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Details on building a hyperbaric chamber for intracellular recording in brain tissue slices

Jay B. Dean,^1,3 Daniel K. Mulkey,¹ and James D. Arehart²

¹Department of Physiology and Biophysics,
Wright State University School of Medicine,
College of Science and Mathematics;
²Supervisor (retired), Instrument Shop, College of Engineering,
Dayton, Ohio 45435 (U.S.A.)

This technical report contains supplemental materials for the Innovative Techniques paper entitled Continuous intracellular recording from mammalian neurons exposed to hyperbaric helium, oxygen, or air (J. B. Dean and D. K. Mulkey, Journal of Applied Physiology, 89: 807-822, 2000). To obtain a copy of this technical report order NAPS Document XXXXX from NAPS c/o Microfiche Publications, PO Box 3523, Grand Central Station, New York, NY 10017.

³ Correspondence:

Abstract

Procedures are outlined for building a hyperbaric chamber that can be used for intracellular electrophysiology in rat brain slices. Included in this discussion are listings of the materials, and their dimensions, that were used to build the pressure containment vessel, equipment sled and high-pressure sample cylinders. Specific details are also provided on the fluid lines, gas lines and electrical lines that connect the hyperbaric chamber to the control panel. Procedures for certifying the structural integrity of the pressure vessel and establishing its pressure rating are also included. In addition, there is a description of the brain slice chamber, within which a brain slice can be positioned at a fluid-gas interface so that test gases can be applied over the surface of the slice, or alternatively, submerged in various perfusion media containing different gas tensions. This technical material supplements the Innovative Techniques paper in Journal of Applied Physiology by Dean & Mulkey (18), which describes how to use the hyperbaric chamber for studying the effects of increased hydrostatic pressure and increased gas partial pressure on neurons in the mammalian central nervous system (mCNS).

Introduction

Exposure to hyperbaric pressure, and breathing hyperbaric gases, can adversely affect the function of the mCNS (1, 2, 7, 22). The cellular mechanisms by which hyperbaric conditions alter neuronal function can be studied by making an intracellular recording of neuronal activity in an in vitro tissue preparation of the mCNS while changing ambient pressure and gas partial pressures. Specifically, intracellular recording allows the investigator to observe changes in membrane potential, membrane conductance, firing rate, action potential and afterhyperpolarization waveforms, and synaptic activity, which correlate with changes in ambient pressure and/or gas partial pressure.

Intracellular recordings have been made successfully in several robust, excitable tissue preparations of the invertebrate CNS and neuromuscular junction while changing ambient pressure (3-6, 8-10, 12-14, 23, 24, 30, 31, 36, 37, 39, 40). In contrast, maintaining an intracellular recording in the mCNS, while changing ambient pressure, has proven to be technically challenging and largely unsuccessful (33-35). Two major obstacles in these earlier studies of the mCNS were the inaccessibility of the microelectrode once the pressure chamber was sealed, which hindered replacement of a broken or plugged microelectrode, and mechanical disturbances of the tissue slice and microelectrode that occurred when sealing the hyperbaric chamber or while compressing or decompressing the chamber (33-35).

In the companion paper (18) to this technical document, we described procedures for using a hyperbaric chamber that has surmounted earlier technical obstacles. Our hyperbaric chamber design has a readily accessible interior to facilitate rapid replacement of a microelectrode. It also has sufficient mechanical stability so that an intracellular recording of a mammalian neuron can be maintained while sealing the pressure vessel and during manipulation of ambient pressure. Our initial findings, using this new hyperbaric chamber design, indicate that it is technically feasible to record intracellularly from a large sample of mammalian neurons over a physiologically significant range of pressure (18, 29).

This paper contains technical material that describes, in detail, how to build the hyperbaric chamber and certify its structural integrity. A comprehensive description of the design and construction of the pressure chamber, and the necessary testing and certification procedures, is warranted because previous reports were incomplete regarding these important details (11, 21, 25, 26, 32, 35, 38, 40). This fact alone hindered our initial attempt at designing a hyperbaric chamber for intracellular recording of mammalian neurons, and resulted in lost time, materials and money. Moreover, a pressure chamber designed specifically for intracellular electrophysiology is not commercially available. The purpose of this technical communication, in conjunction with our other report (18), is to assist the investigator desiring to build a comparable hyperbaric chamber to be used for intracellular electrophysiological studies of the mCNS.

