Computed tomography as a method of estimating bone mineral content in horses

COMPUTED TOMOGRAPHY AS A METHOD OF ESTIMATING BONE MINERAL CONTENT IN HORSES Karen L. Waite, MS~; Brian D. Nielsen, PhD~; and Diana S. Rosenstein, DVM 2

SUMMARY With the advancement of technology, more tools and methods are being made available for the assessment of bone density in the live animal. Computed tomography (CT) is one such tool. In a CT scan, x-rays are passed through a subject and detectors record transmitted energy. The CT computer then constructs cross-sectional images from this data to represent the internal anatomy. The first objective of this study was to investigate CT as a means of estimating bone mineral content of the equine third metacarpal bone. Our hypothesis was that the cross-sectional area of the third metacarpal bone, as measured on CT images, would correlate significantly with ash weight of that section. Our second objective was to use CT to measure and compare bone density in the dorsal, palmar, lateral and medial cortices of the equine third metacarpus. We hypothesized that CT would detect differences in density by cortex. Twelve cadaver thoracic limb specimens, from horses of varying age, breed and prior use, were scanned on a CT 9800 (GE Medical Systems). Scanned sections of the limb were then skinned, dried, ether-extracted and ashed in a muffle furnace. Bone mineral content was expressed as grams of ash/cm section of bone. The image recorded by the CT was developed and assessed using an imaging photodensitometer and a corresponding software package to estimate the area of each bone section. Correlation was determined using SAS (6.12). Differences in bone density by cortex were calculated using a simple t-test. The maximum, minimum and mean estimates of third metacarpal bone cross-sectional area as determined by CT were 519, 351 and 423 mm 2, respectively, while the maximum, minimum and mean ash weights were 8.9, 4.8 and 6.4 g/cm, respectively. Estimates of bone mineral content by ash and cross-sectional area as determined by CT were significantly correlated (r = .91, P = .0001). There was no difference in CT density numbers between lateral and medial cortices, however the dorsal cortex was lower in density than the lateral (P < .05) and the palmar cortex was lower in density than all other cortices (P < .05). This data suggests that in a research setting, CT Authors' addresses: 1Departments of Animal Science and 2Large Animal

Clinical Science, Michigan State University, East Lansing, Michigan 48824-1225

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is an effective method by which to calculate cross-sectional area and, hence, estimate bone mineral content. Computed tomography was also able to detect differences in bone density by cortex. Key words: c o m p u t e d t o m o g r a p h y , bone, third metacarpal

INTRODUCTION Research has shown that there is a positive correlation between failure stress and ash content in the third metacarpal bone of the horse. ~ E1 Shorafa I and others also determined that there is a positive correlation between failure stress and cortical area in the third metacarpus of the horse, suggesting that as ash content and/or cortical area increase, so too does the bone's ability to withstand stress. Ash content may be the most definitive means by which to estimate bone mineral content, however, the terminal nature of the procedure makes it impractical in most equine research situations. As technology advances, more tools and methods are made available for the assessment of bone density in the live animal? ,3,4 Computed tomography is one such tool, and has been used with increased frequency to quantitate bone mineral density in human osteoporosis research. In addition, CT has gained wide acceptance as a veterinary diagnostic tool, used frequently to view the cranial and abdominal regions. 5 Computed tomography is an imaging modality in which x-rays are passed through a subject and detectors record transmitted energy, converting it to a quantifiable number or linear attenuation coefficient. The CT is able to report attenuation values for sectional images based on variation in tissue density of as little as 1%.6 The CT computer then constructs cross-sectional images from this data to represent the internal anatomy. Each point or pixel of the CT image has its own linear attenuation coefficient which is converted mathematically to a CT number (Houndsfield unit). The CT number represents the tissue density at a given point or pixel. Density numbers are generally expressed on a scale o f - l , 0 0 0 to 2,000 HU. Reported CT numbers for water, air and bone are 0, -1,000 and 1,000+HU, respectively. 6 There are a limited number of published studies employing CT as a means of measuring bone density in

