A watertight acrylic-free titanium recording chamber for electrophysiology in behaving monkeys

Abstract

Neurophysiological recording in alert monkeys requires the creation of a permanent aperture in the skull for repeated insertion of microelectrodes. Most laboratories use polymethyl methacrylate to attach a recording chamber over the skull opening. Here, we describe a titanium chamber that fastens to the skull with screws, using no polymethyl methacrylate. The gap between the base of the chamber and the skull is filled with hydroxyapatite, forming a watertight gasket. As the chamber base osseointegates with the skull, the hydroxyapatite is replaced with bone. Rather than having a finite lifetime, the recording chamber becomes more firmly anchored the longer it is in place. It has a small footprint, low profile, and needs little maintenance to control infection. Toilette consists of occasional application of betadine to clean the scalp margin, followed by application of neomycin, polymyxin, and bacitracin ointment. Antibiotic is also placed inside the chamber to suppress bacterial proliferation. Thickening of the dura within the chamber can be prevented by regular application of mitocycin C and/or bevacizumab, an antibody against vascular endothelial growth factor. By conducting an e-mail survey, this protocol for chamber maintenance was compared with procedures used in 37 other vision research laboratories. Refinement of appliances and techniques used for recordings in awake monkeys promises to increase the pace of scientific discovery and to benefit animal welfare.

Fig. 1.

A: lateral view of a Macaca mulatta skull from an animal that developed an infection between the polymethyl methacrylate (PMMA) headcap and the skull. Postmortem revealed extensive thinning of bone that was covered by PMMA. Many screw holes had enlarged sufficiently to release the screws. The two trephine holes were enlarged, with thinned edges. In contrast, excessive bone proliferation was observed at the perimeter of the headcap (arrow). B: view of the chamber interior from inside the skull in another macaque. The base of a stainless steel chamber and the surrounding mass of blue and green PMMA is visible through the trephination. Bone resorption occurred around the chamber base, opening a gap.

Fig. 2.

A: orthographic drawing of the acrylic-free chamber. Inner diameter of the chamber bore is 19 mm. B: rendering of cross section through the chamber and lid mounted on a trephined bone surface. The cross-sectioned lid has been rotated slightly to show the silicone o-ring (orange) and the V-shaped groove that mates with a nylon-tipped set screw to hold the lid in place. The lid's vent is shown, sealed with a countersunk screw. A ring of hydroxyapatite seals the chamber to the skull. C: detail cross section of the chamber base, showing the 2 × 2.5 mm chamfer that is packed with hydroxyapatite following implantation. The screw hole is countersunk with a spherical profile of radius 2.5 mm to match the shape of “Synthes style” bone screw heads.

Fig. 3.

Chamber implantation procedure. A: one screw has been placed, and the pilot hole for the second is being drilled. The scalp is retracted with a muscle hook, and the drill bit is sheathed in a 12-G tube that leaves a limited length of drill bit exposed. Holes are drilled slowly by hand while holding the drill in a tap-wrench. B: once all screws are in place but prior to trephination, the hydroxyapatite gasket is formed. It is visible as a white ring around the floor of the chamber. C: following implantation, the scalp is sutured tightly around the sealed chamber.

Fig. 4.

Chamber interior immediately following trephination, 2 wk following chamber implantation. When allowed to dry slightly, the dura becomes semitransparent, and the large blood vessels that run along sulci can be visualized. Photographs taken at this point are useful for planning electrode penetrations to avoid surface vessels. V1, striate cortex; LS, lunate sulcus; V4, visual area 4.

Fig. 5.

Postmortem view of titanium headpost implantation in an adult macaque. The headpost was in place for 3 yr and 8 mo.

Fig. 6.

A: view of the chamber 16 mo after implantation. Fur surrounding the chamber has been trimmed. B: animal in Fig. 3, photographed 4 yr after implantation. The scalp has receded from the chamber, exposing the anterior foot and its screw head. However, the scalp remains healthy with scant exudate along the margin around the chamber.

Fig. 7.

