Neuronal injury may cause an irreversible damage to cellular, organ and organism function. While preventing neural injury is ideal, it is not always possible. There are multiple etiologies for neuronal injury including trauma, infection, inflammation, immune mediated disorders, toxins and hereditary conditions. We describe a novel laser application, utilizing femtosecond laser pulses, in order to connect neuronal axon to neuronal soma. We were able to maintain cellular viability, and demonstrate that this technique is universal as it is applicable to multiple cell types and media.

Neurons are cells within the nervous system that process and transfer information through electrical impulses and chemical signals1,2. This type of communication between neurons occurs in a specific interphase called a synapse2. The Central Nervous System (CNS) which includes the brain and the spinal cord, as well as the Peripheral Nervous System (PNS) operate in a similar pattern utilizing a neuronal synaptic network as a means of intercellular communication. It is imperative to have intact neuronal circuits in order to maintain normal organ and organism function. The typical neuronal structure includes cell body (soma), dendrites, and an axon. Dendrites are structures arising from the cell body branching multiple times. An axon is a cellular extension arising from the cell body which can traverse a long distance in human beings and in other species. The axon may branch multiple times and connect to multiple cells prior to termination. There is an average of 300 trillion synapses in the adult brain3.

Neural communication is usually transferred through one neuron’s axon to a dendrite or cell body of another neuron. However, there are other connection options based on different neuronal cell structures: for example, axon to axon, dendrite to dendrite, etc4. The cell membrane of the soma and the axon is electrically conducting through activation of voltage-gated ion channels4. These calcium, sodium, potassium, and chloride ion channels generate the electrical signals propagated in the axons.

Neurons are highly specialized cells that can no longer divide. Neurogenesis is very limited in adulthood; hence, nerve injury has a major impact on the ability to maintain normal function. There are a few recognized patterns to peripheral nerve damage post injury: Wallerian degeneration, segmental demyelination and axonal degeneration5,6,7,8,9,10. Wallerian degeneration develops after transection of the nerve and injury to the axon and its myelin sheath, where distal to the transection both the axon and the myelin will degenerate. A conduction block is observed one week after the injury. Regrowth is possible depending upon preservation of the basement membrane of the cells that are producing myelin (Schwann cells) and approximation of the nerve ends5,6. This type of damage may cause atrophy of the muscle innervated by the damaged neuron. Segmental demyelination is the result of damage restricted to the myelin sheath. As the axon is preserved, no end point muscle degeneration is expected or observed6,7. Axonal degeneration is caused by damage to the neural cell body resulting in distal death of the axon. Muscle atrophy will develop unless re-innervation occurs from adjacent nerves, however recovery may be only partial8,9,10. Experimental work on nerve injury repair has been carried out in the last decade and a few approaches have been investigated. These techniques included: bridging a gap by utilizing growth permissive matrix placed across the site of injury to allow axonal growth11, creating new network via stem cell therapy11, providing neurotropic support in order to stimulate sprouting of spared axons or enable regeneration of injured axons11,12. Alternative techniques attempted to overcome myelin associated growth inhibitors11,12, and scar-associated growth inhibitors11,12. Molecular protection of cells was used to avoid from the cascade of biochemical events that lead to cell death post injury12. These investigational treatments were encouraging but had partial success in animal models.

It is of paramount importance to develop a precise means of selectively connecting specific axons to neuron cell body. Such a leap in scientific method will open up doors to unparalleled research frontiers in neurology, cell biology, biochemistry, and electrophysiology. Connecting neurons, before or right after injury, enables the preservation of the viability of the neural network, thereby allowing complex pathophysiological processes, such as neurogenesis, Wallerian degeneration, segmental demyelination, and axonal degeneration to be further understood. Understanding the complex pathophysiological processes and the time frame available in order to prevent conductivity block and axonal death makes it necessary to develop techniques that enable the connection of nerve ends as soon as possible post injury, and maintain the viability of a healthy neural network. We describe a novel laser application to physically reattach severed neurons right after injury. This method may potentially allow further prevention of a conductivity block. Moreover, it may trigger studies questioning the hypothesis whether physical attachment and approximation of the nerve ends will stimulate recovery.

To date, a method to connect neuron ends does not exist. Assessment of axonal growth and regeneration is currently performed via either immunolabeling, where specific proteins that are involved in known regeneration pathways are labeled and monitored or via anterograde and retrograde tracing to visually trace neural connections from their termination/source to their source/termination. These imaging methods are utilized to trace the neuronal projections from one location to various targets in the nervous system, and it allows researchers to study the natural process of axonal regeneration. However, the above mentioned techniques are limited to studying only the natural healing processes of neurons. Thus, control on selection and isolation of neurons, in order to study regeneration of specific neurons, is not available. Knowledge gained from such studies will allow researchers to develop new therapies for, currently, irreversible neuronal injuries and diseases.

A prime candidate method for connecting specific neurons that fulfills such key applications is femtosecond laser pulse technology. This versatile technology has been utilized for very precise cell manipulation, such as optoporation, cell nanosurgery, cell isolation, and embryo transfection13,14. Removal or ionization of material is confined to less than a diffraction limited spot size, with no damage to surrounding material. Femtosecond laser pulses have also been used as a tool to study neuron regeneration by severing neurons and axons15. This method allows creating of precise injury that enables the studies on axonal injury and regeneration at the single cell level15. More recently, it was demonstrated for the first time that this technology can be used to “reverse” cell cutting or isolation, by performing cell-cell attachment16. However, physical connection of single neurons has not been performed thus far.

In this communication we present a method for neuron connection, using femtosecond laser pulses. By physically connecting single axons and neurons right after injury, it will allow researchers to develop new methods of studying the effects of neuron connection on neuronal regeneration, progression of Wallerian degeneration, and the existence of cellular communication, to further our understandings of these phenomena. This effective neuronal connection method should allow the user to select single cells for isolation, connection, and cutting. The technique is shown to be universal and applicable to multiple cell types and their media.

We developed a novel neuron connection method using ultrashort femtosecond laser pulses (illustrated in Fig. 1a). Precise tuning of the laser parameters allowed us to induce a process called hemifusion at the contact point of two phospholipid membranes (illustration of the contact point is seen in Fig. 1b). To achieve neuron connection, the laser intensity and aiming accuracy required are 1.7(±0.08)×1012 W/cm2, and ±0.5 μm, respectively, within the membranes hemifusion location. Exposure to near infrared femtosecond laser pulses induces molecular rearrangement of the phospholipid bilayers via multiphoton and avalanche ionization processes. The high electron and ion density at the laser beam focal point leads to an ultrafast reversible destabilization of the phospholipid molecules. Since the membrane’s exterior surface is permeable to both photo-induced ions and electrons, these can cross over to the central nonpolar region of the phospholipid bilayer and break the bonds of the fatty acid tails, as illustrated in Fig. 1c. At the end of this destabilization process, the ionized phospholipid molecules seek equilibrium state, and form new bonds with nearby ions, as seen in Fig. 1d. Only the phospholipid molecules that are located at the cell membrane contact point cross-link with phospholipid molecules of the adjacent cell membrane. The cross-linking process leads to the formation of a single, shared, phospholipid bilayer (i.e. hemifused membrane), which is the underlying mechanism that takes place in this neuron connection method and provides a strong attachment.

Read more here: http://www.nature.com/articles/srep20529

Katchinskiy, N. et al. Novel Method for Neuronal Nanosurgical Connection. Sci. Rep. 6, 20529; doi: 10.1038/srep20529 (2016).

Advertisements