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Background: The activation of the growth hormone receptor (GHR) is a major determinant of body growth. Defective GHR signaling, as seen in human Laron dwarfism, resulted in low plasma IGF-1 concentrations and limited growth, but also marked absence in the development of breast cancer and type 2 diabetes. In vitro, we identified a small molecule (C#1) that inhibits the translation of GHR mRNA to receptor protein. Methods: Before its application in humans as a potential anticancer drug, C#1 was tested in animals to evaluate whether it could be administered to achieve a plasma concentration in vivo that inhibits cell proliferation in vitro without causing unwanted toxicity. To evaluate the efficacy and toxicity of C#1, a group of six intact female Beagle dogs was treated daily each morning for 90 days with an oral solution of C#1 in Soiae oleum emulgatum at a dose of 0.1 mg/kg body weight. During treatment, dogs were closely monitored clinically, and blood samples were taken to measure plasma C#1 concentrations, complete blood counts (CBC), clinical chemistry, and endocrinology. At the end of the treatment, dogs were euthanized for gross and histopathological analysis. An additional group of six female Beagle dogs was included for statistical reasons and only evaluated for efficacy during treatment for 30 days. Results: Daily administration of C#1 resulted in a constant mean plasma concentration of approximately 50 nmol/L. In both groups, two out of six dogs developed decreased appetite and food refusal after 4-5 weeks, and occasionally diarrhea. No significant effects in CBC or routine clinical chemistry were seen. Plasma IGF-1 concentrations, used as biomarkers for defective GHR signaling, significantly decreased by 31% over time. As plasma growth hormone (GH) concentrations decreased by 51% as well, no proof of GHR dysfunction could be established. The measured 43% decrease in plasma acylated/non-acylated ghrelin ratios will also lower plasma GH concentrations by reducing activation of the GH secretagogue receptor (GHSR). C#1 did not directly inhibit the GHSR in vivo , as shown in vitro. There were no significant effects on glucose, lipid, or folate/homocysteine metabolism. Conclusions: It is concluded that with daily dosing of 0.1 mg C#1/kg body weight, the induction of toxic effects prevented further increases in dosage. Due to the concomitant decrease in both IGF-1 and GH, in vivo inhibition of GHR could not be confirmed. Since the concept of specific inhibition of GHR synthesis by small molecules remains a promising strategy, searching for compounds similar to C#1 with lower toxicity should be worthwhile.

Earlier, we described a breed-specific inflammatory myopathy in Dutch Kooiker dogs (Het Nederlandse Kooikerhondje), one of the nine Dutch breeds. The disease commonly manifests itself with clinical signs of difficulty walking, muscle weakness, exercise intolerance, and/or dysphagia. In nearly all dogs' creatine kinase (CK) activity was elevated. Histopathology reveals the infiltration of inflammatory cells within the skeletal muscles. The objective of this study was to further investigate and characterize the histopathological changes in muscle tissue and immunophenotype the inflammatory infiltrates. FFPE fixed-muscle biopsies from 39 purebred Kooiker dogs were included and evaluated histopathologically according to a tailored classification scheme for skeletal muscle inflammation. As in other breed-related inflammatory myopathies, multifocal, mixed, and predominantly mononuclear cell infiltration was present, with an initial invasion of viable muscle fibres and the surrounding stroma leading to inflammation, necrosis, and tissue damage. Immunophenotyping primarily revealed lymphohistiocytic infiltrates, with CD3+ T-cells being the predominant inflammatory cell type, accompanied by CD8+ cytotoxic T-cells. The concurrent expression of MHC-II class molecules on myofibres suggests their involvement in initiating and maintaining inflammation. Additionally, CD20+ B-cells were identified, though in lower numbers compared to T-cells, and IBA-1-positive macrophages were frequently seen. These findings suggest a breed-specific subtype of polymyositis in Kooiker dogs, akin to other breeds. This study sheds light on the immune response activation, combining adaptive and innate mechanisms, contributing to our understanding of polymyositis in this breed.

Background: Osteochondrosis dissecans (OCD) of articular process joints (APJs) is involved in cervical vertebral compressive myelopathy (CVM). Biomechanical forces, important in development of OCD, depend on joint conformation. Oval and flat APJ surfaces are considered normal., Objectives: To identify and grade gross shape variation of cervical and cranial thoracic APJ surfaces and determine association with histological evidence of osteochondrosis., Study Design: Case series., Methods: Eight hundred and four cervical and cranial thoracic APJ surfaces of 30 foals were evaluated for shape(s) and grades, and were correlated with osteochondrosis., Results: Three top view shapes (oval, pointed, elongated) and seven lateral view shapes (flat, convex, concave, stepped, bevelled, folded edge, raised edge) were regularly encountered. The oval top view shape was most common. Flat and bevelled were the most common lateral view shapes. General shape grade of caudal articular surfaces was significantly higher than of cranial surfaces. The combinations of an oval top view shape and the lateral view shapes folded edge, concave, or flat with additional raised edge and/or folded edge (flat +), were more likely to have OC than oval with convex, bevelled or flat lateral view shapes (normal vs. oval and folded, odds ratio [OR] 2.49 [95% confidence intervals (CIs) 1.13-5.67]; normal vs. oval and flat +, OR 2.77 [95% CI 1.15-6.85]; oval and convex vs. oval and folded, OR 3.20 [95% CI 1.35-8.20]; oval and convex vs. oval and flat +, OR 3.56 [95% CI 1.43-9.54]; oval and bevelled vs. oval and concave, OR 2.02 [95% CI 1.14-3.60]; oval and bevelled vs. oval and folded, OR 3.50 [95% CI 1.91-6.60]; oval and bevelled vs. oval and flat +, OR 3.90 [95% CI 2.00-7.70])., Main Limitations: Most foals (21/30) were less than 1 month old. Lack of observer reliability scores for shape and shape grade., Conclusion: APJs shape might contribute to CVM by increased likelihood to have OC., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: Advanced bipolar tissue sealer/dividers provide the most reliable and efficient means of tissue dissection and blood vessel sealing in laparoscopic surgery and the techniques are continuously improved. In veterinary practice, cost-effectiveness is of major impact, leading to re-use of instruments designed and sold for single use. Two high-end devices were evaluated and compared in a highly standardized laparoscopic ovariectomy procedure in dogs: The new generation Ligasure Maryland Sealer/Divider (LMSD) with improved atraumatic curved jaw shape for delicate tissue handling and dissection and non-stick nanocoating, and the new-generation Articulating Enseal G2 (AENG2) with several proclaimed features improving surgical performance, including articulation of the forceps tip; improved tissue compression during sealing; unique offset electrode configuration; and specific nanoparticle coating minimizing thermal spread and tissue sticking. Twenty-one client-owned dogs admitted for elective laparoscopic ovariectomy were randomly assigned to one of two groups: ovariectomy using AENG2 on the left ovary and LMSD in the right ovary or vice-versa. Procedural video recordings were used to assess ovarian ligament fat score, smoke formation, occurrence of bleeding, and excision duration. Excised tissues were examined histopathologically and collateral thermal damage was scored in three anatomic zones: suspensory ligament, vascular pedicle, and uterine junction. Tissue sealers were used repeatedly following standardized cleaning protocol with instrument washing machine and ethylene oxide gas sterilization and the number of uses until device failure was recorded., Results: Excision times were significantly increased for AENG2 (median 01:35 min) compared to LMSD (median 01:00 min). Minor hemorrhage from incomplete sealing occurred in 3 sites in 2 patients (2x AENG2, 1x LMSD) and was not significantly different between groups. Smoke production as scored on videos and thermal tissue damage scores on histopathology also did not differ between AENG2 and LMSD. Both vessel sealers could be re-used repeatedly., Conclusion: AENG2 provides a good alternative to LMSD in laparoscopic ovariectomy, with only minor differences in measured variables. Subjectively, the articulating feature of AENG2 did not improve surgical performance in laparoscopic ovariectomy and the use of LMSD appeared more straight-forward for this specific procedure. However, differences in operating these devices may be subject to personal preference., (© 2023. The Author(s).)

