics—the use of light to control living brain cells [2]. Optogenetics normally involves the stimulation of genetically modified brain cells with blue light. The trouble is that blue light doesn’t penetrate brain tissue well.

That’s where Han’s so-called upconversion nanoparticles (UCNPs) came in. They attempt to get around the normal limitations of optogenetic tools by incorporating certain rare earth metals. Those metals have a natural ability to absorb lower energy infrared light and convert it into higher energy visible light (hence the term upconversion).

But could those UCNPs also serve as miniature antennae in the eye, receiving infrared light and emitting readily detected visible light? To find out in mouse studies, the researchers injected a dilute solution containing UCNPs into the back of eye. Such sub-retinal injections are used routinely by ophthalmologists to treat people with various eye problems.

These UCNPs were modified with a protein that allowed them to stick to light-sensing cells. Because of the way that UCNPs absorb and emit wavelengths of light energy, they should to stick to the light-sensing cells and make otherwise invisible infrared light visible as green light.

Their hunch proved correct, as mice treated with the UCNP solution began seeing in infrared! How could the researchers tell? First, they shined infrared light into the eyes of the mice. Their pupils constricted in response just as they would with visible light. Then the treated mice aced a series of maneuvers in the dark that their untreated counterparts couldn’t manage. The treated animals also could rely on infrared signals to make out shapes.

The research is not only fascinating, but its findings may also have a wide range of intriguing applications. One could imagine taking advantage of the technology for use in hiding encrypted messages in infrared or enabling people to acquire a temporary, built-in ability to see in complete darkness.

With some tweaks and continued research to confirm the safety of these nanoparticles, the system might also find use in medicine. For instance, the nanoparticles could potentially improve vision in those who can’t see certain colors. While such infrared vision technologies will take time to become more widely available, it’s a great example of how one area of science can cross-fertilize another.

References:

[1] Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae. Ma Y, Bao J, Zhang Y, Li Z, Zhou X, Wan C, Huang L, Zhao Y, Han G, Xue T. Cell. 2019 Feb 27. [Epub ahead of print]

[2] Near-Infrared-Light Activatable Nanoparticles for Deep-Tissue-Penetrating Wireless Optogenetics. Yu N, Huang L, Zhou Y, Xue T, Chen Z, Han G. Adv Healthc Mater. 2019 Jan 11:e1801132.

Links:

Diagram of the Eye (National Eye Institute/NIH)

Infrared Waves (NASA)

Visible Light (NASA)

Han Lab (University of Massachusetts, Worcester)

NIH Support: National Institute of Mental Health; National Institute of General Medical Sciences


Moving Closer to a Stem Cell-Based Treatment for AMD

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In recent years, researchers have figured out how to take a person’s skin or blood cells and turn them into induced pluripotent stem cells (iPSCs) that offer tremendous potential for regenerative medicine. Still, it’s been a challenge to devise safe and effective ways to move this discovery from the lab into the clinic. That’s why I’m pleased to highlight progress toward using iPSC technology to treat a major cause of vision loss: age-related macular degeneration (AMD).

In the new work, researchers from NIH’s National Eye Institute developed iPSCs from blood-forming stem cells isolated from blood donated by people with advanced AMD [1]. Next, these iPSCs were exposed to a variety of growth factors and placed on supportive scaffold that encouraged them to develop into healthy retinal pigment epithelium (RPE) tissue, which nurtures the light-sensing cells in the eye’s retina. The researchers went on to show that their lab-grown RPE patch could be transplanted safely into animal models of AMD, preventing blindness in the animals.

This preclinical work will now serve as the foundation for a safety trial of iPSC-derived RPE transplants in 12 human volunteers who have already suffered vision loss due to the more common “dry” form of AMD, for which there is currently no approved treatment. If all goes well, the NIH-led trial may begin enrolling patients as soon as this year.

Risk factors for AMD include a combination of genetic and environmental factors, including age and smoking. Currently, more than 2 million Americans have vision-threatening AMD, with millions more having early signs of the disease [2].

AMD involves progressive damage to the macula, an area of the retina about the size of a pinhead, made up of millions of light-sensing cells that generate our sharp, central vision. Though the exact causes of AMD are unknown, RPE cells early on become inflamed and lose their ability to clear away debris from the retina. This leads to more inflammation and progressive cell death.

As RPE cells are lost during the “dry” phase of the disease, light-sensing cells in the macula also start to die and reduce central vision. In some people, abnormal, leaky blood vessels will form near the macula, called “wet” AMD, spilling fluid and blood under the retina and causing significant vision loss. “Wet” AMD has approved treatments. “Dry” AMD does not.

But, advances in iPSC technology have brought hope that it might one day be possible to shore up degenerating RPE in those with dry AMD, halting the death of light-sensing cells and vision loss. In fact, preliminary studies conducted in Japan explored ways to deliver replacement RPE to the retina [3]. Though progress was made, those studies highlighted the need for more reliable ways to produce replacement RPE from a patient’s own cells. The Japanese program also raised concerns that iPSCs derived from people with AMD might be prone to cancer-causing genomic changes.