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the dog 5,7 and horse. 8 Markel et al? employed a technique termed quantitative computed tomography which uses a reference phantom to compare known density values to that of the patient. Thompson et al., 8 used an image processing system and commercially available software to process CT images obtained from the first phalanges of a 2-year-old Thoroughbred horse. Neither of these studies consider bone mineral content as a function of cross-sectional area. Consequently, the first objective of this study was to investigate CT as a means of estimating bone mineral content of the equine third metacarpal bone. Our hypothesis was that cross-sectional area of the third metacarpal bone, as measured on CT images, would correlate significantly with the ash weight of that section. Nielsen et al. 9 reported that 2-year-old Quarter Horses in race training experienced fewer injuries when mass was greater in the lateral and medial cortices of the third metacarpal, relative to the palmar cortex as determined through radiographic photodensitometry. It is possible that these cortical changes were not only volumetric, but compositional as well. Computed tomography may provide the means by which to non-invasively identify differences in the cortical density of a given bone. Consequently, the second objective of this study was to use CT to measure and compare tissue density in the dorsal, palmar, lateral and medial cortices of the equine third metacarpus. We hypothesized that CT would detect differences in density by cortex.

MATERIALS AND METHODS Computed Tomography To test our hypothesis, 12 equine cadaver thoracic limbs from horses of varying age, breed and use were collected within 8 h postmortem, and were frozen for future analysis. Limbs were scanned using a CT 9800 (GE Medical Systems, Milwaukee, WI) (120 KV, 120 mA, 2 s scan time). An initial scout view was taken to determine the location of the nutrient foramen as a landmark. Two images were then recorded in 5-mm sections immediately prior to and at the nutrient foramen. Prior to removal of the limb from the machine, the starting and ending point of the images were scored on the limb with a knife. This 1-cm thick section of bone was then removed from the limb with a saw for determination of ash content.

Figure 1. Computed tomography scan of equine third metacarpal bone. actual bone area was determined algebraically by multiplying the 50-mm reference mark on the film by the adjusted cross-sectional bone area and dividing by the length of the reference mark on the image.

Determining Density by Cortex To determine CT density numbers for the dorsal, palmar, lateral and medial cortices, the image location was selected from an initial scout view image of each limb, at the level of the nutrient foramen. From the transverse images, a region of interest (.01 cm 2) in each cortex was randomly selected and the CT density (HU) was reported by the CT 9800 computer for that region. Density numbers for the dorsal, palmar, lateral and medial cortices were recorded and analyzed for difference.

Ash Determination Bone sections were ashed using a modified version of the method described by Meakim et al.4 Sections were skinned and second and fourth metacarpal bones were removed. Third metacarpal sections were oven-dried at 150~ for 12 h and were then weighed. Fat was removed from the bone sections by ether-extraction in a Soxhlet apparatus. Following extraction, bone sections were allowed to air dry until the ether smell was undetectable and were again weighed. Finally, bone sections were burned in a muffle furnace at 500~ for 6 h, weighed to determine grams of ash /1-cm section and weights recorded.

Estimating Cross-Sectional Area Recorded CT images were developed and scanned into a Bio-Rad GS-700 Imaging Densitometer (Hercules, CA). Multi-Analyst software (Bio-Rad, Hercules, CA) was then used to calculate cross-sectional surface area. The endosteal (EM) and periosteal (PM) cortical margins of the MclII section were traced (Figure l) and a profile including area was generated for each trace. The adjusted cross-sectional bone area was calculated by subtracting EM from PM. The

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Statistical Analysis The correlation between ash weight and cross-sectional area as determined using CT was calculated using the Correlation procedure of SAS (6.12). Regression analysis and differences between mean CT density numbers were also analyzed using SAS (6.12).

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y = 0.021x - 2.538, r2=.91

/

8

/

i

means by which researchers can compare third metacarpus cross-sectional area in groups of horses, or in a single horse over time.

J"

-

~ 7 84 9

ash

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area (ram2) Figure 2 , Equine third metacarpal ash (g/cm) and cross sectional area (mm 2) as determined by CT.

