- Email: [email protected]
Ultrasonic characterization of aging in skin tissue
Ultrasound in Med. & Biol., Vol. 6, pp. 369-375 Pergamon Press Ltd., 1980. Printedin Great Britain
ULTRASONIC CHARACTERIZATION OF AGING IN SKIN TISSUE P. K. BHAGATand WASSON KERRICK Wenner-Gren BiomedicalResearch Laboratory,Department of Mechanical Engineering,University of Kentucky,Lexington,KY 40506, U.S.A. and R. W. WAREt V e t e r a n s Medical Center, Le xi ngt on, K Y 40506, U.S.A.
(First received 3 April 1979; and in final form 27 February 1980)
Abstract--The propagation velocity (c) and attenuation coefficient (a) of ultrasound were measured at room temperature in skin tissues excised from twenty aged (27 months) and eight young (2 months) Bar Harbor strain female mice. Frequencies used were 2.25, 5.0, 7.5 and 10.0 MHz. Both velocity and attenuation data were obtained using a pulse-echo technique. Transit times were measured with a universal counter timer to 10 nsec resolution, and corrected for the "dead time" due to the transducer matching layer. Aged skin had significantly lower values of c and a at all frequencies than did the corresponding young tissues. This preliminary study suggests that the tissue changes which collectively constitute aging may, in some instances, be predictably associated with measurable changes in the acoustic properties of those tissues.
Key words: Tissue aging, Ultrasonic pulse echo system, Attenuation coefficient, Velocity of propagation, Frequencydependence of acoustic parameters.
INTRODUCTION
The velocity of propagation of an acoustic wave in a medium is largely dependent upon its density and bulk modulus whereas attenuation is due to viscoelastic properties, the presence of interfaces with differing acoustic impedances, inherent inhomogeneities and tissue geometry. Biological tissues undergo structural and biochemical changes under a variety of physiological and pathological conditions which can lead to changes in density, bulk modulus, distribution of scattering centers and composition within the tissues. These changes, therefore, can be reflected as variations in the measured acoustic parameters, the velocity of propagation and attenuation. Noninvasive measurement of acoustic parameters, therefore, offers the possibility of differential diagnosis of tissue state. The use of ultrasonic energy is especially attractive for this purpose since it has been shown to be nontoxic at levels normally required for diagnostic applications (Hill, 1968; Woodward et al., 1970; Curzen, 1972). tPresent Address: Veterans Medical Center, Kerrville, TX 78028, U.S.A.
Organs such as kidney and other tissues, most notably skin, undergo marked functional changes with aging (Bakerman, 1969). These changes could be brought about by such factors as changes in fat and water content and alterations in the amounts and integrity of connective tissues. These changes may affect the density and elasticity of the tissue which can be detected as variations in the velocity of propagation c (ms -~) and attenuation coefficient, a (db cm-J). The study reported here was an attempt to find a correlation between tissue age and variations in the acoustic parameters. The experimental approach was simple and straightforward and velocity and tissue attenuation were the only parameters measured. The frequencies employed ranged from 2.25 to 10 MHz, covering the frequency range of present day ultrasonic diagnostic equipment. MATERIALS
Two groups of Bar Harbor strain B7D2F1 female mice were used. One group consisted of eight young mice aged approximately 1.82.1 months (average mass -- 21.6 _+2.0 g), the other of twenty old mice aged 26.5 to 27.3
369
370
P. K. BHAGAT et al.
months (41.3_+8.6g). Mice were chosen as the experimental animals because of the ready availability of a group of naturally aged subjects, coupled with the comparative ease and inexpense of acquiring young mice of the same sex and strain. The young mice were fed Purina mouse chow ad lib with tap water during the interim ranging from 1.0 to 2.6 weeks between shipment and sacrifice. The older mice were fed special diets containing varying calcium/phosphorous ratios, with distilled water, for 25 months preceding sacrifice. The food intake of these aged mice was regulated so that all four diet groups had an equal dietary calcium intake. Appendix A gives additional information concerning the food preparation and diets of the aged mice. Acoustic measurements in skin tissue were made after folding to obtain four thicknesses (this allowed the time between echoes to exceed the three/~sec minimum gate delay of instrumentation used). The choice of skin as a test tissue despite the difficulty of working with it was made for the following reasons: (i) age-related compositional and structural changes are more pronounced in skin than in many tissues, (ii) the aging of skin has been rather extensively studied and (iii) for noninvasive work the skin is certainly the most easily accessible tissue (Bakerman, 1969; Robert and Robert, 1973). Mice were sacrificed by injection with roughly 0.5 ml of Brevital Sodium and a rectangle (1.5 x 2.0 cm or larger) of skin removed from each mouse promptly after respiration ceased. The skin was placed in Hank's Balanced Salt Solution (HBSS) after removal of the hair with one or more applications of a depilatory (Surgex Hair Remover Cream). The HBSS bathing the tissues provided an approximately isotonic environment for the tissue intercellular field. Failure to take this precaution can result in a rapid change in measured velocity as the tissue imbibes or expels water. The boundary between skin and other attached tissues can be quite vague, so special effort was made to prepare all skin samples similarly as follows: Samples were cut from the same part of each mouse's body. Muscle tissue is readily distinguishable from skin upon eye examination. Muscle and any fat suspected of not
actually being a part of the skin were pulled/peeled off the skin using forceps.
