Name: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments (Video) | JoVE Journal
Uploaded: 2007-07-30T00:00:00.000+00:00
Duration: 16 min 30 s
Description: Learn to apply light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic neuroscience experiments. This method enables direct cranial bonding without screws, reducing inflammation risks.

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5 minutes

We thank our colleagues in RIKEN-BSI for their kindly supports and fruitful advices during this work. I would like to express my sincere appreciation to Professor Shigemi Mori, Professor G bor Juh sz, Drs. Kiyoji Matsuyama, Katalin A K kesi and rp d Dobolyi who advised me to study microdialysis method during the graduate course at the Graduate University for Advanced Studies, Japan. Finally, we thank Mr. Chris Sarnecky and Dr. Akitsugu Takasu for their supports in video recording.

Step 1. Preparation of probe implantation
<ol>
<li>Anesthesia (not shown in the video) Anesthetize each bird with an interperitoneal injection of a ketamine (12.5 mg/kg body weight) and xylazine (25 mg/kg) mixture to produce a steady level of surgical anesthesia. </li>
<li>Fix a head to a stereotaxic frame Use Xylocaine(TM) jelly to reduce local pains of ear bars' pressure. The head of bird is fixed in a stereotaxic apparatus consisting of ear bars and a beak holder that holds the head at a 45&deg; angle. During the stereotaxic surgery for implantation, warm the body of bird by surgical pad with thermal control (temperature = 34~36&deg;C). </li>
<li>Disinfect surgical area Disinfect the surgical field with isodine(TM) prior to the surgery. </li>
</ol>
Step 2. Stereotaxic positioning
<ol>
<li>Reference point alignment Align dummy probe (glass micropipette) to the Lambda (Y-point) and mark Y-point with the dye-filled pipette. Remaining Dextran-TMR of the last tracer study was used in the video. </li>
<li>Incision of the skin Remove skin and expose the surface of the cranium. Then, clean the surface of the cranium with cotton swabs. </li>
<li>Positioning of dummy cannula (in horizontal direction) Move the dummy cannula to the target site (A-P axis and Lateral coordination) </li>
<li>Small craniotomy and incision of dura mater. Use a&nbsp;fine forceps for craniotomy and a fine injection needle (30 G) for incisions of the dura mater. Then, clean the surface of cranium. </li>
<li>Final positioning (implantation) of dummy cannula Locate the tip of the guide cannula&nbsp;at 0.30 mm above the microdialysis probe-intended target region. Finally, clean the surface of cranium again, because a clean and dry surface is required for tough bonding. </li>
</ol>
Step 3. Pretreatment of the cranial surface
(The similar procedure for "direct filling restoration" of the dental product shall be employed.)
<ol>
<li>Primer treatment <ol>
<li>Mix PRIMAR liquid A with B on a dish.</li>
<li>Apply the mixture on the surface of the cranium with a small brush.</li>
<li>Leave for 30 seconds.</li>
<li>Dry with mild air stream. (Do not wash.) </li>
</ol></li>
<li>Application of bonding agent <ol>
<li>Apply the bonding agent on the surface of the cranium with a small brush.</li>
<li>Apply a gentle air stream. (Do not wash.)</li>
<li>Light-cure for 20 seconds. </li>
</ol></li>
</ol>
Step 4. Mounting composite resin
<ol>
<li>Mount resin with plain plugger If a Plain plugger is not available, a micro-spatula can be used instead. </li>
<li>Light curing</li>
<li>Incremental mounting Incremental mounting is recommended to ensuring complete reaction of the resin. For incremental mounting, apply bonding agent before every mounting. </li>
<li>Mold the composite resin and light-curing for final hardening.</li>
<li>End of the surgery</li>
</ol>
Clean the surgical area, apply antibiotic (Gentamicin ointment), and remove the animal from the stereotaxic flame. Then, keep&nbsp;the animal in a thermo-controlled chamber (temperature = 33-35&deg; C) for post-anesthetic recovery.
As shown in the video, no abnormal behavior was observed in any of the animals on the next day after the surgery.

