La cirugía estereotáxica de supervivencia en los roedores

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Summary

El seguimiento de los niveles de neurotransmisores extracelular en las regiones cerebrales distintas de animales libres de movimiento además de informaciones sobre el vínculo entre la liberación de neurotransmisores y la conducta. Microdiálisis in vivo, junto con detección electroquímica ofrece anatómica excelente y la resolución de químicos, así como información sobre cómo la neurotransmisión basal se ve alterada por las manipulaciones farmacológicas o fisiológicas.

Cite this Article

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Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. Survivable Stereotaxic Surgery in Rodents . J. Vis. Exp. (20), e880, doi:10.3791/880 (2008).

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Abstract

La capacidad de medir los niveles extracelulares basales de los neurotransmisores en el cerebro de los animales despiertos permite la determinación de los efectos de los diferentes retos sistémicos (farmacológica o fisiológica) para el sistema nervioso central. Por ejemplo, uno puede medir directamente cómo las proyecciones de la dopamina del cerebro medio animal responde a la dopamina-la liberación de fármacos, como los estímulos d-anfetamina o naturales como la comida. En este video, te mostramos cómo implantar cánulas guía de orientación sitios específicos en el cerebro de la rata, la forma de insertar e implantar una sonda de microdiálisis y el uso de cromatografía líquida de alto rendimiento, junto con detección electroquímica (HPLC-CE) para medir los niveles extracelulares de oxidables neurotransmisores y metabolitos. Introducción Local precisa de fármacos a través de la sonda de microdiálisis permite un trabajo refinado en la especificidad del sitio en el mecanismo de un compuesto s de la acción. Esta técnica ha anatómica excelente y la resolución de química, pero sólo el tiempo de resolución modesta como muestras de microdiálisis se procesan cada 20-30 minutos para asegurar niveles detectables de neurotransmisores. Complementarias ex herramientas vivo (es decir, cortar y de cultivo celular, electrofisiología) puede ayudar con la monitorización en tiempo real de la neurotransmisión.

Protocol

Resumen

Dos meses de edad, los ratones C57BL/6J edad media o equivalente o tres meses de edad promedio de edad de las ratas Sprague Dawley o equivalente son anestesiados con ketamina (60 mg / kg ip para las ratas, 100 mg / kg ip de ratones) y xilazina (10 mg / kg, ip, ya sea para las especies). La sedación se controla mediante una pizca punta suave reflejo de retirar demostrado en Walantus et al. (JOVE, 6, 2007) y Szot et al. (JOVE, 9, 2007). Termorregulación puede ser proporcionada a través de una almohadilla eléctrica thermostatregulated (ALA Instruments Inc.) y monitoreados a través de un termómetro rectal. La cabeza rapada de pelo y limpiar con yodo antes de la incisión. Después de incisión en la piel (2 cm de largo para las ratas, 1 cm de largo para los ratones) y la eliminación de todos los tejidos blandos de la superficie del cráneo, la colocación de la cánula guía se determina en relación a bregma. Un agujero de 6 mm se perfora el cráneo con un taladro a pilas para cirugía de roedores (Herramientas de Bellas Science, Inc.). Hay que tener cuidado para que la broca no penetra a través de las membranas meníngeas o los vasos sanguíneos. Skull se implanta con bilaterales de 5 mm calibre 21 ejes de acero inoxidable guía que conduce al núcleo accumbens posterior, el cuerpo estriado dorsal o la corteza prefrontal. Las coordenadas estereotáxicas se establecen de acuerdo con Franklin y Paxinos, 1997 (el cerebro del ratón en coordenadas estereotáxica, Academic Press) o Paxinos y Watson, 2006 (el cerebro de rata en coordenadas estereotáxica, Academic Press). Los implantes se fijan con cemento dental. Un bolo de Ringer lactato de la solución salina al 0,9% se da al final de la cirugía (5ml SC en ratas y 1 ml SC en ratones después de los fluidos se calientan a la temperatura normal del cuerpo) para prevenir la deshidratación. La buprenorfina (0.1-0.5mg/kg SC) se administra dos veces al día y, luego, en una función de las necesidades, si el animal parece estar en el dolor. El tratamiento local con antibióticos (bacitracina pomada) y el tratamiento con antibióticos sistémicos (penicilina 100.000 UI / kg IM cada 12 horas durante las primeras 48 horas después de la operación) si se administran después de la operación las infecciones.