METHODS
The pulse-echo technique used for determination of propagation velocity (c) and attenuation coefficient (~) in tissues is schematically shown in Fig. 1. The tissue specimen was placed on a glass microscope slide submerged in a plexiglass tank of HBSS at ambient temperature (20.5°C). All tissues were examined using ultrasonic transducers with resonant frequencies nominally at 2.25, 5.0, 7.5 and 10.0 MHz. These immersion-type piezoelectric transducers (Aerotecb Laboratories, gamma-type pencil probes, narrow band, 20% bandwidth, 0.953cm diameter) were shock excited with a narrow pulse (200 volts peak, duration 60nsec, p r f = 4 K H z ) from an Aerotech Ultrasonic Transducer Analyzer (UTA-3)t. The UTA-3 also supplied a calibrated attenuator, receiver-amplifier, peak detector and gating networks. The input receiver sensitivity is 5 mv with a frequency bandwidth of 24.6MHz and a lower frequency cutoff at 400 KHz. The receiver gain is 40db with an output impedance of 50 ~ and the peak detector has a 5% linearity in the range 50 my-1 volt. Measurements were made with the ultrasonic beam perpendicular to the surface of the glass slide. This orientation was achieved by adjusting the stereotaxic device, which supported the transducer, to the position at which the received reflection from the glass slide was of maximum amplitude when no tissue was interposed between transducer and glass slide. It was checked at distances ranging from the thickness of the thinnest specimen to approximately three times the thickest specimen size. The signal received after one reflection from the glass slide, 2.5 mm thick, (i.e. after two passages through tissue or HBSS) was amplified, gated and the pulse transit time obtained with a Tektronix model 7B15 universal counter timer (10 nsec resolution). Distances were measured using a micrometer (resolution 5 ~m) assisted by an electricalcontinuity checking procedure as explained below. Data were obtained for the tissues of each mouse in the following manner: at each tSubsidiary of KB-Aerotech, Lewistown, Pennsyl- frequency, the values of pulse transit time (t), calibrated attenuator reading (A), the assovania.
371
Ultrasonic characterization of aging in skin tissue Aerotech TransducerAnalyzer (UTA-3) _ - I
I r
Calibrated Attenuator
Range Gate
I HBSS
~
I~
Tissue SpecimenS r-Glass Microscope~
Tektronix Universal Counter Timer (7B15)
ger•
Trig
Synch. I
Time Out
Fig. I. Schematic of ultrasonic pulse echo system for measurement of acoustic parameters.
ciated peak amplitude (positive going) of the received signal (V), and the micrometer reading (p) were recorded with the transducer touching (visual observation) and centered over the specimen. The micrometer reading with the tissue in place, p~, indicates the position at which its tip is in contact with the vertical metal arm bracketed to the transducer probe (Fig. 1). In this position, the ohmmeter indicated zero resistance or continuity. After the acoustic measurements had been made, the tissue was removed and the transducer was repositioned at the same micrometer reading aided by electrical contact between the transducer attached arm and the micrometer. As shown schematically in Fig. 2, this implies Pl = P2. ( U s e of electrical continuity to note the precise contact point provides a very sensitive and reliable method of maintaining the same position with and without tissue). Finally, the transducer was repositioned at micrometer reading P3 and transit time, t, temperature, T, of the HBSS (19.35-< T-<21.81°C) and the time elapsed post mortem (~--<8.25hr) recorded. These data were sufficient to compute c and ot for each tissue at each frequency. Transit time corrections attributable to the transducer matching layer at the interrogating ultrasonic frequency were applied when making velocity computations (Bhagat et al., 1977). Details of transit time correction technique are given in the paper by Papadakis (1972). 2(/93 -- P2)
Velocity in HBSS, CHBSS- ( t 3 - t2)
~ ~
\\\\\\~
~\\\\\\\\\\\~
~bols: p: Micrometer reading V:
Peak voltage of received signal
t:
Measured pulse t r a n s i t time
~m: Correction f a c t o r for transducer matching l a y e r A:
dg s e t t i n g of c a l i b r a t e d a t t e n u a t o r of UTA-3
T:
Temperature of HBSS (°C)
Fig. 2. Details of velocity and attenuation coefficient measurement scheme.
Specimen thickness, d = ( t 2 - ~'m)CHass/2 2d Velocity in tissue sample, c - (tl - ~'m) = CHBSS( h -,,) (t, - zzm) where ~'m = transit time error correction due to transducer matching layer. Attenuation coefficient, a, _ [ ( A 2 - A 0 + 2 0 log10(
2d
VJ V 0 ]
372
P. K. BHAOAT et al.
where ( A 2 - A l ) = d i f f e r e n c e in attenuator setting b e t w e e n positions 2 and 1 in db.
deviations of the m e a s u r e m e n t s . In general, for both age groups the values of c and a increased with frequency. The d e p e n d e n c e of a on f r e q u e n c y appears to be described by an equation of the f o r m a = kf" where k and n are constants. (This equation f o r m has been suggested by several authors (e.g. Wells, 1975) and was used to curve fit our data). The
RESULTS AND DISCUSSION
Figures 3 and 4 show the velocity of propagation, c, and the m e a s u r e d attenuation coefficient, a, as a function of f r e q u e n c y for skin. T h e error bars indicate the standard
1~70
1550
1530
1510
17 AGED SPECIMENS
1490
8 YOUNGSPECIMENS
-
,
I
,
J
2
,
L
4
,
i
6
,
, I
8
FREQUENCY
10
0'IHZ)
Fig. 3. Comparison of propagation velocity in aged and young skin.
t
30
20
§
10
0
-
- 17 AGED SPECINENS
- 8 yo~
I
I
2
,
l
4
,
I
G
,
sPec~.~Ns I
8
,
I
10
FREQUENCY (MHZ)
Fig. 4. Comparison of attenuation coefficient in aged and young skin.
