Amy E. Yeager

EFFECT OF ADIPOSE-DERIVED NUCLEATED CELL FRACTIONS ON TENDON REPAIR IN HORSES WITH COLLAGENASE-INDUCED TENDINITIS

OBJECTIVE To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS 8 horses. PROCEDURES Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 106 MSCs or 10 × 106 AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis.

Accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography.

The length of canine gestation is 65 days from the luteinizing hormone (LH) surge. Early and accurate determination of canine gestational age is useful for predicting and managing parturition. We performed a retrospective study on fetal measurements obtained by transabdominal ultrasonographic examination of 83 bitches (32 breeds) to estimate gestational age. Gestational age was estimated using two published tables correlating either (1). embryonic vesicle diameter (EVD), crown-rump length (CRL), body diameter (BD), and biparietal diameter (HD) to the LH surge in mid-gestational beagles or (2). BD and HD to parturition in late-gestation retrievers. Parturition date was predicted by obtaining the difference between the gestational age estimate and 65 days. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (<or=9 kg), medium (>9-20 kg), large (>20-40 kg), and giant (>40 kg). Mean+/-S.D. litter size (LS) was calculated for each BW group. The BW groups were then divided into small, average, or large LS groups. The accuracy of the prediction was not affected by LS but was affected by maternal body weight for small and giant BW groups only. When adjusted for weight, the accuracy of prediction within +/-1 day and +/-2 day intervals was 75 and 87%, respectively. Using stepwise logisitic regression, the most accurate prediction of parturition date was obtained when fetuses were measured at 30 days after the LH surge, regardless of body weight or LS. Parturition date predictions made after 39 days of gestation using only biparietal and BD fetal measurements were <50% accurate within +/-2 days.

Association between the preovulatory luteinizing hormone surge and the early ultrasonographic detection of pregnancy and fetal heartbeats in Beagle dogs.

The size of the gestational sac and embryonic mass as well as the embryonic heartbeat were examined ultrasonographically from Day 16 to 25 of pregnancy in 15 beagle bitches, using a 7.5 MHz transducer. Results were more consistent when gestational age was based on the day of the preovulatory luteinizing hormone (LH) surge than on the day of first breeding. The gestational sac was first detected at 17 to 20 d after the LH surge, when it was 1 to 2 mm in diameter and 1 to 4 mm in length. The diameter and length of the gestational sac increased exponentially. At Day 25, the mean diameter was 8.2 +/- 0.3 mm (7 to 9 mm) and mean length was 20.3 +/- 1.1 mm (14 to 24 mm). Embryonic mass and heartbeat were first detected at 23 to 25 d after the LH surge. The embryonic heartbeat was detected on the day of or the day after detection of the embryonic mass, at which time the embryonic mass was 1 to 4 mm in length and was located at the periphery of the gestational sac.

Integration of woodchuck hepatitis and N‐myc rearrangement determine size and histologic grade of hepatic tumors

Integrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N‐myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks (Marmota monax) chronically infected with WHV. Fifty‐five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N‐myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N‐myc rearrangement. Fifty‐one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N‐myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N‐myc rearrangements were present. Twenty‐four grade 3 HCCs had integrations of WHV DNA in 79% and N‐myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N‐myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N‐myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV infection. (HEPATOLOGY 2004;39:1008–1016.)

COMPARISON OF RADIOGRAPHY AND ULTRASONOGRAPHY FOR DIAGNOSING SMALL‐INTESTINAL MECHANICAL OBSTRUCTION IN VOMITING DOGS

A cross-sectional study was performed on acutely vomiting dogs to compare the accuracy of radiography and ultrasonography for the diagnosis of small-intestinal mechanical obstruction and to describe several radiographic and ultrasonographic signs to identify their contribution to the final diagnosis. The sample population consisted of 82 adult dogs and small-intestinal obstruction by foreign body was confirmed in 27/82 (33%) dogs by surgery or necropsy. Radiography produced a definitive result (obstructed or not obstructed) in 58/82 (70%) of dogs; ultrasonography produced a definitive result in 80/82 (97%) of dogs. On radiographs, a diagnosis of obstruction was based on detection of segmental small-intestinal dilatation, plication, or detection of a foreign body. Approximately 30% (8/27) of obstructed dogs did not have radiographic signs of segmental small-intestinal dilatation, of which 50% (4/8) were due to linear foreign bodies. The ultrasonographic diagnosis of small-intestinal obstruction was based on detection of an obstructive lesion, sonographic signs of plication or segmental, small-intestinal dilatation. The ultrasonographic presence or absence of moderate-to-severe intestinal diameter enlargement (due to lumen dilatation) of the jejunum (>1.5 cm) was a useful discriminatory finding and, when present, should prompt a thorough search for a cause of small-intestinal obstruction. In conclusion, both abdominal radiography and abdominal ultrasonography are accurate for diagnosing small-intestinal obstruction in vomiting dogs and either may be used depending on availability and examiner choice. Abdominal ultrasonography had greater accuracy, fewer equivocal results and provided greater diagnostic confidence compared with radiography.

Serial ultrasonographic appearance of postpartum uterine involution in Beagle dogs.

Postpartum changes in uterine shape, architecture, echogenicity and diameter were determined during the serial examinations of five beagle bitches. During postpartum Week 1, the uterine horns were tubular structures composed of multiple layers of various echogenicity and had multiple, discrete enlargements with hypoechoic centers at placental sites. Diameters ranged from 1.1 to 3.8 cm at placental site enlargements, and 0.5 to 1.4 cm between enlargements. Uterine involution appeared to be completed by 15 wk post partum. At 15 weeks the uterine horns of each dog were uniform hypoechoic, tubular structures without enlargements and had a reduced diameter of 0.3 to 0.6 cm. These ultrasonographic findings are similar to previously reported gross and light microscopic descriptions of canine uterine involution.