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies. To identify druggable pathways driving PPGL development, we performed RNA sequencing on PPGLs ( n = 19) and normal adrenal medullas (NAMs; n = 10) of dogs. Principal component analysis (PCA) revealed that PPGLs clearly clustered apart from NAMs. In total, 4,218 genes were differentially expressed between PPGLs and NAMs. Of these, 232 had a log 2 fold change of >3 or < -3, of which 149 were upregulated in PPGLs, and 83 were downregulated. Compared with NAMs, PPGLs had increased expression of genes related to the cell cycle, tumor development, progression and metastasis, hypoxia and angiogenesis, and the Wnt signaling pathway, and decreased expression of genes related to adrenal steroidogenesis. Our data revealed several overexpressed genes that could provide targets for novel therapeutics, such as Ret Proto-Oncogene ( RET ), Dopamine Receptor D2 ( DRD2 ), and Secreted Frizzled Related Protein 2 ( SFRP2 ). Based on the PCA, PPGLs were classified into 2 groups, of which group 1 had significantly higher Ki67 scores ( p = 0.035) and shorter survival times ( p = 0.04) than group 2. Increased expression of 1 of the differentially expressed genes between group 1 and 2, pleiotrophin ( PTN ), appeared to correlate with a more aggressive tumor phenotype. This study has shed light on the transcriptomic profile of canine PPGL, yielding new insights into the pathogenesis of these tumors in dogs, and revealed potential novel targets for therapy. In addition, we identified 2 transcriptionally distinct groups of PPGLs that had significantly different survival times., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 van den Berg, Kooistra, Grinwis, Nicoli, Golinelli, Stammeleer, van Wolferen, Timmermans-Sprang, Zandvliet, van Steenbeek and Galac.)

Adrenalectomy is the treatment of choice in case of functional adrenal tumors and malignant adrenal incidentalomas. Laparoscopic adrenalectomy (LA) in dogs has gained popularity in recent years, however, clinical studies on large patient populations are scarce. This retrospective study describes perioperative and recurrence data, survival, and prognostic factors in 70 dogs that underwent LA or open adrenalectomy (OA) in our hospital between 2008 and 2022. Diagnosis was based on history, clinical signs, endocrine function tests and advanced diagnostic imaging. Laparoscopic adrenalectomy was performed in 42 dogs ( n = 27 naturally occurring hypercortisolism, n = 4 pheochromocytoma, n = 1 pheochromocytoma with concurrent hypercortisolism, n = 10 incidentaloma) and OA in 28 dogs ( n = 22 hypercortisolism, n = 3 pheochromocytoma, n = 3 incidentaloma). Bilateral adrenalectomy was performed in 8/70 dogs. Surgical duration of LA and OA did not differ significantly in unilateral and bilateral procedures ( P = 0.108 and P = 0.101, respectively). Systemic hypertension occurred in 7/41 and 1/28 dogs during LA and OA, respectively ( P = 0.130). Hypotension occurred in 2/41 and 4/28 dogs during LA and OA, respectively ( P = 0.214). A total of 40/42 dogs in the LA group and 27/28 in the OA group survived to discharge ( P = 0.810). Mean hospital stay was significantly shorter ( P = 0.006) after LA (1.5 days, range 1-3) than after OA (2.2 days, range 1-4). No significant differences were demonstrated between LA and OA groups in recurrence of adrenal-dependent endocrine disease ( P = 0.332), disease-free period ( P = 0.733) and survival time ( P = 0.353). The disease-specific 1-, 2- and 3-year survival rates were 95, 89, and 89% after LA and 92, 88, and 81% after OA. Tumor size was significantly associated with the occurrence of a recurrence. In addition, tumor size had a negative effect on the disease-free period and survival time. This study shows a favorable outcome of both LA and OA in dogs. Based on low perioperative complication rate, short hospitalization time and long-term outcomes comparable to OA in selected cases, the less invasive laparoscopic approach is considered the preferred technique., Competing Interests: KB is employed by IVC Evidensia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 van Bokhorst, Galac, Kooistra, de Grauw, Teske, Grinwis and van Nimwegen.)