Después de la cirugía, los animales son alojados individualmente con comida y agua ad libitum disponibles. Al menos una semana se permite para la recuperación antes de microdiálisis y la eutanasia. Tras la recuperación de la cirugía, los animales son trasladados a una jaula de microdiálisis y sondas de microdiálisis se insertan y se consolidó en los ejes de guía que se han instalado durante la cirugía. Sonda de inserción no causa dolor o malestar debido a que la sonda está pasando por alto tejido de la piel, los músculos y las meninges a través del eje de guía. Por lo tanto, la inserción de la sonda se realiza sin anestesia y los efectos de anestesia inducida por el comportamiento de la neuroquímica o se evitan. Dejamos que las sondas de estabilizar durante 12 horas y luego iniciar el muestreo cada 30 minutos por otro 12.08 horas, dependiendo del experimento. Hacemos un seguimiento de comportamiento locomotor del animal a través de fotocélulas o registro manual de movimiento por el experimentador. Microdialysate muestras se inyectan en una cromatografía líquida de alta con detección electroquímica (HPLC-CE), instrumento para la detección y análisis neuroquímico. Buscamos efectos sobre la neuroquímica basal y el comportamiento del aparato locomotor. Al final del experimento, el animal es sacrificado por una sobredosis de ketamina sistémica (200 mg / kg ip) y xilazina (20 mg / kg, ip). Entonces, el corazón es perfundido con solución salina al 0,9% seguido de paraformaldehído al 4%. Los cerebros se retiran, congeladas y cortadas a lo largo de las vías de la sonda de microdiálisis para verificar la colocación de la sonda precisa.

Procedimiento

  1. Coloque el instrumento estereotáxico y todos los materiales necesarios. Asegúrese de que el área y los instrumentos se limpian y se esterilizan.

  2. Afeitarse la piel con maquinilla de afeitar eléctrica. Van desde las orejas hasta justo entre los ojos, mover de afeitar en diferentes direcciones para limpiar con eficacia la zona de la piel. Aplicar povidona / yodo a la zona de afeitado, sino proteger los ojos de ella.

  3. Montar el animal en el aparato estereotáxico mediante la colocación de las barras de oído en el conducto auditivo externo y el ajuste en su lugar. En primer lugar montar un bar de oído en el conducto auditivo externo, y luego mantenerlo en su lugar y se deslizan en la barra de la otra oreja. Usted sabe que está en la ubicación correcta cuando la cabeza ya no puede ser movido de lado a lado. Asegurar la boca con el montaje anterior de la estereotáxica y asegúrese de que la cabeza esté a nivel con una regla. Coloque la regla en posición vertical con respecto a la plataforma del instrumento estereotáxico y comprobar si hay un ángulo de 90 ° entre el gobernante y el centro de la cabeza del animal). Confirmarlo mediante el instrumento estereotáxico si ofrece esa capacidad.

  4. Hacer una incisión anterior / posterior en el cuero cabelludo con un bisturí estéril que se extiende desde la lambda para justo entre los ojos del animal. Use pinzas hemostáticas esterilizados para desprender la piel y mantener la incisión abierta. El uso de varios hisopos de algodón estériles, secar la superficie del cráneo al descubierto.

  5. Coloque la cánula guía en su montura, encontrar bregma en el cráneo, y la posición del derecho cánula guía en este lugar. Anote las coordenadas anterior / posterior y lateral. Desde bregma, encontrar las coordenadas correctas necesarias para la colocación de la sonda con la ayuda del atlas estereotáxico. La posición de la cánula guía para las coordenadas correctas, añadiendo o restando de bregma. Traiga su cánula guía hacia abajo hasta que toque el cráneo, y luego grabar esta coordenada ventral. Haga una marca de lápiz con un lápiz estéril en este lugar en el cráneo, que es donde se le de perforación.

  6. Retire la cánula guía y esterilizar su broca. Con cuidado, perfore un agujero en la marca del lápiz hasta llegar a través de la anchura del cráneo. Consulte con la cánula guía para ver si podía limpiar el agujero sin tocar los lados. Mantenga la perforación y el control hasta que la cánula puede borrar en un camino recto. Una vez que se hace el agujero, use una aguja estéril para golpear suavemente las meninges con el fin de permitir la inserción de la cánula sin obstrucciones.