<ol><li>Adell, A., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+Brain+microdialysis%3A+Principles+and+applications%5BTitle%5D)+AND+%22In+Vivo+Neuromethods%3A+Neuromethods%22%5BJournal%5D)">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. Boulton, A., Baker, G., Bateson, A. 32, Humana Press Inc. Totowa. 325-357 (1998).</li>
<li>Kendrick, K. Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKendrick%2C+K+%22Microdialysis+in+the+Neuroscience%22">Microdialysis in the Neuroscience</a>. Robinson, T., Justice, J., Bateson, A. 7, Elsevier. Amsterdam. 327-348 (1991).</li>
<li>Oakley, B., Schafer, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOakley%2C+B+%22Experimental+Neurobiology%3A+A+laboratory+Manual%22">Experimental Neurobiology: A laboratory Manual</a>. , University of Michigan Press. Ann Arbor. (1978).</li>
<li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study%5BTitle%5D)+AND+%22Soc.+Neurosci+Abst%22%5BJournal%5D)">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li>
<li>Boulton, A., Baker, G. The relation of brain electrical activity to behavior. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBoulton%2C+A+%22In+Vivo+Neuromethods%3A+Neuromethods%22">In Vivo Neuromethods: Neuromethods</a>. Robinson, T., Bateson, A. 32, Humana Press Inc. Totowa. 325-357 (1998).</li>
<li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch. Proceeding of the 2nd International Symposium on Mobiligence, , 101-104 (2007).</li>
</ol>

Experiments were undertaken in accordance with the guidelines for animal experimentation of the brain science institute, RIKEN (RIKEN-BSI) and the institute's animal ethics committee approved them.

Compared to the application of conventional self curing acrylic resins, the introduced technique has several important advantages:
1. Direct bonding: 
Self curing acrylic can be attached to the bone for a while but it does not adhere to the bone. Therefore, anchorage screws are required to fix a mounting base (See Figure 2A). In contrast, light curing resin can bond to the bone directly so screws are not required (Figure 2B). Therefore, size of mounting base can be reduced considerab...

<table><tbody><tr><td>Bengalese finches</td><td>Animal</td><td></td><td></td><td>Two adult male , Body weight: 14.2 g and 14.8 g</td></tr><tr><td>Microdialysis probe</td><td>Tool</td><td>EICOM</td><td></td><td>total weight of probe, guide cannula, cap nut and dental adhesive resin: 0.65 to 0.78 g.</td></tr><tr><td>Light-cured dental adhesive resin system</td><td></td><td>Kuraray Medical Inc</td><td></td><td>+Light cured composite resin (CLEARFIL AP-X). +Dual-cure dental adhesive system with the self-etching primer and bonding agent (CLEARFIL Liner Bond II&amp;#931;).</td></tr><tr><td>Surgical instruments</td><td></td><td></td><td></td><td>Stereotaxic frame, surgical microscope, thermal controlled surgical pad and thermal controlled recovery chamber. Plain plugger for handling composite resin. Microspatula or sharp curette to ablate periosteum and muscles.Injection syringe and needle (30G) for inducing anesthesia.Small scissors. Fine forceps for making small craniotomy.</td></tr><tr><td>Light-unit</td><td></td><td>Rolence Enterprise Inc</td><td>Ultra-Lite 500E</td><td></td></tr><tr><td>Microscope</td><td></td><td>Konan Medical</td><td>KOM300</td><td>with C-mount adaptor </td></tr><tr><td>3- CCD camera</td><td>Tool</td><td>Hitachi</td><td>HV-D30</td><td></td></tr></tbody></table>

