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Bu Makalede

  • Özet
  • Özet
  • Giriş
  • Protokol
  • Sonuçlar
  • Tartışmalar
  • Açıklamalar
  • Teşekkürler
  • Malzemeler
  • Referanslar
  • Yeniden Basımlar ve İzinler

Özet

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

Özet

Bu protokol endüstri standardı step motorları ve CNC kontrol yazılımı kullanarak, yaklaşık 1.000 $ (matkap hariç) için bir robot (CNC) stereotaksik alet varolan stereotaksik enstrüman yükseltmek için gerekli tasarım ve yazılım içerir. Her eksen değişken hız kontrolü vardır ve aynı anda veya bağımsız çalıştırılabilir. Robotun esneklik ve açık kodlama sistemi (g-kodu) ticari sistemleri tarafından desteklenmeyen özel görevleri yetenekli olun. Uygulamaları, matkap delikleri, keskin kenar kraniyotomi, kafatası incelmesi ve düşürücü elektrotlar ya da kanül arasında, ancak bunlarla sınırlı değildir. Basit ameliyatlar için g-kodlama yazma hızlandırmak amacıyla, bireylerin hiçbir programlama bilgisine sahip bir cerrahi tasarlamak için izin özel komut geliştirdik. Ancak, motorlu stereotaksi dışında en iyi şekilde kullanıcılar için, matematiksel programlama ve G-Kodlama (basit prog bilgili olması faydalı olacaktır) CNC işleme için sıkıştırma.

Önerilen matkap hızı 40.000 rpm daha büyüktür. Step motor çözünürlüğü 0,346 ° / Adım yönelik 1.8 ° / Step, olduğunu. Standart stereotaksi 2.88 mm / adım çözünürlüğe sahip. Önerilen maksimum kesme hızı 500 mm / sn 'dir. Önerilen maksimum koşu hızı 3,500 mm / sn 'dir. Önerilen maksimum matkap boyutu HP 2'dir.

Giriş

Stereotaktik kemirgen cerrahi hasraı 1, iyontoforez 2, mikrotel implantasyon 3, stimülasyon 4, ve ince kafatası görüntüleme 5 dahil nörobilim uygulamalar geniş bir yelpazede kullanılmaktadır. Ancak, doğru stereotaktik ameliyat için dik bir öğrenme eğrisi ve insan hatası, yüksek olasılık içeren bu teknikleri uygulamak isteyenler karşıya önemli engeller vardır. İnsan hata ölçüm ve hesaplama arızaları, hem de insan hareketlerinin düşük doğruluk ve tekrarlanabilirliğini içerir. Bu karıştırıcı hataları azaltmak için bir çaba, stereotaktik cerrahlar tüm cerrahi işlemler denekler genelinde aynı yerine getirilmesini sağlayan bir sistemden yararlanacak. Hatalarının azaltılması da araştırmacılar hayvan deneklerin kullanımını hayvan deney için 6 Ulusal Sağlık Enstitüleri birincil hedefi en aza indirmek mümkün kılan bir yöntemdir. İdeal bir dünyada, tüm stereotactic ameliyatları deneyler içinde mükemmel tekrarlanabilir olması ve laboratuarları arasında olacaktır. Bu sorunu çözmek için, şirketlerin ölçümleri okumak için yeni ultra-hassas stereotaxics ve dijital görüntüler geliştirdik. Insan hareketi hataları kaldırmak için, motorlu mikro manipülatörler ve stereotaxics ticari olarak üretilen, ancak yüksek maliyet sınırlı bir bütçe ile bir laboratuvara engelleyici olabilir. Ayrıca, kendi yazılım tamamen özel ve yeni bir cerrahi türü karşılamak için araştırmacı tarafından değiştirilemez.

Insan hatası soruna uygun bir çözüm endüstri standardı CNC ekipman kullanarak, bir laboratuvar mevcut model bir robot stereotaksi inşa etmektir. Çünkü gelişen CNC hobi topluluğu, malzemeleri daha az pahalı bilimsel ekipman daha vardır. Bu da son derece esnek ve ucuz bir doğru CNC steryotaksik enstrüman, oluşturmanıza olanak verir. CNC işleme ve G-Code, individu temel bilgilere sahipals tescilli yazılım sınırlamaları olmadan, onlar hayal herhangi stereotaktik cerrahi programlayabilir. Ve, basit ameliyatlar için g-kod üretimini hızlandırmak için, bu protokol kullanıcı açısından içinde ameliyatları (keskin kenar kraniotomi, ince kafatası pencere, delik delme ve implant düşürücü) tasarım ve menüleri tıklayın sağlar yazılım içerir. Bu programlar çıktı CNC yazılımı doğrudan çalıştırılabilir olabilir tamamlanmış bir g-kodu.

