Materials and Methods

The protocols for these studies were approved by the Institutional Animal Care and Use Committee of Tufts University and by the Radiation Safety and Health Hazards Committee in accordance with state and federal regulations for the use of radiopharmaceuticals.

The study was performed in 2 phases. In phase 1, the safety and blood-tissue clearance of ^99mTc-EBl in horses were determined and a ^99mTc-EBl imaging protocol was established. In phase 2, the use of ^99mTc-EBl for detection of experimentally induced inflammation was evaluated.

Animals used in phase 1—Six adult female horses (3 Thoroughbreds and 3 Standardbreds) that were aged 5 to 15 years (mean age, 11.5 years) and weighed 395 to 608 kg (mean weight, 504.9 kg) were used. All horses were determined to be healthy prior to ^99mTc-EBl administration on the basis of physical examination findings and results of a CBC and serum biochemical analyses.

^99mTc-EBl preparation—A fresh solution of EB1 (10 mg/mL) was prepared in sterile 1M sodium acetate solution (pH, 6.0). One milligram (0.1 mL) of EB1 was added to 1.48 to 2.2 GBq (40 to 60 mCi) of ^99mTc-pertechnetate. This was immediately followed by the addition of 1.5 to 4 μL of a fresh solution of stannous chloride (prepared at a concentration of 4 mg/mL with 0.01M HCl containing 1 mg of ascorbate/mL). After thorough mixing, the preparation was left at room temperature (approx 24°C) for 30 minutes. The labeling efficiency and radiochemical purity were determined via ITLC^d with acetone^e and via paper chromatography^f with saline (0.9% NaCl) solution, respectively, by use of 1 to 2 μL of the undiluted sample for each procedure. On completion, chromatography strips were cut in half and activities in the upper and lower portions were determined in a dose calibrator. A radiochemical purity of > 90%, as indicated by both techniques, was required for administration. Sterile saline (2.2 to 2.4 mL) solution was added to the ^99mTc-EBl preparation; the entire sample was drawn into a 3- or 5-mL syringe, and the activity was measured by use of a dose calibrator. Following administration, the activity remaining in the syringe and IV catheter was counted in the dose calibrator to allow determination of the actual activity administered.

Imaging equipment—Scintigraphy was performed by use of a large field-of-view planar scintillation camera⁸ that was equipped with a high-resolution collima-tor. Postacquisition nuclear medicine software^h was used for image display and analysis.

Phase 1 procedures—Each horse was allowed to acclimate to an indoor environment for 4 hours prior to ^99mTc-EBl injection. Forced exercise was not performed and blankets and limb wraps were not applied prior to scanning. A physical examination, a CBC, and serum biochemical analyses were performed the day before (day 0) and 1 and 5 days after injection with ^99mTc-EB1. To reduce movement during image acquisition, each horse was sedated with detomidine hydrochlorideⁱ (0.005 to 0.01 mg/kg, IV, as needed). Intravenous catheters^j (16 gauge; 14 cm) were placed in both jugular veins in an aseptic manner.

Each horse received an empirical dose of 1.1 GBq (30 mCi) of ^99mTc-EBl via the right jugular vein catheter. After each injection of ^99mTc-EBl, 10 mL of saline solution containing heparin was used to flush the catheter. The total dose of ^99mTc-EBl administered was determined by subtracting the residual activity of counts in the syringe and catheter (assessed immediately after injection) from the activity of counts in the syringe of ^99mTc-EBl preparation prior to injection. Blood samples (2 mL) were collected from the left jugular vein catheter at 1, 3, 6, 10, 15, 30, 60, 120, and 240 minutes and 24 hours after radiopharmaceutical injection and placed into weighed tubes. The percentage of the injected dose per milliliter of blood was calculated over time to determine the clearance of ^99mTc-EBl from blood. At the same time that blood samples were collected, urine samples (5 mL) were collected by use of a Foley^k urinary catheter from 2 of the 6 horses.

