Subchondral defects resembling osteochondrosis dissecans in joint surfaces of the extinct saber-toothed cat Smilodon fatalis and dire wolf Aenocyon dirus.

Skeletal disease may hamper the behavior of large predators both living and extinct. We investigated the prevalence of osteochondrosis dissecans (OCD), a developmental bone disease affecting the joints, in two Ice Age predators: the saber-toothed cat Smilodon fatalis and dire wolf Aenocyon dirus. As published cases in modern Felidae and wild Canidae are rare, we predicted that subchondral defects resembling OCD would be rare in the extinct predators. We examined limb joints in juvenile and adult S. fatalis: 88 proximal humeri (shoulder), 834 distal femora (stifle), and 214 proximal tibiae. We also examined limb joints in juvenile and adult A. dirus: 242 proximal humeri, 266 distal femora, and 170 proximal tibiae. All specimens are from the Late Pleistocene Rancho La Brea fossil locality in Los Angeles, California, USA. While the Smilodon shoulder and tibia showed no subchondral defects, subchondral defects in the Smilodon femur had a prevalence of 6%; most defects were small (<7mm); and nine adult stifles with defects also showed osteoarthritis. Subchondral defects in the A. dirus femur had a prevalence of 2.6%; most defects were large (>12mm); and five stifles further developed mild osteoarthritis. Subchondral defects in the A. dirus shoulder had a prevalence of 4.5%; most defects were small, and three shoulders developed moderate osteoarthritis. No defects were found in the A. dirus tibia. Contrary to our prediction, we found a high prevalence of subchondral defects in the stifle and shoulder of S. fatalis and A. dirus resembling OCD found in humans and other mammals. As modern dogs affected by OCD are highly inbred, this high prevalence in the fossil taxa may suggest that they experienced inbreeding as they approached extinction. The deep-time history of this disease supports the need for monitoring of animal domestication, as well as conservation, to avoid unexpected surges in OCD under conditions like inbreeding.

Suspected Gallbladder Torsion in a Juvenile German Shepherd Dog.

An 11 mo old intact female German shepherd dog was presented with a 1 day history of vomiting, abdominal pain, and lethargy. A complete gallbladder torsion with secondary bile leakage was diagnosed during exploratory laparotomy. The dog underwent cholecystectomy at the clearly demarcated border of the viable cystic duct and recovered with no complications. Torsion was possible as a result of the complete absence of normal connective tissue attachments to the hepatic fossa, in human medicine called a wandering gallbladder. No other abnormalities were found on complete abdominal exploration. Very few cases describe torsion of the cystic duct as a cause for extrahepatic biliary disease and secondary bile peritonitis in dogs.

Relationship of preoperative neurologic score with intervals to regaining micturition and ambulation following surgical treatment of thoracolumbar disk herniation in dogs.

OBJECTIVE To estimate the times required for dogs to regain micturition and ambulation following surgical treatment for thoracolumbar Hansen type I intervertebral disk extrusion (IVDE) and determine whether these variables were associated with preoperative modified Frankel score (MFS). DESIGN Retrospective case series with nested cohort study. ANIMALS 54 dogs weighing < 20 kg (44 lb) that were surgically treated for Hansen type I IVDE at a referral hospital between January and December 2015. PROCEDURES Medical records and CT and MRI data were reviewed. Information was collected regarding dog signalment, type and duration of clinical signs, preoperative MFS, degree of spinal cord compression, type of medical and surgical treatment provided, and intervals from surgery to regaining micturition and ambulation (outcomes). Collected data were evaluated for correlations with outcomes. RESULTS Mean ± SD interval from surgery to regaining micturition was 4.1 ± 4.4 days, and mean interval from surgery to regaining ambulation was 13.8 ± 25.1 days. These intervals differed significantly. Significant negative correlations with MFS were identified for interval to regaining micturition (r = -0.63) and interval to regaining ambulation (r = -0.64). No other correlations with outcome were identified. CONCLUSIONS AND CLINICAL RELEVANCE The amount of time required for dogs to regain micturition and ambulation following surgery for thoracolumbar IVDE was correlated with preoperative severity of clinical signs, as reflected by preoperative MFSs. This information should be useful for pre- and postoperative decision-making and setting of expectations for owners of and clinicians treating affected dogs.

