Arch Otorhinolaryngol (1984) 239:243-247 Archives of Oto-Rhino-Laryngology �9 Springer-Verlag 1984

Prolonged Maintenance of Endocochlear Potential by Vascular Perfusion with Media Devoid of Oxygen Carriers*

T. Kobayashi 1, M. Rokugo, D. C. Marcus, T. H. Comegys, and R. Thalmann

Department of Otolaryngology, Washington University Medical School, 517 S. Euclid Ave., St. Louis, MO 63110, USA

Summary. A method is described for maintaining the cochlear potentials of the guinea pig via arterial perfusion of the surviving inner ear with an artificial medium devoid of oxygen carriers or oncotic agents. The endocochlear potential (EP) can be maintained at a normal level for periods in excess of 5 h ; the responses of the EP to anoxia and to furosemide closely approximate those seen in the intact animal. This preparation may represent a simplified method for carrying out selected arterial perfusion experiments in the surviving inner ear.

Key words: Endocochlear potential - Vascular perfusion

Introduction

We have previously reported the maintenance of the endocochlear potential (EP) and cochlear microphonics (CM) of the guinea pig for prolonged periods of time via perfusion of the surviving inner ear using synthetic media containing artificial oxygen carriers [4]. With this preparation we were able to reproduce characteristic responses of the cochlear potentials occurring in the whole animal. In addition, a host of experiments were carried out, which can only be done using artificial blood substitutes. Examples of these are peffusion omitting electrolytes or metabolic substrates, and evaluating the response to addition or substitution of substances one at a time [1-3].

We report here our recent finding that prolonged maintenance of the EP can be accomplished in the guinea pig with media lacking oxygen carriers and oncotic agents if perfusion rates are increased substantially.

* Supported by NIH grant NS 06575 and NSF grant BNS-8118772 1 Present address: Department of Otolaryngology, Tohoku University School of Medicine, 1-1

Seiryo-machi, Sendai, Japan Offprint requests to: R. Thalmann, MD

244 T. Kobayashi et al.

Method

The surgical technique has been described in detail in a previous report [4]. In essence, perfusion was carried out via a catheter inserted into the basilar artery; all vessels except the left anterior inferior cerebellar artery were clamped, restricting flow to the inner ear and a portion of the brain. Effiux was provided by sectioning all vessels of the neck (i.e., decapitation after initiation of perfusion). The composition of the perfusion medium is shown in Table 1. Prior to loading the perfusion system the perfusate was equilibrated with a gas mixture of 95% 02 and 5% CO2. Following equilibration the pH of the fluid was about 7.4; the osmolality was 295 mosmol/kg H20.

The electrophysiological technique has also been reported previously [4]. The EP was recorded from the scala media of the basal turn, via the round-window approach. The CM were recorded from a single electrode in the scala vestibuli of the basal turn, using a frequency of 2,000 Hz.

Results and Discussion

In earlier experiments based on the use of perfluorocompounds as oxygen carriers and polyols as oncotic agents, a flow rate of about 300 ~tl/min was found to be adequate for prolonged maintenance of the cochlear potentials, without leading to an undue increase in the pressure (typically 60-100 mm Hg) of the system [4]. In the present experiments, using the above-mentioned medium devoid of oxygen carriers and oncotic agents, perfusion at this flow rate resulted in a steady decline of the potentials. However, when the flow rate was increased to about 1 -1 .5 ml/min, the potentials remained stable. In fact, it was possible to maintain the EP to within 2 - 3 mV of the original level for over 5 h. The back pressure, although somewhat higher than in the earlier experiments (80-120 mm Hg), did not increase significantly during the entire course of the experiment.

We have observed in the past, in numerous long-term experiments, that the negative (component of the) EP is especially vulnerable, and can be compromised in spite of a normal-appearing (positive) EP. It is therefore important to note that when perfusion was arrested after 5 h (Fig. 1), the typical response of the EP to ischemia was seen: the potential dropped precipitously, reaching a level of about - 3 2 mV, which was comparable to the negativity seen in systemic ischemia of the whole animal. Notice also the rapid restitution of the

mmol/1

NaC1 125.0 KCI 3.5 NaHCOs 25.0 CaC12 1.3 MgC12 �9 6H20 1.14 NaHzPO4 - I=/20 0.51 Glucose 3.3 Urea 2.1

This solution is equilibrated with 95% 0 2 - 5 % CO~ immediately before use; the resultant pH is about 7.4

Table 1. Composition of arterial perfusion medium

Prolonged Maintenance of Endocochlear Potential 245

EP to near the original level following resumption of perfusion, and maintenance of a stable level thereafter. Noteworthy, however, is the lack of an "overshoot", as has been observed in the recovery of the EP from anoxia in the intact animal, and in preparations using "artificial blood" containing an oxygen carrier and oncotic agent. It could be surmised that the oxygen supply in the present experiments is at a near-marginal level.

In the experiment illustrated in Fig. 1, the CM remained at the original level for 2 h, with a subsequent gradual decline of 2 -3 dB over the next 3 h (not shown). The CM were monitored in these experiments merely as an adjunct to our primary objective, the determination of the behavior of the EP. The CM were measured at a steady level of 50 dB SPL, and since neither thresholds nor maxima were determined systematically during the course of the experiment, no definitive statement about the maintenance of the CM can be made at this time.

