Hearing Research, 62 (1992) 57-62 © 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00
57
HEARES 01784
Influence of experimentally elevated blood viscosity on the auditory nerve-brainstem evoked response and threshold D a p h n a L i d a n a, S a u l Y e d g a r b, H . B e n A r o n s o n c a n d H a i m S o h m e r a Departments of a Physiology and b Biochemistry, Hebrew Unirersity - Hadassah Medical School, Jerusalem, Israel and c Department of Anesthesiology, Hadassah Unicersity Hospital, Jerusalem, Israel (Received 20 December 1991; Revision received 20 April 1992; Accepted 4 May 1992)
Blood viscosity, due to its effect on blood flow, is one of the determinants of oxygen delivery. Therefore the influence of elevated blood viscosity on hearing was studied in rats using the auditory brainstem response (ABR) threshold, wave 1 latency, brainstem transmission time (BTI') and wave 1/4 amplitude ratio. Whole blood viscosity (WBV) was elevated by 15-21% in two different ways: elevating the hematocrit (Polycythemia) by acclimation in a hypobaric chamber, or elevating the plasma viscosity by infusing a solution of Polyvinylpyrrolidone-360 (PVP). ABR was recorded before and 24 h after the blood viscosity was elevated, so that each rat served as its own control. Paired t-tests showed that there was no statistically significant difference in the ABR parameters in each of the groups as a consequence of blood viscosity elevation. In conclusion~ the elevation of WBV to this degree for this duration, using two different techniques had no effect either on the function of the auditory nerve and the more peripheral sites, or on the central auditory pathway as studied by ABR. Blood viscosity; Auditory nerve-brainstem response; Polycythemia; Hematocrit
Introduction
It has been suggested that increased viscosity of blood would have a deleterious effect on the auditory system. This hypothesis is based on clinical reports of hearing loss in patients suffer,.'ng from conditions in which blood viscosity was elevated (Davis and Nilo, 1965; Af'ffi and Tawfeek, 1971; Wells et al., 1977). A sensorineural hearing loss has been reported as a symptom in a patient with elevated viscosity due to Polycyt~emia Vera (Davis and Nilo, 1965). In this case hearing threshold and discrimination scores were improved following phlebotomy, and the improvement was explained as being due to a reduction in viscosity following withdrawal of blood. Dormandy (1970) showed that a 10% fall in viscosity in dogs induced by plasma exchange transfusion was accompanied by a 20% rise in cardiac output. Cases of Waldenstrom's Macroglobulinaemia, a disorder with clinical manifestations related to hyperviscosity of the circulating intravascular macroglobulin lgM (Salmon, 1988), were reported to have cochlear hearing loss, characterized by a sudden onset and sequential involvement of both ears (Af'Lfiand Tawfeek, 1971; Wells et al., 1977). Here
Correspondence to: H. Sohmer Department of Physiology, Hebrew University-Hadassah Medical School, P.O.B. 1172, Jerusalem 91010, Israel. Fax: (972) 2-439736.
also, hearing was improved to some extent after treatment which lowered viscosity such as alkylating agents and plasmaphoresis. The effects of the reduction of blood viscosity by hemodilution were investigated in guinea pigs by Hultcrantz and Nuttall (1987) and Nuttall et al. (1988), and the cochlear blood flow was found to increase as hematocrit decreased. Poiseuille's law states that the fit,w of blood is inversely proportional to its viscosity in vessels having fixed dimensions perfused at a constant pressure (Jandie, 1987). This relationship was determined from studies with tubes having rigid walls, using a Newtonian fluid and a constant flow. This clearly is not the case when one considers the flow of blood in vascular beds, in which complicated vascular geometry, pulsatile flow and the anomalous viscosity of blood (since blood is not a Newtonian fluid) induce numerous modifications in this relationship (Burton, 1972; Charlesworth, 1981; Jandle, 1987). Nevertheless, using Po~euille's law as a reasonable, first approximation (Charlesworth, 1981), it is clear that an increment in blood viscosity would reduce blood flow. Since the inner ear transduction mechanism is sensitive to hypoxia (Gaf-ni and Sohmer, 1976; Sohmer et al., 1986, 1989), the hearing loss seen in patients with hyperviscosity may be due to the decreased blood flow to the ear. The purpose of this study was to investigate, in carefully controlled experiments on rats, the effects of elevated blood viscosity on auditory nerve and brain-
58
stem evoked response (ABR) parameters, including threshold. In addition, an attempt was made to determine whether enhanced blood oxygen capacity could offset possible elevations of auditory threshold due to hyperviscosity.