It was recently shown that targeting extracellular vimentin (eVim) is safe and effective in preclinical models. Here, we report the safety and efficacy in client-owned dogs with spontaneous bladder cancer of CVx1, an iBoost technology-based vaccine targeting eVim in combination with COX-2 inhibition. This was a single-arm prospective phase 1/2 study with CVx1 in 20 client-owned dogs with spontaneous UC which involved four subcutaneous vaccinations with CVx1 at 2-week intervals for induction of antibody titers, followed by maintenance vaccinations at 2-month intervals. Additionally, daily cyclooxygenase (COX)-2 inhibition with meloxicam was given. The response was assessed by antibody titers, physical condition, abdominal ultrasound and thorax X-ray. The primary endpoints were the development of antibody titers, as well as overall survival compared to a historical control group receiving carboplatin and COX-2 inhibition with piroxicam. Kaplan-Meier survival analysis was performed. All dogs developed antibodies against eVim. Titers were adequately maintained for the duration of this study. A median overall survival of 374 days was observed, which was 196 days for the historical control group ( p < 0.01). Short-term grade 1-2 toxicity at the injection site and some related systemic symptoms peri-vaccination were observed. No toxicity was observed related to the induced antibody response. A limitation of this study is the single-arm prospective setting. CVx1 plus meloxicam consistently induced efficient antibody titers, was well tolerated and showed prolonged survival. The results obtained merit further development for human clinical care.

Objectives: The success of the ex vivo machine perfusion of pig livers used for preclinical research depends on organ quality and availability. In this study, we investigated whether livers obtained from slaughterhouses are suitable and equivalent to livers obtained from laboratory pigs., Methods: Livers were obtained from slaughterhouse pigs stunned by electrocution or CO 2 inhalation and from laboratory pigs. For the latter group, 45 minutes of warm ischemia was mimicked for a subgroup, ensuring a valid comparison with slaughterhouse-derived livers., Results: Livers from CO 2 -stunned pigs showed lower indocyanine green clearance and bile production, higher blood lactate and potassium concentrations, and higher alanine aminotransferase activities than electrically stunned pigs. Furthermore, livers from electrically stunned pigs, and livers from laboratory pigs, subjected or not to warm ischemia, showed similar performance in terms of perfusion and metabolism., Conclusion: For an ex vivo liver model generated using slaughterhouse pigs, electrical stunning is preferable to CO 2 stunning. Livers from electrically stunned slaughterhouse pigs performed similarly to laboratory pig livers. These findings support the use of livers from electrically stunned slaughterhouse pigs, which may therefore provide an alternative to livers obtained from laboratory pigs, consistent with the principle of the 3Rs.

Background: A high perinatal mortality rate in the Stabyhoun breed prompted one of the Dutch breeding organizations to start an investigation. Preductal segmental tubular aortic hypoplasia is an extremely rarely documented congenital vascular anomaly in dogs, and it is suspected to be the result of constriction of ectopic ductal tissue in the aortic wall at birth., Methods: Over a period of 18 months, Stabyhoun puppies that were stillborn, died or were euthanized before 3 weeks of age were submitted to post-mortem examination at the reporting institution. Pathologic findings were documented., Results: Eight Stabyhoun puppies were submitted during the study period. In five of them, a severe preductal segmental tubular aortic hypoplasia was found. Two of the five puppies were stillborn, and three died spontaneously or were euthanized., Conclusions: Preductal tubular aortic hypoplasia was found in an unusually high frequency in the examined Stabyhoun puppies. Because the condition is believed to cause clinical signs only after birth, this anomaly cannot explain the death of the stillborn puppies. However, it might be responsible for cardiogenic pulmonary edema in the postnatal period. Routine dissection of the great vessels in perinatally deceased puppies would help to establish the prevalence of congenital anomalies of the aorta.

Background: Chronic low back pain, a leading contributor to disease burden worldwide, is often caused by intervertebral disc (IVD) degeneration. Modic changes (MCs) are MRI signal intensity changes due to lesions in vertebral bone marrow adjacent to degenerated IVDs. Only a few studies described the histopathological changes associated with MC to date. MC type 1 is suggested to be associated with bone marrow infiltration of fibrovascular tissue, type 2 with fatty infiltration, and type 3 with bone sclerosis in humans., Methods: This study investigated whether the dog can be a valuable animal model to research MCs, by examining the prevalence, imaging, and histological characteristics of lumbar MCs in dogs (340 dogs, 2496 spinal segments)., Results: Logistic regression analysis indicated that the presence of lumbosacral MCs was associated with age and disc herniation (annulus fibrosis protrusion and/or nucleus pulposus extrusion). According to MRI analysis, MCs were mostly detected at the lumbosacral junction in dogs. Most signal intensity changes represented MC type 3, while previous spinal surgery seemed to predispose for the development of MC type 1 and 2. Histological analysis (16 dogs, 39 spinal segments) indicated that IVDs with MCs showed more histopathological abnormalities in the endplate and vertebral bone marrow than IVDs without MCs. Mostly chondroid proliferation in the bone marrow was encountered, while the histologic anomalies described in humans associated with MCs, such as fibrovascular or fatty infiltration, were scarcely detected., Conclusions: Dogs spontaneously develop MCs, but may exhibit other pathological processes or more chronic bone marrow pathologies than humans with MCs. Therefore, more research is needed to determine the translatability of the MCs encountered in dog low-back-pain patients., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

The Dutch Kooiker dog (het Nederlandse Kooikerhondje) is one of nine Dutch dog breeds. As of 1960, a number of heritable diseases have been noted in this breed. One is an inflammatory myopathy that emerged in 1972, with numbers of affected dogs gradually increasing during the last few decades. The objective of this paper is to describe clinical signs, laboratory results, electromyography and histopathology of the muscle biopsies of the affected dogs. Method: Both retrospectively as well as prospectively affected Kooiker dogs were identified and categorized using a Tiered level of Confidence. Results: In total, 160 Kooiker dogs-40 Tier I, 33 Tier II and 87 Tier III-were included. Clinical signs were (1) locomotory problems, such as inability to walk long distances, difficulty getting up, stiff gait, walking on eggshells; (2) dysphagia signs such as drooling, difficulty eating and/or drinking; or (3) combinations of locomotory and dysphagia signs. CK activities were elevated in all except for one dog. Histopathology revealed a predominant lymphohistiocytic myositis with a usually low and variable number of eosinophils, neutrophils and plasma cells. It is concluded that, within this breed, a most likely heritable inflammatory myopathy occurs. Further studies are needed to classify this inflammatory myopathy, discuss its treatment, and unravel the genetic cause of this disease to eradicate it from this population.

