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Determination of the regional cochlear blood flow in the rat cochlea using non-radioactive microspheres and serially sectioned cochleas
HRR 00540
Determination of the regional cochlear blood flow in the rat cochlea using non-radioactive microspheres and serially sectioned cochleas Hans-Christian
Larsen ‘, Clarence
Angelborg
’
and Norma
Slepecky
’
The regional blood flow to the rat cochlea has been studled using a method which combines the microsphere method with ohvzrvation of serially-sectioned. plastic-embedded cochleah. Direct quantltation of the microspheres I” a reference blood sample and in the different vascular areas of the cochlea allows the analysis of blood flop patterns with respect to the different capillary beds. inner car. blood flow. microsphere
Introduction Many methods have been applied to the investigations of cochlear blood flow including direct in vivo microscopy [13,11], impedance plethysmography [12,16] and the study of the kinetics of perilymph production [9]. Quantitative measurements of the total cochlear blood flow have been obtained using radioactive microspheres [2.10]. More recently modifications of this method, where nonradioactive microspheres are observed in soft surface preparations or in serial sections, have been used to study the regional cochlear blood flow [5.15,14,3]. The aim of the present experiments, using the microsphere method combined with serial sectioning of plastic embedded cochleas, was to study the regional blood flow in specific vascular areas of the rat cochlea. Materials and Methods Eight cochleas from four albino rats were used in this study. The rats were anesthetized with thiobutylbarbital, 120 mg/kg body weight, intraperitoneally. Constant body temperature was maintained with a heating pad. The femoral arteries were catheterized to obtain a reference blood sample and blood pressure measurements. Prior to the 037x-5Y5i/84/$03.00
’ 1984 Elsevier Science Publishers
B.V.
injection of microspheres, the left ventricle of the heart was catheterized transcutaneously using a metal cannula and the site of the catheter was assured by measuring blood acid-base balance (Radiometer, ABL 2, Copenhagen, Denmark). Non-radioactive microspheres (11.7 f 1.7 pm diam.; NEN, Mass., U.S.A.), dissolved in saline and TweenR and prewarmed to body temperature. were injected into the heart over a period of 10 s. The volume and the number of microspheres injected can be seen in Table I. Reference blood samples were taken for 1 min by free flow [l] to determine the cochlear blood flow. The animals were killed by intracardial injection of potassium chloride and the temporal bones dissected out of the skull. After fixation in phosphate-buffered 2.5% glutaraldehyde. the temporal bones were decalcified in 10% EDTA. post-fixed in phosphate-buffered 1% 0~0, and processed for embedding in soft Spurr or Araldite. 22 pm thick serial sections were taken in a plane parallel to the modiolus. The microspheres were counted in each section using a light microscope and the distribution of the microspheres in the capillary beds was noted. To determine the total cochlear blood flow. the number of microspheres in the reference blood samples were counted using a light microscope
12X TABLE
I
THE NUMBER BLOOD FLOW
OF MICROSPHERES IN THE EXAMINED
Rat No.
Cochlea
No. of microspheres per cochlea
Cochlear blood flow (mg/cochlea/min)
1
Right Left Right Left Right Left Right Left
294 249 296 333 371 421 161 274
1.35 1.15 1.54 1.74 1.20 1.36 1.78 3.04
2 3 4
Fig. 1. Microspheres
in cross-sections
lamina portion of cochlear partition;
AND THE TOTAL EIGHT COCHLEAS
and a Burger counting chamber. From this data and the number of microspheres counted in the cochleas, the cochlear blood flow was determined
[Il. To determine the regional cochlear blood flow, the capillary beds were divided into the following vascular areas: the modiolus, which includes the arterioles and the spiral ganglion; the region of the cochlear purtition, which includes the vascular beds within the osseous spiral lamina, the spiral limbus, the vessels of the tympanic lip and the vessel of the basilar membrane; and the luteral wull. which includes the radiating arterioles in the roof of scala vestibuli, the spiral ligament, the vessel of the
of rat cochlea, 22 pm thick, unstained. Arrows indicate microspheres. (a) In osseous (b) in modiolus and spiral ligament; (c) in tympanic lip; (d) in stria vascularis.
spiral
vestibular membrane, the spiral prominence and the stria vascularis. Some of the vascular beds were further subdivided into more specific regions. The rr~~diolus region remains the same. The cochlear partition is divided into the osseous spirtil lumincl which includes the spiral limbus, and the vessels near the nqun uf Corti which include the tympanic lip vessels running at the inferior surface of the osseous spiral lamina under the inner pillar cells and the vessels of the basilar membrane. The lateral wall vascular area is divided into the rudiuring cwterioh of Scala vestibuli, the spirul iigument which includes the vessel of the vestibular membrane atid the spiral prominence, and the strirr cusculuris.