373
Ultrasonic characterization of aging in skin tissue
aged skin tissue had significantly (student's t-test, confidence level 0.99) lower values of c and a at all frequencies than did the corresponding young tissues. The effect of tissue density, donor's physical condition and age on average velocity and attenuation coefficient in skin are depicted in Figs. 5 and 6. It should be noted that ps and Prmss are the densities of skin tissue and HBSS respectively; standard deviations are not shown for reasons of clarity and small sample size. We observed that one of the three emaciated mice had a tumor in the abdominal area and the other two had enlarged growths near the kidney. These gross physical disorders were not differentiated in terms of acoustic properties in this study. Velocity differences between young and aged tissues are significant at all frequencies with velocities in young tissues higher. The velocity of plane longitudinal waves through an infinite homogeneous medium of uniform density, p, is given by c = (E/p) °5, where E is the (adiabatic) modulus of elasticity. The analogous expression for Newtonian liquids is c = (K/p) °'~, where K is the adiabatic bulk modulus. Although not directly applicable to the small tissues examined, these equations suggest that the noted lower velocity in aged skin could result from either (a) an increase
1550
1540
"~
]530
'O" ~'~C~AT~(3 SPtC~NS) • OLDMICE
Os,O~ss (7 SPtCIMEJ~S)
• {,W~ (17 SPECIMENS) A PS'~HBSS(13 SPECIMENS)
~D
1519
1500
1490
2,25
,
i
5
7,5
,
i0
FRE(~JL'~Y (FIZZ)
Fig. 5. Effect of tissue density, d o n o r ' s physical condition and age on average propagation velocity in skin.
t D e n s i t y was not m e a s u r e d , but rather, qualitatively identified by observing w h e t h e r the s p e c i m e n floated (P < m a s s , true for m o s t aged tissue) or sank (p > PHBSS, true for all y o u n g tissues).
YOLINGMICE
35
OLD MICE
~"
(8 SPECIMENS)
EMACIAll~ (3 SPECItIENS) I~ pS'pl~3SS(7SPECIF~NS) 0 NORMAL (17 SPECIMENS) pS
30
/
/
25
;a ..9
20
5
10
2,25
5
7,5
10
FREQUENCY (M~Z)
Fig. 6. Effect of tissue density, d o n o r ' s physical condition and age on average attenuation coefficient in skin.
in density or (b) a decrease in some "resilience factor" related to E and K. However, the average density in aged skin samples were observed to be lower than in young skint, and the average density of the body is known to decrease with age (Bakerman, 1969). Therefore, it seems to suggest that the "resilience factor" in aged skin was appreciably less than that in the young tissues. The skin very obviously changes as it ages. The upper layer of dermis owes its flexible strength to its several fibrous components (connective tissues), with collagen being the most significant and abundant of these. The collagen content of skin generally increases for roughly four decades in humans, and then begins to decline. During this period of decreasing collagen mass, the ability of the collagen fibers to withstand stress decreases due to gradual lipid intercalation into the fibers and loss of some of the fiber-stabilizing covalent cross-links between the individual tropocollagen molecules making up the fibers (Bakerman, 1969; Robert and Robert, 1973). These factors are probably some of the underlying reasons for the common observation that aged skin has undergone a decrease in "elasticity" and an increase in "fragility". These factors would also be expected to (a) lower the density, due to
374
P. K. BHAGAT et al.
increased fat content, and (b) lower the "resilience factor" with age, in agreement with what was observed during or suggested by this study. According to the values tabulated by Goldman and Heuter (1956), and Chivers and Hill (1975), the velocity in fat tissue is about 1460m/sec, well below that for skin as a whole. Goss et al. (1978) in a compilation of empirical ultrasonic properties also list the velocity of sound in fat tissue as being in the range 1400-1490 m/sec. A value of 1540m/sec is reported by Rich et al. (1966) but the temperature at which the measurements were made is not given. The attenuation data given by Goldman and Heuter (1956), Gavrilov (1972), and Chivers and Hill (1975) as well as the compilations by Goss et al. (1978) for fat tissue ranges from 0.43 to about 1.0dB/cm/MHz, less than would be expected for whole skin based on attenuation in other tissues. Thus, assuming that the lower density observed for the aged skin is attributable primarily to an increased fat content, a lower velocity and attenuation in the aged tissues would be expected on this basis alone. The experimental results further support this idea in that of all the subgroups of old mice considered, both velocity and attenuation were highest in the three emaciated mice.