The gamma camera was first positioned dorsally in the region of the kidneys and urinary bladder. Dynamic image acquisition (1 frame/s) was performed for 5 minutes (total of 300 frames) immediately following ^99mTc-EB1 injection. Dynamic images were stored on a 128 × 128-pixel matrix until analysis. Next, sequential 1-min-ute static images were obtained of the left carpal region (lateral view) every 5 minutes for the first 30 minutes after injection with ^99mTc-EBl. One-minute static images of the left side of the body were then acquired, beginning with lateral views of the metacarpal region, sinuses, thyroid gland, thorax, abdomen, pelvis, tarsal and thoracolumbar regions, followed by dorsal views of the sacroiliac region. The imaging sequence was repeated in the same order on all horses at 1, 2, 4, and 24 hours after injection. All static images were acquired by use of a 512 × 512-pixel matrix and stored on a hard drive until analysis.

Phase 1 images were subjectively evaluated by the authors to determine an optimal imaging protocol for phase 2. Images that were motion blurred were eliminated, and images in which anatomic features were clearly visible were further evaluated. Assessments included the presence or absence of the radiopharmaceutical as well as the intensity and homogeneity of uptake within background tissues. All adequate images obtained at various times after injection with ^99mTc-EB1 during phase 1 were then examined to determine the optimal imaging window. To monitor for adverse effects, a physical examination, CBC, and serum biochemical analyses were repeated for each horse on days 1 and 5 after injection with ^99mTc-EBl.

Animals used in phase 2—Two horses from phase 1 were randomly selected to be used in phase 2. A recovery period of at least 1 month was allowed to elapse prior to the onset of phase 2. A physical examination was performed daily on days 0 through 3, and a CBC and serum biochemical analyses were performed on days 0 and 2 after injection with ^99mTc-EBl.

Phase 2 procedures—An IV catheter^ (16 gauge; 14 cm) was placed in the left jugular vein by use of aseptic technique to allow for radiopharmaceutical injection. Each horse was sedated with detomidine hy-drochloride¹ (0.005 to 0.01 mg/kg, IV, as needed) to reduce movement during image acquisition. The ^99mTc-EB1 preparation procedure performed in phase 1 was followed in phase 2; however, the dose of ^99mTc-EBl was increased to 2.2 GBq (60 mCi) to improve image quality. In addition, phase 2 images were acquired on the basis of count density instead of time to allow direct comparison with the soft tissue-phase images obtained after administration of the bone agent ""Tc-HDP¹ as well as to improve image quality

Imaging sequence—The same imaging protocol (positioning and acquisition) was used in all acquisitions on days 1 and 2 for both ^99mTc-EBl and ^99mTc-HDP treatments. A perfusion study was performed involving a dynamic image acquisition (300 frames at 1 frame/s) of the right and left proximal metacarpal regions (dorsal view) during and immediately following either ^99mTc-EB1 or ^99mTc-HDP injection. Next, a dorsal static image of the same area was acquired for 300,000 counts. Lateral static images of the right proximal metacarpal region, followed by the left proximal metacarpal region, right neck region, and left neck region, were then acquired for 150,000 counts each.

For each horse, a baseline ^99mTc-EBl scan was performed on day 1. As described, 2.2 GBq (60 mCi) of ^99mTc-EBl was administered via the left jugular vein catheter, and the imaging sequence was performed immediately after injection and again 2 hours later.

On day 2, experimentally induced foci of inflammation were created via injection of 2% mepivacaine hydrochloride^1,11 (200 mg [10 mL], IM, once) into the musculature of the right side of the neck, approximately 15 cm dorsal to the transverse process of vertebral body C4. A second mepivacaine injection (10 mL) was administered in the right forelimb in the perineural region of the lateral palmar nerve, axial to the head of the fourth metacarpal bone (ie, consistent with the location of a high palmar nerve block). Cobalt Co 57 point markers were placed adjacent to the mepivacaine injection sites in the neck region to help identify the areas of injection during imaging.

Six hours after mepivacaine injection on day 2, ^99mTc-EBl imaging was performed as described; these images were designated as early images. Eight hours after mepivacaine injection, additional images (designated as late images) were acquired just prior to bone agent injection. Eight and a half hours after mepivacaine injection (ie, 2.5 hours after injection with ^99mTc-EBl), a 3-phase bone scan was performed involving 7.4 GBq (200 mCi) of ^99mTc-HDP Beginning immediately after ^99mTc-HDP injection, vascular and soft tissue-phase images were acquired in the same sequence as described. Two hours after injection with ^99mTc-HDR static bone-phase images of the same areas were obtained.

