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Journal of Pharmacological and Toxicological Methods 64 (2011) 246–250

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Journal of Pharmacological and Toxicological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox

Original article

A simple and inexpensive method to fabricate a cannula system for intracranial injections in rats and mice Dadasaheb M. Kokare a, Gajanan P. Shelkar a, Chandrashekhar D. Borkar a, Kartik T. Nakhate a, Nishikant K. Subhedar b,⁎ a b

Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University Campus, Amravati Road, Nagpur, Maharashtra, 440 033, India Indian Institute of Science Education and Research (IISER), First Floor, Central Tower, Sai Trinity Building, Garware Circle, Sutarwadi, Pashan, Pune, Maharashtra, 411 021, India

a r t i c l e

i n f o

Article history: Received 2 June 2011 Accepted 5 August 2011 Keywords: Stereotaxic cannulation Guide cannula Dummy cannula Internal cannula

a b s t r a c t Introduction: Stereotaxic administration of neuroactive agents, either in ventricles, or targeted at specific intracranial sites, is a widely employed strategy for neurological studies in rodents. Surgical implantation of cannula on the skull is particularly useful in chronic treatments. We describe a simple, inexpensive and reliable method to fabricate a cannula system for delivery of drugs at the targeted sites in the brain of rat or mouse. Methods: The system consists of a guide cannula made from a hypodermic needle (24 gauge), a stainless steel wire (30 gauge) that serves as a dummy cannula, and an internal cannula made of stainless steel needle (30 gauge) taken from a hypodermic syringe. The cannula can be implanted by routine stereotaxic procedure and used for acute or chronic drug administration to conscious, free moving animals. Results: With a view to test the system for accuracy, the guide cannula was stereotaxically implanted, and neuropeptide Y was directly delivered into the lateral ventricle. These rats showed a significant increase in food intake. Another set of rats were cannulated for chronic protocol, wherein ethanol was delivered directly into the ventral tegmental area. In operant chamber, these rats showed increased ethanol self-administration. The proposed cannula takes around 5 min to fabricate and costs less than a dollar. Conclusion: We feel that it may serve as an economical and reliable tool in neuropharmacological and neurobehavioral studies. © 2011 Elsevier Inc. All rights reserved.

1. Introduction

2. Materials and methods

Site specific administration of neuroactive substances, pharmacological agents and neuronal tracers, etc., is a routine practice in neurological studies. To this end, stereotaxic cannula implantation is widely undertaken particularly for chronic treatments. Although some methods for the fabrication of cannula system are previously reported (Crane & Glick, 1979; Dougherty & Ellinwood, 1981; Packard, Pohorecky, & Brick, 1984), these seem complex and time consuming. A good cannula system is expected to fulfill the following criteria. It should (1) guarantee a straight implantation at the targeted site, (2) be easy to perform, 3) be non-toxic to the brain tissues, and 4) be cost effective. In our laboratory, we have fabricated a simple cannula system which can fulfill all the above criteria. We provide a list of required raw materials, method of fabrication, and evidence in support of successful implants.

2.1. Fabrication of cannula system

⁎ Corresponding author. Tel.: + 91 20 25908055. E-mail address: [email protected] (N.K. Subhedar). 1056-8719/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2011.08.002

The cannula system consists of the following three components. (1) A guide cannula to be implanted on the skull. (2) A dummy cannula or stylet or obturator made of a stainless steel wire that passes through the guide cannula and blocks the passage when the cannula is not in use. (3) The internal cannula or injector cannula or infusion cannula which is introduced through the guide cannula and serves to deliver the fluid into the target brain region. Fine serrated knife, file and vernier calipers are the tools required, and the procedure of the cannula system fabrication is described below. 2.1.1. Fabrication of guide cannula 1. A 24 gauge hypodermic needle (Becton Dickinson India Pvt. Ltd., India; Fig. 1A) was used for making the guide cannula. The procedure is described below and graphically shown in Fig. 1B–F. 2. Remove the white cement (Fig. 1B) and the smooth surface hub (Fig. 1C) manually with the serrated edge of a sharp knife. This provides the flat base that can sit snugly on the skull. Using the same knife, cut the stainless steel barrel to the desired length

D.M. Kokare et al. / Journal of Pharmacological and Toxicological Methods 64 (2011) 246–250


Fig. 1. Steps involved in the fabrication of the guide cannula are graphically represented. From a 24 gauge hypodermic needle (A), white cement part was removed (B), and this was followed by the removal of the smooth surface hub (C), cutting of stainless steel barrel to desired length (D), and removal of the top funnel portion (E). One wing may be trimmed (F) if the cannulae are to be used for adjacent bilateral implants.