Materials and Methods

We incorporated certain features from several previous hyperbaric chambers into our design (11, 21, 25, 26, 35, 38), but we did not completely mimic any one style of chamber because other features were incompatible with making intracellular recordings in mammalian brain slices (18). Our pressure chamber most closely resembles two previous horizontal chambers (11, 35); however, it differs primarily in the style of closure, the equipment sled that carries the brain slice chamber and microdrives, and the design of the high-pressure sample cylinders that are used for studies of CNS oxygen toxicity (29).

Figures 1, 2, and 3 show different views of the pressure chamber.¹ Each component of the pressure chamber and its exact dimensions are identified as numbered items 1-28 in Figs. 1, 2, and Table 1. The pressure chamber is formed from a steel horizontal vessel (no. 25). Welded to its back end is a slip-on flange (no. 23), to which is bolted the blind flange (no. 24) using twenty hex bolts and nuts (no. 26). Each bolt is tightened to 600 foot pounds. A gasket seated between the slip-on flange and blind flange makes the rear of the pressure vessel gas tight. The front of the chamber seals with a horizontal double-bolt yoke closure (nos. 1a-e). The closure, which includes the door (no. 1a), two yoke halves (no. 1b), two pressure-warning devices (no. 1c), hub (no. 1d) and two horizontal yoke bolts (no. 1e), is welded onto the front of the horizontal vessel. The front lip of the hub and door are clamped together in the closed position with the two yoke halves; an O-ring fits into a groove machined into the face of the hub (Fig. 4A, n). The door can be sealed or opened in ~20 secs at 1 atmosphere absolute (ATA; i.e., room pressure) by closing or opening the yoke halves (no. 1b) with a manually operated chain and sprocket drive attached to the horizontal yoke bolts (18). This style of door works well since it can be closed and sealed while intracellular recording without mechanical disturbance of the microelectrode and tissue slice (18). The closure (nos. 1a-e) is available in a range of sizes and pressure ratings. All components used to construct our pressure containment chamber (nos. 1-28), including the door closure mechanism, were manufactured and assembled by Tube Turns Technologies.¹

Table 1 lists the nineteen openings drilled into the blind flange and horizontal vessel. National pipe thread (NPT) fittings ranging from ¼" to 1" i.d. (nos. 3-22) were welded into these openings to accommodate electrical cables, fluid and gas lines, and light pipe. In addition, two 6.0" i.d. openings in the top of the horizontal vessel, one on either side of the longitudinal midline axis, accommodate two window assemblies (Fig. 1B, no. 2). Each window frame was welded to the pressure vessel at an ~30º angle relative to vertical (Fig. 3A). An O-ring fits into a groove milled into the window frame (Fig. 3B). A 2.0"-thick acrylic window rests on the O-ring in the window frame. The window cap rests on top of the acrylic window, held by eight ½" x 4" steel bolts, each tightened to 350 foot pounds (Fig. 3C).

The equipment sled (discussed below) slides along two 23"-long stainless steel tracks welded to the floor, constructed from 0.5" o.d. steel rod, separated by 6.0" (Fig. 4A, o). The front of each runner is welded to the bottom of the interior floor, 5.0" in from the front edge of the hub (Fig. 4A).

The pressure chamber was welded to a saddle assembly (Figs. 1 & 2, no. 27) made from 3/8" rolled steel plate. Each side of the saddle was cut out (8" x 11") to accommodate an electrical connector panel. The pressure vessel and saddle assembly bolts (eight total, 4 bolts per side, 3/8" x 3 ¾ ") to a cart (no. 28) constructed from 2" x 2" x 1/8" square steel tubing with a ½"-thick top steel plate. The cart rolls on four 6.0" diameter x 2.0" wide polyurethane casters (1,000 pound capacity each). The front two casters swivel 360º.

In another hyperbaric chamber, we would suggest the following three design modifications based on our experiences to date. First, enlarging the inner diameter of the pressure chamber by several inches would be useful when attempting to reposition the microdrives to accommodate different styles of in vitro tissue chambers. Second, we would replace the slip-on flange and blind flange at the rear of the horizontal vessel with another horizontal double-bolt yoke closure. The hinged door of the closure can be drilled and furnished with NPT fittings to accommodate electrical and plumbing lines. While there is no need to access the interior of the chamber through the rear with any regularity, it would be beneficial to have this capability when installing and servicing equipment or making repairs. Finally, we would recommend that all four casters on the cart are of the swivel type. This would facilitate steering the hyperbaric chamber when installing it in the research laboratory.