Phase 2 images were subjectively assessed by 2 investigators (LGK and MS) for areas of abnormal radiopharmaceutical uptake (presence or absence), intensity (mild, moderate, or intense), extension (focal or diffuse), and definition (well-defined or ill-defined). Quantitative analysis was performed with software that allowed ROIs to be manually drawn around the mepivacaine injection sites. A background ROI of identical size was drawn at a location several centimeters from the lesion in similar and apparently normal tissue. The activity within each ROI was expressed as total counts. Target (lesion)-to-background ratios were calculated for each ROI, and the ratio of ^99mTc-HDP uptake to ^99mTc-EBl uptake on day 1 (baseline) and day 2 (after mepivacaine administration) was compared.

Following radiopharmaceutical injection and scintigraphic procedures, all horses remained under quarantine for 24 hours. Horses were released from quarantine once radioactivity levels measured at skin contact were < 2.0 mR/h.

Results

Phase 1—All horses tolerated the scanning procedures well with no observable physiologic alterations associated with ^99mTc-EBl injections. All physical examination findings were considered normal, and results of CBCs and serum biochemical analyses were within reference limits at all time points on days 0, 1, and 5. Labeling efficiency achieved with ITLC and acetone ranged from 90.1% to 98.2% (mean ± SD, 96.1 ± 3.08%). Radiochemical purity achieved with paper chromatography and saline solution ranged from 95.6% to 98.7% (mean, 97.6 ± 1.18%).

Assessment of radioactivity of blood samples collected from all 6 horses revealed that ^99mTc-EBl was rapidly cleared from blood with an α phase (rapid elimination phase) of 2.2 minutes and a β phase (slow elimination phase) of 58 minutes. Assessment of radioactivity in urine samples collected from 2 horses confirmed rapid accumulation of activity within the kidneys and urinary bladder; the maximum activity in urine was evident at 30 minutes after injection.

Dynamic image acquisition performed during the first 5 minutes after injection to assess perfusion of the kidneys and urinary bladder revealed high immediate accumulation of well-defined activity in these organs. The sequential 1-minute static images of the left carpal region (lateral views) that were acquired during the first 5 to 30 minutes after injection with ^99mTc-EBl indicated that immediate uptake was distributed throughout the soft tissues (Figure 1). Among all 6 horses, mean number of counts in the carpal region increased steadily to 20,808 counts at 10 minutes and peaked at 21,056 counts at 30 minutes; activity remained high at 1 hour (19,597 counts) and then gradually decreased (16,280 counts at 2 hours and 10,542 counts at 4 hours). The value at 4 hours after injection was approximately half that detected at 10 minutes.

Sequential 1-minute static scmtigraphic images (A-F) of the left carpal region (lateral view) of a horse obtained at 5-minute intervals after IV injection with ^99mTc-EB1 (1.1 GBq [30 mCi]; phase 1). Notice that uptake of radiopharmaceutical at the 5-minute time point is comparable to that at the 30-minute time point, which enables immediate image acquisition. In each panel, dorsal is to the left; the arrow ndicates the area of the accessory carpal bone.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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Sequential 1-minute static scmtigraphic images (A-F) of the left carpal region (lateral view) of a horse obtained at 5-minute intervals after IV injection with ^99mTc-EB1 (1.1 GBq [30 mCi]; phase 1). Notice that uptake of radiopharmaceutical at the 5-minute time point is comparable to that at the 30-minute time point, which enables immediate image acquisition. In each panel, dorsal is to the left; the arrow ndicates the area of the accessory carpal bone.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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Sequential 1-minute static scmtigraphic images (A-F) of the left carpal region (lateral view) of a horse obtained at 5-minute intervals after IV injection with ^99mTc-EB1 (1.1 GBq [30 mCi]; phase 1). Notice that uptake of radiopharmaceutical at the 5-minute time point is comparable to that at the 30-minute time point, which enables immediate image acquisition. In each panel, dorsal is to the left; the arrow ndicates the area of the accessory carpal bone.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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In the 1-minute static images of the carpal and metacarpal regions; sinuses; thyroid gland; thorax; abdomen; pelvis; and tarsal, thoracolumbar, and sacroiliac regions obtained 30 to 60 minutes after injection with ^99mTc-EBl, homogeneous distribution of the agent within the soft tissues was evident. Uptake within these tissues was high at 1 hour and remained uniform at 2 and 4 hours after radiopharmaceutical injection; there was no apparent uptake in bone at any time point. Radioactivity was minimally detectable in these regions at 24 hours after injection with ^99mTc-EBl (Figure 2). Despite uniform uptake in soft tissues, overall image quality was subjectively judged as lair with too few counts present to provide detailed images. In addition mild persistence of the injected radiopharmaceutical in the vasculature was occasionally detected during the first hour after injection.

