Application of 3D Printing Technology to Produce Hippocampal Customized Guide Cannulas

Abstract

Implantation of guide cannulas is a widely used technique to access specific brain areas. Although commercially available, the need to personalize these implants and the high cost prompted us to design open-source customized devices taking advantage of 3D printing technology. Our cannulas consisted in a 3D-printed head mount designed according to the Paxinos coordinates to reach the CA1 area of the hippocampus. To cut guide cannulas to the proper length, we designed and realized an original 3D-printed linear motion apparatus. Polylactic acid thermoplastic polymer was used as printing material. Homemade or commercial cannulas were implanted in 4- to 6-month-old wild-type mice and intrahippocampal injections of amyloid-β peptide at different concentrations were performed. In vivo behavioral studies of novel object recognition indicated that results obtained with homemade versus commercial devices were comparable. Methylene blue injections and Nissl staining confirmed the correct localization of cannulas in the CA1 area of mouse hippocampus. Our method allows a fast manufacturing of hippocampal cannulas preserving the required precision at very low cost. Furthermore, this system can be easily modified to produce cannulas to target other brain areas. In conclusion, 3D printing might be used as a useful and versatile technology to realize open-source customized devices in neuroscience laboratories.

Keywords: 3D printing; behavioral studies; brain cannulas; hippocampus; neuroscience method; open source apparatus.

Figure 1.

Representation of head mount and guides. a, Photograph of a cannula of our production. b, Rendered image of an assembled cannula. c, Exploded isometric view of the components and key parameters of a guide cannula. d, Technical drawing of the head mount, including all of the relevant dimensions. All measures are expressed in millimeters. e, Left and right guides measured in 26 cannulas have the correct expected length of 1330 μm. Data are expressed as mean ± SEM.

Figure 2.

Linear motion-cutting system. a, Rendered representation of the cutting system. b, Colored highlight of the single components, as follows: (1) light blue, the cutting frame, formed by a base to host the final part of the linear rail and a support for the handheld rotary tool (Dremel); (2) pink, the linear rail, consisting of two 8 mm steel bars held by 3D-printed components to form a sliding carriage; (3) violet, the cut length regulator allowing determination of the length of the guides; and (4) green, the cannula holder placed on top of the carriage to host and hold the head mount in place during the cutting process. c–e, Photographs with front, top, and closeup views of the actual apparatus built in our laboratory.

Figure 3.

Descriptive images of the cutting frame and the linear rail. a, Rendered image of the isolated cutting frame clamped to the table. b, Schematic drawing of the cutting frame. The rotary tool support is mounted on the base and fixed to a structure that can be moved to modify the position of the cutting blade and host air vents to avoid overheating. The rotary tool is equipped with a 38 mm circular diamond-coated blade. c, Rendered image of the linear rail and the base of the cutting frame, both clamped to the table. d, Schematic drawing of the linear rail that is composed of two parallel 8 × 270 mm bars with a 46 mm interaxial distance. The carriage consists of a base with two blind holes hosting three LM8UU linear ball bearings, two on one side and one on the other side to ensure a straight alignment of the 8 mm bars. The base is equipped with a 3D-printed handle to move the carriage back and forth along the linear rail. Another carriage is mounted on the top of the base by four socket head cap M3 screws.

Figure 4.

Cutting length regulator. a, Rendered image of the cutting length regulator, consisting of two main parts, both 3D printed. b, Schematic drawing of the cutting length regulator. The moving part is inserted into its base by two trapezoidal slots. c, Rendered exploded view of the cutting length regulator. d, Schematic drawing of the exploded view in c. The displacement of the carriage is granted by the following two components: an M6 threaded rod, passing through the base and the carriage, with a self-locking M6 nut; an antibacklash system composed of two M3 socket head cap screws supporting two springs pushing from the surface of the base to the inside of the carriage. The carriage can be moved by turning the screw and is held in place by the tension of the springs. Two holders are placed on the side of the carriage fixing it to the base.

Figure 5.

Cannula holder. a, Rendered image of the cannula holder. b, Schematic drawing of the cannula holder formed by a base and a lid. The base, designed to host the cannula head mount, shapes its octagonal form and presents with grooves for the guide cannulas to stay in place. c, Rendered exploded view of the cannula holder. d, Schematic drawing of the exploded view in c. The cannula holder hosts 10.2 × 2.2 mm cylindrical neodymium magnets. Two magnets are inserted on the base of the cannula holder, two on the lid, and one in front of the guides.

Figure 6.

Illustrated step-by-step process of the cutting procedure. a, Positioning of cannula body in its holder with the open side facing the end limit wall. The head mount body should be placed into its housing shaped in the cannula holder oriented as shown in the insert. b, Regulation of the cutting length. Carriage is pushed underneath the blade (multitool turned off), and its relative position is adjusted to the blade. For optimal positioning, the blade is as close as possible to the cannula without touching it. c, Positioning of the steel guides and the head mount shell. The carriage is placed back into a safe position. Steel tubes (spinal needles) are inserted into the head mount through the appropriate guides until they reached the limit stop represented by the wall behind the cannula. A drop of superglue is placed on the cannula body and is covered by the shell part of the cannula to let the head mount be assembled. d, Positioning of the magnetic lid to ensure a tight compression on the head mount ensured by the magnets. e, Cutting of the steel guides at the appropriate length. The rotary tool (Dremel) is turned on at ∼5000 rpm. The main carriage is pushed to the rotating blade cutting the guides. After cutting, the carriage is moved back to the starting position, the rotary tool is stopped, and the lid is removed to extract the cannula.

Figure 7.

Intrahippocampal cannula validation. a, Mouse brain after cannula removal. Guide holes are equidistant from the interhemispheric fissure. b, Injections of methylene blue in the right hemisphere reaching the CA1 area. c, Nissl staining of the coronal section of a mouse left hemisphere. The cannula lesion is visible above the hippocampus. d, In mice implanted with commercial cannulas, intrahippocampal injections of 200 pm Aβ increased the discrimination index, whereas injection of 200 nm Aβ impaired it. e, The same results are obtained in mice infused through our homemade cannulas. f, g, Total exploration time is not modified by treatment in mice injected by commercial (f) or homemade (g) cannulas. *p < 0.05; **p < 0.01. Data are expressed as the mean ± SEM.