Modhus
Fig. 2. Schematic drawing diatrlbution pnrtilion
Coch.
of a cochlear
of the microspheres
and the lateral
limit\ on the mean.
Part.
wall.
Lat.‘Wall turn and the relative
in the modiolus. Bars indicate
the cochlear
95% confidence
Results
All animals tolerated the experimental procedures well and the acid--base balance taken prior to the microsphere injections were within the normal range. No significant change of the blood pressure was caused by the microsphere injections. In the light microscope, the microspheres are easy to see, both when counting them in the reference blood sample and when counting them and noting their position within the sections of the cochlea (Fig. 1). In the cochlea, if a microsphere was cut in two by the knife, this could be recognized by the fact that the cut microsphere appeared lighter in colour, and by the fact that the two parts were at exactly the same place in the cochlea in two adjacent sections.
Fig. 3. Schematic drawing distribution
of a cochlear turn and the relative
of the microspheres
in: M. the modiolus; OS, the
o~sews spiral lamina: OC, the vessels near the organ of Cortl: RA.
the radiating
arterioles:
ligament.
Bars indicate
spiral mean.
SV. the stria vascularls; 95% confidence
SL. the
limits on the
The total number of microspheres could easily be quantitated and the total cochlear blood flow calculated (Table I). Regional blood flow can be calculated when the capillary beds are divided into the three large vascular areas: the modiolus. the cochlear partition and the lateral wall. This division is diagrammed in Fig. 2 and the pattern of the relative distribution of the microspheres is graphically displayed. The greatest proportion of the microspheres is in the lateral wall (77%), less in the modiolus (15%) and least in the cochlear partition (8%). When the quantitative blood flow in these capillary areas is calculated, the mean values + S.D. for the eight cochleas are (mg/ml): modiolus 0.25 f 0.07, cochlear partition 0.13 _t 0.04, lateral wall 1.26 + 0.59. When the vascular areas are further subdivided as diagrammed in Fig. 3, the relative distribution of the microspheres is as follows: 15% in the modiolus, 7% in the osseous spiral lamina, 1% in the vessels near the organ of Corti (there were no microspheres under the tunnel of Corti in the vessel of the basilar membrane), 10% in the radiating arterioles, 49% in the spiral ligament and 18% in the stria vascularis. Discussion By viewing serial sections from plastic-embedded cochleas in the light microscope, it is possible to observe the distribution of microspheres within the cochlea. They are easy to see even in osmicated tissue and their sites within the different vascular beds are easy to distinguish. By such direct observation of serial sections and by quantitating the number of microspheres in the cochlea and in the reference blood sample, we have shown that it is possible to calculate the total cochlear blood flow. The results we have obtained correlate well with those obtained by previous studies using radioactively labeled microspheres [S]. Using this method, it is also possible to get information on the regional cochlear blood flow. When the cochlea is divided into large vascular areas (the modiolus, the cochlear partition and the lateral wall), the pattern of microsphere distribution, and thus the regional blood flow, in the rat cochlea is similar to that seen in the guinea pig [3]. The greatest proportion of the microspheres. and
thus most of the blood flow. is in the lateral wall (77% in the rat, 57% in the guinea pig), and the least is in the vessels near the organ of Corti in the cochlear partition (8% in the rat, 8% in the guinea pig). In the rat there are no microspheres found under the tunnel of Corti. A similar pattern has been found in the rabbit [15]. The results in the rat cochlea differ from those in the guinea pig in that a relatively smaller proportion of the microspheres is found in the modiolus (15% in the rat, 35% in the guinea pig). It cannot be determined whether this reflects a smaller amount of blood flow in the modiolus of the rat or if it results from a difference in the anatomy of the cochlear vessels for example, a difference in vessel diameter or the presence in the guinea pig of a large number of spring coiled arterioles which are surrounded by smooth muscle cells [4]. When the cochlea is subdivided into smaller vascular regions and the results obtained in the rat are compared with those obtained in the guinea pig. further differences become apparent. While the relative distribution of the microspheres in the cochlear partition was similar, it is found on further subdivision that in the rat fewer microspheres are trapped in the vessels close to the organ of Corti than in the region of the osseous spiral lamina closer to the modiolus. In the guinea pig there are microspheres present in the vessel of the basilar membrane under the tunnel of Corti. This distribution may again be a function of the anatomy of the cochlear vessels or it may reflect differences in the cochlear vasculature; for example. in the rat the vessel of the basilar membrane is not often seen [7]. The rat, unlike the guinea pig, has a relatively smaller proportion of microspheres trapped in the radiating arterioles (10% in the rat, 20% in the guinea pig), and a relatively larger proportion in the stria vascularis (18% in the rat. 10% in the guinea pig) and spiral ligament (49% in the rat, 27% in the guinea pig). Since the microspheres flow with the blood until they reach and become lodged in vessels whose diameters approximate their own, these results suggest that some of the microspheres become trapped in the radiating arterioles on their way to the lateral wall. Thus, when studying the regional blood flow. the diame-