One-minute static scmtigraphic images of the left tarsal region (lateral view) of a horse obtained at 1, 2, 4, and 24 hours after IV injection with ^99mTc-EB1 (1.1 GBq; phase 1). In each panel, dorsal is to the left; the arrow indicates the area of the calcanean tuberosity.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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One-minute static scmtigraphic images of the left tarsal region (lateral view) of a horse obtained at 1, 2, 4, and 24 hours after IV injection with ^99mTc-EB1 (1.1 GBq; phase 1). In each panel, dorsal is to the left; the arrow indicates the area of the calcanean tuberosity.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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One-minute static scmtigraphic images of the left tarsal region (lateral view) of a horse obtained at 1, 2, 4, and 24 hours after IV injection with ^99mTc-EB1 (1.1 GBq; phase 1). In each panel, dorsal is to the left; the arrow indicates the area of the calcanean tuberosity.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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Phase 2—For either horse in phase 2, no abnormalities were detected via physical examination, CBC, or serum biochemical analyses following IV administration of the 2.2-GBq dose of ^99mTc-EBl on day 1 or 2. At this higher specific activity (compared with the dose used in phase 1), labeling efficiency achieved with ITLC and acetone ranged from 92% to 98% (mean ± SD, 96.25 ± 2.49%). Radiochemical purity achieved with paper chromatography and saline solution ranged from 94% to 98% (mean ± SD, 95.75 ± 1.48%).

Baseline ^99mTc-EBl scans performed on day 1 were compared with images obtained during phase 1. In all views acquired on a count basis (150,000 counts/limb) following administration of a 2.2-GBq dose, count densities were increased, compared with findings in phase 1 images that were acquired on a time basis (1-minute static images) following administration of a 1.1-GBq dose (Figure 3). The overall quality ol phase 2 images was lmproved, compared with phase 1 images. Photopenic areas of bone were easily identifiable, particularly in the distal limb (Figure 4).

—Lateral scintigraphic images of the left metacarpal region of a horse obtained after IV injection of 1.1 GBq (30 mCi) of ^99mTc-EB1 (A; 1-minute static image; phase 1) and after IV injection of 2.2 GBq (60 mCi) of ^99mTc-EB1 (B; image acquired for 150,000 counts; phase 2). Overall image quality was improved by use of the phase 2 protocol. Notice the well-defined photopenic areas associated with bone structures (B). In each panel, dorsal is to the left; the arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic images of the left metacarpal region of a horse obtained after IV injection of 1.1 GBq (30 mCi) of ^99mTc-EB1 (A; 1-minute static image; phase 1) and after IV injection of 2.2 GBq (60 mCi) of ^99mTc-EB1 (B; image acquired for 150,000 counts; phase 2). Overall image quality was improved by use of the phase 2 protocol. Notice the well-defined photopenic areas associated with bone structures (B). In each panel, dorsal is to the left; the arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic images of the left metacarpal region of a horse obtained after IV injection of 1.1 GBq (30 mCi) of ^99mTc-EB1 (A; 1-minute static image; phase 1) and after IV injection of 2.2 GBq (60 mCi) of ^99mTc-EB1 (B; image acquired for 150,000 counts; phase 2). Overall image quality was improved by use of the phase 2 protocol. Notice the well-defined photopenic areas associated with bone structures (B). In each panel, dorsal is to the left; the arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic image of the distal portion of a normal forelimb of a horse obtained after IV injection of 2.2 GBq of ^99mTc-EB1 (phase 2). The arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic image of the distal portion of a normal forelimb of a horse obtained after IV injection of 2.2 GBq of ^99mTc-EB1 (phase 2). The arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic image of the distal portion of a normal forelimb of a horse obtained after IV injection of 2.2 GBq of ^99mTc-EB1 (phase 2). The arrow indicates the area of the proximal sesamoid bones.

