The advancement of a wide-variety of scientific technologies and techniques over the past century has resulted in an explosion of knowledge in regards to the human body and its’ inter-workings. The development of neuroimaging technology has allowed researchers to understand many of the processes involved in communication within the central nervous system. However, what remains to be understood about the brain far outweighs what is currently known.
Due to unique structure features of brain cells that challenge researchers’ ability to clearly image the complex interconnections of the brain, the brain has persisted as a system of the human body with much left to understand. The innovative CLARITY method has broken these previous barriers to brain imaging and gives researchers access to uncharted parts of the brain. The access granted by this new method has the potential to dramatically increase the understanding of biological and chemical processes in the brain and represents a crucial step towards conquering the mysteries of the brain.
Computerized and magnetic technologies revolutionized neuroimaging in the late 20th century with the development of CT and later MRI scanning. The development of such neuroimaging technologies has provided scientists with a greater understanding of the functions of the different areas of the brain and important information regarding the activity of the brain’s neural network. However, while existing neuroimaging technologies have allowed scientists to visual the activity of the brain’s neural network or study the structure and function of individual axon’s that comprise such network, they lack the ability to comprehensively map axonal connections within and beyond the brain. This comprehensive mapping of the brain is necessary to extend scientists’ understanding of the organization of the brain’s neural network at an axonal level.
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The composition and structure of brain cells posed as the most significant challenge in the development of a technology capable of taking a comprehensive image of the brain’s internal network. To image the brain without structural destruction, light must penetrate the outer layers of the brain to allow visualization of internal tissue. However, lipid dense brain cell membranes act as a barrier that prevents light inside the brain by deflecting light protons at random directions (Deisseroth 2016). Therefore, successful internal imaging of the brain’s neural network is nearly impossible without bypassing these lipid molecules.
The ability to remove lipids from the brain to allowing imaging is challenged by both the density of lipids in the brain and their crucial role in providing structural support and facilitating fast axon communication. The brain is composed of both white and gray tissue matter, which vary with respect to composition and function and highlight the challenge of breaking through the barrier of light-blocking lipid cells in the brain. While gray matter composes the outer layer of the cerebral hemispheres, white matter is found deeper inside the brain and is formed by axons that connect different areas of gray matter (O’Brien & Sampson 1965). The significance of this tissue breakdown within the brain is due to differences in lipid composition of the different tissues.
Results from a study by Dr. John O’Brien and Dr. Lois Sampson of the University of Southern California School of Medicine indicate that the lipid composition of gray and white matter is 36-40% and 49-66% respectively (O’Brien & Sampson). Additionally, the myelin sheaths wrapped around axons to increase the speed of axon potentials and thus communication between neurons, were found to have a lipid composition of 78-81%. These results emphasize the density of lipids in the brain, indicating that successful internal visualization of the brain would not be possible without a method of lipid removal that also addressed the structural and functional roles of lipids in the brain.
In order to address the challenges to successful internal, intact brain imaging, Dr. Karl Deisseroth of Stanford University worked to create a replacement cytoskeleton for brain cells that would allow for transparency while maintaining the functional role of lipids, thus permitting their removal. He and his team found a successful replacement in acrylamide-based hydrogels, which once attached to protein and nucleic acid structures (Chung et al. 2013) would provide structural support allowing for lipid removal by diffusion (Reveles Jensen & Berg 2017) or electrophoresis (Lee, E. 2016). With the lipids having been successfully replaced, the gel-tissue hybrid allows the introduction of fluorescent markers to visualize axons deep within the brain tissue – an intact, internal visualization of the brain.
As previously addressed, the benefits of developing a neuroimaging method that makes the brain transparent to optical imaging are numerous. The internal manipulation of brain tissue at the cellular level with hydrogels allows for comprehensive, three-dimensional imaging of the brain’s subcellular structures without the dismantling necessitated by prior techniques used to observe the same tissue at only a two-dimensional level. The abandonment of dismantling allows for the preservation of the neural network throughout the brain.