Table 1 summarizes the skin data available in the literature obtained from human, dog, pig and mouse (present study). It can be observed that human data (39-44 yr old) of 4.5 db/cm for 2.9 MHz compares favorably with 4.03db/cm obtained for old mice at 2.25MHz in the present study. No real difference could be discerned between diet groups with respect to attenuation and velocity measurements and the data for aged mice include 17 mice excepting the three emaciated ones. The small sample size for any diet group and the variability from animal to animal precluded the possibility of drawing conclusions about diet induced differences. SUMMARY AND CONCLUSIONS
The propagation velocity and attenuation coefficient of ultrasound were measured at room temperature, in skin tissues excised from young (2 months) and aged (27 months) Bar Harbor strain female mice, in the frequency range of 2.25-10 MHz. Our data suggests that the aged skin has significantly lower values of velocity and attenuation at all frequencies compared to that for the young tissues. The results from this pilot project suggest that the anatomical and biochemical changes recognized as tissue aging may be associated with changes in the acoustic properties of
Table 1. Attenuation and velocity of propagation of ultrasound in skin Species
T i s s u e State
Temperature
Frequency
~ (db/cm)
c (ms)
Human Human
Fresh Fresh
40 23
2.1 3.5 7.3 9.2
1498
Human Human (39-44 yr)
Refrigerated
40
0.97 1 3 5 5 0.57 0.97 1.7
Dog Dog
Fresh Refrigerated
40 40
2.9 0.97 0.57 0.97 1.7
Pig Unspecified Mouse
In vivo
Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh
(young) (old) (young) (old) (young) (old) (young) (old)
Body temperature 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5
2.9 4.8 5 I 2.25 2.25 5 5 7.5 7.5 10 10
1540 0.5 2.4 2.7 4.5 1.4 1.8 1.4 3.2 6.2 6.5
Oka (1977) Dussik and (1956)
Fritch
G o a n s et al. (1977) N a k a i m a et a1.(1976)
Oka (1977) N a k a i m a et al. (1976)
1720 3.3 5. ! 9 4.03 9.56 5.21 15.56 7.6 34.03 17.70
Source
1540 1512 1543 1511 1543 1510 1536 1515
G o a n s et al. (1977) Gavrilov (1972) Present study
Ultrasonic characterization of aging in skin tissue
these tissues. If additional studies confirm this, then this may eventually lead to the use of noninvasive ultrasonic techniques for estimating the biological age of certain tissues. REFERENCES
Hill, C. R. (1%8) The possibility of hazard in medical and industrial applications of ultrasound. Br. J. Radiol. 41, 561-569. Woodward, B., Pond, J. B. and Warwick, R. (1970) How safe is diagnostic sonar. Br. J. Radiol. 43, 719-725. Curzen, O. (1972) The safety of diagnostic ultrasound. Practioner 209, 822-827. Bakerman, Seymour (Ed) (1969) Aging Life Processes. Thomas Springfield, Illinois (1%9). Robert, L. and Robert, B. (Ed) (1973) Aging of connective tissue--skin. Proc. l l t h Int. Colloquium Dermochemistry. Aging of Skin. in Frontiers o f Matrix Biology, Vol. 1. S. Karger AG, Basel, Switzerland. Bhagat, P. K., Kadaba, M. P., Ware, R. W. and Cockerill, W. P. (1977) Acoustic parameters of freshly excised tissues of Sprague-Dawley rats. Ultrasonics 15, 179-182. Papadakis, E. P. (1972) Ultrasonic diffraction loss and phase change for broadband pulses. J. Acoust. Soc. Am. 52, 847-849. Wells, P. N. T. (1975) Absorption and dispersion of ultrasound in biological tissues. Ultrasound Med. Biol. 1,369-376. Goldman, D. E. and Heuter, T. F. (1956) Tabular data on the velocity and absorption of high frequency sound in mammalian tissues. J. Acoust. Soc. Am. 28, 35-37. See also Goldman, D. E. and Heuter, T. F. (1957) Errata: Tabular data of the velocity and absorption of high frequency sound in mammalian tissues. J. Acoust. Soc. Am. 29, 655. Chivers, R. C. and Hill, C. R. (1975) Ultrasonic attenuation in human tissue. Ultrasound Med. Biol. 2, 25-29. Goss, S. A., Johnston, R. L. and Dunn, F. (1978) Comprehensive compilation of empirical ultrasonic properties of mammalian tissues. J. Acoust. Soc. Am. 64, 423-457. Rich, C., Klinik, E., Smith, R. and Graham, B. (1%6) Measurement of bone mass from ultrasonic transmission time. Proc. Soc. Exp. Biol. Med. 123, 282-285. Gavrilov, L. R. (1972) Treatment of tissue with ultrasound. Soc. Phys. Acoust. 17, 287-301. Oka, M. (1977) Progress in studies of the potential use of medical ultrasonics. Wakayama Med. Rep. 20, 1-50. Dussik, K. T. and Fritch, D. J. (1956) Determination of sound attenuation and sound velocity in structures constituting the joints, and of the ultrasonic field distribution within the joints on living tissues and anatomical preparations, both in normal and pathological conditions. Public Health Service, N.I.H. Project A454, Progress Report. Goans, R. E., Cantrell, J. H., Jr. and Meyers, F. B. (t977) Ultrasonic pulse echo determination of thermal injury in deep dermal burns. Med. Phys. 4, 259-263. Nakaima, N., Aoyama, H. and Oka, M. J. (1976) Supplementary study on the ultrasonic absorption of human soft tissues. J. Wakayama Med. Soc. 27, 107115, (in Japanese).
375
Shah, B. G., Kirschnaro, V. G. and Draper, H. H. (1%7) The relationship of Ca and P nutrition during adult life and osteoporosis in aged mice. J. Nutr. 92, 30-42.
Acknowledgements--This research was partially supported by the Division of Medical Research Services, Veterans Administration Hospital, Lexington, and the University of Kentucky Research Foundation. We gratefully acknowlege assistance of Dr. P. Thornton who supplied the young and aged mice used in this study. APPENDIX A
Diets of aged mice A semi-purified diet, described by Shah et al. (1967) was used with the calcium (Ca) and phosphorous (P) levels varied by manipulating the cornstarch, CaCOs, and Na2NPO4 ratios. All diets were thoroughly mixed using a twin-shell blender mixer. This mixture was then combined with an equal weight of 0.1% agar-warmed solution, mixed until homogeneous, and allowed to cool in the refrigerator. Cooling resulted in a "cheese-like" consistency which the mice consumed readily. This procedure provided a means of reducing feed wastage (which was minimal), and it prevented the mice from selecting specific ingredients and ignoring others. The feed was mixed weekly and kept in the refrigerator at 4°C. Diets for the various groups were controlled as shown below. Diet on Odd Days Group Low-Low L ow -Med Low-High Control
Diet on Even Days
%Ca
%P
%Ca
%P
0.0 0.0 0.0 0.6
0.1 0.3 0.6 0.3
1.2 1.2 1.2 0.6
0.3 0.3 0.3 0.3
NOTES: 1. Food intake was regulated so that all groups had an equal dietary calcium intake. 2. Distilled water was continuously available. 3. The mice were kept on this diet regimen for 25 months.