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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In the early and late images obtained by use of ^99mTc-EB1 at 6 and 8 hours after injections of mepivacaine on day 2, the mepivacaine injection sites in both the neck and proximal metacarpal regions were associated with areas of well-defined radiopharmaceutical uptake (mild to moderate intensity; Figure 5). Quantitative analysis of the ROIs obtained in the areas of the mepivacaine injection sites of both horses had increased uptake of both ^99mTc-EBl and ^99mTc-HDP, compared with background ROIs in similar tissue and with ROIs of the respective regions in baseline images obtained by use of ^99mTc-EBl on day 1. For the 2 horses, the mean number of background-corrected counts within the ROIs in the neck region in the early and late ^99mTc-EBl-derived images and ^99mTc-HDP-derived soft tissue scans were similar (Table 1); however, the number of counts obtained in the ROI during the bone phase of the ^99mTc-HDP procedures were considerably higher. Counts (background corrected) in the proximal metacarpal region followed a similar pattern in the early and late ^99mTc-EB 1-derived images and ^99mTc-HDP-derived soft tissue images. However, during the bone phase of the ^99mTc-HDP procedure, the mean number of counts began to decrease. Mean T: B ratios were also similar for the early and late ^99mTc-EB 1-derived images and ^99mTc-HDP-derived soft tissue images for both the neck and proximal metacarpal regions, whereas a considerably higher T:B ratio was associated with the bone phase of the ^99mTc-HDP procedure in the neck region only.

—Lateral scintigraphic images of the right carpal-proximal metacarpal region (A-C) and right-sided neck region (D-F) obtained after IV injection of 2.2 GBq of ^99mTc-EB1 in a horse without (day 1; baseline) or with (day 2) experimentally nduced soft tissue inflammation of the neck and forelimb (phase 2); on day 2, 7.4 GBq (200 mCi) of ^99mTc-HDP was subsequently administered IV Inflammation was nduced on day 2 via injection of mepivacaine (200 mg [10 mL]) into the musculature of the right side of the neck (approx 15 cm dorsal to the transverse process of vertebral body C4) and in the perineural region of the lateral palmar nerve, axial to the head of the fourth metacarpal bone (ie, consistent with the location of a high palmar nerve block). A—Day 1 baseline image of the forelimb region after administration of ^99mTc-EB1. B—Day 2 image of the forelimb region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. C—Day 2 image of the forelimb region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). In panels B and C, notice the areas of increased radio-pharmaceutical uptake in the location of the forelimb mepivacaine injection site (arrow). D—Day 1 baseline image of the neck region after administration of ^99mTc-EB1. E—Day 2 image of the neck region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. Two cobalt Co 57 markers were placed dorsal to the mepivacaine injection site (area of increased radiopharmaceutical uptake [arrow]) to denote injection location. Focal intense areas of uptake on the ventral aspect of the neck represent activity within the thyroid gland (arrowhead) and residua radioactivity remaining in the IV catheter (chevron). F—Day 2 image of the neck region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). Increased radiopharmaceutical uptake remains at the mepivacaine injection site (arrow) and within the IV catheter (chevron).

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic images of the right carpal-proximal metacarpal region (A-C) and right-sided neck region (D-F) obtained after IV injection of 2.2 GBq of ^99mTc-EB1 in a horse without (day 1; baseline) or with (day 2) experimentally nduced soft tissue inflammation of the neck and forelimb (phase 2); on day 2, 7.4 GBq (200 mCi) of ^99mTc-HDP was subsequently administered IV Inflammation was nduced on day 2 via injection of mepivacaine (200 mg [10 mL]) into the musculature of the right side of the neck (approx 15 cm dorsal to the transverse process of vertebral body C4) and in the perineural region of the lateral palmar nerve, axial to the head of the fourth metacarpal bone (ie, consistent with the location of a high palmar nerve block). A—Day 1 baseline image of the forelimb region after administration of ^99mTc-EB1. B—Day 2 image of the forelimb region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. C—Day 2 image of the forelimb region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). In panels B and C, notice the areas of increased radio-pharmaceutical uptake in the location of the forelimb mepivacaine injection site (arrow). D—Day 1 baseline image of the neck region after administration of ^99mTc-EB1. E—Day 2 image of the neck region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. Two cobalt Co 57 markers were placed dorsal to the mepivacaine injection site (area of increased radiopharmaceutical uptake [arrow]) to denote injection location. Focal intense areas of uptake on the ventral aspect of the neck represent activity within the thyroid gland (arrowhead) and residua radioactivity remaining in the IV catheter (chevron). F—Day 2 image of the neck region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). Increased radiopharmaceutical uptake remains at the mepivacaine injection site (arrow) and within the IV catheter (chevron).