  7. A continuación, utilizando un taladro de mano, hacer seis agujeros para tornillos de cabeza: dos por delante del orificio de la cánula, dos laterales para el agujero de la cánula, y posterior a los lados dos. Esterilizar seis tornillos y colocarlos en el cráneo hasta que estén bien anclados en.

  8. Limpie la cánula guía con etanol y la solución salina, montar y bajar lentamente a la coordenada correcta ventral. Asegúrese de que los lados no se toquen y que va a la perfección vertical.

  9. Coloque el tornillo de anclaje medial y detrás de los tornillos de cabeza posterior y mantenerla en su lugar con unas pinzas. Mezcle un lote delgada de cemento dental líquido y cubrir la cánula guía, tornillos, y el resto del cráneo con una espátula estéril. Hacer otro lote, esta vez más grueso, y cubren por completo la zona y el tornillo de la cánula y el anclaje suficiente para asegurarla.

  10. A medida que el cemento se vuelve más grueso y antes de que solidifique, separar la piel de la Copa del cemento y el molde de la copa de cemento con la espátula para asegurarse de que la tapa de cemento es lisa por todas partes y no irrita la piel después.

  11. Deje que el cemento dental se seque por completo antes de retirar al animal del aparato. Retire la hemostatos. Aplique bacitracina todo el camino alrededor de la tapa de cemento.

  12. Una vez que el animal está fuera del instrumento estereotáxico, que se inyectan con 0,25 ml de penicilina IM (intramuscular), seguido de 1 ml de solución salina SC (subcutánea).

  13. Colocar el animal en su propia jaula y el monitor hasta que se tome conciencia antes de volver a su habitación para recuperarse.

  14. Observar a los animales hasta que se recupere de la anestesia en el día de la cirugía y al día después de la operación, hasta el final del experimento, los signos de infección y la evaluación del dolor / angustia. Esto incluye los fines de semana y días festivos. Escaso movimiento espontáneo, la vocalización de socorro durante la manipulación, postura encorvada, diarrea, hinchazón y pus en el área de la headmount, y la falta de alimentación / consumo, son signos de dolor y angustia. La buprenorfina (0.1-0.5mg/kg SC) se administra dos veces al día, y luego, en una función de las necesidades, si el animal parece estar en el dolor. El tratamiento local con antibióticos (bacitracina pomada) y el tratamiento con antibióticos sistémicos (penicilina 100.000 UI / kg IM cada 12 horas durante las primeras 48 horas después de la operación) si se administran después de la operación las infecciones. Si alguno de estos síntomas persisten después de la administración de buprenorfina, el líquido adicional, y el tratamiento antibiótico en las 12 horas de cirugía, el animal es sacrificado.

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Discussion

Microdiálisis in vivo es la herramienta de elección para la medición de múltiples neurotransmisores y metabolitos en sitios distintos del cerebro de un animal vivo. Sin embargo, sólo controla los niveles extracelulares de neurotransmisores y que no ofrece el tiempo de resolución de monitor de exocitosis de neurotransmisores en tiempo real. A través de una versión de la técnica llamada "red de flujo", la concentración de neurotransmisores real en un lugar determinado se puede calcular, que a su vez puede dar las medidas exactas de la frecuencia de la recaptación de los neurotransmisores a través de transportadores de membrana plasmática.

Microdiálisis es ideal para ilustrar las diferencias en los niveles basales de neurotransmisor extracelular entre los diferentes grupos de animales (es decir, diferentes genotipos) y en el desciframiento de los efectos de las drogas u otras manipulaciones en la liberación de neurotransmisores.

La introducción de ensayos alternativos para HPLC-CE, como la electroforesis capilar de zona (CZE), junto con detección fluorescente se ha incrementado el tiempo de resolución de microdiálisis in vivo en pocos minutos por muestra.

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Acknowledgements

Con el apoyo de DK065872 (PEV), una familia Smith Premio de la Fundación de Excelencia en Investigación Biomédica (PEV), F31 DA023760.

Materials

Materials are described in the protocol document.