With these challenges in mind, the NEI team led by Kapil Bharti and Ruchi Sharma have designed a more robust process to produce RPE tissue suitable for testing in people. As described in Science Translational Medicine, they’ve come up with a three-step process.

Rather than using fibroblast cells from skin as others had done, Bharti and Sharma’s team started with blood-forming stem cells from three AMD patients. They reprogrammed those cells into “banks” of iPSCs containing multiple different clones, carefully screening them to ensure that they were free of potentially cancer-causing changes.

Next, those iPSCs were exposed to a special blend of growth factors to transform them into RPE tissue. That recipe has been pursued by other groups for a while, but needed to be particularly precise for this human application. In order for the tissue to function properly in the retina, the cells must assemble into a uniform sheet, just one-cell thick, and align facing in the same direction.

So, the researchers developed a specially designed scaffold made of biodegradable polymer nanofibers. That scaffold helps to ensure that the cells orient themselves correctly, while also lending strength for surgical transplantation. By spreading a single layer of iPSC-derived RPE progenitors onto their scaffolds and treating it with just the right growth factors, the researchers showed they could produce an RPE patch ready for the clinic in about 10 weeks.

To test the viability of the RPE patch, the researchers first transplanted a tiny version (containing about 2,500 RPE cells) into the eyes of a rat with a compromised immune system, which enables human cells to survive. By 10 weeks after surgery, the human replacement tissue had integrated into the animals’ retinas with no signs of toxicity.

Next, the researchers tested a larger RPE patch (containing 70,000 cells) in pigs with an AMD-like condition. This patch is the same size the researchers ultimately would expect to use in people. Ten weeks after surgery, the RPE patch had integrated into the animals’ eyes, where it protected the light-sensing cells that are so critical for vision, preventing blindness.

These results provide encouraging evidence that the iPSC approach to treating dry AMD should be both safe and effective. But only a well-designed human clinical trial, with all the appropriate prior oversights to be sure the benefits justify the risks, will prove whether or not this bold approach might be the solution to blindness faced by millions of people in the future.

As the U.S. population ages, the number of people with advanced AMD is expected to rise. With continued progress in treatment and prevention, including iPSC technology and many other promising approaches, the hope is that more people with AMD will retain healthy vision for a lifetime.

References:

[1] Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, Li Y, Stoddard J, Stankewicz C, Wan Q, Zhang C, Campos MM, Miyagishima KJ, McGaughey D, Villasmil R, Mattapallil M, Stanzel B, Qian H, Wong W, Chase L, Charles S, McGill T, Miller S, Maminishkis A, Amaral J, Bharti K. Sci Transl Med. 2019 Jan 16;11(475).

[2] Age-Related Macular Degeneration, National Eye Institute.

[3] Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration. Mandai M, Watanabe A, Kurimoto Y, Hirami Y, Takasu N, Ogawa S, Yamanaka S, Takahashi M, et al. N Engl J Med. 2017 Mar 16;376(11):1038-1046.

Links:

Facts About Age-Related Macular Degeneration (National Eye Institute/NIH)

Stem Cell-Based Treatment Used to Prevent Blindness in Animal Models of Retinal Degeneration (National Eye Institute/NIH)

Kapil Bharti (NEI)

NIH Support: National Eye Institute; Common Fund


Studying Color Vision in a Dish

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Credit: Eldred et al., Science

Researchers can now grow miniature versions of the human retina—the light-sensitive tissue at the back of the eye—right in a lab dish. While most “retina-in-a-dish” research is focused on finding cures for potentially blinding diseases, these organoids are also providing new insights into color vision.

Our ability to view the world in all of its rich and varied colors starts with the retina’s light-absorbing cone cells. In this image of a retinal organoid, you see cone cells (blue and green). Those labelled with blue produce a visual pigment that allows us to see the color blue, while those labelled green make visual pigments that let us see green or red. The cells that are labeled with red show the highly sensitive rod cells, which aren’t involved in color vision, but are very important for detecting motion and seeing at night.


A Ray of Molecular Beauty from Cryo-EM

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Rhodopsin

Credit: Subramaniam Lab, National Cancer Institute, NIH

Walk into a dark room, and it takes a minute to make out the objects, from the wallet on the table to the sleeping dog on the floor. But after a few seconds, our eyes are able to adjust and see in the near-dark, thanks to a protein called rhodopsin found at the surface of certain specialized cells in the retina, the thin, vision-initiating tissue that lines the back of the eye.

This illustration shows light-activating rhodopsin (orange). The light photons cause the activated form of rhodopsin to bind to its protein partner, transducin, made up of three subunits (green, yellow, and purple). The binding amplifies the visual signal, which then streams onward through the optic nerve for further processing in the brain—and the ability to avoid tripping over the dog.


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