Methods
The experiments were conducted on 3 month old male Sabra rats weighing 230-300 g ( m e a n - 258 g). Blood viscosity was elevated in the rats in two different ways. One group was placed into a hypobaric chamber which simulated the conditions of altitude for a period of 3-5 days inducing erythropoiesis (Yoffey et al., 1968). This was called the Polycythemic-ABR group (N = 16). The chamber was maintained at a barometric pressure of about 350 mmHg, and the partial pressure of oxygen was therefore about 65-70 mmHg. The chamber was opened each day for one hour to replenish food and water for the rats. In a second group of rats, Polyvinylpyrrolidone - 360 (PVP) macromolecules were infused. These molecules induce an elevation of the relative viscosity of solutions in-vitro (Scott, 1983), and of plasma viscosity in vivo (Yedgar et al., 1985). This was called the PVP group ( N = 14). The rats were anesthetized with 60 mg/kg Sodium Pentobarbital (IP) and local anesthesia (1% Lignocaine Hydrochloride) was used as necessary in each operation in addition to the general anesthesia. The femoral vein was cannulated and infused with PVP-360 (Sigma Chemical) solution in physiological saline. PVP-360 was chosen because of its limited effect on blood osmolarity and consequently blood volume, and also because satisfactory results could be obtained by applying a relatively low concentration (8%), and by infusing only a minimum volume (0.4-0.45 ml) compared to other possible agents such as Dextran 200. The PVP infusion was set at a flow rate of 0.04 ml/min using an infusion pump (Harvard apparatus, USA, model 470). This rate was halved whenever necessary, depending on the animal's condition. The volume infused was determined so as to elevate the viscosity of the blood to a level similar to that reached in the Polycythemic-ABR group. This would enable a comparison of the ABR results in these two groups with similar elevated blood viscosity but different blood oxygen capacities. Total infusion time was 15 min. A rat was excluded from the study if bleeding occurred during the dissection or the infusion. At the end of the infusion the ~'emoral vein was tied and the skin sutured, and the operated rats were returned to their cage for 24 h. Auditory brainstem responses (ABR) were recorded before and after the blood viscosity was elevated, so that each rat contributed control values at
its normal viscosity. The results were statistically tested with paired t-tests.
ABR recordings ABR was elicited in ether anesthetized rats in response to 20/s, alternating polarity ( N - - 128 or 256), click stimuli. Recording subdermal needle electrodes were applied to the scalp and fore-paw (ground at hindpaw). Rectal temperature was maintained at 36.537.5°C. The recorded electrical activity was filtered (bandpass 200-2000 Hz), amplified and averaged by means of a Microshev-C.ERA 100. The values of wave latencies and amplitudes were measured with the cursor at 120 dB pe SPL. ABR replications were made at each intensity and the ABR threshold was defined as the lowest click intensity (in steps of 5 dB) at which repeatable waves could be recorded. A control recording was made 24 h before either placing the rats into the hypobaric chamber (Polycythemic-ABR group), or infusion with PVP solution (PVP group), and the experimental recording was made 24 h after the removal from the chamber or after the infusion. ABR thresholds were evaluated by three independent observers, and the differences between their threshold determinations were no greater than 5 dB.
