1Department of Neuroscience, Section Anatomy, University Medical Center Groningen, University of Groningen, 2Animal Welfare Office, University of Groningen
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Fornari, R. V., Wichmann, R., Atsak, P., Atucha, E., Barsegyan, A., Beldjoud, H., et al. Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience. J. Vis. Exp. (59), e3528, doi:10.3791/3528 (2012).
Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.
Note: Antiseptic techniques should be employed during the whole procedure. All the instruments and materials (cotton-tipped swabs, gauze, etc.) that will be used during the surgery should be sterilized by autoclaving. A surgical mask, hair bonnet and sterile gloves should be worn. The working area and the stereotaxic apparatus should be cleaned thoroughly, and disinfected with a 70% ethanol solution.
1. Pre-surgical procedures
3. Post-surgical care
4. Representative Results
To determine whether the various modifications to our surgical procedure, particularly with respect to oxygen supplementation and the continuous monitoring of blood oxygenation levels, heart rate and body temperature, enhanced the animal's survival and improved its general condition after surgery, we compared the non-survival rate of 20 cohorts of animals (consisting of 20 rats each) that underwent surgery after we implemented these modifications with the non-survival rate of 24 cohorts (20 rats each) operated with the standard protocol. As is shown in Figure 1a, the non-survival rate was significantly reduced in the sample of cohorts that was operated with the modified protocol (P < 0.05; Mann-Whitney U test, two tailed). Moreover, as is shown in Figure 1b, post-surgical weight loss of rats operated with the modified protocol was also significantly reduced as compared to that of rats operated with the standard procedure (post-operative day 1: P < 0.05; post-operative day 2: P < 0.01; post-operative day 3: P = 0.17; Student t-tests).
Figure 1. Effect of surgical modifications on non-survival rate and post-surgical weight loss. (A.) Non-survival rate of rats operated with the modified protocol as compared to that of rats operated with the standard protocol. The non-survival rate (median ± interquartile ranges) was calculated as the percentage of rats, per cohort of 20 rats, that did not survive surgery. *p < 0.05, Mann-Whitney U test two tailed (n = 20 cohorts for the modified protocol and 24 cohorts for the standard protocol). (B.) Weight loss (mean ± SEM as percentage of pre-surgery weight) during the first (POD1), second (POD2) and third (POD3) post-operative day. *P < 0.05, **P < 0.01, Student t-test (n = 60 per group).
The main purpose of this video presentation is to familiarize behavioral neuroscientists with the basic principles of stereotaxic surgery. Researchers who are already performing stereotaxic surgery might also benefit from this video and consider some of the procedural refinements for use in their own laboratory. An ever-increasing knowledge about optimizing surgical techniques1-3, the development of new anesthetics and analgesics for use in human and veterinary medicine4,12, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to implement new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery. We observed, as shown for a representative sample of animals, an overall increased survival rate and a significantly reduced post-surgical weight loss. Such a reduced post-surgical weight loss might reflect a smaller burden of the surgical procedure on the animal and, consequently, result in a more active animal in the immediate aftermath of surgery. Whether it also has beneficial effects on its long-term health is not clear. However, a remarkable observation was that removing the oxygen supply temporarily led to a marked and reliable decrease in blood oxygen saturation levels, which could drop even below 50% (See the video presentation for a demonstration hereof). It is conceivable that prolonged inadequate blood levels of oxygen, possibly as occurring in animals not provided with any oxygen supplementation during surgery, might result in hypoxia with long-term behavioral consequences and also negatively impact the outcome and/or quality of behavioral experimentation. We do not know whether such a depression of oxygen blood supply during surgery is specific to the anesthetic protocol used in our laboratory (i.e., a mixture of ketamine and dexmedetomidine) or whether it is a more general phenomenon associated with injectable anesthetics. The use of inhalation anesthesia, with a mixture of ambient air and oxygen, might be an alternative method to overcome the depression of blood oxygen levels.
The authors have nothing to disclose.
The authors thank Dr. Peter Gerrits for the drawings used in the video.
|Alcohol 70%||VWR international|
|Atropine sulfate 0,5||Teva Pharmachemie|
|Bulldog haemostatic clamp||Aesculap|
|Cannulas||Component Supply Co.|
|Chlorhexidine 0.5%||APP Pharmaceuticals|
|Cleaning powder||Alconox, Inc.|
|Cotton sticks||Hartmann BV|
|Durogrip Naaldvoerder converse 130mm||Aesculap|
|Durotip Fijne schaar ret.sp/st 110 mm||Aesculap|
|Heating pad||Harvard Apparatus|
|Insect pins (stylets)||Entosphinx (Czech Republik)|
|Ketamine 10% (ketamine)||Alfasan|
|Micro-halsted straight tip||Aesculap|
|NaCl 0,9%||Baxter Internationl Inc.|
|Needles (25G)||Terumo Medical Corp.|
|Oximeter||Edan Instruments, Inc.|
|Pentobarbital||Pharmacy of the UMCG|
|Rimadyl||Pfizer Pharma GmbH|
|Scalpel blade No. 23||Swann-Morton|
|Scalpelholder NR. 4 133 mm||Aesculap|
|Screw driver||Any Supplier|
|Simplex Rapid (dental cement)||Kemdent|
|Skull screws||Component Supply Co.|
|Spongestan special||Johnson & Johnson|
|Surgical forceps 100mm||Aesculap|
|Suture material Safil 5/0||Aesculap|
|Syringe 10 ml||Omnifix|
|Syringe 1ml||Terumo Medical Corp.|