ULTRASONIC CHARACTERIZATION OF AGING IN SKIN TISSUE P. K. BHAGATand WASSON KERRICK Wenner-Gren BiomedicalResearch Laboratory,Department of Mechanical Engineering,University of Kentucky,Lexington,KY 40506, U.S.A. and R. W. WAREt V e t e r a n s Medical Center, Le xi ngt on, K Y 40506, U.S.A.
(First received 3 April 1979; and in final form 27 February 1980)
Abstract--The propagation velocity (c) and attenuation coefficient (a) of ultrasound were measured at room temperature in skin tissues excised from twenty aged (27 months) and eight young (2 months) Bar Harbor strain female mice. Frequencies used were 2.25, 5.0, 7.5 and 10.0 MHz. Both velocity and attenuation data were obtained using a pulse-echo technique. Transit times were measured with a universal counter timer to 10 nsec resolution, and corrected for the "dead time" due to the transducer matching layer. Aged skin had significantly lower values of c and a at all frequencies than did the corresponding young tissues. This preliminary study suggests that the tissue changes which collectively constitute aging may, in some instances, be predictably associated with measurable changes in the acoustic properties of those tissues.
Key words: Tissue aging, Ultrasonic pulse echo system, Attenuation coefficient, Velocity of propagation, Frequencydependence of acoustic parameters.
INTRODUCTION
The velocity of propagation of an acoustic wave in a medium is largely dependent upon its density and bulk modulus whereas attenuation is due to viscoelastic properties, the presence of interfaces with differing acoustic impedances, inherent inhomogeneities and tissue geometry. Biological tissues undergo structural and biochemical changes under a variety of physiological and pathological conditions which can lead to changes in density, bulk modulus, distribution of scattering centers and composition within the tissues. These changes, therefore, can be reflected as variations in the measured acoustic parameters, the velocity of propagation and attenuation. Noninvasive measurement of acoustic parameters, therefore, offers the possibility of differential diagnosis of tissue state. The use of ultrasonic energy is especially attractive for this purpose since it has been shown to be nontoxic at levels normally required for diagnostic applications (Hill, 1968; Woodward et al., 1970; Curzen, 1972). tPresent Address: Veterans Medical Center, Kerrville, TX 78028, U.S.A.
Organs such as kidney and other tissues, most notably skin, undergo marked functional changes with aging (Bakerman, 1969). These changes could be brought about by such factors as changes in fat and water content and alterations in the amounts and integrity of connective tissues. These changes may affect the density and elasticity of the tissue which can be detected as variations in the velocity of propagation c (ms -~) and attenuation coefficient, a (db cm-J). The study reported here was an attempt to find a correlation between tissue age and variations in the acoustic parameters. The experimental approach was simple and straightforward and velocity and tissue attenuation were the only parameters measured. The frequencies employed ranged from 2.25 to 10 MHz, covering the frequency range of present day ultrasonic diagnostic equipment. MATERIALS
Two groups of Bar Harbor strain B7D2F1 female mice were used. One group consisted of eight young mice aged approximately 1.82.1 months (average mass -- 21.6 _+2.0 g), the other of twenty old mice aged 26.5 to 27.3
369
370
P. K. BHAGAT et al.
months (41.3_+8.6g). Mice were chosen as the experimental animals because of the ready availability of a group of naturally aged subjects, coupled with the comparative ease and inexpense of acquiring young mice of the same sex and strain. The young mice were fed Purina mouse chow ad lib with tap water during the interim ranging from 1.0 to 2.6 weeks between shipment and sacrifice. The older mice were fed special diets containing varying calcium/phosphorous ratios, with distilled water, for 25 months preceding sacrifice. The food intake of these aged mice was regulated so that all four diet groups had an equal dietary calcium intake. Appendix A gives additional information concerning the food preparation and diets of the aged mice. Acoustic measurements in skin tissue were made after folding to obtain four thicknesses (this allowed the time between echoes to exceed the three/~sec minimum gate delay of instrumentation used). The choice of skin as a test tissue despite the difficulty of working with it was made for the following reasons: (i) age-related compositional and structural changes are more pronounced in skin than in many tissues, (ii) the aging of skin has been rather extensively studied and (iii) for noninvasive work the skin is certainly the most easily accessible tissue (Bakerman, 1969; Robert and Robert, 1973). Mice were sacrificed by injection with roughly 0.5 ml of Brevital Sodium and a rectangle (1.5 x 2.0 cm or larger) of skin removed from each mouse promptly after respiration ceased. The skin was placed in Hank's Balanced Salt Solution (HBSS) after removal of the hair with one or more applications of a depilatory (Surgex Hair Remover Cream). The HBSS bathing the tissues provided an approximately isotonic environment for the tissue intercellular field. Failure to take this precaution can result in a rapid change in measured velocity as the tissue imbibes or expels water. The boundary between skin and other attached tissues can be quite vague, so special effort was made to prepare all skin samples similarly as follows: Samples were cut from the same part of each mouse's body. Muscle tissue is readily distinguishable from skin upon eye examination. Muscle and any fat suspected of not
actually being a part of the skin were pulled/peeled off the skin using forceps.