(Fig. 1D). The length depends on the ventral coordinates described for the rat or mouse brain (Paxinos & Franklin, 2001; Paxinos & Watson, 1998). Verify the length of the barrel using a vernier scale. For implanting the cannula directed at the lateral ventricle (LV) in the brain of rat (240–260 g), the barrel may be cut to 3.5 mm, or 8 mm, if the cannula is to be targeted at the posterior ventral tegmental area (VTA). 3. Flatten the cut end of the barrel tip with a fine file. 4. With the help of the knife, remove the rounded funnel portion of the cannula. The guide cannula is now ready for implantation on the skull (Figs. 1E, 2C). 5. If bilateral cannulations are required, and if the two implantation sites are close to the mid-sagittal plane (~0.4 mm), one of the four wings of both the cannulae may be removed (Fig. 1F) using the razor blade. While implanting on the skull, each cannula should be arranged such that the winged part is directed laterally (Fig. 2D).

2.1.2. Fabrication of dummy cannula 1. Take a 30 gauge stainless steel wire (purchased from local electronic market) and insert one end into the guide cannula till it protrudes 0.5 mm on the other side of the guide cannula. From the upper end cut the wire about 4–5 mm above the level of guide cannula with scissor (Fig. 2A, B). 2. Bend the outer end of dummy cannula at 90° so that it will not slip through the guide cannula or cause any injury to fore paws as the animal grooms itself. 2.1.3. Fabrication of internal cannula 1. It is prepared from a 30 gauge stainless steel needle attached to a disposable hypodermic insulin syringe (Becton Dickinson India Pvt. Ltd., India) (Fig. 3A).

Fig. 2. A 30 gauge stainless steel wire (A), employed as a dummy cannula was inserted through the guide cannula (B). The position of guide, and dummy cannulae, directed at lateral ventricle [− 0.8 mm posterior, + 1.3 mm lateral to midline and 3.5 mm ventral with respect to bregma (Paxinos & Watson, 1998)] (C), and bilaterally at the ventral tegmental area [– 5.8mm posterior, ±0.7 mm lateral to midline and 8.0 mm ventral with respect to bregma (Paxinos & Watson, 1998)] (D) is shown.


D.M. Kokare et al. / Journal of Pharmacological and Toxicological Methods 64 (2011) 246–250

Fig. 3. Steps involved in the fabrication of internal cannula using a 30 gauge insulin syringe (A). The rectangle indicates the part that was separated (B), and then the upper plastic portion was removed to obtain the internal cannula (C).

2. Separate the stainless steel needle with some holding plastic portion still intact (Fig. 3B) from the body of the syringe using a sharp razor blade. 3. Cut the needle away from the base of the pedestal (Fig. 3C) such that the tip extends 0.5 mm beyond the respective guide cannula. 4. Insert the needle in a polyethylene (PE10) tube and seal with superglue (Fig. 4A), allow it to dry, and check for blockade or leakage by injecting double distilled water using microliter syringe. 5. The internal cannula is now ready for use (Fig. 4B). 2.2. Evaluation of the cannula using rat model Different groups of rats were subjected to the stereotaxic surgery to implant the guide cannula into the LV or VTA. The method has been described previously (Bhisikar, Kokare, Nakhate, Chopde, & Subhedar, 2009; Deo, Dandekar, Upadhya, Kokare, & Subhedar, 2010; Kamdi, Nakhate, Dandekar, Kokare, & Subhedar, 2009; Kokare, Patole, Carta, Chopde, & Subhedar, 2006). Briefly, rats anesthetized with intraperitoneal injection of thiopentone sodium (45 mg/kg), were placed in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA) and under aseptic conditions, a guide cannula was implanted unilaterally into the LV or VTA. The co-ordinates used for LV are −0.8 mm posterior, +1.3 mm lateral to midline and 3.5 mm ventral, and for VTA, these are −5.8 mm posterior, −0.7 mm lateral to midline and 8.0 mm ventral