The pressure containment vessel, upon completion, was inspected for structural integrity, hydrostatically-pressure tested, and certified by the appropriate authority to insure the safety of the operator and avoid structural damage to the chamber. In the U.S., these standard procedures are outlined in the American Society of Mechanical Engineers (ASME) code rules, Section VIII, Division 1, for Pressure Vessels.² The first step in the certification process requires identification of all materials used to construct the pressure containment vessel by their mill specifications; i.e., yield, tensile, % elongation, % reduction of area, and hardness. These values were obtained at the time the steel components were purchased from the mill (steel used for the horizontal vessel, slip-on flange, blind flange, window assemblies and closure mechanism) or the vendor (hex nuts & bolts used to attach blind flange to slip-on flange). Next, after assembling the components of the pressure containment vessel, all welded joints were inspected for structural imperfections using radiography, magnetic particle testing, or liquid penetrant testing. Then the pressure vessel was hydrostatically-pressure tested by filling the inner chamber completely with water and compressing it with air for at least 15 minutes to 1.5x the maximum working pressure (MWP). All phases of the certification procedure were documented and a product identification label and ASME label were affixed to the exterior of the pressure vessel. Our pressure chamber was designed for a MWP of 975 pounds per square inch gauge (psig) pressure (=67.3 ATA) and a hydrostatic test pressure of 1,480 psig (=101.7 ATA). No additional drilling or welding of the pressure containment vessel can be done after the hydrostatic pressure test without invalidating ASME certification. All of the above inspections and tests were conducted at Tube Turns Technologies, Inc. prior to shipping the chamber to Wright State University, where it was then outfitted with plumbing and wiring connections, the equipment sled, and control panel.

The equipment sled was constructed from an aluminum optical bench plate (No. C3640, Edmund Scientific Co., Barrington, NJ) measuring 24" x 12" x ½", trimmed down to 24" x 10" x ½" (Fig. 4B). It slides 14" out of the pressure chamber, allowing easy access to the recording microelectrode, brain slice, and other accessories (Fig. 5A). Four Teflon runners (3 ¾" x 1 1/8" x 1 ½"), one mounted at each corner of the sled (Fig. 5A, dd), attach the equipment sled to the tracks (Fig. 4A, o). A 1½ "-long stop screw extends beneath the sled, in front of the left rear runner (Fig. 4B, y). The stop screw catches on the front end of the left track when the sled is extended from the chamber. A laboratory jack with an adjustable height of 2.4-4.2" (model 271; Newport Corp., Irvine, CA) bolts to the sled and supports the brain slice chamber (Figs. 4B & 5A, q & r).

Figure 6 shows a schematic overview of the plumbing lines used for delivering artificial cerebral spinal fluid (aCSF) to and from the brain slice. It also shows the gas lines used for compression of the brain slice and for testing neuronal chemosensitivity to hyperbaric O₂ and hypercapnia in the interface slice, as opposed to a submerged slice (18); see below. The aCSF, warmed to 37ºC and aerated with 5% CO₂ + 95% O₂ at 1 ATA, is pumped to the brain slice (2-5 ml/min) inside of the pressure chamber using one of two high pressure liquid chromatography (HPLC) pumps (18). An HPLC pump draws aCSF through a stainless steel 10 µm filter, attached to 1/8" o.d. low-pressure tubing. Each HPLC pump connects to the pressure chamber via high-pressure (4,000 psig) stainless steel and poly ether ether ketone (PEEK) tubing, (0.03" i.d. x 1/16" o.d.). All connections between segments of stainless steel and PEEK tubing are made with high-pressure stainless steel unions unless stated otherwise (Alltech Associates Inc., Deerfield, IL). In addition, all fluid (and gas) lines penetrate the wall of the pressure chamber, uninterrupted, through a drilled out Swagelok tube fitting. Each aCSF line enters through a separate Swagelok and NPT fitting (Fig. 1, nos. 21 & 22). Tables 1 and 2 summarize each fluid, gas and electrical line penetrating the chamber wall. Items listed in Tables 1 and 2 are cross-referenced with their NPT fittings shown in Figs. 1 & 2.