METHODS
The pulse-echo technique used for determination of propagation velocity (c) and attenuation coefficient (~) in tissues is schematically shown in Fig. 1. The tissue specimen was placed on a glass microscope slide submerged in a plexiglass tank of HBSS at ambient temperature (20.5°C). All tissues were examined using ultrasonic transducers with resonant frequencies nominally at 2.25, 5.0, 7.5 and 10.0 MHz. These immersion-type piezoelectric transducers (Aerotecb Laboratories, gamma-type pencil probes, narrow band, 20% bandwidth, 0.953cm diameter) were shock excited with a narrow pulse (200 volts peak, duration 60nsec, p r f = 4 K H z ) from an Aerotech Ultrasonic Transducer Analyzer (UTA-3)t. The UTA-3 also supplied a calibrated attenuator, receiver-amplifier, peak detector and gating networks. The input receiver sensitivity is 5 mv with a frequency bandwidth of 24.6MHz and a lower frequency cutoff at 400 KHz. The receiver gain is 40db with an output impedance of 50 ~ and the peak detector has a 5% linearity in the range 50 my-1 volt. Measurements were made with the ultrasonic beam perpendicular to the surface of the glass slide. This orientation was achieved by adjusting the stereotaxic device, which supported the transducer, to the position at which the received reflection from the glass slide was of maximum amplitude when no tissue was interposed between transducer and glass slide. It was checked at distances ranging from the thickness of the thinnest specimen to approximately three times the thickest specimen size. The signal received after one reflection from the glass slide, 2.5 mm thick, (i.e. after two passages through tissue or HBSS) was amplified, gated and the pulse transit time obtained with a Tektronix model 7B15 universal counter timer (10 nsec resolution). Distances were measured using a micrometer (resolution 5 ~m) assisted by an electricalcontinuity checking procedure as explained below. Data were obtained for the tissues of each mouse in the following manner: at each tSubsidiary of KB-Aerotech, Lewistown, Pennsyl- frequency, the values of pulse transit time (t), calibrated attenuator reading (A), the assovania.
371
Ultrasonic characterization of aging in skin tissue Aerotech TransducerAnalyzer (UTA-3) _ - I
I r
Calibrated Attenuator
Range Gate
I HBSS
~
I~
Tissue SpecimenS r-Glass Microscope~
Tektronix Universal Counter Timer (7B15)
ger•
Trig
Synch. I
Time Out
Fig. I. Schematic of ultrasonic pulse echo system for measurement of acoustic parameters.
ciated peak amplitude (positive going) of the received signal (V), and the micrometer reading (p) were recorded with the transducer touching (visual observation) and centered over the specimen. The micrometer reading with the tissue in place, p~, indicates the position at which its tip is in contact with the vertical metal arm bracketed to the transducer probe (Fig. 1). In this position, the ohmmeter indicated zero resistance or continuity. After the acoustic measurements had been made, the tissue was removed and the transducer was repositioned at the same micrometer reading aided by electrical contact between the transducer attached arm and the micrometer. As shown schematically in Fig. 2, this implies Pl = P2. ( U s e of electrical continuity to note the precise contact point provides a very sensitive and reliable method of maintaining the same position with and without tissue). Finally, the transducer was repositioned at micrometer reading P3 and transit time, t, temperature, T, of the HBSS (19.35-< T-<21.81°C) and the time elapsed post mortem (~--<8.25hr) recorded. These data were sufficient to compute c and ot for each tissue at each frequency. Transit time corrections attributable to the transducer matching layer at the interrogating ultrasonic frequency were applied when making velocity computations (Bhagat et al., 1977). Details of transit time correction technique are given in the paper by Papadakis (1972). 2(/93 -- P2)
Velocity in HBSS, CHBSS- ( t 3 - t2)
~ ~
\\\\\\~
~\\\\\\\\\\\~
~bols: p: Micrometer reading V:
Peak voltage of received signal
t:
Measured pulse t r a n s i t time
~m: Correction f a c t o r for transducer matching l a y e r A:
dg s e t t i n g of c a l i b r a t e d a t t e n u a t o r of UTA-3
T:
Temperature of HBSS (°C)
Fig. 2. Details of velocity and attenuation coefficient measurement scheme.
Specimen thickness, d = ( t 2 - ~'m)CHass/2 2d Velocity in tissue sample, c - (tl - ~'m) = CHBSS( h -,,) (t, - zzm) where ~'m = transit time error correction due to transducer matching layer. Attenuation coefficient, a, _ [ ( A 2 - A 0 + 2 0 log10(
2d
VJ V 0 ]
372
P. K. BHAOAT et al.
where ( A 2 - A l ) = d i f f e r e n c e in attenuator setting b e t w e e n positions 2 and 1 in db.
deviations of the m e a s u r e m e n t s . In general, for both age groups the values of c and a increased with frequency. The d e p e n d e n c e of a on f r e q u e n c y appears to be described by an equation of the f o r m a = kf" where k and n are constants. (This equation f o r m has been suggested by several authors (e.g. Wells, 1975) and was used to curve fit our data). The
RESULTS AND DISCUSSION
Figures 3 and 4 show the velocity of propagation, c, and the m e a s u r e d attenuation coefficient, a, as a function of f r e q u e n c y for skin. T h e error bars indicate the standard
1~70
1550
1530
1510
17 AGED SPECIMENS
1490
8 YOUNGSPECIMENS
-
,
I
,
J
2
,
L
4
,
i
6
,
, I
8
FREQUENCY
10
0'IHZ)
Fig. 3. Comparison of propagation velocity in aged and young skin.
t
30
20
§
10
0
-
- 17 AGED SPECINENS
- 8 yo~
I
I
2
,
l
4
,
I
G
,
sPec~.~Ns I
8
,
I
10
FREQUENCY (MHZ)
Fig. 4. Comparison of attenuation coefficient in aged and young skin.