With transparency, the resolution of fiber activation and visualization by fluorescent labeling dramatically increases (Deisseroth 2016). Higher resolution allows for both visualization of internal structure in addition to regional activation, providing researchers with the ability to directly map specific communication pathways inside the brain. Additionally, while developed to create transparency in brain tissue, the CLARITY method is applicable to other biological tissue, making it a powerful technique for gathering information about the human body as a whole (Reveles Jensen & Berg 2017).
Although revolutionary in its’ ability to visualize the neural network of the human brain, the CLARITY method is restricted by several limitations in regards to time and complexity. Once the hydrogel cytoskeleton has been implemented inside the tissue, the density of the hydrogel membrane slows the removal of inhibiting molecules such as lipids, causing clearing and fluorescent labeling to be a time-consuming step in the protocol. (Lee, E. 2016)
It should be noted that several other research groups have developed alternate protocols for hydrogel development that adjusts the porous density to increase the speed of clearing. Since its initial development, modifications in CLARITY protocol have included the use of electrophoresis to decrease elution time to address the mentioned time disadvantage. However, the difficulty of successfully implementing this change in method is high due to its immense complexity and risk of damaging tissue when voltage is too high. Finally, tissue-hydrogel hybrids often expand the original tissue, which prevents direct application of internal mapping to a reference brain.
The knowledge that can be gained from access to the intact internal network of the brain is most significant with regard to the potential applications of this new knowledge. This technique dissipates many of the barriers preventing advancement of clinical, behavioral and cognitive research. With respect to clinical research, abnormalities in neural connection and organization are responsible for countless clinical diseases and disorders.
The combination of structural and functional information may allow for a more direct understanding of the interaction between the structural and function of the brain’s neural network. Better understanding of the processes involved in action and cognition can be used to in turn increase understanding abnormality or failure of such processes. This more specific understanding may help specify steps needed to find a cure for neurodegenerative diseases such as Huntington’s or Parkinson’s disease in addition to treatments for psychiatric disorders such as schizophrenia.
The modern understanding of the human body may seem exponentially greater than what was known only a few decades ago, but the development of CLARITY represents a crucial step towards furthering our understanding of the brain and is an excellent reminder of the necessity for innovation in science. Advancements in science reveal the need for answers to questions not previously considered. There is not an endpoint to what is known in science and innovation is necessary to reveal all that is left to be understood.
The development of CLARITY represents a challenge to complacency with current research techniques and methods and a challenge to the types of questions and projects that are pursued. Innovation is achieved by the removal of belief in limitations that suggest any finality in science. In addition to ‘big ideas’, the execution of innovation is only made possible by collaboration, openness and determination. CLARITY emphasizes foremost that success in innovation does not occur instantly and requires the inevitability of failure throughout development.
Additionally, collaboration served as a crucial part of CLARITY’s initial development, but also has continued to be highlighted in the continued improvement in protocol by researchers across the world. CLARITY represents the collaborative effort of many determined individuals who challenged the believed limitations of neuroimaging to develop a revolutionary technique with the potential to exponentially increase understanding of the brain and with endless possibilities of application within scientific research.
Reference List
- Chung, K et al. (2013). Structural and molecular interrogation of intact biological systems. Nature, 497, 332-337. Retrieved from https://www.nature.com/articles/nature12107
- Deisseroth, K. (2016). A look inside the brain. Scientific American, 30-37.
- Du, H., Hou, P., Zhang, W., & Li, Q. (2018). Advances in CLARITY-based tissue clearing and imaging. Experimental and therapeutic medicine, 16(3), 1567-1576. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122402/
- Lee, E et al. (2016). ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Scientific reports, 6. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707495/
- O’Brien, J.S., Sampson, E.L., (1965). Lipid composition of the normal human brain: gray matter, white matter, and myelin. Journal of Lipid Research, 6, 537-544. Retrieved from http://www.jlr.org/content/6/4/537.full.pdf+html
- Reveles Jensen, K.H., Berg, R.W. (2017). Advances and perspectives in tissue clearing using CLARITY. Journal of Chemical Neuroanatomy, 86, 19-34. Retrieved from https://www.sciencedirect.com/science/article/pii/S0891061817300984?via%3Dihub