Tüm, motorlu stereotaksik yükseltme bir açık kaynak platformu esneklik ve düşük maliyetli korurken, ameliyatları doğruluğu ve tekrarlanabilirliği artan bir ilgi var olanlar için idealdir.

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Protokol

  1. Wire the bipolar stepper motors by screwing the wires into the connectors supplied with the driver board. Wire colors on bipolar stepper motors are standardized (Figure 1).
    Note: The described stepper motors have a resolution of 1.8°/step, geared to 0.346°/step. A standard stereotax has 3 mm/360° of travel. The final resolution is 2.88 μm/step. The motors are also capable of fractional stepping.
    1. Connect the green wire to A+, connect the black wire to A-, connect the red wire to B+ and connect the blue wire to B-.
  2. Slide the couplers over the stepper motors, being careful to align the mounting holes, and secure them using 12x M3 Socket Head Screws (20 mm) (Figure 2).
    1. Ensure that the couplers are firmly attached to the motors.
      Note: The 3D models do not include threads. The parts are labeled with thread size, but they must be tapped after they are manufactured.
  3. Remove the set screws from the thumb grips on all 3 axes of the stereotaxic instrument using a small hex key. The thumb grips are threaded, so turn them counter-clockwise for removal. Keep the PTFE washers in place on the arm (Figure 3).
  4. Screw the threaded end of the collars onto the threaded rods of the stereotaxic instrument's arms (Figure 3).
    1. Ensure there is no gap between the collars and PTFE O-rings. This guarantees that coordinates are maintained when the robot changes directions.
    2. Secure the collars onto the threads of the stereotaxic arms using 3x NF10-32 (1/4 inch) cup point set screws.
    3. Slide each motor and coupler over the collars & stereotaxic arms. Ensure that the motors sit flush with the arms, and the set screw holes on the collars line up with the flat portion of the motor shafts (Figure 4).
    4. Secure the couplers to the stereotax using the mounting holes and 6x NF10-32 (1/2 inch) cup point set screws (Figure 4).
    5. Secure the collars to the motor shafts using 3x NF10-32 (1/4 inch) set screws (Figure 4).
  5. Prepare the CNC driver board by setting each of the controller pins to half stepping.
    Note: This stepper motor driver comes as an exposed circuit board. A case may be built, although it is not necessary. Also, a number of different bipolar stepper motor drivers may be used. If so, ensure that the setup instructions are followed for the specific board purchased.
    1. Align all 6 pins per stepper motor in the same way. Half-stepping allows for double the step resolution in Degrees/step (Figure 5).
    2. Flip pin 1 to the on position, pin 2 to the off position, pin 3 to the on position, pin 4 to the off position, pin 5 to the on position, and pin 6 to the off position (Figure 5).
  6. Plug the motors (X - Y - Z) into the stepper motor driver, along with the 12 V power supply. The correct placement is marked on the driver. Also, attach the stepper driver to a computer's serial port using a DB25cable (Figure 6).
  7. Install CNC milling software onto a personal computer (this will need to be located in a surgical area) following the default instructions. Once installed, open the software to begin configuration.
    1. Configure the software to communicate with the stepper motors.
      Note: The following instructions are intended for use only with the TB6560 stepper motor driver.
    2. Click through the software's menus as follows. Open →Config→ Ports and Pins→Output Signals. Fill in the prompt to match Figure 7 and hit apply.
    3. Click through the software's menus as follows. Open→Config→Ports and Pins→Input Signals. Fill in the prompt to match Figure 8 and hit apply.