Figure 2 shows a typical response of the EP to 2 mM furosemide: a rapid decline of the EP similar to that seen in experiments using "artificial blood" can be seen [4]. When control solution was re-introduced at the time indicated, the EP continued to decline for a short period, due to the "dead space" in the system; the maximum negativity attained was -31 mV. Subsequently, the EP exhibited the typical biphasic recovery, with a rapid rise during the first 6 rain,

100

50

E a2 uu 0

#

I I 10 / / 2 9 0

off

I 3 0 0

\ I / / i i

310 350 360

Minutes

-50 on

Fig. 1. Behavior of the endocochlear potential (EP) in the guinea pig during long-term arterial perfusion of the surviving inner ear with a medium devoid of oxygen carriers and oncotic agents. After 5 h of perfusion the EP remains within 2 mV of its original level in the intact animal. At the indicated time (off) perfusion was interrupted, effectively creating a situation of ischemic anoxia. The EP declined rapidly to about -32 mV, similar to the situation, in the intact animal. When perfusion was recommenced (on), the EP immediately increased, achieving almost complete recovery

246 T. Kobayashi et al.

followed by a more gradual rise until a stable level 2 mV below the original was reached after nearly 2 h. Subsequent interruption of perfusion resulted in a typical decline of the EP to about -30 mV (not shown), indicating that the negative (component of the) EP was also still intact.

It should be stressed that these results were obtained in spite of the absence of an oncotic agent. Inclusion of various oncotic agents in the perfusion media at physiological concentrations resulted in increased viscosity, with unacceptably high back pressures at flow rates sufficient to maintain the potentials.

In another series of experiments, a hypo-osmotic medium (275 mosmol/kg H20) of composition identical to that used in the preparation of "artificial blood" [4] but without FC-47 and Pluronic F-68, was perfused; similar prolonged maintencance of the potentials (over 4 h) was obtained.

It needs to be shown whether vascular perfusion of the surviving inner ear with media containing no oxygen carrier or oncotic agent is biologically equivalent to perfusion with the perfluorocompound and polyol-containing media in all respects. Careful anatomical and metabolic evaluations, as used in the previous studies [1, 4], must obviously be carried out. Furthermore, the behavior of the CM must be more rigorously evaluated.

100 2 mM furosemide

E

0 I I I I control j ~ 60 120

Minutes

-50 Fig. 2. Response of the EP in the guinea pig to 2 m M furosemide. Perfusion conditions are similar to those described for Fig. 1. Af ter 30 min of perfusion of the surviving inner ear with a med ium devoid of oxygen carriers and oncotic agents, the perfusate was switched to one of similar composit ion, but with 2 m M furosemide added. A n immedia te decline is seen. At the indicated time (control) the perfusion was changed back to the original medium. The EP continues to decline for a short t ime (due to the "dead space" in the system) to a level of about - 4 0 mV, then exhibits a biphasic "recovery period" similar to that seen in systemic application of the drug in the intact animal

Prolonged Maintenance of Endocochlear Potential 247

The main reason that we are reporting our findings at this early stage is to make other researchers aware of the possibility that at least selected vascular perfusion experiments can be carried out without the necessity for resorting to media containing artificial oxygen carriers, the preparation of which is technically difficult and fraught with potential sources of artifact. One definite limitation of the approach that may temper its overall usefulness is that we cannot add oncotic agents. Nevertheless, the fact in itself that the EP (and probably the CM) can be maintained without an oncotic agent in the perfusate, is most noteworthy and difficult to interpret.

We have to realize, of course, that both conditions, with and without perfiuorocompounds and oncotic agents, are highly artificial. For instance, when evaluating drug effects using "artificial blood" as the perfusion vehicle, the possibility of the drug complexing with the oncotic agent or oxygen carrier must be taken into account; in addition, perfluorocompounds and polyols seem to adhere to capillary walls and/or to enter into cells. Thus, it must also be recognized that drug permeability and entry into tissues may be affected. The perfluorocompound-containing artificial blood may also release trace amounts of free fluoride, the potential toxicity of which is well documented.

These problems are not encountered in the present preparation. However , the absence of albumin or other proteins creates a whole new set of complicating factors, which must be taken into account. Just as an example, we had to reduce the amount of calcium in the perfusate in order for it to correspond to the ionized fraction present in blood. Furthermore, in the study of other endogenous and exogenous substances that may in part bind to protein or which depend upon protein for transport, the virtually complete absence of binding capacity and the effects thereof must be carefully considered.

References

1. Kambayashi J, Kobayashi T, DeMott JE, Marcus NY, Thalmann I, Thalmann R (1982) Effect of substrate-free vascular perfusion upon cochlear potentials and glycogen of the stria vascularis. Hear Res 6:223-240

2. Kambayashi J, Kobayashi T, Marcus NY, DeMott JE, Thalmann I, Thalmann R (1982) Minimal concentrations of metabolic substrates capable of supporting cochlear potentials. Hear Res 7:105-114

3. Wada J, Kambayashi J, Marcus DC, Thalmann R (1979) Vascular perfusion of the cochlea: effect of potassium-free and rubidium-substituted media. Arch Otorhinolaryngol 225 : 79-81

4. Wada J, Paloheimo S, Thalmann I, Bohne BA, Thalmann R (1979) Maintenance of cochlear function with artificial oxygen carriers. Laryngoscope 89:1457-1473

Received June 15, 1983/Accepted July 18, 1983