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%-20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log 2 fold change was >2 or < -2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans., (© 2022 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Background: Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs)., Hypothesis/objectives: To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI)., Animals: Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs., Methods: Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively., Results: The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P < .001) and NAI (P = .002). The PCC dogs had significantly higher pMN than HC dogs (P < .001), but not higher than NAI dogs (P = .29). Using the upper reference limit, pNMN (>3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99)., Conclusions and Clinical Importance: With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

This case description reports an adult dog with multiple rib fractures that was initially suspected to be the victim of non-accidental trauma, but eventually was diagnosed with severe chronic interstitial nephritis and renal secondary hyperparathyroidism leading to fibrous osteodystrophy and multiple pathological fractures. The importance of the specific expertise of a dedicated expert panel on animal abuse is discussed. This case illustrates the significance of the identification of normal, pathological, and breed-related variations within an animal in addition to forensic expertise, emphasizing that the presence of multidisciplinary teams in an expert panel on animal abuse is crucial., (© 2022. The Author(s).)

Introduction: Minimally invasive microbrachytherapy is in development to treat solid tumors by intratumoral injection of (radioactive) holmium-166 ( 166 Ho) microspheres (MS). A high local dose can be administered with minimal damage to surrounding tissue because of the short soft tissue penetration depth of 166 Ho beta radiation. We aimed to prospectively evaluate the safety and efficacy of 166 Ho microbrachytherapy in client-owned canine patients with soft tissue sarcomas (STS)., Methods: We included seven dogs with STS not suitable for local excision due to tumor size and/or location. 166 HoMS were suspended in a carrier fluid and multiple needle-injections were performed in predetermined tumor segments to maximize tumor coverage. Tumor response was evaluated using 3D caliper and CT measurements. Follow-up further included monitoring for potential side effects and registration of subsequent treatments and survival, until at least two years after treatment., Results: Delivered radioactive doses ranged from 70 to 969 Gy resulting in a mean tumor volume reduction of 49.0 ± 21.3% after 33 ± 25 days. Treatment-related side effects consisted of local necrosis ( n = 1) and ulceration of the skin covering the tumor ( n = 1), which resolved with basic wound care, and surgical excision of residual tumor, respectively. Residual tumor was surgically resected in six patients after 22-93 days. After a mean follow-up of 1,005 days, four patients were alive, two patients were euthanized because of unrelated causes, and one patient was euthanized because of disease progression after the owner(s) declined subsequent surgical treatment., Conclusion: 166 Ho microbrachytherapy was a safe and effective neoadjuvant treatment option for canine patients with STS., Competing Interests: Author JN is co-founder and part-time scientific advisor of Quirem Medical which has been acquired by Terumo Europe NV in July 2020. He is entitled to certain milestone payments from Terumo which are related to Quirem's financial, operational, and regulatory performance in the future. Furthermore, he is inventor on the patents related to radioactive microspheres that are assigned to University Medical Center Utrecht Holding BV, Quirem Medical or BASF Corp. The activities of author JN within Quirem Medical are approved and supported by the Board of Directors of the Radboudumc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morsink, Nijsen, Grinwis, Hesselink, Kirpensteijn and van Nimwegen.)

Background: We previously reported a familial thyroid follicular cell carcinoma (FCC) in a large number of Dutch German longhaired pointers and identified two deleterious germline mutations in the TPO gene associated with disease predisposition. However, the somatic mutation profile of the FCC in dogs has not been investigated at a genome-wide scale., Results: Herein, we comprehensively investigated the somatic mutations that potentially contribute to the inherited tumor formation and progression using high depth whole-genome sequencing. A GNAS p.A204D missense mutation was identified in 4 out of 7 FCC tumors by whole-genome sequencing and in 20 out of 32 dogs' tumors by targeted sequencing. In contrast to this, in the human TC, mutations in GNAS gene have lower prevalence. Meanwhile, the homologous somatic mutation in humans has not been reported. These findings suggest a difference in the somatic mutation landscape between TC in these dogs and human TC. Moreover, tumors with the GNAS p.A204D mutation had a significantly lower somatic mutation burden in these dogs. Somatic structural variant and copy number alterations were also investigated, but no potential driver event was identified., Conclusion: This study provides novel insight in the molecular mechanism of thyroid carcinoma development in dogs. German longhaired pointers carrying GNAS mutations in the tumor may be used as a disease model for the development and testing of novel therapies to kill the tumor with somatic mutations in the GNAS gene., (© 2022. The Author(s).)

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

Thyroid carcinomas (TCs) originating from follicular cells of the thyroid gland occur in both humans and dogs, and they have highly similar histomorphologic patterns. In dogs, TCs have not been extensively investigated, especially concerning the familial origin of TCs. Here, we report familial thyroid follicular cell carcinomas (FCCs) confirmed by histology in 54 Dutch origin German longhaired pointers. From the pedigree, 45 of 54 histopathologically confirmed cases are closely related to a pair of first-half cousins in the past, indicating a familial disease. In addition, genetics contributed more to the thyroid FCC than other factors by an estimated heritability of 0.62 based on pedigree. The age of diagnosis ranged between 4.5 and 13.5 years, and 76% of cases were diagnosed before 10 years of age, implying an early onset of disease. We observed a significant higher pedigree-based inbreeding coefficient in the affected dogs (mean F, 0.23) compared to unaffected dogs (mean F, 0.14), suggesting the contribution of inbreeding to tumour development. The unique occurrence of familial thyroid FCC in this dog population and the large number of affected dogs make this population an important model to identify the genetic basis of familial thyroid FCC in this breed and may contribute to the research into pathogenesis, prevention and treatment in humans., (© 2021 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NB A was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic., (© 2021 Irati Beltrán Hernández et al., published by De Gruyter, Berlin/Boston.)