with respect to bregma (Paxinos & Watson, 1998). Guide cannulae were secured to the skull with the help of stainless steel jewelry screws and held fast with rapidly polymerizing dental acrylic cement (DPI-RR Cold Cure, acrylic powder, Dental Product of India, Mumbai) to the surface of the skull. After the hardening of acrylic cement, the animal was removed from stereotaxic frame. A dummy cannula was introduced into the guide cannula to allow the guide cannula to maintain patency. After surgery, rats were housed individually and allowed to recover for 7 days. After the recovery period, separate sets of LV cannulated rats were injected (5 μl/rat) with potent orexigenic agent, neuropeptide Y (NPY, Sigma, 0.04–0.16 ng/rat) at the onset of the dark phase. The control rats were given artificial cerebrospinal fluid (aCSF). Before administration, the syringe and the tubing were filled with double distilled water and a small air bubble was introduced into the tubing to separate the infusion fluid from the double distilled water. The displacement of air bubble inside the tubing helped to monitor the precise flow of the solution during injection. Immediately after the injections, the preweighed food pellets were offered and cumulative food intake was measured (g) at 2, 4 and 6 h post-injection time-points. Separate groups of VTA cannulated rats were trained in operant chamber (Coulbourn Instruments, USA) to self-administer ethanol solution (100–300 mg%) into the VTA (Rodd et al., 2004), while control rats were given aCSF. Each lever press delivered 100 nl of aCSF or ethanol into the VTA over a period of 5 sec followed by 5 sec timeout interval. The numbers of lever pressings for the intra-VTA aCSF or ethanol infusion were recorded for the duration of 30 min. Additional groups of rats were implanted with commercially available cannulae, targeted at the LV and VTA, and above experimental protocols were repeated. In all the experiments, dummy cannula was removed and replaced manually, each day, with a view to maintaining the patency of guide cannula and to desensitize the rats to injections. All procedures employed in the present study were approved by Institutional Animal Ethics Committee and carried out under strict compliance with Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India, New Delhi, India.

2.3. Cannula placement verification Following termination of the above experiments, the rats fitted with intra-LV or intra-VTA cannulae were euthanized with an overdose of intraperitoneal injection of thiopentone sodium (65 mg/kg). The brains were removed, sectioned in coronal plane using a cryostat, stained with cresyl violet, and examined under the light microscope for the placement of cannula tip and tissue damage (Fig. 5A, B).

Fig. 4. The internal cannula was attached to the polyethylene (PE10) tube (A) and introduced through the guide cannula for intracranial injection to rat (B).

D.M. Kokare et al. / Journal of Pharmacological and Toxicological Methods 64 (2011) 246–250


Fig. 5. Coronal sections of rat brain, showing passage of a guide cannula (asterisk) directed towards the lateral ventricle (LV, A) or ventral tegmental area (VTA, B). IP, interpeduncular nucleus; ml, medial lemniscus; SN, substantia nigra.

3. Results and discussion Earlier attempts at fabricating the guide cannula used stainless steel tubing that was cut to the desired length and polished prior to application (Crane & Glick, 1979). Subsequently, stainless steel barrel obtained from hypodermic needle, embedded in molded dental acrylic cement, was used as a guide cannula (Dougherty & Ellinwood, 1981). Packard et al. (1984) used leur lock base of plastic syringe and stainless steel needle was embedded into leur lock, fixed with dental cement and used as a guide cannula. However, these methods seem complex, tedious and time consuming. In contrast, the proposed method is simple, economical and time saving. The commercial cannulae along with stylet project considerably over the head (~12 mm) and therefore, are more likely to be dislodged by the rat as it grooms itself. Our cannula projects only 6 mm over the skull and are less likely to be dislodged. However, our guide cannula is quite similar to the commercial cannula in terms of the outer diameter. We tested the placement of the proposed guide cannula by stereotaxically implanting them into the LV and VTA, and the accuracy was revealed in microscopy sections. The cannulae were precisely located in the respective target area and no tissue damage, beyond the path of the cannula, was noticed (Fig. 5A, B). The cannula can be used for unilateral, and with a little modification, for the bilaterally symmetrical implantations, even if each is as close as ~0.4 mm to the mid-sagittal plane. Using these cannulae, it is possible to target regions like VTA/medial forebrain bundle and nucleus accumbens shell in the same animal. This may be necessary for intracranial self-infusion, intracranial brain stimulation and intra-LV infusion by osmotic pump with parallel injections in other areas. Dummy cannulae used in the present study were made of a simple wire of desired length and could be replaced each time the internal cannula was introduced during multiple injections. Commercially available cannula system consists of screw cap for dummy cannula which requires extra care while tightening and loosening the dummy cap from the guide cannula, and sometimes the guide cannula may detach from the skull, if excess force is applied. Using the proposed cannula system, we administered NPY into the LV and observed a highly significant increase in the cumulative food intake in a dose related fashion (Fig. 6A). In operant chamber studies, rats showed a concentration dependent increase in the lever pressings for ethanol self-administration into the VTA (Fig. 6B). Comparable results were obtained in the experiments in which commercially obtained cannulae were employed. The results are in agreement with those reported in earlier studies (Nakhate, Dandekar, Kokare, & Subhedar, 2009; Rodd et al., 2004). The cannulae fabricated in our

laboratory were also similar to the commercial in terms of precision in location and caused minimum damage to brain tissues. As in the case of the commercial cannula, our cannula could be used for over 1 month with no loss of patency and employed for targeting almost any part of the rat brain. For example, in our experiment on the self-administration of ethanol in the operant chamber, the cannula was connected and