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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—Lateral scintigraphic images of the right carpal-proximal metacarpal region (A-C) and right-sided neck region (D-F) obtained after IV injection of 2.2 GBq of ^99mTc-EB1 in a horse without (day 1; baseline) or with (day 2) experimentally nduced soft tissue inflammation of the neck and forelimb (phase 2); on day 2, 7.4 GBq (200 mCi) of ^99mTc-HDP was subsequently administered IV Inflammation was nduced on day 2 via injection of mepivacaine (200 mg [10 mL]) into the musculature of the right side of the neck (approx 15 cm dorsal to the transverse process of vertebral body C4) and in the perineural region of the lateral palmar nerve, axial to the head of the fourth metacarpal bone (ie, consistent with the location of a high palmar nerve block). A—Day 1 baseline image of the forelimb region after administration of ^99mTc-EB1. B—Day 2 image of the forelimb region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. C—Day 2 image of the forelimb region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). In panels B and C, notice the areas of increased radio-pharmaceutical uptake in the location of the forelimb mepivacaine injection site (arrow). D—Day 1 baseline image of the neck region after administration of ^99mTc-EB1. E—Day 2 image of the neck region obtained by use of ^99mTc-EB1 6 hours after mepivacaine injection. Two cobalt Co 57 markers were placed dorsal to the mepivacaine injection site (area of increased radiopharmaceutical uptake [arrow]) to denote injection location. Focal intense areas of uptake on the ventral aspect of the neck represent activity within the thyroid gland (arrowhead) and residua radioactivity remaining in the IV catheter (chevron). F—Day 2 image of the neck region obtained by use of ^99mTc-HDP 8.5 hours after mepivacaine injection (2.5 hours after ^99mTc-EB1 injection). Increased radiopharmaceutical uptake remains at the mepivacaine injection site (arrow) and within the IV catheter (chevron).

Citation: American Journal of Veterinary Research 69, 5; 10.2460/ajvr.69.5.639

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In addition, compared with baseline ^99mTc-EBl findings (day 1), there was a 6.6-fold increase in the number of counts (733 vs 110) at the mepivacaine injection site in the neck at 6 hours (early images). At 8 hours after mepivacaine injection (late images), an 8-fold increase in uptake (number of counts, 889 vs 110) over the baseline ROI was evident. In the proximal metacarpal region, a 9.2-fold increase in the number of counts (2,523 vs 274) was detected in the early ^99mTc-EBl ROIs obtained on day 2, compared with baseline value on day 1. Late images (8 hours after mepivacaine injection) revealed an 8.6-fold increase in number counts (2,378 vs 274) in the proximal metacarpal region, compared with baseline value on day 1.

Discussion

In the horses of the present study, no observable abnormalities developed subsequent to administration of ^99mTc-EBl at a dose of 1.1 or 2.2 GBq. These data are in accordance with findings of other studies^11–15 in which ^99mTc-EBl was administered to mice and humans with no resultant adverse effects. Although the number of animals used in our investigation was small, the lack of adverse effects detected in the present study and in studies involving other species leads the authors to believe that ^99mTc-EBl can be used safely in the general horse population.

Acceptable labeling efficiencies of > 90% were achieved in phases 1 and 2 of the present study The importance of performing quality control with ITLC is to detect the presence of free pertechnetate within the sample because unbound pertechnetate can be taken up by tissues such as the stomach, thyroid and salivary glands, and choroid plexus,^17,18 which may affect interpretation of images obtained near these areas. In 2 horses, the labeling efficiencies with ITLC (90.1% and 92%) were near the lower limits of the acceptable range. The exact cause of this is not known; however, the presence of 8% to 10% free ^99mTc-pertechnetate within the ^99mTc-EB1 injectate did not decrease subjective assessment of image quality.