Inside the pressure chamber, both aCSF lines attach to Flex-Connect™ high-pressure tubing (Figs. 4A & 5A, j), which in turn, attaches to a single solenoid valve (model 9-539-900/VAC-1250-PSIA; General Valve Corp., Fairfield, NJ). This allows selection between two different experimental media (Figs. 4B & 5A, v). Flex-Connect™ (tubing 0.03" i.d. x 1/16" o.d.) has a relaxed coil length of 15 cm and a fully extended coil length of 75 cm, which allows both aCSF lines to elongate as the equipment sled is extended out of the pressure chamber (Fig. 5A, j). The outflow port of the solenoid valve connects to a thermoelectric Peltier assembly, which is used to make final adjustments to aCSF temperature (16, 27). Inside the pressure chamber, stainless steel tubing (1/16" o.d.) and tygon tubing (2 mm o.d.) connect the Peltier assembly to the brain slice chamber. This is a friction fit connection and does not require a high-pressure stainless steel union (Fig. 4B, v & w). The dead space volume from the solenoid valve, where the two media lines converge, to the brain slice bath, is 1.8 ml. The design of the brain slice chamber is discussed below and presented in Fig. 5B.

Waste aCSF is drawn from the inner bath of the brain slice chamber to the outer bath via a filter paper wick (Fig. 6; see also Fig. 3A in Dean & Mulkey (18)). The outer bath drains by gravity through a 3/16" o.d. line into a waste reservoir (Fig. 4B, x). The waste reservoir is emptied through Flex-Connect™ tubing (Fig. 4A, k) and stainless steel tubing. An externally mounted ¼-turn ball valve is used to open or close the waste line. We currently use a waste reservoir that is 1/3 the size of that shown in Fig. 4B, x, in order to accommodate a second motorized micromanipulator.

Helium (100%) is used to increase barometric pressure (P_B) up to a maximum simulated depth of ~700 meters sea water. Three helium cylinders (grade 4.7, 300 cu. ft. @ 2,600 psig per cylinder) are interconnected with two CGA 580 tees and three 24" pigtails. Compressed air (79% N₂ + 21% O₂) is also used to pressurize the chamber in some experiments (18). Three air cylinders (industrial grade, 232 cu. ft. @ ~2,000 psig per cylinder) are interconnected by two CGA 590 tees and three 24" pigtails. A valve selects either helium or air as the compression medium (not shown). A vacuum pump (½ hp, 1725 rpm) produces hypobaria up to a maximum simulated altitude of ~40,000 feet. Hypobaria is applied in either helium or air atmospheres (28).

Figure 6 shows a schematic of the control panel that connects the helium cylinders, air cylinders and vacuum pump to the pressure chamber, and identifies the main components (items a-j) used to regulate gas flow and to measure gauge pressure. The pressure chamber connects to the control panel by four 3/4" o.d. Swagelok flexible metal hoses (series H04); one hose each for hyperbaric pressure, hypobaric pressure, venting to 1 ATA, and monitoring gauge pressure (Table 1 & Fig. 2A, nos. 13-15 & 17). The control panel uses 3/8" (course control) and 1/8" (fine control) stainless steel tubing and a series of valves to regulate gas flow to and from the pressure chamber, and 3/8" tubing for gauge pressure lines; however, we rarely use the 1/8" o.d. lines and these could be omitted in another version of the control panel. A metal shield placed over the compression line inlet (Fig. 4A, l), held in place with a magnet, directs helium/air downward, and under the equipment sled, during compression so that gas does not blow directly on the micromanipulators.

Hyperbaria was measured using a 3D® Precision gauge (series 25545-31B21-HCJ, range 0-1,500 psig; Amron International Diving Supply, Inc., Escondido, CA) placed in series with a pressure transducer (PX302-3KGV; Omega Engineering, Stanford, CT) connected to a strain gauge panel meter (DP205-S, Omega Engineering). Hypobaria was measured using a pressure gauge (30HG0, range -30 in. Hg to 0 vacuum; Amron International Diving Supply, Inc.) placed in series with a pressure transducer (PX603-30VAC5V; Omega Engineering) connected to a second strain gauge panel meter (DP205-S, Omega Engineering). Each strain gauge panel meter has an analogue output for sending pressure signals to a chart recorder, computer and magnetic tape.