DOWNLOAD MATERIALS LIST

References

  1. Bungay, P. M., Newton-Vinson, P., Isele, W., Garris, P. A., Justice, J. B. Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma. J. Neurochem. 86, 932-946 (2003).
  2. Chen, K. C. Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters. Journal of Theor. Biology. 238, 863-881 (2006).
  3. Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats. FASEB Journal. 8, 2740-2746 (2008).
  4. Pothos, E. N., Creese, I., Hoebel, B. G. Restricted eating with weight loss selectively decreases extracellular dopamine in the nucleus accumbens and alters dopamine response to amphetamine, morphine and food intake. The Journal of Neuroscience. 15, 6640-6650 (1995).

Comments

37 Comments

  1. If I undertand the study correctly, it would be interesting to report the results of rate of reuptake for neurotransmitter, which is the main purpose of the empriment. 

    Reply
    Posted by: Anonymous
    October 10, 2008 - 6:14 PM
  2. Calculations of the reuptake rate of neurotransmitter can indeed be accomplished through net-flux microdialysis. However, the primary objective is the measurement of basal extracellular levels of neurotransmitters and their metabolites.

    Reply
    Posted by: Anonymous
    October 10, 2008 - 6:45 PM
  3. The authors have wonderfully demonstrated how to perform the stereotaxic experiment in rats. However they should have added few more words on how the ear bars should be adjusted so that it shows equal readings on both the sides before the opening of skull. In my experience I have noted that before opening the skull, one should make sure that both the vernier scales of the ear bars show almost correct readings in order to make sure the skull is on the right path, failure of which might lead to the miscalculation of the stereotaxic coordinates. Thanks and Regards, Rajesh S Omtri.

    Reply
    Posted by: Anonymous
    October 12, 2008 - 5:30 PM
  4. Correct placement of the ear bars is clearly a practice effect. We usually have one of the ear bars tight in position, then insert the tight ear bar in the ipsilateral ear canal, hold it in place and slowly insert the loose ear bar on the contralateral side before tightening it down. It is desirable that the skull is centered in between the ear bars. The skull surface must be always level (parallel to the platform of the stereotaxic instrument and at 90° to the guide of the microdialysis cannula) and skin at the incision surface should be flat and present no humps. These problems occur if the ear bars are inserted incorrectly (not in the ear canal). Correct ear bar placement can be identified by gently trying to move the head of the animal up and down and left to right. Before tightening the incisor bar, up or down movement but not lateral movement should be possible. Correct placement of the ear bars in the ear canal is the most important prerequisite for accurate stereotaxic placement. Emmanuel Pothos 

    Reply
    Posted by: Anonymous
    October 12, 2008 - 6:16 PM
  5. I am a scientist and I find it very hard to see such a gruesome procedure like this one. There should be a clear label on the content of videos as they can be quite disturbing, and they shouldn't be automatically broadcasted on the main website page. I wonder if the editors of this journal seriouly consider the possibility of risks the authors might face by being attacked by animal activists, and if the Principal Investigators of similar papers are held liable for exposing their students' identity to those groups while making these videos. This message dŒs not intend to diminish the value of the present work, but to bring this serious issue to the attention of the editors and the authors who appear on the video.

    Reply
    Posted by: Anonymous
    October 20, 2008 - 3:55 AM
  6. All procedures described in this article have been reviewed by the Institutional Animal Care University Committee at Tufts Medical Center and approved as compliant with federal and state standards of animal care. JoVE also conducted a veterinary review of the article before publication; nothing was "automatically broadcasted" as the viewer claims. Animals are anesthetized before any type of brain surgery, carefully monitored for appropriate depth of anesthesia and hydration during the procedure and diligently followed up through postoperative care with analgesic medication and antibiotics until full recovery. Stereotaxic brain surgery is one of the most sophisticated procedures in live and survivable animal surgery and it normally involves minimal pain for the operated animal because of the conditions set in place as described above. Stereotaxic brain electrode placement is a procedure that has been routinely used even for humans (i.e. Parkinson's disease patients) and such operations have been repeatedly broadcasted over the Internet from several hospitals for educational purposes. In some of these cases, the discomfort of the patient is so minimal that general anesthesia is not used and the patient is awake during surgery and able to respond to questions from the surgeons, who use the patient's response to assess the accurate placement of the electrode in the brain. The whole process in animals or humans is elegant, effective and high technology driven, not gruesome and painful. We appreciate the concern of the viewer about safety issues, but scientists have to take responsibility for their own work and it is not appropriate to publish it anonymously, being in this journal or elsewhere. Otherwise, the whole concept of the validity of peer-reviewed research and accountability of authors for their work is negated. There are numerous pieces of published work in different journals, including dissection videos, autopsies of animal tissue, images of animals etc. that can potentially be used by extremists to target the authors. Censoring scientific journals and scientists cannot eliminate this possibility.