In phase 1, the blood clearance of ^99mTc-EBl was rapid (α phase, 2.2 minutes; β phase, 58 minutes). Rapid clearance of ^99mTc-EBl from blood and diffusion into the extracellular fluid compartment is a desirable property for a soft tissue-imaging agent, thereby allowing immediate image acquisition. Although most of the ^99mTc-EBl was rapidly cleared from the vasculature to the kidneys and urinary bladder, mild persistence of the radiopharmaceutical within the vasculature was occasionally detected. This may have been a result of circulatory factors associated with individual horses. A similar finding of vascular persistence has been associated with other ^99mTc-based pharmaceuticals such as ^99mTc-HDP during its soft tissue phase.

In the horses of the present study, urine accumulation of ^99mTc-EBl was rapid; peak accumulation was detected within 30 minutes after injection. Urine collection was performed on only 2 horses because of the inherent risks of collecting and handling radioactive urine. In addition, urine leakage around the catheter made it difficult to quantify the exact amount of ^99mTc-EBl activity in the urine. The dynamic images of the kidneys and urinary bladder acquired during the first 5 minutes after injection with ^99mTc-EBl also indicated high immediate accumulation of the radiopharmaceutical in these tissues. These findings were anticipated because of the high blood flow to the kidneys and the use of a ^99m Tc-based radiopharmaceutical.¹⁹

The anatomic areas selected for examination in phase 1 were chosen on the basis of the potential clinical relevance of lesions (eg, limbs, pelvis, sinus, and thoracolumbar and sacroiliac regions) or accumulation (thyroid gland) or excretion (salivary glands, stomach, kidneys, and urinary bladder) of ^99mTc-based compounds at those sites.^17,18

Evaluation of phase 1 images revealed that accumulation of ^99mTc-EBl within soft tissues in the study horses was immediate and persisted through the 4-hour postinjection period. Sequential images of the carpal region indicated that the greatest number of counts was present from 10 minutes to 1 hour after injection. Adequate image quality with clear depiction of the anatomic features was maintained at 2 hours, at which time there was only an 18% decrease in counts, compared with 1-hour image findings. By 4 hours, only 54% of the counts present at 1 hour remained, and images were of correspondingly poorer quality This finding suggests that optimal image quality is achieved within the first hour after radiopharmaceutical injection and that adequate image quality is achieved for as long as 2 hours after injection. Image acquisition can be performed at 4 hours; however, a longer acquisition time is likely to be required to obtain images of diagnostic quality. At time points beyond 4 hours, image quality was poor because of the long acquisition time required to obtain a suitable number of counts. The exact mechanism by which ^99mTc-EBl persists in soft tissue is not known. Uptake within soft tissues of the limb, such as the carpal and tarsal regions and the distal portion of the limb, was anatomically identifiable because of the photopenic areas of bone in the images. In contrast, it was much more difficult to identify anatomic landmarks in images of other portions of the body because photopenic areas of bone were obscured by a relatively greater amount of overlying soft tissue. If a body area other than a limb is the site of interest, the use of radioactive surface markers may be helpful in identifying a site of inflammation.

For phase 2 of the present study, mepivacaine was selected as the source of experimentally induced inflammation because local anesthetic agents are known to cause inflammation at the site of IM or perineural injection that can be detected during soft tissue scin-tigraphy^20–22 Results of our study indicated that inflammation induced via local anesthetic injections at 2 locations within the body (neck and proximal meta-carpal region) could be detected by use of ^99mTc-EBl. This finding was supported by subjective assessment of images and quantitative descriptive analysis of background-corrected counts and T:B ratios obtained from ROIs. The small number of horses in phase 2 precluded statistical analysis of the count data. Accumulation of activity at the mepivacaine injection sites persisted throughout the 2-hour imaging period following ^99mTc-EB1 injection. The amount of ^99mTc-EBl uptake was similar in both the early (immediate) and late (2-hour) images. These findings may differ in clinical settings depending on the location of inflammation (abdomen or thorax vs limb) as well as the duration (acute vs chronic) and etiology (inflammation vs infection) of inflammation present and size of the lesion. There was no apparent difference in the number of counts or T: B ratios between the 2-hour (late) ^99mTc-EB 1-derived images (performed immediately prior to ^99mTc-HDP injection) and the ^99mTc-HDP-derived soft tissue images. The exact cause for this finding remains undetermined; however, it is possible that uptake of ^99mTc-HDP in the lesion was negligible. Inclusion of a greater number of horses in the study might have allowed a difference to be detected.