ter of the microspheres relative to that of the radiating arterioles becomes increasingly important. Consequently the diameter of the microspheres used in the present experiment may be too large to permit calculation of the regional cochlear blood flow in the subdivisions of the lateral wall. To do this it may be necessary to use smaller microspheres. Taking the above discussion into account, the regional blood flow to this area is still not what would he expected based on the assumption that the stria vascularis is thought to have a higher metabolic demand than the spiral ligament. Even if all the microspheres trapped in the radiating arterioles were to distribute to the stria, the relative distribution would be less in the stria (28%) than in the spiral ligament (49%). The distribution in the guinea pig would be almost equal (30% in the stria and 27% in the spiral ligament). In the rabbit. where the number of microspheres trapped in the radiating arterioles is low (3%). the relative distributi~~n of microspheres in the stria (35%) is also less than that in the spiral ligament (43%) 1151. These results in the lateral wall of the rat cochlea are not totally unexpected if one considers the vasculature of this region, where more radiating arteriole branches appear to supply the vessels of the spiral prornilletl~e than the stria vascularis (‘71. However. whether the results in the lateral wall reflect different demands on cochlear blood flow, different ways in which the microspheres are distributed, or differences in vascular anatomy has yet to be deterlliined. Frotn the above discussion it seems reasonable to assume that in attempting to analyze the distribution of microspheres when the cochlea is divided into small vascular areas, and when trying to compare the regional cochlear blood flow between different experimental animals. there are several issues that must be addressed. First. the ideal size for the microspheres may not be the same for all animal species. Furthermore. it must be noted that when the cochlea has been subdivided into small vascular areas, fewer microspheres are used to calculate regional cochlear blood flow than were used to calculate the total cochlear blood flow. Care must be used when interpreting the results since the sampling error may be large and the variation in data considerable [6]. Experiments are
in progress to clarify these issues. Our results show that the method described here for calculating total cochlear blood flow by direct observation of the microspheres in plasticembedded, serially-sectioned cochleas gives results which correlate well with the results obtained using radioactive microspheres. Regional blood flow values are easily obtained and the relative distribution of microspheres within the capillary beds displays a constant pattern. When the distribution of microspheres is analyzed by dividing the cochlea into large vascular areas. there is a similar pattern when regional blood flow in the rat is compared to previous results obtained in the guinea pig and the rabbit. Upon further subdivision. the pattern of the distribution of microspheres to the stnaller vascular regions differs between animal species. Our results show that by using 11.7 pnt microspheres it may not be possible to compare regional blood flow across animal species. However. within one species. it will be possible to analyze changes in regional blood flow as reflected by a redistrihution of the microspheres in the cochlea resulting from experimental conditions, for example trauma or drugs. Acknowledgements
This research was supported by a grant from the Swedish Medical Research Council (No. 17X04782). The authors would like to thank John Maines and Ed Dixon of the Instrument Development Shop for their help with technical problems, Steve Falen for help with computer equipment. Anne and Else Slepecky for help with quantitating microspheres. and Pat Kane for secretarial assistance. References 1 Aim. A. and Bill, A. (1972): The oxygen supply to the retma. II. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on weal and retinal blood flow in cats. Acta Physiol. &and. X4. 306. 2 Angelborg, C., Hultcranrz, E. and kgerup, 8. (197’7): The cochlear blood flow. Acta Otolaryngol. X3. 92. 3 Angelborg. C.. Larsen. H.C. and Slepecky, N. (19X4): Regional cochlear blood flow studied hy observation of micwspheres in serial sections. Ann. Otol. Rhinol. Laryngoi. (in prw).