    Reply
    Posted by: Anonymous
    October 20, 2008 - 8:30 AM
  7. Stereotaxic surgery should be performed under aseptic conditions. The surgeon shoud have a cap, mask, and surgery gloves. She should not be touching non sterile items while doing surgery, i.e. pens, cannula etc. Ophthalmic ointment is essential. Hemostats are not good  skin retractors as they damage tissue. There are antibiotics that can be given subcutaneously, which is easier and less painful to give.

    Reply
    Posted by: Anonymous
    February 2, 2009 - 4:18 PM
  8. There is not such a thing as sterile stereotaxic surgery in living animals. The mere presence of a living animal on the table with its fur, bodily fluids etc. negates sterile conditions. Doing the procedure under a culture hood with negative air flow is also not advisable as it limits access to the animal from all angles, it makes it more difficult for the animal to maintain appropriate temperature due to the air flow and it contaminates the hood area, which is counterintuitive particularly if the hood is used for cell cultures. The most appropriate actions are to sterilize the components used for the surgery (i.e. cannula and skull screws) prior to use, sterilize all insrtruments before surgery and during surgery as needed and maintain as clean of an environment as possible in the incision area by shaving away the fur and treating with povidine prior to the incision. Gloves should be used, face mask and cap will not hurt but none of the above will ensure sterile conditions. There is a variety of skin retractors available, we have not found that hemostats are worse than others in damaging tissue. Antibiotics given subcutaneoulsy are acceptable, but not as long lasting as those given intramuscularly. In any case, the easiest antibiotic to administer is bacitracin, right around the headcup of the animal. Emmanuel  

    Reply
    Posted by: Anonymous
    April 22, 2009 - 5:43 PM
  9. Suggestion for dental cement:  My lab uses a UV dental acrylic that is much easier to handle.  The acrylic sets when exposed to a UV light in about 10 seconds, and we do not need to use bone screws to secure the cap.  However, I'm guessing that the UV acrylic is more expensive.  Its available from Pentron. Oh, and don't forget eye lube.

    Reply
    Posted by: Anonymous
    November 1, 2008 - 10:56 PM
  10. We have tried in the past to use dental cements that their manufacturer claims do not require head screws.  We were not convinced. In many cases the cement head cup came off as one piece as we were trying to implant the microdialysis probe. Using sterile head screws is the best way to ensure that the cement cup will be securely attached to the skull. Any other method shaves off about ²0 min from each surgery but it increases the probability that the cup will come off and waste the entire procedure. Suppliers do tend to charge more for cements that supposedly work without head screws so in the long run this is not cost effective.  Eye lube as an eye protectant is indeed a very good precaution for this procedure. 

    Reply
    Posted by: Anonymous
    November 1, 2008 - 11:12 PM
  11. Nice demonstration of stereotaxic surgery in rats. I think that the best way to control that the skull is perfectly flat (parallel to the platform) would to check the height coordinates at the bregma and at the lambda using the canula as recommended in the stereotaxic atlas. That might not be a problem for ICV canulation since the ventricle are quite big but for canulation in a specific structure or nucleus it is critical.  I usually use only 4 screws but I guess 6 are necessary for a microdialysis probe. Also, do you calculate the coordinates from the surface of the skull or from the dura ?

    Reply
    Posted by: Anonymous
    December 2, 2008 - 11:30 PM
  12. We calculate steotactic coordinates from the skull surface.

    Reply
    Posted by: Anonymous
    December 3, 2008 - 12:11 AM
  13. how can i download (Survivable Stereotaxic Surgery in Rodents) thanks

    Reply
    Posted by: Anonymous
    December 18, 2008 - 5:18 AM
  14. Hi.  Please contact us at support@jove.com.