In contrast, when viewing the bone phase images following ^99mTc-HDP injection, additional accumulation of radioactivity was evident because both the number of counts and T:B ratios were considerably increased, compared with values for the late ^99mTc-EB 1-derived images and ^99mTc-HDP-derived soft tissue images. This finding was consistent in the neck region of both horses used in phase 2, but was not evident in the proximal metacarpal region of either horse. It has been theorized that altered ion permeability subsequent to IM injection with mepivacaine results in increased accumulation of radiopharmaceutical within muscle tissue and cells.^20,23 Uptake and retention of the reduced technetium-diphosphonate complex are also functions of the calcium content in tissue.^24,25 On the basis of these principles, it has also been proposed that the release of calcium from damaged muscle or exposure of calcium binding sites on proteins within damaged muscle may be a contributing factor; however, the exact mechanism remains unknown.²⁶ It is possible that delayed imaging with ^99mTc-EBl, performed at 10.5 hours after injection with mepivacaine (at the time when ^99mTc-HDP-derived bone phase imaging was performed in the present study), would also reveal continued uptake of ^99mTc-EB1 within the lesion.

Overall image quality and contrast tissue resolution between bone and soft tissues were substantially improved in phase 2 by increasing the dose of ^99mTc-EBl to 2.2 GBq (60 mCi) and the count density to 150,000 counts/frame from the 1.1-GBq (30-mCi) dose and 1-minute static acquisition used in phase 1. Also, the 2.2-GBq dose of ^99mTc-EBl was a sufficiently low radiation dose to horses and personnel that a bone scan could be conveniently performed on the same day as a ^99mTc-EB1 scan. Doses higher than 2.2 GBq (60 mCi) may preclude the option of performing a bone scan on the same day.

In the present study, there were several limitations, including the small number of horses used in both phases 1 and 2, which limited our ability to complete statistical analyses. Furthermore, during phase 2, a washout period of at least 24 hours between ^99mTc-EBl and ^99mTc-HDP imaging procedures would have been ideal. As the phase 1 results of our study indicated, imaging procedures can be performed at 4 hours following ^99mTc-EBl injection. In phase 2, images of the ^99mTc-HDP-derived soft tissue phase were obtained approximately 2.5 hours after ^99mTc-EBl administration. Therefore, residual ^99mTc-EBl activity may have impacted the ^99mTc-HDP-derived soft tissue images. Finally, it would have been beneficial to assess whether ^99mTc-EBl continued to accumulate in the areas of the mepivacaine injection sites after the 2-hour postinjection time point. In the present study, however, the ^99mTc-EBl and ^99mTc-HDP scans were performed on the same day, which would be a preferred and practical protocol for clinical settings.

On the basis of results of our study, a recommended protocol would be to administer ^99mTc-EBl IV to horses at a dose of 2.2 GBq (60 mCi) and to begin imaging procedures immediately following injection of the area of interest. Images should be acquired for a constant count rate of 150,000 counts for a single limb, 300,000 counts for 2 limbs, and 250,000 counts for other core body regions. If a bone scan is to be performed on the same day, then delayed imaging should always be performed just prior to injection of the bone agent because this will help to determine the amount of background radiation present from ^99mTc-EBl-associated activity. For improved image quality, the bone agent should not be injected until at least 4 hours after ^99mTc-EBl injection.

Results of our investigation suggest that ^99mTc-EBl is safe for use in horses at an empirical dose of 2.2 GBq (60 mCi). This agent was also rapidly cleared from blood and had immediate uniform uptake in normal soft tissues with no apparent uptake in bone. Findings indicated that imaging could be performed beginning immediately after injection with ^99mTc-EBl and can continue for as long as 4 hours, thereby allowing full-body soft tissue scanning to be performed if required. In addition, ^99mTc-EBl also detected early, mild inflammation that was experimentally induced via mepivacaine injection 6 hours prior to obtaining images. Although assessment of a greater number of horses would be needed to statistically validate the use of ^99mTc-EBl as a soft tissue inflammation marker, the potential clinical use of ^99mTc-EBl as a complementary technique to traditional bone scanning is apparent. Further studies are needed to investigate the clinical applications of this agent.