Surgical Site Infection Rate after Hemilaminectomy and Laminectomy in Dogs without Perioperative Antibiotic Therapy.

OBJECTIVE: The aim of this study was to retrospectively review the surgical site infection (SSI) rate in dogs undergoing laminectomies without perioperative antibiotics, and compare those data with the expected infection rate for clean surgical wounds in dogs undergoing similar procedures. METHODS: This was a retrospective single-centre study composed of dogs that underwent hemilaminectomies or laminectomies for thoracolumbar disc herniation or lumbosacral disease during a 2-year period (during 2015 and 2016). All incisional complications within 30 days were recorded and divided into superficial, deep or organ/space infections. Those dogs that received perioperative or postoperative antibiotics due to non-related comorbidities and those with incomplete medical records during the study period were excluded. RESULTS: Of 221 consecutive hemilaminectomy and laminectomy procedures, 154 were included in this research study. One superficial wound infection was recorded and treated with antimicrobials. Overall, the SSI rate was 0.6%, while the expected SSI rate in clean operative wounds in dogs and cats is 2.0 to 4.8%. The SSI rate in human spinal surgery is 0.7 to 4.3%. CLINICAL SIGNIFICANCE: Considering the low incidence of SSI in our study group, the routine use of perioperative antibiotic prophylaxis in dogs undergoing laminectomy procedures should be reconsidered to help address the global problem of bacterial resistance.

Complete Cranial Iliac Osteotomy to Approach the Lumbosacral Foramen.

An approach using a complete cranial iliac osteotomy (CCIO) to access the lumbosacral (LS) foramen in dogs from lateral was developed using cadavers and applied in a clinical patient with degenerative lumbosacral stenosis (DLSS). The foraminal enlargement in the cadavers and the patient was documented on postoperative CT scans. The preoperative CT scan of the patient showed moderate cranial telescoping of the sacral roof and a moderate central disk protrusion, leading to moderate to severe compression of the cauda equina. In addition, there was lateral spondylosis with consequential stenosis of the right LS foramen. The right L7 nerve had lost its fat attenuation and appeared thickened. After a routine L7S1 dorsal laminectomy with a partial discectomy, a CCIO was performed, providing good access to the LS foramen and the adhesions around the proximal L7 nerve caudoventral to the foramen. The osteotomy was stabilized with a locking plate and a cerclage wire. The dog recovered well from the procedures and after 36 h, the dog walked normally and was discharged from the hospital. Eight and 16 weeks later, the signs of the DLSS had markedly improved. From these data, it can be concluded that the CCIO is a useful approach to the LS foramen and intervertebral disk in selected patients with DLSS, giving good access to the structures around the LS foramen.

Epidural Gas Accumulation in Connection with Canine Degenerative Lumbosacral Disease.

Three dogs were presented with lumbosacral hyperesthesia. Computerized tomography scans were performed in all the cases, and magnetic resonance imaging was also performed in cases 1 and 3. There was intervertebral disc (IVD) protrusion causing nerve root compression and epidural gas accumulation in all the three cases. The gas-filled cystic structures in cases 1 and 3 were within the spinal canal; in case 2, the gas was within the disc protrusion. The IVD vacuum phenomenon is relatively common in dogs, but the formation of an epidural gas accumulation in cases of a lumbar disc protrusion is rare. The clinical significance of these epidural gas accumulations is unknown. Two of the dogs were treated surgically, improved after surgery, and showed no signs of pain in the follow-up examinations.

Surgical management of myelopathy caused by a solitary spinal osteochondroma in a young cat.