Hemorheologic measurements Whole blood viscosity (WBV) The rats had to be sacrificed in order to obtain a sufficient quantity of blood for this measurement. On the other hand, the rats with elevated hematocrit (altitude acclimation in the hypobaric chamber) were also used in an additional study (noise induced temporary threshold shifts). Therefore WBV was determined in two additional groups: (a) Rats that had undergone the same procedure of acclimation in the hypobaric chamber as the Polycythemic-ABR group, and their WBV values were assumed to represent the viscosity in that group. These rats are referred to as Polycythemic-Visc group. (b) A control group that had normal blood viscosity which was measured as a control value. The WBV of the PVP rats was measured at the end of the experimental ABR recordings in the same animals. The viscosity of a fluid is defined as the ratio of the shear stress to the shear rate it produces (Dormandy, 1981). In this study the WBV was determined by using a Brookfield Digital Viscometer, model LVTDCP (Brookfield Laboratories Inc. USA), which is a rotational viscometer. The blood sample is placed into a sample cup which is surrounded by a water jacket to maintain constant temperature (37°C). A shallow cone comes into contact with the surface of the sample, and is rotated by a variable speed motor to which it is connected by a spring suspension. The torque in this
59
spring gives a measure of the drag applied to the cone by the blood, and the calculated shear stress is displayed at the top of the viscometer. Since blood is a non-Newtonian fluid, the shear stress is a nonlinear function of shear rate and therefore the viscosity varies with the shear rate (Burton, 1972; Chien et al., 1984). For this reason, different speeds of rotation are used, exposing the sample to different shear rates. Viscosity measurements were made at shear rates of 230, 115, 46 s -t, and for each of them, three values were determined and averaged. The viscosity in centipoise (cP) was calculated from the shear stress divided by the shear rate. Three milliliters of blood were drawn from the heart of sodium-pentobarbital anesthetized rats into a sterile blooJ collecting tube (Sherwood Medical) containing liquid EDTA-K3. To avoid excessive trauma to the blood during withdrawal, a wide bore needle (21 gauge) and minimum suction were used. The homogeneity of the blood was maintained by gentle mixing and 10 rain after the withdrawal of blood, a 1 ml sample was taken for viscosity measurement. Hematocrit measurement
Blood was taken from the tail of each rat in the control state, i.e.. 24 h before any experimental procedure, and in the experimt:atal state, i.e., 24 h after either removal from the hypobaric chamber (Polycythemic groups) or PVP infusion (PVP group). Hematocrit levels were determined in duplicate by centrifuging the sample for 10 min in a Clay Adams Microhematocrit Centrifuge. Also blood from the heart, used for viscosity measurement, was sampled for hematocrit determination in the same fashion.
Results
No physiological or behavioral change could be observed in the PVP rats 24 h after infusion; they moved about freely in their cage and showed no signs of distress. All the rats which were placed in the hypobaric chamber lost about 10% of their body weight (20-30g) since they ate very little in the hypoxic conditions, but apart from that, they also appeared normal. Two groups of rats were identically acclimated in the hypobaric chamber under the same conditions in order to make them polycythemic, as mentioned above: a) the Polycythemic-ABR rats in which ABR was recorded (and then served in a different project so that they were not sacrificed for viscosity measurement), and b) the Polycythemic-Visc rats which were sacrificed and served only for blood viscosity determination. These groups were compared to each other as to weight (24 h aRer removal from the chamber), hematocrit and hematocrit change, in order to show that they
TABLE I Weight, hematocrit and hematocrit change ( = difference/hematocrit before) in the Polycythemic-ABR and Polycythemic.Visc groups Weight Hematocrit (g) (%)
Hematocrit change (%)
after chamber
before chamher
after chamber
Polycythemic-ABR X ( N = 16) SD
227.6 19.9
43.8 2.4
54.2 2.5
24.1 9.0
Polycythemic-Visc ( N = 19)
X SD
221.7 12.7
43.7 2.9
53.2 3.6
22.1 7.5
t-test *
P
57
HEARES 01784
