Materials

Many fiber-shaped dialysis materials have been successfully used in fabrication of microdialysis probes. Popular ones are regenerated cellulose, cellulose acetate, cellulose ester, polysulfone, etc.28 These membranes vary in their permeability limits, diameter, wall thickness, etc. When choosing a dialysis membrane, the primary consideration should be the permeability limit and its performance in vivo. Certain membrane materials (such as polysulfone) offer excellent in vitro recovery numbers for a given surface area, but may perform poorly in vivo, and others are vice versa. The regenerated cellulose membranes are usually rugged enough to sustain the handling required under common laboratory conditions. If the membrane must be wet in order to stay patent, threading it inside the plumbing tubes and gluing it can be tricky. Flaccidity and handling of the membrane in dry and wet conditions should be considered. The membrane should be firm enough that it can be conveniently placed inside the target tissue. Also important factors are availability of diameters that are suitable for the target tissue, and their ability to withhold internal fluid pressures. Such information is usually available from the membrane manufacturer.

The material and dimensions for the inlet and outlet connecting tubing can vary depending upon the material and dimensions of the membrane. The choice of material and diameter of connecting tubing also depends upon the flow rate to which the finished probe is going to be subjected. The fluid pressure inside the dialysis area depends upon the flow rate, diameter, and length of the tubes connected to it, as well as viscosity of the fluid. The relationship between the fluid pressure P inside extremely narrow bore cylindrical tubing is given by the following equation:

where 8/n is a constant of proportionality, V is the viscosity of the fluid, L is the length of the tubing, Q is the flow rate, and R is the radius of the tubing. Physiological fluids, such as ACSF and Ringer's solution, have similar viscosity. Therefore, the length and diameter are the only factors one can manipulate. Ideally, one should have access to the dialysate as soon as it leaves the fiber but this is practically impossible. Thus, one should choose tubes with the shortest length and smallest diameter. According to the above equation, the pressure inside the fiber can be estimated and some theoretical numbers are calculated in Table 11.1.

TABLE 11.1

Pressure (mmHg) Inside the Fiber Due to the Outlet Tube

Diameter Length _Flow Rate (Ml/min)

TABLE 11.1

Pressure (mmHg) Inside the Fiber Due to the Outlet Tube

Diameter Length _Flow Rate (Ml/min)

(Mm)

(cm)

0.05

1

2

3

4

5

25

100

6,532

13,064

26,129

39,193

52,258

65,322

50

100

408

817

1,633

2,450

3,266

4,083

75

100

81

161

323

484

645

806

100

100

26

51

102

153

204

255

200

100

2

3

6

10

13

16

While membranes can be engineered to withstand very high internal fluid pressure, there are other reasons why it is important to use combinations of outlet tubing material to keep the internal fluid pressure in the optimum range. Because the membrane is porous, high internal fluid pressure can push fluid outside the fiber, thereby causing a sweating effect. Moreover, at high pressures there is an increase in the osmotic pressure inside the membrane, which can reduce recovery of extracellular chemicals into the dialysate. Good results can be obtained if pressure inside the fiber does not exceed 500 mmHg. Also, for the tubing inert material such as Teflon, PEEK, fused silica, and polyimide are ideal (see Table 11.2 for sources of materials). Other plastic materials can offer similar results but some (such as poly-vinyl chloride or PVC) can leak plasticizer compounds into the dialysate, which can seriously affect the analysis. Metal or other reactive material (such as ordinary plastic or glass) should be avoided in the plumbing, as well as in the collection tube, because

TABLE 11.2 Microdialysis Supply Vendors

ESA, Inc. Bedford, MA

Bioanalytic Systems, Inc. West Lafayette, IN

Harvard Apparatus Holliston, MA

CMA/Microdialysis Solna, Sweden of reactivity with the perfusion medium or dialysate and the potential for trapping the sampled substances.

The injury to the tissue at the site where the probe is inserted for prolonged periods depends upon the material used in constructing of the probe and the duration for which the probe invades the tissue. The brain reacts very little to the actual fiber.2930 The stiffness of the probe, particularly probes that are constructed with stainless steel, can increase trauma to the brain. It is notable that all earlier studies that reported excessive gliosis around the inserted portion of the probe after prolonged placement3132 were conducted when the microdialysis technique was in its infancy and probes were made out of stainless steel material. In recent reports, probes made out of thin fused silica tubes without a rigid support structure have been reported to induce far less damage to the brain, as seen from postmortem histol-ogy.2933 Furthermore, recent observations indicate that there is no significant difference in the basal levels of dopamine if the probe is reinserted in the same area after 1 week.25

Figures 11.4 and 11.5 depict some of the types of microdialysis probes, guide cannulae, and stylets that are currently in use for sampling from animals. Besides the variations in the tip configuration of radial probes (loop-type, concentric, and side-by-side types discussed above), there are numerous ways in which the fiber tip is joined to the connecting tubes and subsequently plumbed to the rest of the system. In some probes the inlet and outlet tubes are ported out via a rigid plastic mount, which also serves as a probe holder. In contrast, other probes (homemade, as well as some commercial ones) consist only of a slender tube but as an option can be secured to a probe holder by the user (Figure 11.4A, B, C). The probe holder in either configuration supports the delicate body of the probe and is used to secure it to the guide cannula (Figure 11.4E). Moreover, if the probe is used with anesthetized animals, the probe holder secures the probe to the arm of the stereotaxic equipment. The fixed length probe holder offers an advantage in that it does not require any configuration prior to use. However, this fixed probe length design suffers from a lack of flexibility, as it does not allow the investigator to adjust the length of the probe projecting from it. Commonly available probes are designed to fit into a guide with a 1 cm guide tube (Figure 11.4D, E and Figure 11.5, left), which is usually sufficient for most targets in a rat brain. Such fixed-length matching guides and probes work very well for targets located far ventral to the dorsal surface of the skull. But for those targets that are located near the dorsal surface of the skull, such a guide cannula may be too long. On the other hand, there are probes that can be adjusted in the probe holder to allow their use with short guide cannulae that do not penetrate the dura (Figure 11.5, right). Although the nonpenetrating guide cannulae

FIGURE 11.4

Various designs of microdialysis probes, probe holders, and guides. (A) Radial dialysis probe; (B) probe holder; (C) radial dialysis probe affixed to a probe holder; (D) probe guide; (E) radial dialysis probe affixed to the probe holder, with the probe and holder placed in the probe guide; (F) stylet; (G) stylet in probe guide.

FIGURE 11.4

Various designs of microdialysis probes, probe holders, and guides. (A) Radial dialysis probe; (B) probe holder; (C) radial dialysis probe affixed to a probe holder; (D) probe guide; (E) radial dialysis probe affixed to the probe holder, with the probe and holder placed in the probe guide; (F) stylet; (G) stylet in probe guide.

FIGURE 11.5

Commonly used microdialysis guide cannulae. The cannula on the left has the long, penetrating guide tube projecting from the body. The guide is designed to penetrate the dura with the end of the guide tube being placed immediately above the area to be dialyzed. The cannula on the right has the short, nonpen-etrating guide tube. The short guide tube is positioned above the dura such that the dura must be punctured before the dialysis probe is inserted.

offer the flexibility of adjusting the ventral position, they require an additional step of precise affixation to the probe holder.

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