4 Axelsson, A. (1968): The vascular anatomy of the cochlea in the guinea pig and in man. Acta Otolaryngol. Suppl. 243. 5. 5 Axelsson, A., Angelborg, C. and Larsen, H.C. (1983): The microsphere-surface technique for evaluation of cochicar vessels and circulation. Acta Otolaryngol. 95, 297. 6 Buckberg, G.D.. Luck, J.C.. Payne, O.O., Hoffman, J.I.E.. Archie, J.P. and Fixler. D.E. (1971): Some sources of error in measuring regional blood flow with radioactive microspheres. J. Appl. Physiol. 31, 598. I Hornstrand, C., Axelsson. A. and Vertes, D. (1980): The vascular anatomy of the rat cochlea. Acla Otolaryngol. 89, 1. E. and Angelborg, C. (1978): Cochlear blood 8 Hultcrantz, circulation studied with microspheres. Otol. Rhinol. Laryngol. 40, 65. and co&fear 9 Kellerhals. B. (1979): Per~iymph production blood flow. Acta Otoiaryngol. 87. 370. blood flow and hearing 10 Larsen, H.C. (1981): Cochlear related to changes in osmotic and hydrostatic pressure in
11 12
13
14
15
I6
the inner ear. Acta Universnatts Up.taltens~\. D~\aertatt~tn from the Faculty of Medicine. Lawrence, M. (1971): Blood flop through the basilar mcmbtane capillaries. Acta Otoiaryngoi. 71. 106. Morimitsu. T. (1960): Observation of the cochlenr blood circulation in guinea pig by the Impedance plethysmography. Otologia Fukuoka Suppl. 6, 437. Pcrlman, H.B. and Kimura. R.S. (1955): Observations 01 the living blood vessels of the cochlea. Ann Otol. Rhino1 Laryngol. 64. 1176. Prazma, .I., Rodgers. G.K.. and Pillsbury, H.C. (1983) Cochlear blood flow: Effect of noise. Arch Otolaryngol. 109, 611. Slepecky. N., Larsen. H.C. and Angelborg. C. (1983) Cochlear blood flow - a computerized study using microspheres and serial secttoning. Presented at the 20th Workshop on loner Ear Biology, Geilo, Norway. Suga, F. and Snow, J.B. (1969) Adrenergic control of cochlear blood flow. Ann. Otol. Rhinol. Laryngol. 7X. 358.
Determination of the regional cochlear blood flow in the rat cochlea using non-radioactive microspheres and serially sectioned cochleas Hans-Christian
Larsen ‘, Clarence
Angelborg
’
and Norma
Slepecky
’
The regional blood flow to the rat cochlea has been studled using a method which combines the microsphere method with ohvzrvation of serially-sectioned. plastic-embedded cochleah. Direct quantltation of the microspheres I” a reference blood sample and in the different vascular areas of the cochlea allows the analysis of blood flop patterns with respect to the different capillary beds. inner car. blood flow. microsphere
Introduction Many methods have been applied to the investigations of cochlear blood flow including direct in vivo microscopy [13,11], impedance plethysmography [12,16] and the study of the kinetics of perilymph production [9]. Quantitative measurements of the total cochlear blood flow have been obtained using radioactive microspheres [2.10]. More recently modifications of this method, where nonradioactive microspheres are observed in soft surface preparations or in serial sections, have been used to study the regional cochlear blood flow [5.15,14,3]. The aim of the present experiments, using the microsphere method combined with serial sectioning of plastic embedded cochleas, was to study the regional blood flow in specific vascular areas of the rat cochlea. Materials and Methods Eight cochleas from four albino rats were used in this study. The rats were anesthetized with thiobutylbarbital, 120 mg/kg body weight, intraperitoneally. Constant body temperature was maintained with a heating pad. The femoral arteries were catheterized to obtain a reference blood sample and blood pressure measurements. Prior to the 037x-5Y5i/84/$03.00
’ 1984 Elsevier Science Publishers
B.V.
