Breakthrough(s): Imaging Neuronal Function via Osmosis?

Even today, in our hyper-technological society, we find ourselves at a loss when it comes to the diagnosis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. This major, unmet need represents a glaring shortcoming in an era of abundant medical advances.

However, a recently preprinted paper has provided an encouraging glimmer of hope. By using osmotic swelling as a proxy for neuronal function, Pfäffle and colleagues have demonstrated a novel means of optically imaging neuronal activity in living human beings (1).

We all have cities of knowledge nestled into our brains. Such is why diseases of the nervous system are so terrifying. They mange to sneak up on us, like a Trojan Horse, threatening to destroy the very thing we hold most sacred. (Image above is a painting by the author)

Societal Impact: Confronting the unknown that shrouds diseases of the brain and nervous system.

A hallmark of many neurodegenerative diseases is clandestine progression. Diseases such as Alzheimer’s disease are known to worsen silently, beneath a veil of secrecy. This characteristic, silent progression casts neurodegenerative diseases in a grim light. Without adequate intervention during this ‘silent’ phase, the nervous systems of unsuspecting (and oftentimes otherwise healthy) individuals can undergo irreparable damage. Accordingly, people with a family history of Alzheimer’s disease may helplessly wonder: ‘When will my mental faculties begin to slip away?’

Assessing neuronal function in living human beings: Where are we today?

Scientific advances have yielded a plethora of means via which to record neuronal function. Innovative genetically-encoded sensors, such as proteins that fluoresce in response to neuronal activity, have succeeded in catapulting the field of neuroscience to new and exciting heights. However, due to invasive genetic manipulations and tedious experimental paradigms, these flashy approaches have failed to translate into novel diagnostic tools.

Regarding the assessment of neuronal activity in living humans, electroencephalography (EEG) and functional MRI (fMRI) represent two approaches dominating the current clinical landscape. EEG, which records the electrical activity of a person’s brain via scalp electrodes, affords outstanding temporal resolution. As such, EEG is commonly used to diagnose conditions in which the brain’s innate rhythmicity becomes perturbed (e.g. epilepsy). However, due to the fact that EEG acquires its signal from surface electrodes, it suffers from impoverished spatial resolution.

Comparatively, fMRI offers much improved spatial resolution, but does so at the cost of temporal fidelity. Accordingly, volumetric nature of fMRI allows clinicians to image, in three dimensions, the function of specific brain regions. To assess brain function, fMRI relies upon the notion that changes in blood oxygen level serve as a proxy of neuronal activity. This imposes yet a further lag upon the signal obtained via fMRI, as changes in blood oxygenation considerably trail the antecedent neural impulses. Nonetheless, fMRI remains the gold-standard technique via which to image brain function in living humans.

In addition to assessing changes in blood oxygenation, MRI is capable of dynamically assessing the diffusion of water within biological tissues. Towards this end, a widely-used MRI scanning technique, termed diffusion tensor imaging, is often used to trace out the tracts of axon bundles that traverse the brain. Pursuantly, diffusion tensor imaging is principally used to investigate brain connectivity (e.g. How are certain brain regions ‘wired together’?).¹

Figure 1: A colorful depiction of diffusion tensor imaging as provided by Ref. 2. The arbitrary colors are used to identify distinct nerve fiber bundles as they traverse a subject’s brain.

Expanding upon the diffusional prowess of MRI, an innovative MRI technique, diffusion functional MRI (diffusion fMRI), offers a more direct means of assessing neuronal function than fMRI (2).

The basis of diffusion fMRI rests upon the notion that neuronal excitation produces an osmotic gradient which forces water to transiently diffuse into neurons, thereby causing the excited neurons to momentarily swell.² Consequently, diffusion fMRI can faithfully discern brain function by detecting regions in which diffusion is transiently decreased.³

Figure 2. A diffusion fMRI image in response to a flickering visual stimulus. The orange-yellowish voxels, which are located predominately in the vicinity of the primary visual cortex, indicate areas of decreased diffusion.

Nonetheless, a persistent shortcoming of MRI is that MRI remains an intrinsically noisy imaging technique. Furthermore, when compared with optical imaging techniques, MRI offers a spatial resolution that is quite poor. As such, MRI-based approaches are not ideally suited to detect the subtle indications that reveal themselves in early-stage neurodegenerative disease.

Looking into the Eye (and Towards the Future)

To faithfully detect the subtle functional alterations that occur at the onset of neurodegeneration, we need to start thinking optically: How can we see neuronal (dys)function?

Unfortunately, one cannot, under typical circumstances, hope to gain direct optical access to the brain. However, both of your eyes are equipped with millions of intricately connected neurons, many of which are wired directly to your brain (i.e. retinal ganglion cells). Moreover, millions of years of evolutionary optimization have engineered our eyes to transmit light and focus it upon the retina. As such, the retina provides an ideal tissue in which to see neuronal function!

The eyes’ optical accessibility, and their innate connectivity to the brain, have led numerous investigators to inquire whether the eye might faithfully serve as a ‘window to the brain’, thereby providing an advantageous perch upon which to survey the health of the central nervous system (3).

Specifically, in the context of Alzheimer’s disease, such inquiries have yielded a definitive yes, providing strong evidence that the retina undergoes marked anatomical alterations in the wake of Alzheimer’s disease. In particular, it has been shown that the retinal nerve fiber layer thins as a function Alzheimer’s disease progression (4). Such thinning of the retinal nerve fiber layer may provide an indication that Alzheimer’s disease causes retinal ganglion cells, which connect the eyes and brain, to undergo atrophy and/or cell death.

