Novel Method for Neuronal Nanosurgical Connection

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.

Scientific RepoRts | 6:20529 | DOI: 10.1038/srep20529 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 transfection 13,14 . 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 axons 15 . This method allows creating of precise injury that enables the studies on axonal injury and regeneration at the single cell level 15 .
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 attachment 16 . 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)× 10 12 W/cm 2 , and ± 0.5 μ m, 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.
To demonstrate our neuron connection method, we show that this technique can be used on any number and types of neurons by its implementation on two neuron types: P19, and Neuro2A. Neurons were grown in culture, and suspended in DMEM solution right before connection (see online methods). Selected neurons for connection were identified, isolated and brought into contact using an optical tweezer such that the protruding axon of one neuron touched another neuron's soma. In order to ensure that the neurons do not naturally stick to each other, the cells were left touching for a period of time, and then pulled apart by the optical tweezer. The neurons did not show any signs of natural connectivity. The neurons were brought into contact once more, and then femtosecond laser pulses were delivered to the axon and cell soma connection point (see methods) in order to induce an attachment. To validate that a connection was achieved, one of the neurons was moved inside the suspension dish using optical tweezer (see methods), and it was found that all neurons followed a corresponding path, twisted, and rotated as a single entity, without showing any sign of detachment. Figure 2a depicts the attachment of two Neuro2A cells (see methods), where the axon of cell (i) is attached to the soma of (ii).
Connection of single neuron to multiple neurons is a fundamental requirement for assembling a chain of neurons, and to maintain neuronal connectivity and continuity. As shown in Fig. 2b, two axons from Neuro2A (i) were attached to Neuro2A (ii) and (iii). The cells are shown, in Fig. 2c, after being moved and oriented approximately 30° relative to their original orientation.
Figure 2d-f depict a femtosecond laser induced connection of P19 axon to P19 soma (see methods). Here, connection of groups of targeted neurons is demonstrated. Two groups of four P19 cells were identified, as shown in Fig. 2d. The axon of neuron (i) came in contact and was connected with (ii), using femtosecond laser pulses. In  Several groups of neurons were attached in order to demonstrate the proposed neuron connection method. One to two 15 ms pulse trains (i.e. 1.2 × 10 6 pulses) were necessary to achieve attachment, with 90% success rate. Throughout our observations and rigorous manipulations, the cells remained viable and firmly attached without showing signs of deterioration in attachment strength, validating long term viability and attachment prospects. Moreover, previously reported cell-cell attachment method 16 , and long term viability experiments 17,18 also confirm that the laser parameters used for this method fall within a safe range for preserving cell viability and attachment. We envisage that femtosecond laser-induced neuronal nanosurgical connection method can potentially provide a scientific leap that will open up new frontiers in the studies of the effects of connecting neurons, right before or after injury. The preservation of the viability of the neural network will allow researchers to study new complex pathophysiological processes, such as neurogenesis, Wallerian degeneration, segmental demyelination, and axonal degeneration. This will allow further development of new therapies for neuronal injuries and disease.

Materials and Methods
Cell Cultures. P19 mouse teratocarcinoma cells 19 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 7.5% bovine serum and 2.5% fetal calf serum. For neuronal differentiation, P19 cells were cultured in petri dishes (to allow formation of embryoid bodies) at a density of 10 5 cells/ml in the presence of 1 μ M all-trans-retinoic acid (RA) (Sigma R2625) (Day 0) 20 . On Day 2, the medium was replaced with fresh DMEM supplemented with serum and 1 μ M RA. On Day 4, the embryoid bodies were trypsinizsed and broken down into single cells. These single cells were plated on coverslips and cultured in DMEM plus 10% fetal calf serum. On Day 6, the cells were treated with 5 μ g/ml Ara-C (Sigma C1768) in order to remove any remaining proliferating cells. The cells completed neuronal differentiation by Day 8, at which stage long neurite projections could be observed. Neuro2A (mouse neuroblastoma cells) were cultured in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μ g/ml). At confluence, cells were trypsinized and plated on coverslips.
Setup Characteristics. The neuron connection was achieved by using sub-10 femtosecond laser pulses, with 800 nm central wavelength that was delivered from a Ti:Sapphire laser oscillator at a repetition rate of 80 MHz. The near infrared pulses were coupled to an upright Nikon Eclipse 80i optical microscope and directed towards the cells. A 60× (numerical aperture (NA) = 1) water immersion microscope objective was used, in order to focus the laser pulses and to image the cells. A high NA microscope objective is required to achieve high-resolution imaging and to focus the beam to an effective spot size of ~600 nm. At the focal spot, the optimum laser pulse train average power, energy, and intensity are: 200 mW, 2.5 nJ/pulse, and 1.71 × 10 12 W/cm 2 , respectively. Ideal irradiation time is 15 ms pulse train (i.e. 1.2 × 10 6 pulses). The neuron connection experiments were imaged and recorded in real-time using a color charge-coupled device (CCD) camera. The images used in this communication were taken from the video recordings after the completion of the experiment. Experimental Procedure. P19 and Neuro2A cells were placed inside a glass dish with DMEM solution and mounted on a motorized x-y-z nano-translation stage for precise movement control of the cell culture. Trypsin solution was added to each dish in order to release the neurons from the bottom of the plate. After 10 minutes, the dish was shaken by hand in order to suspend the neurons in the solution. Neurons with identifiable axons and that are not attached to large groups have been identified and selected for connection. The selected neurons were brought into contact using an optical tweezer, such that an axon of one neuron touches the soma of the other neuron. The optical tweezer was made collinear with the femtosecond laser pulse train. Once the desired contact region between the axon and cell soma was identified, it was precisely targeted by the femtosecond laser pulse train for a duration of 15 ms. The mechanical integrity of the connected neurons was then assessed using an optical tweezer. This was performed by trapping one of the neurons to the optical tweezer's focal spot, and moving the trapped cell(s) in various paths. In order to verify that proper attachment was obtained, three criteria were assessed: (1) no detachment due to twisting, and drag forces (2) movement of all cells due to trapping of any cell in the group, and (3) movement of all cells as a single unit. We confirmed the physical cellular attachment by following the translation of the trapped cell as an integral unit together with the other cells without detaching from each other. In order to verify that the connected neurons did not move only due to their proximity to the optical trap, groups of four neurons were attached. Using this technique we were able to ensure that the neurons are located far enough from the optical trapping spot, where the attraction forces to the laser tweezers are too weak to pull an individual cell.