    Reply
    Posted by: Anonymous
    April 17, 2009 - 11:09 AM
  15. ı can not understand that why u r doing such as these trials for understanding brain mechanism, cuz I believe that if somethings can not explained naturally, we also can not understand exactly

    Reply
    Posted by: Anonymous
    January 7, 2009 - 7:54 AM
  16. Hey, it is obvious that you are not Dr. Ayla Arslan, then who are you? it seems like you are one of her students using her name as a nick as she always recommend JoVE during her Biopsychology lectures. :)))))))

    Reply
    Posted by: Anonymous
    October 31, 2009 - 6:14 PM
  17. Hi                 I am khalid a Ph.D scholar in deptt of pharmacy,university of Peshawar Pakistan.Its really great contribution to science and i eally enjoyed and learnt alot from the movie of Survivable Stereotaxic Surgery in Rodents thanks indeed and keep up this great work. khalid rauf

    Reply
    Posted by: Anonymous
    February 26, 2009 - 10:56 AM
  18. Wow, you guys really do not knwo what you are doing. Why would you use the archaic acrylic dental cement when you could use Glass Ionomer Luting Cement? Why didnt you anesthiatize with O² delivered isofluorine? Why did you not sue a stereotaxic drill? Why was the cement applied so sloppy? Why do you not use a digittal display for the coordinates, it ensures much more precise surgeries.

    Reply
    Posted by: Anonymous
    March 12, 2009 - 9:38 PM
  19. Hi Dave,

    How dŒs the Glass Ionomer Luting Cement compare with the Light-cured Dental Adhesive Resin listed in this journal by Okamura lab?

    Look forward to hearing from  you.

    Thanks,

    Jim

    Reply
    Posted by: Anonymous
    March 15, 2009 - 12:35 PM
  20. In our hands, dental acrylic is the only cement that ensured headcups stayed on for several weeks when used in combination with 6 skull screws. Emmanuel

    Reply
    Posted by: Anonymous
    April 22, 2009 - 4:04 PM
  21. Do you use any preanesthetic medications?

    Reply
    Posted by: Anonymous
    September 19, 2009 - 12:51 PM
  22. Usually not, if the animal suffers from CRD (chronic respiratory disease) you can pretreat with atropine to facilitate breathing. However, CRD is an indication of substandard conditions in the animal colony (infrequent change of bedding, poor air flow etc.). If you have animals with CRD, consult with your veterinarian to improve your facility and check on your source for laboratory animals, whether commercial or another lab, for facility conditions as well.

    Reply
    Posted by: Anonymous
    October 18, 2012 - 6:29 PM

  23. I am an undergraduate at the University of California, Santa Barbara doing an Honor's thesis project on the rat dorsal Raphe nucleus. In my project, I need to implant a cannula into the dRN, but am concerned about profuse sagittal sinus bleeding if I go through the midline. I noticed in other papers they often go into the DRN at about a 30 degree angle, in order to avoid this issue and also to avoid the cerebral aqueduct. As the angled cannula is a more complicated procedure, for me it would be easiest to place the cannula at the midline, and I'm wondering what's the best way to deal with these issues, such as how bleeding is stopped or slowed down, how it can be avoided, how many animals I can expect to lose, etc. Any advice would be much appreciated!

    Reply
    Posted by: Anonymous
    October 27, 2009 - 3:54 PM
  24. The angled approach is the best, but if you encounter sagittal sinus bleeding make sure to put in place large cotton tips from a sterile bag, press gently for a few minutes to slow down blood flow and leave on until blood has clotted. Then very carefully remove cotton tip to avoid breaking the blood clot. Although this bleeding would be fatal in humans, it usually is not fatal in rats. Emmanuel

    Reply
    Posted by: Anonymous
    October 18, 2012 - 6:34 PM
  25. nice job.
    just some comments:
    the membrane (after cuting the skin to expose the skull) should be carefully and totally removed - this decrease the chances of the acrylic fall off.
    if you do a small cut, 1 or ² screw would be enough.
    another important thing, regarding guide cannula is that it should be obstruct after surgery so that no reflux happens and nothing enters for this hole - if this happens you can loose all you surgery. If, when you try to put a needle inside for drug injection (p.ex) and it dŒsnt enters, you can use a some H²O² to open it (in case of blood coaguation)

    Reply
    Posted by: Anonymous
    December 11, 2009 - 4:52 PM
  26. We use obdurators to seal off guide cannulas post-op. We avoid using only 1-² screws no matter what the size of the incision, this is clearly inadequate anchoring for the headcup and it will come off in a matter of a few days at best. It really pays off to anchor the headcup with as many screws as you can. Emmanuel