Venting the chamber from either hyperbaric or hypobaric pressure to 1 ATA was done with 3/8" (course) and 1/8" (fine) parallel lines and valves that terminate in a high pressure muffler (type P10/1400; Amron International Diving Supply, Inc.). A pressure-relief valve in the top of the chamber (Fig. 1B, no. 3; model M5159B-2M[L]-975; Amron International Diving Supply, Inc.) opens at the MWP (975 psig) to prevent over pressurization of the hyperbaric chamber. If the pressure-relief valve fails, then opening the vent and pressure-warning devices (Fig. 2B, no. 1c) would rapidly decompress the chamber. The pressure-warning devices also function to prevent accidental opening of the closure mechanism during a 'dive'.

Figure 5B shows the brain slice recording chamber that was adapted from a previous design (16).³ The tissue slice can be maintained one of two ways using this style of brain slice chamber. 1) Interface brain slice with a test gas line—The slice is placed at the fluid-gas interface so that various test gases can be blown over the surface of the slice to rapidly change tissue PO₂ and/or PCO₂ at 1 ATA (15, 17, 20). The gas line used for this is labeled in Fig. 6 as 'test gas'. In this configuration, gas mixture passes over the surface of the brain slice at 270-300 ml/min, supplied by a 200 cu. ft. cylinder (~2,000 psig). The gas cylinders use high-pressure regulators (Concoa 806-1135, output range 0-1,500 psig), which maintains inflow pressure greater than hyperbaric chamber pressure. The 1/16" stainless steel gas line connects in series, respectfully, with a multi-turn valve, a flow meter⁴ and check valve, before penetrating the wall of the pressure vessel (Fig. 1B, no. 20). Inside the hyperbaric chamber, the test gas line connects to a 1/16" Flex-Connect™ tubing (Fig. 4A, i), which in turn, connects by friction fit to 2 mm o.d. tygon tubing. The tygon tubing enters the brain slice chamber and makes two loops in the outer bath before terminating at the same level as the top of the inner slice bath, 25-28 mm from the surface of the brain slice (Fig. 5B). Continuous flow of gas over the brain slice, during compression of the chamber, is confirmed by monitoring and adjusting the flow meter. Gas flow is also confirmed by observing the rate of bubbling in the outer bath of the slice chamber of a secondary gas line teed-off from the primary gas line (Fig. 5B). The vent for the hyperbaric chamber was always left open to room pressure, except during a pressure test, to avoid slow build up of O₂ and P_B inside the pressure vessel caused by the small volume of gas flowing through this line. The volume of O₂ added to the sealed pressure chamber from the test gas line is small and measurements made in our laboratory show that it poses no significant problem during 15-30 minutes of compression (i.e., with the vent closed). If, however, the chamber is sealed for 2 hours then the O₂ level inside the chamber increases from nominally zero to ~50%, which is a concern (see below, Safety Issues).

In the interface slice configuration, using 100% O₂ as the test gas, the tissue slice can be made hyperoxic as the hyperbaric chamber is compressed with 100% helium. As P_B increases, O₂ supplied from the test gas line is driven into the brain slice, increasing tissue PO₂. If 95% O₂ + 5% CO₂ is used, the brain slice can be made hyperoxic and hypercapnic during helium compression (unpublished findings, D.K. Mulkey, R.A. Henderson, & J.B. Dean). Using a slice that is maintained at the fluid-gas interface is a much quicker method for administering HBO₂ compared to using the high-pressure sample cylinder and the submerged brain slice because no fluid line dead space is involved (18, 29). With the interface slice, however, it is more difficult to study the effects of pressure per se, since it requires constantly lowering the fractional concentration of O₂ in the test gas mixture, while increasing ambient pressure, in order to maintain constant tissue PO₂. Consequently, most of our research to date has used the submerged brain slice preparation without the test gas line.