Cross section of the chamber and a tool for removal of the lid. The tool resembles an inverted cup with a hole for a freely turning screw in the center of its base. A Teflon washer (white) is placed under the screw head. A stubborn lid can be quickly and easily removed as follows: the retaining set screw and vent screw are removed, and the extraction tool (magenta) is placed over the chamber (cyan). The tool rests on the chamber's shoulder. A socket head cap screw is placed through a clearance hole at the tool's center and into the threaded vent hole of the lid (yellow). Rotation of the screw pulls the lid out of the chamber bore without straining the chamber or headpost attachments to the skull.

Fig. 8.

System to provide axial support to prevent buckling of glass-insulated electrodes as they penetrate dura. A schematic cross section of the chamber and grid insert (Crist Instrument, Hagerstown, MD) is shown. The procedure for electrode penetration is as follows. Remove the lid and place liquid agarose (Sigma A0169) at 38°C inside the chamber. Immediately insert the grid so that excess agarose is displaced through the grid holes, and the entire chamber between the bottom surface of the grid and the dura is filled. Align the grid with a fiduciary on the chamber rim. Tighten the set screw to hold the grid in place. Insert the blunt 23-G outer guide tube (yellow) into the selected grid hole and gently push it down, through the agarose, until it contacts the dural surface. When gently pressed against the dura, the tube will “bounce back” to a constant depth. Using a graduated periodontal probe, measure the length of the tube that protrudes from the grid. Select a spacer (blue) of the same length (±0.5 mm) from a set of precut 19-G tubes. Remove the 23-G guide tube, pass it through the spacer, and reinstall it into the grid. The flared end of the 23-G guide tube (inset 1) will prevent it from passing through the spacer and advancing any deeper. As a result, the tip of the guide tube will be held in a stable position against the surface of the dura (inset 2). The electrode (cyan) is sheathed inside a blunt 31-G tube (red) that is attached to the micromanipulator. This 31-G tube is the same length as the 23-G guide tube. Align the 31-G tube using the X/Y stage and insert it into the 23-G guide tube. Now, the electrode can be driven out of the 31-G tube. Because it is supported axially, it cannot buckle, and its sharp tip will penetrate thickened dura. Furthermore, the agarose will prevent lateral movement of the guide tubes and minimize “tenting” of the dura that could otherwise result in electrode fracture.

Fig. 9.

Fluoroscopic images of an acrylic-free chamber taken to test the seal between chamber and skull. A: side view taken prior to filling the chamber with contrast agent. B: top view with 5 ml of contrast agent in the bore. The contrast agent was left in the chamber for 45 min and then removed. C: high-sensitivity (shorter exposure) image taken immediately after removal of the contrast agent. There is no evidence of leakage. D: side view after contrast agent removal. Seepage of the agent between the scalp and the skull would be visible as a dark line in this image but not in A.

Fig. 10.

Assessment of bevacizumab to reduce vascularization of dura and granulation tissue in the chamber. A: photograph of chamber interior following a period of >3 mo without removal of the lid. B: the same chamber interior 1 wk after applying two 0.5-mg doses of bevacizumab in 0.2 ml on consecutive days. The dura mater was wiped gently with a sterile Q-tip to remove loose tissue, causing little bleeding.

Fig. 11.

Postmortem examination of the titanium/bone interface in a monkey implanted for 16 mo. A: high-magnification photograph of one chamber foot, showing the extent of new bone growth around and over the metal. B: view inside the bore of the chamber, focused at its base. A ridge of new bone (arrow) has covered the circular face of the trephination to extend inside the chamber bore. C: the chamber has been removed, and the outer skull surface photographed. The impression of the chamber's foot print is clearly evident because bone has grown to close the gap between chamber and skull. D: high-magnification view of the region outlined with a white box in C. The bone surface is textured with concentric 50-μm-wide grooves. These are an imprint of the machined metal surface. Inset shows the surface of the chamber's circular chamfer at the same magnification.

Fig. 12.

Histological analysis of remodeled bone. A slice of the skull was cut from the specimen shown in Fig. 11 (between black lines in C). It was decalcified, and thin sections were cut and stained for hematoxylin and eosin. A: transmitted light micrograph of the bone section (pink). The foot plate and screw cross sections have been drawn in without obscuring the tissue section. B: dark-field illumination micrograph of the region outlined by the black box in A. Its histological appearance, with Haversian canals and concentric layers of lamellae, is characteristic of cortical bone. This location, under the chamber's chamfer was originally filled with hydroxyapatite paste.