    4. Click through the software's menus as follows. Open→Config→Ports and Pins→Motor Outputs. Fill in the prompt to match Figure 9 and hit apply.
    5. Click through the software's menus as follows. Open→Config→Motor tuning. Fill in the prompt to match Figure 10 and click Save Axis Settings. Repeat the previous step for all 3 axes using the same values.
  8. Calibrate the stereotax to the scale of the CNC software.
    Note: The software is designed for standard milling machines, so its unit of measure will not be proportional to the travel of a stereotaxic instrument.
    1. Set the motors' velocity to 1 inch per minute, and "jog" the stereotaxic instrument's Z-axis with PgUp/PgDn to the nearest millimeter.
      Note: The maximum recommended jogging speed is 3,500 µm/sec and the maximum recommended cutting speed is 500 µm/sec.
    2. Zero the Z-axis, and Jog the stereotaxic instrument 1 mm. The distance traveled on the Z-axis in Mach3 is the "Scaling Constant". Machine coordinates are determined by multiplying skull coordinates (mm) by the "Scaling Constant".
    3. Perform random tests of all 3 axes by programming them to travel some known distances, and ensure the movements are accurate. If the stereotax travel is too far or short, modify the scaling constant accordingly.
      Note: Once scaling is complete, the included custom scripts may be used to generate g-code for surgeries. However, it is highly recommended that users become familiar with g-code before attempting to auto-generate surgeries. This is imperative for troubleshooting and modifying automated surgeries.
  9. Attach the micro motor drill to the stereotaxic instrument using the extra large probe holder. Note: The minimum recommended drill bit speed is 40,000 rpm.
  10. Auto-generate G-Code for a sharp edge craniotomy with 3 skull screw holes.
    1. Place all of the custom scripts from the software table into a single folder on a PC.
    2. Open the script "SharpEdgeCraniotomy.m"and run the code.
    3. Select Both to the prompt "What Type of Surgery Will You be Performing?" (Figure 11).
    4. Select Custom, to define the corners of the skull window. Fill in each prompt to match Figure 12.
    5. Define the X and Y positions of the craniotomy corners. Each coordinate must be entered in correct order, according the example in Figure 13.
    6. Enter 3 in the prompt to produce 3 skull holes (Figure 14).
    7. Select Define Using Coordinates, and enter the coordinates of each hole from the template in Figure 15.
      Note: If precise coordinates are not important, there is an option to point and click the holes' positions onto an image of a rat skull. Positions will be automatically generated.
    8. Define the drilling parameters. For the first tests surgery, accept the default values.
      Note: These values are dependent on the stepper motors, and the animal's skull. Every rat breed and target location has a slightly different skull thickness. For the initial few surgeries using this device, be prepared to test the drilling depths and remove any remaining skull pieces manually. The values can then be modified for future surgeries (Figure 16).
    9. Name the g-code; it will be automatically generated and saved to the working directory.
  11. Load the g-code into the CNC milling software and a test skull into the stereotaxic instruments ear bars.
    1. Manually jog the drill bit to Bregma using the arrow keys. Use a slow jog speed (~5 inch/m) to ensure accuracy.
    2. Start the drill bit rotating at greater than 38,000 rpm.
    3. Press CycleStart; the stereotax will perform many passes of the same cut, at different depths. Between each pass, the stereotax will pause, so the surgeon may continue or abort cutting. Press Continue Cycle (Alt-R) to continue cutting passes.