CASE SUMMARY: A 10-month-old, male castrated, domestic shorthair cat was presented with fast-progressing ataxia of the pelvic limbs. MRI and CT were performed, revealing a bony proliferation at T11, with mass effect and laterodorsal compression of the spinal cord. After hemilaminectomy and the removal of the bony mass, the cat recovered uneventfully. At the 1 year follow-up, the cat did not show any neurological deficits or signs of recurrence. RELEVANCE AND NOVEL INFORMATION: Solitary osteochondroma as a cause of neurological deficits in the pelvic limb has been described in dogs and humans, but, to the best of our knowledge, there are no reported feline cases described in the literature, in which CT and MRI were performed in combination with the successful removal of the lesion and an excellent outcome for the patient.

Lameness caused by an extradural lumbosacral foraminal synovial cyst in three German Shepherd Dogs.

Three German Shepherd Dogs that were presented for investigation of chronic unilateral hindlimb lameness and pain in the lumbosacral region were diagnosed with an intraspinal, extradural synovial cyst and reactive fibrosis protruding into the foramen of the lumbosacral articulation using magnetic resonance imaging and histology. This extradural mass compressed the nerve root in the foramen and the cauda equina. During a dorsal laminectomy and unilateral partial foraminotomy, the cyst and the fibrotic tissue were removed with the aid of a 2.4 mm 30° arthroscope for visualization of the foramen. The fibrotic tissue surrounding the cysts was in all cases confluent with the annulus of the intervertebral disc. The histological examination confirmed the diagnosis of a synovial cyst in all three cases by finding inflamed synovial membrane in the samples from the wall of the cyst as well as reactive fibrosis and cartilaginous metaplasia in the surrounding tissue. The three patients improved after the surgery and were pain free during the follow-up evaluations.

RNA interference targeting hypoxia inducible factor 1alpha reduces post-operative adhesions in rats.

BACKGROUND: To investigate the use of RNA interference mediated gene down-regulation targeting hypoxia inducible factor 1alpha (HIF-1alpha) and plasminogen activator inhibitor 1 (PAI-1) in an effort to prevent abdominal adhesion formation. MATERIALS AND METHODS: Real time PCR and a PAI-1 protein activity assay were used in vitro to determine the efficacy of small interfering RNAs (siRNAs). For in vivo experiments, 57 white female rats were operated to generate ischemic and serosal injury to the uterine horns, and treated with saline, siRNA(Lamin A/C) (negative control), siRNA(HIF-1alpha), siRNA(PAI-1), or siRNA(HIF-1alpha) plus siRNA(PAI-1). The cationic polyer poly(ethylenimine) (PEI) was used as the delivery vehicle for all siRNAs delivered in vivo. Adhesions were analyzed by a blinded surgeon 8 days post-surgery. RESULTS: After in vitro transfection with siRNA, at least 69% gene down-regulation was obtained for all siRNAs tested. In vitro siRNA-mediated down-regulation of HIF-1alpha, PAI-1 or their simultaneous delivery resulted in a significant decrease of PAI-1 protein activity (at least P < 0.05). Administration of 4 nmol siRNA(HIF-1alpha)/PEI complexes after injury to the uterine horns achieved a statistical reduction of post-operative adhesion formation with a reduction by 52% (P < 0.05). Delivery of 4 nmol siRNA(PAI-1)/PEI complexes and the simultaneous delivery of 2 nmol siRNA(HIF-1alpha) plus 2 nmol siRNA(PAI-1), resulted in a reduction of abdominal adhesion by 36% and 42%, respectively, with the reduction being statistically significant when compared directly to the saline control (P < 0.01). CONCLUSION: These data show that administration of siRNA/PEI complexes within the peritoneal cavity can be used to prevent post-operative abdominopelvic adhesions.

Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis.