Influence of experimentally elevated blood viscosity on the auditory nerve-brainstem evoked response and threshold D a p h n a L i d a n a, S a u l Y e d g a r b, H . B e n A r o n s o n c a n d H a i m S o h m e r a Departments of a Physiology and b Biochemistry, Hebrew Unirersity - Hadassah Medical School, Jerusalem, Israel and c Department of Anesthesiology, Hadassah Unicersity Hospital, Jerusalem, Israel (Received 20 December 1991; Revision received 20 April 1992; Accepted 4 May 1992)
Blood viscosity, due to its effect on blood flow, is one of the determinants of oxygen delivery. Therefore the influence of elevated blood viscosity on hearing was studied in rats using the auditory brainstem response (ABR) threshold, wave 1 latency, brainstem transmission time (BTI') and wave 1/4 amplitude ratio. Whole blood viscosity (WBV) was elevated by 15-21% in two different ways: elevating the hematocrit (Polycythemia) by acclimation in a hypobaric chamber, or elevating the plasma viscosity by infusing a solution of Polyvinylpyrrolidone-360 (PVP). ABR was recorded before and 24 h after the blood viscosity was elevated, so that each rat served as its own control. Paired t-tests showed that there was no statistically significant difference in the ABR parameters in each of the groups as a consequence of blood viscosity elevation. In conclusion~ the elevation of WBV to this degree for this duration, using two different techniques had no effect either on the function of the auditory nerve and the more peripheral sites, or on the central auditory pathway as studied by ABR. Blood viscosity; Auditory nerve-brainstem response; Polycythemia; Hematocrit
Introduction
It has been suggested that increased viscosity of blood would have a deleterious effect on the auditory system. This hypothesis is based on clinical reports of hearing loss in patients suffer,.'ng from conditions in which blood viscosity was elevated (Davis and Nilo, 1965; Af'ffi and Tawfeek, 1971; Wells et al., 1977). A sensorineural hearing loss has been reported as a symptom in a patient with elevated viscosity due to Polycyt~emia Vera (Davis and Nilo, 1965). In this case hearing threshold and discrimination scores were improved following phlebotomy, and the improvement was explained as being due to a reduction in viscosity following withdrawal of blood. Dormandy (1970) showed that a 10% fall in viscosity in dogs induced by plasma exchange transfusion was accompanied by a 20% rise in cardiac output. Cases of Waldenstrom's Macroglobulinaemia, a disorder with clinical manifestations related to hyperviscosity of the circulating intravascular macroglobulin lgM (Salmon, 1988), were reported to have cochlear hearing loss, characterized by a sudden onset and sequential involvement of both ears (Af'Lfiand Tawfeek, 1971; Wells et al., 1977). Here
Correspondence to: H. Sohmer Department of Physiology, Hebrew University-Hadassah Medical School, P.O.B. 1172, Jerusalem 91010, Israel. Fax: (972) 2-439736.
also, hearing was improved to some extent after treatment which lowered viscosity such as alkylating agents and plasmaphoresis. The effects of the reduction of blood viscosity by hemodilution were investigated in guinea pigs by Hultcrantz and Nuttall (1987) and Nuttall et al. (1988), and the cochlear blood flow was found to increase as hematocrit decreased. Poiseuille's law states that the fit,w of blood is inversely proportional to its viscosity in vessels having fixed dimensions perfused at a constant pressure (Jandie, 1987). This relationship was determined from studies with tubes having rigid walls, using a Newtonian fluid and a constant flow. This clearly is not the case when one considers the flow of blood in vascular beds, in which complicated vascular geometry, pulsatile flow and the anomalous viscosity of blood (since blood is not a Newtonian fluid) induce numerous modifications in this relationship (Burton, 1972; Charlesworth, 1981; Jandle, 1987). Nevertheless, using Po~euille's law as a reasonable, first approximation (Charlesworth, 1981), it is clear that an increment in blood viscosity would reduce blood flow. Since the inner ear transduction mechanism is sensitive to hypoxia (Gaf-ni and Sohmer, 1976; Sohmer et al., 1986, 1989), the hearing loss seen in patients with hyperviscosity may be due to the decreased blood flow to the ear. The purpose of this study was to investigate, in carefully controlled experiments on rats, the effects of elevated blood viscosity on auditory nerve and brain-
58
stem evoked response (ABR) parameters, including threshold. In addition, an attempt was made to determine whether enhanced blood oxygen capacity could offset possible elevations of auditory threshold due to hyperviscosity.