373
Ultrasonic characterization of aging in skin tissue
aged skin tissue had significantly (student's t-test, confidence level 0.99) lower values of c and a at all frequencies than did the corresponding young tissues. The effect of tissue density, donor's physical condition and age on average velocity and attenuation coefficient in skin are depicted in Figs. 5 and 6. It should be noted that ps and Prmss are the densities of skin tissue and HBSS respectively; standard deviations are not shown for reasons of clarity and small sample size. We observed that one of the three emaciated mice had a tumor in the abdominal area and the other two had enlarged growths near the kidney. These gross physical disorders were not differentiated in terms of acoustic properties in this study. Velocity differences between young and aged tissues are significant at all frequencies with velocities in young tissues higher. The velocity of plane longitudinal waves through an infinite homogeneous medium of uniform density, p, is given by c = (E/p) °5, where E is the (adiabatic) modulus of elasticity. The analogous expression for Newtonian liquids is c = (K/p) °'~, where K is the adiabatic bulk modulus. Although not directly applicable to the small tissues examined, these equations suggest that the noted lower velocity in aged skin could result from either (a) an increase
1550
1540
"~
]530
'O" ~'~C~AT~(3 SPtC~NS) • OLDMICE
Os,O~ss (7 SPtCIMEJ~S)
• {,W~ (17 SPECIMENS) A PS'~HBSS(13 SPECIMENS)
~D
1519
1500
1490
2,25
,
i
5
7,5
,
i0
FRE(~JL'~Y (FIZZ)
Fig. 5. Effect of tissue density, d o n o r ' s physical condition and age on average propagation velocity in skin.
t D e n s i t y was not m e a s u r e d , but rather, qualitatively identified by observing w h e t h e r the s p e c i m e n floated (P < m a s s , true for m o s t aged tissue) or sank (p > PHBSS, true for all y o u n g tissues).
YOLINGMICE
35
OLD MICE
~"
(8 SPECIMENS)
EMACIAll~ (3 SPECItIENS) I~ pS'pl~3SS(7SPECIF~NS) 0 NORMAL (17 SPECIMENS) pS
30
/
/
25
;a ..9
20
5
10
2,25
5
7,5
10
FREQUENCY (M~Z)
Fig. 6. Effect of tissue density, d o n o r ' s physical condition and age on average attenuation coefficient in skin.
in density or (b) a decrease in some "resilience factor" related to E and K. However, the average density in aged skin samples were observed to be lower than in young skint, and the average density of the body is known to decrease with age (Bakerman, 1969). Therefore, it seems to suggest that the "resilience factor" in aged skin was appreciably less than that in the young tissues. The skin very obviously changes as it ages. The upper layer of dermis owes its flexible strength to its several fibrous components (connective tissues), with collagen being the most significant and abundant of these. The collagen content of skin generally increases for roughly four decades in humans, and then begins to decline. During this period of decreasing collagen mass, the ability of the collagen fibers to withstand stress decreases due to gradual lipid intercalation into the fibers and loss of some of the fiber-stabilizing covalent cross-links between the individual tropocollagen molecules making up the fibers (Bakerman, 1969; Robert and Robert, 1973). These factors are probably some of the underlying reasons for the common observation that aged skin has undergone a decrease in "elasticity" and an increase in "fragility". These factors would also be expected to (a) lower the density, due to
374
P. K. BHAGAT et al.
increased fat content, and (b) lower the "resilience factor" with age, in agreement with what was observed during or suggested by this study. According to the values tabulated by Goldman and Heuter (1956), and Chivers and Hill (1975), the velocity in fat tissue is about 1460m/sec, well below that for skin as a whole. Goss et al. (1978) in a compilation of empirical ultrasonic properties also list the velocity of sound in fat tissue as being in the range 1400-1490 m/sec. A value of 1540m/sec is reported by Rich et al. (1966) but the temperature at which the measurements were made is not given. The attenuation data given by Goldman and Heuter (1956), Gavrilov (1972), and Chivers and Hill (1975) as well as the compilations by Goss et al. (1978) for fat tissue ranges from 0.43 to about 1.0dB/cm/MHz, less than would be expected for whole skin based on attenuation in other tissues. Thus, assuming that the lower density observed for the aged skin is attributable primarily to an increased fat content, a lower velocity and attenuation in the aged tissues would be expected on this basis alone. The experimental results further support this idea in that of all the subgroups of old mice considered, both velocity and attenuation were highest in the three emaciated mice.