Activation of vascular endothelial growth factor (VEGF) receptor-3 (VEGFR-3) by VEGF-C initiates lymphangiogenesis by promoting lymphatic proliferation and migration. However, it is unclear whether VEGFR-3 signaling is required beyond these initial stages, namely during the organization of new lymphatic endothelial cells (LECs) into functional capillaries. Furthermore, the role of VEGFR-2, which is also expressed on LECs and binds VEGF-C, is unclear. We addressed these questions by selectively neutralizing VEGFR-3 and/or VEGFR-2 for various time periods in an adult model of lymphangiogenesis in regenerating skin. While blocking either VEGFR-2 or VEGFR-3 with specific antagonist mAbs (DC101 and mF4-31C1, respectively) prior to lymphatic migration prevented lymphangiogenesis, blocking VEGFR-3 subsequent to migration did not affect organization into functional capillaries, and VEGFR-2 blocking had only a small hindrance on organization. These findings were confirmed in vitro using human LECs and anti-human antagonist mAbs (IMC-1121a and hF4-3C5): both VEGFR-2 and -3 signaling were required for migration and proliferation, but tubulogenesis in 3D cultures was unaffected by VEGFR-3 blocking and partially hindered by VEGFR-2 blocking. Furthermore, both in vitro and in vivo, while VEGFR-3 blocking had no effect on LEC organization, coneutralization of VEGFR-2, and VEGFR-3 completely prevented lymphatic organization. Our findings demonstrate that cooperative signaling of VEGFR-2 and -3 is necessary for lymphatic migration and proliferation, but VEGFR-3 is redundant with VEGFR-2 for LEC organization into functional capillaries.

Low-field Mri and arthroscopy of meniscal lesions in ten dogs with experimentally induced cranial cruciate ligament insufficiency.

Little is known about the magnetic resonance imaging (MRI) appearance of canine meniscal lesions. The aim of this study is to describe the MR appearance of meniscal lesions in dogs with experimentally induced cranial cruciate ligament (CCL) deficiency. The pilot study revealed dogs weighing approximately 10 kg to be too small for meniscal evaluation on low-field MRI. In the main study, dogs weighing approximately 35 kg were used. The left CCL was transected and low-field MRI was performed regularly until 13 months post-surgery. Normal menisci were defined as grade 0. Intrameniscal lesions not reaching any surface corresponded to grade 1 if focal and to grade 2 if linear or diffuse. Grade 3 lesions consisted in linear tears penetrating a meniscal surface. Grade 4 lesions included complex signal changes or meniscal distortion. Between 2 and 13 months post-surgery, all dogs developed grade 4 lesions in the medial meniscus. Most of them corresponded to longitudinal or bucket handle tears on arthroscopy and necropsy. Two dogs showed grade 3 lesions reaching the tibial surface of the lateral meniscus on MRI but not in arthroscopy. Such tears are difficult to evaluate arthroscopically; MRI provides more accurate information about the tibial meniscal surface. Grades 1 and 2 lesions could not be differentiated from presumably normal menisci with our imaging technique. An MRI grading system better adapted to canine lesions has yet to be developed. MRI is a helpful tool for the diagnosis of complete tears in the canine meniscus, especially in larger dogs.

Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines.

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

Treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 delivered from a fibrin matrix.

OBJECTIVE: To report the results of the treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 (nglBMP-2) delivered from a designed fibrin matrix. STUDY DESIGN: Experimental trial in rodents and prospective clinical study in dogs and cats with nonunion fractures. ANIMALS: Twenty adult female, albino, Sprague-Dawley rats; 8 client-owned cats and dogs. METHODS: After development of a fibrin matrix and evaluation of nglBMP-2 in a rodent femoral defect model, 8 consecutive long bone nonunion fractures (no progression in healing in > or = 3 months), were treated using 300 microg nglBMP-2 in a liquid fibrin precursor, injected into the defect gap after fracture revision and stabilization, or through a stab incision into the fracture site. The fibrin matrix was designed to clot in the wound after 60 seconds and to release the nglBMP-2 continuously over several days. RESULTS: Using only fibrin gel, 7% of the rat femoral defect was filled with new formed bone compared with 79% defect filling using 2 microg nglBMP-2 (P=.006). Five and 10 microg nglBMP in fibrin resulted in union of all femoral defects with complete filling of the gap with new bone. Bony bridging and clinical healing was achieved in 7 patients within 24 weeks of administration of nglBMP-2. CONCLUSIONS: Application of nglBMP-2 in a functional matrix can induce bone healing. Controlled release of nglBMP-2 from a fibrin matrix mimics the natural fracture hematoma. CLINICAL RELEVANCE: nglBMP-2/fibrin can successfully replace a cancellous bone autograft in fracture treatment with an associated reduction in graft donor site morbidity and surgical time.