    Reply
    Posted by: Anonymous
    October 18, 2012 - 6:46 PM
  27. Oh! another thing...
    would be really good to use local anesthetic with vasoconstrictor before cutting the scalpe.
    this will minimize animal nociception and will avoid excessive bleeding.
    but not toooooo much, other wise wont be god to animals, and we also see some increase of infeccion

    Reply
    Posted by: Anonymous
    December 11, 2009 - 4:57 PM
  28. The authors and our attending veterinarian would like to add the following information to the article, which was not otherwise clearly stated or shown and may be of help to readers and viewers:
    1) After anesthesia and prior to surgery, eye lubricant was applied to protect the corneas of the animals.
    ²) Prior to inserting the ear bars into the rats' ears, lidocaine gel was applied to provide analgesia.
    3) All rats did receive an initial dose of buprenorphine following the surgeries and then were given subsequent doses on an as needed basis. This was not clear in the video or text.
    4) The dose of penicillin given was 100,000 IU/kg.
    All of the above measures were approved in our IACUC protocol for this procedure and our attending veterinarian has reviewed and verified these additional comments.

    Reply
    Posted by: Anonymous
    April 28, 2010 - 3:07 PM
  29. is it better that ketamine is better than halothan as a anesthetic agent

    Reply
    Posted by: Ravi S.
    May 14, 2010 - 2:16 AM
  30. my just quest is how the hole in the skull is closed/filled? Did you leave it for natural tissue growth or use gelatin or something else.thanks

    Reply
    Posted by: Anonymous
    May 14, 2010 - 9:48 AM
  31. In our case the guide cannula leaves very little space to add anything else. Some of my colleagues are using bone wax or gelatin for larger openings. Emmanuel

    Reply
    Posted by: Anonymous
    October 18, 2012 - 6:41 PM
  32. No flash please

    Reply
    Posted by: Anonymous
    August 8, 2010 - 1:31 PM
  33. Overall a nice video, but there are a few things that should be done to improve aseptic technique. The eyes need to be lubricated prior to shaving to protect them from the hair and from dessication. To reduce infections a surgical drape should be used, along with a surgical mask. Lastly, pointing with a sterile instrument would have been better.

    Reply
    Posted by: Anonymous
    November 5, 2010 - 6:34 PM
  34. It is a very nice presentation, I would like to add a little in it . When the animal id fixed with ear bars and the scale on ear bar and the scale of sterotaxic base should be equidistant
    See in video your demo point 0²:51.

    Varsha

    Reply
    Posted by: Anonymous
    May 24, 2011 - 3:35 AM
  35. Im a 4th year Psych Honours student doing a project that needs me to implant canulas into the infralimbic. I just did my second practice surgery today, and it was terrible. The cannulas were mislocated, it took me an hour to put in 4 bone screws, and they went through the skull, and the dental cement ran into its eyes, and Im just glad that rat was put down before it woke up because there is no way it would have survived. I've always been sort of clumsy, and I have to do 35 of these, and half my year is gone and I dont have time to come up with another project.
    So yeah, Im freaking out right now,

    Reply
    Posted by: Anonymous
    May 30, 2011 - 8:58 AM
  36. Hi Nadia,
    It seems you do not have adequate training and supervision to perform this procedure. It is essential that a member of your laboratory team with extensive experience in stereotactic surgery, if not the primary investigator directly and the head veterinarian for your institution's animal facility, should go over things with you multiple times and actively do the procedure with you before any further attempts. You have to ensure the animal's welfare, lack of pain and recovery during and after the procedure if you wish to be anywhere close to acceptable standards. In my opinion, brain stereotactic surgery is an advanced procedure that should be used only with the maximum of caution and the best of training for undergraduate projects. If the animal facility or your faculty supervisor do not have the time or the skills to train you properly, then it would be best to choose something else for your thesis project. Best, Emmanuel

    Reply
    Posted by: Anonymous
    May 30, 2011 - 10:46 AM
  37. Hi everyone,

    Just a quick question, after attaching the ear bar I have realized that it takes me quite a while after making an incision in the skull to expose the bregma and lambda. Any suggestions?

    Reply
    Posted by: Jin P.
    November 5, 2012 - 6:41 PM

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