injection of microspheres, the left ventricle of the heart was catheterized transcutaneously using a metal cannula and the site of the catheter was assured by measuring blood acid-base balance (Radiometer, ABL 2, Copenhagen, Denmark). Non-radioactive microspheres (11.7 f 1.7 pm diam.; NEN, Mass., U.S.A.), dissolved in saline and TweenR and prewarmed to body temperature. were injected into the heart over a period of 10 s. The volume and the number of microspheres injected can be seen in Table I. Reference blood samples were taken for 1 min by free flow [l] to determine the cochlear blood flow. The animals were killed by intracardial injection of potassium chloride and the temporal bones dissected out of the skull. After fixation in phosphate-buffered 2.5% glutaraldehyde. the temporal bones were decalcified in 10% EDTA. post-fixed in phosphate-buffered 1% 0~0, and processed for embedding in soft Spurr or Araldite. 22 pm thick serial sections were taken in a plane parallel to the modiolus. The microspheres were counted in each section using a light microscope and the distribution of the microspheres in the capillary beds was noted. To determine the total cochlear blood flow. the number of microspheres in the reference blood samples were counted using a light microscope
12X TABLE
I
THE NUMBER BLOOD FLOW
OF MICROSPHERES IN THE EXAMINED
Rat No.
Cochlea
No. of microspheres per cochlea
Cochlear blood flow (mg/cochlea/min)
1
Right Left Right Left Right Left Right Left
294 249 296 333 371 421 161 274
1.35 1.15 1.54 1.74 1.20 1.36 1.78 3.04
2 3 4
Fig. 1. Microspheres
in cross-sections
lamina portion of cochlear partition;
AND THE TOTAL EIGHT COCHLEAS
and a Burger counting chamber. From this data and the number of microspheres counted in the cochleas, the cochlear blood flow was determined
[Il. To determine the regional cochlear blood flow, the capillary beds were divided into the following vascular areas: the modiolus, which includes the arterioles and the spiral ganglion; the region of the cochlear purtition, which includes the vascular beds within the osseous spiral lamina, the spiral limbus, the vessels of the tympanic lip and the vessel of the basilar membrane; and the luteral wull. which includes the radiating arterioles in the roof of scala vestibuli, the spiral ligament, the vessel of the
of rat cochlea, 22 pm thick, unstained. Arrows indicate microspheres. (a) In osseous (b) in modiolus and spiral ligament; (c) in tympanic lip; (d) in stria vascularis.
spiral
vestibular membrane, the spiral prominence and the stria vascularis. Some of the vascular beds were further subdivided into more specific regions. The rr~~diolus region remains the same. The cochlear partition is divided into the osseous spirtil lumincl which includes the spiral limbus, and the vessels near the nqun uf Corti which include the tympanic lip vessels running at the inferior surface of the osseous spiral lamina under the inner pillar cells and the vessels of the basilar membrane. The lateral wall vascular area is divided into the rudiuring cwterioh of Scala vestibuli, the spirul iigument which includes the vessel of the vestibular membrane atid the spiral prominence, and the strirr cusculuris.
Modhus
Fig. 2. Schematic drawing diatrlbution pnrtilion
Coch.
of a cochlear
of the microspheres
and the lateral
limit\ on the mean.
Part.
wall.
Lat.‘Wall turn and the relative
in the modiolus. Bars indicate
the cochlear
95% confidence
Results
All animals tolerated the experimental procedures well and the acid--base balance taken prior to the microsphere injections were within the normal range. No significant change of the blood pressure was caused by the microsphere injections. In the light microscope, the microspheres are easy to see, both when counting them in the reference blood sample and when counting them and noting their position within the sections of the cochlea (Fig. 1). In the cochlea, if a microsphere was cut in two by the knife, this could be recognized by the fact that the cut microsphere appeared lighter in colour, and by the fact that the two parts were at exactly the same place in the cochlea in two adjacent sections.
Fig. 3. Schematic drawing distribution
of a cochlear turn and the relative
of the microspheres
in: M. the modiolus; OS, the
o~sews spiral lamina: OC, the vessels near the organ of Cortl: RA.
the radiating
arterioles:
ligament.
Bars indicate
spiral mean.