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

In chronic experiment of electrophysiology or microdialysis, self curing dental ress have been used widely as a mounting base of electrode or microdialysis probe. Since these acrylics do not bond to the bone surface screws have been used as anchors. However, in small experiment, animals like FIEs or mouth, there are cranium, are very fragile and cannot successfully hold ankles.Sometime these ankles screws cause infection and inflammation of the tissue. In this report, we propose a new application of right curing dental as his raising for mounting base. This material allow direct bonding to the cranium.Therefore, Anchorage screws are not required and surgical field can be reduced considerably. Total weight of a probe, A guide canula a cabinet and mounting base was less than one gram. Past experiences show the bonding effect maintain more than two months.This technique can also be utilized for other experiment animals like mouth, fish and so on. Two implementations in two bars were video recorded. In this video demonstration, the best shot at each step of the protocol were chosen and used.These are stereotaxic apart and summer controlled recovery chamber. These are hundred instruments for surgery. Prime PR on the left and micro problem.Its accessories on the right. If a prime program is not available, a micro spatula can be used instead. This is a set of right curing dental resin.AP primer and a bonding agent were kept in these drug bottles. We chose this product because it is a self acting and dual cure system. If you choose alternatives, please modify the detailed procedure According to a product instructions, gun like shaped instrument on the left side is a visible right curing unit.You can assemble handmade LED light unit as shown on the right. Please refer the documentation for the detail. Following Surgical anesthesia was induced further of the surgical area was removed and head was fixed to the stereotactic frame.Prior the insertion of EVAs DI injury was put to tips of EVAs to prevent from pain. The head of animal was fixed to the frame and head angle was adjusted appropriately. The infect surgical air with ine Iodine, a grass Micropipet was fixed on electrode holder.This micropipet was used for making a reference point in bad experiment. The Lambda Y point was used as the reference point. Small skin incision was made to expose the reference point.Mark the Spot with a grass micropipet filled with a dye. Cut the skin to expose cranial surface, Wipe the surface of bone with cotton swab to eliminate blood breath. Green and dried surface is required for tough bonding.Following a markup target position, small craniotomy was made by fine Forceps. You can also use microdot or bone forceps to make a craniotomy. Then gross micro pipe was removed from a holder and guide, canula or electrode was fixed to the holder.Tip of guide Canula was aligned to the reference Point and then carefully moved to the target point. The smallest incision of ger Matter was made by 30 gauge injection. Needle guide can was moved to the Target position.The second screening of bone surface was Supp applied. This primer consists of rigid A And B.One drop of each rigid was dropped on a dish and mixed by a brush Swab the primer onto the area of contact. The surface of the Guide canula was also swabbed.Mild air bro was provided to spread and dry the primer change the brush and swab a bonding agent onto the area. This bonding agent is right sensitive so that this should be covered during the procedure. The surface of the guide canula was also swabbed.The mild air was provided and visible blue light was eliminated to accelerate chemical reaction of the bonding agent. Orange Coral transparent seed should be used to protect eyes of operator. Eyes of animals should not be given direct righting if necessary.Aluminum hole is used to protect eyes of animals. A Small amount of composite resin was pasted on the cranial surface and guide canula by plain plug. Then visible blue right was eliminated to cure The resin For incremental mounting.A bonding agent was applied before every mounting. The area of surgery was righted continuously because of video recording. However, it is better to avoid strong right because it may shorten a window of time when the materials are pable.Incremental mounting and righting are recommendable for ensuring complete reaction of the regime. You can reform the shape until visible. Right was given Antibiotics or ment was applied to the surgical area to prevent infections.Now the surgery was completed, so stereo adapter was released from the electrode holder. Stereo adapter was removed. DAMing cane was inserted instead and the animal was released from the stereotactic Frame.Finally, the animal was moved to the summer control chamber for recovery from the anesthesia. It was the day after the surgery Compared to the application of conventional self curing. S.The introduced technique have several important advantages.First, it bond to the bone directly. Self curing acrylic can be attached to the bone who are wire, but it does not adhere to the bone. In contrast, light curing raising can bond to the bone directory.It also adhere to some materials including plastic and metals if you use metal primer with it. This feature bonding to variable materials allow as a wide range of application. Second, easy to handle Conventional res processing time window is relatably short.Within a few minutes it has a high fluidity and it is not easy to handle at fast. However, once chemical reaction started, reaction is accelerated by exothermic. Heat and resin became harder in short time.The processing time windows for right curing resin are significantly wider and adjustable. Basically, resins keep constantly from the beginning to the moment when it is right. Cured third reaction does not generate heat.Curing reaction of self curing resin is exosome. When the mounting base is thick, this heat accumulate and made damage. The tissue reaction of light carrying resin does not generate heat and material is biocompatible.Cost of the materials and special equipment like light unit can be a problem. In the supplemental document we introduce a hundred made LED Light unit fit does not cost much. Pretty fast document for details.This technique is applicable to implementation of electrode or micro probe in other experimental animals such as fish, mouse, rabbit, cat at monkey.

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Learn to apply light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic neuroscience experiments. This method enables direct cranial bonding without screws, reducing inflammation risks.

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

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