RESULTS Cross-sectional area of the MclII as determined using CT ranged from 351 to 519 mm 2, with a mean area of 423 mm 2. Ash values ranged from 4.8 to 8.9 g/cm, with a mean of 6.4 g/cm. Ash weight per cm section, and crosssectional area, as determined by CT, exhibited a linear pattern (Figure 2) and were strongly correlated (r2 = .91, P = .0001), as expected. In addition to determining the correlation between ash weight and cross-sectional area, CT density numbers (HU) in dorsal, palmar, lateral and medial cortices of the third metacarpus were generated and analyzed (Table 1). While there was no difference in density numbers between the lateral and medial cortices, the dorsal cortex was lower in density than the lateral cortex (P < .05) and the palmar cortex was lower in density than all other cortices (P < .05).

DISCUSSION

B o n e D e n s i t y by C o r t e x The current study also generated CT numbers (HU) for the dorsal, palmar, lateral and medial cortices of the equine McIII and found differences in density by cortex similar to those reported by Nielsen et al. 9 using radiographic photodensitometry in 2-yr-old Quarter Horses. The dorsal, lateral and medial cortices were found to be greater in density than the palmar cortex in the present study. Nielsen et al. 9 determined that horses not experiencing a bone-related injury had a greater ratio of lateral and medial optical density to palmar optical density than horses that experienced a bone related injury. Similarly, Larkin and Davies l~ determined that 2-yr-old Thoroughbreds in preparation for their first race did not experience shin soreness when a radiographic index of bone proportion was 3.28 or greater. This index represents the ratio of the dorsal cortical width, multiplied by the ratio of cortex to medulla in the dorso-palmar plane. No attempt was made, however, to identify the type or quality of bone. The CT method described in the present study could potentially be used not only to determine cortical width or mass, shown to be predictors of shin soreness in horses, but could also give an indication of bone quality. This could allow one to determine if a given horse was at a higher risk for bone related injury. More research in this area is needed. The technique reported for determining density numbers was adequate for small numbers of CT images or transverse sections of bone. One possible drawback to the method is that while it is possible to measure bone density by cortex, one is only able to generate data for the chosen region of interest as opposed to an area encompassing the entire cortex. Thompson et al. 8 describe a CT software program capable of analyzing bone density of a larger area. This program or one similar to it will be necessary for large scale studies involving bone density and compositional changes in bone as measured by CT.

Cross-Sectional Area The ash weight and cross-sectional area of the equine MclII measured in this study were similar in value to those reported by Meakim et al.4 and El Shorafa et al. 1, respectively. In addition, there was a strong correlation between ash weight and MclII cross-sectional area as determined by CT (r2 = .91, P = .0001). El Shorafa et al. 1 have shown that ash content and cross-sectional area are good indicators of bone strength; hence, CT may provide a non-invasive Table 1, Average CT density by Cortex Variable N Mean (HU) Lateral 12 1850 Medial 12 1832 ab Dorsal 12 1809b Palmar 12 1718 c

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se 9.85 9.97 11.34 13.01

CONCLUSIONS The next step is to attain the software necessary to anaiyze bone density by CT in a more detailed manner. This would make it possible to further study differences and changes in bone density within a given bone. Although its lack of portability makes it useful primarily in a hospital or research setting, future studies could involve investigating density changes in the growing animal or in healing equine bone. Changes in bone density accompanying changes in strain due to different levels of exercise could also be studied using CT. While in its early stages from an equine research perspective, CT is an effective and intriguing means by which

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to measure equine third metacarpus cross-sectional area and bone density by cortex, in a research setting.

REFERENCES 1. El Shorafa WM, Feaster JP and Ott EA: Horse metacarpal bone: age, ash content, cortical area and failure stress interrelationships. J. Anim. Sci. 49:979, 1979. 2. Williams SN, McDowell LR, Lawrence LA, Wilkinson NS, Ferguson PW and Warnick AC: Criteria to evaluate bone mineralization in cattle: I1. Non-invasive techniques. J. Anim. Sci. 69:1243, 1991. 3. Lawrence LA and Ott EA: The use of non-invasive techniques to predict bone mineral content and strength in the horse. Proc. 9th Equine Nutr. Exer. Phys. Symp., 1985. 4. Meakim DW, Ott EA, Asquith RL and Feaster JP: Estimation of mineral content of the equine third metacarpal by radiographic