Deletion mutants of BMP folding variants act as BMP antagonists and are efficient inhibitors for heterotopic ossification.

UNLABELLED: Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or have undergone total hip replacement. In this report, stable folding variants of the natural occurring osteoinductive BMPs were shown to act as inhibitors for heterotopic ossification. The most effective BMP folding variant construct performed even better than the natural occurring BMP antagonist Noggin because it also inhibited calcium deposition of pre-osteoblastic cells. INTRODUCTION: Signal transduction through receptor and ligand binding depends on the proper folding of all partners, especially when it involves the formation of a heterotetramer. In the case, the receptor binding of the ligand can be uncoupled from signal transduction, and folding variants of a ligand can be developed into antagonists of the natural bioactivity of the ligand. Here we present a deletion mutant of a bone morphogenetic protein (BMP) folding variant capable of inhibiting the bone-inducing action of natural occurring BMPs. MATERIALS AND METHODS: Deletion mutants and site-directed mutants of BMP folding variants were generated and tested for their ability to reduce alkaline phosphatase activity and mineralization in a pre-osteoblastic cell line. In vivo activity of the optimized folding variant was determined in a heterotopic ossification model in rodents and in two Xenopus laevis model systems. Biosensor interaction analysis was used to determine the affinity of the optimized BMP folding variant to the extracellular domain of BMP receptors. RESULTS: In vitro and in vivo tests in rodents revealed that the structural elements of the wrist epitope combined with finger 2 and a positive charge proximal to the tip of this finger are sufficient to induce osteoinhibition with deletion mutants and folding variants of mature BMP-4. The inhibitor designed to suppress heterotopic ossification showed BMP antagonist activity in embryos and animal caps of X. laevis. Binding studies of the inhibitor to ectodomains of type I and type II BMP receptors revealed a concentration-dependent binding, especially to the high-affinity BMP receptor. CONCLUSIONS: Deletion mutants of BMP folding variants are a new form of BMP antagonists and act through competition with osteoinductive BMP for BMP receptor binding. The excellent in vivo performance of the optimized folding variant is because of its ability to block signaling of endogenous BMPs deposited in the extracellular matrix even more effectively than the natural occurring BMP antagonist Noggin.

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid.

Graft copolymers were designed that could spontaneously bind to biological surfaces and block subsequent recognition and adhesion at those surfaces. Phenylboronic acid (PBA) moieties in the polymer backbone provided binding to surfaces, forming reversible covalent complexes with cis-diols found in many biological molecules. Pendant poly(ethylene glycol) (PEG) side chains sterically protected those surfaces from subsequent interactions with other proteins and cells. The PEG and PBA grafting ratios on these poly-L-lysine-graft-(PEG;PBA) copolymers [PLL-g-(PEG;PBA)] were varied, and the polymers were tested in models relevant to undesirable wound-healing responses such as peritoneal adhesion formation and posterior capsule opacification. PLL-g-(PEG;PBA) polymers spontaneously coated tissue culture polystyrene and completely blocked rabbit lens epithelial cell adhesion to the surface over a wide range of PEG grafting ratios. PLL-g-(PEG;PBA)s with optimal grafting ratios were able to coat adsorbed serum proteins or extracellular matrices and block cell spreading on the surfaces at 4 h, although the effect was lost within 24 h. The polymer also enhanced the efficacy of surgical lysis of peritoneal adhesions in rats. The reversible covalent complexes formed by the PBA moieties on the copolymer backbone were more effective at binding biological surfaces than electrostatic interactions formed via a copolymer lacking the PBA moieties, that is, PLL-g-PEG.