2) Submerged brain slice without a test gas line—This is the configuration we have used primarily because we can easily differentiate the effects of pressure per se, using the inert gas helium, from those of increased PO₂ (18, 29). In the submerged brain slice preparation, the primary and secondary test gas lines (Fig. 6) are removed. Thus, no additional O₂ or CO₂ are admitted to the hyperbaric chamber. The PO₂ and PCO₂ of the aCSF are set at 1 ATA before delivery into the pressure chamber, or at >1 ATA using small (1 L volume) high-pressure sample cylinders (Swagelok/Whitey Sample Cylinder, 304L SS Dot-3A 1800, MWP= 1,800 psig). In the latter case, aCSF flows into the pressure chamber via a small positive pressure differential (0.06-0.3 atmospheres) from the sample cylinder to the hyperbaric chamber (25, 26).

The sample cylinder, reported in Dean & Mulkey (18), is sealed at each end using a ball valve. Two quick disconnect attachments enable the sample cylinder to be easily inserted in or removed from the aluminum mounting rack (18). Figure 7 shows two views of the sample cylinder and its components. An O₂-CO₂ gas mixture is used to pressurize the cylinder containing 200-700 ml of aCSF, and to supersaturate perfusate with O₂ while keeping pH essentially constant (18). We use a l L cylinder; however, the sample cylinder is available in a wide range of volumes. Specific components used for assembling the sample cylinder, pressure transducer and fluid/gas lines are identified in Fig. 7.

Table 2 summarizes the electrical signals entering and leaving the pressure chamber and the type of pressure sealing assemblies used. Wire assemblies B, C and D enter the pressure chamber through the blind flange (Table 1 and Fig. 2A, nos. 9, 10 & 16). Wire assembly A contains connections for both electrophysiology headstages and exits the pressure chamber through the side of the horizontal vessel (Fig. 1B, no. 5). Each wire assembly terminates in its own electrical connector box, which is fastened to the interior of the pressure chamber with silicone compound (Fig. 4A, a-d).

Membrane potential (V_m) and current are measured using an Axoclamp 2A amplifier (Axon Instruments Inc., Foster City, CA); see also (33, 35). The total length of each Axoclamp 2A headstage lead is 22', beginning at the head stage and ending at its input into the back of the amplifier. One headstage is used to measure membrane potential and the other headstage is used to measure bath/tissue pH. The 10' lead of each headstage is left intact and plugs into an interior connector panel (Fig. 4A, a). Two additional 1' lengths of cable couple the connector panel to the interior end of wire assembly A (Fig. 4A, e). Two additional 6' lengths of cable connect the exterior end of wire assembly A to an external connector box. Two 5' extension cables (EX-1; Axon Instruments Inc.) link the external connector box to ME 1 and 2 inputs in the back of the Axoclamp 2A intracellular amplifier.

The saddle supporting the pressure chamber (Figs. 1 & 2, no. 27) attaches to the common ground in the laboratory, a copper water pipe, via a ½" wide grounding strap. Inside the chamber, a 2' length of grounding strap is secured with a strong magnet to an unpainted portion of the chamber's interior wall (Figs. 4A, g). The other end of the grounding strap attaches to a steel screw (anodized coating removed) mounted to the equipment sled, which serves as the grounding point for electrical equipment inside the pressure chamber.

Despite the long length of the lines connecting the headstage to the amplifier, we rarely had electrical noise problems. However, early on, we had difficulty with large fluctuations of V_mduring compression/decompression that had to be identified and corrected. In the first experiments, a silver wire, coated with molten AgCl (Aldrich 22,792-7), was placed in the inner bath beneath the brain slice to serve as the reference electrode. This style of reference electrode, however, was pressure sensitive in an unpredictable and intermittent fashion. Voltage artifacts usually appeared as transient depolarizations as P_B increased and transient hyperpolarizations as P_B decreased. However, in some cases, the V_m shift was not transient, but was sustained while at pressure. We observed these voltage artifacts with and without air bubbles in the microelectrode, in low resistance micropipettes (3-6 M), and with both slow and fast rates of compression and decompression. We attributed voltage fluctuations to a pressure-induced change in offset potential between the reference AgCl electrode and the intracellular microelectrode. It is unclear why changes in ambient pressure cause intermittent changes in the offset potential when using a AgCl wire reference electrode. This problem was corrected using a combination agar and AgCl wire combination electrode as the indifferent electrode (18).