A collagenase gel/physical defect model for controlled induction of superficial digital flexor tendonitis

REASONS FOR PERFORMING STUDY A consistent and clinically relevant model for the induction of core lesions confined to the mid-metacarpal superficial digital flexor tendon (SDFT) has not been previously reported. Injection of bacterial collagenase is commonly used but often results in large, irregular and inconsistent lesions that disrupt the superficial tendon layers and epitenon. OBJECTIVE To develop and evaluate a new injection technique for collagenase induction of SDFT injury. METHODS Collagenase gel was injected into a physical columnar defect created by longitudinally placing a curved 16 gauge 8.89 cm needle in the mid-metacarpal SDFT in a randomly selected forelimb of 10 horses. A placebo treatment injection was performed 1 week later. Serial ultrasound examinations were performed. Horses were subjected to euthanasia at 2 (n = 2), 4 (n = 2), 8 (n = 4) and 16 (n = 2) weeks post treatment injection. Post mortem magnetic resonance imaging and histological analysis were performed. Gene expression (18S, SCX, TNC, TNMD, COL1A1, COL3A1, COMP, DCN, MMP1, MMP3 and MMP13), total DNA, glycosaminoglycan and collagen content were determined for experimental tendons (n = 10) and unaffected tendons (n = 9). RESULTS Mid-metacarpal SDFT core lesion induction was successful in all tendons with consistent lesion cross-sectional area and minimal epitenon disruption. Histology confirmed loss of normal tendon architecture after tendonitis induction and subsequent healing of the tendon core lesion. Compared with gene expression in unaffected tendons, several tested genes were significantly upregulated (COL1A1, COL3A1, TNMD, SCX, TNC, MMP13), while others showed significant downregulation (COMP, DCN, and MMP3). CONCLUSION Compared with the previously used direct injection of collagenase, this injection technique was easily performed and induced more consistent lesions that were mid-metacarpal and did not disrupt the epitenon. POTENTIAL RELEVANCE This model will allow for objective assessment of therapies for tendon regeneration in the mid-metacarpal SDFT prior to clinical trials and routine clinical application.

Intralesional injection of insulin-like growth factor-I for treatment of superficial digital flexor tendonitis in Thoroughbred racehorses: 40 cases (2000-2004).

OBJECTIVE-To evaluate outcome after intralesional injection of insulin-like growth factor-I (IGF-I) for treatment of superficial digital flexor (SDF) tendonitis in Thoroughbred racehorses. DESIGN-Retrospective case series. ANIMALS-40 Thoroughbred racehorses. PROCEDURES-Medical records of racehorses with SDF tendonitis treated within 13 weeks after injury by intralesional injection of IGF-I (25 or 50 μg every other day for 4 or 5 treatments) were reviewed. Outcome was determined via analysis of race records, owner follow-up, and examination. RESULTS-Mean age of the horses was 3.1 years (range, 2 to 7 years), and time from injury to treatment ranged from 8 to 90 days. Mean ± SD approximate lesion length on admission was 15.6 ± 6.0 cm, and mean percentage cross-sectional area of the tendon affected was 26 ± 18%. Twenty-six of the 40 horses underwent desmotomy of the accessory ligament of the SDF tendon. Echolucency was reduced in 23 of 26 horses by the end of the treatment period. Twenty-one of 34 (62%) horses for which race data were available raced at least once after treatment, including 10 (30%) horses that raced between 1 and 4 times and 11 horses (32%) that raced ≥ 5 times. Thirteen of 28 (46%) horses had a recurrence of tendonitis or developed tendonitis elsewhere. CONCLUSIONS AND CLINICAL RELEVANCE-Results suggested that in Thoroughbred racehorses with SDF tendonitis, intralesional injection of IGF-I led to a decrease in ultrasonographic lesion severity, but treated horses had only a moderate prognosis for return to racing.

DIAGNOSTIC SENSITIVITY OF SUBJECTIVE AND QUANTITATIVE LARYNGEAL ULTRASONOGRAPHY FOR RECURRENT LARYNGEAL NEUROPATHY IN HORSES

Recurrent laryngeal neuropathy (RLN) is the most common cause of laryngeal hemiplegia in horses and causes neurogenic atrophy of the intrinsic laryngeal muscles, including the cricoarytenoideus lateralis muscle. Recurrent laryngeal neuropathy results in paresis to paralysis of the vocal fold and arytenoid cartilage, which limits performance through respiratory compromise. Ultrasound has previously been reported to be a useful diagnostic technique in horses with RLN. In this report, the diagnostic sensitivity of subjective and quantitative laryngeal ultrasonography was evaluated in 154 horses presented for poor performance due to suspected upper airway disease. Ultrasonographic parameters recorded were: cricoarytenoideus lateralis echogenicity (subjective and quantitative), cricoarytenoideus lateralis thickness, vocal fold movement, and arytenoid cartilage movement. Ultrasonographic parameters were then compared with laryngeal grades based on resting and exercising upper airway endoscopy. Subjectively increased left cricoarytenoideus lateralis echogenicity yielded a sensitivity of 94.59% and specificity of 94.54% for detecting RLN, based on the reference standard of exercising laryngeal endoscopy. Quantitative left cricoarytenoideus lateralis echogenicity values differed among resting laryngeal grades I-IV. Findings from this study support previously published findings and the utility of subjective and quantitative laryngeal ultrasound as diagnostic tools for horses with poor performance.