Merkel cell carcinoma (MCC) is in humans and cats a malignant cutaneous neuroendocrine carcinoma, whereas in dogs it possibly has a more benign behaviour. It may be cytologically confused with round cell tumours such as lymphoma because of its striking cytomorphologic similarity. Although MCC is considered to arise from Merkel cells, recent findings indicated that primitive (epi-)dermal stem cells, early B-cells or dermal fibroblasts were the origin of human MCC. The aim of our study was to evaluate a possible lymphoid origin in feline and canine MCCs. Specific analysis of CD3, PAX-5, KIT and PARR assay were performed in 3 feline and 3 canine MCCs. All MCCs (6/6) were negative for CD3 and PAX-5. KIT was expressed in all MCCs (6/6). Assessment of clonality by PARR assay exhibited a polyclonal B- and T-cell receptor rearrangement in all five cases tested. In conclusion, a lymphoid origin of feline and canine MCCs could not be demonstrated. This is in contrast with human MCCs, that often express early B-cell lineage markers., (© 2020 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald = 4.83x10-9) and 19 (Pwald = 2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To describe the signalment, ophthalmic examination findings, and follow-up of dogs affected with a previously unreported retrocorneal pigmentary lesion., Materials and Methods: Retrospective record evaluation spanning 2009-2019., Results: Retrocorneal pigmentary lesions were described in 34 patients (46 eyes). German Shepherds (n = 7), Jack Russel terriers (n = 5), and terrier crosses (n = 4) made up 16/34 (47.1%) of the cases. The mean age was 13.5 years (range 1.4-14.2 years), and 16/30 (53.3%) dogs were female. Most dogs were affected unilaterally (22/34 (64.7%)), the others bilaterally, and 5/34 (14.7%) were referred for it while the others were incidentally diagnosed. The lesions affected the ventral, peripheral, inner cornea and had a round/undulated leading edge. The number of corneal clock hours affected was known for 41/46 (89.1%) eyes and involved 1-3 clock hours in 32/41 (78.1%) eyes, 4-6 in 6/41 (14.6%), 7-9 in 2/41 (4.9%), and 10 in 1/41 (2.4%). The central cornea was affected in 9/46 (19.6%) eyes, and in 5/9 (55.6%), the median corneal clarity score was G2 (scale: G0-G4). The commonest additional findings included free-floating uveal cysts (11/34 dogs, 32.4%), cataracts (6/34 dogs, 17.6%), and primary glaucoma (5/34 dogs, 14.7%). Gonioscopy was available in 16/34 (47.1%) dogs and was normal except in primary glaucoma cases. Follow-up was documented in 13/34 (38.2%) dogs with a mean follow-up of 17 months (range: 5-26 months). Lesion progression was documented in 6/13 (46.2%) dogs., Conclusions: Retrocorneal pigmentation occurs as a slowly progressive lesion of older dogs that could impact vision. Histological studies of affected eyes are warranted., (© 2020 American College of Veterinary Ophthalmologists.)

Objective: To evaluate agreement in results obtained with an MRI-based grading scheme and a macroscopic observation-based grading scheme when used to assess intervertebral disk (IVD) degeneration in cats., Sample: 241 MRI and 143 macroscopic images of singular IVDs in 44 client-owned cats (40 cadaveric and 4 live)., Procedures: Singular images of IVDs were obtained of live cats admitted for treatment of suspected neurologic disease (MRI images of IVDs) and of cadavers of cats euthanized for reasons unrelated to spinal disease (MRI and macroscopic images of IVDs) at the Small Animal Hospital, Vetsuisse Faculty, Zurich, Switzerland, between January 12, 2015, and October 19, 2015. The IVD images were randomized and evaluated twice by 4 observers for each grading scheme. Inter- and intraobserver reliability for the grading schemes was assessed with Cohen weighted κ analysis. Agreement and correlation between results obtained with the 2 grading schemes were determined with Cohen weighted κ and Spearman correlation coefficient (ρ) analyses, respectively., Results: Inter- and intraobserver agreement between results was substantial to almost perfect (mean weighted κ, 0.66 to 0.83 and 0.71 to 0.86, respectively) for the MRI-based grading scheme and moderate to substantial (mean weighted κ, 0.42 to 0.80 and 0.65 to 0.79, respectively) for the macroscopic observation-based grading scheme. Between the 2 grading schemes, agreement in results was moderate (mean ± SE weighted κ, 0.56 ± 0.05), and the correlation was strong (ρ = 0.73)., Conclusions and Clinical Relevance: Results indicated that the MRI-based and macroscopic observation-based grading schemes used in the present study could be used reliably for classifying IVD degeneration in cats.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1 -deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease., Competing Interests: The authors declare no conflict of interest. B.S. and L.C.P. are members of the EASL Consortium for Regenerative Hepatology.

Objective: To determine whether surgical removal of urachal anomalies improves the outcomes of dogs with recurrent lower urinary tract disease (LUTD) and bacterial urinary tract infection (BUTI)., Study Design: Retrospective study., Animals: Thirty-three dogs with urachal anomalies and recurrent LUTD or BUTI., Methods: Medical records of dogs with LUTD or BUTI and a diagnosis of urachal anomaly treated by partial cystectomy were reviewed. A minimum follow-up of 9 months was required for inclusion., Results: Median age at onset of clinical signs was 12 months (range, 1 month to 10 years). Urachal anomalies were detected with histopathology in 20 of 28 (71%) dogs. At a median follow-up of 22 months (range, 9-114), 21 of 28 (64%) dogs were free of signs of LUTD. Nine (27%) dogs exhibited reduced signs of LUTD; in three (9%) dogs, no clinical improvement was observed. Among the 25 dogs with confirmed preoperative BUTI, 22 clinically improved with surgery., Conclusion: Partial cystectomy reduced the long-term severity of clinical signs and risk of recurrence of LUTD or BUTI in dogs with confirmed or suspected urachal anomalies., Clinical Significance: Partial cystectomy should be considered as an adjunct to the treatment of LUTD and BUTI in dogs., (© 2019 The Authors. Veterinary Surgery published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Surgeons.)