Methods
The experiments were conducted on 3 month old male Sabra rats weighing 230-300 g ( m e a n - 258 g). Blood viscosity was elevated in the rats in two different ways. One group was placed into a hypobaric chamber which simulated the conditions of altitude for a period of 3-5 days inducing erythropoiesis (Yoffey et al., 1968). This was called the Polycythemic-ABR group (N = 16). The chamber was maintained at a barometric pressure of about 350 mmHg, and the partial pressure of oxygen was therefore about 65-70 mmHg. The chamber was opened each day for one hour to replenish food and water for the rats. In a second group of rats, Polyvinylpyrrolidone - 360 (PVP) macromolecules were infused. These molecules induce an elevation of the relative viscosity of solutions in-vitro (Scott, 1983), and of plasma viscosity in vivo (Yedgar et al., 1985). This was called the PVP group ( N = 14). The rats were anesthetized with 60 mg/kg Sodium Pentobarbital (IP) and local anesthesia (1% Lignocaine Hydrochloride) was used as necessary in each operation in addition to the general anesthesia. The femoral vein was cannulated and infused with PVP-360 (Sigma Chemical) solution in physiological saline. PVP-360 was chosen because of its limited effect on blood osmolarity and consequently blood volume, and also because satisfactory results could be obtained by applying a relatively low concentration (8%), and by infusing only a minimum volume (0.4-0.45 ml) compared to other possible agents such as Dextran 200. The PVP infusion was set at a flow rate of 0.04 ml/min using an infusion pump (Harvard apparatus, USA, model 470). This rate was halved whenever necessary, depending on the animal's condition. The volume infused was determined so as to elevate the viscosity of the blood to a level similar to that reached in the Polycythemic-ABR group. This would enable a comparison of the ABR results in these two groups with similar elevated blood viscosity but different blood oxygen capacities. Total infusion time was 15 min. A rat was excluded from the study if bleeding occurred during the dissection or the infusion. At the end of the infusion the ~'emoral vein was tied and the skin sutured, and the operated rats were returned to their cage for 24 h. Auditory brainstem responses (ABR) were recorded before and after the blood viscosity was elevated, so that each rat contributed control values at
its normal viscosity. The results were statistically tested with paired t-tests.
ABR recordings ABR was elicited in ether anesthetized rats in response to 20/s, alternating polarity ( N - - 128 or 256), click stimuli. Recording subdermal needle electrodes were applied to the scalp and fore-paw (ground at hindpaw). Rectal temperature was maintained at 36.537.5°C. The recorded electrical activity was filtered (bandpass 200-2000 Hz), amplified and averaged by means of a Microshev-C.ERA 100. The values of wave latencies and amplitudes were measured with the cursor at 120 dB pe SPL. ABR replications were made at each intensity and the ABR threshold was defined as the lowest click intensity (in steps of 5 dB) at which repeatable waves could be recorded. A control recording was made 24 h before either placing the rats into the hypobaric chamber (Polycythemic-ABR group), or infusion with PVP solution (PVP group), and the experimental recording was made 24 h after the removal from the chamber or after the infusion. ABR thresholds were evaluated by three independent observers, and the differences between their threshold determinations were no greater than 5 dB.