Fig. 6. (A) Cumulative food intake by the different sets of rats injected with aCSF (5 μl/rat) or NPY (0.04–0.16 ng/rat) via the cannula system directed at the lateral ventricle. The food intake was measured (g) at 2, 4 and 6 h post-injection time-points. Higher doses resulted in significant increase in food intake. (B) Separate groups of rats, implanted with cannula directed at the ventral tegmental area (VTA), were subjected in operant chamber to self-administer aCSF or ethanol (100–300 mg%) into the VTA for a period of 30 min. The number of lever pressings for ethanol self-administration was recorded during the 30 min of the protocol. The data represent mean ± standard error of mean for each group. The results were analyzed using one-way ANOVA followed by post-hoc analysis of significance with Bonferroni's multiple comparison test. *p b 0.05, **p b 0.01, ***p b 0.001 vs respective aCSF.


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disconnected to the infusion system, each day, for 3–4 weeks to the freely moving rat. In spite of these rigorous handlings, the integrity of the cannula was not compromised. All the components of the cannula system described herein can be easily procured from the market. Fabrication of the cannula, that cost less than a dollar, may be recommended particularly if a large number of rats are to be cannulated. Fabrication of a single cannula does not take much time, with little practice it could be fabricated in less than 5 min. Finally, it may be mentioned that the proposed cannula system is equally suitable for use in mice. Acknowledgment This work was supported by grants from the Department of Biotechnology, Government of India, New Delhi, India (BT/PR14022/ MED/30/324/2010). References Bhisikar, S. M., Kokare, D. M., Nakhate, K. T., Chopde, C. T., & Subhedar, N. K. (2009). Tolerance to ethanol sedation and withdrawal hyper-excitability is mediated via neuropeptide Y Y1 and Y5 receptors. Life Sciences, 85, 765–772. Crane, L. A., & Glick, S. D. (1979). Simple cannula for repeated intracerebral drug administration in rats. Pharmacology, Biochemistry, and Behavior, 10, 799–800.

Deo, G. S., Dandekar, M. P., Upadhya, M. A., Kokare, D. M., & Subhedar, N. K. (2010). Neuropeptide Y Y1 receptors in the central nucleus of amygdala mediate the anxiolyticlike effect of allopregnanolone in mice: Behavioral and immunocytochemical evidences. Brain Research, 1318, 77–86. Dougherty, G. G., Jr., & Ellinwood, E. H., Jr. (1981). A simple multiple-cannula headpiece for the rat. Physiology & Behavior, 26, 879–900. Kamdi, S. P., Nakhate, K. T., Dandekar, M. P., Kokare, D. M., & Subhedar, N. K. (2009). Participation of corticotropin-releasing factor type 2 receptors in the acute, chronic and withdrawal actions of nicotine associated with feeding behavior in rats. Appetite, 53, 354–362. Kokare, D. M., Patole, A. M., Carta, A., Chopde, C. T., & Subhedar, N. K. (2006). GABAA receptors mediate orexin-A induced stimulation of food intake. Neuropharmacology, 50, 16–24. Nakhate, K. T., Dandekar, M. P., Kokare, D. M., & Subhedar, N. K. (2009). Involvement of neuropeptide Y Y1 receptors in the acute, chronic and withdrawal effects of nicotine on feeding and body weight in rats. European Journal of Pharmacology, 609, 78–87. Packard, K., Pohorecky, L. A., & Brick, J. (1984). A simple cannula for intraventricular drug administration in rodents. Journal of Neuroscience Methods, 10, 139–143. Paxinos, G., & Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates (2nd edition). California: Academic Press. Paxinos, G., & Watson, C. (1998). The rat brain in stereotaxic coordinates. London: Academic Press. Rodd, Z. A., Melendez, R. I., Bell, R. L., Kuc, K. A., Zhang, Y., Murphy, J. M., et al. (2004). Intracranial self-administration of ethanol within the ventral tegmental area of male wistar rats: Evidence for involvement of dopamine neurons. Journal of Neuroscience, 24, 1050–1057.

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