Table 1 summarizes the skin data available in the literature obtained from human, dog, pig and mouse (present study). It can be observed that human data (39-44 yr old) of 4.5 db/cm for 2.9 MHz compares favorably with 4.03db/cm obtained for old mice at 2.25MHz in the present study. No real difference could be discerned between diet groups with respect to attenuation and velocity measurements and the data for aged mice include 17 mice excepting the three emaciated ones. The small sample size for any diet group and the variability from animal to animal precluded the possibility of drawing conclusions about diet induced differences. SUMMARY AND CONCLUSIONS
The propagation velocity and attenuation coefficient of ultrasound were measured at room temperature, in skin tissues excised from young (2 months) and aged (27 months) Bar Harbor strain female mice, in the frequency range of 2.25-10 MHz. Our data suggests that the aged skin has significantly lower values of velocity and attenuation at all frequencies compared to that for the young tissues. The results from this pilot project suggest that the anatomical and biochemical changes recognized as tissue aging may be associated with changes in the acoustic properties of
Table 1. Attenuation and velocity of propagation of ultrasound in skin Species
T i s s u e State
Temperature
Frequency
~ (db/cm)
c (ms)
Human Human
Fresh Fresh
40 23
2.1 3.5 7.3 9.2
1498
Human Human (39-44 yr)
Refrigerated
40
0.97 1 3 5 5 0.57 0.97 1.7
Dog Dog
Fresh Refrigerated
40 40
2.9 0.97 0.57 0.97 1.7
Pig Unspecified Mouse
In vivo
Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh
(young) (old) (young) (old) (young) (old) (young) (old)
Body temperature 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5
2.9 4.8 5 I 2.25 2.25 5 5 7.5 7.5 10 10
1540 0.5 2.4 2.7 4.5 1.4 1.8 1.4 3.2 6.2 6.5
Oka (1977) Dussik and (1956)
Fritch
G o a n s et al. (1977) N a k a i m a et a1.(1976)
Oka (1977) N a k a i m a et al. (1976)
1720 3.3 5. ! 9 4.03 9.56 5.21 15.56 7.6 34.03 17.70
Source
1540 1512 1543 1511 1543 1510 1536 1515
G o a n s et al. (1977) Gavrilov (1972) Present study
Ultrasonic characterization of aging in skin tissue
these tissues. If additional studies confirm this, then this may eventually lead to the use of noninvasive ultrasonic techniques for estimating the biological age of certain tissues. REFERENCES
Hill, C. R. (1%8) The possibility of hazard in medical and industrial applications of ultrasound. Br. J. Radiol. 41, 561-569. Woodward, B., Pond, J. B. and Warwick, R. (1970) How safe is diagnostic sonar. Br. J. Radiol. 43, 719-725. Curzen, O. (1972) The safety of diagnostic ultrasound. Practioner 209, 822-827. Bakerman, Seymour (Ed) (1969) Aging Life Processes. Thomas Springfield, Illinois (1%9). Robert, L. and Robert, B. (Ed) (1973) Aging of connective tissue--skin. Proc. l l t h Int. Colloquium Dermochemistry. Aging of Skin. in Frontiers o f Matrix Biology, Vol. 1. S. Karger AG, Basel, Switzerland. Bhagat, P. K., Kadaba, M. P., Ware, R. W. and Cockerill, W. P. (1977) Acoustic parameters of freshly excised tissues of Sprague-Dawley rats. Ultrasonics 15, 179-182. Papadakis, E. P. (1972) Ultrasonic diffraction loss and phase change for broadband pulses. J. Acoust. Soc. Am. 52, 847-849. Wells, P. N. T. (1975) Absorption and dispersion of ultrasound in biological tissues. Ultrasound Med. Biol. 1,369-376. Goldman, D. E. and Heuter, T. F. (1956) Tabular data on the velocity and absorption of high frequency sound in mammalian tissues. J. Acoust. Soc. Am. 28, 35-37. See also Goldman, D. E. and Heuter, T. F. (1957) Errata: Tabular data of the velocity and absorption of high frequency sound in mammalian tissues. J. Acoust. Soc. Am. 29, 655. Chivers, R. C. and Hill, C. R. (1975) Ultrasonic attenuation in human tissue. Ultrasound Med. Biol. 2, 25-29. Goss, S. A., Johnston, R. L. and Dunn, F. (1978) Comprehensive compilation of empirical ultrasonic properties of mammalian tissues. J. Acoust. Soc. Am. 64, 423-457. Rich, C., Klinik, E., Smith, R. and Graham, B. (1%6) Measurement of bone mass from ultrasonic transmission time. Proc. Soc. Exp. Biol. Med. 123, 282-285. Gavrilov, L. R. (1972) Treatment of tissue with ultrasound. Soc. Phys. Acoust. 17, 287-301. Oka, M. (1977) Progress in studies of the potential use of medical ultrasonics. Wakayama Med. Rep. 20, 1-50. Dussik, K. T. and Fritch, D. J. (1956) Determination of sound attenuation and sound velocity in structures constituting the joints, and of the ultrasonic field distribution within the joints on living tissues and anatomical preparations, both in normal and pathological conditions. Public Health Service, N.I.H. Project A454, Progress Report. Goans, R. E., Cantrell, J. H., Jr. and Meyers, F. B. (t977) Ultrasonic pulse echo determination of thermal injury in deep dermal burns. Med. Phys. 4, 259-263. Nakaima, N., Aoyama, H. and Oka, M. J. (1976) Supplementary study on the ultrasonic absorption of human soft tissues. J. Wakayama Med. Soc. 27, 107115, (in Japanese).
375
Shah, B. G., Kirschnaro, V. G. and Draper, H. H. (1%7) The relationship of Ca and P nutrition during adult life and osteoporosis in aged mice. J. Nutr. 92, 30-42.
Acknowledgements--This research was partially supported by the Division of Medical Research Services, Veterans Administration Hospital, Lexington, and the University of Kentucky Research Foundation. We gratefully acknowlege assistance of Dr. P. Thornton who supplied the young and aged mice used in this study. APPENDIX A
Diets of aged mice A semi-purified diet, described by Shah et al. (1967) was used with the calcium (Ca) and phosphorous (P) levels varied by manipulating the cornstarch, CaCOs, and Na2NPO4 ratios. All diets were thoroughly mixed using a twin-shell blender mixer. This mixture was then combined with an equal weight of 0.1% agar-warmed solution, mixed until homogeneous, and allowed to cool in the refrigerator. Cooling resulted in a "cheese-like" consistency which the mice consumed readily. This procedure provided a means of reducing feed wastage (which was minimal), and it prevented the mice from selecting specific ingredients and ignoring others. The feed was mixed weekly and kept in the refrigerator at 4°C. Diets for the various groups were controlled as shown below. Diet on Odd Days Group Low-Low L ow -Med Low-High Control
Diet on Even Days
%Ca
%P
%Ca
%P
0.0 0.0 0.0 0.6
0.1 0.3 0.6 0.3
1.2 1.2 1.2 0.6
0.3 0.3 0.3 0.3
NOTES: 1. Food intake was regulated so that all groups had an equal dietary calcium intake. 2. Distilled water was continuously available. 3. The mice were kept on this diet regimen for 25 months.