Hemorheologic measurements Whole blood viscosity (WBV) The rats had to be sacrificed in order to obtain a sufficient quantity of blood for this measurement. On the other hand, the rats with elevated hematocrit (altitude acclimation in the hypobaric chamber) were also used in an additional study (noise induced temporary threshold shifts). Therefore WBV was determined in two additional groups: (a) Rats that had undergone the same procedure of acclimation in the hypobaric chamber as the Polycythemic-ABR group, and their WBV values were assumed to represent the viscosity in that group. These rats are referred to as Polycythemic-Visc group. (b) A control group that had normal blood viscosity which was measured as a control value. The WBV of the PVP rats was measured at the end of the experimental ABR recordings in the same animals. The viscosity of a fluid is defined as the ratio of the shear stress to the shear rate it produces (Dormandy, 1981). In this study the WBV was determined by using a Brookfield Digital Viscometer, model LVTDCP (Brookfield Laboratories Inc. USA), which is a rotational viscometer. The blood sample is placed into a sample cup which is surrounded by a water jacket to maintain constant temperature (37°C). A shallow cone comes into contact with the surface of the sample, and is rotated by a variable speed motor to which it is connected by a spring suspension. The torque in this
59
spring gives a measure of the drag applied to the cone by the blood, and the calculated shear stress is displayed at the top of the viscometer. Since blood is a non-Newtonian fluid, the shear stress is a nonlinear function of shear rate and therefore the viscosity varies with the shear rate (Burton, 1972; Chien et al., 1984). For this reason, different speeds of rotation are used, exposing the sample to different shear rates. Viscosity measurements were made at shear rates of 230, 115, 46 s -t, and for each of them, three values were determined and averaged. The viscosity in centipoise (cP) was calculated from the shear stress divided by the shear rate. Three milliliters of blood were drawn from the heart of sodium-pentobarbital anesthetized rats into a sterile blooJ collecting tube (Sherwood Medical) containing liquid EDTA-K3. To avoid excessive trauma to the blood during withdrawal, a wide bore needle (21 gauge) and minimum suction were used. The homogeneity of the blood was maintained by gentle mixing and 10 rain after the withdrawal of blood, a 1 ml sample was taken for viscosity measurement. Hematocrit measurement
Blood was taken from the tail of each rat in the control state, i.e.. 24 h before any experimental procedure, and in the experimt:atal state, i.e., 24 h after either removal from the hypobaric chamber (Polycythemic groups) or PVP infusion (PVP group). Hematocrit levels were determined in duplicate by centrifuging the sample for 10 min in a Clay Adams Microhematocrit Centrifuge. Also blood from the heart, used for viscosity measurement, was sampled for hematocrit determination in the same fashion.
Results
No physiological or behavioral change could be observed in the PVP rats 24 h after infusion; they moved about freely in their cage and showed no signs of distress. All the rats which were placed in the hypobaric chamber lost about 10% of their body weight (20-30g) since they ate very little in the hypoxic conditions, but apart from that, they also appeared normal. Two groups of rats were identically acclimated in the hypobaric chamber under the same conditions in order to make them polycythemic, as mentioned above: a) the Polycythemic-ABR rats in which ABR was recorded (and then served in a different project so that they were not sacrificed for viscosity measurement), and b) the Polycythemic-Visc rats which were sacrificed and served only for blood viscosity determination. These groups were compared to each other as to weight (24 h aRer removal from the chamber), hematocrit and hematocrit change, in order to show that they
TABLE I Weight, hematocrit and hematocrit change ( = difference/hematocrit before) in the Polycythemic-ABR and Polycythemic.Visc groups Weight Hematocrit (g) (%)
Hematocrit change (%)
after chamber
before chamher
after chamber
Polycythemic-ABR X ( N = 16) SD
227.6 19.9
43.8 2.4
54.2 2.5
24.1 9.0
Polycythemic-Visc ( N = 19)
X SD
221.7 12.7
43.7 2.9
53.2 3.6
22.1 7.5
t-test *
P