Objective: To report the long-term outcome of nine dogs treated for caudal cervical spondylomyelopathy (CCSM) with surgical spinal fusion., Study Design: Short case series., Animals: Nine large-breed dogs., Methods: Medical records of dogs treated for disc-associated CCSM (2013-2016) were reviewed. The surgery objective was spinal distraction by implantation of a SynCage and fixation with two Unilock plates. Follow-up included the Helsinki pain score questionnaire, neurological grading, radiography, computed tomography (CT), and micro-CT (μCT) with subsequent histopathology (two dogs)., Results: Clinical follow-up was obtained between 9 and 51 months (27.4 ± 13.4 months). The Helsinki pain score and neurological Griffith score improved (P < .01) in all dogs and in eight of nine dogs, respectively. According to CT, the volume of bone (mean ± SD) through the cage was 79.5% ± 14.3%, including compact bone (53.0% ± 23.4%). Subsidence was seen in one of nine dogs. Implant failure was evident in four dogs, and plates were removed in two dogs. In seven of nine dogs, infraclinical pathology was observed in adjacent segment, associated with implants engaging adjacent intervertebral discs. Radiographic evidence of bony fusion between vertebral bodies was noted in all dogs. Spinal fusion was confirmed by μCT and histopathology in two cervical spine segments that became available at 22 and 40 months postoperatively., Conclusion: Instrumented spinal fusion in dogs with disc-associated CCSM resulted in owner satisfaction and radiographic evidence of interbody spinal fusion in all dogs., Clinical Significance: The fusion distraction technique reported here can be used to achieve spinal fusion with a good long-term outcome., (© 2019 The Authors. Veterinary Surgery published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Surgeons.)

Four outbreaks of leukoencephalomyelopathy in colonies of SPF cats on a long-term diet of irradiated dry cat food were observed in the Netherlands between 1989 and 2001. As a primary defect in myelin formation was suspected to be the cause of the disease and myelin consists mainly of lipids and their fatty acids, we investigated the fatty acid composition of the white matter of the spinal cord of affected and control cats and of irradiated and non-irradiated food. The irradiated food had low levels of alpha-linolenic acid compared to linoleic acid as well as a high total omega-6:omega-3 ratio of 7:1 in the irradiated and of 2:1 in the non-irradiated food. The white matter of the spinal cord showed low levels of linoleic acid and absence of alpha-linolenic acid in affected cats as well as absence of lignoceric and nervonic acid in both affected and control cats. These abnormalities in fatty acid composition of the white matter of the spinal cord may reflect an increased need for alpha-linolenic acid as a substrate for longer chain omega-3 fatty acids to compose myelin and thus indicate a particular species sensitivity to dietary deficiency in omega-3 polyunsaturated fatty acids, particularly alpha-linolenic acid in cats. Our findings indicate that abnormalities in fatty acid metabolism in myelin play an essential role in the pathogenesis of this acquired form of leukoencephalomyelopathy in cats., (© 2019 The Authors. Journal of Animal Physiology and Animal Nutrition Published by Blackwell Verlag GmbH.)

A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15% to 20% of cases. The differentiation between an adrenocortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs' survival. Moreover, in human medicine the inter-observer variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and inter-observer agreement scores (intra-class correlation or Cohen's kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivariate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm and + 3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring., (© 2019 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Background: Iron toxicosis is rarely reported in horses and chronic excessive oral iron intake has not been reported to cause clinical symptoms in equids., Objectives: This case series describes 21 genetically unrelated horses and one donkey with chronic iron overload causing haemochromatosis and hepatopathy., Study Design: Case series., Methods: All equids showing clinical signs compatible with chronic liver disease presented to Utrecht University and diagnosed with iron overload and haemochromatosis based on histopathological evaluation of liver tissue and/or blood transferrin saturation levels of >80% and proof of excess dietary iron intake due to excess iron content in drinking water were included., Results: This study included 22 equids. All tested animals (n = 19) had transferrin saturation >80% and 21 of 22 had increased gamma-glutamyltransferase (γGT). Ultrasonography revealed rounded liver margins in five out of six horses and increased echogenicity in 4/6. Histological examination of liver tissue of 12 animals showed hepatitis, fibrosis and haemosiderin accumulation in macrophages and hepatocytes. Post-mortem examination also revealed haemosiderin accumulation in other organs in all seven examined animals. High iron content in drinking water was identified as the source of iron overload in all cases. All animals were housed under the same conditions for a minimum of 9 years prior to diagnosis of haemochromatosis. Of 22 animals, 13 survived until 1 January 2018, ranging from 17 to 79 months post diagnosis., Main Limitations: Histology of liver tissue was not available for 10 of 22 cases., Conclusions: Chronic iron overload can lead to haemochromatosis and hepatopathy in equids. Development of disease is slow and clinical signs are nonspecific. Long-term excessive iron intake in equids should be avoided. If animals drink from natural water sources, it is important to test the water for iron content. The Summary is available in Spanish - see Supporting Information., (© 2018 EVJ Ltd.)

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D ( SDHB and SDHD ) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln ( n = 1) and SDHD p.Lys122Arg ( n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways., Competing Interests: The authors declare no conflict of interest

Background: Focal seizures with fear as a primary ictal manifestation, their diagnostic challenges, and impact on quality of life are well described in human medicine. Reports focusing on ictal fear-like behavior in animals are scarce., Objective: To describe the clinical and histopathological characteristics of a novel focal epilepsy in Boerboel dogs., Animals: Five client-owned Boerboel littermates presented for evaluation of sudden episodes of severe fear-related behavior., Methods: Clinical examination, complete blood cell count, routine blood biochemistry, and urinalysis were performed in all dogs. Magnetic resonance imaging (MRI) scans of the brain were performed in 3 affected Boerboels. In addition, in 2 affected Boerboels, metabolic screening, cerebrospinal fluid (CSF) analysis, and necropsy were performed., Results: Onset of signs was 3 months of age in all affected Boerboels. All Boerboels howled loudly, had an extremely fearful facial expression and trembled during seizures. All affected Boerboels also had autonomic or motor signs. Results of laboratory investigations, diagnostic imaging, and metabolic screening were generally unremarkable. Histopathology showed moderate numbers of single large vacuoles in the perikaryon of neurons throughout the brain, specifically in the deeper cerebral cortical regions. Family history, pedigree analysis, and the homogenous phenotype were suggestive of autosomal recessive inheritance., Conclusions and Clinical Importance: The observed paroxysmal fear-related behavior represents a newly recognized hereditary focal epilepsy in dogs with distinctive clinical and histopathologic features. Veterinarians should be aware that sudden episodes of unusual behavior can represent focal epilepsy., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