SV. the stria vascularls; 95% confidence
SL. the
limits on the
The total number of microspheres could easily be quantitated and the total cochlear blood flow calculated (Table I). Regional blood flow can be calculated when the capillary beds are divided into the three large vascular areas: the modiolus. the cochlear partition and the lateral wall. This division is diagrammed in Fig. 2 and the pattern of the relative distribution of the microspheres is graphically displayed. The greatest proportion of the microspheres is in the lateral wall (77%), less in the modiolus (15%) and least in the cochlear partition (8%). When the quantitative blood flow in these capillary areas is calculated, the mean values + S.D. for the eight cochleas are (mg/ml): modiolus 0.25 f 0.07, cochlear partition 0.13 _t 0.04, lateral wall 1.26 + 0.59. When the vascular areas are further subdivided as diagrammed in Fig. 3, the relative distribution of the microspheres is as follows: 15% in the modiolus, 7% in the osseous spiral lamina, 1% in the vessels near the organ of Corti (there were no microspheres under the tunnel of Corti in the vessel of the basilar membrane), 10% in the radiating arterioles, 49% in the spiral ligament and 18% in the stria vascularis. Discussion By viewing serial sections from plastic-embedded cochleas in the light microscope, it is possible to observe the distribution of microspheres within the cochlea. They are easy to see even in osmicated tissue and their sites within the different vascular beds are easy to distinguish. By such direct observation of serial sections and by quantitating the number of microspheres in the cochlea and in the reference blood sample, we have shown that it is possible to calculate the total cochlear blood flow. The results we have obtained correlate well with those obtained by previous studies using radioactively labeled microspheres [S]. Using this method, it is also possible to get information on the regional cochlear blood flow. When the cochlea is divided into large vascular areas (the modiolus, the cochlear partition and the lateral wall), the pattern of microsphere distribution, and thus the regional blood flow, in the rat cochlea is similar to that seen in the guinea pig [3]. The greatest proportion of the microspheres. and
thus most of the blood flow. is in the lateral wall (77% in the rat, 57% in the guinea pig), and the least is in the vessels near the organ of Corti in the cochlear partition (8% in the rat, 8% in the guinea pig). In the rat there are no microspheres found under the tunnel of Corti. A similar pattern has been found in the rabbit [15]. The results in the rat cochlea differ from those in the guinea pig in that a relatively smaller proportion of the microspheres is found in the modiolus (15% in the rat, 35% in the guinea pig). It cannot be determined whether this reflects a smaller amount of blood flow in the modiolus of the rat or if it results from a difference in the anatomy of the cochlear vessels for example, a difference in vessel diameter or the presence in the guinea pig of a large number of spring coiled arterioles which are surrounded by smooth muscle cells [4]. When the cochlea is subdivided into smaller vascular regions and the results obtained in the rat are compared with those obtained in the guinea pig. further differences become apparent. While the relative distribution of the microspheres in the cochlear partition was similar, it is found on further subdivision that in the rat fewer microspheres are trapped in the vessels close to the organ of Corti than in the region of the osseous spiral lamina closer to the modiolus. In the guinea pig there are microspheres present in the vessel of the basilar membrane under the tunnel of Corti. This distribution may again be a function of the anatomy of the cochlear vessels or it may reflect differences in the cochlear vasculature; for example. in the rat the vessel of the basilar membrane is not often seen [7]. The rat, unlike the guinea pig, has a relatively smaller proportion of microspheres trapped in the radiating arterioles (10% in the rat, 20% in the guinea pig), and a relatively larger proportion in the stria vascularis (18% in the rat. 10% in the guinea pig) and spiral ligament (49% in the rat, 27% in the guinea pig). Since the microspheres flow with the blood until they reach and become lodged in vessels whose diameters approximate their own, these results suggest that some of the microspheres become trapped in the radiating arterioles on their way to the lateral wall. Thus, when studying the regional blood flow. the diame-