Another pressure-related artifact was a large and erratic drift of V_m during compression and decompression. We determined that the aberrant reading was due to a change in the fluid level of the outer bath in the brain slice caused by one of the following: 1) leakage of aCSF from the brain slice chamber through an improperly sealed gas line or fluid line (if the silicone sealant that adjoins the line to the slice chamber is not fully cured, or if air bubbles are trapped inside the sealant, then repeated bouts of compression and decompression eventually initiate leaks in the sealing compound); or 2) reduced flow of aCSF through the drain pipe, caused by an air lock in the line connecting the outer bath to the waste reservoir (Fig. 5B, 'drain').

An external zoom stereoscope (Meiji EMZ-5TR) mounts to the chamber's cart via an articulated arm on a ½" x 48" stainless steel rod. A 0.3x supplementary lens (Meiji MA530) increases the total working distance of the stereoscope to 23.5 cm for visualizing the brain slice and microelectrode inside the pressure chamber (18). The interior of the chamber is illuminated with an externally mounted, 250 watt tungsten halogen Canty lamp (model HYL 250-LS; Amron International Diving Supply) attached to a ¾" x 7" lexan pipe passing through a Swagelok tube fitting (Fig. 2A, no. 6) in the top aspect of the blind flange. Internally, a flexible dual arm fiber optic connects to the lexan light pipe (Fig. 4A, m).

The system used to regulate temperature of the brain slice during compression and decompression was already described (18). The heat exchanger, made from ¼" o.d. copper tubing, penetrates the wall of the pressure chamber via a Swagelok fitting (Figs. 4A & 5A, h). Inside the hyperbaric chamber, the radiator connects to the copper water lines with unions. In another version of the chamber, we would enlarge the internal heat exchanger to improve thermoregulation of the air temperature inside the hyperbaric chamber during compression and decompression. This could be accomplished by running additional water-perfused copper coils the length of the chamber, beneath the equipment sled.

Built-in safe guards, already discussed in Dean & Mulkey (18), prevent compression of the vessel beyond its MWP. The other safety concern is working with a large fractional concentration of O₂ in a hyperbaric environment (hyperbaric oxygen, HBO₂). HBO₂ is dangerous because of the potential threat of fire or explosion. Three potential sources for igniting materials maintained in HBO₂ are static electricity, sparking from electrical equipment inside the chamber, and adiabatic compression. Two recommended brochures for further information on fire safety include: 1) NFPA 53M, 1990, Fire hazards in oxygen enriched atmospheres, 1 National Fire Protection Association, Inc., Batterymarch Park, Box 9101 Quincy, MA; and 2) Technical Bulletin No. 5, Oxygen Systems, The Swagelok Companies, Cincinnati, Ohio, August 1993).

The pressure containment vessel, including the window assemblies and closure mechanism, were designed, built and certified by Tube Turns Technologies, Inc., under the direction of Mr. Eugene Reiter, a hyperbaric engineer. The saddle assembly, cart, equipment sled, control panel, sample cylinders, and brain slice chamber were built by two model makers at the Wright State University Instrument Shop. The electrical wiring for the hyperbaric chamber, and construction of the temperature controller were done by two electrical technicians at the Wright State University Electronics Shop.

Footnotes

¹ We recommend for user safety, and reason of liability, that you enlist the services of a qualified pressure vessel engineer and adhere to the American Society of Mechanical Engineers (ASME) guidelines (see footnote 2) when designing and evaluating a comparable pressure chamber. We worked with Engineers at Tube Turns Technologies, Inc.; 2900 W. Broadway, Box 32160, Louisville, KY 40232 U.S.A..

² 1997 ASME Boiler and Pressure Vessel Code. Section VIII, Division 1. American Society of Mechanical Engineers, United Engineering Center, 345 East 47th Street, NY, NY 10017, 677 pp.

³ The brain slice chamber reported in Dean & Boulant (16) also used three water-perfused thermodes positioned beneath the nylon mesh for changing tissue temperature in various regions of the brain slice. The thermodes were not included in the brain slice chamber we currently use.

⁴ The flow meter insures constant gas flow over the surface of the brain slice during compression and decompression. This flow meter has a MWP of only 200 psig. Consequently, we are limited to test pressures of < 200 psig when using the interface brain slice. The flow meter, however, can be replaced with another that has a MWP= 5,800 psig (FTO-1AINAUGHC-5 Turbine Flowmeter; Flow Technology, Inc.; Phoenix, AZ).

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