Figure 3: An optical coherence tomogram of the author’s retina. The retinal nerve fiber layer, the inner most layer (e.g. the closest layer to the front of the eye), is shown delineated via curly bracket. In the direction of the optic nerve head, the nerve fiber layer thickens as more and more axons fasiculate into constituent bundles. Such thickening is observable in the left-to-right direction.

Crucial steps have undoubtedly been made towards elucidating that our eyes can indeed serve as portals through which neurodegenerative disease may be surveyed. Unfortunately, previous approaches have yet to thoroughly emphasize a functional imaging component. Correspondingly, prior efforts have failed to adequately reveal indications of the subtle dysfunction that forebears neurodegenerative disease.

A Functional Breakthrough: Optically Imaging Neural Activity via Osmosis!

To lift the veil of secrecy under which neurodegenerative disease progresses, and survey neuronal health within living human beings, functional imaging must be performed optically.

Unto this pursuit, Pfäffle et al. elucidate that functionally-induced, osmotically-driven swelling of retinal ganglion cells can be detected via optically. Using a technique that is aptly termed ‘functional optical coherence tomography’, their approach relies upon the notion that as cells become swollen with water, the optical properties of the tissue change. Specifically, swollen cells minutely lengthen the distance light must traverse. A Figure from Pfäffle et al. illustrating this novel finding is shown below:

Figure 4: Traces indicative of retinal ganglion cell function. The vertical dashed line at t=0 represents the time visual stimulus delivery. Subsequent increases in optical path length (Δ𝓁) provide osmotic indications of ganglion cell function.

By functionally imaging retinal ganglion cells in a living human subject, Pfäffle and colleagues have provided a bolus of promise. An innovation of this nature notches the initial first cut through which a river of diagnostic and/or therapeutic advances may foreseeably flow.

Further developments along the lines of Pfäffle et al. will fundamentally improve how we diagnose neurodegenerative diseases!

Translational Avenues: Inroads to Clinical Feasibility

One crucial question remains: How can advancements spurred by the work of Pfäffle et al. become commonplace in neurology clinics across the world?

On first glance, this appears a daunting proposition; technical limitations aside, the trek through regulatory red-tape is an arduous one. However, ~30 years ago, MRI was a primitive laboratory research tool. Fast-forward to the present, and you’d be hard-pressed to find a modern hospital that doesn’t have an MRI scanner.

In this regard, I sense that ophthalmic imaging technologies might be on a similar trajectory to MRI. Specifically, optical coherence tomography (OCT) devices have revolutionized the field of ophthalmology by providing rapid, non-invasive means via which to acquire three-dimensional images of the eye. Consequently, over the past 20 years, OCT has made its way into a larger and larger proportion of ophthalmology clinics.

Moreover, OCT has itself evolved. Just as MRI expanded into the realm of functional imaging, so has OCT! Within the last decade, methods for functionally assessing blood flow in the retina have been implemented using OCT. In its short time since being clinically introduced (FDA approved in 2015), OCT angiography has vastly improved how retinal vasculature is imaged in both health and disease.

Figure 5: An optical coherence tomography angiogram of the author’s eye. OCT angiography is a functional imaging technique, and via motion-contrast imaging, visualizes only blood vessels through which blood cells are actively flowing.

As evidenced by Pfäffle et al., however, the functional-imaging capabilities of OCT reach far beyond angiography. The efforts of Pfäffle and colleagues provide paradigm shifting evidence that OCT can be used to assess neuronal function. Consequently, OCT is on the cusp of fundamentally improving how devastating diseases such as Alzheimer’s disease are diagnosed.

By markedly enhancing diagnostic capabilities, OCT will provide clinicians the ability to survey subtle functional changes months, and even years, before symptoms are likely to occur. Via widening the therapeutic window to unprecedented widths, it is only a matter of time before efficacious strategies to combat once-untreatable diseases begin to emerge!

Notes:

¹ The lipid-rich myelin sheaths that surround the brain’s nerve fibers tend to constrain the diffusion of water along the length of the fibers. Thus, localized regions of highly directional diffusion provide an indication that nerve fibers are present.

² This neuronal swelling is due to an osmotic gradient that is generated in response to a neuron’s electrical activity. To change their membrane potential (a.k.a. voltage), neurons transiently change the concentration of positive ions within the cell. As neurons spike, sodium ions charge inward, and in doing so make the inside of the neuron more ‘salty’. This action creates a momentary osmotic gradient, forcing water to flow into the cell to compensate for said ‘saltiness’.

³ Water becomes momentarily concentrated within intracellular compartments as opposed to freely diffusing around the extracellular space. This creates a ‘diffusional contrast’ of sorts.

References (click/tap to the citations to access full text articles)

1. Pfäffle, C., Hillmann, D., Spahr, H., Kutzner, L., Burhan, S., Hilge, F., … & Hüttmann, G. (2018). Functional imaging of ganglion and receptor cells in living human retina by osmotic contrast. arXiv preprint arXiv:1809.02812.
2. Le Bihan, D., & Iima, M. (2015). Diffusion magnetic resonance imaging: what water tells us about biological tissues. PLoS Biology, 13(7), e1002203.
3. London, A., Benhar, I., & Schwartz, M. (2013). The retina as a window to the brain — from eye research to CNS disorders. Nature Reviews Neurology, 9(1), 44.
4. Kirbas, S., Turkyilmaz, K., Anlar, O., Tufekci, A., & Durmus, M. (2013). Retinal nerve fiber layer thickness in patients with Alzheimer disease. Journal of Neuro-Ophthalmology, 33(1), 58–61.