No standardized treatment guidelines are reported in veterinary medicine for dogs with large pituitary corticotroph neoplasms causing neurological signs, and such dogs usually have a short overall survival. When these dogs undergo pituitary surgery and the tumor regrows there are few reports of subsequent treatments. A 7 yr old male Maltese diagnosed with pituitary-dependent hypercortisolism developed seizures in conjunction with a large pituitary corticotroph adenoma and underwent transsphenoidal hypophysectomy. After 3 yr of clinical remission, hypercortisolism recurred, and trilostane therapy was initiated. One year later, the dog developed new neurological signs and computed tomography revealed regrowth of a large pituitary mass that was then treated with radiation therapy. The dog lived disease-free for 3 more yr. At postmortem examination, a more aggressive pituitary neoplasm than the one examined at the time of surgery was found, which is suggestive of malignant transformation into a carcinoma despite the absence of convincing metastasis.

Background: Chronic hepatitis (CH) in dogs is common and has the tendency to progress to liver cirrhosis (LC). Circulating microRNAs might have the potential as markers for disease progression., Objectives: To investigate whether concentration of specific microRNAs in serum correlate with the stage and grade of CH in Labrador Retrievers., Animals: Twenty-two Labrador Retrievers with histological CH (n = 8), LC (n = 7), and normal liver (NL, n = 7)., Methods: In this retrospective study, serum concentrations of miR-122, miR-29a, miR-133a, miR-181b, and miR-17-5p were measured by quantitative real-time PCR and evaluated using univariate linear regression in dogs. A multivariate model was fit including the grade of hepatitis and the stage of fibrosis., Results: Of the 5 microRNAs, only circulating miR-122 and miR-29a were significantly associated with the grade of hepatitis and the stage of fibrosis. A positive correlation was identified between the grade of hepatitis with miR-122 (r s  = 0.79, P < .001) and miR-29a (r s  = 0.78, P < .001). Both miR-122 (r s  = 0.81, P < .001) and miR-29a (r s  = 0.67, P < .001) showed a significant positive correlation with the stage of fibrosis. MiR-122 concentrations were significantly higher in the CH (P < .01) and LC groups (P < .001) compared to the NL group. MiR-29a concentrations were significantly higher in the CH (P < .001) and LC (P < .001) groups compared to the NL group., Conclusions and Clinical Importance: Circulating miR-122 and miR-29a concentrations might be useful for monitoring the response to treatment and progression of canine CH., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

With the exception of classic functional adenomas in dogs and horses, pituitary lesions are infrequently described in the veterinary literature. Approximately 10% of pituitary glands from asymptomatic humans contain abnormalities, but the equivalent proportion in small animals is unknown. Pituitary glands from 136 dogs and 65 cats collected during routine necropsies were examined to determine the prevalence of pituitary lesions and their histopathological diagnosis. Lesions were characterized in sections stained with hematoxylin and eosin, periodic acid-Schiff (PAS), Gordon and Sweet's and reticulin stains, and immunohistochemistry for adrenocorticotropic hormone (ACTH), growth hormone, melanocyte stimulating hormone-α, and prolactin. Pituitary abnormalities were identified in 36 of 136 (26.4%) dogs and 10 of 65 (15.3%) cats. Cystic changes were the most common lesion, occurring in 18 (13.2%) dogs and 8 (12.3%) cats. Pituitary neoplasia was detected in 14.1% (12/85) of middle-aged and old dogs; 1 (1.5%) cat had pituitary nodular hyperplasia. PAS and reticulin stains helped differentiate ACTH-immunoreactive adenomas from hyperplastic nodules: adenomas contained PAS-positive intracytoplasmic granules and loss of the normal reticulin network. One dog had a pituitary carcinoma with infiltration into the thalamus. Other pituitary abnormalities included secondary metastases (2 dogs) and hypophysitis (4 dogs, 1 cat). In most cases, the lesion appeared to be subclinical and could be considered incidental, whereas clinical manifestations were apparent in only 4 dogs (2.9%) and none of the cats with pituitary lesions. Pituitary abnormalities are common in dogs and cats, and their clinical relevance requires further investigation.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

Equine intervertebral disc degeneration is thought to be rare and of limited clinical relevance, although research is lacking. To objectively assess pathological changes of the equine intervertebral disc and their clinical relevance, description of the normal morphology and a practical, biologically credible grading scheme are needed. The objectives of this study are to describe the gross and histological appearance of the equine intervertebral discs and to propose a grading scheme for macroscopic degeneration. Spinal units from 33 warmblood horses were grossly analyzed and scored. Of the 286 intervertebral discs analyzed, 107 (37%) were assigned grade 1 and grade 2 (considered normal) and were analyzed histologically. A nucleus pulposus and an annulus fibrosus could be identified macroscopically and histologically. Histologically, the nucleus pulposus was composed of a cartilaginous matrix and the annulus fibrosus of parallel collagenous bands. A transition zone was also histologically visible. Intra- and inter-observer reliability scores were high for all observers. Higher grades were associated with greater age. Gross changes associated with equine intervertebral disc degeneration (grades 3-5)-that is, yellow discoloration, cleft formation (tearing), and changes in consistency of the nucleus pulposus-were largely similar to those in humans and dogs and were most prevalent in the caudal cervical spine. Equine intervertebral disc degeneration was not associated with osteophyte formation. Changes of the vertebral bone were most common in the thoracolumbar spine but were not correlated with higher grades of intervertebral disc degeneration. Thus, changes of the vertebral bone should be excluded from grading for equine intervertebral disc degeneration.