ter of the microspheres relative to that of the radiating arterioles becomes increasingly important. Consequently the diameter of the microspheres used in the present experiment may be too large to permit calculation of the regional cochlear blood flow in the subdivisions of the lateral wall. To do this it may be necessary to use smaller microspheres. Taking the above discussion into account, the regional blood flow to this area is still not what would he expected based on the assumption that the stria vascularis is thought to have a higher metabolic demand than the spiral ligament. Even if all the microspheres trapped in the radiating arterioles were to distribute to the stria, the relative distribution would be less in the stria (28%) than in the spiral ligament (49%). The distribution in the guinea pig would be almost equal (30% in the stria and 27% in the spiral ligament). In the rabbit. where the number of microspheres trapped in the radiating arterioles is low (3%). the relative distributi~~n of microspheres in the stria (35%) is also less than that in the spiral ligament (43%) 1151. These results in the lateral wall of the rat cochlea are not totally unexpected if one considers the vasculature of this region, where more radiating arteriole branches appear to supply the vessels of the spiral prornilletl~e than the stria vascularis (‘71. However. whether the results in the lateral wall reflect different demands on cochlear blood flow, different ways in which the microspheres are distributed, or differences in vascular anatomy has yet to be deterlliined. Frotn the above discussion it seems reasonable to assume that in attempting to analyze the distribution of microspheres when the cochlea is divided into small vascular areas, and when trying to compare the regional cochlear blood flow between different experimental animals. there are several issues that must be addressed. First. the ideal size for the microspheres may not be the same for all animal species. Furthermore. it must be noted that when the cochlea has been subdivided into small vascular areas, fewer microspheres are used to calculate regional cochlear blood flow than were used to calculate the total cochlear blood flow. Care must be used when interpreting the results since the sampling error may be large and the variation in data considerable [6]. Experiments are
in progress to clarify these issues. Our results show that the method described here for calculating total cochlear blood flow by direct observation of the microspheres in plasticembedded, serially-sectioned cochleas gives results which correlate well with the results obtained using radioactive microspheres. Regional blood flow values are easily obtained and the relative distribution of microspheres within the capillary beds displays a constant pattern. When the distribution of microspheres is analyzed by dividing the cochlea into large vascular areas. there is a similar pattern when regional blood flow in the rat is compared to previous results obtained in the guinea pig and the rabbit. Upon further subdivision. the pattern of the distribution of microspheres to the stnaller vascular regions differs between animal species. Our results show that by using 11.7 pnt microspheres it may not be possible to compare regional blood flow across animal species. However. within one species. it will be possible to analyze changes in regional blood flow as reflected by a redistrihution of the microspheres in the cochlea resulting from experimental conditions, for example trauma or drugs. Acknowledgements
This research was supported by a grant from the Swedish Medical Research Council (No. 17X04782). The authors would like to thank John Maines and Ed Dixon of the Instrument Development Shop for their help with technical problems, Steve Falen for help with computer equipment. Anne and Else Slepecky for help with quantitating microspheres. and Pat Kane for secretarial assistance. References 1 Aim. A. and Bill, A. (1972): The oxygen supply to the retma. II. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on weal and retinal blood flow in cats. Acta Physiol. &and. X4. 306. 2 Angelborg, C., Hultcranrz, E. and kgerup, 8. (197’7): The cochlear blood flow. Acta Otolaryngol. X3. 92. 3 Angelborg. C.. Larsen. H.C. and Slepecky, N. (19X4): Regional cochlear blood flow studied hy observation of micwspheres in serial sections. Ann. Otol. Rhinol. Laryngoi. (in prw).
4 Axelsson, A. (1968): The vascular anatomy of the cochlea in the guinea pig and in man. Acta Otolaryngol. Suppl. 243. 5. 5 Axelsson, A., Angelborg, C. and Larsen, H.C. (1983): The microsphere-surface technique for evaluation of cochicar vessels and circulation. Acta Otolaryngol. 95, 297. 6 Buckberg, G.D.. Luck, J.C.. Payne, O.O., Hoffman, J.I.E.. Archie, J.P. and Fixler. D.E. (1971): Some sources of error in measuring regional blood flow with radioactive microspheres. J. Appl. Physiol. 31, 598. I Hornstrand, C., Axelsson. A. and Vertes, D. (1980): The vascular anatomy of the rat cochlea. Acla Otolaryngol. 89, 1. E. and Angelborg, C. (1978): Cochlear blood 8 Hultcrantz, circulation studied with microspheres. Otol. Rhinol. Laryngol. 40, 65. and co&fear 9 Kellerhals. B. (1979): Per~iymph production blood flow. Acta Otoiaryngol. 87. 370. blood flow and hearing 10 Larsen, H.C. (1981): Cochlear related to changes in osmotic and hydrostatic pressure in
11 12
13
14
15
I6
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