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Sonuçlar

The end result of the surgery designed in the methods will be a rat skull with a sharp edge craniotomy, and 3 skull holes (Figure 17). Note that the skull used to demonstrate the surgery was much wider than the prototypical rat skull. The sharp edge craniotomy may be used to insert a microwire array into the brain, for high density neural recordings. The CNC stereotax may also be used to lower the array with great precision. Software is included in this protocol that allows the surgeon to define the para...

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Tartışmalar

The use of automated surgery equipment helps to eliminate some of the most common problems in neuroscience research. First, the tool paths are 100% reproducible. Every cut is guaranteed to be in the same location relative to Bregma. Second, it should reduce experimenter error. Although many researchers are highly skilled surgeons, it takes an exceptional amount of practice to become even a competent surgeon. This device will allow new students to quickly and easily perform highly accurate surgeries. Third, motorized surg...

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Açıklamalar

The authors have no competing financial interests to disclose.

Teşekkürler

This study was supported by the National Institute on Drug Abuse Grants DA 006886, and DA 032270.

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Malzemeler

NameCompanyCatalog NumberComments
1x Standard U Frame StereotaxKopfKopfThis protocol should work with most existing stereotaxic devices.
3x 12 V, 1.6 A, 233 oz-inch Geared Bipolar Stepper MotorPhidgetsRobot ShopAny high torque geared stepper motor should do. 
1x 3 Axis CNC Stepper Motor Driver Board ControllerToshibaEbayAny 3 Axis CNC driver should do. Linked Item includes Mach3 CNC software. 
2x Arm Couplers: medial-lateral (ML) & dorsal-ventral (DV)custom machinedPart DrawingsThese must be machined by your local machine shop. (costs will vary)
1x anterior-posterior (AP) Couplercustom machinedPart DrawingsThese must be machined by your local machine shop. (costs will vary)
3x Motor to Stereotax Collarcustom machinedPart DrawingsThese must be machined by your local machine shop. (costs will vary)
View in Browser
12x NF10-32 Cup Point Set ScrewsMcMaster Carr½” LengthYou will need 6 of each.
¼” Length
12x M3 Socket Head Screws (20 mm)McMaster Carr20mm LengthYou will need 4 for each motor
1x Micro-Motor Drill Buffalo DentalX50Any Micromotor drill will work.  At least 38,000 rpm recommended
1x 12 V DC Power Supply12 Volt Adapters12v DC – 7 AmpAny 12 V DC PSU should work (ensure amperage rating is higher than the sum of the motors’ amperage).
1x Extra Large Probe HolderStoeltingStoelting
1x Grade B Rat SkullSkulls UnlimitedSkulls Unlimited
Mach 3 MillArtSoft USATrial DownloadAny Standard CNC controlling software should work.
Surgery DesignerKevin Coffey David BarkerMATLAB File ExchangeThese codes are available to modify. We accept no responsibility for your use or modification of code.

Referanslar

  1. Yin, H. H., Knowlton, B. J., Balleine, B. W. Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning. Eur. J. Neurosci. 19 (1), 181-189 (2004).
  2. West, M. O., Woodward, D. J. A technique for microiontophoretic study of single neurons in the freely moving rat. J. Neurosci. Methods. 11 (3), 179-186 (1984).
  3. Peoples, L. L., West, M. O. Phasic firing of single neurons in the rat nucleus accumbens correlated with the timing of intravenous cocaine self-administration. J. Neurosci. 16 (10), 3459-3473 (1996).
  4. Wolske, M., Rompre, P. P., Wise, R. A., West, M. O. Activation of single neurons in the rat nucleus accumbens during self-stimulation of the ventral tegmental area. J. Neurosci. 13 (1), 1-12 (1993).
  5. Bozza, T., McGann, J. P., Mombaerts, P., &Wachowiak, M. In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron. 42 (1), 9-21 (2004).
  6. Pitts, M. Office of Laboratory Animal Welfare. Institutional animal care and use committee guidebook. , (2002).
  7. Yoon, T., Otto, T. Differential contributions of dorsal vs. ventral hippocampus to auditory trace fear conditioning. Neurobiol. Learn. Mem. 87 (4), 464-475 (2007).
  8. Root, D. H., et al. Differential roles of ventral pallidum subregions during cocaine self-administration behaviors. J. Comp. Neurol. 521 (3), 558-588 (2012).
  9. Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat. Protoc. 5 (2), 201-208 (2010).
  10. Feng, L., Sametsky, E. A., Gusev, A. G., Uteshev, V. V. Responsiveness to nicotine of neurons of the caudal nucleus of the solitary tract correlates with the neuronal projection target. J. Neurophysiol. 108 (7), 1884-1894 (2012).
  11. Babaei, P., Soltani Tehrani,, B,, Alizadeh, A. Transplanted Bone Marrow Mesenchymal Stem Cells Improve Memory in Rat Models of Alzheimer's Disease. Stem Cells Int. 2012, 369417(2012).

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Keywords Open sourceRobotic Stereotaxic InstrumentCNC Stereotaxic InstrumentStepper MotorsCNC Controlling SoftwareVariable Speed ControlCustom TasksDrilling HolesSharp Edge CraniotomiesSkull ThinningLowering Electrodes Or CannulaG codingMathematical ProgrammingG CodingDrill SpeedStepper Motor ResolutionStereotax ResolutionCutting SpeedJogging SpeedDrill Bit Size

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