photometry. J. Anim. Sci. 53:1019, 1981. 5. Markel MD, Morin RL, Wikenheiser MA, Robb RA and Chao EYS: Multiplanar quantitative computed tomography for bone mineral analysis in dogs. Am. J. Vet. Res. 52:1479, 1991. 6. GE Medical Systems: Introduction to CT. Milwaukee, Wl, 1995. 7. Cline JL, Czarnecki-Mauldent GL, Losonsky JL, Sipe CR and Easter, RA. Effect of increasing dietary vitamin A on bone density in adult dogs. J. Anim. Sci. 75:2980-2985, 1997. 8. Thompson KN, Cheung TK and Putnam M: Computerized bone density analysis of the proximal phalanx of the horse. Equine Pract. 18:26, 1996. 9. Nielsen BD, Potter GD, Morris EL, Odom TW, Senor DM, Reynolds JA, Smith WB and Martin MT: Changes in the third metacarpal bone and frequency of bone injuries in young Quarter Horses during race training - observations and theoretical considerations. J. Equine Vet Sci. 17:541-549, 1997. 10. Larkin NC, Davies HMS: The application of a radiographic index to the prevention of dorsal metacarpal disease in Thoroughbred racehorses. Pferdeheilkunde 12: 595-598, 1996.

SALIVA COLLECTION AND RELATIONSHIP BETWEEN LACTATE CONCENTRATION IN BLOOD AND SALIVA OF EXERCISING HORSES Arno Lindner, Dr med vet; Susanne Marx, Dr sci agr; Silke Kissenbeck, Dr oec; Hermann Mosen, Dipl Ing sci agr

INTRODUCTION

SUMMARY

The aims of this study were to examine whether sufficient volumes of saliva can be collected for lactate analysis in exercising horses and to establish the relationship between Nood and saliva lactate concenWation. Saliva was coUected for 30 seconds after exercise with a commercially available cotlon swab Salivette~ from two sites: At the height of the 3rd premolar in the maxilla (upper site) and under the tongue at the level of the frenulum linguae (lower site). Blood was taken from the jugular vein. Horses were submitted to two types of standardized exercise on a treadmill: Continuous exercise and an incremental exercise step test. The average (• standard deviation) volume of saliva sampled at the upper site was 390 • 280 Ill, and at the lower site 450 • 310 pl. The lactate concentration in saliva after continuous exercise sampled at the upper site was 2.21 • 1.97 mmol/1, and at the lower site it was 3.76 • 2.31 mmol/l. There was no significant correlation between lactate concenwation in blood and upper site saliva, and blood and lower site saliva after continuous exercise and during incremental exercise step test. Authors' address: ArbeitsgruppePferd, c/o Im Eichholz 10, 53127 Bonn, Germany

Acknowledgements: The treadmill and the horses were kindly provided by Haflinger Gest0t Kieffer. This work was possible through the financial support of Verein zur FSrderung der Forschung im Pferdesport, Hoeveler Spezialfutterwerke,ScienceConsult and Sarstedt.

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Blood lactate concentration is frequently used in horses as a parameter to evaluate performance, l Blood collection is invasive, and often trainers and owners of horses have reservations against this method. Saliva can be collected non-invasively in horses at rest.z 3, 4, 5 However, it is not known whether in exercising horses sufficient volume of saliva can be collected to run analytical prt~xlures. Saliva has been analyzed in humans for a long time because its composition resembles that of blood.6 It is generally used as a subsWate for the diagnosis of hormonal imbalances, neurological diseases, drug abuse, and for adapting medication levels.6,7 Also, it has been demonsWated in the horse that there is a good correlation between saliva and blood cortisol concenlration 6 and that saliva can be used for the detection of drugs. 8 However, data on lactate concentration in saliva and on the relationship to blood lactate concentration has not been published. The aims of this study were: 1. To examine whether sufficient volumes of saliva can be collected for lactate analysis in exercising horses; 2. To determine the concenlrafion oflac~e in saliva of exercising horses; 3. To establish the relationship between blood and saliva lactate concentration. MATERIAL AND METHODS

Ten 2-year-old horses (six Haflingers and four Standardbreds, body weight 415 • 22 kg; mean + standard deviation) were used. All exercise workouts and standardized exercise step tests (SET) were done on a treadmill with a 17% incline. Horses were submitted to two JOURNAL OF EQUINE VETERINARY SCIENCE