Chronic low back pain is a common clinical problem in both the human and canine population. Current pharmaceutical treatment often consists of oral anti-inflammatory drugs to alleviate pain. Novel treatments for degenerative disc disease focus on local application of sustained released drug formulations. The aim of this study was to determine safety and feasibility of intradiscal application of a poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-bpoly(ε-caprolactone-co-lactide) PCLA-PEG-PCLA hydrogel releasing celecoxib, a COX-2 inhibitor. Biocompatibility was evaluated after subcutaneous injection in mice, and safety of intradiscal injection of the hydrogel was evaluated in experimental dogs with early spontaneous intervertebral disc (IVD) degeneration. COX-2 expression was increased in IVD samples surgically obtained from canine patients, indicating a role of COX-2 in clinical IVD disease. Ten client-owned dogs with chronic low back pain related to IVD degeneration received an intradiscal injection with the celecoxib-loaded hydrogel. None of the dogs showed adverse reactions after intradiscal injection. The hydrogel did not influence magnetic resonance imaging signal at long-term follow-up. Clinical improvement was achieved by reduction of back pain in 9 of 10 dogs, as was shown by clinical examination and owner questionnaires. In 3 of 10 dogs, back pain recurred after 3 months. This study showed the safety and effectiveness of intradiscal injections in vivo with a thermoresponsive PCLA-PEG-PCLA hydrogel loaded with celecoxib. In this set-up, the dog can be used as a model for the development of novel treatment modalities in both canine and human patients with chronic low back pain., (Copyright © 2017 John Wiley & Sons, Ltd.)

A 12-year-old male Yorkshire Terrier was presented because of decreased appetite. Physical examination revealed mammary gland swelling and galactorrhea. Contrast-enhanced computed tomographic scanning of the skull indicated an enlarged pituitary gland, compatible with a pituitary tumor. The serum prolactin concentration was markedly elevated. One week after the start of treatment with the dopamine agonist cabergoline, the serum prolactin concentration normalized and the galactorrhea resolved. Cabergoline was administered for approximately 4 months and then discontinued. Subsequently, serum prolactin concentration increased again, and mammary gland swelling and galactorrhea reappeared. The dog was euthanized 10 months after the first detection of the galactorrhea because of problems not directly related to pituitary disease. Postmortem examination revealed an infiltrative adenoma of the pituitary gland with immunolabeling for prolactin. The clinical and histopathologic findings indicated the diagnosis of a functional prolactinoma in a male dog.

Since the seminal work by Hans-Jörgen Hansen in 1952, it has been assumed that intervertebral disc (IVD) degeneration in chondrodystrophic (CD) dogs involves chondroid metaplasia of the nucleus pulposus, whereas in nonchondrodystrophic (NCD) dogs, fibrous metaplasia occurs. However, more recent studies suggest that IVD degeneration in NCD and CD dogs is more similar than originally thought. Therefore, the aim of this study was to compare the histopathology of IVD degeneration in CD and NCD dogs. IVDs with various grades of degeneration (Thompson grade I-III, n = 7 per grade) from both CD and NCD dogs were used (14 CD and 18 NCD dogs, 42 IVDs in total). Sections were scored according to a histological scoring scheme for canine IVD degeneration, including evaluation of the presence of fibrocyte-like cells in the nucleus pulposus. In CD dogs, the macroscopically non-degenerated nucleus pulposus contained mainly chondrocyte-like cells, whereas the non-degenerated nucleus pulposus of NCD dogs mainly contained notochordal cells. The histopathological changes in degenerated discs were similar in CD and NCD dogs and resembled chondroid metaplasia. Fibrocytes were not seen in the nucleus pulposus, indicating that fibrous degeneration of the IVD was not present in any of the evaluated grades of degeneration. In conclusion, intervertebral disc degeneration was characterized by chondroid metaplasia of the nucleus pulposus in both NCD and CD dogs. These results revoke the generally accepted concept that NCD and CD dogs suffer from a different type of IVD degeneration, in veterinary literature often referred to as chondroid or fibroid degeneration, and we suggest that chondroid metaplasia should be used to describe the tissue changes in the IVD in both breed types.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Selective removal of the pituitary adenoma has not been advocated in dogs with pituitary-dependent hypercortisolism because the pituitary adenoma is usually not visualized on routine computed tomography (CT). Dynamic pituitary CT scanning is aimed at the detection of the pituitary flush and, indirectly, at the presence and position of the adenoma. The first aim of this retrospective study was to compare findings of a multiple slice dynamic scanning protocol with those of a single slice dynamic protocol using a single slice CT scanner. The second aim was to compare the CT findings with surgical findings, and surgical findings with histopathological findings. Computed tomography with single and multiple slice dynamic scanning protocols was performed in 86 dogs with pituitary-dependent hypercortisolism. Thirty dogs underwent transsphenoidal hypophysectomy and pituitary specimens were collected as tumor, normal, mixed and neurohypophyseal samples and processed for histology. The pituitary flush was not detected more frequent in multiple slice dynamic scanning series than in single slice dynamic scanning series. However, in non-enlarged pituitaries, the flush was seen significantly more frequently than in enlarged pituitaries. Prediction of the nature of the tissue during hypophysectomy by the surgeon was inconclusive. In conclusion, when using a single slice CT scanner, both single or multiple slice dynamic scanning protocols can be used for localization of the neurohypophyseal flush, and, indirectly, the adenoma. However, based on this study, the aim of surgery in dogs with pituitary-dependent hypercortisolism remains total adenohypophysectomy, and when the neurophypophysis is recognized, it may be left in situ., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A retrospective radiographic study was performed to investigate the prevalence of diffuse idiopathic skeletal hyperostosis (DISH) and spondylosis deformans (spondylosis) in 2041 purebred dogs and to determine association with age, gender and breed. Four cases of DISH provided information on the appearance of canine DISH. The prevalence of DISH and spondylosis was 3.8% (78/2041) and 18.0% (367/2041), respectively. Of dogs with DISH, 67.9% (53/78) also had spondylosis, whereas 14.0% (53/367) of dogs with spondylosis also had DISH. Dogs with DISH and/or spondylosis were significantly older than those without spinal exostosis. The prevalence of DISH and spondylosis was 40.6% (28/69) and 55.1% (38/69), respectively, in Boxer dogs. Nineteen smaller breeds were not affected by DISH, but showed signs of spondylosis; only standard Poodles appeared not to be affected by either disorder. Radiography, computed tomography (CT), magnetic resonance imaging (MRI), and/or histopathology were used to investigate four DISH cases. It was concluded that spondylosis and DISH can co-occur in dogs. DISH has probably been previously under-diagnosed and mistaken for severe spondylosis. The diagnosis can be made using radiography, CT or MRI. On histology, DISH can be distinguished from spondylosis by the location (ventral longitudinal ligament) and extent of new bone formation., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)